(anura: bufonidae): uma anÁlise integrativa para …

ii

Instituto Nacional de Pesquisas da Amazônia -INPA

Programa de Pós-graduação em Genética, Conservação e Biologia Evolutiva

REVELANDO PADRÕES EVOLUTIVOS DO GÊNERO AMAZOPHRYNELLA

(ANURA: BUFONIDAE): UMA ANÁLISE INTEGRATIVA PARA REVELAR

PROCESSOS DE DIVERSIFICAÇÃO NA AMAZÔNIA

ROMMEL ROBERTO ROJAS ZAMORA

Manaus, Amazonas

Dezembro, 2018

iii

ROMMEL ROBERTO ROJAS ZAMORA

REVELANDO PADRÕES EVOLUTIVOS DO GÊNERO AMAZOPHRYNELLA

(ANURA: BUFONIDAE): UMA ANÁLISE INTEGRATIVA PARA REVELAR

PROCESSOS DE DIVERSIFICAÇÃO NA AMAZÔNIA

TOMAS HRBEK

Marcelo Gordo

Tese apresentada ao Instituto

Nacional de Pesquisas da

Amazônia como parte dos

requisitos para obtenção do título

de Doutor em Genética,

Conservação e Biologia Evolutiva

Manaus, Amazonas

Dezembro, 2018

v

Z25 Zamora, Rommel Roberto Rojas

Revelando padrões evolutivos do gênero Amazophrynella

(Anura: Bufonidae): Uma análise integrativa para revelar

processos de diversificação na Amazônia / Rommel Roberto

Rojas Zamora --- Manaus : [s.n.], 2018. v, 180 f. : il. color.

Tese (Doutorado) --- INPA, Manaus, 2019.

Orientador : Tomas Hrbek.

Coorientador: Marcelo Gordo.

Área de concentração : Genética, Conservação e Biologia

Evolutiva.

1.Amazophrynella. 2.Biogeografia. 3. Bufonidae. I. Título.

CDD

597.8

Sinopse:

Estudou-se a sistemática, taxonomia e diversificação do

gênero Amazophrynella (Anura). Reconstruímos uma

hipótese filogenética do gênero, atualizou-se seu status

taxonômico e inferiu processos sobre sua diversificação.

Palavras-chave: Herpetologia, diversidade, biogeografia,

identificação

vi

Dedicatória

Dedico este trabalho aos meus pais, Roberto e Elvira, por cuidar com empenho e

bom exemplo sua descendência

vii

Agradecimentos

Imensa e extremamente agradecido aos meus Pais: Roberto e Elvira e aos

meus irmãos Rosa e Carlo pelo apoio sem condições até encontrar o meu próprio

caminho na vida;

Agradeço aos meus orientadores Tomas Hrbek e Marcelo Gordo pelos

constantes ensinamentos;

Professora Izeni Pires Farias por me abrir as portas do Laboratório de

Evolução e genética animal-LEGAL;

Vinicius Carvalho, Alexandre Almeida que me ajudaram no início de este

processo desde que cheguei ao Brasil e Maria Tereza no ultimo estagio desta tese;

Meus amigos do LEGAL: Fabio, Valeria, Joiciane, Luciana, Juliana, Priscila,

Carol, Luceia, Mario, Israela, Roberta, Jessica, Elciomar, Sandra, Guta, Fabricio,

Rodrigo e Igor do LABEbo;

Aos meus amigos do projeto Sauim, Leandro Sauim, Leandro Capitão,

Tainara S., Aline M., Raiclicia, Erika Marina Del valle, Daisuke, Edson mucura;

Aos meus amigos das Chepas: Marco, Andy, Cilnio, Tony, Piero, Giu, Chuck

Morris, Cristian, Josep y Carlos oruga pela constante ajuda e risos;

Aos cantantes e grupos que me acompanharam com sua música durante este

doutorado, principalmente The Doors, Los Prisioneiros, Los Destellos, Juaneco, Pink

Floyd, Los Ramones, Ozzy e The ventures;

Meus tios: Ronald, Michel, Marcos, Nora, Silvia, Coco, Pablo, Percy, Elda,

Helencith, Moises; Jomber, Karina.

Aos meus queridos vovós in memoriam: Melitón e Micaela e Jorge e Otília por

seu exemplo;

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior- CAPES pela

bolsa de estudo, Universidade federal do Amazonas-UFAM e Instituto Nacional de

Pesquisas da Amazônia- INPA.

viii

Epígrafe

“Quero dizer agora o oposto do que eu disse antes,

Eu prefiro ser essa metamorfose ambulante

Do que ter aquela velha opinião formada sobre tudo...”

Raul Seixas

ix

Resumo geral

A exploração irracional de recursos naturais, destruição de habitats,

aquecimento global, doenças entre outras ameaças naturais e antropogênicas são

responsáveis pela redução da diversidade do mundo. Assim, delimitar espécies e

identificar processos evolutivos que originaram a diversidade biológica na terra,

especialmente em regiões hotspot de diversidade como a Amazônia, é um dos

maiores desafios científicos da atualidade. Em este sentido, é sumamente

importante documentar a diversidade biológica dentro de um espaço e tempo

histórico antes que desapareça. Este doutorado se centrou em revelar a diversidade

de espécies do gênero Amazophrynella (Anura: Bufonidae) usando diferentes

caracteres evolutivos e reconstruir seus padrões filogenéticos e história

biogeográfica. Integramos características morfológicas (quantitativas e qualitativas),

genéticas (sequências de DNA), comportamentais (cantos de acasalamentos) e

ecológicas (valores ambientais) para delimitar espécies do gênero Amazophrynella.

Usamos as relações filogenéticas de genes mitocondriais e genômicos (ddrad-seq)

entre Amazophrynella (Amazônia) e seu gênero irmão Dendrophryniscus (Mata

Atlântica) para reconstruir a divergência histórica entre estas duas áreas e discutir

subsequentes eventos de diversificação na Amazônia. Nossos resultados

evidenciaram uma grande subestimação da riqueza de espécies do gênero

Amazophrynella, sendo descritas 5 novas espécies, assim também, descrevemos os

parâmetros espectrais e temporais dos cantos de acasalamento de 4 espécies e

redescrevemos a espécie tipo do gênero: A. minuta. As reconstruções

biogeográficas permitiram conhecer o tempo histórico de divergência entre a

Amazônia e Mata Atlântica (Eoceno), e detectar um padrão basal Leste-Oeste

(Mioceno) seguido por uma diversificação Norte-Sul dentro da Amazônia. Este

trabalho atualizou o status taxonômico e sistemática do gênero Amazophrynella e

propõe um modelo complexo de diversificação de anfíbios no neotrópico, dominado

por episódios de fragmentação de populações ancestrais por mecanismos de

vicariância e influência de fatores ecológicos em sua diversificação.

Palavras chave: Amazônia, biogeografia, conservação, sistemática, taxonomia

x

Abstract

Revealing evolutionary patterns of genus Amazophrynella (Anura: Bufonidae): an

integrating analysis to discover diversification processes in the amazon basin

Irrational exploitation of natural resources, habitat destruction, global warming,

diseases and other natural and anthropogenic threats are responsible for reducing

the diversity of the world. Thus, delimiting species and identifying evolutionary

processes that originated the biological diversity in the earth, especially in high

diversity areas of as the Amazon, is one of the greatest scientific challenges of the

present times. It is extremely important to document biological diversity within a

historical space and time before it disappears. This PhD focused on revealing the

diversity of species of the genus Amazophrynella (Anura: Bufonidae) using different

evolutionary traits and reconstructing their phylogenetic patterns and biogeographic

history. We integrated morphological characteristics (quantitative and qualitative),

genetic (DNA sequences), behavioral (mating songs) and ecological (environmental

values) to delimit species of the genus Amazophrynella. We used the phylogenetic

relationships of mitochondrial and genomic genes (ddrad-seq) between

Amazophrynella (Amazon) and its sister genus Dendrophryniscus (Atlantic Forest) to

reconstruct the historical divergence between these two areas and discuss

subsequent diversification events in the Amazon. The results showed a great

underestimation of the richness of species of the genus Amazophrynella, being

described 5 new species, we also describe the spectral and temporal parameters of

the mating songs of 4 species and we redraw the type species of the genus: A.

minuta. The biogeographic reconstructions allowed to know the historical time of

divergence between the Amazon and Atlantic Forest (Eocene), and to detect a basal

East-West (Miocene) pattern followed by a North-South diversification within the

Amazon. We updated the taxonomic and systematic status of the genus

Amazophrynella and proposes a complex model of diversification of amphibians in

the neotropics, dominated by episodes of fragmentation of ancestral populations by

mechanisms of vicariance and influence of ecological factors in their diversification.

Keywords: Amazon, biogeography, conservation, systematics, taxonomy.

xi

Sumario

INTRODUÇÃO GERAL ..................................................................................... 1

Estado da arte do gênero Amazophrynella ..................................................... 6

História natural ............................................................................................. 6

História taxonômica ..................................................................................... 6

Sistemática de Amazophrynella ................................................................. 12

Problemática ................................................................................................. 13

Hipóteses ...................................................................................................... 14

OBJETIVOS ..................................................................................................... 14

Geral ............................................................................................................. 14

Específicos .................................................................................................... 14

REFERÊNCIAS BIBLIOGRÁFICAS……………………………………………….15

CAPITULO I. Uncovering the diversity inside the Amazophrynella minuta complex:

integrative taxonomy reveals a new species of Amazophrynella (Anura, Bufonidae)

from southern Peru……………………………………………………...…...……….23

CAPITULO II. A Pan-Amazonian species delimitation: high species diversity within

the genus Amazophrynella (Anura: Bufonidae)………………………..………….64

CAPITULO III. Description of the advertisement call of four species of

Amazophrynella (Anura: Bufonidae)……………………………………………….149

CAPITULO IV. Redescription of the Amazonian tiny tree toad Amazophrynella

minuta (Melin, 1941) (Anura: Bufonidae) from its type Locality…………………157

CAPITULO V. Diversification in Amazonian through the historical biogeography of

“terra firme” tiny tree toads Amazophrynella (Anura: Bufonidae)……………….190

CONCLUSÕES GERAIS...................................................................................218

xii

Lista de Figuras

Figura 1. Número de espécies ao redor do mundo, nota-se a presença de uma

maior abundância na Amazônia...................................................................................1

FIGURA 2. Protocolo de delimitação de espécies pelo critério da taxonomia

integrativa. Imagem obtida de Padial et al. (2010).......................................................3

FIGURA 3. Distribuição geográfica do gênero Amazophrynella (Anura:

Bufonidae)....................................................................................................................4

FIGURA 4. Espécies nominais de Amazophrynella: A) A. amazonicola, B) A. matses;

C) A. minuta; D) A. bokermanni; E) A. vote; F) A. manaos......................................... 5

FIGURA 5. Padrões morfológicos de Amazophrynella minuta. A) Rio Curaray, Peru

(Foto: Gagliardi); B) Rio Tahuayo, Peru (Foto: Medina); C) Taracuá, Brasil; D) Rio

Imiri, Peru; E) Reserva Yasuni, Equador (Foto: Ron)..................................................7

FIGURA 6. Padrões morfológicos de Amazophrynella bokermanni. A) Juruti, Brasil

(Foto: ©Gordo); B) Juruti, Brasil (Foto: ©Gordo); C) Rio Xingu, Brasil (©Peloso); D)

Rio Tapajós, Brasil (© Oliveira)....................................................................................8

FIGURA 7. Padrões morfológicos de Amazophrynella vote. A) Município de

Aripuanã, Brasil; B) Parque Nascente do Lago Jari, Brasil; C) Igarapé Açuã, Brasil,

D) Fazenda São Nicolau, Brasil. Fotos: ©Ávila............................................................9

FIGURA 8. Padrões morfológicos de Amazophrynella manaos. A) Campus da

Universidade Federal do Amazonas-UFAM, Brasil; B) Reserva CEPEAM, Brasil; C)

Reserva de Ducke, Brasil; D) Presidente Figueiredo, Brasil......................................10

FIGURA 9. Distribuição esquemática das seis espécies nominais de

Amazophrynella. Azul: A. amazonicola; Amarelo: A. matses; Celeste: A. minuta;

Vermelho: A. manaos; Rojo: A. bokermanni; Laranja: A. vote. Espaços sem cores

não apresentam dados para o gênero.......................................................................11

FIGURA 10. Relações filogenéticas do gênero Amazophrynella obtidas de Rojas et

al., (2015). Análise de Máxima verossimilhança do gene 16S DNAmt

...................................................................................................................................12

1

INTRODUÇÃO GERAL

Estima-se a existência de 6.5 milhões espécies terrestres e 2.2 milhões de

espécies oceânicas e que quase 30% das espécies do mundo ainda não foram

descritas (Mora et al., 2011). A Amazônia é considerada a área com maiores

registros de espécies de flora e fauna do mundo (Jenkins et al., 2013) (Figura 1),

sendo que, explicar a origem da sua diversidade tem intrigado naturalistas e

evolucionistas ao longo do tempo (Ribas et al., 2012).

FIGURA 1. Número de espécies ao redor do mundo, nota-se a presença de uma

maior abundância na Amazônia.

Entender os padrões e processos que geraram a biodiversidade é essencial

para o desenvolvimento de estratégias de conservação (Xu et al., 2008) e fornece

pistas para o entendimento da distribuição da biodiversidade em diversos grupos

taxonômicos (Webb, 2000; Webb et al., 2002; Vitt et al., 1999). Alfred Russel

Wallace (Wallace, 1852) postulou que os padrões de diversidade de vertebrados são

gerados pelos rios Amazônicos, desde então diversas hipóteses alternativas tem

sido propostas, visando explicar a distribuição e origem da diversidade (Haffer, 1969;

2

Fjeldsa, 1995; Endler, 1997; Colinvaux, 2001; Marrgoi e Cerqueira, 1997; Nores,

1999).

Reconstruções geológicas da Amazônia sugerem uma grande dinâmica ao

longo do tempo (Hoorn et al., 2010). Diversos estudos sugerem que os padrões de

diversidade atual não surgiram pela influência de um único evento diversificador,

mas sim pela combinação de eventos históricos, tais como: a reorganização

geológica (Hoorn et al., 2010) e modificações paleogeográficas (Rull, 2008) durante

o Neógeno, e eventos ecológicos: como mudanças cíclicas climáticas do Pleistoceno

(Haffer e Prance, 2001).

É conhecido que a distribuição da fauna Amazônica não ocorre ao acaso e

segue um padrão geral em áreas de endemismo que são delimitadas pelos grandes

interflúvios (Silva et al., 2005). Essas áreas possuem congruência de táxons

espacial e filogeneticamente com um conjunto próprio de linhagens e com trajetórias

evolutivas diferenciadas (Silva et al., 2005). Smith et al. (2014) reconheceram nove

grandes áreas de endemismo para aves na Amazônia: Guiana, Imeri, Napo,

Inambari, Rondônia, Tapajós, Xingu, Belém e Huallaga, sendo reconhecida também

a área do interflúvio Rio Negro/Solimões- Jaú (Borges e Silva, 2012).

Processos como extinção, vicariância e dispersão são reconhecidos como

processos naturais de diversificação ao longo da evolução dos taxa (Wiens e

Donoghue, 2004). De maneira geral, a especiação simpatrica (competição entre

populações com posterior divergência de nicho ecológico) e alopátrica (presença de

uma barreira geográfica) são atribuídos como os tipos de especiação mais comuns

na Amazônia (Rull, 2008; Antonelli et al., 2010, 2018). Assim, com o rápido

crescimento do conhecimento filogenético, processos de diversificação e eventos

evolutivos (Donoghue, 2008; Antonelli et al., 2010; Smith et al., 2014) novas

percepções sobre os padrões de diversidade no neotrópico foram questionadas,

como por exemplo a existência de complexos de espécies.

Diferentes grupos taxonômicos de vertebrados distribuídos na Amazônia são

considerados como complexos de espécies: mamíferos (Galimberti et al., 2015);

aves (Fernandes et al., 2013); répteis (Miralles e Carranza, 2010); peixes (Machado

et al., 2018) e anuros (Gehara et al., 2014) caracterizados por apresentar várias

3

linhagens evolutivas independentes nomeadas como uma única espécie através de

sua distribuição geográfica. Levando em consideração que o número de espécies

reconhecidas para a Amazônia encontra-se atualmente subestimada (Fouquet et al.,

2007), delimitar limites entre espécies torna-se fundamental para a conservação da

diversidade biológica.

Atualmente a comunidade científica aceita o fato de que o processo de

delimitação de espécies precisa ser multicaracteres (Padial et al., 2010), usando

caracteres morfológicos: (ex. caracteres diagnósticos merísticos, morfométricos e

diferenças estatísticas); pre- zigóticos (análises dos parâmetros espectrais e

temporais de cantos de acasalamento); ecológicos (requerimentos ambientais,

sobreposição de nicho, comportamento) e genéticos (monofilia, técnicas de

coalescência como GMYC, BPTP, bGMYC, distâncias genéticas, caracteres

diagnósticos genéticos) entre outros (ex. Angulo e Reichle, 2008; Ortega-Andrade et

al., 2015; Zahng et al., 2013). A integração desses critérios operacionais, permite

uma estabilidade taxonômica em grupos morfologicamente conservados ou aqueles

que apresentam alta plasticidade fenotípica, evitando assim a subestimação ou

superestimação da diversidade de espécies (Hebert et al., 2003; Hebert et al., 2005;

Simões et al., 2008; Elmer et al., 2013; Rojas et al., 2016) (Figura 2).

FIGURA 2. Protocolo de delimitação de espécies pelo critério da taxonomia

integrativa. Imagem obtida de Padial et al. (2010).

Em comparação com outras Classes de vertebrados, os Anfíbios,

particularmente espécies da Ordem Anura, constituem um excelente grupo para

4

estudar a diversidade subestimada e inferir padrões biogeográficos por possuírem

uma diversidade críptica o pseudocriptica (Funk et al., 2012; Cornils e Held, 2014),

representam complexos de espécies com várias linhagens independentes (Gehara

et al., 2014), pouco explorados taxonomicamente, baixa capacidade de dispersão

vs. aves e mamíferos (Vences e Wake, 2007), são sensíveis as mudanças

ambientais e eventos históricos (Godinho e Da Silva, 2018), possuem alta taxa

reprodutiva, dependem da qualidade do habitat e possuem ambas fases de vida

(aquática e terrestre) (Vences e Wake, 2007).

Além de ser um grupo propicio para inferir padrões evolutivos e pouco

conhecido taxonomicamente , estima-se que 40% das espécies de Anuros se

encontram em perigo de extinção (Collins, 2010), e que, além da destruição de seus

habitats, o fungo Batrachochytrium dendrobatidis encontra-se diminuindo

progressivamente as populações nos Andes e Amazônia (Becker et al., 2016;

Catenazzi e von May, 2014).

Neste contexto encontra-se o gênero Amazophrynella, este gênero

compreende anfíbios anuros pertencentes à família Bufonidae. O gênero apresenta

distribuição Pan- amazônica, sendo distribuído nos territórios da Bolívia, Peru,

Equador, Colômbia, Venezuela, Brasil, Suriname e Guiana Francesa e Guiana

(Fouquet et al., 2012a; Rojas et al., 2018a) (Figura 3).

FIGURA 3. Distribuição geográfica do gênero Amazophrynella (Anura: Bufonidae).

5

O gênero Amazophrynella é irmão de Dendrophryniscus, que possui espécies

restritas à Mata Atlântica do Brasil. As espécies de Amazophrynella encontram-se

restritas em alturas que variam entre 50-708 metros desde o nível do mar. No início

deste doutorado eram reconhecidas 06 espécies ao longo de toda sua distribuição

geográfica, sendo que 06 novas espécies foram descritas no processo deste

trabalho (Rojas et al., 2016, 2018a) (Figura 4).

FIGURA 4. Espécies nominais de Amazophrynella: A) A. amazonicola, B) A. matses;

C) A. minuta; D) A. bokermanni; E) A. vote; F) A. manaos.

6

Estado da arte do gênero Amazophrynella

História natural

Amazophrynella significa “pequenos Amazônicos” (Fouquet et al., 2012a). As

espécies apresentam esse nome porque são de tamanhos pequenos e ocorrem na

Amazônia. Os machos adultos apresentam entre 13-16 milímetros (mm), enquanto

as fêmeas entre 15-25 mm. São espécies de terra firme, encontrados

preferencialmente na serapilheira de bosques secundários e primários, as espécies

apresentam dimorfismo sexual evidente, sendo as fêmeas maiores que os machos,

sua reprodução ocorre durante a época de chuvas (Novembro - Fevereiro) (Rojas et

al., 2014, 2018b).

Os machos cantam em frequências altas (> 3000 Hz) próximos de pequenas

poças d` água de pouca profundidade durante a manhã ou crepúsculo, sendo

possível encontrar abundância de indivíduos agrupados ao redor de pequenos

corpos d`água com abundantes folhas, galhos, árvores ou arbustos (comunicação

pessoal do autor). Os ovos são pigmentados e são depositados em raízes de

árvores e arbustos ou debaixo da serapilheira úmida (Ávila et al., 2012; Rojas et al.,

2015, 2016). Os girinos são pequenos e vivem em pequenos grupos, são apenas

descritos para duas espécies: A. siona e A. manaos.(Menin et al., 2014; Rojas et al.,

2018a).

História taxonômica

A primeira espécie do gênero descrita foi Amazophrynella minuta (Melin,

1941) como Atelopus minutus, a localidade tipo é Taracuá, São Gabriel da

Cachoeira, estado do Amazonas, Brasil. A descrição foi muito breve e a diagnose

morfológica generalizada (Melin, 1941; Rojas et al., 2018c). Posteriormente

McDiarmid (1971) mudou para Dendrophryniscus minutus. Historicamente o nome

de A. minuta foi usado para indivíduos distribuídos em toda a Amazônia durante

muito tempo como o Peru, Equador, Brasil, Guiana francesa (ex. Duellman 1978;

Zimmerman e Rodrigues 1990; Magnusson e Hero 1991; Rodrigues e Duellman

1993; Duellman e Mendelson 1995; Fouquet et al., 2012a) desde o ano 1941 até o

2012 (Figura 5).

7

FIGURA 5. Padrões morfológicos de Amazophrynella minuta. A) Rio Curaray, Peru

(Foto: Gagliardi); B) Rio Tahuayo, Peru (Foto: Medina); C) Taracuá, Brasil; D) Rio

Imiri, Peru; E) Reserva Yasuni, Equador (Foto: Ron).

Posteriormente foi descrita Amazophrynella bokermanni (Izecksohn, 1992)

como uma espécie do gênero Dendrophryniscus. Esta espécie possui o I dedo da

mão maior ou igual ao II, sendo uma característica das espécies dentro deste

complexo (Rojas et al., 2018a). Foram reportadas populações do rio Tapajós e Rio

trombetas, estado do Pará, Brasil (Àvila et al.,2012), Urucu, município de Coari,

Amazonas e no baixo rio Purus (Waldes et al., 2013), mas esta população foi

8

confundida com A. vote (observação pessoal do autor). Esta espécie apresenta

profundas divergências morfológicas (Figura 6).

FIGURA 6. Padrões morfológicos de Amazophrynella bokermanni. A) Juruti, Brasil

(Foto: ©Gordo); B) Juruti, Brasil (Foto: ©Gordo); C) Rio Xingu, Brasil (©Peloso); D)

Rio Tapajós, Brasil (© Oliveira)

No ano 2012 foi proposto o gênero Amazonella (Fouquet et al., 2012b) mas

foi substituído por Amazophrynella (Fouquet et al., 2012a) para as duas espécies de

Dendrophryniscus (D. minutus e D. bokermanni) distribuídos na Amazônia. As

espécies de Dendrophryniscus da Mata Atlântica do Brasil mantiveram sua

identidade taxonômica.

9

O 2012 ano foi descrita A. vote Ávila, Carvalho, Gordo, Ribeiro & Morais 2012.

Esta espécie distribui-se nos municípios de Cotriguaçu; Colniza, Aripuanã; Parque

Estadual do Guariba, Manicoré; Reserva Biológica de Jaru, Ji-Paraná; Lago Açaí,

Novo Aripuanã; Parque Estadual do Matupiri, Manicoré; Cachoeirinha, Manicoré;

Parque Nacional Nascentes do Lago Jari, Tapauá; Igarapé Açuã. O principal caráter

de identificação morfológica é o padrão de coloração ventral e o tamanho (Ávila et

al., 2012); mas além de sua descrição, não se conhece nada sobre seus padrões

ecológicos, variação morfológica, entre outras características. Esta espécie também

apresenta variações fenotípicas (Figura 7).

FIGURA 7. Padrões morfológicos de Amazophrynella vote. A) Município de

Aripuanã, Brasil; B) Parque Nascente do Lago Jari, Brasil; C) Igarapé Açuã, Brasil,

D) Fazenda São Nicolau, Brasil. Fotos: ©Ávila

10

Amazophrynella manaos Rojas, Carvalho, Gordo, Ávila, Pires & Hrbek 2014

(Figura 8) apresenta como localidade tipo o campus da Universidade Federal do

Amazona-UFAM, Brasil, e sua distribuição geográfica abrange as localidades de

Mineração Taboca, Reserva Florestal Adolpho Ducke, Presidente Figueiredo,

Reserva ZF-2, REBIO Uatumã, RDS Uatumã e Parque Estadual Rio Negro Setor

Sul, Rio Cuieiras. Estudos prévios indicam uma linhagem evolutiva independente

dentro de essa espécie localizada na Guiana Francesa (Fouquet et al., 2012a).

FIGURA 8. Padrões morfológicos de Amazophrynella manaos. A) Campus da

Universidade Federal do Amazonas-UFAM, Brasil; B) Reserva CEPEAM, Brasil; C)

Reserva de Ducke, Brasil; D) Presidente Figueiredo, Brasil.

11

Posteriormente foram descritas Amazophrynella amazonicola Rojas,

Carvalho, Ávila, Pires, Gordo & Hrbek 2015 e A. matses Rojas, Carvalho, Ávila,

Pires, Gordo & Hrbek 2015, estas duas últimas espécies encontram-se distribuídas

dentro do departamento de Loreto, Perú (Rojas et al., 2015). Se conhece pouco

sobre sua história natural e extensão de sua distribuição geográfica. Estas duas

espécies formam parte do complexo A. minuta.

FIGURA 9. Distribuição esquemática das seis espécies nominais de

Amazophrynella. Azul: A. amazonicola; Amarelo: A. matses; Celeste: A. minuta;

Vermelho: A. manaos; Rojo: A. bokermanni; Laranja: A. vote. Espaços sem cores

não apresentam dados para o gênero.

Entre os trabalhos realizados com o gênero em estudo, podemos citar

Duellman (1978), Rodrigues e Duellman (1994), Duellman e Mendelsom (1995) e De

la Riva et al. (2010). Mas estes trabalhos focam-se principalmente, em apresentar

pontos de distribuição geográfica de A. minuta e A. bokermanni. Com respeito as

outras espécies de Amazophrynella, não existem discussões taxonômicas e estudos

de variação populacionais dos seus aspectos morfológicos, genéticos e aspectos de

história natural.

12

Sistemática de Amazophrynella

A primeira hipótese filogenética do gênero reconheceu duas espécies

nominais: A. minuta e A. bokermanni, e duas linhagens evolutivas independentes: A.

aff. minuta “western Amazonian” e A. sp “Guianas” (Fouquet et al., 2012a). Uma

árvore mais completa (Rojas et al., 2014) mostrou a existência de um complexo de

espécies dentro de A. minuta e populações não descritas relacionadas com A.

manaos.

Na hipótese filogenética de Rojas et al. (2015) identifica-se unidades

evolutivas independentes pertencentes ao complexo de A. minuta e A. manaos,

sendo descritas A. amazonicola e A. matses (Figura 10). Posteriores estudos em

sistemática e taxonomia do gênero foram realizados durante o processo deste

trabalho, onde foram descritas A. javierbustamantei (Rojas et al., 2016- ver capitulo

I) e A. teko, A. siona, A. xinguensis e A. moisesii (Rojas et al., 2018- ver capitulo II).

FIGURA 10. Relações filogenéticas do gênero Amazophrynella obtidas de Rojas et

al., (2015). Análise de Máxima verossimilhança do gene 16S DNAmt baseado em

480 pares de base.

13

Uma das características atuais de Amazophrynella e a existência de

distâncias genéticas elevadas. Rojas et al. (2014) através do uso do gene

mitocondrial 16S RNA observaram elevadas divergências moleculares (≤4% e

≥15%) entre as espécies conhecidas.

As elevadas divergências moleculares, junto com as características de vida destas

pequenas espécies, assim como o conhecimento da existência de uma diversidade

subestimada neste gênero considerado como críptico (Fouquet et al., 2007, Fouquet

et al., 2012a, Rojas et al., 2014), podem supor que as populações das espécies de

Amazophrynella possam representar espécies novas.

Problemática

Com o advento de análises genéticas coalescentes do sequenciamento

mitocondrial e de nova geração (next generation sequences- ddrad) junto com o uso

do protocolo de delimitação de espécies da taxonomia integrativa e baixo o conceito

unificado de espécies (De Queiroz, 2007), uma nova era para taxonomia tornou-se

possível, já que com as relações sistemáticas do gênero Amazophrynella atualizada

será possível inferir hipóteses mais exatas da influência dos eventos históricos,

biogeográficos e ecológicos em linhagens corretamente delimitadas. Esta

abordagem tornará possível novas interpretações da história biogeográfica das

espécies e permitirá conhecer a diversidade escondida de anuros e outros taxas

Amazônicos.

Historicamente as principais dificuldades em conhecer a diversidade do

gênero Amazophrynella foi a diagnose morfológica generalizada da espécie tipo: A.

minuta (Melin, 1941), ausência de amostras para análises comparativas

morfológicas, genéticas e do conhecimento básico da sua história natural e

comportamento reprodutivo (ex. cantos de acasalamentos). Adicionando-se a

inexistência de uma hipótese filogenética completa do gênero que permita identificar

linhagens evolutivas independentes, abordar a sistemática molecular junto a

taxonomia integrativa e entender seus padrões biogeográficos para revelar

processos de diversificação na Amazônia.

14

Hipóteses

No presente estudo as hipóteses são de que (1) o gênero Amazophrynella

apresenta uma diversidade subestimada de espécies sendo que o número de

espécies atuais não reflete a sistemática atual; (2) os atuais padrões de distribuição

das linhagens de Amazophrynella apresentam uma radiação evolutiva influenciada

por eventos históricos e ecológicos do Neógeno (Mioceno-Plioceno).

OBJETIVOS

Geral

Revisar a sistemática e taxonomia do gênero Amazophrynella e estudar os

padrões e processos envolvidos na origem de sua diversidade na Amazônia.

Específicos

Reconstruir a filogenia de Amazophrynella e usar a taxonomia integrativa para

delimitar espécies e propor rearranjos taxonômicos com o intuito de revelar a

diversidade escondida de anuros neotropicais;

Investigar a história evolutiva e diversificação em Amazophrynella visando

entender a divergência entre Amazônia e Mata Atlântica e procurando padrões

biogeográficos de anuros na Amazônia.

15

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CAPITULO I

Uncovering the diversity inside the Amazophrynella minuta complex: integrative

taxonomy reveals a new species of Amazophrynella (Anura, Bufonidae) from

southern Peru. Rojas, R.R., Chaparro, J.C., Carvalho, V.T. De, Ávila, R.W., Farias,

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24

Uncovering the diversity inside the Amazophrynella minuta complex (Anura,

Bufonidae): integrative taxonomy reveals a new species from southern Peru

Rommel R. Rojas1,2*, Juan C. Chaparro3, Vinícius Tadeu De Carvalho2,4, Robson W.

Ávila5, Izeni Pires Farias2, Tomas Hrbek2 and Marcelo Gordo6

1 Programa de Pós-graduação em Genética Conservação e Biologia Evolutiva-

Instituto Nacional de Pesquisas da Amazônia-INPA, Av. André Araújo, 2936,

Manaus, Brazil, 2 Laboratório de Genética e Evolução Animal, Departamento de

Genética, ICB, Universidade Federal do Amazonas, Av. Gen. Rodrigo Octávio

Jordão Ramos, 3000, Manaus, Brazil, 3 Museo de Historia Natural, Universidad

Nacional de San Antonio Abad del Cusco, Peru, 4 Programa de Pós-Graduação em

Biodiversidade e Biotecnologia, Av. Gen. Rodrigo Octávio Jordão Ramos, 3000,

Manaus, Brazil, 5 Universidade Regional do Cariri, Centro de Ciências Biológicas e

da Saúde, Departamento de Ciências Biológicas, Campus do Pimenta, Rua Cel.

Antônio Luiz, 1161, Bairro do Pimenta, Crato, Brazil, 6 Departamento de Biologia,

ICB, Universidade Federal do Amazonas, Av. Gen. Rodrigo Octávio Jordão Ramos,

3000, Manaus, Brazil.

*Corresponding autor: [email protected]

25

Abstract.

A new species of the genus Amazophrynella (Anura, Bufonidae) is described from

the departments of Madre de Dios, Cusco and Junin in Peru. An integrative taxonomy

approach is used. A morphological diagnosis, morphometrics comparisons,

description of the advertisement call, and the phylogenetic relationships of the new

species are provided. Amazophrynella javierbustamantei sp. n. differs from other

species of Amazophrynella by: intermediate body-size (snout-vent length 14.9 mm

in males, n = 26 and 19.6 mm in females, n = 20), tuberculate skin texture of body,

greatest hand length of the Amazophrynella spp. (3.6 mm in males, n = 26 and 4.6

mm in females, n = 20), venter coloration yellowish, tiny rounded black points

covering the venter, and thirteen molecular autapomorphies in the 16S RNA gene. Its

distribution varies from 215 to 708 m a.s.l. This discovery highlights the importance of

the remnant forest in preserving the biodiversity in Peru, and increase in seven the

species formally described in the genus Amazophrynella.

