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Identifying monophyletic groups within Bugula

sensu lato (Bryozoa, Buguloidea) http://www.producao.usp.br/handle/BDPI/49614

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Identifying monophyletic groups within Bugula sensu lato(Bryozoa, Buguloidea)KARIN H. FEHLAUER-ALE, JUDITH E. WINSTON, KEVIN J. TILBROOK, KARINE B. NASCIMENTO &LEANDRO M. VIEIRA

Submitted: 5 December 2014Accepted: 8 January 2015doi:10.1111/zsc.12103

Fehlauer-Ale, K.H., Winston, J.E., Tilbrook, K.J., Nascimento, K.B. & Vieira, L.M. (2015).Identifying monophyletic groups within Bugula sensu lato (Bryozoa, Buguloidea). —ZoologicaScripta, 44, 334–347.Species in the genus Bugula are globally distributed. They are most abundant in tropicaland temperate shallow waters, but representatives are found in polar regions. Seven speciesoccur in the Arctic and one in the Antarctic and species are represented in continental shelfor greater depths as well. The main characters used to define the genus include bird’s headpedunculate avicularia, erect colonies, embryos brooded in globular ooecia and branchescomprising two or more series of zooids. Skeletal morphology has been the primary sourceof taxonomic information for many calcified bryozoan groups, including the Buguloidea.Several morphological characters, however, have been suggested to be homoplastic at dis-tinct taxonomic levels, in the light of molecular phylogenies. Our purpose was to investigatethe phylogenetic interrelationships of the genus Bugula, based on molecular phylogeneticsand morphology. A Bayesian molecular phylogeny was constructed using original and previ-ously published sequences of the mitochondrial genes cytochrome c oxidase subunit 1 (COI)and the large ribosomal RNA subunit (16S). Morphological characteristics from scanningelectron and light microscopy were used to confirm the clades detected by the molecularphylogeny. Our results suggest that the genus is composed of four clades, for which we pro-vide diagnoses: Bugula sensu stricto (30 species), Bugulina (24 species), Crisularia (23 species)and the monotypic Virididentula gen. n. Ten species could not be assigned to any of thosegenera, so they remain as genus incertae sedis. Our findings highlight the importance ofusing molecular phylogenies in association with morphological characters in systematic revi-sions of bryozoan taxa.Corresponding author: Karin H. Fehlauer-Ale, Laborat�orio de Bentos, Centro de Estudos do Mar,Universidade Federal do Paran�a, Avenida Beira-Mar, s/n, Caixa Postal 61, CEP 83255-976,Pontal do Sul, Pontal do Paran�a, PR, Brazil. E-mail: [email protected] H. Fehlauer-Ale, Laborat�orio de Bentos, Centro de Estudos do Mar, Universidade Federal doParan�a, Avenida Beira-Mar, s/n, Caixa Postal 61, CEP 83255-976, Pontal do Sul, Pontal doParan�a, PR, Brazil and Laborat�orio de Sistem�atica e Evoluc�~ao de Bryozoa, Centro de BiologiaMarinha, Universidade de S~ao Paulo, Rodovia Manoel Hyp�olito do Rego, km 131,5 Praia doCabelo Gordo, CEP 05588-000, S~ao Sebasti~ao, SP, BrazilJudith E. Winston, Smithsonian Marine Station, 701 Seaway Drive, Fort Pierce, FL 34949,USA. E-mail: [email protected] J. Tilbrook, Oxford University Museum of Natural History, Parks Road, Oxford, OX13PW, UK. E-mail: [email protected] B. Nascimento, Laborat�orio de Sistem�atica e Evoluc�~ao de Bryozoa, Centro de BiologiaMarinha, Universidade de S~ao Paulo, Rodovia Manoel Hyp�olito do Rego, km 131, 5 Praia doCabelo Gordo, CEP 05588-000, S~ao Sebasti~ao, SP, Brazil. E-mail: [email protected] M. Vieira, Departamento de Zoologia, Centro de Ciencias Biol�ogicas, Universidade Federalde Pernambuco, Av. Prof. Moraes Rego 1235, Cidade Universit�aria, CEP 50670-901, Recife,Pernambuco, Brazil and Laborat�orio de Sistem�atica e Evoluc�~ao de Bryozoa, Centro de BiologiaMarinha, Universidade de S~ao Paulo, Rodovia Manoel Hyp�olito do Rego, km 131,5 Praia doCabelo Gordo, CEP 05588-000, S~ao Sebasti~ao, SP, Brazil. E-mail: [email protected]

ª 2015 Royal Swedish Academy of Sciences, 44, 3, May 2015, pp 334–347334

Zoologica Scripta

IntroductionBryozoans are suspension feeding, colonial and mostly ses-sile aquatic invertebrates, represented by approximately5870 described living (Bock & Gordon 2013) and 15 000fossil species (Gordon 2009). Within the order Cheilosto-mata, Bugula Oken, 1815 (Buguloidea, Bugulidae) is one ofthe most familiar and well-studied genera, represented bymore than 80 valid species (Bock & Hayward 2014).Species in the genus Bugula are globally distributed,

inhabiting shallow water in tropical to cold water regions(Kluge 1962; Hayward 1995; Hayward & Ryland 1998;Winston & Woollacott 2008; Vieira et al. 2012), with somerepresentatives reported from deeper waters of the conti-nental shelf and beyond (Hayward 1981). Some species areknown from fouling communities and also may form well-established introduced populations (Mackie et al. 2006;Johnson & Woollacott 2012; Karlson & Osman 2012;Fehlauer-Ale et al. 2014); others are apparently endemic toparticular regions (Ryland 1960; Winston & Woollacott2008; Vieira et al. 2012).The genus Bugula has a long and complicated taxonomic

