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Instituto Nacional de Pesquisas da Amazônia – INPA
Programa de Pós-Graduação em Genética, Conservação
e Biologia Evolutiva
Filogenia e biogeografia de três famílias de aves do
Neotrópico
Mateus Ferreira
Manaus, Amazonas
Fevereiro, 2018
Mateus Ferreira
Filogenia e biogeografia de três famílias de aves do
Neotrópico
Orientador: Dra. Camila Cherem Ribas
Tese apresentada ao Instituto Nacional de
Pesquisas da Amazônia como requisito para
obtenção do grau de doutor em Genética,
Conservação e Biologia Evolutiva.
Manaus, Amazonas
Fevereiro, 2018
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Folha reservada para a banca julgadora da versão final da tese 1
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Ficha Catalográfica 3
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Agradecimentos 5
Agradeço primeiramente a minha orientadora Camila Ribas, pela paciência e confiança 6
que depositou em mim durante esses anos de orientação. Sem sombra de dúvidas, esse trabalho 7
não seria possível sem essa amizade e parceria. 8
Ao meu co-orientador, Joel Cracraft, com quem tive a sorte de trabalhar durante o meu 9
doutorado sanduíche. Pelas excelentes conversas e orientações sobre biogeografia e sobre os 10
padrões e processos que moldaram a diversidade de aves no mundo. 11
À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) e ao 12
programa de Pós-Graduação em Genética, Conservação e Biologia Evolutiva, do Instituto 13
Nacional de Pesquisas da Amazônia, pela concessão da bolsa de doutorado no país e bolsa 14
sanduíche (# 88881.133440/2016-01), que tornaram este projeto possível. 15
Aos curadores e responsáveis pelas coleções científicas que gentilmente cederam 16
material para que este trabalho fosse desenvolvido: Fátima Lima e Antonita Santana (MPEG); 17
Marlene Freitas (INPA); Nate Rice (ANSP), Cristina Miyaki (LGEMA), Donna Dittman e 18
Robb Brumfield (LSU), Paul Sweet e Tom Trombone (AMNH), Mark Robbins (KU), John 19
Bates e Ben Marks (FMNH), Brian K. Schmidt (USNM), Sharon Birks (UWBM). E, a todas as 20
pessoas envolvidas nas expedições de coleta dessas institutições. 21
Ao projeto “Dimensions US-Biota: Assembly and evolution of the Amazon biota and 22
its environment: an integrated approach”, um projeto financiado conjuntamente pela Fundação 23
de Amparo à Pesquisa de São Paulo (FAPESP #2012/50260-6) e pelo National Science 24
Fundation (NSF DEB 1241056). Cujo apoio e financiamento foram essenciais para a execução 25
das várias etapas desse doutorado. 26
A todos os colegas do EBBA, pela constante ajuda e pelas excelentes discussões e 27
incentivos, e pelo café, especialmente pelos cafés: Robs, Fernanda, Rafael, Claudinha, Érico, 28
Erik, Lídia, Renatinha, Jessica, Nelson, Carol, Waleskinha e todo mundo que passou por aqui. 29
Ao pessoal que me aguentou durante esse doutorado: Maricota, Leandro, Marina, Ana, 30
Marizita, Derek, Miquéias, Pedro, Cadu e Manu. Em especial à Romina, pela caminhada lado 31
a lado durante toda a execução desse projeto, pelos puxões de orelha quando eu precisei e por 32
ter me aguentado todo esse período. 33
Ao pessoal do LTBM, Giselle e Paula, pela excelente companhia, pelos cafés e ajudas 34
quando precisei. 35
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To everyone who received me at the AMNH during my sandwich fellowship: Lydia, 36
Bill, Tom, Paul, Gabi, Brian, Luke, and Peter. A special thanks to Jessica and Laís for all the 37
support and friendship during my time in NY. 38
Também gostaria de agradecer ao Laboratório Nacional de Computação Científica 39
(LNCC/MCTI) por fornecer recursos de computação de alto desempenho através do 40
supercomputador SDumont, fundamentais para as análises realizadas neste estudo. 41
Por fim, um agradecimento especial para a minha família, que me apoiou 42
incondicionalmente em todo esse percurso, e cuja ajuda foi essencial para a finalização deste 43
doutorado. 44
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“Nothing in biology makes sense except in the light of evolution” 65
Theodosius Dobzhansky 66
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“Life and Earth evolve together” 68
Leon Croizat 69
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Resumo 72
O Neotrópico é uma das regiões com os maiores índices de biodiversidade do planeta e muito 73
tem se questionado sobre a origem de tamanha diversidade. Acredita-se que os padrões de 74
diversidade atual dentro da região sejam um resultado da complexa história geomorfológica e 75
climática da região. Entre os eventos geomorfológicos mais discutidos estão o soerguimento 76
dos Andes e consequente reestruturação da drenagem continental, e o fechamento do Istmo do 77
Panamá, que permitiu a troca intercontinental de biotas. Neste trabalho foram selecionadas três 78
famílias de aves do Neotrópico. A família Trogonidae tem uma distribuição Pantropical, 79
ocorrendo também nas regiões subtropicais e tropicais da África e Ásia, no entanto, a maior 80
diversidade encontra-se justamente na região Neotropical. As famílias Bucconidae e Galbulidae 81
são duas famílias irmãs endêmicas do Neotrópico. Foram selecionadas amostras de todas as 82
espécies e quase todas as subespécies descritas para os três grupos. Para as espécies amplamente 83
distribuídas foram selecionadas amostras ao longo de toda a distribuição e uma análise prévia 84
para verificar a estrutura filogeográfica de cada grupo, com base nesses resultados, foram 85
selecionadas amostras para o sequenciamento de milhares de loci de regiões Ultra Conservadas 86
(Ultraconserved Elements, UCE). Dessa forma, compilamos três estudos nessa tese. No 87
primeiro capítulo, foi estudado um complexo de aves da família Galbulidae associada aos 88
ambientes de areia branca na região Amazônica. Através da comparação entre marcadores 89
moleculares com diferentes métodos de herança, DNA mitocondrial e nuclear (UCE), pudemos 90
observar um conflito entre esses dois marcadores. Através deste conflito foi possível propor um 91
modelo de diversificação para os ambientes de areia branca na região. No segundo capítulo 92
analisamos a diversificação global da família Trogonidae, com o auxílio dos UCEs 93
reconstruímos a relação filogenética entre todas as espécies da família e estimamos uma árvore 94
datada da diversificação de Trogonidae. No terceiro e último capítulo, analisamos os padrões 95
de diversificação das famílias Galbulidae e Bucconidae através de uma abordagem 96
filogeográfica e filogenética. Neste trabalho pudemos observar que a diversidade do grupo se 97
encontra claramente subestimada. 98
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Abstract 102
The Neotropical region has one of the highest biodiversity index in the planet and several 103
hypotheses have been proposed to explain the origin of such diversity. Currently, landscape 104
and climatic evolution are credited to be the two main processes responsible for shaping the 105
patterns. Landscape evolution includes, for example, the Andean uplift and consequent 106
continental drainage reconfiguration, and the closure of the Isthmus of Panama, which 107
allowed the Great American Biotic Interchange. In the present study we selected three 108
Neotropical families of birds. Trogonidae has a Pantropical distribution, members of this 109
family inhabit tropical and subtropical regions of Africa, Asia, however, the highest diversity 110
is currently found in the Americas. Galbulidae and Bucconidae are sister families and 111
endemics to the Neotropics. WE sampled all species and almost all subspecies currently 112
recognized for this three families, and for widespread species we thoroughly sampled 113
throughout their distributions to uncover hidden phylogeographic patterns. Based on these 114
results, we selected the samples to sequence thousands of Ultraconserved Elements (UCE). 115
Thus, we compiled three studies for this thesis. In the first chapter, we studied one Galbulidae 116
species complex associated with the Amazonian White-sand environments. We compared 117
between molecular markers that have different heritage systems, the mtDNA and nuDNA 118
(UCE), where we recovered contrasting histories between markers, and based on these results 119
we proposed a diversification model for the White-sand environments. In the second chapter, 120
we analyzed the global diversification of Trogonidae, employing thousands of UCE loci to 121
propose a phylogenetic hypothesis between all species currently recognized, and we also 122
estimated a fossil calibrated time tree for Trogonidae diversification. At last, in the third 123
chapter, we analyzed the diversification patterns for Galbulidae and Bucconidae using a 124
phylogeographic/phylogenetic approach. In this chapter it was clear how these groups 125
diversity in underestimated by currently taxonomic approach. 126
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Sumário 128
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Agradecimentos .................................................................................................. iv 130
Resumo ............................................................................................................... vii 131
Abstract ............................................................................................................. viii 132
Introdução Geral ................................................................................................. 1 133
Objetivos............................................................................................................... 7 134
Capítulo 1 ............................................................................................................. 8 135
Abstract ............................................................................................................................................ 10 136
1. Introduction ............................................................................................................................. 11 137
2. Methods .................................................................................................................................... 13 138
2.1. Taxon sampling ................................................................................................................. 13 139
2.2. DNA extraction, amplification and sequencing................................................................. 14 140
3. Results ...................................................................................................................................... 17 141
3.1. Sanger sequencing and haplotype networks ..................................................................... 17 142
3.2. mtDNA genome and time tree ........................................................................................... 18 143
3.3. UCE sequencing, RAxML and Species trees ..................................................................... 18 144
4. Discussion ................................................................................................................................. 19 145
4.1. mtDNA and nuDNA incongruence .................................................................................... 19 146
4.2. Biogeography of WSE avifauna ........................................................................................ 23 147
4.3. Evolution in the White-sand environments ........................................................................ 24 148
5. Conclusion ................................................................................................................................ 26 149
Acknowledgements .......................................................................................................................... 27 150
Funding ............................................................................................................................................ 27 151
References ........................................................................................................................................ 28 152
Capítulo 2 ........................................................................................................... 38 153
Abstract ............................................................................................................................................ 39 154
Introduction ..................................................................................................................................... 40 155
Results .............................................................................................................................................. 43 156
UCE sequencing........................................................................................................................... 43 157
Phylogenetic inference ................................................................................................................ 43 158
Time-calibrated tree .................................................................................................................... 44 159
Discussion ......................................................................................................................................... 44 160
Phylogenomic contribution to the reconstruction of Trogonidae diversification .................. 44 161
Diversification and biogeography of Trogons ........................................................................... 46 162
Africa and Asia diversification ................................................................................................... 48 163
Neotropical diversification ......................................................................................................... 50 164
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Conclusion ........................................................................................................................................ 52 165
Materials and Methods ................................................................................................................... 53 166
Taxon sampling and DNA extraction ........................................................................................ 53 167
UCE and exons assembly ............................................................................................................ 53 168
Phylogenetic relationships and species tree analysis ................................................................ 54 169
Dating analysis ............................................................................................................................. 55 170
Acknowledgements .......................................................................................................................... 55 171
References ........................................................................................................................................ 56 172
Capítulo 3 ........................................................................................................... 71 173
Abstract ............................................................................................................................................ 72 174
Introduction ..................................................................................................................................... 73 175
Material and Methods ..................................................................................................................... 75 176
Sampling and DNA isolation ......................................................................................................... 75 177
Phylogeographic structure and UCE sampling ............................................................................ 76 178
UCE assembly ............................................................................................................................... 76 179
Phylogenetic relationship .............................................................................................................. 77 180
Results .............................................................................................................................................. 77 181
Phylogeographic results ................................................................................................................ 77 182
UCE sequencing ............................................................................................................................ 78 183
Phylogenetic results ...................................................................................................................... 78 184
Discussion ......................................................................................................................................... 79 185
Phylogenetic results ...................................................................................................................... 79 186
Galbulidae systematics .................................................................................................................. 80 187
Bucconidae systematics ................................................................................................................. 83 188
Conclusion ........................................................................................................................................ 88 189
Acknowledgements .......................................................................................................................... 88 190
References ........................................................................................................................................ 89 191
Síntese Geral .................................................................................................... 107 192
Referências Bibliográficas .............................................................................. 108 193 194
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Introdução Geral 200
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O Neotrópico é uma das regiões biogeográficas com uma das maiores biodiversidades 202
do mundo (Jetz et al., 2012; Holt et al., 2013), mesmo que uma grande parcela dessa diversidade 203
ainda seja desconhecida (Kier et al., 2005; Hopkins, 2007; Barrowclough et al., 2016). Na 204
região Neotropical, os biomas Mata Atlântica, Cerrado e Amazônia despontam como hotspots 205
de biodiversidade altamente ameaçados pela ação humana (Myers et al., 2000; Mittermeier et 206
al., 2003; Colombo e Joly, 2010). Em especial para a região Amazônica, que abrange mais de 207
40% da área total do Neotrópico, desde que Wallace (1852), fez suas primeiras observações 208
sobre a importância dos rios na delimitação da distribuição de diferentes espécies de primatas, 209
vários trabalhos foram realizados demonstrando a importância dos afluentes do rio Amazonas 210
na estruturação da diversidade alfa da região (Vanzolini e Willians, 1970; Cracraft, 1985; 211
Haffer, 1985; Ávila-Pires, 1995). A comparação e aparente congruência dos padrões de 212
distribuição geográfica permitiu a elaboração de algumas hipóteses sobre quais processos 213
poderiam ter dado origem a esses padrões (revisões em Haffer (1997) e Leite e Rogers (2013)), 214
incluindo as variações climáticas do Pleistoceno, em especial o Último Máximo Glacial (LGM 215
– Last Glacial Maximum, ca. 20.000 anos) (Haffer, 1969; Brown et al., 1974); a influência das 216
incursões marinhas (Nores, 1999; 2004); e a formação dos rios da bacia Amazônica (Bates et 217
al., 2004; Ribas et al., 2012). Contudo, essas hipóteses foram formuladas com base apenas na 218
distribuição geográfica dos táxons, com o advento da filogeografia (Avise et al., 1987; Avise, 219
2009) e técnicas de datação molecular (Bromham e Penny, 2003; Bromham et al., 2017) novas 220
teorias foram propostas e além da congruência entra a distribuição geográfica o tempo de 221
diversificação também passou a fazer parte da comparações (Donoghue e Moore, 2003). Como 222
consequência, a teoria dos refúgios associados aos eventos climáticos do LGM foi parcialmente 223
rejeitada, já que as espécies se mostraram mais antigas que os ciclos glaciais mais recentes 224
(Colinvaux et al., 2000; Bush e Oliveira, 2006). As incursões marinhas, por outro lado, seriam 225
muito antigas para explicar a origem das espécies (Hoorn, 1993), favorecendo o modelo da 226
hipótese dos rios como barreira. 227
Atualmente, no entanto, o que sabemos sobre a complexidade da diversidade Amazônica 228
sugere que mais de um processo está por trás de sua origem (Bush, 1995; Bates et al., 2008; 229
Smith et al., 2014). Todos os eventos que moldaram a paisagem do Neotrópico ao longo do 230
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tempo podem ter influenciado a diversificação da biota, por exemplo: A) o fim do “isolamento 231
esplêndido” (Simpson, 1980; Dacosta e Klicka, 2008) após o estabelecimento do Istmo do 232
Panamá (Haug e Tiedeman, 1998; Coates e Stallard, 2013; Lessios, 2015; Odea et al., 2016). 233
B) O soerguimento da cadeia de montanha dos Andes (Garzione et al., 2008; Hoorn et al., 2010; 234
Horton, 2018), que influenciou drasticamente não só a drenagem da bacia Amazônica (Hoorn 235
e Wesselingh, 2010; Latrubesse et al., 2010; Shephard et al., 2010; Nogueira et al., 2013; Hoorn 236
et al., 2017), como também o clima de todo o continente (Hartley, 2003; Ehlers e Poulsen, 237
2009; Insel et al., 2009). C) A influência das flutuações climáticas do Pleistoceno também 238
voltou a fazer parte das discussões, especialmente com relação ao estabelecimento de diferentes 239
regimes de precipitação dentro do continente (Cheng et al., 2013; Wang et al., 2017). 240
Dessa forma, faz-se necessário investigar não somente a evolução do modelo através das 241
variáveis biológicas, mas também quais processos físicos podem ter influenciado a sua 242
diversificação (Baker et al., 2014). Por exemplo, o estabelecimento do atual curso 243
transcontinental do rio Amazonas, ainda bastante discutido na literatura, varia entre o final do 244
Mioceno (10 – 7 Ma) (Hoorn e Wesselingh, 2010; Hoorn et al., 2017), início do Plioceno (~5 245
Ma) (Latrubesse et al., 2010), ou ainda, durante o Pleistoceno (2,5 Ma) (Nogueira et al., 2013; 246
Rossetti et al., 2015). Nesse sentido, estudando um gênero de aves (Psophiidae: Psophia) que 247
é restrita aos ambientes de terra-firme, e dessa forma suscetível às mudanças na drenagem da 248
Amazônia, Ribas et al. (2012) propuseram um modelo de diversificação da fauna de terra firme 249
ao correlacionar os eventos de diversificação das espécies do gênero ao estabelecimento de 250
barreiras associadas aos principais afluentes da bacia, favorecendo o modelo do 251
estabelecimento do rio Amazonas durante o Pleistoceno. O modelo proposto por Ribas et al. 252
(2012) sugere que para compreender os fatores que influenciaram a evolução da paisagem, 253
como o efeito da formação de um determinado rio na diversificação de espécies de terra-firme, 254
deve-se buscar padrões congruentes, temporais e espaciais, de diversificação em grupos que 255
serão de fato afetados diretamente pela barreira (e.g. Polo, (2015)). Em contraponto, análises 256
que buscam explicar a diversificação na Amazônia através de um único processo, como por 257
exemplo, a importância dos rios como barreira utilizando uma ampla gama de táxons com 258
nichos variados (Oliveira et al., 2017; Santorelli et al., 2018; Smith et al., 2014) tendem a 259
refutar esta teoria, já que diferentes organismos respondem de diferentes maneiras aos 260
processos e eventos históricos. Dessa forma, aceitando que a diversificação na Amazônia é 261
inerentemente complexa, o teste de hipóteses deve ser feito de maneira dirigida, ou seja, deve-262
se buscar grupos taxonômicos que tenham sido potencialmente influenciados pelas barreiras 263
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em questão. Só assim será possível estabelecer a importância biológica de um determinado 264
evento e gerar dados importantes para o estabelecimento dos modelos de evolução 265
geomorfológica da região (Baker et al., 2014). 266
Essa iluminação recíproca entre os processos físicos e bióticos, no entanto, só é possível 267
levando em consideração o fato de que qualquer evento de diversificação só pode ser 268
correlacionado com um evento biogeográfico se duas condições forem respeitadas: 1) as 269