Resumen.

Describimos una nueva especie del género Amazophrynella (Anura, Bufonidae) del

Perú de los Departamen- tos de Madre de Dios, Cusco y Junin de Peru. Utilizamos

un método de taxonomía integrativa. Obtuvimos la diagnosis morfológica,

comparaciones morfométricas, descripción del canto de reproducción y las rela-

ciones filogenéticas de la nueva especie. A. javierbustamantei sp. n. difiere de las

otras Amazophrynella spp. por poseer tamaño medio (Hocico-cloaca en machos 16.9

mm, n = 26 y en hembras 19.6 mm, n = 20); textura de la piel tuberculada; tamaños

de las manos mayores (3.6 mm en machos, n = 26 y 4.6 mm en hembras, n = 20);

coloración ventral amarillento-pálida, pequeños puntos redondos de color negro en

el vientre y por trece autopomorfias moleculares en el gen 16S RNA. Su distribución

varía desde 215 m hasta 708 m a.s.n.m. Este descubrimiento resalta la importancia

de los remanentes de la selva Peruana en térmi- nos de conservación, e incrementa

en siete las especies formalmente descritas en del género Amazophrynella.

26

Resumo.

Descrevemos uma nova espécie do gênero Amazophrynella (Anura, Bufonidae) dos

departamentos de Madre de Dios, Cusco e Junin do Peru. Utilizamos um método de

taxonomia integrativa. Apresentamos a diagnose morfológica, comparações

morfométricas, descrevemos o canto de anúncio e geramos uma hipó- tese

filogenética da nova espécie. Amazophrynella javierbustamantei sp. n. difere das

outras Amazophrynella spp. por possuir tamanho médio (Comprimento rostro-cloacal

16.9 mm em machos, n = 26 e 19.6 mm em fêmeas, n=20); textura da pele

tuberculada; tamanhos das mãos maiores (3.6 mm em machos, n = 26 e 4.6 mm em

fêmeas, n = 20); coloração ventral amarelo-clara, coberta por pequenos pontos

redondos pretos e por treze autapomorfias moleculares no gene 16S RNA. Sua

distribuição varia entre os 215 m até os 708 m a.n.m. Nossa descoberta aumenta a

importância dos remanescentes da floresta Peruana em termos de conservação e

incrementa em sete as espécies formalmente descritas no gênero Amazophrynella.

Keywords

Amphibian, Tree Toad, conservation, South of Peru, integrative taxonomy

Palabras claves

Anfibios, Sapo del árbol, conservación, Sur del Perú, taxonomía integrativa

Palavras chaves

Anfíbios, Sapo do arvore, conservação, Sul do Peru, taxonomia integrativa

27

Introduction

Until 2012, two species of Amazophrynella were placed in the genus

Dendrophryniscus Jimenez de la Espada, 1868. Fouquet et al. (2012a) recognized

that species of Den- drophryniscus from the Amazon and Atlantic rainforests

represented morphologically and genetically deeply divergent lineages, and thus the

authors proposed a new genus: Amazophrynella Fouquet, Recorder, Texeira,

Cassimiro, Amaro, Camacho, Damace- no, Carnaval, Moritz & Rodrigues, 2012 for

the Amazonian species A. minuta and A. bokermanni.

In the following years, an additional four new species of the genus were

described: A. vote Ávila, Carvalho, Gordo, Ribeiro & Morais, 2012 and A. manaos

Rojas, Carvalho, Gordo, Ávila, Farias & Hrbek, 2014 based on morphology; A.

amazonicola and A. matses Rojas, Carvalho, Gordo, Ávila, Farias & Hrbek, 2015,

based on morphology and genetic data (Ávila et al. 2012; Rojas et al. 2014, 2015).

Species of the genus are distributed in nine South American countries: Bolivia, Peru,

Ecuador, Colombia, Venezuela, Guiana, French Guiana Brazil, and presumably in

Suriname (Frost et al. 2015).

Using a phylogenetic analysis based on mitochondrial and nuclear genes

(Fouquet et al. 2007, 2012a), the existence of three independent evolutionary

lineages was discovered within the nominal species A. minuta from Ecuador and

French Guianas; subsequently, other independent evolutionary lineages were

discovered from Brazil and Peru (Rojas et al. 2014, 2015). The difficulties in

delimiting species within the A. minuta species complex resides in the relatively

generalized diagnosis (see Melin 1941) and the poor geographic sampling. For these

reasons, historically, the name A. minuta has been used for individuals distributed

throughout the Amazonian biome (e.g. Du- ellman 1978; Zimmerman and Rodrigues

1990; Magnusson and Hero 1991; Rod- rigues and Duellman 1993; Duellman and

Mendelson 1995; Fouquet et al. 2012a).

Thus, taxonomy and systematics of populations that are currently part of the A.

minuta complex remains largely unresolved (Rojas et al. 2014), in turn limiting the

knowledge of the true taxonomic diversity of the genus (Ávila et al. 2012; Rojas et al.

2014, 2015). Given this scenario, herein is described an additional new species of

28

Amazophrynella from the departments of Madre de Dios, Cusco and Junin, Peru,

founded on the prin- ciples of integrative taxonomy. Morphological, morphometric,

bioacoustic and phylogenetic relationships are provided as evidence for the

existence of the new taxon.

Material and methods

Morphology

Forty-eight specimens previously identified as Amazophrynella minuta (Melin,

1941), deposited at the Museo de Historia Natural del Cusco, Universidad Nacional

de San Antonio Abad del Cusco (MHNC) and Museo de Historia Natural de la

Universidad Nacional Mayor de San Marcos (MHNSM) were analyzed. This material

was compared with twenty preserved specimens of A. minuta from the type locality

(Taracuá mission, on the right bank of the Uaupés River, municipality of São Gabriel

da Cach- oeira, Brazil), deposited in the Collection of Amphibians and Reptiles of the

Instituto Nacional de Pesquisas da Amazônia–INPA, Brazil (INPA-H). Further

comparisons were made with three syntypes deposited at the Naturhistoriska

Museet, Göteborg, Sweden (NHMG), and the original description of the species

(Melin 1941).

Additionally five preserved specimens of Amazophrynella bokermanni

(Izecksohn, 1993) from near the type locality (Juruti, 30 Km from type locality), the

holotype and paratypes of A. manaos deposited in the Collection of Amphibians and

Reptiles of the Instituto Nacional de Pesquisas da Amazônia–INPA, Manaus,

Amazonas, Brazil (INPA- H), the holotype of Amazophrynella vote, deposited in the

Coleção Zoológica de Ver tebrados of the Universidade Federal de Mato Grosso–

UFMT, Cuiabá, Mato Grosso, Brazil (UFMT-A), seventeen paratypes deposited in the

Collection of Amphibians and Reptiles of the Instituto Nacional de Pesquisas da

Amazônia–INPA, Manaus, Amazo- nas, Brazil (INPA-H), and the holotype and

paratypes of A. amazonicola and A. matses, deposited at the Museo de Zoologia de

la Universidad Nacional de la Amazonia Peruana (MZUNAP) were analyzed (see

Appendix 1 listing all the revised specimens). Morphological character analyses were

carried out according to Cruz and Fussi- nato (2008) and Fouquet et al. (2012a). Sex

was determined by gonad analysis.

29

Measurements were carried out with a digital caliper following Kok and Kala-

mandeen (2008) and Duellman (1978). SVL (snout-vent length) from the tip of the

snout to the posterior edge of the cloaca; HL (head length) from the posterior edge of

the jaw to the tip of the snout; HW (head width), the greatest width of the head,

usually at the level of the posterior edges of the tympanum; ED (eye diameter); IND

(internarinal distance), the distance between the edges of the nares; SL (snout

length) from the anterior edge of the eye to the tip of the snout; HAL (hand length)

from the proximal edge of the palmar tubercle to the tip of Finger III; UAL (upper arm

length) from the edge of the body insertion to the tip of the elbow; THL (thigh length)

from the vent to the posterior edge of the knee; TL (tibia length) from the outer edge

of the knee to the tip of the heel; TAL (tarsal length) from the heel to the proximal

edge of the inner metatarsal tubercle; FL (foot length) from the proximal edge of the

inner metatarsal tubercle to the tip of Toe IV. Diagnosis of characters follow Chaparro

et al. 2015.

Statistical analysis. We used a total of 80 adult males of the Amazophrynella minuta

species complex (numbers of individuals and populations of origin in parentheses): A.

minuta sensu stricto (n = 23, from Taracuá), A. amazonicola (n = 15, from Puerto

Almendras and Fazenda Zamora); A. matses (n = 13, from Nuevo Salvador) and the

new species of Amazophrynella (n = 29, from Tambopata, Nuevo Arequipa, Candamo

and Inambari). All morphometric measures were log10 transformed to conform to

requirements of normality (Hayek et al. 2001). The effect of size was removed from

all variables by regressing them against SVL and using the residuals of each variable

in a Principal Component Analysis (PCA). Significance of morphometric differences

was tested with Multivariate Analysis of Variance (MANOVA) with the two first

principal components being treated as dependent variables and species as

independent variables. The first two principal components were used since they

explained the majority of observed variation in shape. A Discriminant Function

Analysis (DFA) was performed to test classification of individuals in predicted groups.

All the statistical analysis were performed in R (R Development Core Team 2011)

adopting a 5% significance cut-off. PCA was used to detect groups representing

putative cryptic species and DFA was subsequently applied to identify the set of

characters that best diagnose those groups (Padial and De la Riva 2009).

30

Additionally we noted large size in the HAL of the new species of Amazophrynella,

and we used an Analysis of Variance (ANOVA) of the original data (from A. minuta,

A. matses, A. amazonicola and the new species) to statistically support this

hypothesis

Molecular data

Laboratory procedure. Total DNA was extracted from muscle tissue using standard

phenol/chloroform extraction (Sambrook et al. 1989). A 480 bp fragment of the 16S

rDNA was PCR amplified using the 16Sar and 16Sbr primers (Palumbi 1996). Ampli-

fication was carried out under the following conditions: 60 s hot start at 92 °C

followed by 35 cycles of 92 °C (60 sec), 50 °C (50 sec) and 72 °C (1.5 min). Final

volume of the PCR reaction was 12 μl and contained 4.4 μL ddH2O, 1.5 μL of 25 mM

MgCl 21.25 μL of 10 mM dNTPs (2.5mM each dNTP), 1.25 μL of 10x buffer (75 mM

Tris HCl, 50 mM KCl, 20 mM (NH42SO4), 1 μL of each 2 μM primer, 0.3 μL of 5

U/μLDNA Taq Polymerase (Biotools, Spain) and 1 μL of DNA (about 30 ng/μL).

Sequenc- ing reactions were carried out according to the manufacturer’s

recommendation for the ABI BigDye Terminator cycle sequencing mix, using 16Sa

primer and an annealing temperature of 50 °C. Sequencing reactions were

precipitated using standard EDTA/ EtOH protocol, and resolved in an ABI 3130xl

automatic sequencer.

Phylogenetic analysis. We obtained 16S rDNA sequence data from two specimens of

the new species (Accession numbers: KR905184, KR905185), two paratypes of A.

vote (Accession numbers: KF433970, KF433971), two specimens of A. bokermanni

(Accession numbers: KF433975, KF433976), two topotypic specimens of A. minuta

(Accession numbers: KF792834, KF792836), two paratopotypes of A. matses

(Acces- sion number: KP681688, KP681689), the holotype and one paratopotype of

A. amazonicola (Accession number: KP681868, KP681669) and two paratypes of

A. manaos (Accession number: KF433954, KF433957) deposited in the tissue

collection of the Laboratório de Evolução e Genética Animal of the Universidade

Federal do Amazonas (CTGA-ICB/UFAM). The dataset also included two sequences

of A. sp. aff. minuta (Accession number: AY326000, DQ158420) from Darst and

Canatella (2004), Pra- muk (2006) and two sequences of A. sp. aff. manaos

31

(Accession number: EU201057, JN867570) from Fouquet et al. (2007). As outgroups

we used species of the sister taxon Dendrophryniscus (see Table 2 for samples

information). Sequences were aligned using the Clustal W algorithm (Thompson et

al. 1996) implemented in BioEdit (Hall 1999) and alignment was adjusted as

necessary against the secondary structure of the 16S rDNA. The existence of

lineages in a phylogenetic tree-based context (Baum and Donoghue 1995) was

performed using Maximum Like- lihood analysis (Felsenstein 1981) in the program

Treefinder (Jobb 2008) using the GTR+I+G model of substitution, selected via Akaike

information criterion as implemented in Modeltest 3.7 (Posada 2006). Phylogenetic

support was assessed via 10 000 non-parametric bootstrap (Felsenstein 1985).

Additionally uncorrected pairwise genetic distances between linages identified by

phylogenetic inference of Amazophrynella were calculated in MEGA 5.05 (Tamura et

al. 2007).

Molecular species delimitation. Evolutionary lineages are diagnosed by

discontinuities in character variation among lineages, and correspond to phylogenetic

species. The existence of lineages is therefore a necessary and sufficient

prerequisite for inferring the existence of a species under the different

conceptualizations of the Phylogenetic Species Concept (PSC) (Cracraft 1983; Baum

and Donoghue 1995; De Queiroz 2007). The existence of lineages in a non-tree-

based context (Cracraft 1983) was inferred using Population Aggregation Analysis

performed at the level of an individual (Davis and Nixon 1992; Rach et al. 2008)

using the dataset with the Amazophrynella minuta species complex: A. matses, A.

minuta, A. amazonicola and the new species. The analyses were performed in the

program R (R Development Core Team 2011).

Bioacoustics

We analyzed one advertisement call obtained from the CD of Frogs of

Tambopata, Peru (Macauly Library of Natural Songs and Cornell Laboratory of

Ornithology) by the authors Cocroft et al. (2001) from the Natural Reserve of

Tambopata, a locality of occurrence of the new species. The call was edit with the

software Audacity 1.2.2 for Windows (Free Software Foundation Inc. 1991). The

spectral and temporal parameters of the recording were analyzed in the software

32

Raven Pro. 1.3 for Windows (Cornell Laboratory of Ornithology). The advertisement

call was obtained from one male in a temperature 25 °C (Crocoft et al. 2001). We

measured the following quantitative parameters: call duration (seconds); pulses per

call; length of silence between calls (seconds); dominant frequency (kHz);

fundamental frequency (kHz) and time to peak at maximum frequency (seconds).

Results

Phylogenetic analysis and systematics

In the resulting phylogeny, the six nominal species of Amazophrynella were

recognized as monophyletic (Fig. 1). In the genus we can distinguish two

monophyletic groups: One clade (bootstrap support = 100) formed by the species: A.

manaos, A. bokermanni and A. vote and another represented by the species of the

A. minuta “species complex” (bootstrap support = 98): A. minuta, A. amazonicola, A.

matses and the new species described herein. In the first clade the Amazophrynella

species: A. manaos is sister taxon of the possible new specie from the Guiana

Shield: A. sp. aff. manaos (bootstrap support= 91), and both are sister to A.

bokermanni (bootstrap support= 98). Amazophrynella vote is sister of A. bokermanni

+ (A. manaos + A. sp. aff. manaos) with a bootstrap support of 81. The second clade

corresponding to the A. minuta “species complex”, A. amazonicola is sister of A.

minuta + A. sp. aff. minuta from western Amazonia (bootstrap support= 99). Our

analysis further highlighted the occurrence of a new monophyletic lineage (A.

javierbustamantei sp. n.) showing sister relationship with A. matses (bootstrap

support = 96), both being in turn sister group of A. amazonicola + (A. minuta + A. sp.

aff. minuta) with a bootstrap support of 99.

Smallest uncorrected 16S rDNA p-distances estimated between phylogenetic

linages was observed between A. minuta and A. sp. aff. minuta (= 3%). Greatest

interspecific distance (= 14%) was observed between Amazophrynella

javierbustamantei sp. n. and A. bokermanni and was comparable to divergence

observed between A. manaos and A. minuta. Within the “A. minuta” species complex,

the new species shows a high degree of genetic divergence from A. minuta (= 7%),

A. amazonicola (= 9%) and minor genetic distance with their sister taxon A. matses

(= 3%) (see all pairwise genetic distance values summarized in Table 3). According to

33

the Population Aggregation Analysis, the newly identified lineage was also

diagnosable by thirteen molecular autapomorphic characters (Table 4) leading us to

the conclusion that this lineage corresponds to a new species.

Morphometric analysis

Comparative analysis of quantitative morphological data allowed us to

distinguish Amazophrynella sp. n. from the other members of the A. minuta “species

complex”. The first two principal components extracted by the PCA account for

48.56% of the variation found in the dataset. The first component (PC1) explained

24.93% of total variation. In the first principal component axis, A. amazonicola is

distinguished from the other species due to its larger size (SVL = 14.9 ± 0.7 mm, see

Table 1), sharing relative size with A. minuta sensu stricto (SVL = 13.5 ± 0.6 mm, see

Table 1), the species A. matses is distinguish by having the smallest size of the

genus (SVL range= 12.1 ±0.6 mm, see Table 1), and shares this characteristic with

Amazophrynella sp. n. (SVL = 14.9 ± 0.9 mm, see Table 1) (Fig. 2). The second

component explains 23.63% of the variation. This axis represents a shape variation

vector; in this axis Amazophrynella javierbustamantei sp. n. is well distinguished from

the three formally described species, sharing more similarity with A. matses (Table 5).

All the species of the group are significantly different in shape (MANOVA,

F24.3, Pillae´s trace < 0.001). The discriminate function analysis (DFA) found

specimens correctly classified in 56.6% of cases and a moderate prior probability of

groups (A. minuta = 28.75%, A. amazonicola = 18.75%, A. matses = 16.25% and A.

javierbustamantei sp. n. = 36.25%). The variables that contributed most to the

classification were HAL, SVL and TAL (Table 6). The differences in HAL were

significant (ANOVA, F45.27, P < 0.001) among all the species of A. minuta “species

complex” (see Fig. 1) and reveals Amazophrynella javierbustamantei sp. n. as the

species with the largest HAL (Fig. 3).

Morphological description

Amazophrynella javierbustamantei sp. n.

http://zoobank.org/A946B949-1D1F-4FF5-B722-0B33435EE610

34

Holotype (Fig. 4). MHNC 8331 (Genbank 16S rRNA: KR905184). Adult male,

collected at Quebrada Guacamayo (12°54'24.5"S; 69°59'32.7"W, 215 m a.s.l.) km

105 of the highway Puerto Maldonado–Cusco City, District Inambari, Province

Tambopata, Department Madre de Dios, Peru, on 27 October 2009 by Juan C.

Chaparro and Oscar Quispe.

Paratypes (Fig. 5). Twenty-two specimens (males= 09, females= 13). MHNC

8363, MHNC 8245, MHNC 8238, adult males, MHNC 8316, MHNC 8484, MHNC

8362, MHNC 8354, adult females, collected with the holotype (12°28'25"S,

69°12'36"W, 205 m a.s.l.). MHNC 11001, adult male, MHNC 11002, MHNC 11003,

MHNC 11004, adult females collected by E. Aguilar on 17 May 2009, from La Pampa

km 107 highway Puerto Maldonado–Cusco City, Department Madre de Dios

(12°40'14.14"S, 72°27'30"W, 250 m a.s.l.). MHNSM 17993, adult male collected by

A. Angulo in 1999; from Province Manu, locality of Inambari, Depart- ment Madre de

Dios (13°02'29.28"S, 70°22'46.65"W, 306 m a.s.l.). MHNSM 25651, adult female,

collected by D. Rodriguez on April 2007, from Province La Convención, locality of

Camana, Department Cusco (12°05'9.25”S, 73°03'2.61”W, 680 m a.s.l.). MHNC

9939, MHNC 9940, adult females, collected by J. Delgado on 17 January 2010 from

Province La Convención, locality of Mapi, Department Cusco (11°31'19.17”S,

73°28'29.83”W, 708 m a.s.l.). MHNC 9387, adult male, collected by G. Estrada on 21

January 2010, from locality of Tambo Poyeni near Quebrada Mayapo, Department

Junin (11°19'29.9”S, 73°32'16.7"W, 388 m a.s.l.). MHNC 9754, MHNC 9756, adult

males, MHNC 9626, MHNC 9679, MHNC 9680, MHNC 9757, adult females,

collected by A. Pari on January 2010, from locality of Tsoroja, Department Junin

(11°18'56.06”S, 73°32'32.11”W, 399 m a.s.l. and 11°23'14.50”S , 73°29'43.00”W, 450

m a.s.l.).

Diagnosis. The new species is part of Amazophrynella based on molecular

phylogenetic relationships (Fig. 1) and morphological synapomorphies (Fouquet et al.

2012a). Amazophrynella javierbustamantei sp. n. is characterized by: (1) skin on

dorsum tuberculate, with many subconical tubercles disperse on arms, legs, head

and body; ventral skin coarsely areolate, throat and chest aerolate; (2) tympanic

membrane and tympanic annulus not apparent through the skin; (3) snout long,

subacuminated, protruding in lateral views; (4) upper eyelid with smaller tubercles,

35

cranial crests absent; (5) dentigerous process of vomers absent; (6) vocal sac, vocal

slits and nuptial pads absent; (7) finger I shorter than finger II, tips of digits rounded;

(8) fingers lacking lateral fringes; (9) ulnar tubercles present; (10) heel bearing eight

or more small low tubercles, tarsus with small tubercles and lack of folds; (11) plantar

surfaces of feet bearing one metatarsal tubercle, the inner 2.5x larger than the outer,

outer subconical; supernumerary plantar tubercles round and low; (12) toes lacking

lateral fringes; webbing basal; toe III equal than toe V, tips of digits rounded; (13)

dorsally is dark brown to light brown, and gray to black in some, ventrally, cream with

yellow to orange marks, with black to dark brown spots; (14) SVL 16.39–22.25 mm in

females, 12.79–16.42mm in males; (15) hand length is the greatest of all species of

Amazophrynella: 3.6 mm in males (n= 26) and 4.6 mm in females (n=20), see Fig. 3;

(16) thirteen molecular autapomorphies in the 16S rDNA gene.

Comparison with other species. Amazophrynella javierbustamantei sp. n. (Figs

4, 5, 6) differs in the following character states (states of other species in

parentheses). From A. minuta (Fig. 6A) by having body skin texture tuberculate

(roughly granular); relative abundance of spiny granules on the forelimbs (prickly

warty skin on axillary region of the forelimbs); absence of large warts on dorsum

(presence of large warts); throat and chest cream-grayish (light brown); posterior side

of belly color pale orange yellowish with tiny rounded black or dark brown spots

(throat and the whole belly in- tensely orange yellowish); tiny rounded black spots

covering the belly (irregular black ocelli or blotches); metatarsal tubercle rounded

(oval). From A. bokermanni (Fig. 6B) relative size of fingers, with finger I shorter than

II (I>II); snout vent length smaller in males (15.8 mm) and females (22.25 mm) (A.

bokermanni with maximum 22 mm SVL in males and 28 mm SVL in females, see

Izecksohn 1993); smaller snout in males, with 2.2 mm SL, n = 26 (2.7 mm SL, n = 5;

see Table 1); posterior side of belly color pale orange yellowish with tiny rounded

black or dark brown spots (white coloration with small black dots). From to A. vote

(Fig. 6C) snout subacuminated in dorsal view (rounded); posterior side of belly color

pale orange yellowish with tiny rounded black or dark brown spots (ventral color

pattern reddish brown, with presence of small white dots). From A. manaos (Fig. 6D)

snout subacuminated (snout truncate); dorsal skin finely granular (dorsal surfaces

granular); throat and chest grayish (dark coloration); posterior side of belly color pale

36

orange yellowish with tiny rounded black spots (venter cream with black spots or

stripes). From to A. matses (Fig. 6E) snout subacuminated (snout slightly truncate),

edges of nasal protrusion not dilated (dilated in ventral view); shape of palmar

tubercle rounded (palmar tubercles elliptical); finger tips unexpanded (expanded),

rounded tiny black spots covering the belly (medium-sized black ocelli or streaks);

coloration of the belly pale yellow (belly completely yellow). From A. amazonicola

(Fig. 6F) by the absence of small triangular protrusion on the tip of the snout in both

dorsal and ventral views (presence); body surface granular (finely granular), dorsum

uncovered with medium-sized granules scattered irregularly (covered with medium-

sized granules scattered irregularly); posterior side of belly color pale orange

yellowish with tiny rounded black or dark brown spots (orange yellowish with dark red

and brown blotches).

Description of the holotype. Body slender, head triangular, slightly longer than

wide; head length 35.5% of SVL, head width 30.9% of SVL. Snout long, subacumi-

nate in dorsal view, protruding in lateral view; canthus rostralis straight and loreal

region vertical; without papilla; snout length 39.0% of head length; tympanic

membrane and tympanic annulus not apparent through the skin, skin of the tympanic

area covered by round sub-conical warts; vocal sac externally not visible, vocal slits

absent; eyes prominent 23.8% of head length; upper eyelid covered with small

tubercles; those close to the external margin aligned in a more or less distinct row;

nostril closer to snout than to eyes; internarial distance smaller than eye diameter;

presence of a line of small spiny granules from the outer edge of the mouth to upper

arm, choanas small and circular. Dorsal skin finely tuberculate with several large

tubercles scattered sub-conical tubercles on upper arm; texture of ventral skin

granular, covered by rounded granules. Dorsolateral surfaces, granular, with

presence of large rounded tubercles. Forelimbs slender, upper arm length 29.6% of

SVL; edges of lower arm and upper arm finely tu-berculate with several large sub-

conical and spiny granules; hand length 76.5% of upper arm length; fingers slender,

tips not expanded; relative length of fingers I<II<IV<III; supernumerary tubercles and

accessory palmar tubercles present, palmar large and rounded, supernumerary

tubercles low, small rounded; subarticular tubercles rounded and small, one tubercle

on fingers I, II and IV and two on finger III; fingers I and II basally webbed; indistinct

37

nuptial pad. Hind limbs slender; ventral skin from thigh to tarsus covered by spiny

tubercles, foot length 66% of thigh length; relative length of toes I<II<V<III<IV: inner

metatarsal tubercle oval, 2.5× larger than outer; outer metatarsal tubercles small,

rounded; subarticular tubercles present, rounded, present one on fingers I, II, and

two on fingers III, V and three on finger IV; and tip of toes not expanded.

Measurements of the holotype (in millimeters). SVL 15.1; HW 4.6; HL 5.3; SL 2.1; ED

1.2; IND 1.0; UAL 4.4; HAL 3.4; THL 8.1; TAL 8.1; TL 4.5; FL 5.3.

Coloration of the holotype. In life: dorsum of the holotype mostly light brown

with dark brown in the dorsum; dorsolaterally creamish-brown with scattered black

blotches; dorsal surfaces of hands and feet creamish-brown, and gray on arms and

legs; belly creamish-gray with black dots, and the throat gray; fingers, toes and

plantar surfaces reddish-black; groin with orange marks; iris with a bronze ring;

cloaca with orange flap, black pupil and bronze iris. In alcohol: dorsum brownish-

grey; venter cream with black and brown dots; orange surfaces turned cream, with a

white longitudinal stripe on upper jaw extending from nostril to forearm.

Variation. The new species is phenotypically variable. In some individuals (e.g.

MHNC 8245 and MHNC 11002, see Fig. 5) patterning on the dorsum varies, with

these specimens presenting brown chevrons extending from the head to the vent.

Some individuals showed a white line extending from the tip of the nose to the upper

arm. Another specimen (MHNC 9739, see Fig. 5) presented a yellow pale coloration

in the axillary region (in ventral view). In some individuals, the coloration of the throat

ex- tended onto the chest (e.g. MHNC 11002, MHNC 9739 and MHNC 8245, see Fig.

5). The pale yellow coloration of the belly surface may extend from thighs to the chest

or just to the middle of the belly (e.g. MHNC 8362, see Fig. 5 and Fig. 7B). In some

individuals, the thighs are abundantly covered by rounded tiny spots extending to the

shank (Fig. 7B). In preserved specimens the dorsum becomes light brown and the

belly coloration vary from white to yellow pale (e.g. MHNSM 31255 and MHNSM

17993, see Fig. 5). The color of the finger becomes pale red and in other individuals

the red coloration of the fingers became brown or orange (Fig. 5).

Bioacoustics. The following values are presented as: min-max (average ± SD,

number of notes). The call is a trill type call issued during continuous and regular in-

38

tervals (Fig. 8). Each note had a duration of between 0.03 to 0.08 seconds (0.05 ±

0.01 seconds, n = 20). The number of pulses varied between 8 to 18 pulses per note

(10.4 ± 2.6 pulses/note, n = 20). The silence between notes varied from 0.4 to 1.6

seconds (0.8 ± 0.3 seconds, n = 20). The dominant frequency varied from 3962.1 to

3789.8 kHz (3927.6 ± 70.7 kHz, n = 20), and coincides with the fundamental

frequency. Time to peak amplitude was around 0.014 to 0.04 seconds (0.02 ± 0.01

seconds, n = 20).

Distribution, ecology and conservation. Amazophrynella javierbustamantei sp.

n. is known from the Department of Cusco, in the lower Urubamba river basin and

De- partment of Madre de Dios (Inambari, Candamo and Nueva Arequipa) in Peru

(Fig. 9). Its distribution can vary from 215 m a.s.l. to 708 m a.s.l. Additional

specimens were recorded at Los Amigos Biological Station , Tapir Lodge, and

Explorers Inn, in Tambopata National Reserve. Individuals were active during the day,

jumping on leaf litter, at night they were sleeping on leaves around 30 cm above

ground. This species breeds close to the edges of permanent oxbow lakes, males

call during the day while perched above streams in tangles (Cocroft et al. 2001).

Three of the localities, km 105, 107 and 117 of the highway Puerto Maldonado–

Cusco, Department Madre de Dios, show evidence of serious environmental impacts

due to illegal gold mining activities, with forest and soil removed, and environmental

pollution via organic and inorganic chemicals and heavy metal (specially mercury)

poisoning. In addition, the new species is distributed inside of territories where oil

companies are operating. On the other hand, the species is present in two protected

areas, the Tambopata Natural Reserve and Machiguenga Communal Reserve. The

conservation status of this species remains unknow, but was listed in 2008 as Least

Concern on the IUCN red list (2015), because it was confused with Amazophrynella

minuta, and because Amazophrynella minuta s.l. had wide distribution at that time,

apparent tolerance of a certain degree of habitat modification, presumed large

population, and because it is unlikely to be declining, and thus did not qualify for

listing in a more threatened category. With recent studies the genus, the species

complex of Amazophrynella minuta, was split in five species, three of them are now

formally described for Peru (Amazophrynella matses, A. amazonicola and A.

javierbustamantei sp. n.). The recognition of these new species will require the

39

reevaluation of the conservation status of these species. It should also act as an

impetus for additional field and laboratory studies of Peruvian amphibians, in order to

understand the real conservation status of this fauna.

Etymology. The species is named after Dr. Javier Bustamante, a Peruvian

residing in United States, to whom we dedicate this species in recognition of his

friendship and support of herpetological taxonomy and systematics research and

amphibian conservation in Peru.

Discussion

Taxonomic reviews of Amazonian amphibians suggests that morphological

characters are too conservative to permit delimiting species since closely related

species share similar morphologies, and amphibians in general are morphologically

conservative (e.g., Elmer et al. 2007; Fouquet et al. 2007c; Funk et al. 2011; Padial et

al. 2009). Thus, the use of integrative techniques in taxonomy is revolutionizing the

identification and delimitation of species based on independent lines of evolutionary

evidence (Dayrat 2005; Padial and De la Riva 2009). The use of an integrative

approach not only allows for the discovery and delimitation of new species, it also

helps us to understand the mechanism of species formation. Thus, integrative

taxonomy allows us to have a better understanding of the true scope of anuran

diversity in the Amazon, and it allows us to better understand the processes that

generated this biodiversity.

The taxonomic ambiguity surrounding the name A. minuta and to a lesser

extent A. bokermanni resulted in a severe underestimation of the taxonomic diversity

of this genus. Since the descriptions of A. minuta in 1941 and A. bokermanni in 1993,

the taxonomy of the genus has not been revised, leading to misdiagnoses of other

species as either A. minuta or A. bokermanni due to the relatively generalized

descriptions of these taxa. Three publications since 2012 (Ávila et al. 2012; Rojas et

al. 2014, 2015) described four new species, increasing the taxonomic diversity of the

genus by 200%. All four species were previously classified as populations of a single

species with a large distribution (A. minuta sensu lato). Although striking, the severe

underestimation of taxonomic diversity observed in Amazophrynella and the

existence of multiple lineages in Amazophrynella minuta is nothing particular to this

40

group. Examples of other Ama- zonian species complexes include Rhinella

margaritifera and Scinax ruber, Pristimantis ockendeni, Pristimantis fenestratus,

Engystomops petersi, Hybsiboas fasciatus, Dendropso- phus minutus and

Osteocephalus taurinus (Fouquet et al. 2007; Elmer and Canatella 2008; Padial et al.

2009; Funk et al. 2011; Caminer and Ron 2014; Gehara et al. 2014, Jungfer et al.

2013). The descriptions by Rojas et al. (2014, 2015) were based, in part, on

diagnostic characters observed in the 16S rDNA. This gene is widely used as a DNA

barcode for amphibians, for reliable species identification (Vences et al. 2005,

Fouquet et al. 2007), for evaluating monophyly of species and for discovering

divergent lineages (Pa dial et al. 2009, Crawford et al. 2010; Padial et al. 2010 and

Padial et al. 2012). Based on 16S rDNA analyses, we also have evidence that A.

bokermanni and A. vote represent species complexes (RRRZ, personal observation).

This observation is in addition to the existence of the two candidate species of

Amazophrynella already observed in previous analyses: one from the Guiana Shield

(A. sp. aff. manaos), sister taxon of A. manaos, and another from Ecuador (A. sp. aff.

minuta), sister taxon of A. minuta sensu stricto (Fig. 1).