history. The name, Bugula, was introduced by Oken (1815)to accommodate three species: Cellularia neritina, Cellulariaavicularia and Bugula avicularia (Linnaeus, 1758). Later tax-onomists used different names or grouped species in differ-ent ways. Harmer (1923, 1926) synonymized nine genericnames under Bugula: Acamarchis Lamouroux, 1816 (typespecies Sertularia neritina Linnaeus, 1758; objective juniorsymonym of Bugula, see below); Halophila Gray, 1843 (typespecies Halophila johnstonae Gray, 1843); Avicularia Gray,1848 (junior homonym of spider genus Avicularia Lamarck,1818); Bugulina Gray, 1848 (type species Sertularia avicular-ia Linnaeus, 1758); Crisularia Gray, 1848 (type speciesSertularia fastigata Linnaeus, 1758; =Cellularia plumosaPallas, 1766; see Harmer, 1923); Avicella Van Beneden,1848 (no type species selected); Ornithopora d’Orbigny,1852 (type species Sertularia avicularia); Ornithoporinad’Orbigny, 1852 (type species Ornithoporina aviculariad’Orbigny, 1852; =Avicularia flabellata Gray, 1848; see Har-mer 1923); and Dendrobeania Levinsen, 1909 (type speciesFlustra murrayana Johnston, 1847). Two of these genera,however, are currently considered distinct genera in Bugu-lidae: Dendrobeania (see Hayward & Ryland 1998) andHalophila (see Winston 2005); although Ornithopora waspreviously considered as a synonym of Bugula (Harmer1923), it is an objective junior synonym of Bugulina (seebelow). Crisularia is here considered a distinct taxon (seebelow).In 1956, the International Commission on Zoological

Nomenclature (ICZN), the governing body for animalnames under the International Union of Biological Sci-ences to make decisions about names that violate the rules,

laid down in the International Code made a decision negat-ing the use of Bugula Oken. Opinion 417 (Hemming 1956)stated that names introduced by Oken (1815) were notavailable because Oken’s work was not consistently binomi-nal. To change the long-used name of this speciose andabundant group of bryozoans at this point would have beendisruptive to bryozoan taxonomy. To maintain stability,John Ryland (Ryland 1967) petitioned the Commision ofZoological Nomenclature to make the genus name BugulaOken available for use. Sertularia neritina Linnaeus, 1758;was subsequently designated the type by the ICZN underits plenary powers (Opinion 902); thus, Bugula is an objec-tive senior synonym of Acamarchis. Since then, the genusname Bugula has been applied in its broad sense to specieswith a variety of morphological character states withrespect to branching pattern, spines and ooecia (e.g.Hayward & Ryland 1998; Winston & Woollacott 2008;Vieira et al. 2012).The genus has been characterized by bird’s head pedun-

culate avicularia, erect colonies and branches comprisingtwo or more series of boat-shaped zooids (Ryland 1960).Almost all species have pedunculate bird’s head avicularia,the type species Bugula neritina (Linnaeus, 1758) being aremarkable exception. The patterns of branch bifurcationin biserial species are usually one of three types describedby Harmer (1923): types 3, 4 or 5 (Fig. 1; see also Harmer1926; Winston & Woollacott 2008; Vieira et al. 2012).The basal and lateral walls of zooids are lightly calcified,and most of the frontal wall is membranous, providing flex-ibility to the branches. The majority of species have spineson the distal angles of the zooids; in some species, thespines are merely pointed extensions of the distal edges ofzooids, but in others, they are articulated (Winston &Woollacott 2008). The ooecia may be crescentic to globu-lar or helmet shaped (Ramalho et al. 2005; Winston &Woollacott 2008).Skeletal morphology has been the primary source of taxo-

nomic information for many calcified bryozoan groups (e.g.McKinney & Jackson 1989; Jackson & Cheetham 1990;Hayward & Ryland 1998, 1999; Tilbrook 2006; Cheethamet al. 2007; Vieira et al. 2012, 2013, 2014). Several morpho-logical characters, however, have been recently revealed tobe homoplastic at distinct taxonomic levels in the light ofmolecular phylogenies, suggesting that the overall currentbryozoan classification requires revision. Examples includecalcified body forms distinguishing ctenostomes from chei-lostomes within Gymnolaemata (Fuchs et al. 2009); mor-phology and structure of the frontal wall withinCheilostomata (Tsyganov-Bodounov et al. 2009); skeletalmorphology at ordinal and family levels within Cyclostomata(Waeschenbach et al. 2009); shape of clusters of zooids rotating360° around stolon for the ctenostomes Amathia brasiliensis

ª 2015 Royal Swedish Academy of Sciences, 44, 3, May 2015, pp 334–347 335

K. H. Fehlauer-Ale et al. � Monophyletic groups within Bugula sensu lato

Busk, 1886; and Amathia distans Busk, 1886 (Fehlauer-Aleet al. 2011); development of frontal wall along with ascus andassociated structures involved in lophophore extensionwithin the infraorder Ascophorina (Knight et al. 2011); andlarval characters across the classes Gymnolaemata, Phylacto-laemata and Stenolaemata (Waeschenbach et al. 2012). Onthe other hand, certain morphological characters or suites ofcharacters are phylogenetically informative at one level oranother, for example, the shape of vibracular chamber andooecium defining the cheilostome genus Scrupocellaria sensustricto (Vieira et al. 2014). Thus, an integrative approachbased on supporting molecular phylogenies with morpholog-ical characters seems very useful in clarifying the systematicsof bryozoan taxa.We aimed to investigate the interrelationships of the

genus Bugula, using DNA sequence data from two mito-chondrial (mt) genes: cytochrome c oxidase subunit 1(COI) and large ribosomal RNA subunit (16S). Morpho-logical characters based on scanning electron microscopy(SEM) and light microscopy were taken into account toconfirm the clades detected by the molecular phylogeny.