unidades biológicas utilizadas devem ser comparáveis entre si e devem representar linhagens 270
com uma história evolutiva única; 2) a relação filogenética entre essas linhagens deve 271
representar de fato a história de diversificação do grupo. 272
A primeira condição refere-se ao fato de que as unidades utilizadas no estudo devem 273
representar linhagens independentes. Geralmente, entende-se que espécies devem ser a unidade 274
básica para qualquer estudo de ecologia, evolução, ou biogeografia, no entanto, essa prática 275
pode ser particularmente problemática na Amazônia, uma vez que grande parte das espécies 276
amplamente distribuídas pela região na realidade representam um complexo de linhagens 277
evolutivas independentes (Ribas et al., 2012; D’horta et al., 2013; Fernandes et al., 2013; 278
Fernandes et al., 2014; Hrbek et al., 2014; Boubli et al., 2015; Thom e Aleixo, 2015; Byrne et 279
al., 2016; Carneiro et al., 2016; Boubli et al., 2017; Ferreira et al., 2017; Lima et al., 2017; 280
Ribas et al., 2018). Para aves, em particular, esse déficit entre a taxonomia atualmente 281
reconhecida e a real diversidade está diretamente relacionado ao fato de que a definição daquilo 282
que reconhecemos como espécie ainda é muito influenciado pelo tipo de conceito de espécie 283
utilizado, em especial o conceito biológico de espécie (Mayr, 1942), que implica no 284
reconhecimento de metapopulações isoladas reprodutivamente. No entanto, o reconhecimento 285
de isolamento reprodutivo em populações naturais é particularmente difícil, especialmente em 286
populações alopátricas, onde é impossível observar naturalmente esse contato. Mesmo em 287
populações parapátricas, o contato e estabelecimento de uma zona híbrida não necessariamente 288
ameaça o statu quo das espécies envolvidas (Weir et al., 2015). Especialmente, porque a 289
capacidade de hibridização entre espécies, mesmo distantes, parece ser uma característica 290
sinapomórfica para aves (Grant e Grant, 1992; Gill, 1998; Harrison e Larson, 2014). 291
O conceito de espécie, mesmo sendo um dos assunto centrais para os estudo de evolução 292
e ecologia, permanece ainda sem definição clara e é sem dúvida um dos pontos mais discutidos 293
dentro da biologia (Mayr, 1976; Donoghue, 1985; Isaac et al., 2004; De Queiroz, 2005; Aleixo, 294
2007; Joseph et al., 2008; Aleixo, 2009; De Queiroz, 2011; Cellinese et al., 2012; De Queiroz, 295
2012; Willis, 2017). Ressaltando o impacto dessa escolha entre um conceito ou outro e do nosso 296
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atual conhecimento sobre a taxonomia de aves, um estudo recente demonstrou que a diversidade 297
das aves é, pelo menos, duas vezes maior do que a atualmente reconhecida (Barrowclough et 298
al., 2016). Por exemplo, dentro da Neotrópico, um dos padrões mais observados é a existência 299
de espécies amplamente distribuídas, compostas por diferentes subespécies morfologicamente 300
distintas e geograficamente estruturadas, as quais foram, no entanto, agrupadas dentro de uma 301
mesma espécie devido a existência da possibilidade dessas populações hibridizarem caso 302
entrem em contato. 303
A segunda condição está relacionada aos problemas de conflitos entre a história de um 304
único gene e a história da espécie (Degnan e Rosenberg, 2009; Knowles, 2009). Esse conflito 305
tem se tornado cada vez mais evidente em face do desenvolvimento de técnicas de 306
sequenciamento massivo em paralelo (Metzker, 2010). Apesar de estarem se tornando mais 307
acessíveis, o sequenciamento e análise do genoma completo para organismos não modelo ainda 308
é impraticável para trabalhos que requerem amostragem de muitos indivíduos. Dessa forma, 309
algumas técnicas de se utilizar representações reduzidas foram desenvolvidas. Duas abordagens 310
dominam o cenário atualmente, uma delas é a utilização de enzimas de restrição para sítios 311
específicos ao longo de todo o genoma, denominada RAD-seq (restriction-site-associated DNA 312
sequencing) (Davey et al., 2011); e a outra é a utilização de sondas de RNA desenvolvidas para 313
capturar regiões específicas do genoma (Grover et al., 2012; Lemmon et al., 2012; Lemmon e 314
Lemmon, 2013). Uma das abordagens de sequenciamento de captura é a técnica que utiliza 315
sondas específicas para regiões do genoma ultra conservadas (do inglês, Ultra Conserved 316
Elements, UCE) (Faircloth et al., 2012). Essas regiões ultra conservadas foram selecionadas 317
pois permitem a utilização de um mesmo conjunto de sondas para realizar estudos em diversos 318
níveis taxonômicos, pois apesar das regiões centrais serem altamente conservadas, as regiões 319
flanqueadoras possuem variação suficiente tanto para recuperar relações mais antigas 320
(Mccormack et al., 2012; Crawford et al., 2015; Faircloth et al., 2015), quanto mais recentes 321
(Bryson et al., 2016; Manthey et al., 2016), inclusive utilizadas em radiações adaptativas 322
(Meiklejohn et al., 2016), onde altos níveis de separação incompleta de linhagens (do inglês, 323
Incomplete Lineage Sorting, ILS) sejam esperados (Degnan e Rosenberg, 2006; Oliver, 2013). 324
De modo a tentar então lançar alguma luz sobre os possíveis eventos que moldaram a 325
diversificação da biota Neotropical, foram selecionadas três famílias de aves: Trogonidae, 326
Galbulidae e Bucconidae. As três famílias possuem representantes por toda a região 327
Neotropical, incluindo várias espécies, ou grupo de espécies, amplamente distribuídas. A 328
família Trogonidae tem distribuição Pantropical, estando ausente apenas da região Australiana. 329
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Representantes dessa família, popularmente conhecidos como surucuás, são aves de médio 330
porte e sua dieta varia entre insetívora e onívora, apresentam plumagem com coloração bastante 331
chamativa, e são reconhecidas por serem más dispersoras, não sendo capazes de realizar voos 332
de longa distância (Collar, 2017). Apesar de apresentarem plumagem bastante distinta, a 333
morfologia interna da família é bastante conservada e a sua monofilia nunca foi questionada 334
(Livezey e Zusi, 2007; Collar, 2017). No entanto, a relação entre trogonídeos e outras aves não 335
passeriformes já foi bastante controversa (Cracraft, 1981; Maurer e Raikow, 1981; Monteros, 336
2000; Mayr, 2003; Livezey e Zusi, 2006). Atualmente, aceita-se que a família seja uma das 337
primeiras linhagens a diversificar dentro da radiação de Coracimorphae sendo grupo irmão de 338
todas as outras famílias do grupo Core Landbirds (Jarvis et al., 2015; Prum et al., 2015). 339
Atualmente são reconhecidas 45 espécies (Collar, 2017; Gill e Donsker, 2018; Ramsen et al., 340
2018) distribuídas em sete gêneros. A região Neotropical contém a maior diversidade da 341
família, com quatro gêneros e cerca de 30 espécies. A região Asiática contém dois gêneros e 12 342
espécies, e por último, a região Africana, possui um gênero com três espécies. Apesar da maior 343
diversidade da família ser encontrada no Neotrópico, a existência de fósseis na Europa (Mayr, 344
1999; Kristoffersen, 2002; Mayr, 2005) sugere uma origem no Paleártico e posterior dispersão 345
e colonização da distribuição atual. Diversos trabalhos já tentaram abordar a relação 346
filogenética entre os representantes da família (Monteros, 1998; Johansson e Ericson, 2005; 347
Moyle, 2005; Dacosta e Klicka, 2008; Ornelas et al., 2009; Hosner et al., 2010), entretanto, 348
nenhum foi capaz de resolver a relação entre os gêneros. O último trabalho publicado (Hosner 349
et al., 2010), e o único a incluir representantes de todos os gêneros, recuperou uma parafilia 350
entre regiões geográficas, sugerindo um cenário biogeográfico bem mais complexo, em que a 351
região Neotropical, por exemplo, tenha sido ocupada por pelo menos três linhagens distintas. 352
As famílias Galbulidae e Bucconidae formam um clado já bem estabelecido, tanto com 353
caracteres morfológicos (Livezey e Zusi, 2007), quanto dados moleculares (Hackett et al., 2008; 354
Prum et al., 2015). Dentro da ordem Piciformes, são as únicas famílias com representantes com 355
distribuição exclusivamente neotropical, formando o grupo irmão das outras famílias de 356
Piciformes (Prum et al., 2015). A família Galbulidae é composta por aves de pequeno a médio 357
porte, asas arredondadas e um bico longo e afilado utilizado para capturar insetos durante o 358
voo. Possui 19 espécies distribuídas em cinco gêneros diferentes (Tobias, 2017; Gill e Donsker, 359
2018; Ramsen et al., 2018). As espécies da família são geralmente agrupadas em oito grupos 360
zoogeográficos, seis desses grupos representam complexos de espécies com distribuições 361
alopátricas ou parapátricas, e dois são espécies amplamente distribuídas (Collar, 2017). A 362
6
família Bucconidae também inclui aves de pequeno a médio porte, asas curtas e arredondadas, 363
tendo como característica uma cabeça relativamente grande, atualmente são reconhecidas 35 364
espécies para a família distribuídas em nove gêneros (Gill e Donsker, 2018; Ramsen et al., 365
2018). Os trabalhos de filogeografia desenvolvidos com representantes da família Bucconidae 366
– Malacoptila (Ferreira et al., 2017), Monasa e Nonnula (Soares, 2016) e Nystalus (Duarte, 367
2015) – demonstraram que a diversidade reconhecida pela taxonomia tradicional para esses 368
grupos é subestimada, já que existem muito mais linhagens genéticas geograficamente isoladas 369
do que táxons reconhecidos, demonstrando a importância da condução dos estudos de 370
filogeografia para elucidar a delimitação taxônomica dessas espécies amplamente distribuídas. 371
Dessa forma, o presente trabalho tem por objetivo reconstruir a relação filogenética entre 372
todas as linhagens dessas três famílias de modo a reconstruir a história de diversificação desses 373
três grupos. Para tanto, foram amostrados indivíduos ao longo da distribuição de todas as 374
espécies amplamente distribuídas para uma análise prévia da estrutura genética de cada uma 375
dessas linhagens. Com base nos resultados obtidos previamente foram selecionadas amostras 376
representativas de cada um desses agrupamento, tentando incluir, sempre que possível, um 377
representante para cada um dos táxons reconhecidos. Para essas amostras foram sequenciados 378
mais de 2000 loci de UCE, e com base nessa representação reduzida do genoma foram 379
realizadas análises para a reconstrução filogenética dos grupos. 380
381
382
7
383
384
Objetivos 385
386
O objetivo geral foi investigar a história biogeográfica da região Neotropical com base 387
nas relações filogenéticas entre todos os táxons atualmente reconhecidos para as famílias 388
Trogonidae, Bucconidae e Galbulidae baseado em dados de sequenciamento genômico. Sendo 389
que para isso foi necessário: 390
Capítulo 1: revisar a taxonomia e compreender os processos de isolamento e fluxo 391
gênico em um contexto espacial; 392
Capítulo 2: compreender a origem de táxons Neotropicais em uma família amplamente 393
distribuída; 394
Capítulo 3: compreender a estrutura filogeográfica de espécies amplamente distribuídas 395
em duas famílias Neotropicais para com isso obter uma reconstrução filogenética representativa 396
da diversificação do grupo. 397
398
399
8
400
401
402
403
404
405
406
407
408
409
Capítulo 1 410
411
412
Ferreira, M.; Fernandes, A.M.; Aleixo, A.; Antonelli, 413
A.; Olsson, U.; Bates, J.M.; Cracraft, J.; Ribas, C.C. 414
Evidence for mtDNA capture in the jacamar Galbula 415
leucogastra / chalcothorax species-complex and 416
insights on the evolution of white-sand environments 417
in the Amazon basin. Molecular Phylogenetics and 418
Evolution (no prelo) 419
420
421
422
9
423
424
Manuscript submission to Molecular Phylogenetics and Evolution 425 Contribution type: Original article 426 427 Evidence for mtDNA capture in the jacamar Galbula leucogastra / chalcothorax species-428 complex and insights on the evolution of white-sand environments in the Amazon basin. 429
430 Ferreira, Mateusa*; Fernandes, Alexandre M.b; Aleixo, Alexandrec; Antonelli, Alexandred,e,f; 431 Olsson, Urban d,f; Bates, Jonh M.g; Cracraft, Joelh; Ribas, Camila C.i 432
433 a Programa de Pós-Graduação em Genética, Conservação e Biologia Evolutiva, INPA, 434 Manaus, AM, Brazil 435 b Unidade Acadêmica de Serra Talhada, UFRPE, Serra Talhada, PE, Brazil 436 c Coordenação de Zoologia, MPEG, Belém, PA, Brazil 437 d Department of Biological and Environmental Sciences, University of Gothenburg, SE-413 438 19 Gothenburg, Sweden 439 e Gothenburg Botanical Garden, SE-413 19 Gothenburg, Sweden 440 f Gothenburg Global Biodiversity Centre, Box 461, SE-405 30 Gothenburg, Sweden 441 gDepartment of Ornithology, FMNH, Chicago, IL, USA 442 h Department of Ornithology, AMNH, New York, NY, USA 443 i Coordenação de Biodiversidade, INPA, Manaus, AM, Brazil 444
*Corresponding author 445 446 Correspondence: Mateus Ferreira, Coordenação de Biodiversidade, Instituto Nacional de 447
Pesquisas da Amazônia, CEP 69080-971, Manaus-AM, Brazil 448 E-mail: [email protected] 449
450
10
Abstract 451
Jacamar species are found throughout Amazonia, with several different species occupying 452
forested habitats, but one species-complex, Galbula leucogastra / chalcothorax, inhabits areas 453
of open vegetation, known as white-sand environments (WSE). Previous studies of WSE birds 454
recovered shallow genetic structure in mtDNA coupled with signs of gene flow among WSE 455
areas. Here we characterize diversification of the G. leucogastra/chalcothorax species-complex 456
with dense sampling across its distribution, using mitochondrial DNA and Ultraconserved 457
Elements (UCE) loci. We performed likelihood and Bayesian analysis to recover the 458
phylogenetic relationships among populations using a concatenated approach, as well as a 459
species-tree analysis using *BEAST. The mtDNA results recovered at least six geographically-460
structured lineages in which G. chalcothorax was embedded within lineages of G. leucogastra. 461
In contrast, analysis of UCE data with both concatenated and species-tree approaches recovered 462
G. chalcothorax as sister to all G. leucogastra lineages. We hypothesize that the mitochondrial 463
genome of the Madeira population of G. leucogastra was captured by G. chalcothorax early in 464
their initial divergence, and we suggest how WSE evolution and the co-evolution of mtDNA 465
genes and nuclear genes might have played a role in this rare event. 466
467
Keywords: White-sand environments, Amazonia, Galbulidae, jacamars, mtDNA capture, UCE 468
469
11
1. Introduction 470
White-sand environments (WSE) represent a unique type of habitat within Amazonia. 471
Apart from the continuous forest habitats found all over the basin, the WSE consists of patches 472
of differentiated habitats scattered in the landscape and isolated by the forest matrix (Adeney 473
et al., 2016). WSE consist of a continuum from open non-forested habitats, such as campinas, 474
with a predominance of grass and shrubland, to denser vegetation, called campinaranas and 475
varillales, all associated with sandy soils. This insular characteristic of WSE continues to 476
intrigue researchers as to how the ecosystem and its specialized biota evolved, how it responded 477
to Pleistocene glacial cycles, and whether the specialized biota disperse through the forest 478
matrix among patches of WSE (Brown and Benson, 1977; Anderson, 1981; Capurucho et al., 479
2013; Matos et al., 2016). Besides its characteristic fragmentation, WSE are more 480
physiologically stressful and challenging from an ecological and evolutionary perspective, 481
making them much more taxonomically selective, with overall diversity being smaller when 482
compared with adjacent forest areas (Borges, 2003; Fine et al., 2010; Laranjeiras et al., 2014; 483
Adeney et al., 2016), although several new species endemic to this habitat having recently been 484
described (Whitney and Alonso, 1998, 2005; Alonso and Whitney, 2001; Cohn-Haft and Bravo, 485
2013; Cohn-Haft et al., 2013). Some studies point to a recent and dynamic history for WSE 486
(Latrubesse and Franzinelli, 2002; Rossetti et al., 2012), yet this habitat is usually associated 487
with ancient soils (Adeney et al., 2016). Although some plant species have a loose association 488
with WSE (Fine and Baraloto, 2016), others are tightly associated with them, such as species 489
of Pagamea (Vicentini, 2016). The same can be observed for other organisms (Cohn-Haft, 490
2008; Vriesendorp et al., 2006), especially birds (Borges et al., 2016a; Borges et al., 2016b). 491
Therefore, the combination of persistent white-sand soils with recent climatic and landscape 492
changes must have had an important influence on the evolution and distribution of WSE and of 493
the biota that inhabits them. 494
12
The few phylogeographic studies of WSE birds that have been undertaken, show little 495
genetic diversity with no geographic structure throughout Amazonia (Polytmus theresiae, 496
Matos et al., 2016); or shallow but geographically structured genetic diversity, with significant 497
migration rates between some populations (Tachyphonus phoenicius, Matos et al., 2016; 498
Xenopipo atronitrens, Capurucho et al., 2013). In general, results obtained so far for WSE birds 499
suggest that: (1) black-water flooded forest (igapó), due to similarities to WSE in vegetation 500
structure, may facilitate dispersal between isolated WSE patches; and, (2) Pleistocene glacial 501
periods, especially the Last Glacial Maximum, are temporally correlated with geographical 502
expansion of populations of species specialized in WSE. 503
These studies have been based on mtDNA markers (Capurucho et al., 2013), or on a 504
combination of mtDNA and a single nuclear marker (Matos et al., 2016). Until recently, most 505
phylogeographic studies have employed mtDNA. Its characteristic maternal inheritance, 506
comparatively small effective population size, rapid rate of mutation, and lack of 507
recombination, coupled with the fact that it is easy to amplify and sequence, have long made 508
mtDNA markers ideal for phylogeographic studies (Avise et al., 1987; Avise, 2009). However, 509
there are potential biases and limitations associated with these data (Zink and Barrowclough, 510
2008) and hybridization and introgression could be overlooked (Carling and Brumfield, 2008). 511
Thus, the inclusion of nuclear markers often yields different perspectives. That said, no markers 512
are without biases and the inclusion of autosomal markers entail other problems, such as 513
discordances between gene trees and species-trees (Knowles, 2009), as well as between the 514
history of mtDNA and nuclear markers, especially because the small number of nuclear loci 515
employed usually do not have enough information in recent divergences (Zink and 516
Barrowclough, 2008; Daly-Engel et al., 2012; Toews and Brelsford, 2012; Sloan et al., 2017). 517
These latter studies demonstrated the value of using several different markers to truly 518
understand species/lineage histories, yet until the advent of high throughput parallel sequencing 519
13
techniques, such multi-loci analyses were very time-consuming and uneconomical (Metzker, 520
2010). One of the new genomic markers made accessible by next-generation sequencing 521
technologies is Ultra-Conserved Elements – UCE (Faircloth et al., 2012; McCormack et al., 522
2013). The use of UCEs provides access to large quantities of genomic data to assess 523
relationships at multiple time and taxonomic scales (Faircloth et al., 2012), from very old 524
radiations (Moyle et al., 2016), to more recent ones (Smith et al., 2014; Harvey et al., 2016; 525
Manthey et al., 2016). 526
Here, we investigate a rare pattern of evolutionary diversification in Amazonian WSE 527
avifauna by reconstructing the phylogeography of a jacamar species-complex using genomic 528
data. The jacamars (family Galbulidae) are exclusive to the Neotropics, with 19 species and 5 529
genera, mostly associated with wooded, lowland forest habitat (Stotz et al., 1996; Tobias, 530
2017). In Amazonia, most species are restricted to upland (terra firme) and flooded (varzea and 531
igapó) forests, with only two species (Galbula leucogastra and G. chalcothorax) known to 532
occur in WSE (Borges et al., 2016a). Galbula leucogastra and G. chalcothorax were previously 533
considered subspecies of a single species (Peters, 1948; Haffer, 1974), but were split by Parker 534
and Remsen (1987), based on diagnostic plumage and size differences. A phylogeny of the 535
family, based on multiple gene regions, indicates that G. leucogastra and G. chalcothorax are 536
sister-species with high support (Witt, 2004). Here we first investigate the distribution of 537
mtDNA diversity within these two species by sampling individuals from throughout their 538
distributions. Then, based on these results, we obtained sequences of thousands of genomic 539
markers (UCE) for a subset of samples to reconstruct their history of diversification and make 540
inferences about the evolution of WSE. 541
542
2. Methods 543
2.1. Taxon sampling 544
14
We sampled 35 individuals covering almost the entire distribution of the Galbula 545
leucogastra / chalcothorax (Table S1). As outgroups, we used one sample of G. albicollis (Witt, 546
2004). All tissues sequenced are represented by voucher specimens deposited in ornithological 547
collections in Brazil and the USA (Table S1). 548
2.2. DNA extraction, amplification and sequencing 549
DNA was extracted using a modified phenol-chloroform protocol (Sambrook and 550
Russel, 2001). We used published DNA primers (Sorenson et al., 1999) to amplify and 551
sequence two mitochondrial genes (Cytochrome b [cytb], and NADH subunit 2 [ND2]) for all 552
individuals following standard PCR protocols. For a subset of individuals (see below) we 553
extracted DNA using the DNeasy kit (Qiagen Inc.) following the manufacturer’s protocol, and 554
sent the extracts to RapidGenomics® (Gainsville, FL) for sequencing, using a probe set 555
targeting 2321 loci of Ultra Conserved Elements (UCE) plus 98 conserved exons from genes 556
that were previously used in phylogenetic analysis (Harvey et al., 2017). Some of the exons 557
used were flanked by introns, which are more variable, and were the focus of this capture. More 558
information about the capture and sequencing of UCE loci can be found in Faircloth et al. 559
(2012). 560
2.3. Phylogenetic analysis and haplotype networks 561
Phylogenetic analysis of the mtDNA genes using the complete dataset (cytb and ND2, 562
N=35) was performed using Bayesian Inference (BI) implemented in MrBayes 3.2.6 (Ronquist 563
et al., 2012). Both genes were concatenated and the best partition scheme and substitution 564
model were selected by PartitionFinder 2.1.1 using the Bayesian Information Criteria (BIC) 565
(Lanfear et al., 2016). We partitioned the genes by codon position, considering possible 566
saturation in the codon’s third position. Four parallel simultaneous runs were performed, for a 567
total of 4x107 generations, with trees sampled every 1000 generations. We discarded the first 568
15
10% of trees as burn-in after checking the ESS values of each run in Tracer 1.6 (Rambaut et 569
al., 2014). We used TCS v1.21 (Clement et al., 2000) to reconstruct haplotype networks. 570
2.3.1. UCE and exons assembly 571
Based on the results of the mtDNA, we selected eight samples for UCE sequencing 572
(Table 1). The raw data received from Rapid Genomics were processed using the Phyluce script 573
pack (Faircloth 2016). Sequences with adapter contamination, and those of low-quality, were 574
trimmed using illumiprocessor (Faircloth, 2013) and Trimmomatic (Bolger et al., 2014). After 575
the sequences were ‘cleaned’ we employed Trinity RNASeq assembler r201331110 (Grabherr 576
et al., 2011) to assemble the contigs using a de novo method. The contigs were then compared 577
with the UCE database to identify which UCE loci were sequenced. Since Trinity does not 578
recover information on heterozygote loci we performed a second round of assembly using the 579
contigs that were identified as a reference to map the clean reads back to it using the Bowtie2 580
(Langmead et al., 2009; Langmead and Salzberg 2012) plugin in Geneious R7.1 (Kearse et al., 581
2012). The consensus sequence of each individual, derived from the reads, mapped back to each 582
reference, was called using a threshold of 75% with a depth of at least 5 reads. We then aligned 583