Although the taxonomic status of these candidate species will need to be con-

firmed using morphological and bioacoustics data, it is clear that even with the recent

descriptions, the taxonomic diversity of the genus remains underestimated. While

part of our evidence for the existence of the new species as well as those described

previously by Rojas et al. (2014, 2015) comes from the use of molecular data, the

descriptions make use of other data types and non-molecular diagnoses. Thus these

undiscovered lineages were not truly cryptic (morphologically cryptic), but rather the

result of poor taxonomic knowledge of the group. In this respect, the genus Amazo-

phrynella again is not the exception, but rather the norm.

The species A. javierbustamantei sp. n. is clearly differentiated in multivariate

morphometric space from the other members of the Amazophrynella minuta “species

group” (A. minuta, A. amazonicola and A. matses). Together with the description of

Amazophrynella javierbustamantei sp. n. we also provide advertisement call.

Amazophrynella javierbustamantei sp. n. is only the second species of the genus for

which an advertisement call is known and recorded (see Duellman 1978). Acoustics

can provide evidence of potentially new species with behavioral or pre- mating

41

isolating mechanisms (e.g. De la Riva et al. 1997; Gerhardt 1998; Simões et al. 2008,

Padial and De la Riva 2009; Padial et al. 2012), thus providing evidence of

evolutionary mechanisms that contributed to the species diversity of the genus

Amazophrynella.

The threats to the biological conservation of A. javierbustamantei sp. n. are

evident, with uncontrolled exploration for gold, illegal mining and the destruction of

habitat in the Departments of Madre de Dios and Cusco, probably causing a

significant reduc- tion in the population sizes of the species and fragmenting its

distribution. For these reasons is necessary to analyze the current population status

and trends of this and another amphibian species in this Department of southern

Peru.

Acknowledgements

RRRZ was supported by a fellowship from Coordenação de Aperfeiçoamento

de Pessoal de Nível Superior (Coordenação de Aperfeiçoamento de Pessoal de

Nível Su- perior - CAPES), RRRZ also thanks Nora M. Rojas Ruiz, Silvia Rojas,

Marco Rojas Ruiz for hospitality in Lima, Elda Zamora, José Armas, Micaela Perea,

Ronald Rojas and Miguel Rojas in Iquitos and Edson Caceres, Fababa and Douglas

Giardini in Puerto Maldonado, Peru. For allowing access to the respective

herpetological collections under their care, we are grateful to Richard C. Vogt

(Instituto Nacional de Pes quisas da Amazonônia - INPA), Göran Nilson

(Naturhistoriska Museet, Göteborg - NHMG), Marcos A. Carvalho (Universidade

Federal de Mato Grosso - UFMT) and Juan Carlos Cusi (Museo de Historia Natural

de la Universidad Nacional Mayor de San Marcos - MUSM), Percy Yanque and Rocio

Orellana (Museo de Historia Natural del Cusco - MHNC). We are grateful to

Asociación para la Conservación de la Cuenca Amazonica (ACCA) which partially

funded the expedition to Madre de Dios, under the project Manu-Tambopata

Conservation Corridor,that resulted in the discovery of the new species. VTC was

supported by a fellowship from Fundação de Amparo a Pesquisa do Estado do

Amazonas (FAPEAM), IF and TH were supported by research productivity

fellowships from the Conselho Nacional de Desenvolvi- mento Cientifico e

Tecnológico (CNPq). Collecting permits in Peru were granted by Dirección General

42

Forestal y de Fauna Silvestre (DGFFS) del Ministerio del Me- dio Ambiente (MINAM-

N°-0320, RJ-RCM-N°-001-2009-SERNANP-RCM, RJ- RCA-N°-002-2009-

SERNANP-RCAS, RD-N°-435-2009-AG-DGFFS-DGEFFS and RD-Nº-038-2010-AG-

DGFFS-DGEFFS). We are grateful to Mario Nunes for his assistance with laboratory

work.

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Appendix 1

Specimens examined

Amazophrynella minuta—BRAZIL: Taracuá, Uaupés River: INPA-H 32725, INPA-H

32723, INPA-H 32729, INPA-H32730, INPA-H32736, INPA-H32731 (females) and

INPA-H 32724, INPA-H32728, INPA-H 32733, INPA-H 32735, INPA-H 32722, INPA-H

32738, INPA-H 32737, INPA-H 32739. INPA-H 32720, INPA-H 32732, INPA-H 32726,

INPA-H 32730, INPA-H 32740, INPA-H 32734, INPA-H 32721 (males).

Amazophrynella bokermanni—BRAZIL: Juriti, Pará: INPA-H 31861, INPA-H 31864,

INPA-H 31863, INPA-H 31862, INPA-H 31865, municipality of Juriti, Pará State, Brazil

(50 km from type locality).

Amazophrynella vote—BRAZIL: Fazenda São Nicolau, Cotriguaçu, Mato Grosso

(Holotype: UFMT-A 11138); Madeira River, Manicoré (Paratypes: INPA-H 12256,

12331, 12255, 12342, 12343, 12366, 12267); Aripuanã River, Novo Aripuanã

(Paratype: INPA-H 12326); Parque Estadual do Guariba: Manicoré (Paratypes: INPA-

H 21558); Parque Nacional Nascentes do Lago Jari, Tapauá: Amazonas (Paratypes:

INPA-H 27412, 27417-27419, 27421-27423, 27425-27426).

Amazophrynella manaos—BRAZIL: Campus da Universidade Federal do Amazonas,

Amazonas (Holotype: INPA-H 31866, paratypes: INPA-H 6983, INPA-H 6984, INPA-H

6987, INPA-H 7797); Presidente Figueiredo, Amazonas (Paratypes: INPA-H 29568,

INPA-H 29569, INPA-H 29571, INPA-H 29570, INPA-H 29572, INPA-H 20986; INPA-

H 21217, INPA-H 30577, INPA-H 30575, INPA-H 30573, INPA-H 30572, INPA-H

30576); Reserva Florestal Adolpho Ducke, Amazonas (INPA-H 21028, INPA-H

21170, INPA-H 21060, INPA-H 31866, INPA-H 21007, INPA-H 21008, INPA-H 21009,

INPA-H 21010, INPA-H 21011, INPA-H 21012, INPA-H 21013).

Amazophrynella amazonicola—PERU: Puerto Almendra San Juan Bautista, Loreto

(Holotype: MZUNAP 901, paratopotypes: MZUNAP 906; MZUNAP 915; MZUNAP

110; MZUNAP 907, MZUNAP 917; MZUNAP 889; MZUNAP 910; MZUNAP 911;

MZUNAP 916; MZUNAP 913; MZUNAP 914; paratypes: MZUNAP 906; MZUNAP

915; MZUNAP 110; MZUNAP 907, MZUNAP 917; MZUNAP 889; MZUNAP 910;

MZUNAP 911; MZUNAP 916; MZUNAP 913; MZUNAP 914); 58 km of Iquitos–Nauta

49

highway on Fundo Zamora, San Juan Bautista, Loreto (Paratypes: MZUNAP 908,

MZUNAP 924, MZUNAP 886, MZUNAP 900, MZUNAP 888, MZUNAP 919, MZUNAP

902, MZUNAP 887, MZUNAP 905, MZUNAP 920); Nauta, Maynas (Paratypes:

MZUNAP 918, MZUNAP 909); Fundo UNAP, Maynas, Loreto (Paratype: MZUNAP

242)

Amazophrynella matses—PERU: Nuevo Salvador, Requena, Loreto (Holotype:

MZUNAP 921, paratopotypes: MZUNAP 934, MZUNAP 955 MZUNAP 940, MZUNAP

948 MZUNAP 943, MZUNAP 952, MZUNAP 953, MZUNAP 958, MZUNAP 922,


938, MZUNAP 936); Jenaro Herrera, Requena, Loreto (Paratypes: MZUNAP 928,


935, MZUNAP 950, MZUNAP 937, MZUNAP 939, MZUNAP 941, MZUNAP 942,

MZUNAP 946, MZUNAP 947, MZUNAP 949).

50

Fig. 1. Maximum Likelihood tree of the Amazophrynella species based on the

GTR+I+G model, using 480 bp of 16S rDNA. Numbers below branches represent

bootstrap support with 1000 replications.

51

Fig. 2. Principal Component Analysis (PCA) of the Amazophrynella minuta species

complex. See Table 4 for character loadings on each component.

Fig. 3. Measurement comparison of the Hand Length (HAL) between species of the

Amazophrynella minuta complex.

52

Fig. 4. Holotype of Amazophrynella javierbustamantei sp. nov. (MHNC 8331); A)

dorsal view; B) ventral view; C) dorsolateral view; D) right hand; E) right foot.

53

Fig. 5. Dorsal and ventral view of some paratypes of Amazophrynella

javierbustamantei sp. nov. MHNC 8245; MHNSM 31255; MHNSM 17993 (Adult

males); MHNC 1102, MHNC 9739, MHNC 8362 (Adult females).

54

Fig. 6. Dorsal and ventral morphological comparison between the Amazophrynella

spp. (non-voucher specimens): A) A. javierbustamantei sp. nov.; B) A. minuta; C) A.

bokermanni; D) A. vote; E) A. manaos; F) A. matses; G) A. amazonicola.

55

Fig. 7. Dorsal and ventral variation of Amazophrynella javierbustamantei sp. nov.

(non-voucher specimens): A-C) Nueva Arequipa, Madre de Dios Dept., Peru; B)

Basin of Bajo Urubamba, Cusco Dept., Peru.

56

Fig. 8. Advisement call of Amazophrynella javierbustamantei sp. nov. from the

Tambopata Reserve, Madre de Dios, Peru (207 meters a.s.l.) (©Macauly Library of

Natural Songs and ©Cornell Laboratory of Ornithology) by the authors Crocoft,

Morales and Mc Diarmid (2007). A) Oscilogram and spectrogram by one note; B)

Oscilogram and spectrogram of notes from the advisement call.

57

Fig. 9. Distribution map of Amazophrynella javierbustamantei sp. nov. records in

Peru. Red squares represent paratypes and the star the type locality (where the

holotype has been collected.

58

Table 1. Measurements (mm) of adult male specimens (including the holotype) in the type series of Amazophrynella spp. Mean ±

standard deviation, with ranges in parentheses. Abbreviations are defined in Material and methods.

Variable A. minuta s.s.

(n=15)

A. manaos s.s.

(n=29)

A. bokermanni

(n=5)

A. vote

(n=14)

A. amazonicola

(n=15)

A. matses

(n=13)

A. javierbustamantei sp. nov.

(n=26)

SVL 13.5±0.6 (12.5-14.2)

14.2±0.7 (12.3-15.0)

16.8±1.4 (14.6-18.2)

13.1±0.7 (12.0-14.1)

14.5±0.7 (13.3-15.4)

12.1±0.6 (11.5-13.5)

14.9±0.9 (12.7-16.4)

HW 4.2±0.2 (4.0-4.3) 4.2±0.3 (3.7-4.7)

3.2±0.3 (2.5-3.3)

4.0±0.7 (3.3-4.4)

4.4±0.3 (4.2-4.6) 3.6 ±0.2 (3.1-3.8)

4.2 ±0.2 (3.5-4.7)

HL 4.9±0.2 (4.8-5.3) 5.3±0.3 (4.7-5.6)

3.4±0.4 (2.8-3.8)

4.6±0.3 (4.0-5.2)

5.2±0.3 (5.0-6.2) 4.3 ±0.3 (3.9-4.8)

5.1 ±0.3 (4.4-5.6)

SL 2.3±0.1 (2.2-2.5) 2.7±0.2 (2.3-2.7)

3.0±0.4 (2.2-3.1)

2.1±0.2 (1.9-2.6)

2.4±0.2 (2.2-2.5) 2.0 ±0.3 (1.6-2.3)

2.2 ±0.2 (1.7-2.6)

ED 1.4±0.1 (1.3-1.5) 1.3±0.1 (1.2-1.6)

1.8±0.2 (1.5-2.0)

1.3±0.1 (1.2-1.5)

1.2±0.1 (0.9-1.2) 1.1 ±0.1 (0.9-1.2)

1.3 ±0.1 (1.0-1.6)

IND 1.2±0.1 (1-1.3) 1.1±0.1 (1.0-1.4)

1.4±0.2 (1.0-1.5)

1.1±0.1 (1.0-1.3)

1.2±0.1 (1.0-1.3) 1.0 ±0.1 (0.8-1.2)

0.9 ±0.1 (0.8-1.2)

UAL 3.8±0.2 (3.2-4.1) 3.6±0.4 (2.9-4.1)

5.5±0.6 (5.0-5.6)

3.9±0.5 (2.8-3.9)

4.5±0.3 (4.2-5.3) 3.5 ±0.4 (2.9-4.2)

4.5 ±0.4 (3.8-5.7)

HAL 2.8±0.2 (2.6-3.0) 2.8±0.6 (1.9-2.9)

3.4±0.6 (2.8-4.2)

2.7±0.3 (2.3-3.2)

3.2±0.2 (2.8-3.3) 2.7 ±0.2 (2.3-3.1)

3.6 ±0.4 (2.5-4.5)

59

THL 6.8±0.2 (6.4-7.2) 6.7±0.3 (2.3-3.1)

8.7 ±1.4 (7.2-8.9)

6.5±0.7 (5.4-7.2)

7.7±0.6 (6.3-8.0) 6.2 ±0.4 (5.1-6.3)

7.6 ±0.7 (6.2-9.2)

TAL 6.7±0.3 (6.3-7.1) 6.9±0.6 (4.2-7.3)

8.3±1.0 (6.7-9.2)

5.7±0.7 (4.8-7.0)

7.2±0.6 (6.1-7.9) 5.8 ±0.3 (5.1-6.3)

7.6 ±0.7 (6.2-8.8)

TL 4.1±0.2 (3.8-4.6) 4.6±0.4 (4.3-6.3)

5.4±1.4 (2.9-6.2)

3.8±1.0 (4.2-7.0)

4.2±0.6 (6.3-8.0) 3.8 ±0.2 (3.6-4.3)

4.7 ±0.8 (3.9-8.7)

FL 4.8±0.4 (4.2-5.2) 5.2±0.5 (4.7-6.1)

6.3±1.3 (3.9-7.6)

4.4±0.6 (3.2-5.4)

5.1±0.4 (4.7-6.0) 4.3 ±0.4 (5.5-3.0)

5.7 ±0.6 (4.5-7.2)

60

Table 2. Uncorrected p-distances between Amazophrynella species and one representative of the sister genus Dendrophryniscus.

Molecular distances are based on the 480-bp fragment of 16S rDNA. We included A. minuta s.s. from its type locality and the two

candidate species Amazophrynella aff. manaos and A. aff. minuta from Fouquet et al. (2012a).

16S rDNA 1 2 3 4 5 6 7 8 9 10

1 A. amazonicola

2 A. matses 0.08

3 A. aff. minuta 0.06 0.07

4 A. minuta 0.05 0.08 0.03

5 A. javierbustamantei sp. nov. 0.09 0.03 0.06 0.07

6 A. vote 0.12 0.12 0.12 0.12 0.13

7 A. bokermanni 0.12 0.12 0.11 0.11 0.13 0.10

8 A. manaos 0.12 0.12 0.12 0.14 0.12 0.10 0.08

9 A. aff. manaos 0.12 0.11 0.12 0.13 0.12 0.10 0.07 0.04

10 D. leucomystax 0.19 0.21 0.17 0.18 0.20 0.22 0.18 0.20 0.20

61

Table 3. Species level diagnostic characters observed in the 16S rDNA gene of

Amazophrynella javierbustamantei sp. nov. and other species of Amazophrynella.

First line indicates position of the character within the 16S rDNA gene; (-) indicates a

deletion.

Species 213 232 271 276 470 471 473 474 476 477 478 479 480

A. manaos A C A C A T G T C A A A A

A. vote A T A C C C C T T A A A G

A. minuta C T A A C C C T T A A A G

A. bokermanni A T A C A T G T C A A A A

A. amazonicola C C A C C C C T T A A T G

A. javierbustamantei T G G T T G T G A G C C -

A. matses C T A C C C C T T A A T T

62

Table 4. Character loadings, eigenvalues, and percentages of explained variance for

Principal Components (PC) 1–2. The analysis was based on eleven morphometric

variables of adult males of the Amazophrynella minuta complex (A. minuta sensu

strictro; A. amazonicola; A. matses and A. javierbustamantei sp. nov.)

Variables PC1 PC2

HW 0.462 -0.146

HL 0.455 -0.104

SL 0.374 -0.244

ED 0.261 0.052

IND 0.369 -0.271

UAL 0.139 0.258

HAL -0.032 0.484

THL 0.311 -0.295

TAL 0.314 0.350

TL 0.116 0.364

FL 0.063 0.433

% of variation 24.93 23.63

% 24.93 48.56

63

Table 5. Character loadings of explained variance for Discriminant Function Analysis

(DFA). The analysis was based on twelve morphometric variables of adult males of

the Amazophrynella minuta complex (A. minuta sensu stricto; A. amazonicola; A.

matses and A. javierbustamantei sp. nov.)

Variables Discriminant Function

SVL 6.343

HW -7.628

HL 0.146

SL -5.479

ED -1.175

IND -6.015

UAL 1.313

HAL 5.744

THL -3.871

64

CAPITULO II

A Pan-Amazonian species delimitation: high species diversity within the genus

Amazophrynella (Anura: Bufonidae). Rojas, R.R., Fouquet, A., Ron, S., Hernandez,

E., Melo-Sampaio, P., Chaparro, J., Vogt, R., Carvalho, V., Pinheiro, L., Ávila, R.,

Pires, I., Gordo, M. & Hrbek, T. (2018). PeerJ, 6, e4941.

65

A Pan-Amazonian species delimitation: high species diversity within the genus

Amazophrynella (Anura: Bufonidae)

Rommel R. Rojas1*, Antoine Fouquet2, Santiago R. Ron3, Emil José Hernández-

Ruz4, Paulo R. Melo-Sampaio5, Juan C. Chaparro6,7, Richard C. Vogt8, Vinícius

Tadeu de Carvalho1, Leandra Cardoso Pinheiro9, Robson W. Ávila10, Izeni Pires

Farias1, Marcelo Gordo11, Tomas Hrbek1*

1Laboratory of Evolution and Animal Genetics (LEGAL), Department of Genetics,

ICB, Universidade Federal do Amazonas, Manaus, AM, Brazil

2 USR 3456 LEEISA - Laboratoire Ecologie, Evolution et Interactions des Systèmes

Amazoniens, Centre de recherche de Montabo, Cayenne, French Guiana.

3 Museo de Zoología, Escuela de Biología, Pontificia Universidad Católica del

Ecuador, Quito, Ecuador.

4 Laboratório de Zoologia, Faculdade de Ciências Biológicas, Campus Universitário

de Altamira, Universidade Federal do Pará, Altamira Pará, Brazil.

5 Departamento de Vertebrados, Museu Nacional, Rio de Janeiro, Brazil

6 Museo de la Biodiversidad del Peru, Cusco, Peru.

7 Museo de Historia Natural de la Universidad Nacional de San Antonio Abad del

Cusco, Peru.

8 CEQUA, Coordenação de Biodiversidade, Instituto Nacional de Pesquisas da

Amazônia, Manaus, AM, Brazil.

9 Museu Paraense Emilio Goeldi, Belem, Pará, Brazil.

10 Departamento de Ciências Biológicas, Centro de Ciências Biológicas e da Saúde,

Universidade Regional do Cariri , Crato, Brazil.

11 Departamento de Biologia, ICB, Universidade Federal do Amazonas, Manaus, AM,

Brazil.

* Correspondence: [email protected]; [email protected]

mailto:[email protected]


66

Abstract

Amphibians are probably the most vulnerable group to climate change and

climate-change associate diseases. This ongoing biodiversity crisis makes it thus

imperative to improve the taxonomy of anurans in biodiverse but understudied areas

such as Amazonia. In this study, we applied robust integrative taxonomic methods

combining genetic (mitochondrial 16S, 12S and COI genes), morphological and

environmental data to delimit species of the genus Amazophrynella (Anura:

Bufonidae) sampled from throughout their pan-Amazonian distribution. Our study

confirms the hypothesis that the species diversity of the genus is grossly

underestimated. Our analyses suggest the existence of eighteen linages of which

seven are nominal species, three Deep Conspecific Lineages, one Unconfirmed

Candidate Species, three Uncategorized Lineages, and four Confirmed Candidate

Species and described herein. We also propose a phylogenetic hypothesis for the

genus and discuss its implications for historical biogeography of this Amazonian

group.

67

Introduction

Amphibians are undergoing a drastic global decline (Beebee & Griffiths,

2005). This decline is primarily attributable to habitat destruction, diseases (chytrid

fungus) and global climate change (Collins, 2010). In Amazonia the primary threat is

habitat destruction, although the chytrid fungus has reached the Amazon basin

(Valencia-Aguilar et al., 2015; Becker et al., 2016), and is starting to have an impact

on Amazonian and Andean anurans (Lötters et al., 2005, 2009; Catenazzi & von

May, 2014). Most Amazonian amphibians are thought to have broad, often basin

wide distributions, although their geographic distributions are generally poorly known.

More detailed analyses generally reveal the existence of multiple deeply divergent

lineages, suggesting cryptic diversity. Fouquet et al. (2007) estimated that amphibian

diversity of Amazonia is underestimated by 115%, while Funk et al. (2011) suggest

this underestimate is closer to 150–350%. But even without taking into account the

high levels of crypsis or pseudocrypsis (morphological differences apparent but

overlooked) in widespread Amazonian anurans, Amazonia has the highest diversity

of amphibians on this planet (Jenkins et al., 2013).

Delimiting species and their geographic distributions is therefore crucial for the

understanding of impacts on the biodiversity of Amazonian anurans, and for the

assessment of their conservation status (Angulo & Reichle, 2008). Previous studies

suggest a prevalent conservatism in the morphological evolution of anurans (eg.

Elmer et al., 2007; Robertson & Zamudio, 2009; Vences et al. 2010; Kaefer et al.,

2012; Rowley et al., 2015), thus, species delimitation based solely on morphological

characters may fail to differentiate among species. Conversely, delimiting species

solely based on molecular characters or genetic distances harbors potential pitfalls

that have been well documented (eg. Carstens et al., 2013; Sukumaran & Knowles,

2017). Environmental data also have the potential to provide important information to

taxonomy since species have distinct ecological requirements that determine their

occurrence in time and space (Soberón et al., 2005). Therefore, species delimitation

relying on a pluralistic approach seeking to unite several lines of evidence (Dayrat,

2005; Padial et al., 2010) generally provides robust and consensual taxonomic

hypotheses (eg. Padial & De La Riva, 2009) especially in morphologically conserved

68

groups, i.e. taxonomic groups harboring cryptic or pseudocryptic taxa (Cornils &

Held, 2014).

The frog genus Amazophrynella Fouquet, Recoder, Teixeira, Cassimiro,

Amaro, Camacho, Damasceno, Carnaval, Moritz, & Rodrigues 2012a is distributed

throughout Amazonia, and currently comprises seven small-sized (12.0–25.0 mm)

species (Fouquet et al., 2012b). All species inhabit the forest leaf litter (Rojas et al.,

2015), breed in seasonal pools and have diurnal and crepuscular habits (Fouquet et

al., 2012b; Rojas et al., 2014, 2016).

Until 2012, only two species were recognized: Amazophrynella minuta from

western Amazon and A. bokermanni from eastern Amazon (Fouquet et al., 2012b).

Since 2012 five additional species have been described from western Amazon (A.

vote, A. manaos, A. amazonicola, A. matses and A. javierbustamantei). The

taxonomy of the genus remains, however, far from being resolved (Rojas et al.,

2016). Although molecular phylogenetic analyses in Fouquet et al. (2012b) and Rojas

et al. (2015, 2016) provided evidence for the existence of multiple lineages, the

scarcity of material suitable for morphological and bioacoustic analyses prevented

the description of these lineages as new species.

In this study, we revisit the genus Amazophrynella, include specimens from

new localities, and reconstruct intra- and inter-specific phylogenetic relationships. We

delimit candidate species based on molecular data and subsequently seek support

for these lineages combining qualitative and quantitative morphological data and

environmental evidence. As a result of these analyses, we formally describe four new

species of Amazophrynella from Brazil, Ecuador, French Guiana and Peru, and

identify additional seven candidate species. Additionally, we provide new insights into

the overall phylogenetic relationships for the genus, and discuss biogeographic

history of this Amazonian group.


Protocol for species delimitation

We evaluated the status of populations of Amazophrynella, adhering to the

unified species concept proposed by De Queiroz (2007), that conceptualizes species

69

as lineages of ancestor-descendent populations which maintain their distinctness

from other such lineages and which have their own evolutionary tendencies and

historical fates. We followed the consensus protocol of integrative taxonomy

proposed by Padial et al. (2010). The concept of candidate species adopted in this

study follows the subcategories proposed by Vieites et al. (2009) in using: Confirmed

Candidate Species (CCS) for lineages that present high genetic distance and can be

differentiated by other traits (i.e. morphological data), Deep Conspecific Lineages

(DCL) for lineages that are genetically divergent but not supported by any other

character (these characters being available), Unconfirmed Candidate Species (UCS)

for lineages that are genetically divergent but no additional characters are available

to support this divergence (these characters not available) and Uncategorized

Lineages (UL) for lineages that do not corresponds to any of the above categories.

Focal species and morphological examination

Field work and visits to museum collections were carried out between 2011

and 2017. Field collection of specimens followed the technique of visual encounter

surveys and pitfall-barrier traps (Crump & Scott,1994). Museum acronyms are found

in Sabaj (2016) except for Museo de Biodiversidad del Peru (MUBI; this collection is

part of Museo de Historia Natural, Universidad Nacional de San Antonio Abad,

Cusco, Peru). Collecting permits in Peru were granted by Dirección General Forestal

y de Fauna Silvestre del Ministerio del Medio Ambiente (MINAN; No. AUT-IFS-2017-

055), in Ecuador by Ministerio del Ambiente (MA; 001-1-IC-FAU-DNB/MA) and in

Brazil by the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio; No.

39792-1 and No. 32401). The material of Amazophrynella teko from Mitaraka

(French Guiana) was collected during the “Our Planet Reviewed” expedition,

organized by the MNHN and Pro-Natura International.

We examined topotypical material of Amazophrynella minuta deposited at the

collection of Amphibians and Reptiles of the Instituto Nacional de Pesquisas da

Amazônia–INPA (INPA–H) and three syntypes (NHMG 462, NHMG 463, NHMG 464)

deposited at the Göteborgs Naturhistoriska Museum, Sweden; five specimens of A.

bokermanni (Izecksohn, 1993) from near the type locality (c. 30 Km) deposited at the

INPA collection; the type series of A. vote (Ávila et al., 2012) deposited at the

70

Coleção Zoológica de Vertebrados of the Universidade Federal de Mato Grosso–

UFMT, Cuiabá, Mato Grosso, Brazil (UFMT–A) and INPA; A. manaos (Rojas et al.,

2014) deposited at the INPA; A. amazonicola and A. matses (Rojas et al., 2015)

deposited at the Museo de Zoología–Universidad Nacional de la Amazonia Peruana–

UNAP and A. javierbustamantei (Rojas et al., 2016) deposited at the Museo de

Biodiversidad del Peru (MUBI), Museo de Historia Natural de la Universidad Nacional

Mayor de San Marcos (MHNSM). List of examined specimens is found in Appendix

S1.

Qualitative morphological terminology was according to Kok & Kalamandeen

(2008). Morphological comparison between specimens were made through visual

inspection of diagnostic characters that include: dorsal skin texture, ventral skin

texture, head shape, shape of palmar tubercle, relative length of fingers and venter

coloration (Fouquet et al., 2012b, Rojas et al., 2014, 2015, 2017). We used ventral

incision to perform gonadal analyses . Developmental stages of tadpoles were

determined using Gosner's protocol (1960). Descriptive terminology, morphometric

variables and developmental stages of tadpoles follow Altig & McDiarmid (1999).

Spectral and temporal parameters of advertisement calls (when available) were

analyzed in the software Praat for Windows (Boersma & Weenink, 2006).

Bioacoustics terminology followed Köhler et al. (2017).

Morphological quantitative analyses

Quantitative measurements of body were obtained with a digital caliper (0.1

mm precision) following Kok & Kalamandeen (2008) with the aid of an ocular

micrometer in a Leica stereomicroscope. Measurements were taken from the right

side of specimens, and, if this was not feasible, from the left side. Measurements

were: SVL (snout-vent length) from the tip of the snout to the posterior margin of the

vent; HL (head length) from the posterior edge of the jaw to the tip of the snout; HW

(head width), the greatest width of the head, usually at the level of the posterior

edges of the tympanum; ED (eye diameter); IND (internarinal distance), the distance

between the edges of the nares; SL (snout length) from the anterior edge of the eye

to the tip of the snout; HAL (hand length) from the proximal edge of the palmar

tubercle to the tip of finger III; UAL (upper arm length) from the edge of the body

71

insertion to the tip of the elbow; THL (thigh length) from the vent to the posterior edge

of the knee; TL (tibia length) from the outer edge of the knee to the tip of the heel;

TAL (tarsal length) from the heel to the proximal edge of the inner metatarsal

tubercle; FL (foot length) from the proximal edge of the inner metatarsal tubercle to

the tip of toe IV. We rounded all measurements to first decimal place to avoid

pseudoprecision (Hayek, Heyer & Gascon, 2001).

Principal Component Analyses (PCA) were performed on residuals obtained

by linear-regressing each variable on SVL, thus removing the effects of size. We

used only males specimens because of absence of females in some lineages. The

PCA was used to detect groups representing putative species. We also performed a

discriminant Function Analysis (DFA) to identify morphometric variables that

contribute the most to species separation and to test the classification of specimens

into mtDNA lineages. For DFA we used morphometric size-free data set. To

determine the number of correct and incorrect assignments of specimens to each of

the mtDNA lineages, we jackknifed our data matrix. The significance of differences of

morphological variables among mtDNA lineages was tested using the Kruskal-Wallis

(KW) non-parametric test. All the statistical analyses (PCA, DFA and KW) were

performed in R v3.4.3 (R Development Core Team) using the stats package and

setting the significance cut–off at 5%.

DNA amplification

DNA extraction, gene amplification and sequencing was carried out using

standard protocols (Appendix S2). Sequence data were deposited in GenBank under

the accession numbers MH269714–MH270330 (Table S2a).

Phylogenetic analyses and species delimitation

We collected molecular data for 230 individuals of Amazophrynella from 35

localities, including topotypical material for all nominal species and encompassing the

entire distribution of the genus. We obtained a total of 1430 bp from three

mitochondrial loci [16S rRNA (16S), 480 bp; 12S rRNA (12S), 350 bp; and

Cytochrome oxidase subunit I (COI), 600 pb (see Appendix S4, Table S4a)]. The

edition and alignment of the sequences was performed using Geneious v.6.1.8.

(Kearse et al., 2012) and the Clustal W algorithm (Thompson et al., 2002). We used

72

only unique haplotypes for phylogenetic reconstruction. We concatenated all loci,

treating them as a single partition evolving under the same model of molecular

evolution. The best model of molecular evolution (GTR+G+I) was estimated in

JModelTest (Posada, 2008) and selected using the Akaike Information Criterion–AIC.

Phylogenetic analyses were performed using Bayesian Inference (BI) using MrBayes

3.2.1. (Huelsenbeck & Ronquist, 2001). We generated 107 topologies, sampling

every 1000th topology and discarding the first 10% topologies as burn-in. The

stationarity of the posterior distributions for all model parameters was verified in

Tracer v1.5 (Rambaut & Drummond, 2009). From the MCMC output, we generated

the final consensus tree-maximum clade credibility tree using Tree Annotator v1.6.2

(part of Beast software package). For visualization and edition of the consensus

maximum clade credibility tree, we used the program Figtree v.1.3. (Rambaut, 2009).

We used a Poisson tree processes (PTP) model (Zhang et al., 2013) to infer

the most likely number of species in our dataset, as implemented in the bPTP server

(http://species.h-its.org/ptp/). The PTP model is a simple, fast and robust algorithm to

delimit species using non-ultrametric phylogenies, ultrametricity is not required

because the algorithm models speciation rates by directly using the number of

substitutions. The fundamental assumption is that the number of substitutions

between species is significantly higher than the number of substitutions within

species. In a sense, this is analogous to the GMYC (General Mixed Yule Coalescent)

approach that seeks to identify significant changes in the rate of branching events on

the tree. However, GMYC uses time to identify branching rate transition points,

whereas, in contrast, PTP directly uses the number of substitutions (Zhang et al.,

2013). For input, we used a BI tree estimated by MrBayes. We ran the PTP analyses

using 10⁵ MCMC generations, thinning value of 100, a burn-in of 10%, and opted for

remove the outgroup to improve species delimitation. Convergence of MCMC chain

was confirmed visually. To ensure that the lineages detected using PTP presented

high genetic distance (>3.0%, sensu Fouquet et al., 2007) we calculated uncorrected

p-distance using the 16S mtDNA (Vences et al., 2005) in the program MEGA 7.0

(Kumar, Stecher & Tamura, 2016).