Material and methodsSampling and morphological examination

All necessary permits were obtained for the field studiesdescribed (collecting permit numbers 10186 and 19936SISBIO/ Instituto Chico Mendes de Conservac�~ao da Bio-diversidade). The bryozoans were collected by hand orscuba diving in the intertidal or shallow subtidal from

localities along the coasts of Brazil (SW Atlantic), Spain(NE Atlantic), UK (Bristol Channel), USA (NW Atlantic)and New Zealand (Tasman Sea), and also at greater depths(25–37 m) in Sweden (Kattegat Sea) (Table S1). Samplingdetails are available on the Barcode of Life Database (BOLD)System v3 (Ratnasingham & Hebert 2007) (www.boldsys-tems.org), on the public project BRBRY.The samples were fixed in 92–99% ETOH while still in

the field. In the laboratory, colonies were examined andidentified on a Zeiss SV 11 stereomicroscope (Jena,Germany). The identified material was then stored at−20°C until extractions of genomic DNA (gDNA) wereperformed. Some specimens were mounted on stubs andcoated with a gold–palladium alloy for observation by SEMwith JEOL 6460LV (Akishima-Shi, Tokyo, Japan) andZeiss LEO 440 machines.Several specimens sequenced for this study were depos-

ited as hologenophores (Pleijel et al. 2008) at the Museu deZoologia da Universidade de S~ao Paulo (MZUSP), S~aoPaulo, Brazil. Others are held at the Center for MarineStudies of the Federal University of Paran�a (CEM/UFPR),Pontal do Paran�a, Paran�a, Brazil; Center for Marine Biol-ogy of the University of S~ao Paulo (CEBIMar-USP), S~aoSebasti~ao, S~ao Paulo, Brazil; and San Jose State University,San Jose, California, USA (Table S1; BOLD).

DNA extraction, amplification and sequencing

Total gDNA was extracted with the DNeasy Blood andTissue Kit (QIAGEN, Germantown, MD, USA) from

A B C D

Fig. 1 Bifurcation pattern of species included in present study. A, type 4 found in some Bugula species (Bugula neritina-minima group) andVirididentula gen. n. B, modified type 3 found in some Bugula species (Bugula uniserialis group). C, type 3, found in Crisularia. D, type 5,found in some Bugulina species (A, C, and D redrawn from Ryland, 1960).

336 ª 2015 Royal Swedish Academy of Sciences, 44, 3, May 2015, pp 334–347

Monophyletic groups within Bugula sensu lato � K. H. Fehlauer-Ale et al.

http://www.boldsystems.org

http://www.boldsystems.org

clean zooids containing polypides. Segments of COI (50

and 30 end fragments, henceforth referred as COI-5P andCOI-3P, respectively) and 16S were sequenced for thesampled material (Table S1). Primer details, cycling condi-tions and amplicon sizes are detailed in Table S2. PCRswere carried out in 25 lL volumes using illustra puReTaqReady-To-GoTM PCR Beads (GE Healthcare, Fairfield,CT, USA); each PCR contained ~30 ng of gDNA templateand each primer in 0.8 lM. PCR products were cleanedwith illustra GFXTM PCR DNA and Gel Band PurificationKit (GE Healthcare).Sequencing in both directions was performed in an ABI

PRISM� 3100 Genetic Analyzer, using the BigDye�Ter-minator v3.1 Cycle Sequencing Kit (Applied Biosystems,Foster City, CA, USA). Complementary strands were com-bined and edited with CodonCode Aligner v4 (CodonCodeCorporation, Dedham, MA, USA) and compiled usingGENEIOUS PRO v5.5.6 (Drummond et al. 2011). COIsequences were translated using the invertebrate mt geneticcode in Geneious Pro to check for internal stop codonsand frameshift mutations, none of which were detected.Sequence identity was checked using the basic local align-ment search tool (BLAST) (www.ncbi.nih.gov/BLAST/).Sequences were deposited in GenBank (Table S1; BOLD),and chromatograms were deposited in BOLD.

Phylogenetic analysis

In an attempt to achieve a broad representation of Buguloi-dea in our analyses, we included 47 sequences of COI-5Pand 32 sequences of 16S available in GenBank for thissuperfamily (last search in April 2014). These were addedto the dataset comprising our newly generated sequences(COI-5P, n = 4; 16S, n = 14). Because COI3-P sequencesfor only seven species of Bugula were available on the dateof our last search, we did not include this segment in thephylogenetic analysis, although we deposited our 11 newsequences in GenBank (Table S1).Forty-five nominal species of Buguloidea, including 22

of Bugula (Table S1), comprised the ingroup. The bryozo-ans Amathia distans Busk, 1886 (Ctenostomata, Vesicularii-dae), Crisia pseudosolena (Marcus, 1937) (Stenolaemata,Crisiidae) and Conopeum seurati (Canu, 1928) (Cheilosto-mata, Electridae) were included in the outgroup (TableS1).All sequences were aligned in MAFFT v6.903 (Katoh et al.

2005), using default parameters and allowing designation ofambiguity codes, present in some sequences retrieved from

GenBank. Ambiguously aligned regions for the 16S genewere excluded with Gblocks Server (http://mole-vol.cmima.csic.es/castresana/Gblocks_server.html; Castre-sana 2000), using all the options for a less stringentselection. Gblocks removed 26.5% of the original 16Salignment (637 bp), leaving 468 included positions; 330 ofthese were variable.Substitution saturation tests were performed in DAMBE

(Xia 2013) for the 1st, 2nd and 3rd codon positions ofCOI-5P, using the method of Xia et al. (2003) and Xia &Lemey (2009). The tests indicated little saturation for the1st and 2nd positions, but substitution saturation andtherefore no useful phylogenetic signal for the 3rd position.Thus, we removed this position for the phylogenetic analy-sis, reducing the original alignment from 675 to 450 bp.The default settings of JMODELTEST version 0.1.1 (Guin-

don & Gascuel 2003; Posada 2008) were used to select theoptimal substitution model separately for each gene parti-tion, using the Akaike information criterion (AIC). Themodels TrN+I+G and TIM2+I+G were selected for theCOI and the 16S partitions, respectively. Phylogenetic rela-tionships were evaluated through Bayesian inference (BI) inMRBAYES v3.1.2 (Ronquist & Huelsenbeck 2003). Theequivalent settings of both models were set to nst = 6,rates = gamma and ngammacat = 4. Two runs with fourchains each were performed for each analysis for 15 000 000generations and sampled every 1000 generations, until thestandard deviation of split frequencies had reached a valuebelow 0.01; 25% of the generations were discarded as burn-in. Bayesian posterior probabilities (pp) ≥50% are shown.