each locus using MAFFT (Katoh and Standley, 2013) with default options, and prepared the 584
input matrix for the subsequent analysis. To infer the phylogenetic relationship among all 585
samples we concatenated all the UCE loci and employed RAxML v8.2 (Stamatakis, 2014) 586
under a Maximum Likelihood analysis. Since we recovered almost all UCE loci for each 587
sample, we only used loci that were shared among all samples, with the final matrix having 588
2271 loci. This matrix was analyzed by running RAxML to search for the optimal tree, under 589
the fast hill climbing algorithm, and bootstraping was performed with the autoMRE algorithm 590
in the program. 591
The 98 exons targeted were from 47 different genes. Because some of the sequences 592
included intronic regions, which are prone to indels, de novo assembly was not an option. 593
16
Therefore, we mapped all the probes to the Galbula dea genome, identified the genes that were 594
targeted, and then used the whole gene-sequences to map the reads back following the same 595
approach that we used for UCE loci. Since recombination is not expected to happen inside one 596
gene, all exonic regions recovered belonging to the same gene were considered to be connected 597
in the species-tree (ST) analysis. 598
2.3.2. Mitochondrial genome assembly and time tree 599
As a byproduct of the UCE sequencing we also recovered the complete mtDNA genome. 600
We mapped all the Trinity contigs from each specimen to two reference mtDNA genomes from 601
representatives of close related families, the Downy Woodpecker, Dryobates pubescens (Aves, 602
Picidae; NC_027936.1), and the Ivory-billed Araçari, Pteroglossus azara (Aves, 603
Ramphastidae; DQ780882.1, Prum et al., 2015). After we identified the contigs from each 604
individual we used those contigs to map back the reads of that same specimen, again using 605
Bowtie2 to check for coverage depth. Incongruences found between reads and contigs were 606
checked manually. The complete mtDNA genomes were then aligned using MAFFT (Katoh 607
and Standley, 2013) under default options. The mtDNA genomes downloaded from GenBank 608
were used to import annotations. Coding regions were manually checked for codon translations, 609
and translated protein sequences were compared to check for frame shifts and stop codons. We 610
employed the concatenated coding regions in BEAST 1.8.2 (Drummond et al., 2012) to 611
estimate a time tree calibrated with the cytochrome b mutational rate of 0.0105 (normal 612
distribution, SD=0.0034) substitution.lineage-1.million years-1 (Weir and Schluter, 2008). The 613
best partition scheme and substitution model were selected by PartitionFinder 2.1.1 under the 614
Bayesian Information Criteria (BIC) (Lanfear et al., 2016). Two independent runs of 108 615
generations were performed sampling trees every 1000 generations. Convergence, posterior 616
distributions, and ESS values were checked in Tracer 1.6 (Rambaut et al., 2014). 617
2.3.3. Species-tree analysis 618
17
Considering the possibility that concatenation might result in highly supported but 619
inaccurate results (Kubatko and Degnan 2007; Weisrock et al., 2012, but see Gatesy and 620
Springer 2014), we performed a species-tree analysis, which infers the most likely species-tree 621
based on individual gene trees, using the StarBEAST2 (Ogilvie et al., 2017) template in the 622
BEAST v.2.4.6 package (Bouckaert et al., 2014). Even though StarBEAST2 was developed to 623
deal with huge amounts of data, we selected only the loci that had more than four parsimony 624
informative sites (PIS) among our samples. This latter step reduces the total time of analysis 625
and also avoids including loci lacking phylogenetic signal, which would create noise in the 626
analysis. We employed PartitionFinder2 (Lanfear et al., 2016) to check for the best partition 627
scheme and substitution model. Trees models were unlinked, except for exons from the same 628
gene, in which case we linked tree models across different partitions. We used a Yule model of 629
speciation, and ploidy was set to 2.0, unless genes were from the Z chromosome (in which case, 630
ploidy=1.5). We also included the complete mtDNA as a single locus, with ploidy=1.0. 631
632
633
3. Results 634
3.1. Sanger sequencing and haplotype networks 635
We sequenced 996 bp and 1013 bp, respectively, of the cytb and ND2 dataset. The best 636
partitioning scheme consisted of four partitions (cytb_pos1 = K80+I; ND2_pos2+cytb_pos2 = 637
HKY; ND2_pos3+cytb_pos3 = GTR+G; ND2_pos1 = HKY+I). The BI analysis, and the 638
haplotype network, recovered eight allopatric mtDNA lineages, six of them are well-supported 639
clades, while two of them are represented by a single individual each (Fig. 1). Although all 640
clades corresponding to the allopatric lineages had strong support, basal relationships among 641
them were poorly supported, the only exceptions being the sister relationships between Guiana 642
and Negro clades and between G. chalcothorax and the Madeira lineage of G. leucogastra. 643
18
Haplotypes networks were recovered using the concatenated matrix of cytb and ND2 in 644
which all missing data were discarded (1304 bp). Almost all networks were indicative of recent 645
population expansion, with little to no genetic diversity within lineages, except for the Madeira 646
lineage and for G. chalcothorax, for which we recovered a different haplotype for each 647
specimen. It is worth noting that samples from different banks of the Tapajós River are 648
separated by six mutational steps (Fig. 1: light and dark green), and that samples of G. 649
chalcothorax (Fig.1: light and dark brown) exhibit almost the same number of mutations among 650
them as they do in relation to the haplotypes from the Madeira lineage. 651
3.2. mtDNA genome and time tree 652
We recovered the complete mitochondrial genome from all samples sequenced for 653
UCEs. In contrast to our cytb+ND2 tree, the tree based on all the mtDNA coding genes was 654
highly supported (Fig. 2). Molecular dating indicates that diversification of the mtDNA lineages 655
started in the Middle Pleistocene, at about 1.5 million years ago (mya) (95%HPD = 2.4 - 0.75). 656
Although all nodes were recovered with high support, the first three splits occurred in a short 657
period of time, with short internodes, suggesting a rapid radiation among lineages from 658
southern, northern and western Amazonia (Fig. 2). The earliest divergence is suggested to have 659
been between populations separated by the Amazon River (Fig. 2). In both mtDNA analyses 660
(cytb+ND2 and mtDNA genome), G. chalcothorax was recovered as the sister-group to the G. 661
leucogastra lineage from the west bank of Madeira River, with their divergence dating of 662
around 0.74 mya (95%HPD = 1.21 – 0.38), therefore rendering G. leucogastra paraphyletic. 663
The lineages from the north bank of the Amazon River were also recovered as sister-groups, 664
and diverged roughly around the same time, 0.61 mya (95%HPD = 1 – 0.31). The most recent 665
divergence occurred between lineages separated by the Tapajós River at 0.28 mya (95%HPD = 666
0.47 – 0.13). 667
3.3. UCE sequencing, RAxML and Species trees 668
19
The complete UCE matrix, which included only those loci shared among all samples, 669
contained 2271 UCE loci, with mean locus length of 543.06 bp (see Table 1 for total number 670
of reads, Trinity contigs, UCE and exon loci recovered from each sample; for alignments, total 671
number of loci, and locus information, see Table 2). The concatenated RAxML tree recovered 672
G. chalcothorax as sister to all other samples of G. leucogastra with high bootstrap support 673
(p=100, Fig. 3). Thus, the earliest divergence is here suggested to have occurred between an 674
eastern and a western population, unlike the pattern suggested by the mitochondrial data. The 675
first split within G. leucogastra is between lineages north and south of the Amazon River, 676
followed by a split across the Madeira River (p=100), and then younger splits across the Tapajós 677
(p=96) and the Aripuanã (p=76). 678
For the StarBEAST species-tree we used 124 loci that had more than four parsimony 679
informative sites. The species-tree was identical in topology to the concatenated RAxML UCE 680
phylogeny, with some differences in statistical support, including two nodes without strong 681
support in the species-tree (p<0.95) (Fig. 3). In both the concatenated and the species-trees, we 682
found contrasting differences compared to the mtDNA genome tree. Besides the nature of the 683
earliest split in the complex, the most significant one is that the nuclear data recover G. 684
leucogastra as monophyletic and sister to G. chalcothorax with strong statistical support; the 685
mtDNA genome tree, in contrast, found G. leucogastra to be paraphyletic, and G. chalcothorax 686
as sister to the G. leucogastra Madeira lineage (Fig. 2). Furthermore, in the UCE trees the G. 687
leucogastra Aripuanã lineage (Fig. 3, dark pink) was strongly clustered with samples 688
distributed east of the Madeira River (Fig. 3). 689
690
4. Discussion 691
4.1. mtDNA and nuDNA incongruence 692
20
Historically the Purplish Jacamar (G. chalcothorax) was considered a subspecies of the 693
Bronzy Jacamar (G. leucogastra) (Peters, 1948; Haffer, 1974). Parker and Remsen (1987) 694
proposed that the two taxa be recognized as separate species based on their distinct phenotypes: 695
G. leucogastra is bronzy-green, with some suffused metallic blue, and a white belly, whereas 696
G. chalcothorax is tinged reddish-purple, and has a black belly with only the feathers tips being 697
white. Although these color characters seem to fluctuate across populations, G. chalcothorax is 698
distinctly larger than G. leucogastra (Haffer, 1974). Parker and Remsen (1987) also suggested 699
that Haffer (1974) did not recognize G. chalcothorax as a full species because of the supposition 700
they would interbreed if the two taxa came together, but they also noted (p. 98) that “the absence 701
of major river barriers between their ranges suggests that no interbreeding occurs or would 702
occur”. 703
The structure recovered by the mtDNA data within G. leucogastra, with five well 704
supported mtDNA clades, suggests that current taxonomic treatment misrepresents the diversity 705
within this species, which currently includes only two subspecies: G. l. leucogastra and G. l. 706
viridissima (Griscom and Greeway, 1941). Surprisingly, mtDNA data also revealed that all G. 707
leucogastra specimens from the Madeira clade, the geographically closest to G. chalcothorax 708
is sister to G. chalcothorax with high support, but with no shared haplotypes among species 709
(Fig. 1, 2). In contrast, the UCE concatenated RAxML tree as well as the UCE species-tree 710
recovered G. leucogastra and G. chalcothorax as monophyletic sister species, with the Madeira 711
lineage of G. leucogastra sister to G. leucogastra lineages from SE Amazonia (i.e. Aripuanã 712
and Tapajós lineages, Fig. 3). Multiple explanations have been proposed for conflict in 713
mitochondrial and nuclear histories (summarized in Table 1). 714
In the G. leucogastra / G. chalcothorax diversification mitochondrial capture may have 715
been influenced by the populational and ecological context of differentiation within WSE. After 716
the lineages in the south diverged east and west of the Madeira, the ancestral lineages of G. 717
21
chalcothorax and those of the Madeira met and hybridized. Although it is difficult to determine 718
when the contact started, it ended around 0.74 mya, as shown in our mtDNA time tree. 719
Moreover, even though the ranges of G. leucogastra and G. chalcothorax appear to be currently 720
allopatric (Tobias 2017), they approach each other between the Purus and Juruá rivers (Fig. 1). 721
Therefore, past gene flow may have been possible during drier climatic periods in SW 722
Amazonia (see below) (Mayle et al., 2004; Bush, 2017). mtDNA clades found within G. 723
leucogastra are more structured and differentiated than the clades found within the other WSE 724
birds, but all of them agree in recovering a well supported clade in northern Amazonia, and 725
with the Madeira being an important barrier in the south (Cracraft, 1985; Borges et al., 2012; 726
Ribas et al., 2012; Fernandes et al., 2013; Fernandes et al., 2014; Ferreira et al., 2017). The 727
maintenance of such structured mtDNA lineages may indicate that little or no gene flow is 728
present between the lineages, suggesting that the forest matrix is important for maintaining 729
allopatry. 730
Although mtDNA may reflect species boundaries (Hill, 2017), recent studies have 731
shown a number of cases in which apparent mtDNA paraphyly is not just derived from improper 732
taxonomy (McKay and Zink, 2010) but also from mtDNA introgression among adjacent 733
populations (see also Toews and Brelsford, 2012). For example, a mitochondrial sweep was 734
proposed in the certhiola complex in the Old World warbler genus Locustella, which is 735
comprised of three species (certhiola, ochotensis and pleskei). Phylogenetic studies using 736
mtDNA and nuDNA recovered conflicting results in that pleskei was paraphyletic relative to 737
certhiola and ochotensis on the mtDNA tree, whereas the nuDNA species-tree recovered 738
species monophyly (Drovetski et al., 2015). In addition, these authors found signs of 739
asymmetrical introgression, in which the species expanding its range (ochotensis) appears to 740
have invaded the species with a smaller ranges (pleskei), resulting in mtDNA introgression from 741
the species with large Ne to the one with smaller Ne. nuDNA introgression was in the opposite 742
22
direction, causing the paraphyly observed in the mtDNA tree (Drovetski et al., 2015). A similar 743
scenario was found in the eastern Australian rosellas (Platycercus, Shipham et al., 2017). The 744
three species of the subgenus Violania showed discordances between RADseq data and mtDNA 745
trees. Whereas the RAD trees recovered P. eximius as sister to the clade P. venustus and P. 746
adscitus, the mtDNA phylogeny recovered P. venustus as sister to P. adscitus and P. eximius. 747
Furthermore, when data for the isolated Tasmanian P. e. diemenensis were added, the same 748
relationship as those from the RAD data were recovered, suggesting that the subspecies of P. 749
eximius from the mainland (P. e. eximius) captured the mtDNA from P. adscitus. 750
Although these two documented cases represent examples of how mitochondrial sweeps 751
could occur in populations with known zones of hybridization, genetic and phenotypic data 752
suggest that there is no current hybrid zone corresponding to the conflict between UCE and 753
mtDNA reported here. Furthermore, isolation might lead to co-evolution of mitochondrial and 754
nuclear genes involved in cellular respiration, which could function as a post-zygotic barrier to 755
gene flow, due to Bateson-Dobzhansky-Muller Incompatibility (BDMI) (Orr, 1996). Given the 756
fragmented distribution of WSE in Amazonia, it is possible that the occupation of new patches, 757
or the fragmentation of previously continuous habitats into smaller patches due to landscape 758
evolution, followed by some time in allopatry, could lead to the mtDNA structure we observe 759
nowadays and consequent coevolution with nuclear background. All lineages we recovered for 760
this complex have deep structure in the mtDNA haplotypes, even between lineages with 761
adjacent distributions, such as Guiana and Negro lineages or Aripuanã and Tapajós lineages 762
(Fig. 3). Sex-biased traits, such as differential dispersal, hybrid fitness or mate choice are 763
commonly used to explain discordances between mtDNA and nuDNA (Excoffier, 2009; Toews 764
and Brelsford, 2012). However, in a recent review of this process, Bonnet et al. (2017) 765
simulated several scenarios and observed that the only way to have massive discordance in all 766
simulations, without detectable nuclear introgression, is when there is positive selection acting 767
23
on mitochondrial lineages. Surprisingly, Bonnet et al. (2017) were unable to detect 768
mitochondrial adaptive introgression using Tajima’s D and Fu’s Fs tests, reinforcing the 769
argument that these tests have low statistical power to detect adaptive introgression (Bonnet et 770
al., 2017). In addition, the mtDNA can accumulate deleterious mutations quickly, and in small 771
populations, drift could spread these deleterious mutations across the whole population in short 772
periods of time. Therefore, small populations may accumulate several deleterious mutations 773
and the “defective” mtDNA lineage can be supplanted by a foreign mtDNA lineage (Llopart et 774
al., 2014; Hulsey et al., 2016; Sloan et al., 2017). This hypothesis can be more plausible if 775
effects of the mtDNA sweep are more beneficial than the disadvantageous effects of 776
mitonuclear incompatibilities. This event, combined with the fact that both species occupy 777
different habitats inside the WSE, could explain why we observe the incongruences between 778
mtDNA and nuDNA. 779
4.2. Biogeography of WSE avifauna 780
In phylogeographic studies of the Black Manakin (Xenopipo atronitrens, Pipridae), 781
Capurucho et al. (2013) found the largest mtDNA divergences to correspond to populations 782
found across the Branco and Amazonas rivers. Similar results were observed for the Red-783
shouldered Tanager (Tachyphonus phoenicius, Thraupidae, Matos et al., 2016), but with greater 784
isolation between opposite margins of the Amazon river. The divergence times estimated 785
between northern and southern lineages within X. atronitrens and T. phoenicius were 0.92 and 786
0.88 Ma, respectively, both slightly younger than the mean age estimate we obtained for the 787
first divergence on the mtDNA tree (~1.5 Ma, 95%HPD = 0.75 - 2.4) in G. leucogastra, but 788
with overlap of confidence intervals. Another WSE specialist studied, the Green-tailed 789
Goldenthroat (Polytmus theresiae, Trochilidae), showed no genetic structure, but exhibited 790
signs of recent population expansion (Matos et al., 2016). Signs of recent gene flow among 791
otherwise isolated populations of the aforementioned species contrast with the highly-792
24
structured lineages recovered here. Although we found evidence for an ancient capture event 793
of mtDNA lineages, there is no evidence of current gene flow between G. leucogastra and G. 794
chalcothorax. Xenopipo atronitrens and G. leucogastra/chalcothorax are found in both WSE 795
and black-water flooded forest, T. phoenicius in WSE and savannas, and P. theresiae in WSE, 796
black-water flooded forest and savannas (Borges et al., 2016b). When compared to the other 797
WSE species, G. leucogastra and G. chalcothorax are the only exclusive insectivores, meaning 798
that they need not have as extensive foraging areas as do frugivores or nectarivores (Levey and 799
Stiles, 1992), and hence they are potentially more prone to isolation and differentiation (Burney 800
and Brumfield, 2009). 801
4.3. Evolution in the White-sand environments 802
White-sand environments cover an area of approximately 5% of the Amazon basin 803
(Adeney et al., 2016). They can be covered by different kinds of vegetations, from open 804
grasslands to different types of forest. In general, these communities grow on nutrient poor and 805
highly acidic soils, usually associated with quarzitic sand, even though some clay and silt can 806
also be found with varying amounts of organic matter (Adeney et al., 2016). This complex 807
environment, however, does not share a single history, since different patches of WSE may 808
have different geological origins (Prance and Schubart 1978; Frasier et al., 2008). 809
Podzolization, a natural process in which all nutrients are leached away from the top layers of 810
soil, leaving only sand (Sauer et al., 2007), appears to be a principal cause of in loco formation 811
of the white sand soils, especially in northeastern Amazonia (Nascimento et al., 2004). In 812
central, northwestern, and southern Amazon, white sand soils can be found as fluvial deposits 813
of ancient rivers (Roddaz et al., 2005), or abandoned ancient paleochannels (Latrubesse, 2002; 814
Cordeiro et al., 2016). In western Amazonia, white sand formations date to before the Andean 815
uplift, and are probably a result of westward rivers flowing from the Guiana and Brazilian 816
shields to the Pacific Ocean, during the Early Miocene (Hoorn, 1993). These sandy sediments 817
25
were reorganized and recycled multiple times within the basin during the Andean uplift, giving 818
patches of WSE in western Amazonia a very insular and scattered characteristic, especially 819
because most of these sediments are now covered by more recent clay-rich sediments derived 820
from the Andes. This mosaic of sediments is reflected in soils with distinct edaphic conditions, 821
which influence floristic composition that ultimately influences local bird communities 822
(Pomara et al., 2012). 823
Phylogeographic studies of WSE specialized birds suggest that they have recently 824
occupied the Amazonian WSE from east to west (Whitney and Alonso, 1998; Capurucho et al., 825
2013; Matos et al., 2016). Also, most of WSE birds have sister groups inhabiting other open 826
vegetation habitats and not the adjacent Amazonian humid forest formations, such as terra-827
firme or varzea (Rheindt et al., 2008; Capurucho et al., 2013; McGuire et al., 2014; Matos et 828
al., 2016). This suggests the colonization of Amazonian WSE by lineages that had already 829
evolved in open habitats, instead of ancestral lineages from neighboring humid forest. In this 830
sense, Galbula leucogastra and Galbula chalcothorax are unlike other WSE taxa since all other 831
Galbula species are found in forest habitats (Witt, 2004; Tobias, 2017). 832
The WSE were probably more widespread throughout the continent before Andean 833
uplift, thus extant WSE lineages of birds should be the ones resilient enough to endure the 834
reconfiguration of the Amazon basin (Campbell et al., 2006; Hoorn et al., 2010; Nogueira et 835
al., 2013). The pattern of more genetic diversity in the east we observe today should be then 836
related to the fact that during the Pleistocene climatic cycles, eastern Amazonia experienced 837
greater fluctuations in precipitation (Wang et al., 2017). Although these cyclical oscillations 838
were not enough to replace forest with savannas (Bush, 2017; Wang et al., 2017), they may 839
have affected forest structure (Barthe et al., 2017; Cowling et al., 2001). This could have 840
facilitated contact between different patches of WSE in the East, especially for birds that can 841
use black-water flooded forest, allowing them to expand their distribution and colonize 842
26
previously unoccupied patches of WSE. The paleoclimatic record (Cheng et al., 2013; Wang et 843
al., 2017) suggests that western Amazonia remained as humid as it is today throughout the 844
Pleistocene climatic oscillations, while eastern Amazonia experienced about 42% less rainfall 845
when compared with modern values (Wang et al., 2017). Even though eastern Amazonia 846
experienced drier climate, there is no evidence suggesting replacement of forest by savanna 847
(Bush et al., 2017). So, the existence of WSE in western Amazonia would occur only in 848
scattered patches in recycled quartzite soils reminiscent of ancient fluvial deposits (Hoorn, 849
1993), or as fluvial deposits of ancient rivers (Latrubesse, 2002). This scenario of contracting 850
WSE areas in the west, because of recycling soils during Andean uplift, and WSE expansion in 851
the east, especially during Pleistocene climatic cycles, would probably explain the smaller Ne 852
of G. chalcothorax and the mtDNA capture from G. leucogastra as it expanded its distribution 853
during dry cycles. 854
855
5. Conclusion 856
Here we shown an instance of clear discordance between phylogenetic relationships 857
recovered using mtDNA and nuclear data. Interestingly, nuclear data agrees with current 858
taxonomy, which is based on phenotypic patterns, while the mtDNA relationships seem to be 859
related to an old event of mtDNA capture. The capture event relates to what is currently known 860
about the distinct biogeographical histories of WSE in Eastern and Western Amazonia. While 861
these results raise important issues about apparent mtDNA paraphyly of taxa and the 862