To generate a dated tree in Beast 2.0 (Drummond & Rambaut, 2007), we

selected one representative individual per species. We used a birth and death prior,

http://species.h-its.org/ptp/

73

GTR+I+G evolution model and calibrated the tree using normal distribution following

the divergence time estimates of Fouquet et al. (2012a): crown age of Hyloidea

(mean = 77.0 ± 10 Ma); basal divergence time of Bufonidae (mean = 67.9 ± 12 Ma);

divergence of Atelopus + Oreophrynella vs. other Bufonidae (mean = 60.0 ± 11 Ma);

Nannophryne vs. other Bufonidae (mean = 47.0 ± 8 Ma); Rhaebo vs. other crown

Bufonidae (mean = 40.8 ± 7 Ma) and Dendrophryniscus vs. other crown Bufonidae

(mean = 52.1 ± 9). We generated 107 topologies, sampling every 1000th topology and

discarding the first 10% topologies as burn-in. Stationarity of the posterior

distributions for all model parameters was verified in Tracer v1.5 (Rambaut &

Drummond, 2009). From the MCMC output, we generated the final consensus

maximum clade credibility tree using Tree Annotator v1.6.2 (part of Beast software

package). For visualization and edition of the consensus tree, we used the program

Figtree v.1.3. (Rambaut, 2009).

Environmental analyses

The environmental analyses were undertaken in order to test if delimited

species occur in distinct climatic environments (Soberón et al. 2005). We retrieved

high resolution bioclimatic layers (30 arc–seconds ~ 1 km, present environmental

conditions) using the Community Climate System Model- (CCSM4) from the

WorldClim project (http://www.worldclim.org/) (Hijmans et al., 2005). To avoid

geographic pseudoocurrence of points, localities were filtered using the program

Geographic Distance Matrix Generator 1.2.3. (Ersts, 2014) considering a threshold of

1 km between localities. The localities of each lineage used for analyses are in

Appendix S3, Table S3a.

To identify environmental variables that were most informative and test the

classification of specimens into mtDNA lineages using ecological variables, we

performed Principal Component Analysis (PCA) and Discriminant Function Analysis

(DFA) separately for each lineages/species of the eastern and western clades. The

analyses were performed using the 19 BioClim environmental variables in WordClim.

Probability of correct assignment of individuals to lineages was tested using

jackknife.

Electronic publication of new zoological taxonomic names

http://www.worldclim.org/

74

The electronic version of this article in Portable Document Format (PDF) will

represent a published work according to the International Commission on Zoological

Nomenclature (ICZN), and hence the new names contained in the electronic version

are effectively published under that Code from the electronic edition alone. This

published work and the nomenclatural acts it contains have been registered in

ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life

Science Identifiers) can be resolved and the associated information viewed through

any standard web browser by appending the LSID to the prefix http://zoobank.org/.

The LSID for this publication is: urn:lsid:zoobank.org:pub:1C6046BE-CFC4-4060-

A1CA-0C9C9C1C7A0A. The online version of this work is archived and available

from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.

Results

Phylogenetic and species diversity

The concatenated data resulted in a strongly supported phylogeny (Fig. 1),

with high degree of divergence among putative and nominal species of

Amazophrynella. The PTP model of species delimitation detected a total of eighteen

lineages (posterior probability = 0.48–0.91) (Appendix S4, Fig. S4a) of which seven

are nominal species and 11 are candidate species.

The phylogeny of Amazophrynella recovered the presence of two clades

diverging basally, both strongly supported: one distributed in eastern and other in

western Amazonia (see Fig. 1A). The eastern clade was formed by two strongly

supported subclades, herein called northeastern (NE) and southeastern (SE) clades.

The northeastern clade included three lineages and the southeastern clade seven

lineages. The western clade was formed by two well supported subclades, herein

called northwestern (NW) and southwestern (SW) clades. Both subclades were

composed of four lineages (see Fig. 1A). Uncorrected p-distances for 16S mtDNA

between pairs of sister lineages are presented in Table 1. Each lineage presented

high genetic divergence (>3.0%) compared to its sister taxon and ranged between

3.0–3.2% (3.0 ± 0.1) to 4.0–6.0% (5.0 ± 0.1).

Our timetree recovered Dendrophryniscus as sister taxon of Amazophrynella

(see Appendix S5, Fig. S5a for complete timetree calibration), with a divergence time

http://zoobank.org/

75

estimated at 38.1 Ma (95% HPD: 49.0–29.0 Ma), an Eocene divergence, with strong

support (pp = 1.0, see Fig. 2). Within Amazophrynella the eastern/western

divergence was estimated at 24.8 Ma (95% HPD: 30.0–19.0 Ma), a Late Oligocene

to Early Miocene divergence. Within the eastern clade the SE and NE subclades

diverged during the Early Miocene (20.1 Ma, 95% HPD: 22.0–18.0 Ma). In the

western clade, the split between the NW and SW subclades was estimated at 16.5

Ma (95% HPD= 18.0–13.0 Ma), a Middle Miocene divergence. Divergence time

between each pair of lineages within each of the four above clades varied between

10.8 and 2.1 Ma.

Morphological analyses

A total of 468 specimens (adult males and females) were examined for

comparative morphological analyses (Table 2); these analyses did not include

Amazophrynella aff. matses sp, A.sp2 and A sp3 (see Fig. 1). Measurements of

males and females are presented in Table 3 and Table 4. For morphometric analyses

(Principal Components Analyses-PCA and Discriminant Function Analyses-DFA) we

used 237 adult male specimens (87 from the eastern clade and 148 from the western

clade). The specimens used in morphometric analyses are listed in Appendix S6.

The PCA of the eastern and western clades revealed a grouping of specimens

based on morphometric traits and allowed us to distinguish all the mtDNA lineages in

multivariate space (Fig. 3A and 3B). Character loadings, eigenvalues and percentage

of variance explained for PCA (PC I-II) for morphometric variables for the eastern and

western clades are provided in Appendix S7 and Table S7a-b.

In the eastern clade specimens of each lineage can be successfully separated

based on morphometric traits using PCA (Fig 3A). The first two principal components

extracted by the PCA account for 57.7% of the variation found in the dataset. The

first component (PC1) explained 37.48% of the total variation and the second

component (PC2) explained 20.29% of the variation. Using DFA a total of 80% of

specimens were correctly classified to phylogenetic groups. The number of

individuals correctly assigned to each clade by DFA are presented in Table 5.The

DFA showed that the variables that contributed the most to the morphometric

76

separation were snout length, tarsal length, and head width. Head measurement

traits (head width, head length, snout length, and intranasal distance) explained 93%

of the classification by the first two discriminant axes (Appendix S8, Fig. S8a-B).

Loadings and percentage of variance explained for discriminant axes (F1–2) of

morphometric variables in eastern clade are provided in Appendix S8 and Table

S8a).

In the western clade specimens of each lineage can be successfully separated

based on morphometric traits using PCA (Fig 3B). The first two principal components

extracted by the PCA account for 52.37% of the variation found in the dataset. The

first component (PC1) explained 33.2% of the total variation and the second

component (PC2) explained 19.17% of the variation. Using the DFA a total of 68% of

specimens were correctly assigned to phylogenetic groups. The number of

individuals correctly assigned to each clade by DFA are presented in Table 5. The

DFA showed that the variables that most contributed to the morphometric separation

were eye diameter, hand length, head width and foot length. Head traits (head

length, eye diameter and intranasal distance) and hand traits (hand length) were the

variables that explained 78% of the classification by the first two discriminant axes

(Appendix S8, Fig. S8a-A). Loadings and percentage of variance explained for

discriminant axes (FI–II) of morphometric variables in western clade are provided in

Appendix S8 and Table S8a.

Environmental analyses

We obtained a total of 90 unique localities for final analysis, 43 localities of the

eastern and 47 localities of the western clade, representing the occurrences of all

species but Amazophrynella aff. matses sp, A.sp2 and A sp3 (see Fig. 1). The list of

localities used for environmental analyses and discriminant function analyses are in

Appendix S3 and Table S3a.

The PCA of the eastern and western clades revealed a grouping of specimens

based on environmental traits and allowed us to distinguish all the mtDNA lineages in

the multivariate space (Fig 3C and 3D). Character loadings, eigenvalues and

percentage of variance explained for PCA (PC 1-2) analyses for environmental

77

variables for the eastern and western clades are provided in Appendix S7 and Table

S7c-d.

In the eastern clade specimens of each lineages can be successfully

separated based on environmental traits using PCA (Fig 3C). The first two principal

components extracted by the PCA account for 87.71% of the variation found in the

dataset. The first component (PC1) explained 73.28% of the total variation and the

second component (PC2) explained 14.43% of the variation. A total of 65% of

specimens were correctly classified to their lineage. The numbers of individuals

correctly assigned to each clade by DFA are presented in Table 6. The

environmental variables that most contributed to separating lineages were mean

temperature of the coldest quarter (bio11), maximum temperature of the warmest

month (bio5), mean diurnal temperature range (bio2) and isothermality (bio3)

(Appendix S8, Fig. S8a-C). Loadings and percentage of variance explained per

discriminant axes (F1–2) of environmental variables in the eastern clade are provided

in Appendix S8 and Table S8b.

In the western clade specimens of each lineages can be successfully

separated based on environmental traits using PCA (Fig 3D). The first two principal

components extracted by the PCA account for 95.55% of the variation found in the

dataset. The first component (PC1) explained 95.37% of the total variation and the

second component (PC2) explained 0.18% of the variation. A total of 81% of

specimens were correctly assigned to their candidate species. The numbers of

individuals correctly assigned to each clade by DFA are presented in Table 6. The

environmental variables that most contributed to group separation were annual mean

temperature (bio1), mean diurnal temperature range (bio2), mean temperature of the

warmest quarter (bio10) and mean temperature of the wettest quarter (bio8)

(Appendix S8 and Fig. S8a-D). Loadings and percentage of variance explained for

discriminant axes (F1–2) of environmental variables in the western clade are

provided in Appendix S8 and Table S8b.

Taxonomic decisions

Our data analysis of Amazophrynella suggest the existence of 18 linages of

which seven are nominal species, three Deep Conspecific Lineages, one

78

Unconfirmed Candidate Species, three Uncategorized Lineages and four Confirmed

Candidate Species (Table 2). The four CCSs presented at least one diagnostic

morphological character, monophyly with a strong phylogenetic support using the

standard DNA barcode 16S fragment (Vences et al., 2005) and divergence from its

sister taxon at environmental and morphometric data. Based on these results, herein

we described A. teko sp. nov., A. siona sp. nov. A. xinguensis sp. nov. and A.

moisesii sp. nov.

Species accounts

Amazophrynella teko sp. nov.

urn:lsid:zoobank.org:act:590F41D2-7138-42F8-8509-448602C2D040

Amazonella sp. Guianas (Fouquet et al. 2012a: 829, French Guiana [in part])

Amazophrynella sp. Guianas (Fouquet et al. 2012b: 68, French Guiana [in part])

Amazophrynella sp. Guianas (Rojas et al. 2015: 85, French Guiana [in part])

Amazophrynella sp1. (Fouquet et al. 2015: 365, French Guiana [in part])

Amazophrynella sp. aff. manaos (Rojas et al. 2016: 49, French Guiana [in part])

Holotype (Fig. 4). MNHN 2015.136, adult male, collected at Alikéné (3°13'07''N,

52°23'47''W), 206 m a.s.l., district of Camopi, French Guiana by J.P. Vacher on

March 21, 2015.

Paratypes. Twenty-six specimens (males = 13; females = 13). French Guiana:

District of Saint Laurent du Maroni: Mitaraka layon (2°14'09''N, 54°26' 57''W) 330 m

a.s.l., MNHN 2015.137, MNHN 2015.138, MNHN 2015.139, MNHN 2015.140 (adult

males), MNHN 2015.141, MNHN 2015.142, MNHN 2015.143 (adult females), A.

Fouquet and M. Dewynter between 23 and 28 February 2015; Pic Coudreau du Sud

(2°15'14''N, 54°21'04''W) 360 m a.s.l., MNHN 2015.152 (adult male), MNHN

2015.153 (adult female), M. Blanc on February 2015. Flat de la Waki (3°05'15'' N,

53°24'12''W) 173 m a.s.l., INPA–H 36598 (adult female), J.P. Vacher on April 04,

2014. District of Camopi: Mitan (2°37'42''N, 52°33'15''W) 110 m a.s.l., INPA–H

36596, MNHN 2015.144, MNHN 2015.145, MNHN 2015.146, MNHN 2015.147,

MNHN 2015.148 (adult males), MNHN 2015.149, MNHN 2015.150 (adult females),

79

A. Fouquet and P. Nunes between 20 and 24 March 2015. Alikéné (3°13'07''N,

52°23'47''W) 206 m a.s.l. District of Saint Georges: Saint Georges (3°58'03''N,

51°52'20''W) 76 m a.s.l., MNHN 2015.151 (adult male), A. Fouquet and E. Courtois

on February 2015; Mémora (3°18'47''N, 52°10'49''W) 77 m a.s.l., MNHN 2015.154

(adult male), MNHN 2015.155 (adult female), A. Fouquet and P. Nunes on March 18,

2015; Saut Maripa (3°48'22''N, 51°53'36''W) 51 m a.s.l., INPA–H 36597, INPA–H

36610, INPA–H 36599, INPA–H 36601, INPA–H 36600 (adult females), Antoine

Fouquet and E. Courtois on February 2012.

Diagnosis. An Amazophrynella with (1) SVL12.9–15.8 mm in males, 17.9–21.5 mm in

females (2) snout acute in lateral view; upper jaw, in lateral view, protruding beyond

lower jaw; (3) texture of dorsal skin granular; (4) cranial crest, vocal slits and nuptial

pads absent; (5) dorsum covered by abundant rounded granules; (6) abundance of

granules on tympanic area, on edges of upper arms and on dorsal surface of arms;

(7) ventral skin highly granular; (8) fingers slender, basally webbed; (9) finger III

relatively short (HAL/SVL 0.2–0.22 mm, n = 30); (10) finger I shorter than finger II;

(11) palmar tubercle protruding and elliptical; (12) hind limbs relatively short

(TAL/SVL 0.48–0.49, n = 30); (13) toes slender, basally webbed; in life: (14) venter

cream; small blotches on venter.

Comparison with other species (characteristics of compared species in parentheses).

Amazophrynella teko sp. nov. is morphologically most similar to A. manaos from

which it can be distinguished by: large SVL of males 12.9–15.8 mm, n = 13 (vs. 12.3–

15.0 mm, n = 27, Fig. 5, t = 2.04, df = 16.78, p–value = 0.02); snout acute in lateral

view (truncate); larger THL of males, 53% of SVL, n = 13 (vs. smaller THL, 47.2% of

SVL, n = 27); abundance of granules on tympanic area (absent); smaller hind limbs,

TAL/SVL 0.48–0.49, n = 30 (vs. 0.50–0.51, n = 56). From A. bokermanni by the

relative size of fingers: FI<FII (vs. FI>FII); thumb not large and robust (thumb large

and robust, Fig. 6A vs. 6D). From A. vote by larger SVL of males 12.9–15.8 mm, n =

13 (vs. 10.0–14.2 mm, n = 14, see Fig. 3, t = 4.93, df = 25.91, p–value = 0.001) and

females 17.9–21.5 mm, n = 17 (vs. 13.5–19.1 mm, n = 21); texture of dorsal skin

granular (tuberculate); longer UAL, 33% of SVL (vs. smaller UAL 29.8%); longer hind

limbs, TAL/SVL 0.48–0.49, n = 30 (vs. 0.43–0.44, n = 35); venter coloration cream

(red-brown, Fig. 7B vs. 7F). From A. minuta by snout acute in lateral view (pointed,

80

Fig. 8A vs. 8B); larger snout of males–50% of HL, n = 14 (vs. SL 46% of HL, n = 13);

palmar tubercle elliptical (rounded, Fig. 6A vs. 6G); venter cream (yellow-orange, Fig.

7A vs. 7B). From A. amazonicola by dorsal skin texture granular (finely granular);

absence of small triangular protrusion on the tip of the snout (present, Fig. 8A vs.

8H); palmar tubercle elliptical (rounded); venter coloration cream (venter yellow–

orange). From A. matses by smaller SVL of males 12.9–15.8 mm, n = 13 (vs. 11.4–

13.5 mm, n = 13, Table 3 and Fig. 3, t = 7.89, df = 21.34, p–value = 0.001) and

females 17.9–21.5 mm, n = 17 (vs. 15.6–19.0 mm, n = 18); snout profile acute in

lateral view (truncate); texture of dorsal skin granular (spiculate); venter cream

(venter pale yellow). Compared to A. javierbustamantei by shorter hand, HAL/SVL

0.2–0.22, n = 30 (vs. 0.23–0.24, n = 60); texture of dorsal skin granular (tuberculate);

venter cream (pale orange yellowish); tiny blotches on venter (tiny rounded points,

Fig. 7B vs. 7J). Compared to A. siona sp. nov. by large size SVL of adult males 12.9–

15.8 mm, n = 14 (vs. 11.5–14.7 mm, n = 27, Fig. 5, t = 6.15, df = 18.1, p–value =

0.001) and adult females 17.9–21.5 mm, n = 17, (vs. 16.1–20.0 mm, n = 35) and;

smaller hind limbs, TAL/SVL 0.48–0.49, n = 30 (vs. 0.5–0,52, n = 62); palmar

tubercle elliptical (rounded), venter cream (venter bright red). From A. xinguensis sp.

nov. by FI < FII (vs. FI ≥ FII, Fig. 6A vs. 6C); palmar tubercle rounded (ovoid). From

A. moisesii sp. nov. by venter cream (venter pale yellow); shorter hand, HAL/SVL

0.2–0.22, n = 30 (vs. 0.23–0.25, n = 28).

Description of the holotype. Body slender, elongate. Head triangular in lateral view

and pointed in dorsal view. Head longer than wide. HL 34.4% of SVL. HW 27.8% of

SVL. Snout acute in lateral view and triangular in ventral view. SL 50% of HL. Nostrils

slightly protuberant, closer to snout than to eyes. Canthus rostralis straight in dorsal

view. Internarial distance smaller than eye diameter. IND 33.3% of HW. Upper eyelid

covered with smaller pointed tubercles. Eyes wide, prominent, ED 30.7% of HW.

Tympanum not visible through the skin. Skin around tympanum covered by granules.

Vocal sac not visible. Texture of dorsal skin granular. Texture of dorsolateral skin

granular. Forelimbs slender. Edges of forelimbs with scattered granules, in dorsal

and ventral view. Upper arms robust. UAL 33.1% of SVL. Abundance of granules on

upper arm. HAL about 22.5% of UAL. Fingers basally webbed. Fingers slender, tips

unexpanded. Relative length of fingers: I<II<IV<III. Supernumerary tubercles and

81

accessory palmar tubercles rounded. Palmar tubercle small and rounded.

Subarticular tubercles rounded. Texture of gular region granular. Texture of ventral

skin highly granular. Small granules in the venter. Hindlimbs slender. Edges of the

thigh to tarsus covered by conical tubercles. THL 52.3% of SVL. TAL 45.6% of SVL.

Tarsus slender. TL 29.8% of SVL. FL 70.8%. Relative length of toes: I<II<III<V<IV.

Inner metatarsal tubercle oval. Outer metatarsal tubercles small and rounded.

Subarticular tubercles rounded. Toes slender and elongate. Tip of toes not

expanded, basally webbed. Cloacal opening slightly above midlevel of thighs.

Measurement of the holotype (in mm). SVL: 15.1; HW: 4.2; HL: 5.2; SL: 2.6; ED: 1.6;

IND: 1.4; UAL: 5.0; HAL: 3.4; THL: 7.9; TAL: 6.9; TL: 4.5; FL: 5.6.

Variation (Fig. 9). There is little variation among the examined specimens. Sexual

dimorphism was observed in SVL, with 12.9–15.8 mm (14.7 ± 0.8 mm, n = 13) in

males and 17.9–21.5 mm (19.2 ± 1.8 mm, n =17) in females. Specimens (MNHN

2015.137, MNHN 2015.138, MNHN 2015.139, MNHN 2015.140) present lesser

abundance of granules on arm insertion. In some individuals (MNHN 2015.143) the

ventral and the dorsolateral region present one to three large tubercles. Subarticular

tubercles more protruding and swollen in females. Blotches on belly display different

sizes (larger vs. small, see Fig. 10). In life, venter coloration between cream to off-

white .Palm and sole between light red and orange. In preserved specimens, the

palmar tubercle is more flattened than in life.

Coloration of the holotype (in life). Head black brown, in dorsal view. Dorsum brown.

Flanks brown. Scattered tubercles on flanks white. Dorsal surfaces of upper arm, arm

and hand black. Dorsal surfaces of thighs, tibia, tarsus and foot black. Ventral

surfaces of upper arm, arm and palm cream. Ventral surfaces of thighs cream,

mottled with black blotches. In dorsal view, tarsus and tibia creamy, sole light red.

Gular region brown. Belly cream with black tiny blotches. Posterior region of the thigh

and cloaca with black blotches. Longitudinal white stripe on upper jaw extending from

nostril to tympanum. Iris golden and pupil black.

Color in preservative (~70% ethanol, Fig. 10). Almost the same as color in life. We

noted the progressive loss of dorsal coloration which eventually becomes black. The

82

chest lost its coloration and became less intense. The dark blotches on venter

became less evident. The coloration of the fingers and toes became pale red.

Bioacoustics (Fig. 11). Lescure & Marty (2000) described the advertisement call of

Amazophrynella teko sp. nov. as the call of Dendrophryniscus minutus. We recorded

two individuals at Mitaraka (2°14'09''N, 54°26'57''W) and Alikéné (3°13'07''N,

52°23'47''W), French Guiana. All call parameters described by Lescure & Marty

(2000) show an overlap with our recorded calls. Call trill emitted at regular intervals.

Note duration 0.15–0.19 seconds (0.16 ± 0.01 seconds, n = 29). Fundamental

frequency between 2733.3–3555.3 Hz (3115.3 ± 263.7 Hz, n = 29). Dominant

frequency between 3993.3–4980.8 Hz (4638.4 ± 288.27 Hz, n = 29). Number of

pulses between 10–30 per call (25.5 ± 10.4 pulses/call, n = 29). Time to peak

amplitude between 0.06–0.13 seconds (0.08 ± 0.02 seconds, n = 29). The call has a

downward modulation, reaching its maximum frequency near its beginning.

Distribution and natural history (Fig. 1B). Amazophrynella teko sp. nov. have been

recorded from the district of Saint Laurent du Marioni, Saint Georges and Camopi,

French Guiana, the state of Amapá, Brazil and in the southern region of Suriname

(AF personal observation). It occurs at elevations ranging from 70 m a.s.l. to 350 m

a.s.l. The species is diurnal and crepuscular but is also active at night during peak

breeding period, which normally occurs at the beginning of the rainy season

(January–February). This species shows a conspicuous sexual dimorphism, with

males being much smaller than females. The conservation status of this species

remains unknown. The habitat destruction and pollution must affect their populations;

however, due to its abundance we believe that this species probably needs not be

classified above Least Concern category.

Etymology. The specific epithet is a noun in apposition and refers to the name of the

Teko Amerindians who occupy the southern half of French Guiana; the area

occupied by the Teko tribe also encompasses the type locality.

Amazophrynella siona sp. nov.

urn:lsid:zoobank.org:act:66224D58-8DE0-4D5B-950D-1206FFA4AC11

Atelopus minutus: (Duellman & Lynch 1969: 238, Sarayacu [Ecuador])

83

Dendrophryniscus minutus (Duellman 1978: 120, Santa Cecilia [Ecuador])

Dendrophryniscus minutus (Duellman & Mendelson III 1995: 336, vicinities of San

Jacilllo and Teniente Lopez [Peru])

Amazonela cf. minutus “western Amazonia” (Fouquet et al. 2012a: 829, “western

Amazonia”, Ecuador [in part])

Amazophrynella cf. minutus “western Amazonia” (Fouquet et al. 2012a: 68, “western


Amazophrynella aff. minuta “western Amazonia” (Rojas et al. 2015: 84, “western


Amazophrynella aff. minuta (Rojas et al. 2016: 49, “western Amazonia”, Ecuador [in

part])

Holotype (Fig. 12). QCAZ 27790, adult male, collected at Yasuni National Park,

(0°40'01"S, 76°26'33"W), 200 m a.s.l., Bloque 31, Apaika, Province of Orellana,

Ecuador, by F. Nogales on October 7 2000.

Paratypes. Sixty-six specimens (males = 17, females = 49), Ecuador: Provincia

Sucumbíos: Reserva de Producción Faunística Cuyabeno (0°00'58"S, 76°09'59"W),

203 m a.s.l., QCAZ 52433–34, S. R. Ron; Reserva de Producción Faunística

Cuyabeno (0°00'58"S, 76°09'59"W), 203 m a.s.l, QCAZ 37758–59, QCAZ 37761, L.

A. Coloma; Reserva de Producción Faunística Cuyabeno (0°00'58"S, 76°09'59"W),

203 m a.s.l., QCAZ 6071, QCAZ 6091, QCAZ 6095, QCAZ 6097, QCAZ 6105 (adult

females), QCAZ 6111 (adult males), QCAZ 6113, QCAZ 6118, QCAZ 6127, QCAZ

6128, J. P. Caldwell; Santa Cecilia (0°04'50"S, 76°59'24"W), 330 m a.s.l., QCAZ

4469, QCAZ 4472, M. Crump; Tarapoa (0°07'10"S, 76°20'23"W), 330 m a.s.l., QCAZ

36331, QCAZ 36336, QCAZ 36338, QCAZ 36357, E. Ponce. Provincia Pastaza:

Community of Kurintza (2°03'50"S, 76°47'53"W), 350 m a.s.l., QCAZ 56342 (adult

female), QCAZ 56354, QCAZ 56361 (adult males), D. Velalcázar; A. Villano

community, AGIP oil company (1°30'28"S, 77°30'41"W), 307 m a.s.l., QCAZ 38599,

QCAZ 38679, QCAZ 38722, Galo Díaz; Around Villano community, AGIP oil

company (1°30'28"S, 77°30'41"W), 307 m a.s.l. QCAZ 38642, Y. Mera; Community

of Kurintza (2°03'50"S, 76°47'53"W), 350 m a.s.l., QCAZ 38809 (adult females), F.

84

Varela; Community of Kurintza (2°03'50"S, 76°47'53"W), 350 m a.s.l., QCAZ 54213,

Yerka Sagredo; Bataburo Lodge (1°12'30" S, 76°42'59"W), 260 m a.s.l., QCAZ

39408 (adult female), S. D. Padilla; Lorocachi (1°37'17" S, 75°59'21"W), 229 m a.s.l.,

QCAZ 8902 (adult female), M. C. Terán; Lorocachi (1°37'17"S, 75°59'21" W), 229 m

a.s.l., QCAZ 56165 (adult male), S. R. Ron; Bloque 31 in Yasuni National Park,

(0°56'20"S, 75°50'20"W), 230 m a.s.l, QCAZ 11973, QCAZ 11979, QCAZ 11981

(adult males), G. Fletcher; Canelos (0°29'53"W, 76°22'26"S), 265 m a.s.l., QCAZ

52819, QCAZ 52823, D. Pareja; Canelos (0°29'53"W, 76°22'26"S), 265 m a.s.l.,

QCAZ 17391, L. A. Coloma. Provincia Orellana: Tambococha (0°58'42" S,

75°26'13"W), 194 m a.s.l., QCAZ 55345 (adult female), Fernando Ayala-Varela;

Yasuni National Park, scientific station of the Pontificia Universidad Católica del

Ecuador-PUCE, (0°56'31" S, 75°54'18"W), 203 m a.s.l., QCAZ 51068, E. Contreras;

Yasuni National Park, scientific station of the Pontificia Universidad Católica del

Ecuador-PUCE, (0°56'31" S, 75°54'18"W), 203 m a.s.l., QCAZ 21425, QCAZ 21431

(adult females), J. Santos; Garzacocha (0°45'28"S, 76°00'44"W), 230 m a.s.l., QCAZ

20504 (adult female), M. Díaz; Yuriti (0°33'26"S, 76°48'55"W), 220 m a.s.l., QCAZ

10526, (adult female), M. Read; Kapawi Lodge (2°32'19"S, 76°51'30"W), 257 m

a.s.l., QCAZ 8725, S. R. Ron; Kapawi Lodge (2°32'19"S, 76°51'30"W), 257 m a.s.l.,

QCAZ 25504 (adult males), QCAZ 25533 (adult female), K. Elmer; Fatima, 10 km

from Puyo (1°24'47"S, 77°59'56"W), 1000 m a.s.l., QCAZ 7135 (adult female), M.

Tapia; Provincia Morona Santiago: Pankints (2°54'07"S, 77°53'39"W), 320 m a.s.l.,

QCAZ 46430 (adult female), J. B. Molina. Peru: Department Loreto: Teniente Lopez

(2°35'30.90"S, 76°07'2.84"W), 255 m a.s.l., MUBI 7611, MUBI 7685, MUBI 7686,

MUBI 7698, MUBI 7699, MUBI 7700 (adult females), J. C. Chaparro on October 12,

2008; Jibarito (2°47'55.90"S, 76°0'21.51"W), 236 m a.s.l., MUBI 7786, MUBI 7809,

MUBI 7814 (adult female), J. Delgado on November 5, 2008; Shiviyacu

(2°29'30.92"S, 76°5'18.31"W), 226 m a.s.l., MUBI 14730 (adult female), M. Medina

on June 17, 2008; Jibarito (2°43'51.4"S, 76°01'7.48"W), near Corrientes River, 220 m

a.s.l., MUBI 6292 (adult female), G. Chavez on March 20, 2008.

Referred specimens. USNM 520898, 520900b–01 (adult males), USNM 520896–97,

520899, 520901, 520906 (adult females), collected at Lagarto Cocha River

(0°31'23"S, 75°15'25"W), Province of Loreto, Peru by S. W. Gotte on March 1994.

85

Diagnosis. An Amazophrynella with (1) SVL 11.5–14.7 mm in males, 16.1–20.0 mm

in females; (2) snout acute in lateral view; upper jaw, in lateral view, protruding

beyond lower jaw; (3) texture of dorsal skin finely granular; (4) cranial crests, vocal

slits and nuptial pads absent; (5) small granules from the outer edge of the mouth to

upper arm; (6) ventral skin granular; (7) tiny granules on ventral surfaces; (8) fingers

slender, basally webbed; (9) finger III relative short (HAL/SVL 0.20–0.21, n = 62);

(10) finger I shorter than finger II; (11) palmar tubercle rounded; (12) hind limbs

relatively large (TAL/SVL 0.5–0.52, n =62); (13) toes lacking lateral fingers; in life:

(14) venter reddish brown; yellow blotches on venter.


Amazophrynella siona sp. nov. is most similar to A. amazonicola from which it can be

distinguished by (characteristics of compared species in parentheses): the snout

acute in lateral view (pointed, Fig. 8C vs. 8H), absence of protuberance on the tip of

the snout (present); fingers basally webbed (webbing between FI and FII); yellow

blotches on venter (dark blotches, Fig. 7C vs. 7H). From A. matses by the texture of

dorsal skin granular (spiculate); larger HL, 5.6–7.2 mm in adult males, n = 27 (vs.

4.4–6.2 mm, n = 26, t = 7.21, df = 20.1, p–value = 0.001); snout acute in lateral

(truncate); palmar tubercle rounded (elliptical, Fig. 6B vs. 6F); yellow blotches on

venter (black blotches). From A. minuta by texture of dorsal skin finely granular

(highly granular); small granules from the outer edge of the mouth to upper arm

(small warts); tiny granules cover the venter surfaces (absent); shorter HAL,

HAL/SVL 0.20–0.21, n = 62 (vs. 0.2–0.3, n = 20). Compared to A. javierbustamantei

by shorter hand, HAL/SVL 0.20–0.21, n = 62 (vs. 0.23–0.24 , n = 60); texture of

dorsal skin finely granular (finely tuberculate); snout acute in lateral view

(subacuminate). From A. bokermanni by the relative size of fingers with FI<FII

(FI>FII); thumb not large and robust (large and robust, Fig. 6B vs. 6D). From A. vote

by snout acute in profile (rounded); dorsal skin finely granular (tuberculate); dorsal

coloration light brown (brown); venter bright red (red-brown, Fig. 7C vs. 7F); yellow

blotches on venter (white tiny spots). From A. manaos by present rounded palmar

tubercle (elliptical); snout acute in profile (truncate); venter bright red (white, Fig. 7C

vs. 7G); yellow blotches on venter (black patches). Compared to A. teko sp. nov. by

small SVL of adult males 11.5–14.7 mm, n = 27 (12.9–15.8 mm, n = 14, = 6.15, df =

86

18.1, p–value = 0.001, Fig. 5) and adult females 16.1–20.0 mm, n = 35 (vs. 17.9–

21.5 mm, n = 17); tiny granules cover venter (absent); longer hind limbs, TAL/SVL

0.5–0.52, n = 62 (vs. 0.48–0.49, n = 30); palmar tubercle round (elliptical); venter

bright red (cream). From A. xinguensis sp. nov. by FI<FII (vs. FI ≥ FII, Fig. 6); palmar

tubercle rounded (ovoid); venter bright red (cream). From A. moisesii sp. nov. by

shorter hand, HAL/SVL 0.20–0.21, n = 30 (vs. 0.23–0.25, n = 28); venter bright red

(pale yellow).


and rounded in dorsal view. Head longer than wide. HL 39.6% of SVL. HW 31.3% of

SVL. Snout acute in lateral view and pointed in dorsal view. SL 42.8% of HL. Nostrils

slightly protuberant, closer to snout than to eyes. Canthus rostralis straight in dorsal

view. Internarial distance smaller than eye diameter. IND about 27.6% of HW. Upper

eyelid covered with tiny tubercles. Eye wide, prominent, about 30.3% of HL.

Tympanum not visible through the skin. Skin around tympanum covered by tiny

granules. Vocal sac not visible. Texture of dorsal skin finely granular. Texture of

dorsolateral skin finely granular. Forelimbs slender. Edges of forelimbs with granules,

in dorsal and ventral view. Upper arms robust. UAL 30.5% of SVL. Small granules

from the outer edge of the mouth to upper arm. HAL 72.4% of UAL. Fingers basally

webbed. Fingers slender, tips unexpanded. Relative length of fingers: I<II<IV<III.