Results and discussionPhylogenetic analysis

The BI molecular phylogeny based on the concatenation ofCOI-5P and 16S sequences suggests that the genus Bugulasensu lato is composed of four clades (Fig. 2), as follows.

Bugula Oken sensu stricto. The BI tree detected a moder-ately supported clade (pp, 78%) that includes Bugula ingensVieira, Winston and Fehlauer-Ale, 2012; Bugula migottoiVieira, Winston and Fehlauer-Ale, 2012; the Bugula neriti-na complex [see Fehlauer-Ale et al. (2014) for detailsregarding species delimitation for this complex]; Bugularochae Vieira, Winston and Fehlauer-Ale, 2012; and Bugulasubglobosa Harmer, 1926 (Fig. 2). All the species in thisclade lack articulated spines and exclusively share the pres-ence of long zooids, bifurcating biserial branches with

Fig. 2 COI-5P + 16S Bayesian phylogeny for Bugula sensu lato. Clades of Bugula sensu stricto, Bugulina, Crisularia and Virididentula gen. n.are highlighted in gray, and pp values ≥50% of nodal support are shown. The scale bar indicates branch length in number of substitutionsper site. *Sequences obtained in this study; **misidentified species in GenBank (see Table S1).



http://www.ncbi.nih.gov/BLAST/

http://molevol.cmima.csic.es/castresana/Gblocks_server.html

http://molevol.cmima.csic.es/castresana/Gblocks_server.html

Crisia pseudosolenaKF714793*, KF714809*

Beania mirabilis HQ896144Bicellariella ciliata FJ196101

Bugulina carvalhoi KF714797*

Caberea lata HQ896153

Conopeum seurati KF714792*, KF714808*

Tricellaria occidentalis HQ896152

Amastigia xishaensis HQ896156

Amathia distans JF490058, JF490053

Bugula ingens JQ693390

Bugula neritina Type N KC129811, KC130044

Crisularia turrita AY173428**; KC129850, JX183889; AY633483, AY633950Crisularia plumosa KC129848, JX183888

Bugulina stolonifera AF061433**, AY633944; AY789087; AY173427; KC129849, JX183887

Virididentula dentata AY061742; AY061743; AY061744; AY061745

Bicellariella ciliata edentata KF714788*, KF714795*

Beania hexaceras HQ896142Virididentula dentata KF714798*; KC129718, JX183885

Bugulina fulva KC129719, JX183886

Bugula rochae KF714791*, JQ693391

Virididentula dentata AY633933

Tricellaria inopinata AY061750

Scrupocaberea maderensis AY789103 Bugulopsis monotrypa AY789106

Beania klugei KF714794*

Crisularia purpurotincta KF714790*, KF714802*Crisularia microoecia KF714801*


Licornia diadema HQ896158


Bugula migottoi KC129720, JX183890

Scrupocellaria scruposa FJ196098

Bugulina foliolata KF714799*

Crisularia pacifica AY633474**, AY633938; AY633475, AY633939; AY633476, AY633940;AY633477, AY633941

Beania magellanica JF950422, JF950315

Caberea n. sp. KF714805*

Caberea rostrata JF950414, JF950334**

Bugulina turbinata KF714804*

Bugula neritina Type D KC129836, JX183894

Caulibugula pearsei KF714807*

Crisularia guara KF714789*, KF714800*

Bugulina simplex KF714803*; AY633478, AY633942

Bugula subglobosa HQ896147

Beania plurispinosa JF950431, JF950316

, Amastigia rudis HQ896154

Bugula neritina Type S KC129741, KC129979

Caulibugula armata KF714806*

Beania vegae HQ896141

Pomocellaria varians AF156291

Crisularia bowiei KF714796*

Bugulina flabellata AY633952** ; AY061749; AY633484, AY633951

Crisularia cucullata AY061748

Bugulina turbinata AY633481, AY633947; AY633480, AY633946; AY633482, AY633948

9 5

9 7

9 8

100

100

100

97

100

9 9

100

100

100

100

100

0.2

93

61

65

79

9451

87

89

8953

72 83

100

94

52

89

98

53

60

8994

78

72



bifurcation type 4 (Figs 1a, S1a) or a modified type 3 withuniserial appearance (Figs 1b, S1b; see Vieira et al. 2012),and spherical ooecia attached at the inner distal angle ofthe autozooids (Fig. S1b–d).The congruence between the molecular phylogeny and

shared morphological characters allowed us to define thespecies in this clade as representatives of the genus Bugulasensu stricto, including the type species Bugula neritina. Wealso include in Bugula sensu stricto 25 other species (see theTaxonomic accounts section) for which no molecular dataare available, but which share the suite of morphologicalcharacteristics listed above.