straightforward use of mtDNA relationships in taxonomy, they also show that interesting 863
biogeographic histories can be uncovered when enough data is available, allowing for a 864
comparison with mtDNA. This will be an important contribution of NGS for studies for recent 865
speciation and taxonomy. 866
867
27
868
Acknowledgements 869
We thank the curator and curatorial assistants of the Academy of Natural Science of Drexel 870
University, Philadelphia, USA (ANSP); Field Museum of Natural History, Chicago, USA 871
(FMNH); Instituto Nacional de Pesquisas da Amazônia, Manaus, Brazil (INPA); Lousiana State 872
University Museum of Natural Science, Baton Rouge, USA (LSUMZ); and Museu Paraense 873
Emílio Goeldi, Belém, Brazil (MPEG), for borrowing tissue samples under their care. We thank 874
S. W. Cardiff and N. Rice for helping us with LSUMZ and ANSP specimens, respectively. We 875
are also grateful for all collectors involved in the fieldwork throughout Amazon that make this 876
paper possible. We thank J. M. G. Capurucho and S. H. Borges for early inputs on this paper. 877
878
Funding 879
Support to M.F.’s graduate research was provided by CAPES PhD fellowship, and CAPES 880
PDSE fellowship (# 88881.133440/2016-01), support also from the AMNH Frank M. Chapman 881
Memorial Fund. Support to A.M.F. during his post-doc studies was provided by CNPq 882
(#500488/2012-6). Laboratory and Sanger sequencing costs were partly covered by grants to 883
A. Aleixo (CNPq # 471342/2011-4 and FAPESPA # ICAAF 023/2011) and A.Antonelli from 884
the European Research Council under the European Union’s Seventh Framework Programme 885
(FP/2007-2013, ERC Grant Agreement n. 331024), the Knut and Alice Wallenberg Foundation 886
through a Wallenberg Academy Fellowship, the Swedish Research Council (2015-04857), and 887
the Swedish Foundation for Strategic research. A.Aleixo, C.C.R., J.M.B., J.C. and M.F. also 888
thanks the grant Dimensions US-Biota-São Paulo: Assembly and evolution of the Amazon biota 889
and its environment: an integrated approach, co-funded by the US National Science Fundation 890
(NSF DEB 1241056) to J.C. and the Fundação de Amparo à Pesquisa do Estado de São Paulo 891
(FAPESP grant #2012/50260-6) to Lucia Lohmann. A. Aleixo and C.C.R. are supported by 892
28
CNPq research productivity fellowships. The authors acknowledge the National Laboratory for 893
Scientific Computing (LNCC/MCTI, Brazil) for providing HPC resources of the SDumont 894
supercomputer, which have contributed to the research results reported within this paper. 895
896
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1174
Author contibutions 1175 M.F. and A.M.F. developed the sampling plan, extracted DNA and sequenced all samples. M.F. 1176 performed all analysis. A.A.P., A.A., U.O., J.M.B., J.C. and C.C.R. were involved in intellectual merit, 1177 funding, and writing. All authors participated in writing the manuscript. 1178 Supporting information 1179 Additional supporting information may be found in the online version of this article. 1180 Table S1 Supplementary details of individuals. 1181 1182
34
Table 1 - Samples used for UCE sequencing, their voucher numbers, general locality, number of clean reads 1183 after Illumiprocessor, number of contigs assembled by Trinity, and total UCE loci recovered from Trinity. 1184 1185
Species Museum voucher Locality Clean reads Trinity contigs UCE loci
G. chalcothorax LSUMZ B2803 N of Napo River, Iquitos, Peru 1,524,126 6,537 2,230
G. leucogastra INPA A4182 145 Km WWS of Apuí, AM, Brazil 2,540,148 12,163 2,269
G. leucogastra INPA A4672 Right bank of Jatapú River, AM, Brazil 2,209,895 9,491 2,263
G. leucogastra LSUMZ B35619 Arapiuns River, PA, Brazil 4,394,658 10,853 2,246
G. leucogastra LSUMZ B9608 Nicolás Suarez, Pando, Bolívia 1,677,988 5,074 1,928
G. leucogastra MPEG 59360 Novo Airão, AM, Brazil 2,372,950 6,026 1,957
G. leucogastra MPEG 75618 Right bank of Tapajós River, PA, Brazil 1,346,149 6,117 2,263
G. leucogastra MPEG 73685 Novo Aripuanã, AM, Brazil 1,466,240 6,896 2,227
G. albirostris INPA A064 Amazonas, Brazil 2,809,416 16,718 2,256
1186 Table 2 – Summary of each method, including number of loci, total length, mean length size of each loci, 1187 minimum and maximum length, number of Parsimony Informative sites. 1188 1189
Method Complete Exons Species Tree†
Number of loci 2271 47 124
Total lenght (bp) 1,233,287 47,580 80,085
Mean lenght size (bp) 543.06 849.64 645.85
Min - Max lenght (bp) 118 – 1,305 182 - 3093 347 – 3093
Number of PI sites (mean) 2003 (0.88) 190 (3.39) 744 (6)
†without the mtDNA 1190 1191 Table 3 – Possible causes of conflict in mitochondrial and nuclear DNA histories. 1192 1193
Inferred process Reference
Incomplete lineage sorting Funk and Omland, 2003; McKay and Zink, 2010;
Zink and Barrowclough, 2008
Incomplete sampling Shipham et al., 2015, 2017
Improper taxonomy McKay and Zink, 2010
Adaptive introgression Bock et al., 2014; Dobler et al., 2014
Demography or Sex-biased traits Bonnet et al., 2017; Daly-Engel et al., 2012;
Rheindt and Edwards, 2011; Sloan et al., 2017
1194
35
Figure 1 - Map of sequenced individuals, phylogenetic Bayesian tree recovered, and haplotype networks. The 1195 colors in the tree, map and networks are correspondent, and the tree and networks are based on two mtDNA genes 1196 (2009 bp, cytb and ND2). Posterior probabilities obtained at each node are indicated on the tree, red circles 1197 represent pp=1. The brown labeled points are G. chalcothorax, all other lineages are G. leucogastra. Terminal 1198 names in red are samples used in the UCE analysis. 1199 1200
1201 1202
36
Figure 2 – Chronogram recovered by BEAST using all mtDNA coding genes with a calibration derived from the 1203 mutational rate of the cytb gene (Weir and Schluter 2008). Posterior probabilities obtained at each node are 1204 indicated in the tree, red circles represents pp>98, associated confidence interval (95% HPD) for diversification 1205 time (blue bar), and the median time of divergence. Colors are correspondent with Figure 1. 1206 1207
1208 1209
37
Figure 3 - Comparison between the concatenated UCE RAxML tree (left) and the StarBEAST2 species tree (right). 1210 Bootstrap support for the RAxML tree, and the posterior probability for the StarBEAST species tree, is show near 1211 the nodes. Colors are correspondent with Figure 1. 1212 1213
1214 1215 1216
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Capítulo 2 1228
1229
1230
Ferreira, M.; Aleixo, A.; Bates, J. M.; Cracraft, J.; 1231
Ribas, C. C. Phylogenomics of trogons (Aves: 1232
Trogonidae) shed light on the Quaternary 1233
biogeography of tropical forests and the connections 1234
between Asia, North and South America. Manuscrito 1235
formatado para Molecular Biology and Evolution 1236
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Manuscript submission to Molecular Biology and Evolution 1240
Contribution type: Article 1241
1242
Phylogenomics of trogons (Aves: Trogonidae) shed light on the Quaternary 1243
biogeography of tropical forests and the connections between Asia, North 1244
and South America 1245
1246
Ferreira, Mateus1*; Aleixo, Alexandre2; Bates, John M.3; Cracraft, Joel4; Ribas, Camila C.5 1247 1248 1 Programa de Pós-Graduação em Genética, Conservação e Biologia Evolutiva, INPA, 1249
Manaus, AM, Brazil 1250 2 Coordenação de Zoologia, MPEG, Belém, PA, Brazil 1251 3 Department of Ornithology, FMNH, Chicago, IL, USA 1252 4 Department of Ornithology, AMNH, New York, NY, USA 1253 5 Coordenação de Biodiversidade, INPA, Manaus, AM, Brazil 1254 *Corresponding author 1255 1256
Correspondence: Mateus Ferreira, Coordenação de Biodiversidade, Instituto Nacional de 1257 Pesquisas da Amazônia, CEP 69080-971, Manaus-AM, Brazil 1258
E-mail: [email protected] 1259 1260
1261
Abstract 1262
The pantropical distribution of trogons always drew attention of biogeographers, with 1263
species distributed all over the forests regions of subtropical and tropical Africa, Asia and 1264
America, several studies tried to reconstruct the phylogenetic relationships without, however, 1265
being able to achieve conclusive results. For the first time, all genera and almost all currently 1266
recognized species, 43 out of 45, were sampled and sequenced for thousands of ultraconserved 1267
elements (UCE) to reconstruct the family phylogenetic hypothesis. We analysed the 1268
concatenated dataset using different treatments for missing data with RAxML and ExaBayes, 1269
we also estimated a species tree using SVDquartets. We also estimated a fossil calibrated time 1270
tree for trogons diversification sampling 177 individuals of the Core Landbirds for RAG1 and 1271
RAG2 genes. Our results were congruent among all methods with high nodal support, 1272
disagreement between treatments (Species Tree x concatenated) were observed only at the basal 1273
40
nodes. In general, our results support the monophyly of the different biogeographical regions, 1274
with Apaloderma species being sister to the Asian (Harpactes and Apalharpactes) and the 1275
Neotropical trogons (Euptilotis, Pharomachrus, Priotelus, and Trogon). Trogonidae initial 1276
diversifications occurred around 20 Ma, and continued till the Pleistocene, where most of the 1277
Neotropical species appeared. Based on these results, we proposed how the climate changes 1278
since the Late Oligocene influenced forest distributions and how the establishment of land 1279
bridges between continents helped shape the family diversification. 1280
1281
Introduction 1282
The Trogonidae have some of the most colourful and exquisite plumages among birds. 1283
Representatives of this family, usually known as trogons or quetzals, can be found in forested 1284
tropical and subtropical regions of Africa, Asia and America (Collar 2017). The monophyly of 1285
the family was never questioned due to the morphological homogeneity among species 1286
(Livezey and Zusi 2007; Collar 2017), the most iconic feature that differentiate trogons and 1287
quetzals from other birds is the heterodactyl foot, in which digits 1 and 2 are directed backwards 1288
and the basal half of digits 3 and 4 are fused and directed forward (Maurer and Raikow 1981; 1289
Mayr 2009). However, it is precisely this unique feature that makes trogons so difficult to relate 1290
with extant birds. Despite several attempts to reconstruct the relationship between trogons and 1291
other birds, most of the morphological (Cracraft 1981; Maurer and Raikow 1981; Mayr 2003; 1292
Livezey and Zusi 2007) and the first molecular analyses (Monteros 2000; Hackett, et al. 2008; 1293
McCormack, et al. 2013) were unable to recover conclusive results about their phylogenetic 1294
relationships. Only recently, employing genomic representations, trogons were shown to be a 1295
sister group to a clade containing mousebirds (Coliiformes), cuckoo rollers (Leptosomiformes) 1296
and other Core Landbirds (Jarvis, et al. 2014; Prum, et al. 2015). 1297
41
Although the relationship with other birds is partially resolved, the relationships within 1298
the family are still pending conclusive results. Historically, the genera and species within each 1299
biogeographic region were considered monophyletic. The highest diversity is found in the 1300
Neotropical region, with four genera, Euptilotis, Pharomachrus, Priotelus and Trogon, and ~30 1301
species ranging from southwestern USA to northern Argentina. The Indo-Malaysian region 1302
comprises 2 genera, Apalharpactes and Harpactes, and 12 species, ranging from southern India, 1303
Southeast Asia, Philippines, the Malay Peninsula, Borneo, Philippines, Sumatra and Java, while 1304
the African region includes only one genus, Apaloderma and tree species. Although trogons are 1305
currently found only in tropical and subtropical regions, fossil records indicate that they had a 1306
wider distribution in the past. Two fossils from Europe, Primotrogon wintersteini (Mayr 1999) 1307
from the Middle Oligocene, and ?P. pumilio (Mayr 2005), from the Middle Eocene, are credited 1308
to be sister group to all other extant species (Mayr 2009). Whereas Septentrogon madseni 1309
(Kristoffersen 2002), from the transitional Paleocene-Eocene Fur Formation in north-western 1310
Denmark shares morphological characteristics that put him inside the Trogonidae. The presence 1311
of these fossils in Europe suggests a widespread lineage occurring in regions that are currently 1312
unsuitable for them. The similarity between fossils and extant trogons also indicates that this 1313
lineage suffered little morphological changes through time. This apparent conservatism of 1314
morphological characteristics also makes the inferences of phylogenetic relationships among 1315
extant species difficult. 1316
The first molecular phylogenetic hypothesis for trogons was based on two mitochondrial 1317
genes and included 20 out of the ca. 45 species (Monteros 1998). This study supported the 1318
hypothesis of monophyly of the biogeographic regions, recovering the Neotropical genera sister 1319
to the Asian, with the African clade sister to these two (Monteros 1998). Following studies that 1320
increased the number of genes and/or samples, however, couldn’t recover the monophyly of the 1321
Neotropical genera, nor the relationship among the different regions (Johansson and Ericson 1322
42
2005; Moyle 2005; DaCosta and Klicka 2008; Hosner, et al. 2010). The most recent paper 1323
(Hosner, et al. 2010), and the first one to sample the genus Apalharpactes, recognized six clades 1324
(Apaloderma, Apalharpactes, Harpactes, Pharomachrus/Euptilotis, Priotelus, and Trogon) 1325
with uncertain relationships among them, but showing evidences of Apalharpactes being more 1326
closely related with the African Apaloderma, than to the other Asian genus, Harpactes, 1327
implying a very complex biogeographical pattern, with two independent colonizations of Asia. 1328
A similar pattern suggested for the Neotropical genera, which group three distinct clades 1329
(Hosner, et al. 2010). 1330
This uncertainty regarding phylogenetic relationships so far was probably related to the 1331
scarcity of signal due to a low number of loci employed in previous studies. Genomic analyses 1332
using a reduced representation of the genome can increase phylogenetic information and avoid 1333
confounding the histories of single genes with the species relationships (Degnan and Rosenberg 1334
2009; Knowles 2009). Also, since the correct interpretation of biotic evolution can shed light 1335
on the landscape evolution (Baker, et al. 2014), a robust and well supported phylogenetic 1336
hypothesis is of extreme importance for defining hypothesis in biogeography (Donoghue and 1337
Moore 2003; Lexer, et al. 2013). In this sense, a prominent approach to study systematics using 1338
genomic markers is the use of probes for Ultraconserved Elements (UCE)(Faircloth, et al. 2012; 1339
McCormack, et al. 2012; McCormack and Faircloth 2013; McCormack, et al. 2013; Faircloth, 1340
et al. 2015). These probes, have been employed to reconstruct deep (Faircloth, et al. 2015; 1341
Moyle, et al. 2016; Branstetter, et al. 2017; Esselstyn, et al. 2017) and shallow (Bryson, et al. 1342
2016; Manthey, et al. 2016) phylogenetic relationships, even where high incomplete lineage 1343
sorting is expected, such as in cases of rapid evolutionary radiation (Meiklejohn, et al. 2016). 1344
Therefore, trogons represent a great study model on how genomic representation may 1345
elucidate uncertain phylogenetic relationships, and to understand how the landscape evolution 1346
shaped the family diversification, due to its pantropical geographic distribution and preference 1347
43
for forested habitats. Here, we aim (1) to generate and unprecedent and robust analyses of 1348
phylogenetic relationships within the Trogonidae family, using nearly complete sampling of all 1349
recognized speces and a genomic representation of more than 2,000 UCE loci, (2) to investigate 1350
the monophyly of main biogeographical regions, and (3) to reconstruct a calibrated tree to infer 1351
the timing of diversification, and how it was influenced by the global events on geography and 1352
climate. 1353
1354
Results 1355
UCE sequencing 1356
The reference sequences we extracted from the Apaloderma vittatum genome (Gilbert, 1357
Jarvis, Li, Consortium, et al. 2014) included 2,228 loci. The mean number of sequences for 1358
each individual was 2,080,592, and a mean number of UCE loci was 2,222, with only one toe 1359
pad sample (AMNH 322898) recovering less than 2000 loci (Table 1). The complete matrix 1360
contained 1421 loci, with mean locus length of 510.27 base pairs, and a total of 37,880 1361
parsimony informative (PI) sites, mean of 26.6 per locus (Table 2). The incomplete matrices 1362
with 95% and 75% completeness have 2,210 and 2,217 loci, with mean locus length of 499.77 1363
and 495.95 base pairs, and 55,060 and 57,259 PI sites, with mean of 24.91 and 25.83 sites per 1364
locus (Table 2). 1365
Phylogenetic inference 1366
The tree topologies were congruent among all methods and with high node support, apart 1367
from the SVDq analyses, in which the basal nodes presented low support. The concatenated 1368
RAxML and ExaBayes phylogenies recovered the Asian trogons sister to the Neotropical, and 1369
these two sisters to the African clade with high support (Fig. 1). All the ExaBayes analyses, 1370
including the complete and the two incomplete datasets, recovered the same topology with all 1371
nodes with the maximum posterior probability (Fig. 1). Although the topologies recovered by 1372
44
RAxML trees were congruent with ExaBayes, some of the basal nodes received low support. 1373
The same was observed with SVDq. 1374
Within the Asian group, Apalharpactes was sister to Harpactes, but with low support in 1375
the RAxML (Table 3) analyses. Within Harpactes we recovered three groups: (1) the distinct 1376
H. oreskios; (2) the two small-bodied species H. duvaucelli and H. orrhophaeus; and (3) the 1377
large-bodied species, containing the other species, with clearly defined and high support 1378
supported relationships (Fig. 1). The Neotropical clade was recovered with high nodal support 1379
(Table 3), showing the quetzals, Euptilotis and Pharomachrus, as sister to Priotelus and Trogon 1380
(Fig. 1). Pharomachrus moccino, the only Central America species, is sister to all other 1381
Pharomachrus species. The two Andean species, P. antisianus and P. auriceps, are not closely 1382
related (Fig. 1). Within Trogon, the most diverse genus in the family, we recovered 5 clades, 1383
all of which include species at both sides of the Andes (Fig. 1). 1384
Time-calibrated tree 1385
The concatenated matrix of RAG1 and RAG2 sequences includes 4757 base pairs for 177 1386
representatives of the Core Land birds (Claramunt and Cracraft 2015; Prum, et al. 2015) 1387
(Supplementary Table 1). Phylogenetic analysis of this matrix recovered a well-supported tree. 1388
Trogonidae diversification started in the Early Miocene, the first of four divergence events are 1389
close to each other, around 20 Ma (Fig. 2). While the Asian species originated during the Late 1390
Miocene/ Early Pliocene, most Neotropical species originated during the Late 1391
Pliocene/Pleistocene (Fig. 2). 1392
1393
Discussion 1394
Phylogenomic contribution to the reconstruction of Trogonidae diversification 1395
Recovering basal relationships in the Trogonidae phylogeny has proven to be challenging, 1396
and previous studies have failed to resolve the relationships among genera (Monteros 1998; 1397
45
Mayr 2003; Johansson and Ericson 2005; Moyle 2005), either because of incomplete taxon 1398
sampling or inadequate number of markers. Monteros (1998) using only two mtDNA genes 1399
recovered a tree topology similar to the one we recovered, in which taxa from different 1400
biogeographical regions were monophyletic. However, the relationships among genera were 1401
not well supported, and Apalharpactes was not sampled. Johansson and Ericson (2005), and 1402
then Moyle (2005), increased the sampling and added a few nuclear introns, yet there were few 1403
improvements in phylogenetic resolution. Moyle (2005) recovered a paraphyletic Neotropical 1404
group, with the quetzals being sister to all other genera, and the Asian and African group sister 1405
to each other embedded within Trogon and Priotelus. Johansson and Ericson (2005) based on 1406
a combined analysis of mtDNA and three nuclear introns recovered a topology similar to ours, 1407
however, node support for the Neotropical group, and the node grouping Asia and the 1408
Neotropics, received low to moderate support. Hosner, et al. (2010) were the first to include an 1409
Apalharpactes sample, but their results were also inconclusive, as relationships among genera 1410
were poorly supported and biogeographical groups, except for Africa, were not monophyletic. 1411
Our phylogenetic results were the first to recover with moderate to high support the 1412
relationship of almost all currently recognized species, as our analyses recovered most of the 1413
nodes with high statistical support (Fig. 1). The nodes that did not receive full support at the 1414
base of the tree (Table 3) are connected by short internodes, probably as a result of an ancient 1415
rapid radiation (Whitfield and Lockhart 2007). Recurrent issues arising from rapid radiations 1416
usually include incomplete lineage sorting (ILS), represented by conflict among gene trees due 1417
to successive events of speciation in short periods of time, which can be accentuated by large 1418
population sizes (Oliver 2013; Suh, et al. 2015). ILS probably was also the main cause of low 1419
support in previous studies that employed few genetic markers, as they could have conflicting 1420
histories (Knowles 2009; Oliver 2013) and probably lacked strong phylogenetic signal to 1421
recover the deep phylogenetic relationships (Salichos and Rokas 2013). Evidence of gene tree 1422
46
incongruence was strongly observed in the whole-genome analysis of bird diversification, 1423
where there was no single gene tree that fully corroborated the combined topology (Jarvis, et 1424
al. 2014). However, counterintuitive, increasing the number of markers does not necessarily 1425
means an improvement in poorly supported nodes. Instead, expanding the number of markers 1426
increases the probability of discordance among them (Oliver 2013), and thus, notably in events 1427
of rapid radiation, some divergences are expected not to behave as a fully bifurcating tree, but 1428
more like a network (Bapteste, et al. 2013; Suh, et al. 2015) because most genes will have 1429
discordant histories due to ILS (Degnan and Rosenberg 2006). Therefore, concatenation may 1430
be the best approach when the number of possible sites supporting a relationship is concentrated 1431
in a few loci diluted in a high number of loci affected by ISL (Gatesy and Springer, 2014; 1432
Springer and Gatesy, 2016). Nonetheless, based on our results, after the first events of 1433
diversification, most of nodes were recovered with high statistical support for all analysis, 1434
including the Neotropical node, which means that, even though we probably do not have enough 1435
confidence to allege the correct order of events that trogons went through their initial 1436
diversification, we may still infer some hypothesis based on current distribution and ecology. 1437
Diversification and biogeography of Trogons 1438
Trogons are still-hunting predators feeding on insects or small vertebrates, but most of 1439
Asian and Neotropical species also feed on fruits, with quetzals being mostly frugivores. They 1440
inhabit the midstory and canopy of tropical and subtropical forest, with some species occurring 1441
in forested patches of open habitats (e.g. Trogon curucui). Most species are territorialists, with 1442
small territories, and lack the capacity to fly over long distances, usually flying from perch to 1443
perch in short sallies (Collar 2017). The morphological conservatism of fossils compared to 1444
extant species suggests that trogons have not underwent large ecological shifts (Mayr 1999, 1445
2003; Mayr 2005), hence their historical distribution probably was affected by the distribution 1446
of suitable habitats through time. Although nowadays there is no continuous patch of suitable 1447
47
habitats, i.e. forested habitat, between Africa, Asia and America, during the Early Miocene, due 1448
to a warmer climate, most of the dry land was covered by forest habitats, such as the broad-leaf 1449