Supernumerary tubercles and accessory palmar tubercles rounded. Palmar tubercle

large and rounded. Subarticular tubercles rounded. Texture of gular region finely

granular. Texture of ventral skin granular. Small granules on venter. Hindlimbs

slender. Edges of thigh to tarsus covered by conical tubercles. THL 51.8% of SVL.

TAL 50.6% of SVL. Tarsus slender. TL 29.8% of SVL. FL 60% of THL. Relative

length of toes: I<II<V<III<V. Inner metatarsal tubercle oval. Outer metatarsal

tubercles small and rounded. Subarticular tubercles rounded. Toes slender and

elongate. Tip of toes not expanded, unwebbed. Cloacal opening slightly above

midlevel of thighs.

Measurement of the holotype (in mm). SVL 12.6; HW 3.9; HL 5.0; SL 2.1; ED 1.2;

IND 1.1; UAL 3.8; HAL 2.7; THL 7.2; TAL 6.9; TL 3.9; FL 4.3.

87

Variation (Fig. 13). The new species presents extensive variation among specimens

(eg. https://bioweb.bio/galeria/FotosEspecimenes/Amazophrynella%20minuta/1).

Sexual dimorphism was observed in SVL, with 11.5–14.7 mm (13.0 ± 0.6 mm, n =

29) in males and 16.1–20.8 mm (18.3 ± 0.9 mm, n = 35) in females. Specimens

(MUBI 7686, MUBI 7698, MUBI 7699, MUBI 7700) from Andoas,Peru, present fewer

tubercles on upper arm. Abundance of granules on ventral surfaces varies in density

(eg. QCAZ 21425, QCAZ 21431, QCAZ 20504, QCAZ 10526, QCAZ 46430). Some

individuals (eg. QCAZ 37761, QCAZ 6095, QCAZ 6105) present one to two large

tubercles on dorsolateral region. Specimens from Pastaza (eg. QCAZ 56342, QCAZ

56354, QCAZ 56361, QCAZ 38599, QCAZ 38679, QCAZ 38722) present greater

abundance of granules on dorsum. Some individuals display different sized blotches

on venter, while in other specimens, blotches are absent (Fig. 13C). In life, belly

coloration varies between yellow to light red. The gular region varies from light red to

red. Thighs, shanks, tarsus and feet vary from light red to red, in dorsal view. Palm

and sole color from light red to orange, in ventral view.

Coloration of the holotype (in life). Head brown, in dorsal view. Dorsum mostly brown.

Flanks reddish brown. Dorsal surfaces of upper arm, arm and hand light brown.

Dorsal surfaces of the thighs, tibia, tarsus and foot light brown. Ventral surfaces of

upper arm light red, arm light brown, palm reddish brown. Gular region reddish

brown. Belly bright red with yellow blotches. Axillar region with yellow granules.

Ventral surfaces of thighs, tarsus and tibia reddish brown, sole reddish brown. Iris

golden and pupil black.

Color in preservative (~70% ethanol, Fig. 14). Almost the same as color in life.

Dorsum became brown. We detected a gradual fading of the red and yellow

coloration of the chest and venter. The blotches on venter became less evident.

Fingers and toes became pale red.

Tadpoles (Fig. 15). Duellman & Lynch (1969) described the tadpole of

Amazophrynella siona sp. nov. as Atelopus minutus based on ten individuals at stage

31 and three at stage 40, from Sarayacu, Province of Pastaza, 400 m a.s.l. The

morphological characteristics described by Duellman & Lynch (1969) are similar to

those observed by us. We analyzed ten tadpoles at stage 30. Body ovoid in dorsal

https://bioweb.bio/galeria/FotosEspecimenes/Amazophrynella%20minuta/1

88

view. Total length 11.0–13.2 mm (11.5 ± 0.84 mm). Body length 3.6–4.8 mm (4.2 ±

0.3 mm); depressed in lateral view. Body height 1.2–1.9 mm (1.5 ± 0.2 mm), body

widest posteriorly. Snout rounded in dorsal and lateral view. Eye diameter 0.3–0.5

mm (0.3 ± 0.1 mm). Eye snout distance 0.9–1.4 mm (1.2 ± 0.14 mm). Nostrils small,

closer to eyes than to tip of snout. Inter nasal distance 0.5–0.75 mm (0.6 ± 0.1 mm).

Inter orbital distance 0.5–0.75 mm (0.6 ± 0.09 mm). Spiracle opening single, sinistral

and conical. Spiracle opening on the posterior third of the body. Centripetal wall

fused with the body wall and longer than the external wall. Upper and lower lips bare,

single row of small blunt teeth, sectorial disc absent. Jaw sheaths finely serrated.

Two upper and three lower rows of teeth. Oral disc weight 0.8–1.1 mm (0.9 ± 0.1

mm). Dorsal fin originating on the tail-body junction, increasing in height throughout

the first third of the tail and decreasing gradually in the posterior two thirds of the tail

to a pointed tip, in lateral view. Ventral fin originating at the posteroventral end of the

body, higher at the first third of the tail, decreasing gradually in height toward tail tip.

Tail length 5.4–8.1 mm (6.8 ± 0.9 mm). Tail height 0.9–1.1 mm (0.9 ± 0.1 mm). Body

and tail rosaceous with small dark pointed flecks on body in fixed specimens. In life,

Duellman & Lynch (1969) reported brown body and spotted tail with black and small

brown flecks on caudal musculature, the entire dorsal fin and posterior third of ventral

fin.

Bioacoustics (Fig. 16). The advertisement call of Amazophrynella siona sp. nov. was

described by Duellman (1978) as the advertisement call of Dendrophryniscus

minutus from Santa Cecilia, Ecuador. We analyzed one call from the Reserva de

Producción Faunistica Cuyabeno, Province of Sucumbíos, Ecuador (QCAZ 18833)

(http://zoologia.puce.edu.ec/Vertebrados/Anfibios). The call was recorded one day

after capture, on February 6, 2002. In our analysis all the call parameters from

Duellman (1978) overlap with the call of the new species. Call trill emitted at irregular

intervals. Note duration 0.03– 0.06 seconds (0.013 ± 0.001 seconds, n = 16). The

fundamental frequency 2000–3240.1 Hz (3000.9 ± 101.79 seconds, n = 16).

Dominant frequency 3647.5–4200 Hz (3757.9 ± 138.1 Hz, n = 16). The number of

pulses 23–28 pulses per note (28.5 ± 5.3 pulses/note, n = 16). Time to peak

amplitude 0.01–0.03 seconds (0.02 ± 0.01 seconds, n = 13). The call has a

downward modulation, reaching its maximum frequency almost at the middle.

http://zoologia.puce.edu.ec/Vertebrados/Anfibios)

89

Distribution and natural history (Fig. 1B). Amazophrynella siona sp. nov. have been

recorded from Ecuador, in Provinces of Orellana, Sucumbíos and Pastaza and Peru

in the Province Andoas, northern Loreto Department. It occurs at elevations ranging

from 200–900 m a.s.l. The species is found in the leaf litter of primary and secondary

forest, terra firme or flooded forest, and swamps. It is active during the day; at night

individuals rest on leaves, usually less than 50 cm above ground. It breeds

throughout the year (Duellman, 1978). This species shows conspicuous sexual

dimorphism, with males being much smaller than females. The amplexus is axillar.

Eggs are pigmented; males call from amidst leaf litter. Duellman & Lynch (1969)

reported that this species deposited its eggs in gelatinous strands 245–285 mm long,

with 245–291 eggs. It can be abundant at some sites (eg., Cuyabeno reserve; SRR

pers. obs.) Given its large distribution range (> 20000 km2) which also includes vast

protected areas and locally abundant populations, we suggest assignment this

species to the Least Concern category.

Etymology. The specific epithet is a noun in apposition and refers to the Siona, a

western Tucanoan indigenous group that inhabits the Colombian and Ecuadorian

Amazon. The Siona inhabit the Cuyabeno Lakes region, an area where

Amazophrynella siona sp. nov. is be abundant. While working in his undergraduate

thesis in the early 1990s, SRR lived with the Siona at Cuyabeno. The Siona chief,

Victoriano Criollo, had an encyclopedic knowledge of the natural history of the

Amazonian forest, superior in extent and detail to that of experienced biologists. His

death, a few years ago, represents one of many instances of irreplaceable loss of

traditional knowledge triggered by cultural change among Amazonian Amerindians.

Amazophrynella xinguensis sp. nov.

urn:lsid:zoobank.org:act:55CD4C19-9A39-4DEB-BA6C-F02F9735BB77

Amazophrynella cf. bokermanni (Vaz–Silva et al. 2015: 208, “Volta grande”, Xingu

River, Pará, Brazil)

Holotype (Fig. 17). INPA–H 35471, adult male, collected at the Sustainable

Development Project (PDS) Virola Jatobá (3°10'06'' S, 51°17'54.2''W), 86 m a.s.l.,

municipality of Anapú, state of Pará, Brazil by E. Hernández and E. Oliveira on

December 06, 2012.

90

Paratypes. Twenty two specimens (males = 4, females = 14, immatures = 4). Brazil:

Pará State: Municipality of Senador José Porfírio: Fazenda Paraíso (2°34'37''S,

51°49'50.3''W), 57 m a.s.l., INPA–H 35482, INPA–H 35493 (adult males), INPA–H

35472 (adult female), E. Hernández and E. Oliveira on December 05, 2012.

Municipality of Anapu: PDS Virola Jatobá, (3°10'06''S, 51°17'54.2''W), 86 m a.s.l.,

INPA–H 35484, INPA–H 35485 (adult males), INPA–H 35473, INPA–H 35474,

INPA–H 35475, INPA–H 35476, INPA–H 35477, INPA–H 35478, INPA–H 35479,


INPA–H 3592 (adult females), E. Hernández and E. Oliveira on December 06, 2012.

Municipality of Vitória do Xingu, Ramal dos Cocos (3°09'42.1''S, 52°07'41.9''W), 110

m a.s.l., INPA–H 35486, INPA–H 35487, INPA–H 3588, INPA–H 35489 (immatures),

E. Hernández and E. Oliveira on December 04, 2012.


in females; (2) snout pointed in lateral view; (3) upper jaw, in lateral view, protruding

beyond lower jaw; 4) tympanums, vocal sac, parotid gland and cranial crest not

evident; (5) texture of dorsal skin highly granular; (6) abundance of small tubercles

on dorsum, on upper arm and on arms; (7) texture of ventral skin granular; (8) fingers

I and II basally webbed; (9) finger III relative short (HAL/SVL= 0.20–0.22, n = 18);

(10) thumb larger and robust; (11) finger I larger or equal than finger II, FI = 2.1 vs.

FII = 2.1 in adult males, n = 5 and FI = 2.8 mm, vs. FII = 2.9 mm, in adult females, n

= 13; (12) palmar tubercle ovoid; (13) toes slender, basally webbed; in life: (14)

venter greyish; black dots on venter.


Amazophrynella xinguensis sp. nov. is more similar to A. bokermanni from which it

can be distinguished by: texture of dorsal skin highly granular (granular); relative size

of fingers: FI ≥ FII mean 2.1 mm, in I vs. 2.1 mm in II in A. xinguensis sp. nov. n = 5

(vs. FI > FII, mean 2.2 mm in FI vs. in 2.0 mm FII in A. bokermanni, n = 7, Fig. 6C vs.

6D); shape of palmar tubercle elliptical (rounded); presence of tubercles on dorsum

(absent); dorsal coloration dark brown (light brown); venter light gray (white); gular

region dark brown (grayish brown). From the other species of Amazophrynella the

new species is easily differentiated by having FI ≥ FII (FI < FII in all the other species,

Fig. 6); its greater SVL of males (KW x2 = 108.6, df = 10, p–value = 0.001, Fig. 5) and

91

its protruding ovoid palmar tubercle (vs. A. teko, A. manaos, A. vote, A. minuta, A.

bokermannni, A. javierbustamantei, A. matses, A. Amazonicola, A. siona sp. nov. A.

teko sp. nov., A. moisesii sp.nov. see Fig. 6).

Description of the holotype. Body robust. Elongate. Head pointed in lateral view and

triangular in dorsal view. Head longer than wide. HL 35.5% of SVL. HW 27.1% of

SVL. Snout acute in lateral view and triangular in dorsal and ventral view. SL 64.0%

of HL. Nostrils slightly protuberant, closer to snout than to eyes. Canthus rostralis

straight in dorsal view. Internarial distance smaller than eye diameter. IND about

20.8% of HW. Upper eyelid covered by small granules. Eye prominent, 30.3% of HL.

Tympanum not visible through the skin. Skin around tympanum covered by tiny

granules. Vocal sac not visible. Texture of dorsal skin highly granular. Rounded small

tubercles on dorsum. Texture of dorsolateral skin granular. Forelimbs thick. Edges of

arms of forelimbs with granules, in dorsal and ventral view. Upper arms robust. UAL

28.5% of SVL. Abundance of small tubercles on upper arm. HAL 68.4% of UAL.

Fingers slender, tips unexpanded. Fingers basally webbed on finger II and finger III.

Relative length of fingers: I≥II<IV<III. Supernumerary tubercles rounded. Palmar

tubercle ovoid. Gular region finely granular. Texture of ventral skin granular. Small

granules in the venter. Hind limbs slender. Edges of thigh to tarsus covered by

conical tubercles. THL 52.2% of SVL. Tibias almost the same length as thighs. TAL

48.9% of SVL. Tarsus slender. TL 29.8% of SVL. FL 60.0% of THL. Relative length

of toes: I<II<III<V<IV. Inner metatarsal tubercle oval. Outer metatarsal tubercles

small and rounded. Subarticular tubercles rounded. Toes slender. Tip of toes not

expanded, basally webbed. Cloacal opening slightly above midlevel of thighs.

Measurement of the holotype (in mm). SVL 18.5, HW 5.0, HL 6.0, SL 3.1, ED 2.1,

IND 1.6; UAL 6.6; HAL 4.1, FI 1.9, FII 1.9, THL 9.7, TAL 9.3, TL 5.7, FL 6.4.

Variation (Fig. 18). Sexual dimorphism was observed in SVL, with 17.7–20.0 mm

(18.9 ± 1.0 mm, n = 5) in males and 22.4–26.3 mm (24.1 ± 1.2 mm, n = 13) in

females. Some individuals (i.e. INPA–H 35473, INPA– H 35477, INPA–H 35475)

present one to two large tubercles on dorsolateral region. The granules on ventral

surfaces are greatly abundant in some individuals (eg. INPA–H 35478, INPA–H

35480, INPA–H 35486). The gular region presents black or brown coloration. Dots on

92

venter display different sizes (small to medium) and abundance (Fig. 18D vs 18A). In

life, ventral surfaces from cream to light gray. Thighs, shanks and tarsus between

cream to white coloration, in ventral view. Palm and sole present different tonalities of

orange, in ventral view.

Coloration of the holotype (in life). Head dark brown, in dorsal view. Dorsum mostly

light brown with brown chevrons. Flanks cream. Dorsal surfaces of upper arm, arm

and hand light brown. Dorsal surfaces of thighs, tibia, tarsus and foot brown. Ventral

surfaces of upper arm, arm and palm cream. Ventral surfaces of thighs, tarsus and

tibia cream, sole black. Gular region cream. Belly cream with tiny black blotches.

White line from the tip of snout to cloaca. Iris golden and pupil black.

Color in preservative (~70% ethanol, Fig. 19). In preservative, the coloration is almost

the same than life. The coloration of the dorsum became dark brown. Gular region

and venter became white. The iris loses its coloration. The fingers and toes became

cream.

Distribution and natural history (Fig. 1B). Amazophrynella xinguensis sp. nov. have

been recorded from State of Pará, Brazil, at three localities: PDS Virola Jatoba,

municipality of Anapú, Fazenda Paraiso, municipality of Senador José Porfirio (right

bank of Xingu River) and Ramal dos Cocos, municipality of Altamira (left bank of

Xingu River), all of them in area of influence of the Belo Monte dam. It occurs in

elevations of 86–106 m a.s.l. This species is found amidst leaf litter. The amplexus is

axillar (Fig. 18C). Reproduction occurs in the rainy season in tiny puddles. Males

were found hidden in the leaf litter. Tadpoles and advertisement call are unknown.

The conservation status of this species remains unknown, but the recent construction

of the Belo Monte hydroelectric complex on the Xingu River represents a threat to

the population status of this species.

Etymology. The specific epithet refers to geographic distribution of the species within

the lower Xingu River basin, Brazil.

Amazophrynella moisesii sp. nov.

urn:lsid:zoobank.org:act:9984F3CB-9416-482D-8F63-5D78C8CDC032

Dendrophryniscus minutus (Bernarde et al. 2011: 120 plate 2, Fig. d)

93

Amazophrynella minuta (Bernarde et al. 2013: 224, 227 plate 7 Fig. c; Miranda et al.

2015: 96)

Holotype (Fig. 20). UFAC–RB 2815 adult male, collected in the Parque Nacional da

Serra do Divisor, Igarapé Ramon (7°27'00"S, 73°45'00"W), 400 m a.s.l., municipality

of Mâncio Lima, Acre, Brazil by Moises Barbosa de Souza on 1 January, 2000.

Paratypes. Thirty eight specimens (males = 18, females = 20, Acre, Brazil: Reserva

Extrativista Alto do Juruá (9°03'00"S, 72°17'00"W), 260 m a.s.l., UFAC–RB 823

(adult male), Moisés B. Souza and Adão J. Cardoso on 26 February 1994, UFAC–

RB 878–879 (adult males), Moisés B. Souza and Paulo Roberto Manzani between

16 and 18 July 1994; UFAC–RB 2606–2611 (adult females), Moisés B. Souza and

M. Nascimento between 7 and 8 March 1998. Parque Nacional da Serra do Divisor:

Igarapé Anil (8°59'00"S, 72°29'00"W), 192 m a.s.l., UFAC–RB 1337–1341 (adult

females), UFAC–RB 1343 (adult female), Moisés B. Souza and William Aiache on 10

November 1994; Zé Luiz lake (8°54'00"S, 72°32'00"W), UFAC-RB 1774–1775 (adult

females), Moisés B. Souza and William Aiache between 9 and 10 November 1996;

Igarapé Ramon (7°27'00"S, 73°45'00"W), 400 m a.s.l., UFAC–RB 1375 (adult

female), Moisés B. Souza and William Aiache between 12 and 13 November 1996,

UFAC–RB 2772–2773 (adult females), UFAC–RB 2816–2817 (adult males), Moisés

B. Souza between 18 and 20 January 2000; Môa River (7°30'00"S, 73°36'00"W), 331

m a.s.l, UFAC–RB 1493 (adult male), Moisés B. Souza and William Aiache between

19 and 20 November 1997, UFAC–RB 2687–2697 (adult males), Moisés B. Souza

on 10 January 2000. Floresta Estadual do Gregório, municipality of Tarauacá

(7°59'00"S, 71°22'36.8"W), 240 m a.s.l., UFAC–RB 5678 (adult female), Moisés B.

Souza and Marilene Vasconcelos between 23 and 26 July 2000; Centrinho do Aluísio

site, municipality of Porto Walter UFAC–RB 6273 (adult male), Paulo Roberto Melo

Sampaio, on 8 January 2014. Municipality of Mâncio Lima, Acre (7°23'10.32"S, 73°

3'31.68"W), MNRJ 91670 (field number PRMS 420) (adult female) Paulo Roberto

Melo Sampaio and Evan M. Twomey on 24 March 2016. Amazonas state:

Municipality of Envira (7º31'16.14"S, 70º1'3.84"W), MNRJ 91669 (field number

PRMS 404) (adult female) Paulo Roberto Melo Sampaio and Evan M. Twomey on 12

March 2016.

94


in females; (2) snout acuminate in lateral view, upper jaw, in lateral view, protruding

beyond lower jaw; (3) snout length protuberant, large for the genus (SL/HL= 0.48–

0.5); (4) cranial crest, vocal slits and nuptial pads absent; (5) small tubercles on

upper arms and posterior area of tympanums; (6) texture of dorsal skin tuberculate;

(7) texture of ventral skin highly granular (8) finger III relative large (HAL/SVL 0.23–

0.25, n = 28); (9) fingers slender, basally webbed; (10) finger I shorter than finger II;

(11) palmar tubercle elliptic; (12) hind limbs relatively large (TAL/SVL 0.51–0.53, n =

28); (13) toes slender basally webbed; in life: (14) venter pale yellow; small irregular

dots on venter.


Amazophrynella moisesii sp. nov. is most similar to A. javierbustamantei from which it

can be distinguished by: protruding snout, SL/HL 0.48–0.5, n = 28 (vs. 0.43–0.45, n =

60); snout acuminate, in lateral view (subacuminate); ventral skin highly granular

(coarsely areolate); larger hind limbs, TAL/SVL 0.51–0.53, n = 28 (vs. 0.49–0.51, n =

60); venter bright yellow (pale yellowish orange); small irregular blotches on venter

(tiny rounded points). From the other species of the genus Amazophrynella the new

species is easily differentiated by its large hand, HAL 3.6–5.6 mm (4.62 ± 0.62 mm)

in adult females, 2.5–4.1 mm (3.4 ± 0.52 mm) in adult males (KW x2 = 100.2, df = 10,

p–value = 0.001, Fig. 21); longer SL, adult females 3.4–2.5 mm (3.0 ± 0.2 mm) and

adult males 2.1–3.0 mm (2.6 ± 0.3 mm, KW x2 = 104.3, df = 10, p–value = 0.001, Fig.

22); FI < FII (FI > FII in A. bokermanni, and FI ≥ FII in A. xinguensis sp.nov. - Fig. 6K

vs. 6C and 6K vs. 6D) and venter coloration pale yellow (white, in A. manaos, cream

in A. teko sp. nov., red brown in A. vote and reddish brown in A. siona sp. nov., see

Fig. 7).


and pointed in dorsal view. Head longer than wide. HL 33.8 % of SVL. HW 30.8% of

SVL. Snout prominent, acuminate in lateral view and pointed in dorsal view. SL

50.9% of HL. Nostrils closer to snout than to eyes. Canthus rostralis straight in dorsal

view. Internarial distance smaller than eye diameter. IND about 30.9% of HW. Upper

eyelid covered by abundant granules on borders. Eye prominent, about 35.7% of HL.

Tympanum not visible through the skin. Skin around tympanum covered by small

95

granules. Vocal sac not visible. Texture of dorsal skin tuberculate. Abundance of

granules on dorsum. Dorsolateral skin granular. Forelimbs slender. Edges of

forelimbs covered by small conical granules, in dorsal and ventral view. Upper arms

slender. UAL 35.2% of SVL. Small conical granules from the outer edge of the mouth

to upper arm. Upper arm covered by abundant medium size granules. Large HAL.

HAL 72.9% of UAL. Fingers basally webbed. Fingers slender, tips unexpanded.

Relative length of fingers: I<II<IV<III. Supernumerary tubercles and accessory palmar

tubercles rounded. Palmar tubercle large and elliptic. Subarticular tubercles rounded.

Texture of gular region tuberculate. Texture of ventral skin highly granular. Small

granules on venter. Hindlimbs slender. Thigh to tarsus covered by conical granules

on borders. THL 54.4% of SVL. Tibias almost the same length as thighs. TAL 53.6%

of SVL. Tarsus slender. TL 33.8% of SVL. FL 74.3% of THL. Relative length of toes:

I<II<V<III<V. Inner metatarsal tubercle rounded. Outer metatarsal tubercles small

and rounded. Subarticular tubercles rounded. Toes slender and elongate. Tip of toes

not expanded, basally webbed. Cloacal opening slightly above middle of thighs.

Measurement of the holotype (in mm). SVL 13.6, HW 4.2, HL 5.1, SL 2.6, ED 1.5,

IND 1.3; UAL 4.8; HAL 3.5, THL 7.4, TAL 7.3, TL 4.5, FL 5.5.

Variation (Fig. 23). Phenotypically, the new species present some variation among

specimens. Sexual dimorphism was observed in SVL, with 12.2–15.8 mm (14.3 ± 1.5

mm, n = 15) in males and 16.4–20.9 mm (18.5 ± 1.6 mm, n =15) in females. Some

specimens present greater abundance of granules on dorsum (eg. UFAC–RB 2690).

Some individuals present greater abundance of small tubercles on dorsolateral

region (eg. UFAC–RB 2611, UFAC–RB 2603, UFAC–RB 2689, UFAC–RB 2692).

Another specimen (UFAC–RB 2610) presents brown chevrons extending from the

head to the vent, in dorsal view. Some individuals (eg. UFAC–RB 829) present a line

on dorsum, extending from the tip of the snout to cloaca. The pale yellow coloration

of ventral surfaces may extend from thighs to the chest or just to the middle of the

venter. In some specimens, the irregular black dots on venter vary in abundance and

size (eg. Fig. 24B vs. 24E). In life and preserved specimens, venter coloration

between pale yellow and yellow. In some individuals, the thighs are abundantly

covered by rounded tiny spots extending to the shank (Fig. 24C vs. 24D).

96

Coloration of the holotype (in life). Head brown, in dorsal view. Dorsum mostly light

brown. Flanks cream with scattered small black dots. Dorsal surfaces of upper arm,

arm and hand light brown. Dorsal surfaces of thighs, tarsus and foot light brown.

Ventral surfaces of upper arm, arm and palm cream. Ventral surfaces of thighs,

tarsus and tibia cream with small black dots. Sole light brown. Fingers cream, in

ventral view. Gular region cream with small dots. Venter pale yellow with small dots.

Iris golden and pupil black.

Color in preservative (~70% ethanol, Fig. 24). Nearly the same as color in life. The

dorsum became light brown. We detected a fading of pale coloration of the chest and

venter became cream. The small irregular dots on venter became less evident. The

hand and foot became cream, in ventral view. The gular region and venter became

cream. The iris loses its coloration.

Distribution and natural history (Fig. 1B). Amazophrynella moisesii sp. nov. have

been recorded from Brasil. State of Acre: municipalities of Cruzeiro do Sul, Mâncio

Lima, Porto Walter and Tarauacá; State of Amazonas: municipality of Envira. Peru:

Department of Huanuco, Panguana, Rio Llullapichis. Due to its abundance and

presence in conservation units of Brazil (Floresta Estadual do Gregório, Reserva

Extrativista do Alto Juruá and Parque Nacional da Serra do Divisor) we recommend

the IUCN Least Concern category.

Etymology. The specific epithet refers to Dr. Moisés Barbosa de Souza, a Brazilian

biologist, professor and friend at the Universidade Federal do Acre (UFAC), to whom

we dedicate this species in recognition of his contributions to herpetological research

and amphibian conservation in the state of Acre, Brazil.

Discussion

To date no study that analyzed a broadly distributed Amazonian taxon

confirmed the existence of just one broadly distributed species (eg., Funk et al.,

2012; Jungfer et al., 2013; Fouquet et al., 2014; Caminer & Ron, 2014; Gehara et al.,

2014; Ferrão et al., 2016). In recent years it has become evident that widespread

species in fact represent species complexes characterized by many deeply divergent

lineages, eg. Adenomera andreae, Dendropsophus minutus, Rhinella margaritifera,

Scinax ruber, Pristimantis ockendeni, Pristimantis fenestratus, Engystomops petersi,

97

Boana fasciata, Physalaemus petersii, Leptodactylus marmoratus and

Osteocephalus taurinus (Fouquet et al., 2007; Padial & Riva, 2009; Angulo &

Icochea, 2010; Funk et al., 2012; Jungfer et al., 2013; Caminer & Ron, 2014;

Fouquet et al., 2014; Gehara et al., 2014; Lourenço et al., 2015). These discoveries

imply that public data deposited in, for example GenBank, Gbif or IUCN are often

flawed and that the numerous metaanalyses (Godinho & Silva, 2018) based on such

data may be imprecise or even inaccurate. As a consequence of not recognizing true

taxonomic diversity of anurans, macroecological studies will fail to recognize actual

patterns of geographic structuring, and ultimately will not contribute to our

understanding of the evolutionary and ecological processes that lead to and are

maintaining this diversity.

Our results suggest that the genus harbors more than twice as many species

as current estimates. In the last several years the systematics and taxonomy of the

genus Amazophrynella has begun to be elucidated (Ávila et al., 2012; Rojas et al.,

2014, 2015, 2016). Resulting from these studies, five new species (A. vote, A.

manaos, A. amazonicola, A. matses and A. javierbustamantei—previously mistaken

for A. minuta) were described. With the description of the four new species in this

study, the total number of nominal species reaches 11 (Fig. 25), representing an

important increase in species diversity of the genus. The number of undescribed

species as a percentage of total is concordant with estimates from previous studies

aiming to elucidate the species diversity of Amazonian frogs (eg. Elmer et al., 2007;

Fouquet et al., 2007; Padial et al., 2012; Ron et al., 2012; Caminer & Ron, 2014;

Gehara et al., 2014; Ferrão et al., 2016). Therefore, our study adds to this growing

body of studies, and confirms the hypothesis that the species diversity within

Amazophrynella is much higher than currently accepted. The four CCS described in

our study present clear differences in diagnostic morphological characters,

divergence at ecological requirements and large genetic distance when compared

with their sister taxa. But it should also be clear that our taxonomic decisions were

conservative, and that numerous putative lineages within Amazophrynella still await

formal description. This conservative approach aims to promote taxonomic stability,

but as a consequence continues, albeit to a lesser degree, to underestimate the true

species diversity of Amazonian anurofauna.

98

A limiting factor of our study was the use of a single molecular marker (16S,

12S and COI mtDNA loci). The potential limitations for species delimitation using

mtDNA have been discussed in literature (eg. Ranalla & Yang, 2003; Yang &

Rannala 2010; Dupuis et al., 2012; Fujita et al., 2012). The use of additional nuclear

markers is generally recommended as the use of these unliked markers has the

potential to improve the accuracy of phylogenetic reconstructions and species

delimitation. In spite of having used only mtDNA loci, our study also provides an

extensive new morphological dataset, bioacoustic data and accurate collecting

locality information which allowed us to associate environmental data with each

specimen. All these additional data support and reinforce the inference based on the

mitochondrial genes.

Our phylogenetic analysis also reveals a striking biogeographic pattern with a

basal eastern and western divergence followed by a northern and southern split

within both eastern/western clades (Fig. 2). Our basal east-west pattern dated to the

Miocene and match similar patterns and divergence times detected in other groups of

frogs (Symula et al., 2003; Noonan & Wray, 2006; Funk et al., 2007; Garda &

Cannatella, 2007; Fouquet et al. 2014). Paleoenvironmental reconstructions of

Amazonian history suggest that there was a large lacustrine region in western

Amazon which began to form at the beginning of the Miocene (~24 Ma) (Hoorn et al.,

2010). This lake and marshland system, known as Lake Pebas, existed in

southwestern Amazonia, and was drained first to the north and then to the east

(Hoorn et al., 2010). Paleoenviromental data suggest marine incursions into western

Amazon during the Miocene, and Noonan & Wray (2006), for example, suggest the

importance of these incursions for the diversification of Amazonian anurofauna. In

general, however, marine incursions remain largely untested as a diversifying force

(Noonan & Wray, 2006; Garda & Cannatella, 2007; Antonelli et al., 2009). In addition,

it is reported that in early Miocene, the Purus arch was still active, and was a

prominent landscape feature in central Amazon (Wesselingh & Salo, 2006;

Figueiredo et al., 2009; Caputo & Soares, 2016) thus this geological formation also

could explain the east-west pattern as well. While other hypotheses, such as

Pleistocene refugia have also been proposed to explain this east-west pattern of

diversity (Pellegrino et al., 2011), the Miocene marine incursions have the best

99

temporal concordance with the basal east-west divergence pattern observed in

Amazophrynella and other Amazonian anuran groups.

The northern and southern split within both the eastern and western clades

occurred in early Miocene (~20.1 Ma) in the eastern Amazonia clades, while the

diversification of the western Amazonian clade commenced in the Middle Miocene

(~16.5 Ma). The beginning of the diversification of these clades appears to be

asynchronous and therefore is unlikely attributable to a single event. The more recent

date of diversification of the western clade is likely to have followed the last marine

incursion, i.e. a colonization of newly available habitat in western Amazon from

eastern Amazon. Independent of the absolute timing these divergence events, the

four subclades are restricted to north and south of the Amazon River, a common

pattern in many vertebrates species groups analyzed at the Amazonia-wide scale

(eg. Kaefer et al., 2012; Ribas et al., 2012; Fouquet et al., 2015; Oliveira et al., 2016).

In the case of Amazophrynella species, ecological characteristics such as small body

side, being a terra firme species and being restricted to reproducing in puddles

(Rojas et al. 2016), clearly evidences these species’ inability to disperse across

rivers. This in turn implies that major Amazonian rivers should limit the distributions of

lineages of Amazophrynella, a pattern observed in our phylogeny. However, the role

of rivers in driving diversification of Neotropical frogs remains controversial (see

Vences and Wake 2007 vs. Lougheed et al., 1999). But it is clear that geological and

climatic changes in the Miocene and Pliocene played an important role in the

diversification of Amazonian vertebrates (Bush, 1994; Glor et al., 2001; Da Silva &

Patton, 1998; Symula et al., 2003; Santos et al., 2009; Kaefer et al., 2012; Fouquet et

al., 2014; Gehara et al., 2014). However, only future process-based studies and

biogeographic hypotheses testing will allowed us to reveal the mechanisms (eg.

dispersion, vicariance, founder event) by which Amazophrynella diversified.

100

Acknowledgments

We would like to thank Ariane Silva, from the herpetological collection of the

Instituto Nacional de Pesquisas da Amazônia (INPA) in Manaus, Brazil, to Fernando

Ayala from the Pontificia Universidad Católica de Ecuador (PUCE) in Quito-Ecuador,

and to O. Aguilar and R. Orellana (MUBI) for providing administrative support and

part of the material for this study. R.R. Rojas thanks Alexander Almeida, Ian Pool

Medina, Richard Naranjito Curto for field support and Mario Nunes for laboratory

assistance.