Resurrection of Bugulina Gray and Crisularia Gray. Gray(1848) erected the genera Bugulina Gray, 1848; and Crisu-laria Gray, 1848; to accommodate Cellularia aviculariaLinnaeus, 1758; and Cellularia plumosa Pallas, 1766; respec-tively. Unfortunately, Gray (1848) included neither mor-phological descriptions nor drawings of the two genera.Later authors (Hincks 1880; Harmer 1923, 1926) synony-mized Bugulina and Crisularia with Bugula Oken, based onboth having erect colonies with boat-shaped zooids andpedunculate bird’s head avicularia. However, the BI tree(Fig. 2) and several morphological characters supportGray’s original (1848) taxonomic decisions and conse-quently allow the resurrection of both genera, as follows:Figure 2 depicts Bugulina carvalhoi (Marcus, 1949) comb.

n.; Bugulina flabellata (Thompson in Gray, 1848) comb. n.;Bugulina foliolata (Vieira, Winston and Fehlauer-Ale, 2012)comb. n.; Bugulina fulva (Ryland, 1960) comb. n.; Bugulinasimplex (Hincks, 1886) comb. n.; Bugulina stolonifera(Ryland, 1960) comb. n.; and Bugulina turbinata (Alder,1857) comb. n. These species comprise a well-supportedclade (pp, 100%). Morphologically, the clade members areexclusively characterized by dimorphic avicularia (Fig. S2a,c), and globular ooecia centred in the zooid midline andattached to the distal region of the autozooid (Fig. S2a,b),with the proximal and lateral ectooecial area calcified, butthe rest of the ooecium membranous (Fig. S2b–f); so thatwhen the ooecium is preserved dried, the distal and frontalareas are often shrunken and depressed, showing the calci-fied entooecium underneath, a state distinct from the prox-imal exposed entooecium characteristic of the Crisulariaspecies (see below). Moreover, except for Bugulina avicular-ia (Linnaeus, 1758) and Bugulina stolonifera comb. n. (Fig.S2c,d), whose colonies have biserial branches with variablebranching patterns (in B. stolonifera branching patternstypes 3, 4 and 5 may occur in the same colony; see Maturo1966; Hayward & Ryland 1998), species in this clade arealso characterized by colonies with multiserial branches.As Bugulina avicularia, the type species of the genus Bugu-

lina Gray, has dimorphic avicularia and globular ooecia cen-

tred in the zooid midline (see De Blauwe 2009), as well asbiserial colonies and a variable branching pattern like colo-nies of Bugulina stolonifera, we allocate the species to BugulinaGray. We also assign to Bugulina another 17 species thatwere previously assigned to Bugula Oken, including Bugulinaavicularia, because they share the same morphological attri-butes (see the Taxonomic accounts section), although DNAsequence data are still lacking.The BI molecular phylogeny (Fig. 2) also detected

another well-supported clade (pp, 98%), comprising Crisu-laria bowiei (Vieira, Winston and Fehlauer-Ale, 2012)comb. n.; Crisularia guara (Vieira, Winston and Fehlauer-Ale, 2012) comb. n.; Crisularia pacifica (Robertson, 1905)comb. n.; Crisularia plumosa (Pallas, 1766); Crisularia purp-urotincta Norman, 1868) comb. n.; and Crisularia turrita(Desor, 1848) comb. n. These species share the combinedpresence of bifurcation pattern type 3 (Harmer 1923), likespecies in the Crisularia turrita comb. n. group (Vieira et al.2012) (Fig. 1c), and an ooecium with proximally exposedentooecium and calcification restricted to the distal edge(Fig. S3a–f). Hence, we resurrect Crisularia Gray to accom-modate all six species mentioned above, including the typespecies Crisularia plumosa, and 15 others that exhibit thesesame morphological characters, but for which DNAsequence data are lacking (see the Taxonomic accounts sec-tion). Additionally, although not grouping in the Crisulariaclade (Fig. 2), Crisularia cucullata (Busk, 1867) comb. n. andCrisularia microoecia (Osburn, 1914) comb. n. were alsoaccommodated in this genus because they exhibit the suiteof morphological characters stated above, except that theovicells are partially immersed at distal wall of the zooecia.

Virididentula gen. n. The BI tree (Fig. 2) shows all repre-sentatives of Virididentula dentata (Lamouroux, 1816) comb.n. (formerly Bugula dentata Lamouroux, 1816) to comprisea moderately supported clade (pp, 83%). Similar to thegenus Bugula, Virididentula dentata also has colonies withbranching pattern type 4 (Harmer 1926) (Fig. 1a). Virid-identula dentata, however, is the only species previouslyassigned to Bugula Oken that has jointed outer distal spinesand an ooecium with the ectooecium reduced except at thelateral proximolateral edges, with a membranous frontalarea and smooth entooecium (Fig. S4a,b). The BI molecu-lar phylogeny indicating a clade for this species separatefrom that of Bugula Oken sensu stricto, and the exclusivelyshared suite of morphological characters support the desig-nation of a new monotypic genus, Virididentula gen. n., toaccommodate the former species ‘Bugula dentata’ (see diag-nosis in the Taxonomic accounts section).Virididentula dentata is globally distributed, being

reported from several localities around the Australia–NewGuinea continent, Brazil, Celebes Sea, Hawaii, Japan,



Madeira and South Africa (Ryland 1974; Mackie et al.2002; Lim 2004; Ramalho et al. 2005). Intraspecific mor-phological variation related to avicularia dimorphism, thezooidal position of avicularia and the orientation of ooecia(Ryland 1974; Mackie et al. 2002), and matching divergentlineages based on COI sequences (Mackie et al. 2002) nowsuggest the existence of a complex of cryptic species, ratherthan a single widespread species, for this taxon, strengthen-ing the case for generic segregation by the establishment ofthe genus Virididentula.

Taxonomic accounts

Family BUGULIDAEGenus Bugula Oken, 1815 (Figs 1a,b, S1a,d)Bugula Oken, 1815: 89. Type species: Sertularia neriti-na Linnaeus, 1758; by subsequent designation.Acamarchis Lamouroux, 1816: 132. Type species: Sertularianeritina Linnaeus, 1758; by subsequent designation (see Harmer1923) .Diagnosis. Colony erect or creeping, bifurcating biserial

branches; autozooids elongate, basal and lateral walls lightlycalcified, frontal surface mostly membranous; pedunculatebird’s head avicularia present (except in Bugula neritina),attached to the proximal third of the lateral edge of thezooid; ooecium pedunculate, rarely reduced, spherical,attached to the inner distal margin of zooid; ooecium withtwo calcified layers (ectooecium and entooecium) and aninner membranous ooecial vesicle; ancestrula withoutspines; two bifurcation patterns: (i) type 4 (Bugula neritina-minima group; Fig. 1a; see Winston & Woollacott 2008)and (ii) modified type 3 (Bugula uniserialis group; Fig. 1b;see Vieira et al. 2012), with long proximal gymnocyst.