deciduous (Mixed Mesophytic) forest that covered most of the Northern Hemisphere (Baskin 1450
and Baskin 2016), and forests dominated by deciduous conifers that extended even over the 1451
Article Circle (Jahren 2007; Jahren and Sternberg 2008). 1452
The abundance of forests during the Tertiary is due to both warmer temperatures and 1453
twice the current amount of CO2 concentrations (Zachos, et al. 2001). However, after the 1454
Eocene Climatic Optimum (52 to 50 Ma), in which global mean temperatures were 8-10°C 1455
higher, the world temperature started to cool down with two climatic aberrations, where the 1456
amount of ice in polar regions increased drastically. The first one, known as Oi-1, happened 1457
just above the limits between Eocene and Oligocene (34 Ma) (Zachos, et al. 2001), this 1458
glaciation event caused rapid expansions of Antarctic continental ice-sheets and global 1459
temperatures remained low until a warming trend at the end of Oligocene (Zachos, et al. 2001). 1460
This warm phase that followed extended from the Late Oligocene until middle Miocene (~15 1461
Ma) with the Mid-Miocene Climatic Optimum (17 to 15 Ma) and it was followed by a gradual 1462
cooling, with the culmination in the Glacial cycles throughout the Plio/Pleistocene (Zachos, et 1463
al. 2001). The second aberration, Mi-1, happened during this warm period at the end of the 1464
Oligocene (~23 Ma), and was followed by a series of glaciation events (Zachos, et al. 2001), 1465
period well within the confidence interval for the initial diversification events we recovered in 1466
our time calibrated phylogeny. Both aberrations probably influenced the distribution and rates 1467
of diversification in some groups that have similar distributions as trogons, such as ferns 1468
(Bauret, et al. 2017; Hennequin, et al. 2017), and flowering plants (Li, et al. 2017). Interestingly, 1469
other groups of birds that have similar distributions present different patterns of diversification 1470
than trogons; woodpeckers (Aves: Picidae) and kingfishers (Aves: Alcedinidae) apparently 1471
have dispersed to the New World from the Old World more than once, however these events 1472
48
seem to be younger than those we recovered for trogons, around 15 to 5 Ma for woodpeckers 1473
(Shakya, et al. 2017), and 10 to 5 Ma for kingfishers (Andersen, et al. 2017). This pattern 1474
suggests that dispersal between Asia and America was possible during a long period of time, 1475
probably experiencing cycles of connection and disconnection due to climatic variations 1476
(Zachos, et al. 2001). Therefore, our temporal framework supports an ancestral lineage 1477
distributed over the Palearctic region (Claramunt and Cracraft 2015), with dispersal to Asia, 1478
Africa and America during a short period of time, causing the poorly supported nodes we 1479
observed in our analysis. 1480
Africa and Asia diversification 1481
Even though African and Asian linages are as old as the Neotropical, only 6% and 31% 1482
of species diversity are found in these areas, respectively. Although contentious, there are 1483
probably many reason for the uneven diversity among areas. Monteros (1998) suggests that 1484
competitive exclusion might play a role in this pattern, as African and Asian trogons need to 1485
compete with other groups of frugivores birds, such as mousebirds (Colliformes), hornbills 1486
(Bucerotidae), barbets (Megalaimidae and Lybiidae), turacos (Musophagidae), and several 1487
families of passerines (Irenidae, Pycnonotidae, etc). While the Neotropical trogons are, along 1488
cotingas (Cotingidae) and toucans (Ramphastidae), one of the most important family for seed 1489
dispersal in this region (Collar, et al. 2017). 1490
Inside Africa, except for Apaloderma narina which has six recognized subspecies, the 1491
other two, A. vittatum and A. aequatoriale are monotypic (Collar 2017). However, no 1492
phylogeographic study was conducted to evaluate genetic structure within these species, with 1493
recent studies using other organism as models showing shallow genetic structure probably 1494
originated by aridification of the continent as a response of Plio/Pleistocene climatic 1495
fluctuations (Bowie, et al. 2004; Bowie, et al. 2006; Voelker, et al. 2010). The diversification 1496
event we recovered between A. vittatum and A. narina happened around 7.4 Ma (Fig. 2) and 1497
49
precedes the beginning of the most drastic climatic fluctuations of the Pliocene, making any 1498
assumption of what may have caused this very hard, in particular considering that Africa has 1499
been geomorphologically stable for the last 40 Ma (Potts and Behrensmeyer 1992). Also, A. 1500
vittatum inhabits the montane forests, while A. narina and A. aequatoriale, inhabits the 1501
lowlands, and although we could not sample A. aequatoriale, previous work recovered it as 1502
sister species to A. narina (Hosner, et al. 2010). Suggesting that other mechanisms may be 1503
responsible for Apaloderma species diversification (Moritz, et al. 2000). 1504
In contrast with previous studies (Hosner, et al. 2010), our analyses recovered the 1505
monophyly of Asian trogons. Although the bootstrap support was moderate for this node in the 1506
likelihood analysis, it was recovered with high statistical support in the Bayesian analysis 1507
(Table 3). This suggest that after the initial diversification of the family, at least two Paleartic 1508
lineages (Claramunt and Cracraft 2015) colonized the Sundaland, the continental shelf that 1509
extended from SE Asia and comprises the Malay Peninsula, and the islands of Borneo, Java, 1510
and Sumatra. The time of diversification we found for Apalharpactes and Harpactes is 1511
consistent with the Hymalayan uplift acceleration, derived from India-Asia continental collision 1512
(Hall 2012; Hu, et al. 2017), and with the intermittent glaciations that followed the Mi-1 1513
glaciation at the Oligocene-Miocene boundary (Zachos, et al. 2001). These two events 1514
combined may have shaped Asian trogons diversification, however, making assumptions about 1515
Haparctes diversification involves a very complex history, and it is difficult based on extant 1516
species distribution to make any assumption about possible biogeographic barriers. Current 1517
geography of SE Asia and the Sunda islands can be misleading, the Sunda shelf was once 1518
exposed and covered by forest (Hall 2012; Bruyn, et al. 2014), and sea-level fluctuations were 1519
responsible for islands “formation” and connectivity, especially during the climatic fluctuations 1520
of the Pleistocene (Woodruff 2010). This mechanism is suggested as a possible explanation for 1521
Southeast Asia bird diversification (Lim, Rahman, et al. 2010; Lim, Zou, et al. 2010; Lim, et 1522
50
al. 2017). However, most of the Harpactes diversification events precede the Pleistocene, and 1523
occurred between the Mid-Miocene Climatic Optimum (17-15 Ma) (Zachos, et al. 2001) and 1524
the Early Pliocene, much older than the diversification events of the Neotropical clade, for 1525
example. The only phylogeographic study conducted so far, with the Philippine Trogon 1526
(Harpactes ardens), demonstrated geographical structure among different island matching 1527
subspecies distribution (Hosner, et al. 2014), whereas H. kasumba, H. diardii and H. 1528
erythrocephalus showed little to no genetic variation in the mtDNA for the few samples used 1529
(Hosner, et al. 2010). Therefore, further studies, with broad sampling are necessary to 1530
understand how the Pleistocene climate, and sea level fluctuation, influenced population 1531
structure, which in turn may shed some light on the initial diversification of this genus. 1532
Neotropical diversification 1533
For the first time, Neotropical trogons were recovered as a monophyletic group with high 1534
statistical support (Monteros 1998; Johansson and Ericson 2005; Moyle 2005; Hosner, et al. 1535
2010). Although most of extant diversity is currently found in Central and South America, 1536
trogons arrived first in the Americas through the Beringia Bridge, northwest North America, 1537
and colonized the whole west coast, during a period when there were vast forests covering 1538
North America (Baskin and Baskin 2016). Therefore, tracing back the events related with the 1539
initial divergences would require extensive palaeontological investigation. The overall trend we 1540
observe in this clade diversification is that Central American lineages occupied South America 1541
through the Panamanian Isthmus, and most of divergence events postdate the Mid-Miocene 1542
Climatic Optimum (17-15 Ma), which marks the beginning of the cooling trend that escalated 1543
to the Plio-Pleistocene glaciations. Also during this period, there was extensive orogenic 1544
activity in Mexico, including the uplift of Sierra Madre Occidental (34 – 15 Ma) (Ferrari, et al. 1545
2007) and the formation of the Trans-Mexican Volcanic Belt (35 – 2.5 Ma) (Ferrari, et al. 2000). 1546
Both events triggered climatic changes, which in turn influenced the establishment of major 1547
51
biomes in Mexico (Ferrari, et al. 1999), that have been shown to have influenced diversification 1548
in Amazillia hummingbirds (Ornelas, et al. 2014), and some plants (Lavin, et al. 2004; Becerra 1549
2005; Arakaki, et al. 2011). 1550
Another major event that shaped Neotropical trogons diversification was the 1551
establishment of the connection between North and South America, through the uplift of the 1552
Isthmus of Panama. The Great American Biotic Interchange allowed inter-continental exchange 1553
of biotas that were previously isolated in both continents and is of great importance for shaping 1554
bird assemblages and diversification (Weir, et al. 2009; Smith and Klicka 2010). Early studies 1555
suggested that the connection was only fully established at 3 Ma (Haug and Tiedeman 1998; 1556
Coates and Stallard 2013; Odea, et al. 2016), however, even though contentious in the literature 1557
(Farris, et al. 2011; Montes, et al. 2012; Bacon, et al. 2013; Bacon, et al. 2015a, b; Hoorn and 1558
Flantua 2015; Lessios 2015; Montes, et al. 2015; Odea, et al. 2016), this date was broadly used 1559
as a calibration point in phylogenetic studies attempting to integrate and synthesize patterns of 1560
dispersion across the Isthmus (review in Bacon, et al. (2015a)). Our results suggest that trogon 1561
dispersion across the Isthmus started as early as 6.5 Ma, with the split of Pharomachrus 1562
moccino from the other Pharomachrus species, and happened at least six additional times 1563
within Trogon diversification, all of them after 4 Ma. These results are also supported by a 1564
former study using only one mitochondrial marker for Trogon (DaCosta and Klicka 2008). 1565
Finally, the most notorious accomplishment of Neotropical trogons was to colonize the 1566
Greater Antilles. The genus Priotelus, which includes species endemic to the islands of Cuba, 1567
P. temnurus, and Hispaniola, P. roseigaster, split from Trogon around 17 Ma (Fig. 2). Trogons 1568
are well known for being weak fliers, so the chances of the ancestor of Priotelus to have 1569
dispersed through the ocean to colonize not just one, but two Caribbean islands are low. One 1570
possible explanation is the land bridge that once connected Central America to South America, 1571
known as GAARlandia (Greater Antilles + Aves Ridge) land bridge (Iturralde-Vinent 1994, 1572
52
2006). Although this land connection is credited to be much older (35 – 33 Ma) (Alonso, et al. 1573
2011; Rícan, et al. 2013; Nieto-Blázquez, et al. 2017) than the split of Priotelus and Trogon, 1574
during the Middle-Late Miocene, the emerged islands that were part of the land bridge were 1575
still connected by shallow seas (Iturralde-Vinent 2006), and sea levels fluctuations may have 1576
facilitated the dispersal to these islands. Fabre, et al. (2014) studying Caribbean rodents found 1577
a similar age (16.5 Ma) for the subfamily of rodents that occupy the Greater Antilles. However, 1578
the sister group is from South America, and the authors suggested that the ancestor of this group 1579
colonized the Caribbean Islands via rafting. Our results imply in a more complex scenario for 1580
the Greater Antilles colonization, and further studies are required to evaluate this late 1581
connection. 1582
1583
Conclusion 1584
In this study we recovered the phylogenetic relationships among almost Trogonidae taxa 1585
using a genomic approach. Coupled with our fossil calibrated time tree, we were able to propose 1586
a model of diversification that related not only how the climate change since the Late Oligocene, 1587
but also the connections between continents, shaped the family diversification. The monophyly 1588
of the different biogeographical regions was recovered, and even though some nodes at the base 1589
of the tree received low support, the pattern of rapid radiation is clear at the initial stages of 1590
trogons diversification. Also, even though trogons are currently restricted to subtropical and 1591
tropical regions, they were widespread lineages in the past, and their diversification was 1592
influenced by forest distribution through time. Our results also identified some interestingly 1593
new questions to be pursued: Are Neotropical trogons species really younger than African and 1594
Asian, or is it just a sampling artifact? What was the influence of past sea level fluctuations in 1595
the diversification of Harpactes? Is competition preveting diversification in Apaloderma? 1596
1597
53
Materials and Methods 1598
Taxon sampling and DNA extraction 1599
We sampled 48 individuals comprising all genera and currently recognized species of the 1600
Trogonidae family, except for the African Bare-cheeked Trogon (Apaloderma aequatoriale), 1601
and the narrow endemic Javan Trogon (Apalharpactes reinwardtii) (Collar 2017; Gill, et al. 1602
2018; Remsen, et al. 2018). All samples are represented by voucher specimens deposited in 1603
ornithological collections at the American Museum of Natural History (AMNH), Academy of 1604
Natural Sciences of Drexel University (ANSP), Field Museum of Natural History (FMNH), 1605
Instituto Nacional de Pesquisas da Amazônia (INPA), Kansas University (KU), Laboratório de 1606
Genética e Evolução Molecular de Aves - USP (LGEMA), Louisiana Museum of Natural 1607
History (LSUMZ), Museu Paraense Emílio Goeldi (MPEG), Smithsonian Institution National 1608
Museum of Natural History (USNM) and Burke Museum (UWBM) (Appendix S1). 1609
DNA from fresh tissue was extracted with the DNeasy kit (Qiagen Inc.), following the 1610
manufacture’s protocol. For taxa lacking fresh tissues we cut toepad clips from museum 1611
specimens with a sterile surgical blade and processed in a dedicated room for ancient DNA 1612
(aDNA Lab, AMNH). Toepads were rinsed with 100% ethanol, and ultra-pure water prior to 1613
digestion to remove any inhibitor that could cause problems in downstream procedures. We 1614
then extracted DNA with the DNeasy kit (Qiagen Inc.), replacing the regular silica columns 1615
with the QIAquick columns, to ensure maximum DNA yield. All extracts were sent to Rapid 1616
Genomics (Gainsville, FL) for library prep and target-capture sequence 2321 loci of 1617
Ultraconserved Elements (UCE) plus 98 conserved exons from 46 genes that were previously 1618
employed in phylogenetic analyses (Hackett, et al. 2008; Kimball, et al. 2009; Harvey, et al. 1619
2017). 1620
UCE and exons assembly 1621
54
The raw sequence data were processed with the Phyluce script pack (Faircloth 2016). We 1622
employed illumiprocessor (Faircloth 2013) and Trimmomatic (Bolger, et al. 2014) to remove 1623
adapter contamination and low-quality reads. To assemble a reference genome, we mapped the 1624
UCE and exons probes back to the Apaloderma vittatum genome (Gilbert, Jarvis, Li, 1625
Consortium, et al. 2014) using the script phyluce_probe_run_multiple_lastzs_sqlite, and then, 1626
phyluce_probe_slice_sequence_from_genomes to extract the probe region plus 500 base pairs 1627
from each flanking region. Apaloderma exonic regions were identified based on the Gallus 1628
gallus genes, and annotations of CDS and exons were copied to the reference sequences inside 1629
Geneious version R10.2.3 (Kearse, et al. 2012). With these sequences as a reference we mapped 1630
back the clean reads of each individual employing Bowtie2 (Langmead and Salzberg 2012) 1631
plugin 7.2.1 inside Geneious. The consensus sequences were called with the highest quality 1632
threshold and a depth of at least 4 reads. Each locus was aligned with MAFFT (Katoh and 1633
Standley 2013) under default parameters. 1634
Phylogenetic relationships and species tree analysis 1635
Since the intergeneric relationship among trogons are still mostly unresolved (Monteros 1636
1998; Johansson and Ericson 2005; Moyle 2005; Hosner, et al. 2010), we first performed a 1637
maximum likelihood analyses in RAxML v8.2 (Stamatakis 2014), and a Bayesian Inference 1638
analyses in ExaBayes v.1.4 (Aberer, et al. 2014), using the concatenated matrix with three 1639
treatments for missing data: a complete matrix, where no missing data was allowed, and two 1640
where the missing data was allowed, a 95% and 75% completeness matrix, in which each locus 1641
should have at least 95% or 75%, respectively, of all individuals in the matrix. As outgroups 1642
we selected one mousebird (Colius striatus, (Gilbert, Jarvis, et al. 2014b)), and a roller 1643
(Leptosomus discolor, (Gilbert, Jarvis, et al. 2014a)), suggested by recent studies as the closest 1644
relatives to the Trogonidae family (Jarvis, et al. 2014; Prum, et al. 2015). We also estimated a 1645
species tree using the SVDquartets (Chifman and Kubatko 2014) implemented in PAUP* 1646
55
v4a(build157) (Swofford 2002), that samples quartets of individuals for each gene tree and infer 1647
an unrooted phylogeny, performing a species tree using a coalescent approach. We 1648
exhaustively sampled all quartets and performed a 100 bootstrap to quantify the support for 1649
each node. 1650
Dating analysis 1651
To date the Trogonidae phylogeny we employed the slow evolving recombination-1652
activating genes (RAG-1 and RAG-2) and a dense sampling for the Core Landbirds group 1653
(Telluraves), with the same calibration points used by Claramunt and Cracraft (2015). The 1654
concatenated matrix was partitioned by codon and the best partition and substitution model 1655
schemes were selected by PartitionFinder2 (Lanfear, et al. 2017). 1656
1657
Acknowledgements 1658
The authors thankfully acknowledge all the curators and curatorial assistants of the 1659
American Museum of Natural History, New York, USA (AMNH), Academy Academy of 1660
Natural Science of Drexel University, Philadelphia, USA (ANSP); Field Museum of Natural 1661
History, Chicago, USA (FMNH); Instituto Nacional de Pesquisas da Amazônia, Manaus, Brazil 1662
(INPA); Kansas University (KU), Laboratório de Genética e Evolução Molecular de Aves – 1663
USP (LGEMA), Lousiana State University Museum of Natural Science, Baton Rouge, USA 1664
(LSUMZ); and Museu Paraense Emílio Goeldi, Belém, Brazil (MPEG), Smithsonian Institution 1665
National Museum of Natural History (USNM), for borrowing tissue samples under their care. 1666
We are also grateful for all collectors involved in the fieldwork that make this paper possible. 1667
We thank L. Moraes for early input on this paper. MF acknowledge CAPES for his PhD 1668
fellowship, and CAPES PDSE fellowship (# 88881.133440/2016-01) and the support from the 1669
AMNH Frank M. Chapman Memorial Fund. The authors also thanks the grant Dimensions US-1670
Biota-São Paulo: Assembly and evolution of the Amazon biota and its environment: an 1671
56
integrated approach, co-funded by the US National Science Fundation (NSF DEB 1241056) to 1672
J.C. and the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP grant 1673
#2012/50260-6) to Lucia Lohmann. AA and CCR are supported by CNPq research productivity 1674
fellowships. The authors acknowledge the National Laboratory for Scientific Computing 1675
(LNCC/MCTI, Brazil) for providing HPC resources of the SDumont supercomputer, which 1676
have contributed to the research results reported within this paper. 1677
1678
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Table 1 – Samples used in this study, the museum voucher numbers, locality and geographical coordinates 1966 (when available), number of UCE reads, and loci recovered for each sample. 1967
Species Museum voucher Locality Clean
reads
UCE
loci
Apaloderma vittatum SRP028834 Tanzania: Udzungwa Mts. - 2,228*
Apaloderma narina AMNH DOT-12430 Liberia: Lofa, Ziggida (08°02'15.5"N 9°31'49.5"W) 3,607,056 2,228
Apalharpactes mackloti LSUMZ B-49104 Indonesia: Sumatra 1,664,511 2,220
Apalharpactes mackloti AMNH 633881 Indonesia: Sumatra, Bandar-Baroe (03°15'57.6''N 98°30'49.9''E) 2,758,684 2,080
Harpactes ardens USNM 607340 Philippines: Barrio Via, Sitio Hot Springs, Baggao Mun. (17°50'N,
122°01'E) 1,193,041 2,208
Harpactes diardii AMNH DOT-563 Malaysia: Sabah, Klias Forest Reserve (05°19’34’’N
115°40’25’’E) 3,601,173 2,226
Harpactes oreskios ANSP 16308 Malaysia: Sabah, Mendolong (04°54'27.6"N 115°47'04.5"E) 5,208,017 2,228
Harpactes orrhophaeus AMNH DOT-15159 Malaysia: Sabah, Mt. Lucia (04°27’37.8’’N 117°55’20.4’’E) 4,250,801 2,228
Harpactes duvaucelli LSUMZ B-38592 Malaysia: Sabah, Imbak Valley, ca 60 km S Telupid (5°06’N
117°01’51’’E) 887,312 2,222
Harpactes fasciatus AMNH 778649 India: Dangs, Bhawandagad 5,386,424 2,218
Harpactes erythrocephalus AMNH DOT-12240 Vietnam: Quang Nam, Ngoc Linh Range (15°11’00’’N
108°02’00’’E) 2,126,329 2,224
Harpactes wardii AMNH 307761 Myanmar: Laukkaing 5,151,969 2,198
Harpactes whiteheadi LSUMZ B-52627 Malaysia: Sabah, Tambuman, Mt. Trus Madi (05°35’09’’N
116°29’26’’E) 11,299,280 2,228
Harpactes kasumba AMNH DOT-15326 Malaysia: Sabah, Ulu Tungud Forest Reserve, Melian Range
(05°50’48’’N 117°10’57’’E) 4,264,359 2,228
Euptilotis neoxenus AMNH DOT-11080 USA: Arizona, Ramsey Canyon Preserve (31°26'50.2"N
110°18'25.8"W) 1,955,116 2,186
Pharomachrus pavoninus INPA A-1993 Brazil: Amazonas, Parque Nacional do Jaú (01°49’50’’S
61°35’45’’W) 2,080,592 2,215
Pharomachrus auriceps
hargitti AMNH 175988 Ecuador: Baeza, Arriba (0°27’54’’S 77°53’44.9’’W) 6,034,956 2,210
Pharomachrus auriceps
auriceps FMNH 473723 Peru: Rodriguez de Mendoza (06°S 77°W) 2,620,376 2,221
Pharomachrus fulgidus AMNH 322895 Venezuela: Near village of Junquito on Colonia Tovar Rd
(10°27’23’’N 67°04’31’’W) 4,665,318 1,864
Pharomachrus moccino AMNH 326512 Honduras: Mt Pucca, Gracias (14°34’43’’N 88°38’30’’W) 5,630,314 2,215
Pharomachrus antisianus ANSP 19429 Ecuador: Napo, 12 km NNE El Chaco; Mirador 5,651,764 2,228
Priotelus temnurus ANSP 20257 Cuba 1,644,934 2,220
Priotelus roseigaster KU 8098 Dominican Republic: Parque Nacional Sierra Baoruco, Pueblo
Viejo (18°12’N 71°32’W) 1,431,709 2,221
Trogon clathratus USNM 613996 Panama: Bocas del Toro, Los Planes (08°35’43’’N 82°14’16’’W) 3,200,785 2,158
Trogon mesurus ANSP 19305 Ecuador: Esmeraldas, 20 km ENE Muisne (0°38’51’’N
79°59’59’’W) 7,341,190 2,142
Trogon massena KU 2073 Mexico: Campeche, Silvituc (18°13’48’’N 90°12’W) 1,689,867 2,224
Trogon comptus LSUMZ B-11829 Ecuador: Esmeraldas, El Placer (0°52’N 78°33’W) 2,072,859 2,228
Trogon melanurus INPA A-1955 Brazil: Amazonas, Parque Nacional do Jaú (01°49’50’’S
61°35’45’’W) 2,451,461 2,225
Trogon viridis INPA A-5240 Brazil: Pará, Aveiro, left bank Tapajós River (03°42.3’S
55°35.5’W) 1,893,902 2,226
Trogon chionurus LSUMZ B-28571 Panama: Colón, Achiote Road (09°13’32’’N 80°0’56’’W) 1,879,103 2,225
Trogon melanocephalus USNM 646857 El Salvador: La Paz, Aeropuerto Internacional El Salvador
(13°25’57’’N 89°03’50’’W) 1,521,530 2,224
Trogon citreolus UWBM 101087 Mexico: Michoacán, Lazaro Cardenas, La Mira (18°05.71’N
102°23.71’W) 1,311,613 2,224
Trogon bardii LSUMZ B-71992 Costa Rica: Osa, Los Charces (08°40’19’’N 83°30’19’’W) 2,036,944 2,226
Trogon violaceus MPEG CN437 Brazil: Pará, Alenquer, ESEC Grão-Pará (0°09’S 55°11’W) 1,251,316 2,222
Trogon caligatus LSUMZ B-66270 Peru: Tumbes, El caucho Biological Station (3°49’25’’S
80°15’37’’W) 4,878,667 2,150
Trogon ramonianus INPA A-5449 Brazil: Pará, Santarém, Rio Arapiuns (3°19’S 55°20’W) 2,665,900 2,228
Trogon curucui INPA A-5286 Brazil: Pará, Aveiro, left bank Tapajós River (3°42.3’S
55°35.5’W) 1,157,694 2,221