101

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Table 1. Uncorrected p – distances among mtDNA lineages of Amazophrynella. Molecular distances are based on the 480–bp

fragment of 16S rDNA.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

1 A. amazonicola

2 A. siona sp. nov. 0.07

3 A. aff. minuta sp1 0.08 0.09

4 A. minuta 0.09 0.09 0.02

5 A. matses 0.09 0.13 0.09 0.09

6 A. aff. matses sp1 0.09 0.13 0.09 0.10 0.02

7 A. javierbustamantei 0.09 0.13 0.08 0.08 0.06 0.06

8 A. moisesii sp. nov. 0.08 0.11 0.08 0.08 0.09 0.09 0.06

9 A. vote 0.12 0.15 0.11 0.11 0.13 0.13 0.11 0.10

10 A. aff. vote sp1 0.12 0.15 0.11 0.11 0.12 0.12 0.12 0.11 0.03

11 A. aff. vote sp2 0.12 0.15 0.11 0.11 0.12 0.12 0.12 0.11 0.04 0.03

12 A. bokermanni 0.12 0.14 0.11 0.11 0.12 0.12 0.11 0.11 0.05 0.05 0.06

13 A. sp2 0.12 0.15 0.10 0.11 0.11 0.11 0.11 0.11 0.07 0.08 0.08 0.07

14 A. sp3 0.11 0.14 0.10 0.10 0.11 0.11 0.12 0.10 0.07 0.07 0.07 0.06 0.04

15 A. xinguensis 0.12 0.15 0.11 0.12 0.13 0.13 0.13 0.11 0.07 0.08 0.08 0.07 0.05 0.06

16 A. manaos 0.13 0.15 0.12 0.13 0.11 0.11 0.12 0.12 0.09 0.09 0.08 0.09 0.09 0.09 0.09

17 A. sp1 0.12 0.15 0.11 0.12 0.11 0.12 0.12 0.13 0.11 0.10 0.09 0.10 0.09 0.10 0.10 0.06

115

Table 2. Taxonomic status, congruence and comparison of main diagnostic morphological characters of species identified in

phylogenetic analyses (16S + 12S + COI). Character (-) indicates no data available. CCS= Confirmed Candidate Species;

UCS= Unconfirmed Candidate Species; DCL= Deep Conspecific Lineages; UL= Uncategorized Lineage.

Lineages Status Dorsal skin texture

Ventral skin texture

Head shape

Palmar tubercle

FI vs. FII

Venter coloration

Venter stain

A. manaos CCE Granular Granular Truncate Elliptical I<II White Large blotches

A. teko sp. nov. CCE Highly

granular Highly granular

acute Elliptical I<II Creamy Small blotches

A. sp1 UL Highly

granular Highly granular

acute Elliptical I<II Creamy Small blotches

A. vote CCE Tuberculate Granular Rounded Rounded I<II Reddish-

brown Small dots

A. aff. vote sp1 DCL Tuberculate Granular Rounded Rounded I<II reddish-

brown Small dots

A. aff. vote sp2 DCL Tuberculate Granular Rounded Rounded I<II reddish-

brown Small dots

A. bokermanni CCE Granular Granular Pointed Rounded I>II white Small dots

A. xinguensis sp. nov. CCE Highly

granular Granular Pointed Ovoid I=II Greyish Medium-size dots

A. sp2 UL - - - - - - -

A. sp3 UL - - - - - - -

116

A. matses CCS Spiculate Granular Acute Rounded I<II Yellow Blotches

A. aff. matses sp1 UCS - - - - - - -

A. javierbustamantei CCE Tuberculate Coarsely

areolate Acuminate Rounded I<II Pale

yellow Small dots

A. moisesii sp. nov. CCS Tuberculate Highly

granular Acuminate Elliptical I<II Pale

yellow Tiny points

A. amazonicola CCS Finelly

granular Granular Pointed Rounded I<II Yellow Medium-size blotches

A. siona sp. nov. CCS Finelly

granular Granular Acute Rounded I<II Reddish-

brow Small blotches

A. minuta CCS Highly

granular Granular Pointed Rounded I<II Yellow-

orange Large blotches

A. aff. minuta sp1 DCL Highly

granular Granular Pointed Rounded I<II Yellow-

orange Large blotches

117

Table 3. Descriptive morphometric statistics (in mm) for males of nominal and CCE of Amazophrynella. KW= Kruskal Wallis test, (+)

p-value<0.05.

Variable

A. minuta

(n = 20)

A. matses

(n = 13)

A. javierbustam-antei

(n = 28)

A. moisesii sp. nov (n =15)

A. amazonicola

(n = 15)

A. siona sp. nov. (n = 29)

A. bokerma-nni

(n = 7)

A. xinguensis sp.nov. (n = 5)

A. manaos

(n = 27)

A. teko sp.nov. (n = 13)

A. vote

(n = 14)

KW p-value

SVL 13.5±0.6 12.1±0.6 14.9±0.9 14.3±0.5 14.5±0.7 13.1±0.6 16.3±0.2 18.8±0.9 14.2±0.7 14.8±0.7 13.1±0.7 +

HW 4.2±0.2 3.6±0.2 4.2±0.2 4.3±0.4 4.4±0.3 3.9±0.3 4.8±0.1 5.1±0.2 4.2±0.3 4.5±0.3 4.0±0.7 +

HL 4.9±0.2 4.3±0.3 5.1±0.3 5.4±0.3 5.2±0.3 4.9±2.2 5.7±0.1 6.6±0.2 5.3±0.3 5.3±0.2 4.6±0.3 +

SL 2.3±0.1 2.0±0.3 2.2±0.2 2.6±0.2 2.4±0.2 2.2±0.2 3.0±0.1 3.2±0.1 2.7±0.2 2.5±0.1 2.1±0.2 +

ED 1.4±0.1 1.1±0.1 1.3±0.1 1.6±0.2 1.2±0.1 1.3±0.1 1.7±0.1 2.0±0.1 1.3±0.1 1.5±0.1 1.3± .1 +

IND 1.2±0.1 1.0±0.1 0.9±0.1 1.2±0.1 1.2±0.1 1.1±0.08 1.4±0.1 1.5±0.5 1.1±0.1 1.3±0.1 1.1±0.1 +

UAL 3.8±0.2 3.5±0.4 4.5±0.4 4.8±0.6 4.5±0.3 4.1±0.4 5.4±0.4 6.1±0.5 3.6±0.4 4.8±3.2 3.9±0.5 +

HAL 2.8±0.2 2.7±0.2 3.6±0.4 3.4±0.5 3.2±0.2 2.7±0.2 3.4±0.6 3.7±0.3 2.8±0.6 3.2±0.2 3.0±0.3 +

THL 6.8±0.2 6.2±0.4 7.6±0.7 7.9±0.8 7.7±0.6 7.0±0.4 8.0±0.3 9.5±0.8 6.7±0.3 7.6±0.8 6.5±0.7 +

TAL 6.7±0.3 5.8±0.3 7.6±0.7 7.7±0.9 7.2±0.6 6.6±0.4 7.5±0.3 9.1±0.7 6.9±0.6 7.3±0.5 5.7±0.7 +

TL 4.1±0.2 3.8±0.2 4.7±0.8 5.2±1.2 4.2±0.6 4.1±0.4 4.8±0.4 5.5±0.2 4.6±0.4 4.6±0.4 3.8±1.0 +

FL 4.8±0.4 4.3±0.4 5.7±0.6 5.7± 0.7 5.1±0.4 4.7±0.5 5.6±0.4 6.4±0.2 5.2±0.5 5.5±0.5 4.4±0.6 +

118

Table 4. Descriptive morphometric statistics (in mm) for females of nominal and CCS of Amazophrynella. KW= Kruskal Wallis test,

(+) p-value<0.05.

Variable

A. minuta

(n = 20)

A. matses

(n = 13)

A. javierbustam-antei

(n = 28)

A. moisesii sp. nov (n =15)

A. amazonicola

(n = 15)

A. siona sp. nov. (n = 35)

A. bokerma-nni

(n = 7)

A. xinguensis sp.nov. (n = 13)

A. manaos

(n = 27)

A. teko sp. nov. (n = 17)

A. vote

(n = 14)

KW p-value

SVL 17.4±0.9 17.1±0.7 19.7±1.8 18.5±1.6 18.1±1.1 18.3±0.9 23.4±0.8 24.1±1.2 20.8±2.1 19.2±1.1 16.3±1.6 +

HW 5.1±0.4 4.8±0.4 5.0±0.3 5.1±0.3 5.1±0.4 5.1±0.3 6.4±0.3 6.3±0.3 6.0±0.6 5.4±0.3 4.8±0.4 +

HL 6.0±0.4 5.6±0.3 6.2±0.3 6.4±0.4 6.1±0.4 6.2±0.3 7.9±0.3 7.9±0.3 7.2±0.3 6.5±0.3 5.4±0.4 +

SL 2.7±0.2 2.7±0.3 2.8±0.2 2.9±0.3 1.5±0.2 2.9±0.3 3.6±0.1 3.75±0.2 3.3±0.3 2.9±0.2 2.6±0.3 +

ED 1.7±0.3 1.4±0.2 1.5±0.3 1.9±0.2 1.4±0.1 1.7±0.2 2.2±0.2 2.1±0.1 1.8±0.2 1.8±0.1 1.7±0.2 +

IND 1.4±0.1 1.2±0.2 1.2±0.1 1.4±0.1 1.2±0.1 1.4±0.1 1.6±0.1 1.6±0.1 2.0±0.1 1.5±0.1 1.3±0.1 +

UAL 5.2±0.2 5.2±0.2 6.1±0.6 6.0±0.5 5.5±0.6 5.6±0.4 7.9±0.3 8.0±0.4 5.5±0.3 6.1±0.5 4.9±0.7 +

HAL 3.6±0.3 3.7±0.3 4.6±0.4 4.6±0.5 3.9±0.4 3.9±0.3 4.9±0.2 5.0±0.4 4.4±0.3 4.1±0.3 3.4±0.5 +

THL 8.5±0.9 8.3±0.4 9.6±0.8 9.8±0.4 9.5±0.8 9.4±0.6 11.8±0.7 11.8±0.8 10.2±0.6 9.5±0.5 7.7±0.8 +

TAL 8.4±0.7 8.3±0.4 9.8±0.8 9.6±0.5 9.1±0.7 9.2±0.6 11.0±0.4 11.2±0.6 10.2±0.6 9.4±0.6 7.2±1.0 +

TL 5.4±0.4 5.3±0.4 5.9±0.5 5.7±0.3 5.4±0. 5.7±0.5 6.9±0.4 7.1±0.4 7.1±0.9 5.7±0.4 4.6±0.6 +

FL 6.4±0.7 6.2±0.4 7.2±0.7 7.3±0.7 6.5±0.6 7.0±0.6 8.6±0.5 8.9±0.5 8.1±0.6 7.2±0.62 5.6±0.9 +

119

Table 5. Successful classification in morphological space (males) recovered phylogenetic mt DNA lineages (Eastern and Western

clades). In parenthesis, the percentage of successfully classification. The numbers in the cells represent the numbers of

individuals assigned to each clade by discriminant analyses. UCS and UL were not included.

Lineages

(Eastern clade)

A. manaos (90%)

A. teko sp. nov. (68%)

A. vote (100%)

A. aff. vote sp1 (63%)


A. bokermanni (50%)

A. xinguensis sp. nov. (80%)

A. manaos 27 0 0 0 0 0 0

A. teko sp. nov. 0 15 0 0 0 1 0

A. vote 0 0 13 0 0 0 0

A. aff. vote sp1 1 1 0 7 2 0 0

A. aff. vote sp2 0 0 0 3 0 0 0

A. bokermanni 1 1 0 0 0 3 1

A. xinguensis sp. nov.

1 0 0 0 0 0 4

Lineages

(Western clade)

A. matses (39%)

A. javierbustamantei (79%)

A. moisesii sp. nov. (31%)

A. amazonicola (85%)

A. siona sp. nov. (59%)

A. minuta (74%)

A. minuta sp1

(0%)

A. matses 5 5 0 1 2 0 0

A. javierbustamantei

1 23 1 0 2 2 0

A. moisesii sp. 0 0 4 0 7 0 2

120

nov.

A. amazonicola 0 1 0 22 2 1 0

A. siona sp. nov. 0 2 2 2 16 5 0

A. minuta 0 0 1 2 23 3

A. minuta aff. sp1 0 0 2 0 0 7 0

121

Table 6. Successful classification in environmental space recovered phylogenetic mt DNA lineages (Eastern and Western clades).

In parentheses, the percentage of successful classifications. The numbers in the cells represent the numbers of individuals

assigned to each clade by discriminant analyses. UCS and UL were not included.

Lineages

(Eastern clade)

A. manaos (77%)

A. teko sp. nov. (90%)

A. vote (80%)



A. bokermanni (50%)

A. xinguensis sp. nov. (66%)

A. manaos 7 0 0 0 0 1 0

A. teko sp. nov. 0 10 0 0 0 0 0

A. vote 0 0 4 2 2 0 0

A. aff. vote sp1 1 0 1 2 2 0 0

A. aff. vote sp2 0 0 0 1 1 1 0

A. bokermanni 1 1 0 0 0 2 1

A. xinguensis sp. nov.

0 0 0 0 0 1 2

Lineages

(Western clade)

A. matses (87%)

A. javierbustamantei (100%)

A. moisesii sp. Nov. (62)%)

A. amazonicola (100%)

A. siona sp. nov. (80%)

A. minuta (70%)

A. minuta sp1

(0%)

A. matses 7 0 0 0 0 0 0

A. javierbustamantei 0 6 2 0 0 0 0

A. moisesii sp. nov. 0 0 5 0 0 0 0

A. amazonicola 1 0 0 6 0 0 0

122

A. siona sp. nov. 0 0 0 0 8 2 1

A. minuta 0 0 1 1 7 0

A. aff. minuta sp1 0 0 0 0 1 1 0

123

Figures

Figure 1. Phylogeny and geographic distribution of Amazophrynella. A) Phylogenetic

relationship among nominal and putative species of Amazophrynella based on

Bayesian inference inferred from 1430 aligned sites of the 16S, 12S and COI mtDNA

genes. Numbers in branches represent Bayesian posterior probability. B) Geographic

distribution of Amazophrynella spp. Colors and symbols = occurrence areas for each

clade based on specimens reviewed in collections. Black points = Localities of

genetic collection from specimens. Colors and symbols of clades in the phylogenetic

tree correspond to colors and symbols on the map. Base maps were downloaded as

freely available SRTM maps from https://earthexplorer.usgs.gov/.

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Figure 2. Time calibrated tree of Amazophrynella with posterior probabilities and

mean age. Blue bars represent 95% HPD.

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Figure 3. Principal components analyses (PCA) of morphometric and enviromental

variables: Morphometric PCA: A) Eastern clade, B) Western clade. Environmental

PCA: C) Eastern clade, D) Western clade. Symbols and colors represents the clades

recovered by the phylogenetic analyses (Fig.1). UCS and UL were not include.

126

Figure 4. Holotype of Amazophrynella teko sp. nov. (MNHN 2015.136); A) dorsal

view; B) ventral view; C) dorsal view of the head; D) ventral view of the head; E) left

toe; F) left hand. Photos by Rommel R. Rojas.

127

Figure 5. Measurement comparison of SVL between males of nominal species of

Amazophrynella.

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Figure 6. Comparison of palmar tubercles of nominal species of Amazophrynella. A)

A. teko sp. nov. B) A. siona sp. nov. C) A. xinguensis sp. nov. D) A. bokermanni. E)

A. vote. F) A. amazonicola. G) A. minuta. H) A. matses. I) A. manaos. J) A.

javierbustamantei. K) A. moisesii sp. nov. Elliptical (A, I, J); Rounded (B, E, D, H, F,

G); Ovoid (C). See Table 2. Photos by Rommel R. Rojas.

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Figure 7. Ventral skin coloration of nominal species of Amazophrynella. A) A. minuta.

B) A. teko sp. nov. C) A. siona sp. nov. D) A. xinguensis sp. nov. E) A. bokermanni.

F) A. vote. G) A. manaos. H) A. amazonicola. I) A. matses. J) A. javierbustamantei,

K) A. moisesii sp. nov Large blotches (A, G); medium size blotches (H); small

blotches (B, I, C); small dots (F, E, J); medium size dots (D); tiny points (K). See

Table 2. Photos by Rommel R. Rojas.

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Figure 8. Comparison of head profile of nominal species of Amazophrynella in lateral

view. A) A. minuta. B) A. teko sp. nov. C) A. siona sp. nov. D) A. xinguensis sp. nov.

E) A. bokermanni. F) A. vote. G) A. manaos. H) A. amazonicola. I) A matses. J) A.

javierbustamantei. K) A. moisesii sp. nov. Arrow indicates a small protuberance in the

tip of the snout of A. amazonicola. Pointed (A, H, D, E); acute (B, C, I); truncate (G);

rounded (F); acuminate (K, J). See Table 2. Photos by Rommel R. Rojas.

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Figure 9. Morphological variation in live Amazophrynella teko sp. nov. (unvouchered

specimens). Photos by Antoine Fouquet.

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Figure 10. Morphological variation of preserved specimens of Amazophrynella teko

sp. nov. Adult males: MHNN 2015.138 (A-B); MHNN 2015.152 (C-D); MHNN

2015.139 (E-F). G-L Adult females: MHNN 2015.141 (G-H); MHNN 2015.143 (I-J);

MHNN 2015.150 (K-L). Photos by Rommel R. Rojas.

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Figure 11. Oscillogram and spectrogram of the advertisement call of Amazophrynella

teko sp. nov. A) three notes, B) one note.

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Figure 12. Holotype of Amazophrynella siona sp. nov. (QCAZ 27790); A) dorsal view;

B) ventral view; C) ventral view of head; D) dorsal view of head; E) right hand; F)

right foot. Photos by Rommel R. Rojas.

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Figure 13. Morphological variation of live Amazophrynella siona sp. nov. QCAZ

51068 (A-B); QCAZ 42988 (C-D); QCAZ 42988 (E-F). Photos by Santiago R. Ron.

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Figure 14. Morphological variation of preserved specimens of Amazophrynella siona

sp. nov. Adult males: QCAZ 54213 (A-B); QCAZ 11979 (C-D); QCAZ 18826 (E-F).

Adult females: QCAZ 38679 (G-H); QCAZ 6091 (I-J); QCAZ 52434 (K-L). Photos by

Rommel R. Rojas.

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Figure 15. Tadpole of Amazophrynella siona sp. nov. National Park Yasuni, Ecuador

(QCAZ 24576), stage 30; A) dorsolateral view; B) dorsal view; C) ventral view; D) oral

disc view. Photos by Rommel R. Rojas.

Figure 16. Oscillogram and spectrogram of the advertisement call of Amazophrynella

siona sp. nov. A) three notes, B) one note.

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Figure 17. Holotype of Amazophrynella xinguensis sp. nov. (INPA-H 35471); A)

dorsal view; B) ventral view; C) ventral view of head; D) dorsal view of head; E) right

hand; F) right foot. Photos by Rommel R. Rojas.

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Figure 18. Morphological variation of live Amazophrynella xinguensis sp. nov.

(unvouchered specimens). Photos by Emil Hernández-Ruz.

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Figure 19. Morphological variation of preserved specimens of Amazophrynella

xinguensis sp. nov. Adult males: INPA-H 35482 (A-B), INPA-H 35493 (C-D); INPA-H

35471 (E-F). Adult females: INPA-H 35477 (G-H); INPA-H 35478 (I-J); INPA-H 35479

(K-L). Photos by Rommel R. Rojas.

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Figure 20. Holotype of Amazophrynella moisesii sp. nov. (UFAC-RB 2815); A) dorsal

view; B) ventral view; C) ventral view of head; D) dorsal view of head; E) right hand;

F) right foot. Photos by Rommel R. Rojas.

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Figure 21. Measurement comparison of HAL between males of nominal species of

Amazophrynella.

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Figure 22. Measurement comparison of SL between males of nominal species of

Amazophrynella.

144

Figure 23. Morphological variation in live Amazophrynella moisesii sp. nov.

(unvouchered specimens). Photos by Paulo R. Melo-Sampaio.

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Figure 24. Morphological variations of preserved specimens of Amazophrynella

moisesii sp. nov. Adult males: UFAC-RB 1698 (A-B); UFAC-RB 2694 (C-D); UFAC-

RB 2815 (E-F). Adult females: UFAC-RB 2608 (G-H); UFAC-RB 2610 (I-J); UFAC-

RB 2607 (K-L). Photos by Rommel R. Rojas.

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Figure 25. Confirmed candidate species (CCS) of Amazophrynella: A-B) A. minuta

Photo by Rommel R. Rojas; C-D) A. teko sp. nov. Photo by Antoine Fouquet; E-F) A.

siona sp. nov. Photo by Santiago R. Ron; G-H) A. xinguensis sp. nov. Photo by Emil

Hernándes-Ruz; I-J) A. bokermanni Photo by Marcelo Gordo; K-L) A. manaos Photo

by Rommel R. Rojas. M-N) A. amazonicola Photo by Rommel R. Rojas. O-P) A.

matses Photo by Rommel R. Rojas; Q-R) A. javierbustamantei Photo by Juan Carlos

Chapparro; S-T) A. vote Photo by Robson W. Ávila; U-V) A. moisesii sp. nov. Photo

by Paulo R. Melo-Sampaio.

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CAPITULO III

Description of the advertisement call of four species of Amazophrynella

(Anura:Bufonidae). Rojas, R.R., Carvalho, V., Ávila, R., Kawashita, R., Hrbek, T. &

Gordo, M. (2018). Zootaxa, 4459, 193–196.

148

Description of the advertisement call of four species of Amazophrynella

(Anura:Bufonidae)

ROMMEL R. ROJAS1*, VINICIUS TADEU DE CARVALHO1,2, ROBSON W. ÁVILA2,

RICARDO A. KAWASHITA-RIBEIRO3, TOMAS HRBEK1 & MARCELO GORDO4

1 Laboratório de Genética e Evolução Animal, Departamento de Genética, ICB,

Universidade Federal do Amazonas, Av. Gen. Rodrigo Octávio Jordão Ramos, 6200,

CEP 69077–000, Manaus, AM, Brazil *Corresponding author:

[email protected]

2 Universidade Regional do Cariri (URCA), Campus do Pimenta, Rua Cel. Antônio

Luiz, 1161, Bairro do Pimenta, CEP 63105-100, Crato, CE, Brazil

3 Instituto de Ciências e Tecnologia das Águas, Universidade Federal do Oeste do

Pará, Av. Mendonça Furtado, 2946, CEP 68040-050, Santarém, PA, Brazil.

4 Departamento de Biologia, Instituto de Ciências Biológicas, Universidade Federal

do Amazonas, Av. General Rodrigo Octávio Jordão Ramos, 6200. CEP 69077–000,

Manaus, AM, Brazil



149

Amazophrynella comprises seven small bufonid species with a pan-

Amazonian distribution (Fouquet et al. 2012a, b; Rojas et al. 2016). All species

inhabit the forest leaf litter, breed in seasonal puddles and are diurnally and

nocturnally active (Fouquet et al. 2012b; Rojas et al. 2014; 2015; 2016). Until now

only one nominal species, A. javierbustamantei, and two putative lineages — A. sp.

(Rio Yuyapichis, Peru) and A. aff. minuta (Santa Cecilia, Ecuador) — had their

advertisement calls formally described (Duellman 1978; Schlüter 1981; Rojas et al.

2016). Herein, we described for the first time the advertisement calls from additional

four species of Amazophrynella.

The calls were recorded in uncompressed .wav format, with a Zoom H1 Handy

Recorder (Zoom Corporations, Tokyo, Japan) equipped with an internal microphone,

positioned approximately 1.0–2.5 m from the focal male. All calls were filtered with

Audacity 1.2.2 for Windows (Free Software Foundation Inc., 1991). Praat 4.2.22 for

Windows (Boersma & Weenick, 2006) was used to generate audiospectrograms and

oscillograms at a sampling frequency of 44.0 kHz and 16-bit resolution. Spectral

parameters were analyzed through fast Fourier transformations (FFT) (width 1024

points). We used the Seewave package (Sueur et al. 2008) implemented in the

software R (R Development Core Team, 2008) for figures. Air temperature was

measured immediately after sound recording of each male. Call structures were

visually analyzed in the spectrograms subsequent to which we measured the

following quantitative parameters considered informative in amphibian taxonomy

(Köhler et al. 2017): call duration (s), inter-call interval (s), number of pulses per call,

dominant frequency (Hz), fundamental frequency (Hz), pulse rate (pulses/s) and call

rise time (s) and numbers of harmonics. Call parameter measurements are presented

as range (mean ± standard deviation) throughout the text.

The characterization of the advertisement call of Amazophrynella manaos

Rojas, Carvalho, Gordo, Ávila, Farias & Hrbek, 2014 was based on 20 calls from

three individuals (INPA-H 6983, snout-vent-length (SVL): 13.1 mm; INPA-H 6984,

SVL: 13.6 mm; INPA-H 6987, SVL: 13.9 mm) recorded between 8:30–10:00 h at the

type locality (campus of the Federal University of Amazonas/UFAM , 03°05’37” S,

59°58’26” W), within the city of Manaus, state of Amazonas, Brazil. Air temperature at

the time of recording varied between 26.0 to 27.5°C. The call consisted of a trilled

150

note emitted between regular silent intervals (Fig. 1A). Notes had an upward

amplitude modulation, reaching their maximum intensity near the end of the note.

Note duration ranged between 0.133 and 0.156 s (0.144 ± 0.006 s). Number of

pulses per note ranged from 35 to 51 pulses/note (44.050 ± 5.175 pulses/note). The

inter-note interval was 0.474 to 1.755 s (0.762 ± 0.355 s). The dominant frequency

ranged from 2990.110 to 3427.050 Hz (3338.860 ± 147.153 Hz). The fundamental

frequency ranged from 2617.899 to 3656.613 Hz (3038.247 ± 270.139 Hz). The rise

time ranged from 0.083 to 0.112 s (0.101 ± 0.008 s). Numbers of harmonic varied

from 3 to 5 (4.150 ± 0.587).

The characterization of the advertisement call of Amazophrynella bokermanni

(Izecksohn, 1993) was based on 22 calls from one individual (INPA-H 31861, SVL:

14.2 mm) recorded at 16:00 h around 30 km east from the species type locality

(03°41’20” S, 59°06’19” W), in Juruti, state of Pará, Brazil. Temperature varied from

26°C to 27°C. The call consisted of one trilled note emitted at regular silent intervals

(Fig. 1B). Notes had an upward amplitude modulation increasing in the first half of

the call. Note duration ranged from 0.125 to 0.163 s (0.146 ± 0.011 s). The number of


inter-note silent interval ranged from 0.631 to 1.501 s (0.721 ± 0.177 s). The

dominant frequency varied from 3346.143 to 3831.633 Hz (3635.711 ± 159.738 Hz).

The fundamental frequency ranged from 3048.185 to 3410.97 Hz (3195.456 ±

140.472 Hz). Rise time ranged from 0.041 to 0.091 s (0.061 ± 0.015 s). Number of

harmonics varied from 3 to 6 (4.181 ± 0.795).

The characterization of the advertisement call of Amazophrynella vote Ávila,

Carvalho, Gordo, Kawashita-Ribeiro & Morais, 2012 was characterized based on 30

calls from one individual (UFMT 11141, SVL: 15.3 mm) recorded at 10:00 h at the

type locality, at São Nicolau Farm (09°50’43.3” S, 58°13’10.6” W), in the municipality

of Cotriguaçu, state of Mato Grosso, Brazil. Air temperature varied from 26°C to

28°C. The call consisted of one trilled note emitted between regular silent intervals

(Fig. 1C). Notes had an upward amplitude modulation increasing towards the end of

the call. Note duration ranged from 0.098 to 0.150 s (0.127 ± 0.014 s). The number of


inter-note silent interval ranged from 0.544 to 1.879 s (0.831 ± 0.389 s). The

151

dominant frequency varied between 3084.211 to 3415.444 Hz (3204.169 ± 75.266

Hz), the fundamental frequency from 3250.921 to 3750.712 Hz (3492.608 ± 155.652

Hz). Rise time ranged from 0.054 to 0.120 s (0.087 ± 0.015 s). Number of harmonics

varied from 3 to 4 (3.133 ± 0.345).

The characterization of the advertisement call of Amazophrynella amazonicola

Rojas, Carvalho, Gordo, Ávila, Farias & Hrbek, 2014 is based on 35 calls from one

individual (MZUNAP 917- SVL: 13.8 mm) recorded at 10:00 h at the type locality

Puerto Almendra (3°49'41''S, 73°22'07''W), department of Loreto, Peru. The

recording temperature varied from 25°C to 26°C. The call consisted of one trill note

issued during irregular intervals (Fig. 1D). Notes had a downward modulation,

reaching maximum frequency almost at the beginning. The duration of the notes was

from 0.013 to 0.025 s (0.018 ± 0.003 s). The number of pulses was from 4 to 12 per

note (8.514 ± 2.331 pulses/note). The inter-note interval was from 0.660 to 2.3240 s

(1.617 ± 0.344 s). The dominant frequency varied from 3054.840 to 3521.22 Hz

(3277.398 ± 93.458 Hz). The fundamental frequency varied from 1404.170 to

2893.010 Hz (2354.207 ± 364.419 Hz), sometimes coinciding with dominant

frequency. The time to peak at maximum frequency was from 0.002 to 0.008 s (0.004

± 0.001 s). Number of harmonics varied from 2 to 3 (2.571 ± 0.502).

Analysis of the advertisement calls suggests a conservative structure of the

calls among species of the genus Amazophrynella. The calls of A. manaos, A.

bokermanni and A. vote are much more similar to each other than to that of A.

amazonicola, the main differences being note duration and number of pulses/note,

fundamental and dominant frequencies. The structure and values of call parameters

of the advertisement call of A. amazonicola are similar to those of A. sp. and A. aff.

minuta from Peru and Ecuador (see Duellman 1978; Schlüter 1981) and A.

javierbustamantei (Rojas et al. 2016), the main differences being the values of the

dominant frequency, note intervals and note duration. The advertisement call of A.

manaos was more similar to that of A. bokermanni in number of pulses per note and

note duration, with the main difference between the two species being the values of

dominant frequency and fundamental frequency. The species A. vote had the

greatest note duration and A. amazonicola had the shortest note duration.

Furthermore, similarities in call structure of the advertisement calls are also reflected

152

in the evolutionary relationships among these species, as suggested by the

phylogenetic hypothesis proposed by Rojas et al. (2016). Bioacoustic data are scarce

for Amazophrynella, but are likely to be important in contributing to the resolution of

the taxonomic status of unconfirmed candidate species (UCS, sensu Vieites et al.

2009) within the framework of integrative taxonomy (Padial et al. 2010).

The senior author thanks Tainara V. Sobroza for help in bioacoustics analyses.

RWA thanks CNPq for his productivity research grant (303622/2015-6). MG thanks

Selvino Neckel-Oliveira for support in Juruti Project.

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new species of Amazophrynella (Anura: Bufonidae) from the Southwestern part of

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FIGURE 1. Advertisement call of four species of Amazophrynella. A) A. manaos

(Campus UFAM, INPA-H 6983, 26.0 °C); B) A. bokermanni (Juruti, INPA-H 31861,

27°C); C) A. vote (São Nicolau Farm, UFMT 11141, 27°C); D) A. amazonicola

(Puerto Almendra, MZUNAP 917, 25°C).

156

CAPITULO IV

Redescription of the Amazonian tiny tree toad Amazophrynella minuta (Melin, 1941)

(Anura: Bufonidae) from its type Locality. Rojas, R.R., Fouquet, A., Carvalho, V.,

Ron, S., Chaparro, J., Vogt, R., Ávila, R., Pires, I., Gordo, M. & Hrbek, T. Zootaxa

4482 (3): 511–526.

157

Redescription of the Amazonian tiny tree toad Amazophrynella minuta (Melin,

1941) (Anura: Bufonidae) from its type Locality

Rommel R. Rojas1, Antoine Fouquet2, Vinícius Tadeu De Carvalho1, Santiago Ron3,

Juan Carlos Chaparro4, Richard C. Vogt5, Robson W. Ávila6, Izeni Pires Farias1,

Marcelo Gordo7 & Tomas Hrbek1

1Laboratório de Evolução e Genética Animal (LEGAL), Departamento de Genética,

Instituto de Ciências Biológicas, Universidade Federal do Amazonas, Av. General

Rodrigo Octávio Jordão Ramos, 6200. CEP 69077–000 Manaus, AM, Brazil


Amazoniens, Centre de recherche de Montabo, 275 route de Montabo, BP 70620,

97334 Cayenne, French Guiana.


Ecuador, Av. 12 de Octubre y Roca, Aptdo. 17–01–2184, Quito, Ecuador.

4 Museo de la biodiversidade-MUBI, Peru.

5 CEQUA, Coordenação de Biodiversidade, Instituto Nacional de Pesquisas da

Amazônia, Av. André Araújo, 2936, Aleixo, CEP 69060-001, Manaus, AM, Brazil.

6 Departamento de Química Biológia, Universidade Regional do Cariri, Campus do

Pimenta, Rua Cel. Antônio Luiz, 1161, Bairro do Pimenta, CEP 63105–100, Crato,

CE, Brazil.


do Amazonas, Av. General Rodrigo Octávio Jordão Ramos, 6200. CEP 69077–000

Manaus, AM, Brazil.