Remarks. Busk (1884) distinguished four groups (a, b, cand d) of Bugula based on branch morphology, shape ofavicularia and presence of spines. Harmer (1923) later pro-posed the names Himantozoum Harmer, 1923 and Camptop-lites, Harmer, 1923; for Busk’s (1884) groups a and b,respectively. Among other species assigned to Bugula byBusk, (1884), species of type d belong to Bugula sensustricto, but Bugula margaritifera Busk, 1884, assigned to typec, belongs to Hiamantozoum. Other species assigned toBugula group c belong to Bugulina.Bugula neritina shares morphological features with some

species included in two distinct morphologically definedgroups of Bugula, the Bugula minima group (Winston &Woollacott 2008) and the Bugula uniserialis group (Vieiraet al. 2012). These shared characters include the following:(i) absence of oral spines, (ii) ancestrula without spines, (iii)ooecia with calcified ectooecium and entooecium, and (iv)ooecia attached to inner distal margin of zooid [except inBugula gautieri Ryland, 1962; whose ooecia are reduced

and the embryos are found on the basal wall of the zooecia(Ryland 1962), like those observed in many cheilostomefamilies (Ostrovsky 2013)]. In spite of the absence of avicu-laria in Bugula neritina, the other species in the genus havebird’s head avicularia attached to the lateral edge in theproximal third of the zooid. Most of the remaining speciespreviously assigned to Bugula, easily distinguished by hav-ing distal spines, are here reassigned to three other genera(see below).We suggest that Bugula must be restricted to species

with biserial colonies, type 4 or modified type 3 bifurcation(with uniserial appearance), spherical ooecia attached at theinner distal angle of the autozooid, and zooids withoutarticulated spines (Figs 1a,b, S1a–d). Thirty species areassigned here to Bugula sensu stricto: 19 belonging to theBugula neritina-minima group (for descriptions of andremarks on these species see Harmer 1926; Hastings 1939;Ryland 1962; Prenant & Bobin 1966; Gordon 1986;Winston & Woollacott 2008; Vieira et al. 2012), and 11 tothe Bugula uniserialis group (for descriptions of and remarkson these species see Yanagi & Okada 1918; Hayward 1981;Fransen 1986; Tilbrook et al. 2001; Vieira et al. 2012).

Valid species. Species of the Bugula neritina-minimagroup: Bugula alba Vieira, Winston and Fehlauer-Ale,2012; Bugula capensis Waters, 1887; Bugula ceylonensisWinston and Woollacott, 2008; Bugula crosslandi Hastings,1939; Bugula expansa Hastings, 1939; Bugula gautieriRyland, 1962; Bugula migottoi Vieira, Winston andFehlauer-Ale, 2012; Bugula miniatella Winston and Woolla-cott 2008; Bugula minima Waters, 1909; Bugula neritina(Linnaeus, 1758); Bugula neritinoides Hastings, 1939; Bugulapaternostrae Winston and Woollacott, 2008; Bugula prismat-ica (Gray, 1843); Bugula providensis Winston and Woolla-cott, 2008; Bugula robusta MacGillivray, 1869; Bugularobustoides Winston and Woollacott, 2008; Bugula solorensisWinston and Woollacott, 2008; Bugula subglobosa Harmer,1926; and Bugula vectifera Harmer, 1926. Species of theBugula uniserialis group: Bugula biota Vieira, Winston andFehlauer-Ale, 2012; Bugula decipiens Hayward, 1981; Bugulagnoma Vieira, Winston and Fehlauer-Ale, 2012; Bugulahummelincki Fransen, 1986; Bugula ingens Vieira, Winstonand Fehlauer-Ale, 2012; Bugula protensa Hayward, 1981;Bugula rochae Vieira, Winston and Fehlauer-Ale, 2012; Bu-gula scaphoides (Kirkpatrick, 1890); Bugula scaphoides con-stricta Yanagi and Okada, 1918; Bugula scaphula Tilbrook,Hayward and Gordon, 2001; and Bugula uniserialis Hincks,1884.

Genus Bugulina Gray, 1848 (Figs 1d, S2a–f)Bugulina Gray, 1848: 114. Type species: Sertularia avicular-ia Linnaeus, 1758; by original designation.



Avicularia Thompson in Gray, 1848: 105. Type species: Avicu-laria flabellata Thompson in Gray, 1848; by original designa-tion. Not Avicularia Lamarck, 1818.Ornithopora d’Orbigny, 1852: 321. Type species: Sertulariaavicularia Linnaeus, 1758; by original designation.Ornithoporina d’Orbigny, 1852: 322. No type species designed.Bugula b Busk, 1884: 90 (part).Diagnosis. Colony erect, with bifurcating biserial to

multiserial branches; autozooids elongate, basal and lateralwalls lightly calcified, frontal surface mostly membranous; apedunculate bird’s head avicularium attached to lateral edgeof zooid; dimorphic avicularia present, smaller on innerthan on outer zooids of the branches; ooecium centred inmidline and attached to the distal region of the autozooid,with ectooecium calcified laterally and proximally, membra-nous distofrontally; ancestrula with spines; biserial colonieswith variable bifurcation pattern, often type 5.