Trogon aurantius LGEMA 15782 Brazil: Minas Gerais, RPPN Serra do Caraça (20°07’01’’S
43°29’16’’W) 1,162,924 2,213
Trogon surrucura MPEG SC015 Brazil: Santa Catarina, Blumenau, Vila Itoupava (26°39’59’’S
49°05’41’’W) 2,005,634 2,224
Trogon rufus tenellus LSUMZ B-26564 Panama: Colón, Gamboa (9°09’25’’N 79°45’36’’W) 4,118,529 2,228
Trogon rufus amazonicus INPA A-5284 Brazil: Pará, Aveiro, left bank Tapajós River (3°42.3’S
55°35.5’W) 3,892,857 2,228
Trogon rufus chrysochlorus LGEMA 9557 Brazil: São Paulo, Ubatuba (23°23’24’’S 45°05’24’’W) 1,161,086 2,225
64
Trogon elegans FMNH 434014 El Salvador: Sonsonate: Izalco, Canton Las Laja (13°45’35’’N
89°40’21’’W) 475,853 2,201
Trogon mexicanus FMNH 343220 Mexico: Jalisco, Puerto los Mazos, Sierra de Manantlan
(19°28’09’’N 103°56’51’’W) 1,322,925 2,222
Trogon aurantiiventris LSUMZ B-41625 Panama: Bocas del Toro, Chiriqui (8°47’29’’N 82°12’35’’W) 6,441,454 2,228
Trogon collaris puella FMNH 394272 Mexico: Oaxaca, San Gabriel Mixtepec, Sierra de Miahuatlan
(16°09’56’’N 97°01’29’’W) 292,340 2,114
Trogon collaris collaris MPEG CN450 Brazil: Pará, Alenquer, ESEC Grão-Pará (0°09’S 55°11’W) 1,361,638 2,221
Trogon personatus LSUMZ B-48503 Guyana: Potaro-Siparuni, Kopinang Mountain (4°57’54’’N
59°54’49’’W) 1,826,664 2,228
1968 Table 2 – Summary information of each method, including number of loci, total length of the concatenated 1969 alignment, mean length size per locus, minimum and maximum length, and the total number of the Parsimony 1970 Informative (PI) sites. 1971
Complete 75% 95%
Number of loci 1421 2110 2217
Total lenght 725090 1054512 1099526
Mean length size 510.27 499.77 495.95
Min-max length 259-1145 162-1145 162-1145
Number of PI sites 37,880 55,060 57,259
1972 Table 3 – Node support for recalcitrant nodes in the Trogonidae phylogeny. 1973
RAxML ExaBayes SVDq
75% 95% complete 75% 95% complete 95%
Asian + Neotropical 70 62 84 1.0 1.0 1.0 -
Apalharpactes + Harpactes 60 46 52 1.0 1.0 1.0 -
Neotropical 100 100 100 1.0 1.0 1.0 100
1974
1975
65
Figure 1 – Phylogeny of Trogonidae inferred with ExaBayes summarizing the results from other analyses. The 1976 circle at each node represent the statistical support for the RAxML analyses and the species tree reconstruction 1977 inferred by SVDq. Green lines represent distribution shifts from Central America to South America. Trogon 1978 species were group in five species groups highlighted with grey boxes: “rufus”, “collaris”, “melanurus”, “viridis”, 1979 and “violaceus”. 1980 1981
1982
1983
66
Figure 2 – Time-calibrated phylogeny of Trogonidae inferred from the concatenated dataset of RAG1 and RAG2 1984 genes using BEAST. This tree represents part of the tree calibrated using (Claramunt and Cracraft 2015) 1985 calibrations, complete taxon data in Supplementary Table 1. The basal nodes were constrained to match the UCE 1986 topology, all other nodes have a red circle, if the posterior probability is 1.0, or the posterior is written next to the 1987 node. Timings of major splits are shown next to each node. Blue bars represent the 95% HPD estimates of node 1988 height. Green lines represent distribution shifts from Central America to South America. The top-right figure 1989 represents the whole tree with calibration points as red circles. 1990 1991
1992
1993
67
Supplementary Table S1 – Table containing taxonomic information on all specimens employed in the RAG time 1994 tree. The RAG1 and RAG2 column refers to GenBank accession numbers for these two genes. Taxonomy follows 1995 del Hoyo, et al. (2017). 1996 1997
Order Family Species RAG1 RAG2
Passeriformes Thraupidae Thraupis cyanocephala AY057035 AY443236
Passeriformes Emberizidae Emberiza schoeniclus AY056992 AY443143
Passeriformes Passeridae Passer montanus AF143738 AY443198
Passeriformes Prunellidae Prunella collaris AY057024 AY443213
Passeriformes Dicaeidae Dicaeum aeneum AY443282 AY443139
Passeriformes Regulidae Regulus calendula AY057028 AY443220
Passeriformes Irenidae Irena cyanogaster AY056999 AY443158
Passeriformes Nectariniidae Nectarinia olivacea AY057009 AY443180
Passeriformes Turdidae Catharus ustulatus AY443265 AY443114
Passeriformes Cinclidae Cinclus cinclus AY056985 AY443119
Passeriformes Mimidae Mimus patagonicus AY057005 AY443173
Passeriformes Sturnidae Sturnus vulgaris AY057032 AY443232
Passeriformes Troglodytidae Troglodytes aedon AY057038 AY443241
Passeriformes Certhiidae Certhia familiaris AY056983 AY443115
Passeriformes Sittidae Sitta carolinensis AY443332 AY443227
Passeriformes Sylviidae Sylvia nanna AY057033 AY443233
Passeriformes Pycnonotidae Pycnonotus barbatus AY057027 AY443219
Passeriformes Hirundinidae Hirundo rustica AY443290 AY443154
Passeriformes Aegithalidae Aegithalos iouschensis AY056976 AY443103
Passeriformes Locustellidae Megalurus palustris AY319988 AY799840
Passeriformes Remizidae Remiz pendulinus AY443328 AY443222
Passeriformes Promeropidae Promerops cafer AY443323 AY443212
Passeriformes Monarchidae Monarcha axillaris AY057006 AY443176
Passeriformes Laniidae Lanius excubitor AY443293 AY443160
Passeriformes Artamidae Artamus leucorhynchus AY056980 AY443109
Passeriformes Artamidae Artamus cyanopterus AY443262 AY443108
Passeriformes Artamidae Cracticus quoyi AY443278 AY443135
Passeriformes Vangidae Vanga curvirostris AY057040 AY443244
Passeriformes Platysteiridae Batis mixta AY443263 AY443110
Passeriformes Vireonidae Vireo philadelphia AY057041 AY443245
Passeriformes Melanocharitidae Melanocharis nigra AY057002 AY443167
Passeriformes Melanocharitidae Melanocharis vesteri AY443299 AY443168
Passeriformes Orthonychidae Orthonyx teminckii AY057012 AY443309
Passeriformes Climacteridae Climacteris erythrops AY443268 AY443121
Passeriformes Menuridae Menura novaehollandiae AY057004 AY443171
Passeriformes Furnariidae Furnarius rufus AY056995 AY443149
Passeriformes Rhinocryptidae Scytalopus magellanicus AY443331 AY443226
Passeriformes Thamonophilidae Terenura sharpei JX213518 JX213481
Passeriformes Pipridae Piprites chloris FJ501717 FJ501897
Passeriformes Pipridae Piprites pileata JF970177 KC157559
Passeriformes Pipridae Lepidothrix coronata FJ501655 FJ501835
Passeriformes Pipridae Antilophia galeata FJ501600 FJ501780
Passeriformes Oxyrunchidae Oxyruncus cristatus FJ501689 FJ501878
Passeriformes Cotingidae Cotinga cayana FJ501623 FJ501803
68
Passeriformes Cotingidae Laniisoma elegans FJ501651 FJ501831
Passeriformes Cotingidae Phoenicircus nigricollis FJ501705 FJ501885
Passeriformes Tyrannidae Tyrannus tyrannus AF143739 AY443243
Passeriformes Sapayoidae Sapaoya aenigma DQ320606 DQ320573
Passeriformes Dendrocolaptidae Dendrocolaptes certhia FJ461166 FJ460982
Passeriformes Pittidae Pitta sordida AY443219 AY443206
Passeriformes Acanthisittidae Acanthisitta chloris AY056975 AY443102
Psittaciformes Psittacidae Psittacus erithacus EF517674 EF517687
Psittaciformes Psittacidae Alisterus scapularis KT954426 EF517677
Psittaciformes Psittacidae Melopsittacus undulatus XM_005150647.1 XM_005150646.1
Psittaciformes Psittacidae Micropsitta brujinii EF517673 EF517681
Psittaciformes Psittacidae Amazona aestiva LMAW01003202 LMAW01003202
Psittaciformes Psittacidae Myopsitta monachus DQ143328 -
Psittaciformes Psittacidae Agapornis personata EF517672 EF517679
Psittaciformes Cacatuidae Calyptorhynchus funereus KT954425 EF517680
Psittaciformes Strigopidae Nestor notabilis XM_010020228.1 XM_010020229.1
Falconiformes Falconidae Falco peregrinus AY461399 KT954538
Falconiformes Falconidae Falco cherrug XM_005441067.1 XM_005441068.2
Falconiformes Falconidae Daptrius ater AY461397 KT954537
Falconiformes Falconidae Micrastur gilvicollis AY461403 KT954536
Cariamiformes Cariamidae Cariama cristata XM_009699718.1 XM_009699720.1
Piciformes Ramphastidae Pteroglossus aracari KT954416 KT954525
Piciformes Capitonidae Capito niger KT954414 KT954523
Piciformes Semnornidae Semnornis frantzii KT954415 KT954524
Piciformes Lybiidae Trachyphonus erythrocephalus KT954413 KT954522
Piciformes Lybiidae Lybius hirsutus KT954412 KT954521
Piciformes Megalaimidae Megalaima oorti KT954411 KT954520
Piciformes Picidae Melanerpes carolinus KT954418 KT954527
Piciformes Picidae Picoides pubescens XM_009905561.1 XM_009905562.1
Piciformes Picidae Picumnus cirratus AF295195 -
Piciformes Indicatoridae Indicator variegatus KT954417 KT954526
Piciformes Bucconidae Bucco capensis MPEG_ARA018
Piciformes Bucconidae Nystalus maculatus MPEG_MARJ045
Piciformes Bucconidae Nonnula rubecula INPA_A4705
Piciformes Bucconidae Monasa atra INPA_A8299
Piciformes Bucconidae Chelidoptera tenebrosa MPEG_JTW1160
Piciformes Bucconidae Hapaloptila castanea LSU_12059
Piciformes Bucconidae Micromonacha lanceolata LSU_4489
Piciformes Bucconidae Cyphos macrodactylus MPEG_AMA354
Piciformes Bucconidae Notharchus tectus LSU_28765
Piciformes Bucconidae Hypnellus bicinctus FMNH_339641
Piciformes Bucconidae Nystactes tamatia MPEG_JRT134
Piciformes Bucconidae Notharchus ordii LSU_25460
Piciformes Bucconidae Notharchus hyperrhynchus MPEG_GAPTO296
Piciformes Bucconidae Malacoptila fulvogularis FMNH_321031
Piciformes Bucconidae Malacoptila rufa LSU_103572
Piciformes Galbulidae Jacamalcyon tridactyla MPEG_800
Piciformes Galbulidae Brachygalba lugubris MPEG_293
Piciformes Galbulidae Jacamerops aureus MPEG_JAP375
69
Piciformes Galbulidae Galbacyrhynchus purusianus INPA_A1429
Piciformes Galbulidae Galbula dea INPA_A2288
Piciformes Galbulidae Galbula leucogastra MPEG_AMZ190
Piciformes Galbulidae Galbula ruficauda MPEG_MARJ109
Piciformes Galbulidae Galbula cyanescens MPEG_PUC159
Piciformes Galbulidae Galbula albirostris MPEG_JAP616
Piciformes Galbulidae Galbula cyanicollis MPEG_FLJA056
Coraciformes Alcedinidae Chloroceryle americana KT954422 KT954533
Coraciformes Alcedinidae Halcyon malimbica DQ111819 KT954532
Coraciformes Alcedinidae Alcedo leucogaster DQ111794 KT954531
Coraciformes Momotidae Momotus momota KT954421 KT954530
Coraciformes Todidae Todus angustirostris KT954420 KT954529
Coraciformes Coraciidae Coracias caudata AF143737 AY443126
Coraciformes Brachypteracidae Brachypteracias leptosomus KT954423 KT954534
Coraciformes Meropidae Merops pusillus KT954419 KT954528
Coraciformes Meropidae Merops nubicus XM_008938323.1 XM_008938322.1
Bucerotiformes Upupidae Upupa epops KT954409 KT954517
Bucerotiformes Phoeniculidae Phoeniculus purpureus KT954408 KT954516
Bucerotiformes Bucerotidae Buceros rhinoceros XM_010145185.1 XM_010145184.1
Bucerotiformes Bucerotidae Buceros bicornis KT954407 KT954515
Bucerotiformes Bucerotidae Tockus camurus KT954406 KT954514
Leptosomatiformes Leptosomidae Leptosomus discolor XM_009958543.1 XM_009958545.1
Colliformes Coliidae Colius colius KT954404 KT954512
Colliformes Coliidae Colius striatus XM_010201405.1 XM_010209029.1
Strigiformes Strigidae Strix occidentalis DQ482641 KT954508
Strigiformes Strigidae Ninox novaeseelandiae KT954400 KT954507
Strigiformes Tytonidae Tyto alba XM_009975325.1 XM_009975324.1
Strigiformes Tytonidae Phodilus badius KT954402 KT954510
Accipitrifromes Accipitridae Buteo jamaicensis EF078718 KT954506
Accipitrifromes Accipitridae Elanus caeruleus EF078724 KT954505
Accipitrifromes Pandionidae Pandion haliaetus EF078706 KT954504
Accipitrifromes Sagittaridae Sagittarius serpentarius KT954399 KT954503
Accipitrifromes Cathartidae Cathartes aura EF078766 KT954502
Accipitrifromes Accipitridae Aquila chrysateos XM_011594630.1 XM_011594629.1
Accipitrifromes Accipitridae Haliaeetus albicilla XM_009928640.1 XM_009928639.1
Accipitrifromes Accipitridae Haliaeetus leucocephalus XM_010586008.1 XM_010586006.1
Trogoniformes Trogonidae Apaloderma vittatum XM_009874816.1 XM_009869619.1
Trogoniformes Trogonidae Apaloderma narina AMNH_DOT12430
Trogoniformes Trogonidae Apalharpactes mackloti LSU_49104
Trogoniformes Trogonidae Apalharpactes mackloti AMNH_633881
Trogoniformes Trogonidae Harpactes ardens AY625239 -
Trogoniformes Trogonidae Harpactes ardens USNM_607340
Trogoniformes Trogonidae Harpactes diardii AMNH_DOT563
Trogoniformes Trogonidae Harpactes oreskios AY625238 -
Trogoniformes Trogonidae Harpactes oreskios ANSP_16308
Trogoniformes Trogonidae Harpactes orrhopheus AY625241 -
Trogoniformes Trogonidae Harpactes orrhopheus AMNH_DOT15159
Trogoniformes Trogonidae Harpactes duvaucelli LSU_38592
Trogoniformes Trogonidae Harpactes fasciatus AMNH_778649
70
Trogoniformes Trogonidae Harpactes erythrocephalus AMNH_DOT12240
Trogoniformes Trogonidae Harpactes wardii AMNH_307761
Trogoniformes Trogonidae Harpactes whiteheadii LSU_52627
Trogoniformes Trogonidae Harpactes kasumba AMNH_DOT15326
Trogoniformes Trogonidae Euptilotis neoxenus AMNH_DOT11080
Trogoniformes Trogonidae Pharomachrus pavoninus LSU_4986
Trogoniformes Trogonidae Pharomachrus auriceps hargitti AMNH_175988
Trogoniformes Trogonidae Pharomachrus auriceps auriceps FMNH_473723
Trogoniformes Trogonidae Pharomachrus fulgidus AMNH_322895
Trogoniformes Trogonidae Pharomachrus moccino AMNH_326512
Trogoniformes Trogonidae Pharomachrus antisianus ANSP_19429
Trogoniformes Trogonidae Priotelus temnurus ANSP_20257
Trogoniformes Trogonidae Priotelus roseigaster KU_8098
Trogoniformes Trogonidae Trogon clathratus USNM_613996
Trogoniformes Trogonidae Trogon mesurus ANSP_19305
Trogoniformes Trogonidae Trogon massena KU_2073
Trogoniformes Trogonidae Trogon comptus LSU_11829
Trogoniformes Trogonidae Trogon melanurus INPA_A1995
Trogoniformes Trogonidae Trogon viridis INPA_A5240
Trogoniformes Trogonidae Trogon chionurus LSU_28571
Trogoniformes Trogonidae Trogon melanocephalus USNM_646857
Trogoniformes Trogonidae Trogon citreolus UWBM_101087
Trogoniformes Trogonidae Trogon bardii LSU_71992
Trogoniformes Trogonidae Trogon violaceus MPEG_CN437
Trogoniformes Trogonidae Trogon caligatus LSU_66270
Trogoniformes Trogonidae Trogon ramonianus INPA_A5449
Trogoniformes Trogonidae Trogon curucui INPA_A5286
Trogoniformes Trogonidae Trogon aurantius LGEMA_15782
Trogoniformes Trogonidae Trogon surrucura MPEG_SC015
Trogoniformes Trogonidae Trogon elegans FMNH_434014
Trogoniformes Trogonidae Trogon rufus amazonicus INPA_A5284
Trogoniformes Trogonidae Trogon rufus tenellus LSU_26564
Trogoniformes Trogonidae Trogon rufus chrysochlorus LGEMA_9557
Trogoniformes Trogonidae Trogon mexicanus FMNH_343220
Trogoniformes Trogonidae Trogon aurantiiventris LSU_41625
Trogoniformes Trogonidae Trogon collaris puella FMNH_394272
Trogoniformes Trogonidae Trogon collaris collaris MPEG_CN450
Trogoniformes Trogonidae Trogon personatus LSU_48503
1998
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Capítulo 3 2012
2013
2014
Ferreira, M.; Aleixo, A.; Bates, J. M.; Cracraft, J.; 2015
Ribas, C. C. Phylogeography and phylogenomics of 2016
jacamars (Aves: Galbulidae) and puffbirds (Aves: 2017
Bucconidae) reveal underestimation of species 2018
diversity and recurrent biogeographic patterns in the 2019
Neotropics. Manuscrito formatado para Zoological 2020
Journal of Linnean Society 2021
2022
72
2023
2024 Manuscript submission to Zoological Journal of Linnean Society 2025 Contribution type: Article 2026 2027
Phylogeography and phylogenomics of jacamars (Aves: Galbulidae) and 2028
puffbirds (Aves: Bucconidae) reveal underestimation of species diversity 2029
and recurrent biogeographic patterns in the Neotropics 2030
2031 Ferreira, Mateus1*; Aleixo, Alexandre2; Bates, John M.3; Cracraft, Joel4; Ribas, Camila C.5 2032
2033 1 Programa de Pós-Graduação em Genética, Conservação e Biologia Evolutiva, INPA, 2034 Manaus, AM, Brazil 2035 2 Coordenação de Zoologia, MPEG, Belém, PA, Brazil 2036 3 Department of Ornithology, FMNH, Chicago, IL, USA 2037 4 Department of Ornithology, AMNH, New York, NY, USA 2038 5 Coordenação de Biodiversidade, INPA, Manaus, AM, Brazil 2039 *Corresponding author 2040 2041
Correspondence: Mateus Ferreira, Coordenação de Biodiversidade, Instituto Nacional de 2042 Pesquisas da Amazônia, CEP 69080-971, Manaus-AM, Brazil 2043
E-mail: [email protected] 2044
2045
Short running title: Galbuliformes phylogenomic 2046 2047 2048 2049
Abstract 2050
Galbulidae (jacamars) and Bucconidae (puffbirds) are sister families endemic to the 2051
Neotropical region. Together they comprise 57 species and more than a 100 described 2052
subspecies. Both families have their highest diversity in Amazonia. Within Galbulidae, most 2053
species have restricted and parapatric / allopatric distributions in relation to other closely related 2054
species, while within Buccondiae, species are widespread and polytypic. In this study, we 2055
obtained DNA sequence data for over 400 samples, and used previous published results, of all 2056
widespread species to uncover phylogeographic patterns. Then, based on these results, we 2057
selected and sequenced thousands of Ultraconserved Elements to reconstruct the phylogenetic 2058
relationships among these phylogeographic groups and propose the first phylogenetic 2059
hypothesis for these two families with dense taxon sampling. Our phylogeographic results 2060
recovered phylogeographic breaks in almost all studied groups, most of them associated with 2061
the main tributaries of the Amazon River, and many corresponding to already described 2062
subspecies. We then reconstructed phylogenetic relationships based on over 2,000 UCE loci 2063
using a concatenated approach in a Bayesian Inference framework. Overall, most nodes had 2064
73
high support, and the relationships among genera, species and instraspecific diversity were 2065
discussed. We propose the recognition of all subspecies that received support from the 2066
phylogeographic and phylogenomic approaches as distinct species. We found evidence of 2067
paraphyly of several species and proposed taxonomic changes to deal with that. Also, we 2068
propose a new genus of puffbirds, Cryptobucco gen. nov., to accommodate the paraphyly of 2069
Notharchus species. 2070
2071
Introduction 2072
Species usually are the basic unit of any study in evolutionary biology. Considering they 2073
should represent the lowest and only non-arbitrary rank above individuals, species are the basic 2074
operational unit for comparing any intrinsic evolutionary aspect, such as physiology, behaviour, 2075
morphology, etc. However, we still lack a broad and comprehensive concept for species 2076
recognition (Cellinese, Baum & Mishler, 2012; de Queiroz, 2007; de Queiroz, 2012). In birds, 2077
taxonomy has been historically influenced by the Biological Species Concept (Mayr, 1942; 2078
Mayr, 1976), based on reproductive isolation as the main criterion for species delimitation. 2079
Therefore, since this concept was adopted several distinct allopatric populations were lumped 2080
as subspecies due to morphological similarities pending further investigation to prove the 2081
absence of gene flow (Peters, 1945; Peters, 1948). This implies that allopatric and parapatric 2082
populations, even if diagnosably distinct, should only be recognized as full species if there is 2083
evidence of reproductive isolation (Gill, 2014). 2084
In the Neotropical region, and especially in Amazonia, one of the main issues that 2085
obscures the recognition of diversity patterns is the fact that most widespread species are in fact 2086
complexes of taxa, usually diagnosable and geographically structured, that are lumped under 2087
the same species name due to their morphological similarities and physical isolation. Many of 2088
these polytypic species, when thoroughly sampled, prove to include distinct lineages, 2089
sometimes not even closely related to each other (Bravo, Chesser & Brumfield, 2012; Bravo, 2090
Remsen, Whitney & Brumfield, 2012; Fernandes, Wink, Sardelli & Aleixo, 2014; Isler, Bravo 2091
& Brumfield, 2013; Lopes, Chaves, Aquino, Silveira & Santos, 2017; Lutz, Weckstein, Patane, 2092
Bates & Aleixo, 2013; Ribas, Aleixo, Nogueira, Miyaki & Cracraft, 2012; Ribas, Aleixo, 2093
Gubili, d'Horta, Brumfield & Cracraft, 2018; Tobias, Bates, Hackett & Seddon, 2008). The 2094
recognition of these hidden lineages is critical for appropriate hypothesis formulation in 2095
macroevolution and biogeography (Donoghue & Moore, 2003; Lexer, Mangili, Bossolini, 2096
Forest, Stölting, Pearman, Zimmermann, Salamin & Carine, 2013). For example, Amazonian 2097
74
areas of endemism were recognized based on congruent distribution patterns of bird species 2098
(Borges & Da Silva, 2012; Cracraft, 1985), and have been used as a basis to formulate 2099
hypothesis of biotic diversification in Amazonia (Haffer, 1969; Haffer, 1974; Haffer, 1997). 2100
Considering that any biogeographic study should be based on a taxonomy that correctly 2101
recognizes the evolutionary units included in the studied groups, for the present study we 2102
densely sampled all recognized taxa within two sister families of birds restricted to the 2103
Neotropical region. Galbulidae and Bucconidae form a clade, sometimes recognized in its own 2104
order Galbuliformes, that diverged from all the other Piciformes during the early Eocene and 2105
diverged from each other in the Late Eocene (Prum, Berv, Dornburg, Field, Townsend, 2106
Lemmon & Lemmon, 2015). Although the ancestor was from the Afrotropical region the two 2107
families’ entire diversification happened inside the Neotropical region (Claramunt & Cracraft, 2108
2015). Hence, making these two families excellent models to understand how landscape 2109
evolution of the Neotropical region influenced diversification. However, there are no 2110
phylogenetic hypotheses about relationships within these two families, and the few 2111
phylogeographic studies conducted so far with Bucconidae species showed that the diversity is 2112
highly underestimated by current species limits (Almeida, 2013; Duarte, 2015; Ferreira, Aleixo, 2113
Ribas & Santos, 2017; Soares, 2016). Although Galbulidae species were never subjected to 2114
phylogeographic studies, with 19 species distributed in 5 genera, jacamar distributions were 2115
used as models by Haffer (1974), together with other families, when he proposed his theory for 2116
Amazonian diversification (Haffer, 1974). Haffer recognized eight zoogeographic groups, five 2117
were composed of species complexes, and two were widespread polytypic species. Bucconidae, 2118
in turn, are composed of 38 species distributed in 12 genera. However, half of those species 2119
consist of polytypic groups lumped as subspecies due to morphological similarities. Groups 2120
such as the White-fronted Nunbird, Monasa morphoeus, and the Rusty-breasted Nunlet, 2121
Nonnnula rubecula, are composed of several subspecies, which in fact still underestimate the 2122
phylogeographic structure recovered for them (Soares, 2016). On the other hand, Malacoptila 2123
species are widespread species for which only a few subspecies were described, however, 2124
phylogeographic patterns indicated a great underestimation of taxonomic diversity. For 2125
example, for a single species, the Rufous-necked Puffbird (M. rufa), that only includes two 2126
subspecies described, ten distinct genetic lineages were recovered (Ferreira et al., 2017). Due 2127
to these first results, the present study focused on sampling all named taxa described for these 2128
two families, and sampling all widespread species throughout their distribution to uncover 2129
phylogeographic patterns. Based on these results, we selected samples representing all 2130
75
phylogeographic groups and sequenced thousands of Ultraconserved Elements (UCE) 2131
(Faircloth, McCormack, Crawford, Harvey, Brumfield & Glenn, 2012; McCormack & 2132
Faircloth, 2013; McCormack, Harvey, Faircloth, Crawford, Glenn & Brumfield, 2013) to 2133
recover their phylogenetic relationships. Our aims are (1) to characterize the phylogeographic 2134
patterns and population structure within widespread species, recognizing the cryptic diversity 2135
within them, when present; (2) propose a densely sampled phylogenetic hypothesis for these 2136
two families; and (3) discuss patterns of diversification in the entire clade. 2137
2138
Material and Methods 2139
Sampling and DNA isolation 2140
We sampled 436 individuals from almost all named taxa currently recognized within 2141
Galbuliformes (Gill & Donsker, 2018; Peters, 1948; Piacentini, Aleixo, Agne, Mauricio, 2142
Pacheco, Bravo, Brito, Naka, Olmos, Posso, Silveira, Betini, Carrano, Franz, Lees, Lima, Pioli, 2143
Schunck, do Amaral, Bencke, Cohn-Haft, Figueiredo, Straube & Cesari, 2015; Rassmussen & 2144
Collar, 2002; Remsen, Areta, Cadena, Claramunt, Jaramillo, Pacheco, Pérez-Emen, Robbins, 2145
Stiles, Stotz & Zimmer, 2018 Tobias, 2017), and when available, we used published sequences 2146
to select samples for UCE sequencing. All samples are represented by voucher specimens 2147