158

Abstract

The description of Amazophrynella minuta was published in 1941 by the

Swedish naturalist Douglas Melin based on material from Taracuá (Amazonas state,

Brazil). This description was very brief and based on the morphology of few

specimens with diagnostic characters and color variation not well defined. Moreover,

the type series is currently in poor state of conservation. Consequently, taxonomic

ambiguity surrounds the nominal taxon A. minuta, which hampers the description of

many unnamed congeneric species. Herein, we redescribe A. minuta based on

recently collected specimens from the type locality, we designate a lectotype,

formulate a new diagnosis, provide patterns of morphological variation,

measurements and body proportions

Key words: Amphibians, Brazil, lectotype, systematics, Taracuá, taxonomy

Resumen

La descripción de la especie Amazophrynella minuta fue realizada en 1941

por el naturalista Sueco Douglas Melin en la localidad de Taracuá, estado de

Amazonas, Brasil. La descripción fue muy breve y basada solamente en la

morfología de pocos especímenes. Por ese motivo, los caracteres diagnósticos y el

padrón de coloración no fueron bien definidos en la descripción original, y en la

actualidad la serie tipo se encuentra en malas condiciones de preservación. En este

trabajo, realizamos la redescripción de A. minuta basados en especímenes

recientemente colectados de la localidad tipo, designamos un lectotipo, formulamos

una nueva diagnosis y proporcionamos el patrón de variaciones morfológicas,

medidas morfológicas y proporciones corporales

Resumo

A descrição da espécie Amazophrynella minuta foi publicada no ano 1941

pelo naturalista Sueco Douglas Melin baseado em material proveniente da localidade

de Taracuá, estado do Amazonas, Brasil. A descrição original foi muito breve e

baseada na morfologia de poucos indivíduos. Assim, os caracteres diagnósticos e o

padrão de coloração não foram bem definidos na descrição original, e a serie tipo

encontra-se em condições precárias. Consequentemente há ambiguidade

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taxonômica envolvendo o táxon nominal A. minuta, o que dificulta a descrição de

muitas espécies existentes no gênero Amazophrynella. Neste trabalho, realizamos a

redescrição de A. minuta utilizando espécimes recentemente coletados da localidade

tipo, designamos um lectótipo, formulamos uma nova diagnose morfológica,o padrão

de variação morfológica, medidas morfométricas e proporções corporais.

160

Introduction

The genus Amazophrynella Fouquet, Recoder, Teixeira, Cassimiro, Amaro,

Camacho, Damasceno, Carnaval, Moritz, & Rodrigues 2012a, comprises a group of

small-size bufonids distributed throughout Amazonia (Frost, 2017, Fouquet et al.,

2012b). They inhabit primary forest leaf litter, have diurnal activity and reproduce in

seasonal ponds (Magnusson & Hero, 1991; Ávila et al., 2012; Rojas et al., 2014,

2015). Currently eleven species are described (Rojas et al., 2018)

Douglas Melin described the type species of the genus, Amazophrynella

minuta in 1941, as Atelopus minutus. The description was based on four specimens

collected in 1924 in Taracuá, municipality of São Gabriel da Cachoeira, Amazonas

state, Brazil. The type specimens are deposited in the Göteborgs Naturhistoriska

Museum, Sweden (NHMG 462–465). These specimens are presently in poor state of

conservation, thus, difficult to compare morphologically with other species and

populations of Amazophrynella. In addition, the description is quite brief and lacks

many details in morphological diagnosis, variation, measurements and body

proportions that are perceived today as crucial given the actual diversity of the group.

During the last decade, several studies demonstrated that the genus

Amazophrynella represents a complex of cryptic species (Fouquet et al., 2012b;

Rojas et al., 2016; Rojas et al., 2018) and that many species have been, and still are,

erroneously identified as A. minuta (Fouquet et al., 2012b; Rojas et al., 2016). A

redescription of this species is, therefore necessary to accurately delimit and

describe other species of Amazophrynella.

Preliminary characterization of topotypic A. minuta from the type locality

carried out by Rojas et al. (2014) only provided general morphological characters and

did not describe the morphological variation and body measurements or define the

species’ current distribution. Herein we use extensive morphological, molecular and

bioacoustics data to redescribe A. minuta. We also provide a new combination of

diagnostic characters, and describe morphological variation, reproductive behavior

and geographic occurrence.


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Type locality. The type locality of Amazophrynella minuta is “Taracuá, Rio Uapés,

Brazil” (Melin, 1941) (=Taracuá, 0.10°S, 68.46°W, datum= WGS-84, municipality of

São Gabriel da Cachoeira, Amazonas state, Alto Rio Negro Amerindian Reserve,

Brazil). Topotypical specimens studied herein were collected on August 19 and 20,

2013. See Appendix 1 for examined specimens.

Measurements. The following morphological measurement were taken with a

Mitotuyo digital calliper (0.1 mm precision) with an ocular micrometer in a Zeiss

stereomicroscope: snout-vent length (SVL); head length (HL); head width (HW),

upper eyelid width (UEW); eye diameter (ED); snout length (SL); eye-to-nostril

distance (END); internarinal distance (IND); interorbital distance (IOD); hand length

(HAL); upper arm length (UAL); thigh length (THL); tibial length (TL); tarsal length

(TAL) and foot length (FL) following Kok & Kalamandeen (2008). Sex was determined

by gonadal analysis.

Definition of morphological characters. External morphological nomenclature

follows Kok & Kalamandeen (2008). Main diagnostic characters within

Amazophrynella were defined as follows:

Texture of skin (Figure 1). Most bufonids species are covered with variable–sized

warts (Ford & Cannatela, 1993), defined as, bearing protuberances with keratinized

tip (Kok & Kalamandeen, 2008). We defined texture of skin in Amazophrynella as

follows: tuberculate (Figure 1A), when the species present small sized wart with

conical tips. Granular (Figure 1B), when the species present medium sized warts with

rounded tips. In order to refine these taxonomic character, we considered skin as

“highly granular”, when species present high density of medium sized warts with

rounded tips (Figure 1C) and “finely granular”, when species present low density of

small sized warts with rounded tips (Figure 1D). Spiculate, when the species present

small sized warts with pointed tips (Figure 1E).

Ventral color pattern (Figure 2). We defined ventral color pattern in

Amazophrynella as follows (adapted from Kok & Kalamandeen, 2008): Blotches,

when the species present small to large, irregular light or black markings contrasting

with the background coloration (Figure 2A-D). Dots, when the species present small

or minute, more or less regular light or black markings contrasting with the

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background coloration (Figure 2E-F). Points, when the species present small to

medium-sized roughly round black markings in contrast to the background color

(Figure 2G).

Shape of head. We followed the nomenclature of proposed by Heyer et al. (1990)

page 409.

Shape of palmar and subarticular tubercles (Figure 3). We identified three main

shapes of palmar and subarticular tubercles in species of Amazophrynella, that are

defined as follows: Rounded, when the palmar and subarticular tubercles present a

circular shape, without a distinct point (Figure 3A). Ovoid, when the palmar and

subarticular tubercles present an oval, inverted egg shape, with a tapering or

irregular point (Figure 3B). Elliptical, when the palmar and subarticular tubercles

present an oval shape, with a flattened point (Figure 3C).

Advertisement call. The call was recorded in uncompressed .wav format, with a

Zoom H1 Handy Recorder (Zoom Corporations, Tokyo, Japan) equipped with an

internal microphone, positioned approximately 1.0–2.5 m from the focal male. All

calls were filtered with Audacity 1.2.2 for Windows (Free Software Foundation Inc.,

1991). Praat 4.2.22 for Windows (Boersma & Weenick, 2006) was used to generate

audiospectrograms and oscillograms at a sampling frequency of 44.0 kHz and 16-bit

resolution. Spectral parameters were analyzed through fast Fourier transformations

(FFT) (width 1024 points). Air temperature was measured immediately after each

sound recording. Call structures were visually analyzed in the spectrograms

subsequent to which we measured the following quantitative parameters considered

informative in amphibian taxonomy (Köhler et al., 2017): call duration (s), inter-call

interval (s), number of pulses per call, dominant frequency (Hz), fundamental

frequency (Hz), pulse rate (pulses/s) and call rise time (s) and numbers of harmonics.

Call parameter measurements are presented as range (mean ± standard deviation).

Results

Lectotype designation and redescription justification. Because superficiality in

morphological characters in original description, absent of material other than the

type series and because it is known that the genus Amazophrynella represents a

complex of cryptic species (Fouquet et al., 2012b; Rojas et al., 2018) and many

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similar species may currently be confused by A. minuta, a designation of a lectotype

will aid to taxonomic stability. In addition, that, a redescription will facilitate future

species delimitation within this species complex.

According to the International Code of Zoological Nomenclature (ICZN, 1999)

article 74.1.1: “A lectotype may be designated from syntypes to become the unique

bearer of the name of a nominal species-group taxon and the standard for its

application”. We also follow the recommendations of the article 73C of the ICZN

(2017) to provide data on the lectotype.

Species account

Amazophrynella minuta (Melin, 1941)

Synonymy

Atelopus minutus: Melin (1941: 40, Brazil; Amazonas Taracuá)

Dendrophryniscus minutus: McDiarmid (1971: 39, Brazil, French Guyana, Guyana,

Suriname, Venezuela, Colombia, Ecuador, Peru, Bolivia)

Amazonella minuta: Fouquet et al. (2012b: 832, Brazil, French Guyana, Guyana,

Suriname, Venezuela, Colombia, Ecuador, Peru, Bolivia)

Lectotype. Naturhistoriska Museet, Göteborg, Sweden, NHMG 462, SVL: 13.40 mm,

adult male, collected at Taracuá (0.10°S, 68.46°W) 100 m a.s.l, Amazonas state,

Brazil by Douglas Melin on 1924.

Paralectotypes (Figure 4). NHMG 463, NHMG 465 (adult males), NHMG 464 (adult

female).

Topotypical specimens. INPA–H 32721–34, adult males and INPA-H 32735–40, adult

females collected by Rommel R. Rojas at the same place of lectotype on August 19

and 20, 2013.

Diagnosis. A species of Amazophrynella with: (1) Medium body size for the genus

(see Table 1), adult females 15.0–19.0 mm SVL (n=11), adult males 12.2–14.2 mm

SVL (n=20); (2) snout pointed in lateral view; upper jaw, in lateral view, protruding

beyond lower (3) tympanum, vocal sac, parotid gland and cranial crests

164

inconspicuous; (4) texture of dorsal skin highly granular; (5) abundant small rounded

warts (in part conical) present on dorsal surfaces of forelimbs and hindlimbs; (6)

texture of ventral skin highly granular; (7) fingers slender, basally webbed; (8) finger I

shorter than finger II; (9) finger III relatively long (HAL/SVL 0.2–0.3 mm, n=31); (10)

palmar tubercle elliptical; (11) supernumerary tubercles rounded; (12) long hind limbs

(TAL/SVL 0.4–0.5, n=31); (13) toes slender, basally webbed; (14) toes lacking lateral

fringes ;(15) plantar surfaces of feet bearing one metatarsal tubercle, the inner 2.0x

larger than the outer, outer subconical; supernumerary plantar tubercles rounded;

(16) in life, venter coloration yellow–orange with larger to medium size black blotches

on venter.

Comparisons with other species. Amazophrynella minuta is most similar to A.

amazonicola from which it can be distinguished by (characteristics of compared

species in parentheses) absence of small triangular protrusion at the tip of the snout;

texture of dorsal skin highly granular (granular); palmar tubercle elliptic (rounded).

From A. matses by snout pointed in lateral view; texture of dorsal skin highly granular

(spiculate); venter coloration yellow–orange (pale yellow; Figure 5A vs. 5C). From A.

javierbustamantei by texture of dorsal skin highly granular (tuberculate); palmar

tubercle elliptical (rounded) throat and chest coloration light brown (grayish cream);

large blotches on venter (small points). From A. siona by snout profile pointed

(acute), warts on dorsum (granules) and texture of dorsal skin highly granular (finely

granular). From A. moisesii by snout profile pointed (acuminate), texture of dorsal

skin highly granular (tuberculate), large blotches on venter (tiny points). From A.

bokermanni, A. manaos, A. vote, A. xinguensis and A. manaos the main difference in

the venter coloration yellowish–orange (white in A. manaos, A. bokermanni and A.

teko, red brown in A. vote, brown in A. xinguensis; Figure 5A vs. 5F-J), FI < FII (vs. FI

> FII in A. bokermanni, and FI ≥ FII in A. xinguensis).

Redescription. Body slender, slightly enlarged posteriorly. Sexual dimorphism

observed in SVL, with 12.2–14.7 mm (13.6 ± 0.6 mm, n=20, Table 4) in adult males

and 14.5–19.4 mm (17.5 ± 1.5 mm, n=11) in adult females. Head pointed, longer than

wide: HL 4.6–6.8 mm (5.4 ± 0.5 mm), 36.0% of SVL; HW 3.8–5.8 mm (4.6 ± 0.1 mm),

30.0% of SVL. Snout profile pointed in lateral view; SL 2.1–3.2 mm (2.5 ± 0.3 mm),

46.0% of HL. Canthus rostralis straight and loreal region vertical. Nostrils

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protuberant, closer to snout than to eyes. IND 1.0–1.5 mm (1.2 ± 0.1 mm), 27.0% of

HW. Interorbital and occipital regions flat. Tympanic membrane and tympanic annulus

not apparent. Parotoid glands not visible. Vocal sac not visible in males. Eyes

prominent. Eye diameter 1.2–2.0 mm (1.5 ± 0.2 mm), 29.0% of HL. Eyelid with small

tubercles on borders. Choanae small and circular. Dorsal skin texture highly granular.

Small rounded warts (in part conical) on dorsolateral and lateral surfaces posterior to

eye, on posterior dorsum, and on dorsal surfaces of entire arm. Texture of gular

region, chest and belly highly granular. Forelimbs slender. Forearms robust,

especially in males. UAL 3.5–5.5 mm (4.4 ± 0.7 mm); 29.3% of SVL. Fingers slender.

HAL 2.4–4.5 mm (3.1 ± 0.5 mm); 70.0% of UAL. Edges of forelimbs with scattered

granules, in dorsal and ventral view. Fingers basally webbed. Relative length of

fingers: I < II < IV < III. First finger short, third two times the size of the second, fourth

larger than the first and second. Large elliptical palmar tubercle. Supernumerary

tubercles rounded. Thenar tubercle absent. Tip of fingers unexpanded. Nuptial pad

not evident. Hind limbs slender. Ventral and lateral surfaces of forelimbs granular.

THL 6.3–10.0 mm (7.6 ± 0.1 mm), 50.0% of SVL. TAL 7.4–10.0 mm (7.4 ± 1.0 mm),

49.4% of SVL. TL 3.7–6.7 mm (4.7 ± 0.8 mm); 32.0% of SVL. FL 4.2–8.0 mm (5.5 ±

0.9 mm), 71.1% of THL. Cloacal opening slightly above mid–level of thighs. Grouping

of brownish–yellow granules from the hidden surface of the thighs to the shank.

Basal webbing on foot. Toes lacking lateral fringes. Relative length of toes: I < II < III

< V < IV. First toe very short; second half the size of the third. Elliptical inner

metatarsal tubercle present. Subarticular tubercles visible, more protruding and

swollen in females than males. Foot slender. Tips of toes unexpanded.

Variation. The variation in measurements is presented in Table 1. Specimens from

Japura River (INPA–H 32731, INPA–H 32724, INPA–H 32734) are longer (SL about

1.0 mm) than individuals from the type locality. Some individuals from São Gabriel da

Cachoeira present minor abundance of warts on dorsal surface (i.e. INPA–H32731,

INPA–H 32724 and INPA–H 32734). Mottled warts disseminated on chest and belly

are found in specimens INPA–H32731 (São Gabriel da Cachoeira) and INPA–35496

(Japura River). Some specimens from the type locality (i.e. INPA–H 32728, INPA–H

32735) and from the Japura River (INPA–H 35512), present lower abundance of

warts on arms.

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Coloration in life (Figure 6). Head brown. Dorsum reddish–brown. Flanks light brown.

Dorsal surfaces of the upper arm and arm reddish–brown. Hands and fingers light

brown, in dorsal view. Insertion of the arms present yellow blotches. Dorsal surfaces

of the thighs, tibia, tarsus and feet reddish–brown. Ventral surfaces of upper arm and

arm cream. Palm reddish. Ventral surfaces of thighs mottled cream with small black

blotches. Thighs with a transverse lateral black bar. Posterior region of the thigh and

cloaca yellow, covered by small black blotches. Tarsus with transverse brown bars.

Sole reddish–brown. Gular region and chest grayish–brown. Venter coloration

between yellow to yellow–orange. Venter covered by large to medium size black

blotches. In ventral view, some specimens present yellow blotches at the insertion of

the arms. The specimens INPA–H 32725 (Taracuá) and INPA–H 35494 (Japura

River), display yellow coloration with small black blotches on posterior region of the

thigh near the cloaca. Iris golden and pupil black.

Color in preservative (Figure 7). The color in preservative faded. We noted the

progressive loss of the ventral coloration that became pale yellow. The yellow

coloration on axillary surface disappeared. In ventral view, the color of fingers and

toes lost their reddish intensity and became cream.

Distribution (Figure 8). Amazophrynella minuta is distributed throughout the eastern

border region of the Amazonas state in Brazil, a region also known as “Cabeça do

Cachorro” (“Dog’s head”, English free translation). The species is found in the

following localities: Taracuá (0.10°N, 68.46°W), São Gabriel da Cachoeira (0.16°S,

66.98°W), Cucuí (1.07°N, 66.88°W) and Japura River close to Vila Bittencourt

(1.81°S, 68.98°W), at elevations between 90–105 m a.s.l. In Colombia it is reported

in territories of the Department of Caquetá (0.92°N, 75.67°W) and Vaupés (0.90°S,

69.67°W) (Lynch, 2006) at elevations between 100–200 m a.s.l. (Acosta–Galvis,

2017). In Venezuela (not examined), the species was reported from Raudal de

Danto, Río Cuao (4.53°N, 67.18°W) Amazonas state, at elevation of 105 m a.s.l.

(Rojas–Runjaic et al., 2013).

Ecology. We found all individuals in the morning near a water body. Specimens were

collected in primary “terra firme” forest (“firm land” – non–flooded forest, English free

translation) some in the leaf litter and others among tree roots. Specimens from São

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Gabriel da Cachoeira were collected in a fragment of forest near to the airport; the

individuals were found around a stream with abundant leaf litter. Amplexus is axillar

(Figure 9).

Phylogenetic analysis. The complete phylogenetic relationships of Amazophrynella,

the phylogenetic position of A. minuta and genetic distance between Amazophrynella

spp. were reported in Rojas et al. (2018)- Figure 1 and Table 1).

Advertisement call (Figure 10). The characterization of the advertisement call of

Amazophrynella minuta was based on 14 calls from one individual from São Gabriel

da Cachoeira (INPA-H 32730, SVL: 12.2 mm, recorded between 15:30–16:00 h). Air

temperature at the time of recording varied between 26.0 to 27.0°C. The call

consisted of a trilled note emitted between regular silent intervals. Notes had an

upward amplitude modulation, reaching their maximum intensity near the middle of

the note. Note duration ranged between 0.310 and 0.108 s (0.223 ± 0.068 s).

Number of pulses per note ranged from 18 to 35 pulses/note (26.286 ± 6.557

pulses/note). The inter–note interval was 0.663 to 1.050 s (0.827 ± 0.120 s). The

dominant frequency ranged from 4242.790 to 4562.011 Hz (4419.213 ± 125.642 Hz).

The fundamental frequency ranged from 3201.010 to 3788.520 Hz (3489.211 ±

235.791 Hz). The rise time ranged from 0.045 to 0.083 s (0.059 ± 0.015 s). Number

of harmonics varied from 3 to 5 (4.150 ± 0.587).

Osteology. A detailed review and description of the skeleton of A. minuta were

provided by McDiarmid (1971). In the same publication, McDiarmind (1971) reported

characters that could be tentatively considered as putative genus level

synapomorphies (see Fouquet et al., 2012b).

Discussion

Amazophrynella minuta was described by Melin in 1941 from a few specimens

collected at Taracuá, municipality of São Gabriel da Cachoeira, Amazonas state,

Brazil in 1924. The original description lacked precision in describing morphological

characters such as shape of head, in dorsal and lateral view; texture of dorsal and

ventral skin; formula of fingers and toes; measurements, proportions of body and

details in coloration of the dorsal and ventral surfaces. Since these morphological

characters are important to distinguish and diagnose species of the genus (see

168

Izecksohn, 1993; Ávila et al., 2012; Rojas et al., 2014, Fouquet et al., 2012b), herein,

we provided a detailed description of these characters. Furthermore, with the

identification of diagnostic characters and definition of character states of A. minuta,

this work will facilitate the comparison between species of Amazophrynella and

contribute to future descriptions of the remaining unnamed species in the genus

(Rojas et al., 2018).

Morphological traits and phylogenetic relationships suggest that

Amazophrynella minuta is most closely related to A. amazonicola, A. matses and A.

javierbustamantei with which it notably shares coloration and patterns on the belly

(see Figure 5); however, A. minuta differs from these species by snout profile and

texture of dorsal and ventral skin. From the other species of Amazophrynella, A.

minuta differs in SVL, body proportions, size of FI vs. FII; venter coloration, texture of

dorsal skin, type of palmar tubercle and webbing type on fingers (see Comparisons

with other species for details). The morphological crypsis in Amazonian anurans is a

taxonomic problem that hinders descriptions based only on morphological data, but

can and has been overcome in several groups using an integrated approach to

taxonomy (analyses of advertisement calls, morphology, genetics, tadpoles, etc.)

(sensu Padial et al., 2009; Padial et al., 2012; Caminer & Ron 2014)

The description of the advertisement call of A. minuta will contribute to

distinguishing nominal populations from others (i.e. Colombia, Venezuela and Brazil)

in the aim to delimit new species. As expected, the advertisement call of A. minuta is

similar to other species of Amazophrynella (Rojas et al., 2018). As a genus–level

synapomorphic character the advertisement calls of Amazophrynella present a

simple note that varies between 0.0180 to 0.0146 s; a fundamental frequency

between 2354.207 to 3204.169 Hz; a dominant frequency between 3033.86 to

3635.71 Hz; numbers of pluses ranged from 8 to 52.4 and a rise time from 0.004 to

0.107 s. Unfortunately, we could not collect tadpoles of Amazophrynella minuta,

although we made two expeditions to the remote type locality in July 2012 and again

in August 2014. Within the genus, tadpoles are scarce and basic ecology remains

largely unstudied (Rojas et al., 2016). The description of these (tadpoles and

behavior) biological variables in nominal and putative species of Amazophrynella

may provide important data for future species delimitation within the genus.

169

Genetic and morphological data from populations of A. minuta from Colombia

and Venezuela were not analyzed in this study. Given the propensity of hidden

diversity in this group a careful examination of material from these populations is

needed. Future integrative taxonomic analyses will certainly improve our knowledge

of phylogenetic relationships and taxonomy of Amazophrynella.

170

Acknowledgements

We thank the people of the community of Taracuá, especially Sr. Maximiliano Correia

Menezes (representative of the Amerindian village of Taracuá and FOIRN), Sr.

Gabriel Correia Menezes, Sr. João Filho Menezes and Sra. Vera Correia for

hospitality and logistic support in Taracuá. We thank Junior Menezes, Max Junior

Menezes, and Bene Menezes for field support. Federação de Organizações

Indígenas do Rio Negro/FOIRN allowed access to Ameridian lands. We thank Göran

Nilson and Anders Larsson (Naturhistoriska Museet, Göteborg) for their

consideration and for sending color photographs of the syntypes of A. minuta. Marcia

Lima de Queiroz and Miss Lana helped in the INPA. Mario Nunez helped with the

molecular analyses. Funding for this work came from CNPq/SISBIOTA Processo No.

563348/2010-0 and SISBIOTA/FAPEAM. Coordenação de Aperfeiçoamento de

Pessoal de Nível Superior (CAPES) provided financial support and awarded a PhD

fellowship to R. R. R. This work is part of R.R.R.’s PhD Thesis in Genetics,

conservation and evolutionary biology program of INPA. AF has benefited from an

“Investissement d’Avenir” grant managed by the Agence Nationale de la Recherche

(CEBA, ref. ANR-10- LABX-25- 01). RWA thanks CNPq for his productivity research

grant (303622/2015-6). For funding for specimens collection and laboratory work

SRR thanks SENESCYT, Arca de Noe initiative.

171

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complex: integrative taxonomy reveals a new species of Amazophrynella

(Anura, Bufonidae) from southern Peru. ZooKeys, 71,43–71.


Rojas, R.R., Fouquet, A., Ron, S., Hernandez, E., Melo-Sampaio, P., Chaparro, J.,

Vogt, R., Carvalho, V., Pinheiro, L., Ávila, R., Pires, I., Gordo, M. & Hrbek, T.

(2018). A Pan-Amazonian species delimitation: high species diversity within the

genus Amazophrynella (Anura:Bufonidae). PeerJ, 6, e4941.

https://doi.org/10.7717/peerj.4941



174

Specimens examined.

A. minuta.—BRAZIL: Amazonas State: Uauapés River, Taracuá, INPA–H 32720–23,

INPA–H 32725–26, INPA–H 32728–30, INPA–H 32732, INPA–H 32733, INPA–H

32735–40, NHMG 462, NHMG 463, NHMG 464.; Sao Gabriel da Cachoeira, INPA–

H32731, INPA–H 32724, INPA–H 32734, Japura River, INPA–H 355507–12, INPA–H

35494–98.

A. bokermanni.—BRAZIL: Pará State, Juriti (near 30 km from type locality) INPA–H

31861–65, Amazonas State, Autazes: INPA–35529–30; Pará State: Xingu River INPA

35473–77.

A. vote.—BRAZIL: Mato Grosso State: Cotriguaçu, Fazenda São Nicolau, UFMT–A

11138 (Holotype); UFMT 11136, UFMT 11142, UFMT 11145–50, UFMT 11152–55,

UFMT 4412; Manicoré, Madeira River, INPA–H 12255–56, 12331, 12342–43, 12366–

67 (Paratypes); Parque Estadual do Guariba INPA–H 21558, Novo Aripuanã,

Aripuanã River, INPA–H 12326 (Paratype) INPA–H 35540−50; Estado Amazonas:

Tapauá, Parque Nacional Nascentes do Lago Jari, INPA–H 27412, 27417−19,

27421−23, 27425−26 (Paratypes), Tapauá, Purus river, INPA–H 35551−53,

Manaquiri, INPA–H 35535−39; Estado do Amazonas: Matupiri, INPA–H 31867,

INPA–H 31868, INPA–H 31870–75, INPA–H 31877–80, INPA–H 31882, INPA–H

31883–66.

A. manaos.—BRAZIL: Amazonas State: Campus da Universidade Federal do

Amazonas, INPA–H 31866 (Holotype), INPA–H 6983–64, INPA–H 6987, INPA–H

7797 (paratypes); Presidente Figueiredo, INPA–H 20986, INPA–H 21217, INPA–H

29568–72, INPA–H 30575–77, INPA–H 30572–73 (Paratypes); Reserva Florestal

Adolpho Ducke, INPA–H 21028, INPA–H 21170, INPA–H 21060, INPA–H 31866,

INPA–H 21007–13, INPA–H 20963–INPA-H 20990.

A. matses.—PERU: Department Loreto: Requena, Nuevo Salvador, MZUNAP

921(Holotype), MZUNAP 922–23, MZUNAP 925–27, MZUNAP 934, MZUNAP 936,

MZUNAP 938, MZUNAP 940, MZUNAP 943–44, MZUNAP 948, MZUNAP 952–53,

MZUNAP 955, MZUNAP 958 (paratopotypes); Jenaro Herrera MZUNAP 928–31,


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941–42, MZUNAP 946–47, MZUNAP 949 (Paratypes).

A. amazonicola.—PERU: Department Loreto: San Juan Bautista, Puerto Almendra

MZUNAP 901 (Holotype), MZUNAP 906–07, MZUNAP 910–11, MZUNAP 913–17;

MZUNAP 110; MZUNAP 889 (paratopotypes); 58 km of Iquitos–Nauta highway on

Fundo Zamora, MZUNAP 887–88, MZUNAP 900, MZUNAP 902, MZUNAP 886,

MZUNAP 905, MZUNAP 908, MZUNAP 919–20, MZUNAP 924 (Paratypes); Maynas,

Nauta, MZUNAP 909, MZUNAP 918; Fundo UNAP, MZUNAP 242 (Paratype).

A. javierbustamantei.—PERU: Department Madre de Dios: Tambopata, Quebrada

Guacamayo, MHNC 8331(Holotype), MHNC 8316, MHNC 8238, MHNC 8362–63,

MHNC 8245, MHNC 8484, La Pampa, MHNC 1101–04; Nuevo Arequipa, MHNC

8245, MHNC 8331, MHNC 8238, MHNC 8354, MHNC 8484, Rio Tambopata,

MHNSM 9633, MHNSM 9635, MHNSM 9640–42, MHNSM 9644, MHNSM 9646–48;

Manu, Inambari, MHNSM 17993; La Convencion, Camana, MHNSM 2565; Mapi,

MHNC 9939–40. Departamento: Junin: Tambo Poyeni, MHNC 9387; Tsoroja, MHNC

9626, MHNC 9754, MHNC 9756–57, MHNC 9679, MHNC 9680. Department: Cusco:

Urubamba, Urubamba River, MHNC 9626, MHNC 9686–87.

A. moisesii.—BRAZIL: Acre: Parque Nacional da Serra do Divisor: Igarapé Ramon:

UFAC-H 2815 (Holotype), UFAC-H 1375, UFAC-H 2772–2773, UFAC-H 2603, UFAC-

H 2607, UFAC-H 3573–UFAC-H 3575, UFAC-H 2690, UFAC-H 2692, UFAC-H 2815–

2817, UFAC-H 1698; Igarapé Anil: UFAC-H 1337–1343; Zé Luiz lake: UFAC-H 1774–

1775; Môa river: UFAC-H 1493, UFAC-H 2687–2697. Reserva Extrativista Alto do

Juruá: UFAC-H 822–823, UFAC-H 878–879, UFAC-H 2606–2611 Gregório Forest

Reverse: UFAC-H 5678.

A. teko.—BRAZIL: Amazonas state, Trombetas river: INPA-H 35513–35530, Amapa

state: UFPA AA 604. French Guiana: District of Camopi: Alikéné: MNHN 2015.136

(Holotype). District of Saint Laurent du Maroni: Mitaraka layon: MNHN 2015.137,

MNHN 2015.138, MNHN 2015.139, MNHN 2015.140, MNHN 2015.141, MNHN

2015.142, MNHN 2015.143, Pic Coudreau du Sud: MNHN 2015.152, MNHN 2015,

Flat de la Waki: INPA–H 36598. District of Camopi: Mitan: INPA–H 36596, MNHN

2015.144, MNHN 2015.145, MNHN 2015.146, MNHN 2015.147, MNHN 2015.148,

MNHN 2015.149, MNHN 2015.150. District of Saint Georges: Saint Georges: MNHN

176

2015.151, Mémora: MNHN 2015.154, MNHN 2015.155, Saut Maripa: INPA–H 36597,

INPA–H 36610, INPA–H 36599, INPA–H 36601, INPA–H 36600.

A. xinguensis.—BRAZIL: Para state: Sustainable Development Project (PDS) Virola

Jatobá: INPA–H 35471 (Holotype), INPA–H 35484, INPA–H 35485 INPA–H 35473,



INPA–H 35491, INPA–H 3592, Fazenda Paraiso: INPA–H 35493, INPA–H 35472,

Ramal dos Cocos: INPA–H 35486, INPA–H 35487, INPA–H 3588, INPA–H 35489.

A. siona.—ECUADOR: Province of Orellana: Yasuni National Park: QCAZ 27790

(Holotype), QCAZ 11981, QCAZ 51068, QCAZ 21425, QCAZ 21431, QCAZ 11973,

QCAZ 11979. Provincia Sucumbios: Reserva de Producción Faunística Cuyabeno:

QCAZ 52433–34, QCAZ 37758–59, QCAZ 37761, QCAZ 6071, QCAZ 6091, QCAZ

6095, QCAZ 6097, QCAZ 6105, QCAZ 6111, QCAZ 6113, QCAZ 6118, QCAZ 6127,

QCAZ 6128, Santa Cecilia, QCAZ 4469, QCAZ 4472, Tarapoa: QCAZ 36331, QCAZ

36336, QCAZ 36338, QCAZ 36357. Provincia de Pastaza: Community of Kurintza:

QCAZ 56342, QCAZ 56354, QCAZ 56361, Villano community, AGIP oil company:

QCAZ 38599, QCAZ 38679, QCAZ 38722, Around Villano community, AGIP oil

company: QCAZ 38642, Community of Kurintza: QCAZ 38809, QCAZ 54213,

Bataburo Lodge: QCAZ 39408, Lorocachi: QCAZ 8902, Lorocachi: QCAZ 56165,

Canelos: QCAZ 52819, QCAZ 52823, QCAZ 17391. Provincia Orellana:

Tambococha: QCAZ 55345, Garzacocha: QCAZ 20504, Yuriti: QCAZ 10526, Kapawi

Lodge: QCAZ 8725, QCAZ 25504, QCAZ 25533 10 km from Puyo: QCAZ 7135.

Provincia Morona Santiago: Pankints: QCAZ 46430. PERU: Department Loreto:

Teniente Lopez: MHNC 7611, MHNC 7685, MHNC 7686, MHNC 7698, MHNC 7699,

MHNC 7700, Jibarito: MHNC 7786, MHNC 7809, MHNC 7814. Shiviyacu: MHNC

14730, near Corrientes River: MHNC 6292.

A. moisesii.—BRAZIL: Acre state: Reserva Extrativista Alto do Juruá: UFAC-RB 823,

UFAC-RB 878–879, Parque Nacional da Serra do Divisor: Igarapé Anil: UFAC-RB

1337–1341, UFAC-RB 1343, Zé Luiz lake: UFAC-RB 1774–1775, Igarapé Ramon:

UFAC-RB 1375, UFAC-RB 2772–2773, UFAC-RB 2816–2817, Môa river: UFAC-RB

1493, UFAC-RB 2687–2697, Gregório forest Reserve: UFAC-RB 5678.