Remarks. Oken (1815) included Sertularia avicularia inthe genus Bugula; later, this group was named Bugulina byGray (1848). Ryland (1967) suggested rejection of thename Bugulina by the ICZN due a possible misidentifica-tion of the type species, Sertularia avicularia, a biserial spe-cies, with the multiserial species Avicularia flabellata, type ofAvicularia Thompson in Gray, 1848 (a junior homonym ofAvicularia Lamarck, 1818). However, the Commision ofZoological Nomenclature (1970) rejected the inclusion ofBugulina in the Official Index of Rejected and Invalid Gen-eric Names in Zoology (Opinion 902), and it remainedavailable for use. Bugulina avicularia is characterized by bi-serial colonies and an ooecium with ectooecium calcifiedlaterally and proximally, membranous distofrontally; itsbifurcation pattern is also variable, like those of Bugulinastolonifera. Two other biserial species are also reassigned toBugulina: Bugula tricuspis (Kluge, 1955) comb. n. andBugulina carvalhoi; they are also characterized by having avariable bifurcation pattern and ooecia with a distal mem-branous area. Bugulina carvalhoi is distinct in having somebiserial branches in early growth and forming brancheswith 3–6 series of autozooids.Here, we also assign the multiserial species previously

assigned to Bugula to the genus Bugulina owing to the pres-ence of ooecia with proximal and lateral calcified ectooe-cium and a distal membranous area. The multiserialbranches described for the majority of species, the shape ofthe ooecium and the presence of dimorphic avicularia—absent in Bugulina tricuspis—are easily recognizable mor-phological features that make the species of the genusBugulina distinct from those of Bugula and Crisularia.Twenty-four species are assigned here to Bugulina (for

descriptions and remarks on each species listed, seePackard 1863; Hincks 1886; Robertson 1905; Marcus 1949;

Osburn 1950, 1953; Ryland 1960; Kluge 1962; Prenant &Bobin 1966; Hayward & Ryland 1998; De Blauwe 2009;Vieira et al. 2012).

Valid species. Bugulina angustiloba (Lamarck, 1816)comb. n.; Bugulina aquilirostris (Ryland, 1960) comb. n.;Bugulina avicularia (Linnaeus, 1758); Bugulina borealis(Packard, 1863) comb. n.; Bugulina calathus (Norman, 1868)comb. n.; Bugulina calathus minor (Ryland, 1962) comb. n.;Bugulina californica (Robertson, 1905) comb. n.; Bugulinacarvalhoi (Marcus, 1949) comb. n.; Bugulina ditrupae (Busk,1858) comb. n.; Bugulina eburnea (Calvet, 1906) comb. n.;Bugulina flabellata (Thompson, in Gray, 1848) comb. n.;Bugulina flabellata acuminata (Osburn, 1953) comb. n.;Bugulina foliolata (Vieira, Winston and Fehlauer-Ale, 2012)comb. n.; Bugulina fulva (Ryland, 1960) comb. n.; Bugulinalongirostrata (Robertson, 1905) comb. n.; Bugulina multiseri-alis (d’Orbigny, 1841) comb. n.; Bugulina pedata (Harmer,1926) comb. n.; Bugulina pugeti (Robertson, 1905) comb.n.; Bugulina pugeti kiuschiuensis (Sil�en, 1941) comb. n.;Bugulina simplex (Hincks, 1886) comb. n.; Bugulina spicata(Hincks, 1886) comb. n.; Bugulina stolonifera (Ryland, 1960)comb. n.; Bugulina tricuspis (Kluge, 1955) comb. n.; Buguli-na turbinata (Alder, 1857) comb. n.

Genus Crisularia Gray, 1848 (Figs 1c, S3a–f)Crisularia Gray, 1848: 111. Type species: Cellularia plumosaPallas, 1766; by original designation.Avicella Van Beneden, 1848: 74 (part). No type speciesdesigned.Diagnosis. Colony erect, bifurcating biserial branches,

attached to substrata by long radicles; autozooids elongate,basal and lateral walls lightly calcified, frontal surfacemostly membranous, its plane often tilted towards the axis;a pedunculate bird’s head avicularium attached to the outerlateral edge of zooid; ooecium rounded cap (rarelyreduced), globular, slightly shifted towards inner margin,with calcified ecooecium at the distal edge; ancestrula vase-shaped, with spines; bifurcation pattern type 3.

Remarks. Here, we reassign the species of the Bugulaturrita group (see Vieira et al. 2012) to the genus Crisular-ia, characterized by bifurcation pattern type 3 (Fig. 1c) androunded, cap-shaped ooecia, with distally calcified ectooe-cium (Fig. S3f). Three species (Bugula cucullata Busk, 1867;Bugula cuspidata Hastings, 1943; and Bugula microoeciaOsburn, 1912) are characterized by reduced ooecia (seeHastings 1943; Maturo 1966), but also have bifurcationpattern type 3 like those of Crisularia; they are here reas-signed to Crisularia, although the first and the last werenot placed in the Crisularia group in the BI tree (Fig. 2).Reduction of ooecia is also found in other genera in Bugu-



lidae (Ostrovsky 2013), such as Bugula sensu stricto (seeabove). Twenty-three species are assigned here to Crisular-ia (for descriptions and remarks on each species listed seeHincks 1886; Waters 1900; Osburn 1912, 1950; Harmer1926; Hastings 1943; Maturo 1966; Prenant & Bobin 1966;Castric-Fey 1971; Rao & Ganapati 1974; Androsova 1977;Hayward & Ryland 1998; Vieira et al. 2012).

Valid species. Crisularia aperta (Hincks, 1886) comb. n.;Crisularia bengalensis (Rao & Ganapati, 1974) comb. n.;Crisularia bowiei (Vieira, Winston and Fehlauer-Ale, 2012)comb. n.; Crisularia cucullata (Busk, 1867) comb. n.; Crisu-laria cucullifera (Osburn, 1912) comb. n.; Crisularia cuspidata(Hastings, 1943) comb. n.; Crisularia dispar (Harmer, 1926)comb. n.; Crisularia gracilis (Busk, 1858) comb. n.; Crisular-ia grayi (Maturo, 1966) comb. n.; Crisularia guara (Vieira,Winston and Fehlauer-Ale, 2012) comb. n.; Crisulariaharmsworthi (Waters, 1900) comb. n.; Crisularia hyadesi(Jullien, 1888) comb. n.; Crisularia marcusi (Maturo, 1966)comb. n.; Crisularia microoecia (Osburn, 1914) comb. n.;Crisularia mollis (Harmer, 1926) comb. n.; Crisularia pacifica(Robertson, 1905) comb. n.; Crisularia nana (Androsova,1977) comb. n.; Crisularia plumosa (Pallas, 1766); Crisulariaprenanti (Castric-Fey, 1971) comb. n.; Crisularia purpuro-tincta (Norman, 1868) comb. n.; Crisularia rylandi (Maturo,1966) comb. n.; Crisularia serrata (Lamarck, 1816) comb.n.; Crisularia turrita (Desor, 1848) comb. n.