deposited at the ornithological collections of the American Museum of Natural History 2148
(AMNH), Academy of Natural Sciences of Drexel University (ANSP), Field Museum of 2149
Natural History (FMNH), Instituto Nacional de Pesquisas da Amazônia (INPA), Kansas 2150
University (KU), Laboratório de Genética e Evolução Molecular de Aves - USP (LGEMA), 2151
Louisiana Museum of Natural History (LSUMZ), Museu Paraense Emílio Goeldi (MPEG), 2152
Smithsonian Institution National Museum of Natural History (USNM) and Burke Museum 2153
(UWBM) (Table S1). 2154
DNA from fresh tissue was extracted with the DNeasy kit (Qiagen Inc.), following the 2155
manufacturer’s protocol. For taxa lacking fresh tissues we sampled toe pad clips from museum 2156
specimens at the American Museum of Natural History (AMNH). Toe pads were cut from 2157
specimens with a sterile surgical blade and processed in a dedicated room for ancient DNA 2158
(aDNA Lab, AMNH). They were rinsed with 100% ethanol, and twice with ultra-pure water 2159
prior to digestion to remove any inhibitor that could cause problems in downstream procedures. 2160
We then extracted DNA with the DNeasy kit (Qiagen Inc.), replacing the regular silica columns 2161
with the QIAquick (Qiagen Inc.) columns, to ensure maximum DNA yield. 2162
2163
76
Phylogeographic structure and UCE sampling 2164
Widespread species that lacked previous studies were sampled throughout their 2165
distributions to uncover phylogeographic structure. We amplified one mitochondrial gene 2166
(NADH subunit 2 – ND2) following conventional PCR protocols and sequenced both strands 2167
with BigDye® Terminator v3.1 in an ABI 3130/3130XL automated capillary sequencer 2168
(Applied Biosystems®) following manufacturer’s protocols. The sequences were edited on 2169
Geneious version 10.2.3 (Kearse, Moir, Wilson, Stones-Havas, Cheung, Sturrock, Buxton, 2170
Cooper, Markowitz, Duran, Thierer, Ashton, Meintjes & Drummond, 2012) and aligned with 2171
MAFFT (Katoh & Standley, 2013) under default parameters. We analysed each species 2172
complex independently. Within Galbulidae we analysed five species complexes: 1) 2173
Brachygalba and Jacamaralcyon; 2) Jacamerops; 3) Galbula dea; 4) Galbula cyanicollis, G. 2174
chalcocephala, and G. albirostris; and 5) G. ruficauda, G. pastazae, G. cyanescens, G. 2175
tombacea, and G. galbula. We used a previous study to select samples for G. leucogastra and 2176
G. chalcothorax (Ferreira et al., submitted). For Bucconidae, we gathered data in this study for 2177
five polytypic species or species complexes: 1) Bucco capensis; 2) Cyphos macrodatylus; 3) 2178
Notharchus tectus; 4) Notharchus ordii, N. hyperrhynchus, N. macrorhynchus, N. swainsoni, 2179
and N. pectorales; and 5) Chelidoptera tenebrosa. Sample selection for the genera Monasa, 2180
Nonnula, Malacoptila, and Nystalus was based on previous studies (Almeida, 2013; Duarte, 2181
2015; Ferreira et al., 2017; Soares, 2016). The best evolutionary model for each matrix was 2182
selected by jModelTest 2.1.10 (Darriba, Taboada, Doallo & Posada, 2012). We performed a 2183
Bayesian inference analysis (BI) implemented in MrBayes 3.2.6 (Ronquist, Teslenko, van der 2184
Mark, Ayres, Darling, Hohna, Larget, Liu, Suchard & Huelsenbeck, 2012) with four parallel 2185
simultaneous runs consisting of a total of 4x107 generations, sampling trees every 1000 2186
generations. ESS values, stationarity, and convergence among runs were checked in Tracer 1.6 2187
(Rambaut, Suchard, Xie & Drummond, 2014). Based on these results we selected our samples 2188
for UCE sequencing. All extracts were sent to Rapid Genomics (Gainsville, FL) for library prep 2189
and target-capture sequencing of 2321 loci of Ultraconserved Elements (UCE) (Faircloth et al., 2190
2012; McCormack et al., 2013). 2191
UCE assembly 2192
The raw sequence data were processed with the Phyluce script pack (Faircloth, 2016). 2193
We employed illumiprocessor (Faircloth, 2013) and Trimmomatic (Bolger, Lohse & Usadel, 2194
2014) to remove adapter contamination and low-quality reads. We assembled our targeted 2195
regions using a reference genome for each family. For Bucconidae, we used the Collared 2196
77
puffbird (Bucco capensis), and for Galbulidae, the Paradise jacamar (Galbula dea) genomes. 2197
We mapped the UCE probes back to each genome using the script 2198
phyluce_probe_run_multiple_lastzs_sqlite, and then, phyluce_probe_slice_sequence_from_g-2199
enomes to extract the probe region plus 500 base pairs from each flanking region (Faircloth, 2200
2016). With these sequences as a reference we mapped back the clean reads of each individual 2201
employing Bowtie2 (Langmead & Salzberg, 2012) plugin 7.2.1 inside Geneious version 10.2.3 2202
(Kearse et al., 2012). The consensus sequences were called with the highest quality threshold 2203
and a depth of at least 4 reads. Each locus was aligned with MAFFT (Katoh & Standley, 2013) 2204
under default parameters. 2205
Phylogenetic relationship 2206
Even though the sister relationship between Galbulidae and Bucconidae is well 2207
established (Hackett et al., 2008; Livezey & Zusi, 2007; Prum et al., 2015), we used the 2208
Rhinoceros hornbill (Buceros rhinoceros, Bucerotidae)(Gilbert, Jarvis, Li, Li, Avian Genome 2209
Consortium, Wang & Zhang, 2014b), the Northern Carmine bee-eater (Merops nubicus, 2210
Meropidae)(Gilbert, Jarvis, Li, Li, Avian Genome Consortium, Wang & Zhang, 2014c), and 2211
the Downy woodpecker (Picoides pubescens, Picidae)(Gilbert, Jarvis, Li, Li, Avian Genome 2212
Consortium, Wang & Zhang, 2014a) as outgroups. To recover the phylogenetic relationships, 2213
we performed a Bayesian Inference analysis in ExaBayes v1.4 (Aberer, Kobert & Stamatakis, 2214
2014) employing the concatenated matrix of all UCE loci with 75% completeness, where only 2215
loci that had at least 75% of all individuals were selected. Four parallel chains consisting of 2216
4x107 generations were performed. 2217
2218
Results 2219
Phylogeographic results 2220
With a few exceptions, we obtained the whole ND2 sequence for all samples. 2221
Phylogenetic trees and maps of samples and lineages’ distributions can be found in the 2222
Supplementary Material (Figures S1-S10). Overall, most species complexes contained 2223
phylogeographic structure in the mtDNA that matches known areas of endemism for birds. The 2224
only two widespread species that apparently lacked phylogeographic structure were Cyphos 2225
macrodactylus and Chelidoptera tenebrosa. The phylogeographic breaks were more 2226
conspicuous in birds with stronger association with terra-firme forests [Fig. S2-S4, S6, 2227
Malacoptila spp. (Ferreira et al., 2017), Monasa morphoeus and Nonnula rubecula (Soares, 2228
2016)]. However, species associated with other habitats, such as várzeas, open habitats (i.e. 2229
78
non-forested) or white-sand environments also showed structure [Fig. S1, S5, S8-S9, also 2230
Galbula leucogastra/chalcothorax, Nystactes (Almeida, 2013), Nystalus spp. (Duarte, 2015), 2231
and Nonnula ruficapilla (Soares, 2016)]. Nonetheless, some lineages are represented by a single 2232
individual and additional samples should be collected and analysed to make further 2233
assumptions. It is also worth to note that some species were paraphyletic in the mtDNA. The 2234
most remarkable are the complex Brachygalba lugubris, B. albogularis (Fig. S1), G. albirostris, 2235
G. cyanicolis, and G. chalcocephala (Fig. S4), G. ruficauda, G. cyanescens (Fig. S5); 2236
Notharchus tectus, N. subtectus (Fig. S8); N. hypperhynchus, N. swainsoni, N. macrorhynchus 2237
(Fig. S9). 2238
UCE sequencing 2239
The reference sequences we assembled from the Collared puffbird (Bucco capensis) and 2240
the Paradise jacamar (Galbula dea) genomes included 2226 and 2279 sequences, respectively. 2241
The mean number of sequences was 2,240,885 reads; and a mean number of 2191 UCE loci per 2242
sample (Table S1). The matrix for Galbulidae contained 2165 loci, while for Bucconidae, the 2243
matrix had 2158 loci. 2244
Phylogenetic results 2245
In general, the ExaBayes tree is well supported, with most of the nodes with lower support 2246
found near the tips (Fig. 2, 3). Galbulidae consisted of two clades, the first comprises 2247
Jacamaralcyon and Brachygalba, and the other, Jacamerops, Galbacyrhynchus, and Galbula 2248
(Fig. 1). Within Bucconidae, some genera were paraphyletic. Bucco, that previously included 2249
four species (Gill & Donsker, 2018; Peters, 1948; Piacentini et al., 2015; Remsen et al., 2018), 2250
comprises three distinct genera as previously suggested by morphological characters 2251
(Rassmussen & Collar, 2018): B. capensis Linneus, 1766 is the family and genus type and more 2252
closely related to Nystalus; Cyphos macrodactylus von Spix, 1824, is sister to the clade that 2253
comprises Notharchus, Hypnelus, and Nystactes; and finally, Nystactes tamatia (J. F. Gemelin, 2254
1788), and N. noanamae (Hellmayer, 1909), more closely related with Hypnelus species (Fig. 2255
3). Notharchus was also paraphyletic, with Hypnelus and Nystactes embedded within it. N. 2256
tectus and N. subtectus were sister to Hypnelus, Nystactes, and the remaining Notharchus 2257
species (Fig. 1, 3). 2258
The relationships within genera in the UCE trees (Fig. 2, 3) mostly agreed with the 2259
mtDNA phylogeographic structure. Most notably is the paraphyly of Brachygalba lugubris in 2260
relation to B. albogularis (Fig. 2), and the polyphyletic status of Galbula ruficauda, in which 2261
the lineages from Central America (G. melanogenia), and northern South America (G. pallens, 2262
79
G. ruficauda), including G. pastazae, are sister group to the clade comprising the species group 2263
of G. albicollis (albicollis, chalcocephala, cyanicollis) and G. galbula (galbula, pastazae, 2264
cyanescens, rufoviridis). Also, in contrast with the mtDNA, the two samples of G. cyanescens 2265
are sister to G. heterogyna and G. rufoviridis from the Brazilian Shield, instead of being 2266
embedded between them (Fig. S5). For puffbirds, the UCE tree also recovered the paraphyly of 2267
N. tectus subspecies (Fig. 3), and for the hyperrhynchus group (Fig. S9), we recovered N. 2268
macrorhynchus sister to N. swainsoni and N. hyperrhynchus, rendering the Amazonian group 2269
paraphyletic. 2270
2271
Discussion 2272
Phylogenetic results 2273
Our dense sampling coupled with the use of UCE loci provided good insights about 2274
genera and species relationships. We sampled all species, and almost all subspecies, for the two 2275
families, and characterized the spatial distribution of mtDNA lineages for all widespread 2276
species. Predominantly, our results indicate a severe disparity between currently recognized 2277
species and the potential number of independent evolutionary units within these clades. 2278
Avian taxonomy has historically been greatly influenced by the Biological Species 2279
Concept (BSC), which assumes that reproductive isolation is required for recognition of species 2280
status (de Queiroz, 2005). This condition, can be easily detected in sympatric taxa, however, 2281
for parapatric and allopatric populations, natural observations are very hard to detect. 2282
Consequently, many morphologically distinct taxa have been lumped into species complexes, 2283
pending further analysis to prove them different. Thus, the null hypothesis for species 2284
recognition has been of peer-reviewed publications proving that essential reproductive isolation 2285
is true among allopatric populations. It implies that we should be looking for reasons that 2286
differentiate allopatric populations, either through genetic evidence or some other characteristic 2287
that would lead to reproductive isolation, rather than assuming that they already are 2288
reproductive isolated, because they are not in contact, and looking for evidence proving the 2289
contrary (Gill, 2014). Albeit avian taxonomy and systematics is probably the best known among 2290
vertebrates, there are still many taxa to be described (Barrowclough, Cracraft, Klicka & Zink, 2291
2016), and although species concept, or criteria, are amid one of the most controversial topics 2292
in biology (Aleixo, 2007; Dayrat, Cantino, Clarke & de Queiroz, 2008; de Queiroz, 2012), the 2293
appropriate understanding of a lineage’s evolutionary history is essential to several fields, 2294
including conservation and biogeography (Avendaño, Arbeláez-Cortés & Cadena, 2017; Ribas, 2295
80
Gaban-Lima, Miyaki & Cracraft, 2005; Tobias, Bates, Hackett & Seddon, 2008), especially in 2296
the emergent field of geogenomics (Baker, Fritz, Dick, Eckert, Horton, Manzoni, Ribas, 2297
Garzione & Battisti, 2014). Therefore, we are confident our results provide great insight about 2298
Galbulidae and Bucconidae systematics and will enable future biogeography studies to uncover 2299
how the landscape evolution of South America shaped this group’s diversity. 2300
Galbulidae systematics 2301
Galbulidae currently recognized diversity includes 19 species distributed in 5 genera 2302
(Tobias, 2017). Our results, however, show that this diversity is severely underestimated. In 2303
addition to the fact that most widespread species have genetic lineages structured 2304
geographically, we also found evidence that, at least four species are para- (Brachygalba spp.) 2305
or polyphyletic (Galbula ruficauda complex). Conceding that we recognize all subspecies that 2306
were monophyletic in our analyses and elevate them to species status, the species diversity of 2307
Galbulidae practically doubles, from 19 to 37 species, including at least six new taxa that need 2308
to be formally described. Biogeographically, there is also some noteworthy patterns that arouse 2309
from the mtDNA data. All widespread species presented some degree of genetic structure in 2310
the known areas of endemism in Amazonia (Borges & Da Silva, 2012; Cracraft, 1985). Most 2311
of the larger Amazonian tributaries, including rivers such as the Negro, Madeira, Solimões, and 2312
Amazonas delimit lineages in opposite margins, however, if they were responsible for causing 2313
these divergences still need to be investigated. 2314
According to our phylogenetic hypothesis for Galbulidae, there are now eight main 2315
groups of species: 2316
1. Brachygalba and Jacamaralcyon 2317
Brachygalba and Jacamaralcyon species were recovered as sisters to all other jacamars. 2318
The monotypic Jacamaralcyon species, Jacaramaralcyon trydactyla (Viellot, 1817), is 2319
endemic to the Atlantic Forest, inhabiting semi-deciduous or gallery forest. This species was 2320
recovered as sister to all other Brachygalba species (Fig. 1), which prefer forest edges and open 2321
habitats throughout the Amazon basin and north South America. B. goeringii Sclatter, PL & 2322
Salvin, 1869 and B. salmoni Sclatter, PL & Salvin, 1879 represent two distinct lineages within 2323
Brachygalba radiation (Fig. 2), with very distinct plumages and restricted distributions in 2324
northern South America. B. goeringii was recovered as sister to all other Brachygalba species, 2325
and B. salmoni, as sister group to the species group of B. lugubris (naumburgae, obscuriceps, 2326
lugubris, and melanosterna) and B. albogularis (von Spix, 1824), from the Amazon basin (Fig. 2327
2). Because B. albogularis was embedded within B. lugubris lineages, we recommend that the 2328
81
current subspecies of B. lugubris should now be elevated to species status. This way, we resolve 2329
the paraphyly of B. lugubris, and fully recognize all its diversity. Further studies are necessary 2330
to completely understand B. obscuriceps Zimmer, JT & Phelps, 1947 and B. naumburgae 2331
Chapman, 1931 distributions, especially regarding the relationship between B. lugubris, and B. 2332
l. fulviventris Sclater, PL, 1891 and B. l. caquetae Chapman, 1917. 2333
2. Jacamerops 2334
Jacamerops individuals are so distinct from the other jacamars that were once considered 2335
to belong to a separate subfamily Jacameropinae. Although this treatment is no longer followed, 2336
Jacamerops are by far the bulkiest jacamars, inhabiting the midstory and canopy of continuous 2337
forest in the Amazon basin. Among the four subspecies recognized, J. a. ridgway Todd, 1943 2338
formed a well supported clade in both analyses. (Fig. 2, S2), while J. a. aureus (Statius Müller, 2339
PL, 1776) was monophyletic in the mtDNA analysis (Fig. S2) but paraphyletic in the UCE 2340
analysis, with the two individuals from the Guiana Shield as sister to all other J. aureus 2341
individuals (Fig. 2). Since the type from J. a. aureus is British Guiana (Peters, 1948), we 2342
consider that only this group should be recognized as J. aureus, while the second lineage should 2343
receive a new name (Fig. 2). An interesting biogeographic pattern that arouse from Jacamerops 2344
data was the sister relationship between J. penardi Bangs & Barbour, 1922, from Central 2345
America, and J. isidori Deville 1849, from the Madeira-Solimões interfluve. A similar pattern 2346
was found in the Hylophylax species complex (Fernandes et al., 2014). Finally, J. ridgwayi 2347
Todd, 1943 requires further study to fully evaluate all diversity present in this group, our results 2348
suggest the presence of at least 4 mtDNA lineages, each separated by the main rivers of the 2349
Brazilia Shield. 2350
3. Galbalcyrhynchus 2351
Galbacyrhynchus species are endemic to floodplain forests from Western Amazon. 2352
Galbalcyrhynchus purusianus Goeldi, 1904 was considered conspecific with G. leucotis Des 2353
Murs, 1845, and they were actually considered male and female forms of the same species. 2354
Nonetheless, the parapatric distribution and the apparently lack of intermediate forms (Haffer, 2355
1974) render these two taxa the status of distinct species (Fig. 2). 2356
4. Galbula dea complex 2357
Previously allocated in the genus Urogalba, Galbula dea individuals are the most 2358
morphologically distinct among Galbula species. Our results recovered six distinct mtDNA 2359
lineages (Fig. S3) that matches with the UCE results (Fig. 2), in which four already have 2360
associated names. G. dea (Linnaeus, 1758) from the Guiana Shield; G. brunneiceps (Todd, 2361
82
1943) from the Negro-Solimões interfluve; G. phainopepla (Todd, 1943) from the Solimões-2362
Madeira interfluve; and G. amazonum (Sclater, PL, 1855). The last lineage, from the Madeira-2363
Tapajós interfluve was considered to be part of G. d. brunneiceps (Peters, 1948), however, since 2364
the type locality from G. brunneiceps is Manacapurú, Rio Solimões, Brazil, we suggest that a 2365
new name should be given to this lineage. 2366
5. G. leucogastra/chalcothorax 2367
This complex includes the only jacamars that inhabit white-sand environments (Adeney, 2368
Christensen, Vicentini & Cohn-Haft, 2016) in the Amazon basin. Although highly structured 2369
throughout its distribution (Ferreira et al., submitted) this group lacks morphological 2370
distinctiveness among genetic lineages, thus further systematic and taxonomic work is required 2371
before the proposition of any change in nomenclature. 2372
6. Galbula albirostris, G. chalcocephala, and G. cyanicollis 2373
These tree species were formerly considered conspecifics in G. albirostris Latham, 1790 2374
(Peters, 1948), later Haffer (1974) recognized G. cyanicollis Cassin, 1851, based on the lack of 2375
interbreeding between these two forms. Our results support the recognition of all three species, 2376
with G. albirostris restricted to the Guiana Shield, east of Negro River; G. chalcocephala 2377
Deville, 1849 in between the west bank of lower Negro river, west of Branco River, and north 2378
of Solimões all the way down to the west bank of the upper Ucayali River (Harvey, Seeholzer, 2379
Cáceres A, Winger, Tello, Camacho, Aponte Justiniano, Judy, Ramírez, Terrill, Brown, León, 2380
Bravo, Combe, Custodio, Zumaeta, Tello, Bravo, Savit, Ruiz, Mauck & Barden, 2014); and at 2381
last, G. cyanicollis, along the south bank of Amazon River. This group of species, in contrast 2382
with other jacamars, only inhabits the interior of forests, mainly in terra-firme habitats. Not 2383
surprisingly, the mtDNA showed lineages separated by the main Amazonian tributaries (Fig. 2384
S4). However, some lineages presented some interesting biogeographic patterns, such as the 2385
distinct lineage at the lower portion of Madeira-Tapajós interfluve, that is also found in other 2386
groups of birds, such as Rhegmatorhina berlespchi (Ribas et al., 2018), Malacoptila rufa 2387
(Ferreira et al., 2017), and Glyphorhynchus spirurus (Fernandes, Gonzalez, Wink & Aleixo, 2388
2013). Another pattern, that has not been reported before for birds, is the distinct lineage 2389
between the Purus and Tapajós Rivers (Fig. S4). This is the first evidence of a lineage of and 2390
understory terra-firme bird that has n structure related to the Madeira River. 2391
7. G. melanogenia, G. pastazae, G. pallens, and G. ruficauda. 2392
Although G. melanogenia Sclater, PL, 1852, was first described as a full species, it was 2393
later lumped together with G. rufoviridis Cabanis, 1851 in G. ruficauda Cuvier, 1816 due to 2394
83
morphological similarity (Peters, 1948). Our results, however, recovered this group as sister to 2395
the clade containing G. cyanicolis and G. galbula complex (Fig. 2). Also, G. pastazae 2396
Taczanowski and Berlepsch, 1885, probably the only jacamar to live in high altitudes, is 2397
embedded between G. melanogenia and G. ruficauda (Fig. S5, 2). Therefore, our 2398
recommendation is that G. melanogenia, from Central America and the Pacific coast of 2399
Colombia and Ecuador, along with G. pallens Bangs, 1898 and G. ruficauda Cuvier, 1816 2400
should be recognized as species. Further studies are required to check the validity of G. r. 2401
brevirostris Cory, 1913. 2402
8. Galbula galbula, G. tombacea, G. cyanescens and G. rufoviridis 2403
This group is often regarded as G. galbula (Linnaeus, 1766) species group due to 2404
morphological and ecology similarity. Usually associated with forest edges and floodplains 2405
forest, while G. albirostris species group, its sister clade (Fig. 2), is usually associated with the 2406
interior of terra-firme forests. Despite been associated with floodplain forests, and therefore, 2407
not “bounded” by rivers, there are no previous reports of hybridization among these taxa. We 2408
found, however, that the individual INPA A019 is phenotypically G. tombacea (checked by 2409
M.F.), however, the mtDNA clustered with G. cyanescens Deville, 1849 (Fig. S5). This is the 2410
only reported case of hybrids among this group, the other individual that could be a hybrid - G. 2411
cyanescens, voucher MPEG MAD305 - is phenotypically G. cyanescens (checked by Fátima 2412
Lima), even though the individual was collected in the right bank of Madeira River, supposedly 2413
the limit between distributions of G. cyanescens and G. heterogyna Todd, 1932. Another 2414
important pattern that we can observe in this group is the apparently discordance between the 2415
mtDNA and UCE trees (Fig. S5, 3). Our mtDNA tree recovered G. cyanescens as one lineage 2416
embedded within lineages of G. rufoviridis and G. heterogyna. It also recovered G. rufoviridis 2417
as paraphyletic (Fig. S5). The UCE tree instead, recovered G. cyanescens as sister to G. 2418
heterogyna and G. rufoviridis (Fig. 2). In addition, all samples we sequenced for G. rufoviridis 2419
were recovered as monophyletic and sister to G. heterogyna. Thus, this might be an evidence 2420
of mtDNA capture (Sloan, Havird & Sharbrough, 2017), in which probably G. cyanescens 2421
captured the mtDNA lineage of G. heterogyna. However, further studies are required to 2422
understand the direction and timing of this event. 2423
Bucconidae systematics 2424
Our results showed that, similar to the situation with Galbulidae, Bucconidae diversity is 2425
underestimated. In addition, we found evidence of genera paraphyly. Phylogeographic patterns 2426
recovered for widespread puffbird species varied from little to no genetic structure, as in 2427
84
Chelidoptera and Cyphos, to highly structured, as in Malacoptila (Ferreira et al., 2017), 2428
Nonnula rubecula and N. ruficapilla, and Monasa morphoeus (Soares, 2016). Historically, 2429
apart from the morphologically explicit genera Hapaloptila, Chelidoptera, Malacoptila, 2430
Micromonacha, Monasa, and Nonnula, all other species were lumped in Bucco Brisson, and 2431