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TABLE 1. Comparisons of measurements (in mm) among males and females of

Amazophrynella minuta (for abbreviations of characters see Materials and Methods).

Character/Sex Male

(n=20)

Female

(n=11)

SVL 13.6 ± 0.6 (12.2–14.7) 17.4 ± 0.9 (14.5–19.4)

HW 4.3 ± 0.2 (3.8–4.8) 5.1 ± 0.4 (4.6–5.9)

HL 5.1 ± 0.3 (4.6–5.8) 6.0 ± 0.4 (5.1–6.9)

SL 2.3 ± 0.2 (2.1–2.6) 2.7 ± 0.2 (2.4–2.9)

ED 1.5 ± 0.2 (1.2–2.0) 1.7 ± 0.3 (1.5–1.9)

IND 1.2 ± 0.1 (1.0–1.4) 1.4 ± 0.1 (1.2–1.5)

UAL 4.0 ± 0.4 (3.5–5.3) 5.2 ± 0.2 (4.6–5.5)

HAL 2.8 ± 0.2 (2.4–3.2) 3.6 ± 0.3 (3.0–4.4)

THL 7.0 ± 0.4 (6.3–8.1) 8.5 ± 0.9 (6.8–10.1)

TAL 6.8 ± 0.4 (6.1–8.1) 8.4 ± 0.7 (7.2–10.0)

TL 4.4 ± 0.6 (3.7–6.7) 5.4 ± 0.4 (4.6–6.0)

FL 4.9 ± 0.4 (4.2–5.8) 6.4 ± 0.7 (5.3–8.2)

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Figure 1. Terminology used in this paper to describe texture of dorsal skin in

Amazophrynella. A) Tuberculate; B) granular; C) highly granular; D) finely granular;

E) Spiculate. See Materials and Methods for character definition.

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Figure 2. Terminology used in this paper to describe ventral color pattern in

Amazophrynella. A-B) large black blotches over light background; C-D) small black

blotches over light background; E-F) small black dots over light background; G) small

black points over light background. See Materials and Methods for character

definition

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Figure 3. Terminology used in this paper to describe shape of palmar and

subarticular tubercles in Amazophrynella. A) Rounded; B) oval; C) elliptical. See

Materials and Methods for character definition.

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Figure 4. Lectotype (A= NHMG 462) and paralectotypes (B= NHMG 465, C= NHMG

463) of Amazophrynella minuta (Melin, 1941) from Taracuá, Rio Uaupés, Amazonas

State, Brazil deposited at the Naturhistoriska Museet, Göteborg, Sweden. Additional

pictures of the types are provided in Figure 3 -page 70 in Ávila et al. (2012). Photos

by: Anders Larson.

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Figure 5. Amazophrynella spp. A) A. minuta; B) A. bokermanni; C) A. matses; D) A.

javierbustamanteu; E) A. siona; F) A. bokermanni; G) A. manaos, H) A. vote, I) A.

xinguensis, J) A. teko, K) A. moisesii

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Figure 6. Morphological variation in life of Amazophrynella minuta (Melin, 1941) from

type locality. A-F) adult males; G-I) adult females.

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Figure 7. Morphological variations of preserved specimens of Amazophrynella

minuta (Melin, 1941) from type locality. A-C) adult females; D-F) adult males.

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Figure 8. Geographic distribution of Amazophrynella minuta (Melin, 1941), star

marks the type locality. Brazil, state of Amazonas: 1) São Gabriel da Cachoeira; 2)

Lago Jabo-Cucui; 3-4) Vila Bittencourt, Japura River. Colombia, Department of

Vaupés: 5) estación biológica Cacaparu; 6) Santander; 7) Vaupés River; 8) Mitu;

Department of Amazonas: 10) Restinga isla mariname; 11) La chorrera; Department

of Caqueta:12) Cabanachagra; 13) Caqueta River. Venezuela; State of Amazonas:

14) Raudal de Danto, Río Cuao.

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Figure 9. Axillary amplexus between specimens of Amazophrynella minuta (Melin,

1941)

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Figure 10. Advertisement call of Amazophrynella minuta (Melin, 1941) from São

Gabriel da Cachoeira, State of Amazonas, Brazil. A) Oscillogram and spectrogram

visualizing three notes; B) Oscillogram and spectrogram visualizing one note.

188

CAPITULO IV

Diversification in Amazonian through the historical biogeography of “terra firme”

tiny tree toads Amazophrynella (Anura: Bufonidae). Rojas, R.R., Fouquet, A.,

Carvalho, V., Ron, S., Ávila, R., Pires, I., Gordo, M. & Hrbek, T. Artigo para ser

submetido na revista Molecular phylogenetics and evolution

189

Diversification in Amazonian through the historical biogeography of “terra

firme” tiny tree toads Amazophrynella (Anura: Bufonidae)

Rommel R. Rojas1, Antoine Fouquet2, Vinícius Tadeu De Carvalho1, Santiago Ron3,

Robson W. Ávila4, Izeni Pires Farias1, Marcelo Gordo5 & Tomas Hrbek1

1Laboratório de Evolução e Genética Animal (LEGAL), Departamento de Genética,

Instituto de Ciências Biológicas, Universidade Federal do Amazonas, Av. General

Rodrigo Octávio Jordão Ramos, 6200. CEP 69077–000 Manaus, AM, Brazil


Amazoniens, Centre de recherche de Montabo, 275 route de Montabo, BP 70620,

97334 Cayenne, French Guiana.


Ecuador, Av. 12 de Octubre y Roca, Aptdo. 17–01–2184, Quito, Ecuador.

4 Departamento de Química Biológia, Universidade Regional do Cariri, Campus do

Pimenta, Rua Cel. Antônio Luiz, 1161, Bairro do Pimenta, CEP 63105–100, Crato,

CE, Brazil.


do Amazonas, Av. General Rodrigo Octávio Jordão Ramos, 6200. CEP 69077–000

Manaus, AM, Brazil.


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Abstract

Understand the patterns and processes that generated the high species

diversity in Amazonia is essential to develop conservation strategies and provide

information of biodiversity evolution. The frogs genus Amazophrynella comprises

“terra firme” tiny tree toad with an Pan-Amazonian distribution that present and

striking evolutionary pattern. Here, we employed genomic data to reconstruct their

divergence times, ancestral area distributions, dispersal–vicariance events and

temporal pattern of diversification. Discrete and continuous biogeographic inferences

from multiple models. Lineages diversification and areas reconstructions predicted

similar historical biogeography dynamics for Amazophrynella which may suggest that

extant geographical distributions and diversity patterns were influenced strongly by

long-distance dispersal across geographical barriers. In addition, our results suggest

an old connection between Amazonia and Atlantic forest and illustrate how climatic

and landscape modifications preceding the Miocene shaped the initial diversification

of South American Amazonian frogs.

Key words: Anura, biodiversity, biogeography, conservation, diversification, endemic

areas.

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Introduction

The neotropics present the most extraordinary and fascinating biodiversity in

the earth, as such, is a perfect target for research about the origin of biological

diversity (Rull, 2008). Landscape Paleogene reconstructions suggest a continuous

forest connecting the ancient Amazonia and Atlantic forest during Eocene divided by

the origin of drier savanna vegetation comprising by Cerrado, Caatinga and Chaco

(Morley, 2000; Werneck et al., 2012). Phylogenetic evidence also indicates periods of

forest re-connection between both areas (Batalha-Filho et al., 2013; Capurucho et

al., 2018; Costa, 2003; Dal Vechio et al., 2018).However, despite previous studies

that suggest an ancient (Eocene/Miocene) and recent (Pleistocene) divergence

between organism on each side (Fouquet et al., 2012; Pellegrino et al., 2011)

ancestral scenarios reconstructions and biological diversification in early stages of

forest divergence still poorly understood.

The Amazonian present the largest records of flora and fauna in the world

(Jenkins et al., 2013). Geomorphological and climatic fluctuations during Miocene-

Pleistocene has been proposed as important causes of their megadiversity (Antonelli

et al., 2018; Hoorn et al., 2010). Processes as extinction, vicariance and dispersion

and sympatric and allopatric speciation are attributed as the most common

mechanisms in diversification of Amazon taxa (Antonelli et al., 2018; Rull, 2011;

Wiens and Donoghue, 2004). Another evolutionary feature in Amazonian biota is

their not random phylogeographic distribution - endemism areas (Ribas et al., 2012;

Smith et al., 2014).

This striking pattern has been widely studied through lineages diversification of

mammals, birds and lizards (Oliveira et al., 2016; Patton et al., 2000; Ribas et al.,

2012), but historical reconstructions using non-model taxa as amphibians is scarce

(i.e. Noonan and Wray, 2006; Castro-Viejo et al., 2013; Fouquet et al., 2015;

Godinho and Silva 2018). In addition, most of these studies do not explore whether

the observed patterns of biodiversity are concordant with current models of faunal

differentiation in the Amazon basin. Hence, by studying amphibian evolutionary

history, we can gain greater insight into the history of their habitats and of the other

inhabitants of these areas (Castroviejo-Fisher et al., 2014). However, evolutionary

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history and diversification of Amazonian amphibians is poorly understanding in

neotropics (Castroviejo-Fisher et al., 2014; Fouquet et al., 2007; Gehara et al., 2014;

Godinho and Da Silva, 2018; Santos et al., 2009).

Frogs constitute an excellent group to study species diversification because

their low dispersion abilities compared with birds and mammals (Zeisset and Beebee,

2008), being strong sensitive to environmental and geological modification (Godinho

and Da Silva, 2018), highly depend on habitat quality and have both life stages

(aquatic and terrestrial) (Vences and Wake, 2007). In addition, it is estimated that

almost 40% of species are in danger of extinction (Collins, 2010), and their

populations are progressively decreasing in Andes and Amazonia (Becker et al.,

2016; Catenazzi and von May, 2014). Likewise, only a few frog lineages includes

Amazonian and Atlantic forests counterparts (i.e. Fouquet et al., 2014; Sá et al.,

2018; Thome et al., 2016).

In this study we focus on “terra firme” tiny tree toads of genus Amazophrynella

(Anura: Bufonidae) that constitutes an interesting anuran clade because their

intriguing patterns of phylogenetic evolution (basal east-west followed by a north

south diversification), being distributed by all endemic areas (Rojas et al., 2018) and

being sister clade of endemic species from Atlantic forest (Dendrophryniscus)

(Fouquet et al., 2012a). Amazophrynella comprise seventeen nominal and putative

lineages with a Pan-Amazonian distribution, present small size (snout vent length ≥

26.0 mm) and exhibit high levels of morphological conservation (Rojas et al., 2018).

They are diurnal and crepuscular, found exclusively in leaf litter of primary and

secondary rainforest, their reproduction occurs in small puddles, their eggs are

pigmented and deposited in roots shrubs and under litter (Rojas et al., 2018, 2016,

2015). In summary, Amazophrynella encompasses a taxonomic well resolved group

with a striking phylogenetic and ecological pattern, thus combined with their broad

geographic distribution, make Amazophrynella an ideal species group to study

diversification in Amazonia. But despite this knowledge a comprehensive

biogeographical reconstruction and lineage diversification of genus is still lacking.

Here, using the phylogenetic relationships of Amazophrynella, we explore

lineage diversification and their historical biogeography.

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Taxonomic and molecular sampling

We used an extensive sampling of the genus Amazophrynella in their entire

Pan-Amazonian distribution with a total of 235 terminals from 35 localities from

seventeen lineages including nominal and putative species from Rojas et al. (2018)

(Supporting Information Appendix S1). Genomic data - Single nucleotide polymorphic

(SNPs) DNA sequence data were obtain from a subset of 23 individuals of

Amazophrynella representing the mtDNA clades (putative and nominal species)

previously delimited by Rojas et al. (2018). We also obtain genomic date from two

individuals of genus Dendrophryniscus and Rhinella margartifera as outgroup.

Molecular procedures

Tissue samples (silver or muscle) were preserved in 100% ethanol and kept at

-20°C for DNA extraction. Standard molecular protocols were used from DNA

extracting to sequencing (Sambrook et al., 1989). Amplification of mitochondrial

genes (12S, 16S and COI) was carried out under the following conditions: 60 s hot

start at 92°C followed by 35 cycles of 92°C (60 sec), 50°C (50 sec) and 72°C (1.5

min). Final volume of the PCR reaction for all genes was 12 μl and contained 4.4 μL

of ddH2O, 1.5 μL of 25 mM MgCl2, 1.25 μL of 10 mM dNTPs (2.5mM each dNTP),

1.25 μL of 10x buffer (75 mM Tris HCl, 50 mM KCl, 20 mM (NH4)2SO4), 1 μL of each

2 μM primer, 0.3 μL of 5 U/μL DNA Taq Polymerase (Fermentas, Lithuania) and 1 μL

of DNA (about 30 ng/μL).

Preparation of the library for genomic data was done according to protocol

developed by Peterson et al. (2012) or ddRADseq libraries. After enrichment PCR

and fragment size selection (400 bp) in the Pippin Prep electrophoresis platform

(Saga Science), all samples were grouped in equimolar quantities and sequenced in

the Genetic Sequencer Generation Ion Torrent PGM, using reagents and sequencing

kits 400pb and chip 318. Voucher specimens for the sampled taxa are listed in

Supporting Information Appendix S1.

The reading of genomic data was processed in the PyRAD (Eaton, 2014) and

used to generate a Matrix of SNPs and concatenated sequences. All alleles used

194

had Least 5X of minimum coverage in each locus and frequency in at least 50% of

the individuals. We obtain a total of 4,152,088 fragments, already filtering the poor

quality sequences. A total of 58% deposition of the library enriched with ion sphere

particles (ISPs) was obtained. PyRAD generated a phylip file with 144,146 bp, which

represents 500 loci with 12070 variable sites and 6077 informative sites of

parsimony.

Phylogenetic methods and molecular clock calibration

We implemented Bayesian phylogenetic analyses in the software package

Beast 2.0 and run a StartBeast analysis (Drummond and Rambaut, 2007). Tree

searches were performed assuming both single models of sequence evolution for

each locus, and Markov chain Monte Carlo (MCMC) searches were made for 10

million generations, sampling every 1000 generations, for a total of 10 000 trees. We

estimated model parameters during runs, and estimated Bayesian posterior

probabilities as the proportion of trees sampled; the trees obtained in the first one

million generations were discarded. The best model of molecular evolution

(GTR+G+I) was estimated in JModelTest (Posada, 2008) and selected using the

Akaike Information Criterion–AIC.

We used the uncorrelated relaxed lognormal clock (Drummond et al., 2006),

with a birth and dead tree prior of speciation. We used divergence time constraints,

applying normal distributions to the prior probabilities of the dates, to the following

nodes a crown age of Amazophrynella + Dendrophryniscus vs. others Bufonidae

52.0–32.0 Ma (mean= 40.2 ± 10.0), Amazophrynella + Dendrophryniscus 49.0–29.0

Ma (mean= 38.1 ± Ma) and eastern vs. western Amazophrynella lineages divergence

obtained from Rojas et al. (2018).

From the MCMC output, we generated the final consensus tree-maximum

clade credibility tree- using Tree Annotator v1.6.2 (part of Beast software package).

For visualization and edition of the consensus maximum clade credibility tree, we

used the program Figtree v.1.3. (Rambaut, 2009).

Analysis of historical biogeography

https://onlinelibrary.wiley.com/doi/full/10.1111/jbi.12208#jbi12208-bib-0020

https://onlinelibrary.wiley.com/doi/full/10.1111/jbi.12208#jbi12208-bib-0021

195

To perform ancestral ranges estimation we excluded non Amazophrynella and

Dendrophryniscus taxa, including only one terminal per area from Starbeast.

Inference of biogeographical history on the dated phylogeny of Amazophrynella we

used the package BioGeography with Bayesian (and likelihood) Evolutionary

Analysis in R Scripts–BioGeoBEARS (Matzke, 2013) using the program R (R

Development Core Team, 2011). We compared six biogeographical models

implemented in BioGeoBEARS to determine their fit to our data. Likelihood values of

the models were compared using Likelihood Ratio Test (LRT), and model–fit was

assessed in BioGeoBEARS by comparing weighted Akaike’s information criterion

scores (Matzke, 2013).

The species distribution areas were assigned based on biogeographic regions

Inambari (In), Guyana (Gu), Napo (Na), Rondonia (Ro), Tapajós (Ta), Xingú (Xi) and

Imeri (Im) and Atlantic Forest (AF) (Olson et al., 2001; Ribas et al., 2012; Smith et al.,

2014). For BioGeoBEARS analysis we used a maximum number of areas (8) per

node unconstrained. This option was selected because the distribution range of the

taxa in past was not detected either in Amazonian, Atlantic forests or both areas and

because the speculative influenced of the geomorphological events in

biogeographical mechanisms (vicariance, dispersion or founded effect), the option no

constraint could be made regarding potential ancestral distributions. Other values

were left at default values (Matzke, 2013).

Phylogeographic analysis

In order to reconstruct the phylogeographic history of Amazophrynella

lineages through time in continuous space (by using location coordinate data for

each lineage), we used the Relaxed Random Walk (RRW), approach proposed by

Lemey et al., (2010). The RRW model involves a Bayesian framework, reconstructing

phylogeographic history on a continuous landscape through the standard Brownian

diffusion process using geographical coordinates, phylogenetic genealogy and

continuous trait (Lemey et al., 2010).

The analysis was performed using the mitochondrial and genomic data. We

used a coalescent prior with constant population size and an uncorrelated log–

normal RRW model as well an GTR+G+I model substitution for each mitochondrial

196

and nuclear marked and length of chain of 107 generations and 10% of cut-off was

used for burn-in. Mixing of parameter sampling, Effective Sample Size (ESS) and

convergence were checked in Tracer 1.5. The resulted tree was summarized with

Tree Annotator 1.7.2. The software SPREAD was used to generate a Keyhole

Markup Language (KML) file which was plotted in a Google Earth map

(http://earth.google.com).

Analysis of lineage diversification

The timing and patterns of diversification in “terra firme” tiny tree toads with

lineage-through-time (LTT) plots. LTT plots depict the cumulative (log) number of

nodes as a function of time. We estimated LTT plots on the basis of calibrated

molecular clock chronograms obtained via bayesian analysis. We also tested

whether significant changes occurred in the diversification rates throughout the

evolutionary history of Amazophrynella by implementing various likelihood-based

statistical methods that simultaneously accommodates undersampling of extant taxa,

rate variation over time, and potential periods of declining diversity (Morlon et al.,

2011). The analyses was made using the software package RPanda (Morlon et al.,

2016) implemented in R (R Development Core Team, 2011).

Results

Phylogenetic and divergence times

The age of the most recent common ancestor (MRCA) of Amazophrynella and

its sister lineage Dendrophryniscus is estimated at c. 34.2 Ma (95% HPD: c. 38.2–

30.5 Ma) in Eocene and middle Oligocene, with high node support (posterior= 1).

Within Amazophrynella the eastern/western divergence is estimated at c. 24.8 Ma

(95% HPD: c. 28.3–21.6 Ma) in late Oligocene and early Miocene. Within the eastern

clade the north and south division is estimated at c. 19.7 Ma (95% HPD: c. 22.6–17.0

Ma) in early Miocene. In western clade, the north vs. south split is estimated at c.

16.5 (95% HPD= 19.0–14.0 Ma) in the middle Miocene (Figure 1). Major

diversification events between lineages within each of the four above clades varied

between 5.0 and 1.0 Ma in late Miocene and Pliocene.

Historical biogeography

http://earth.google.com/

197

BioGeo–BEARS analyses of genomic marked were DIVALIKE model including

founder-effect speciation (DIVALIKE+J) as the model that provided the best fit of all

models to our dataset (see in Table 1). The results of the biogeographical model

selected by BioGeo–BEARS are presented in Figure 1.

The biogeographic reconstruction indicates that the MRCA of Amazophrynella

and Dendrophryniscus had a wide distribution range in two areas: Atlantic Forest +

lnambari. Subsequently analysis suggests vicariance between Amazonia vs. Atlantic

Forest. In Amazonian ancestral area were Inambari. Subsequently analysis suggest

a dispersion mechanism from Inambari to Guyana and Imeri. In eastern Amazonia

were detected a dispersion from Guyana to Inambari and from Inambari to Rondonia

and Xingu being Tapajos the last colonized area. In western clade were detected a

dispersion from Imeri to Inambari following by a colonization by dispersion from Imeri

to Napo.

Diversification patterns

The best model to explain Amazophrynella diversification was exponential

variation in speciation with no extinction (Table 2- Figure 2). Phylogeographic

analysis under Brownian motion model (Figure 3) suggested an ancestor of

Amazophrynella and Dendrophryniscus distributed through southern Amazonia +

Cerrado and Atlantic Forest (Figure 3A). This ancestral population remained during

the late Eocene and middle Oligocene c. 44.0–35.0 Ma.

Subsequent scenario showed a widespread ancestral of Amazophrynella

distributed in central Amazonia (Figure. 3B). This ancestral population existing in

middle Oligocene between 35.0–30.0 Ma. The ancestral population of

Amazophrynella split in two demes (eastern and western) around 30.0–20.0 Ma

during late Oligocene and early Miocene (Figure 3C). The two demes expanded and

split in four ancestral population (north and south) that emerged and coexisted in

early Miocene 20.0–15.0 Ma (Figure 3D). Range expansion of four-ancestral

population increase in north-eastern and south-western and Amazonia at 15.0–10.0

Ma (Figure 3E), follow by a north-western and south-eastern range expansion at

10.0–5.0 during late Miocene (Figure 3F). The final expansion of ancestral

populations occurs at 5.0–1.0 Ma during Pliocene (Figure 3G).

198

Discussion

Our results show clear convergence among discrete and continuous

biogeographic inferences from multiple models. Lineages diversification and areas

reconstructions predicted similar historical biogeography dynamics for

Amazophrynella which may suggest that extant geographical distributions and

diversity patterns were influenced strongly by long-distance dispersal across

geographical barriers. The best-fitting model in our diversification analyses are likely

trend of rising diversity with slight variations through time (exponential). Taxon age is

a possible explanation for the observed differences across clades. Thus, because

diversification rates in early adaptive radiation were low (Figure 3) and less niches

were available in initial diversification stages. However, the result should be taken

with caution because the small sample size (n=17 clades) could be a problem

(Freckleton et al., 2002).

Ancestral area reconstruction and diffusion model suggested a connection

from Amazonia to Atlantic forest through south of Cerrado. Hoorn et al. (2010)

suggested that changes in the Amazonian landscape in response to the Andes uplift

could have favored these ancient links. Batalha-Filho et al., (2013). described this

connection during Miocene for birds, but our divergence time estimates were older

(Eocene/Oligocene) presenting a similar pattern with other frogs (i.e De Sá et al.,

2018). Our results contrast with the other two historic connections, along the northern

coast and through current Central Caatinga, called ‘‘young pathway’’ by Batalha-Filho

et al., (2013). Palynological data from Late Pleistocene (10.9–10.5 Ma) of central

Caatinga detected evidences of taxa currently found in Amazonian and Atlantic

Forest, indicating a forest vegetation there along the last glacial cycle (Colinvaux et

al., 2000; Prates et al., 2017).

Our analysis recovers a late Oligocene vicariance between ancestral of

Amazophrynella and Dendrophryniscus. Previously studies suggest climatic

modifications during Eocene and Oligocene that propitiated patches of open

vegetation and changes in floristic composition (Crisci et al., 1991; Jaramillo, 2006,

Grahan, 2010), thus promoting the origin and expansion of a drier savanna

vegetation (Werneck, 2011). These events prevent a long period (44.0-35.0 Ma-

199

Figure 3A) of biotic interchange between Amazonian and Atlantic forest (Batalha-

Filho et al., 2013; Capurucho et al., 2018; Costa, 2003), likely caused ancient

vicariance of forest dwelling amphibians. Generalist species that tolerated dry

environmental conditions could disperse in both wet-forest, as Dendropsophus gr.

minutus, Syncope and Chiasmocleis (Gehara et al., 2014; Sá et al., 2018). However,

Amazophrynella and Dendrophryniscus fail to disperse during forest reconnections

because gallery forest in dry areas must remained unsuitable for ancestral, that

limited their distribution inside areas that presented original or similar niche

conditions that ancestral area, exemplifying as a case of ancestral niche

conservationism.

Initial diversification of Amazophrynella are according with others studies that

pre- date Neogene (Castroviejo-Fisher et al., 2014; Kok et al., 2017; Santos et al.,

2009; Van Bocxlaer et al., 2010). Discrete and continuous models showed a

widespread ancestor in Amazonia, that gradually dispersed from central lowland

Amazonian forests to Guyana and Brazilian Shields, and them diversified during the

last 15.0–10.0 Ma in middle and late Miocene. These diversification scenario follows

the end of marine incursions, high levels of Andean sedimentation and establishment

of modern transcontinental Amazon drainage system (Antonelli et al., 2010; Hoorn et

al., 2010; Rull, 2011). Western Amazonian dispersal routes have most likely occurred

because suitable habitats conditions were available across a wider region after of

marine incursions retractions in the last 10.0–5.0 Ma.

Phylogeographic breaks in eastern vs. western in Amazonia have been widely

detected at different taxonomic levels, including butterflies (Hall and Harvey, 2002);

lizards (Gamble et al., 2008; Glor et al., 2001; Kronauer et al., 2005; Miralles and

Carranza, 2010); frogs (Garda and Cannatella, 2007; Lougheed et al., 1999; Noonan

and Wray, 2006; Symula et al., 2003), birds (Bates et al., 1998); fishes (Farias and

Hrbek, 2008) and mammals (da Silva and Patton, 1993; Steiner and Catzeflis,

2004). Our biogeographic reconstructions suggest an early Miocene (20-15.0 Ma)

east-west divergence. These results are older than previously reported by other

Amazonian taxa as lizards (Gamble et al., 2008; Miralles and Carranza, 2010;

Pellegrino et al., 2011); birds (Eberhard and Bermingham, 2005) and mammals (da

200

Silva and Patton, 1993), but match with time split with other amphibians (i.e. Garda

and Cannatella, 2007; Noonan and Wray, 2006).

Subsequent diversification events in Amazophrynella take place before

Pleistocene dry periods at 5.0-1.0 Mya. Climatic warmer Miocene and the relatively

cooler Pliocene could have favored the high levels of dispersion in lineages. A similar

time estimates diversifications patterns were similar in other species of frogs as

Dendropsophus gr. minutus (Gehara et al., 2014); Rhinella gr. marina (Maciel et al.,

2010); Adenomera gr. andreae (Fouquet et al., 2014); Ranitomeya spp. (Santos et

al., 2009)and Centrolenidae spp. (Castroviejo-Fisher et al., 2014).

Because we coded the presence of taxa in the Amazon region as binary

characters, we ignored distinct distributions within this region. However, is also

apparent that the observed lineages our study are geographically structured, and the

distribution of Amazonian lineages seems to correspond in large part to the proposed

areas of Amazonian endemism (Cracraft 1985; Borges & da Silva 2012). Our

likelihood analysis of discrete biogeographic reconstruction suggested Inambari,

Guyana and Napo as most ancestral areas in Amazonia and a recent colonization in

Tapajos and Imeri. Another striking biogeographic pattern is that one representant of

Inambari (A. aff. vote sp.2) were placed into the Guyana shield clade (Figure 2). This

pattern can be attributed to a long dispersion event (Matzke, 2012); hybridization

and/or the retention of ancestral polymorphic alleles commonly detected in frogs at

genomic scales (Fouquet et al., 2018).

In addition, assuming that extant geographical distributions reflect ancestral

occurrences, it is tempting to suggest that basal east/west diversification in

Amazophrynella took place after populations moved across structural arch (i.e. Purus

arch), or even across marine incursions, and south/north diversification after the

Amazonas river establishment (c. 9.0 Ma -(Hoorn et al., 2017). Future studies should

take into account known areas of endemism for Amazonian amphibians to

investigate the potential impact of historic (i.e. structural arch-Purus arch, marine

incursions, Pleistocene forest fragmentation) and ecological niche evolution in

diversification of Amazonian frogs.

201

Acknowledgements

We thank the people of the community of Taracuá, especially Sr. Maximiliano Correia

Menezes (representative of the Amerindian village of Taracuá and FOIRN), Sr.

Gabriel Correia Menezes, Sr. João Filho Menezes and Sra. Vera Correia for

hospitality and logistic support in Taracuá. We thank Junior Menezes, Max Junior

Menezes, and Bene Menezes for field support. Federação de Organizações

Indígenas do Rio Negro/FOIRN allowed access to Ameridian lands. Marcia Lima de

Queiroz and Miss Lana helped in the INPA. Mario Nunez helped with the molecular

analyses. Funding for this work came from CNPq/SISBIOTA Processo No.

563348/2010-0 and SISBIOTA/FAPEAM. Coordenação de Aperfeiçoamento de

Pessoal de Nível Superior (CAPES) provided financial support and awarded a PhD

fellowship to R. R. R. This work is part of R.R.R.’s PhD Thesis in Genetics,

conservation and evolutionary biology program of INPA. AF has benefited from an

“Investissement d’Avenir” grant managed by the Agence Nationale de la Recherche

(CEBA, ref. ANR-10- LABX-25- 01). RWA thanks CNPq for his productivity research

grant (303622/2015-6). For funding for specimens collection and laboratory work

SRR thanks SENESCYT, Arca de Noe initiative.

202

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Table 1. Log Likelihood scores for each model of biogeographic dispersal run in

BioGeoBEARS (Matzke, 2013) for genomic marked. Note that the DIVALIKE+ J had

the highest log likelihood score and was therefore chosen as the model that

explained the data best.

Model LnL d e j AIC AIC_wt

DEC -36.38 0.003 0.005 0 76.75 4.8e-05

DEC+J -27.09 1.0e-12 6.3e-10 0.054 60.19 0.19

DIVALIKE -31.85 0.003 1.0e-12 0 67.70 0.0045

DIVALIKE+J -25.68 1.0e-12 1.0e-12 0.049 57.35 0.79

BAYAREALIKE -46.38 0.005 0.081 0 96.75 2.2e-09

BAYAREALIKE+J -29.64 1.0e-07 1.0e-07 0.06 65.28 0.015

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Table 2. Log‐likelihood and AIC values of the six diversification models fitted to the

branching times derived from the Bayesian maximum clade credibility chronogram of

Amazophrynella.

Model LogL AICc AICw

Expanding diversity; constant speciation rate -52.679 107.624 0.416

Expanding diversity; exponential variation in speciation rate

-51.388 107.634 0.484

Expanding diversity; linear variation in speciation rate

-54.253 113.365 0.023

Expanding diversity; constant speciation and extintion rate

-52.665 113.176 0.093

Expanding diversity; exponential variation in speciation rate and constant extinction

-52.388 110.623 0.025

Expanding diversity; linear variation in speciation rate and constant extinction

-52.665 113.176 0.024

213

Figure 1. Graphical representation of the historical biogeography of Amazophrynella.

A) Map delimiting the distribution of each geographic area assigned to each lineage

in the BioGeoBEARS analysis. (B) Bayesian time-calibrate from dd Rad´s genomic

topology. Ages/posterior probability are indicated alongside on the upper and below

of the nodes. The 95% confidence intervals for nodes are highlighted by horizontal

light blue bars. Colored squared indicate the ancestral area and biogeographic

events.

214

Figure 2. Semi-logarithmic lineage-through-time (LTT) plot depicting diversification

patterns of Amazophrynella. LTT plots obtained via penalized likelihood (PL) and

exponential curve of best model of diversification are superimposed.

215

Figure 3. Phylogeographic analysis of Amazophrynella using continuous Relaxed Random

Walks (RRW) based on mt DNA and genomic dataset. Red polygons indicate potential area

of occupancy of the MRCA. (A) Ancestral connection between Amazophrynella and

Dendrophryniscus; (B) ancestral of Amazophrynella in Amazonia; (C-D) east - west

expansion; (E-F) north-south expansion; (G) final diversification in Amazophrynella. Map

were generated using Google Earth (earth.google.com).

216

CONCLUSÕES GERAIS

❖ A diversidade de espécies do gênero Amazophrynella se encontrava subestimada.

Através do uso de diferentes linhas de evidências evolutivas propomos a hipóteses

filogenética de ampla abrangência geográfica para gênero e confirmamos a

existência de onze espécies validas, sendo que outras linhagens ainda não foram

confirmadas. A integração de diversos caracteres evolutivos auxilia em manter uma

taxonomia estável, auxiliar nas decisões taxonômicas e delimitações de espécies. No

entanto, ainda existe uma grande diversidade de espécies escondida dentro do

gênero Amazophrynella.

❖ A história biogeográfica de Amazophrynella indica uma quebra populacional entre

Amazônia e Mata Atlântica no Eoceno, uma divisão basal entre o Leste-Oeste da

Amazônia no Oligoceno e uma quebra norte-sul no Mioceno. Nossas reconstruções

ancestrais sugerem um ancestral amplamente distribuído dividido por mecanismos

de vicariância. A divergência entre Amazônia e Mata atlântica foi provavelmente

influenciado pelo levantamento inicial dos Andes que ocasionaram mudanças

ambientais e períodos secos dentro da antiga floresta continua Sul-Americana. Na

Amazônia, diversos eventos geológicos como a elevação do Arco do Purus e

incursões marinhas poderiam ter ocasionado o padrão leste e oeste, no entanto

nossos tempos de divergência não são completamente sincronizados com as

predições de estas hipóteses. Assim mesmo, a divisão entre o clados do norte e sul

não foi ocasionado pela formação do rio Amazonas e poderia representar uma

pseudo- congruência filogenética. Fatores ecológicos e especialização do nicho

ecológico, assim como forças neutras ocuparam um papel principal na diversificação

e dispersão de Amazophrynella na Amazônia.

(anura: bufonidae): uma anÁlise integrativa para …

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