later split in Notharchus and Hypnelus. Currently, although some authors recognize different 2432
genera for former Bucco species (i.e. Cyphos, and Nystactes) (Rassmussen & Collar, 2018), 2433
many others still keep several species within the genus Bucco (Gill & Donsker, 2018; Piacentini 2434
et al., 2015; Remsen et al., 2018). Our results however recovered Bucco as polyphyletic, and 2435
thus, we favor the recognition of Cyphos Spix, 1824 (which has priority over Argicus Cabanis 2436
& Heine, 1863) and Nystactes Gloger 1827. Also, we recovered Notharchus specie as 2437
paraphyletic, with the species group of N. tectus (Boddaert, 1783) as sister to the clade 2438
containing Hypnelus, Nystactes and the other species of Notharchus. One way to resolve this 2439
paraphyly would be to include Hypnelus and Nystactes in the genus Notharchus, however, both 2440
Nystactes and Hypnelus species are morphologically distinct from any of Notharchus species. 2441
Therefore, since the type species of Notharchus is N. hyperrhynchus Sclater, PL, 1856, we 2442
propose a new generic name for this group: 2443
Cryptobucco, gen. nov. Ferreira, Aleixo, Bates, Cracraft & Ribas 2444
Type species: Bucco tectus Boddaert, 1783 2445
Included taxa: Cryptobucco tectus (Boddaert, 1783), comb. nov.; Cryptobucco picatus 2446
(Sclater, PL, 1856), comb. nov.; Cryptobucco subtectus (Sclater, PL, 1860), comb. nov. 2447
Etymology: The genus name Cryptobucco alludes to the fact that this group, first 2448
described as Bucco and then placed in Notharchus, represents a hidden diversity inside 2449
Bucconidae that was until now concealed due to morphological similarity among species of 2450
Notharchus and the new genus. The name is of masculine gender. 2451
1. Bucco capensis and Nystalus 2452
Bucco capensis Linnaeus, 1766 and Nystalus species were recovered as sister to all other 2453
puffbirds. The sister relationship we recovered between B. capensis and Nystalus is validated 2454
by the bill-tip morphology that was previously used to separate former Bucco species in the 2455
genera Cyphos and Nystactes (Rassmussen & Collar, 2018). Our results for B. capensis samples 2456
recovered three clades in the UCE tree (Fig. 3) in contrast to the four clades found in the mtDNA 2457
analysis (Fig. S7). Our UCE analysis also favor the recognition of B. dugandi Gilliard, 1949 2458
and suggest the presence of a new taxon yet undescribed. Nystalus relationships found here 2459
were similar to a previous study that used only one mtDNA marker (Duarte, 2015), which 2460
85
recovered N. maculatus (Gmelin, JF, 1788) and N. striatipectus (Sclater, PL, 1854) as sister to 2461
all remaining species, and N. chacuru (Vieillot, 1816) as sister to N. radiatus (Sclater, PL, 1854) 2462
and the N. striolatus species complex: N. obamai Whitney et al., 2013; N. striolatus (Pelzeln, 2463
1856), and N. torridus Bond & Meyer de Schauensee, 1940. 2464
2. Chelidoptera 2465
The Swallow-winged puffbird, Chelidoptera tenebrosa Pallas, 1782, is by far the most 2466
distinct puffbird, aberrant both in morphology and in ecology. With swallow-like morphology, 2467
they are highly specialized in aerial activity, and its flying proficiency is probably the cause for 2468
the lack of genetic structure we found in the mtDNA (Fig. S10). However, we were unable to 2469
sample UCE from the two toe pad samples, from the subspecies C. t. pallida Cory, 1913, from 2470
Northwest Venezuela; and C. t. brasiliensis Sclater, PL, 1862, from the east coast in Brazil. 2471
3. Monasa and Nonnula 2472
Monasa and Nonnula were the focus of a recent phylogeographic study (Soares, 2016). 2473
Species from both genera presented high levels of genetic structure in the mtDNA, and we 2474
sampled one individual per mtDNA lineage that were uncovered previously. We recovered 2475
Monasa as sister to Chelidoptera, and these two sisters to Nonnula (Fig. 1). Relationships inside 2476
each genus (Fig. 3) were also congruent to Soares (2016). In addition to this previous study, we 2477
were able to sample three toe pads representing three subspecies of M. morphoeus (Hahn & 2478
Küster, 1823): M. m. morphoeus (Hahn & Küster, 1823) from the east coast of Brazil; M. m. 2479
pallescens Cassin, 1860; and M. m. grandior Sclater, PL & Salvin, 1868, both from Central 2480
America. However, their phylogenetic relationship with other subspecies of M. morphoeus was 2481
uncertain (Fig. S11) and further studies are required to fully understand if the phylogeographic 2482
structure found in the mtDNA matches the UCE. For Nonnula, our results support the paraphyly 2483
of N. ruficapilla (Tschudi, 1844), with N. amaurocephala Chapman, 1921 is embedded within 2484
it. Both genera are being studied using broader sampling of individuals and molecular markers. 2485
4. Malacoptila 2486
Malacoptila UCE topology was congruent with the concatenated dataset topology from 2487
Ferreira et al. (2017), placing M. fulvogularis Sclater, PL, 1854 as sister to all other species. 2488
This result changes the previous biogeographic interpretations, and a more detailed study 2489
focusing on this genus is necessary, to fully understand the relationship of Malacoptila species, 2490
including the position of M. mystacalis (Lafresnaye, 1850), that in the concatenated UCE tree 2491
was recovered as sister to all other species (Fig. 3). Since, M. mystacalis UCE contigs were 2492
shorter due to DNA degradation common in toe pad samples (McCormack, Tsai & Faircloth, 2493
86
2016), we assembled a small subset of Malacoptila samples to minimize the effects of missing 2494
data, and yet, M. mystacalis was again, recovered as sister to all other species of Malacoptila 2495
(Fig. S12). Further sampling of this narrow endemic species is required to confirm if this pattern 2496
is true, or an artefact of toe pad sequencing error. 2497
5. Hapaloptila 2498
The monotypic Hapaloptila castanea (Verreaux, J, 1866) was recovered as sister group 2499
to Micromonacha, Cyphos, Cryptobucco, Hypnelus, Nystactes, and Notharchus (Fig. 1, 3). 2500
Very distinct in morphology, this species is specialized in cloud forests, usually above 1,500 2501
m, and even though it can be found in both sides of the Andes, no subspecies was ever described. 2502
The two specimens we samples are from opposite sides of Andes, however a more focused 2503
work on this species is required to understand the relationships among these apparently disjunct 2504
populations. 2505
6. Micromonacha 2506
Micromonacha lanceolata (Deville, 1849) occurs in the middle and upper stories of 2507
forests in both sides of the Andes, usually below 1,500 m. With populations also found in 2508
Panama and Costa Rica. Although no subspecies is currently recognized (Rassmussen & Collar, 2509
2018), populations from Central America were historically recognized in a distinct subspecies 2510
M. l. austinsmithi Dwight and Griscom, 1942. Our results recovered the sample from Panama 2511
as sister to the other two samples from Peru and Brazil, however, we refrain from making any 2512
nomenclatural change pending better sampling of this group to fully understand its diversity. 2513
7.Cyphos 2514
Cyphos macrodactylus Spix, 1824 can only be found east of the Andes, mostly near water 2515
inside terra-firme and varzea forests in Western Amazon. Our phylogeographic sampling 2516
showed almost no genetic structure, only the westernmost sample showed some difference. If 2517
this is, in fact, a phylogeographic structure, or just an artifact in sampling, still needs to be 2518
investigated. The described subspecies C. m. caurensis (Cherrie, 1916) from the Caura River 2519
region, Venezuela, is currently considered undifferentiated from the nominal form (Rassmussen 2520
& Collar, 2018), and probably does not correspond to this phylogeographic break, additional 2521
sampling is required for further assumptions. 2522
8. Cryptobucco 2523
The three species included in the newly described genus Cryptobucco, were first 2524
described as full species, and later lumped and considered conspecific as C. tectus (Boddaert, 2525
1783) (Peters, 1948). Recently, C. subtectus regained its status as full species (Rassmussen & 2526
87
Collar, 2018), but C. tectus and C. picatus are still considered subspecies (Gill & Donsker, 2527
2018; Remsen et al., 2018). Our results recovered C. picatus as sister to a clade containing C. 2528
tectus and C. subtectus, both in the mtDNA and the UCE tree. Biogeographically, implying that 2529
the two forms found in the Amazon, C. picatus and C. tectus, are not sister. Therefore, we 2530
propose the recognition of these taxa as full species and a more extensive work should be carried 2531
out to understand the limits of distribution of both Amazonian species, and if there is any 2532
contact, what are the implications of it. 2533
9. Hypnelus and Nystactes 2534
The sister relationship of Hypnelus and Nystactes is supported by the autapomorphic bifid 2535
bill tip in both genera, that is also present in Notharchus, although less pronounced in the later 2536
(Rassmussen & Collar, 2018). Hypnelus species are restricted to northern South America, with 2537
H. ruficollis (Wagler, 1829) having three subspecies, and H. bicinctus (Gould, 1837), two. Their 2538
specific status has been questioned based on hybridization in part of their distribution (Donegan, 2539
Quevedo, Verhelst, Cortés-Herrera, Ellery & Salaman, 2015), however without a thorough 2540
genetic and geographic sampling, this decision remains questionable. Nystactes noanamae 2541
(Hellmayr, 1909) and the species group of N. tamatia (Gmelin, JF, 1788), form the sister group 2542
of Hypnelus (Fig. 1, 3). Nystactes noanamae, is a restricted-range species, present only in a 2543
small portion of northwest Colombia, and currently considered Near-threatened by IUCN 2544
(Rassmussen & Collar, 2018). Its sister species, N. tamatia, is associated with the flooded 2545
forests in Amazonia, rarely found far from the water, even when in terra firme. Previous 2546
phylogeographic study found six genetic lineages for N. tamatia, one lineage was composed by 2547
only one sample though (Almeida, 2013). Nevertheless, our results corroborate the 2548
relationships previously found, and further studies are being conducted to understand the 2549
relationships and distribution of each lineage (Almeida, 2013). 2550
10. Notharchus 2551
Notharchus species can be grouped into three distinct groups based on distribution and 2552
morphology. Notharchus ordii (Cassin, 1851), as sister to all other species, is restricted to 2553
Amazonia, and unusually uncommon in collections. Its habitat preference and current 2554
distribution is virtually unknown. The sampling we gathered for the mtDNA sequencing 2555
actually represents all tissue samples available, and the apparent phylogeographic structure we 2556
found (Fig. S9) may only represent an artifact of sampling. Notharchus pectorales (Gray, GR, 2557
1846) is restricted to Northwest Colombia and East Panama. The last groups of species, is the 2558
group centered in N. macrorhynchus (Gmelin, JF, 1788). The ND2 analyses recovered a 2559
88
polytomy between the N. swainsoni (Gray, GR, 1846) N. macrorhynchus, and several lineages 2560
of N. hyperrhynchus, including one lineage from Central America (Fig. S9). Our UCE tree, in 2561
contrast, recovered N. macrorhynchus sister to N. swainsoni and N. hyperrhynchus. This result 2562
corroborates the recognition of N. hyperrhynchus and N. swainsoni as full species and renders 2563
the two Amazonian groups as non-sister lineages. Although the two subspecies of N. 2564
hyperrhynchus seem to be paraphyletic in the UCE topology, the geographical relationship 2565
seem to be reasonable, and a reappraisal of this subspecies distribution is desirable in further 2566
studies. 2567
2568
Conclusion 2569
The results presented here corroborate most of the diversity historically described in these 2570
two families, but also hidden patterns that need further investigation. With our thorough 2571
sampling of practically all widespread species and species complexes we were able to recover 2572
the phylogeographic patterns for the entire diversification of jacamars and puffbirds. This study 2573
is the first one to present a phylogenetic hypothesis for this two families employing a genomic 2574
dataset. Based on this tree we resolved some relationships that were obscured by morphological 2575
similarities among taxa, such as the recognition of the different species previously lumped into 2576
Galbula ruficauda, and even described a new puffbird genus to allocate the paraphyletic 2577
Notharchus species. Overall, the results presented here are another instance reinforcing the fact 2578
that Neotropical bird diversity still is underestimated, and that we still need exploratory research 2579
to fully comprehend diversity patterns, especially in the super complex Amazonian Basin, 2580
which will be of extreme importance for future biogeographical interpretations and better 2581
conservation planning. 2582
2583
Acknowledgements 2584
The authors thankfully acknowledge all the curators and curatorial assistants of the 2585
American Museum of Natural History, New York, USA (AMNH), Academy Academy of 2586
Natural Science of Drexel University, Philadelphia, USA (ANSP); Field Museum of Natural 2587
History, Chicago, USA (FMNH); Instituto Nacional de Pesquisas da Amazônia, Manaus, Brazil 2588
(INPA); Kansas University (KU), Laboratório de Genética e Evolução Molecular de Aves – 2589
USP (LGEMA), Lousiana State University Museum of Natural Science, Baton Rouge, USA 2590
(LSUMZ); and Museu Paraense Emílio Goeldi, Belém, Brazil (MPEG), Smithsonian Institution 2591
National Museum of Natural History (USNM), for borrowing tissue samples under their care. 2592
89
We are also grateful for all collectors involved in the fieldwork that make this paper possible. 2593
To J. McKay for helping with some laboratory procedures at the AMNH. MF acknowledge 2594
CAPES for his PhD fellowship, and CAPES PDSE fellowship (# 88881.133440/2016-01) and 2595
the support from the AMNH Frank M. Chapman Memorial Fund. The authors also thank the 2596
grant Dimensions US-Biota-São Paulo: Assembly and evolution of the Amazon biota and its 2597
environment: an integrated approach, co-funded by the US National Science Fundation (NSF 2598
DEB 1241056) to J.C. and the Fundação de Amparo à Pesquisa do Estado de São Paulo 2599
(FAPESP grant #2012/50260-6) to Lucia Lohmann; PEER-USAID Cycle 5 to CCR. AA and 2600
CCR are supported by CNPq research productivity fellowships (#310880/2012-2 and 2601
#308927/2016-8, respectively). The authors acknowledge the National Laboratory for 2602
Scientific Computing (LNCC/MCTI, Brazil) for providing HPC resources of the SDumont 2603
supercomputer, which have contributed to the research results reported within this paper. 2604
2605
2606
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Figure 1 – Phylogeny of the Galbulidae and Bucconidae families inferred with ExaBayes. All nodes in this tree 2778 receive the maximum posterior probability. The two genomes used as reference sequence were included in this 2779 analysis. 2780 2781
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Figure 2 – Phylogeny of the Galbulidae inferred by ExaBayes with the 75% completeness matrix. Node support 2785 is indicated near it, if no support is indicated posterior probability is 1.0. 2786 2787
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Figure 3 – Phylogeny of the Bucconidae inferred by ExaBayes with the 75% completeness matrix. Node support 2791 is indicated near it, if no support is indicated posterior probability is 1.0. 2792 2793
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Figure S1 – Phylogenetic relationship and map with sample distribution of Brachygalba and Jacamaralcyon 2796 species. Phylogenetic tree was recovered by MrBayes using the mtDNA gene ND2 (1041bp). Red circles mean 2797 posterior probabilities of 1.0, values that differs are indicated near the node. Samples highlighted in red were the 2798 samples selected for UCE analysis. The maps contain sample localities and approximate lineage distribution. 2799 Colours are correspondent between the tree and the map. 2800 2801
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Figure S2 – Phylogenetic relationship and map with sample distribution of Jacamerops aureus complex. 2805 Phylogenetic tree was recovered by MrBayes using the mtDNA gene ND2 (1041bp). Red circles mean posterior 2806 probabilities of 1.0, values that differs are indicated near the node. Samples highlighted in red were the samples 2807 selected for UCE analysis. The maps contain sample localities and approximate lineage distribution. Colours are 2808 correspondent between the tree and the map. 2809 2810
2811
2812
2813
98
Figure S3 – Phylogenetic relationship and map with sample distribution of Galbula dea complex. Phylogenetic 2814 tree was recovered by MrBayes using the mtDNA gene ND2 (1041bp). Red circles mean posterior probabilities 2815 of 1.0, values that differs are indicated near the node. Samples highlighted in red were the samples selected for 2816 UCE analysis. The maps contain sample localities and approximate lineage distribution. Colours are 2817 correspondent between the tree and the map. 2818 2819
2820
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99
Figure S4 – Phylogenetic relationship and map with sample distribution of the species complex of G. albirostris, 2822 G. chalcocephala and G. albirostris. Phylogenetic tree was recovered by MrBayes using the mtDNA gene ND2 2823 (1041bp). Red circles mean posterior probabilities of 1.0, values that differs are indicated near the node. Samples 2824 highlighted in red were the samples selected for UCE analysis. The maps contain sample localities and 2825 approximate lineage distribution. Colours are correspondent between the tree and the map. 2826 2827
2828
2829
2830
100
Figure S5 – Phylogenetic relationship and map with sample distribution of the species complex of G. galbula, G. 2831 tombacea, G. cyanescens, G. pastazae, and G. ruficauda. Phylogenetic tree was recovered by MrBayes using the 2832 mtDNA gene ND2 (1041bp). Red circles mean posterior probabilities of 1.0, values that differs are indicated 2833 near the node. Samples highlighted in red were the samples selected for UCE analysis. The maps contain sample 2834 localities and approximate lineage distribution. Colours are correspondent between the tree and the map. 2835 2836
2837
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2839
101
Figure S6 – Phylogenetic relationship and map with sample distribution of the species Bucco capensis. 2840 Phylogenetic tree was recovered by MrBayes using the mtDNA gene ND2 (1041bp). Red circles mean posterior 2841 probabilities of 1.0, values that differs are indicated near the node. Samples highlighted in red were the samples 2842 selected for UCE analysis. The maps contain sample localities and approximate lineage distribution. Colours are 2843 correspondent between the tree and the map. 2844 2845
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Figure S7 – Phylogenetic relationship and map with sample distribution of the species Cyphos macrodactylus. 2849 Phylogenetic tree was recovered by MrBayes using the mtDNA gene ND2 (1041bp). Red circles mean posterior 2850 probabilities of 1.0, values that differs are indicated near the node. Samples highlighted in red were the samples 2851 selected for UCE analysis. The maps contain sample localities and approximate lineage distribution. Colours are 2852 correspondent between the tree and the map. 2853 2854
2855
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Figure S8 – Phylogenetic relationship and map with sample distribution of the species complex of Notharchus 2858 tectus, N. subtectus, and N. picatus. Phylogenetic tree was recovered by MrBayes using the mtDNA gene ND2 2859 (1041bp). Red circles mean posterior probabilities of 1.0, values that differs are indicated near the node. Samples 2860 highlighted in red were the samples selected for UCE analysis. The maps contain sample localities and 2861 approximate lineage distribution. Colours are correspondent between the tree and the map. 2862 2863
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2867
104
Figure S9 – Phylogenetic relationship and map with sample distribution of the species complex of Notharchus 2868 ordii, N. pectorales, N. swainsoni, N. macrorhynchus, and N. hyperrhynchus. Phylogenetic tree was recovered by 2869 MrBayes using the mtDNA gene ND2 (1041bp). Red circles mean posterior probabilities of 1.0, values that 2870 differs are indicated near the node. Samples highlighted in red were the samples selected for UCE analysis. The 2871 maps contain sample localities and approximate lineage distribution. Colours are correspondent between the tree 2872 and the map. 2873 2874
2875
2876
2877
2878
105
Figure S10 – Phylogenetic relationship and map with sample distribution of the species Chelidoptera tenebrosa. 2879 Phylogenetic tree was recovered by MrBayes using the mtDNA gene ND2 (1041bp). Red circles mean posterior 2880 probabilities of 1.0, values that differs are indicated near the node. Samples highlighted in red were the samples 2881 selected for UCE analysis. The maps contain sample localities and approximate lineage distribution. Colours are 2882 correspondent between the tree and the map. 2883 2884
2885 2886
2887
2888
2889
106
Figure S11 – Phylogenetic tree recovered for Monasa using a subset of samples to check for M. mystacalis 2890 phylogenetic affinity. The same tree topology was recovered by RAxML and ExaBayes, the RAxML bootstrap 2891 support were low overall. 2892 2893
2894 2895
Figure S12 – Phylogenetic tree recovered for Malacoptila using a subset of samples to check for M. mystacalis 2896 phylogenetic affinity. The same tree topology was recovered by RAxML and ExaBayes with high support, with 2897 only node receiving bootstrap support different from 100. 2898 2899
2900
2901
2902
2903
2904
107
Síntese Geral 2905
Neste trabalho coletamos dados que nos ajudaram a compreender a relação filogenética 2906
de três famílias de aves do Neotrópico. A utilização de dados de sequenciamento genômico e a 2907
inclusão de amostras representando quase todas as linhagens basais em cada família permitiu 2908
realizar inferências sobre a importância de uma amostragem ampla, tanto num sentido de 2909
amostras, quanto marcadores. No primeiro capítulo pudemos observar o impacto do conflito 2910
entre marcadores moleculares com diferentes padrões de herança, e quais as implicações 2911
biológicas deste conflito. Além disso, através da análise combinada da história dos dois 2912
marcadores foi possível propor um modelo de evolução das áreas de vegetação aberta 2913
relacionadas aos solos de areia branca dentro da bacia Amazônia. No segundo capítulo, 2914
recuperamos a relação filogenética da família Trogonidae utilizando quase todas as espécies 2915
descritas com base em uma matriz com mais de 2.000 marcadores moleculares. Com base 2916
nesses resultados traçamos um modelo de como a evolução do clima desde o final do Oligoceno 2917
e as conexões entre os continentes influenciaram a história de diversificação do grupo. Por fim, 2918
no terceiro capítulo, analisamos a diversidade intraespecífica de duas famílias endêmicas do 2919
Neotrópico e reconstruímos a primeira hipótese de relação filogenética para Galbulidae e 2920
Bucconidae utilizando dados genômicos. Neste capítulo pudemos observar como a percepção 2921
da diversidade nesses grupos é subestimada e influenciada pela taxonomia vigente, e que a 2922
amostragem densa ao longo da distribuição de espécies amplamente distribuídas pode revelar 2923
táxons e padrões ainda desconhecidos. 2924
De modo geral, este trabalho reforça a complexidade dos padrões de diversidade da biota 2925
Neotropical, e que ainda se faz necessário estudos para desvendar esses padrões, em especial 2926
na Amazônia. Além disso, fica claro que a diversidade real da região ainda está mascarada pela 2927
taxonomia vigente e revisões sistemáticas e taxonômicas são necessárias. Só através do 2928
reconhecimento dessa diversidade escondida é que será possível, não só traçar hipóteses sobre 2929
os processos que deram origem a tamanha diversidade, mas também traçar planos de 2930
conservação que reconheçam a história evolutiva de cada um desses grupos. 2931
2932
108
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