Genus Virididentula gen. n. (Figs 1a, S4a,b)Type species. Acamarchis dentata Lamouroux, 1816.Etymology. The genus name is a latinized combination

viridi-, from Latin viridis, green, owing to the green colour ofthe colonies, plus dent-, Latin dens, dentis, tooth, alluding tothe long zooidal spines, and the diminutive Latin suffix -ulus.The name is in the feminine form to parallel that of Bugula.

Diagnosis. Colony erect, strongly coloured, green togreen-blue in colour, with bifurcating biserial branches;bifurcation type 4; autozooids elongate, basal and lateralwalls well calcified, frontal surface mostly membranous andturned towards the axis of the branches; distal articulatedspines present; a pedunculate bird’s head aviculariumattached to the outer lateral edge of the zooid; dimorphicavicularia sometimes present; ooecium closed by an opercu-lum, attached to the distal region of zooids, near to the axis,slightly oblique; ooecium with the ectooecium reduced at thelateral proximal edge, with a broad membranous frontal areaand smooth entooecium; ancestrula with spines.

Remarks. Virididentula gen. n. is also characterized bybranching pattern type 4 (Harmer 1923) as with someBugula species, but it is distinct from Bugula in having

jointed outer distal spines, and ooecium with ectooeciumreduced at lateral proximal edge with a membranous fron-tal area and smooth entooecium.

Valid species. Virididentula dentata comb. n. (Lamouroux,1816).

Genus incertae sedisSome species assigned to Bugula (viz. Bugula aspinosa Liu,1984; Bugula hessei Hasenbank, 1932; Bugula intermediaLiu, 1984; Bugula longissima Busk, 1884; Bugula orientalisLiu, 1984; Bugula philippsae Harmer, 1926; and Bugulasimpliciformis Osburn, 1932 (see Busk 1884; Harmer 1926;Hasenbank 1932; Osburn 1932; Liu 1984) require addi-tional studies based on type specimens and/or DNAsequencing to test their generic status. These species mayeven belong to distinctive genera other than Bugula, Bugu-lina, Crisularia and/or Virididentula because of charactersthat may include a distinct branching pattern, the shape ofthe bird’s head avicularia, a long peduncle on the avicularia,the shape of the ooecia and/or the presence of abfrontalkenozooids along the branches.Brief descriptions, but no recent figures, are known for

two species that may have branching pattern type 3, viz.Bugula lophodendron Ortmann, 1890, (Ortmann 1890: plate1, figure 18) and Bugula tschukotkensis Kluge, 1952 (Kluge1962: figure 116); these species may also belong to Crisu-laria, but their assignment remains uncertain until examina-tion of the type material is carried out.Finally, Bugula umbelliformis Yanagi and Okada, 1918;

could be placed in Bugulina owing to its erect colonies withmultiserial branches and the bird’s head avicularia on theouter lateral margin of autozooids. Despite these similari-ties, Bugula umbelliformis differs in lacking distal spines andooecia. The absence of information on reproductive struc-tures precludes the allocation of Bugula umbelliformis toBugulina. Therefore, we place this species in genus incertaesedis.

List of species of Genus incertae sedis. Bugula aspinosa Liu,1984; Bugula hessei Hasenbank, 1932; Bugula intermediaLiu, 1984; Bugula longissima Busk, 1884; Bugula lophoden-dron Ortmann, 1890; Bugula orientalis Liu, 1984; Bugulaphilippsae Harmer, 1926; Bugula simpliciformis Osburn,1932; Bugula tschukotkensis Kluge, 1952; and Bugula umbelli-formis Yanagi and Okada, 1918.

Misidentified species

In the BI tree, Bugulina turbinata was represented by speci-mens collected in the NE Pacific that were sequenced forCOI-5P and 16S (Table S1); additionally, we generated anew 16S sequence from a specimen collected in the Bristol



Channel (Table S1). The sample from the Bristol Channel(Bugulina turbinata, GenBank KF714804) did not groupwith the three samples from the NE Pacific [Bugulina turbi-nata, GenBank AY633480 (COI-5P), AY633946 (16S);AY633481 (COI-5P), AY633947 (16S); AY633482 (COI-5P), AY633948 (16S); Fig. 2]. Alder (1857) stated the typelocality of this species to be the British coast and foundspecimens at Tenby, a locality near Watwick Bay, wherewe collected our sample. Thus, we suggest that thesequences from specimens from the NE Pacific representanother, previously undetected species of Bugulina thatresembles Bugulina turbinata. Morphological examination ofNE Pacific specimens is thus necessary to further investi-gate their taxonomic status.Additionally, pairwise percentage similarity values calcu-

lated in Geneious Pro (very close to or equal to 100%)and the BI tree (Fig. 2) revealed that the specimensfor some sequences retrieved from GenBank had notbeen correctly identified. These are Bugulina flabellata(GenBank AY633952), Bugulina stolonifera (GenBankAF061433) and Crisularia pacifica (GenBank AY633474),which had been incorrectly assigned to ‘Bugula neritina’,‘Bugula pacifica’ and ‘Bugula dentata’, respectively (TableS1).In a preliminary BI phylogenetic analysis (not shown),

Bicellariella fragilis Seo & Min, 2009 (GenBankHQ896150–HQ89615051; Table S1) grouped in the Bugu-la sensu stricto clade. Bicellariella fragilis, identified by havingjoints, turbinate zooids and very long spines (Seo & Min2009), clearly does not share the diagnostic characters ofBugula sensu stricto. Thus, we removed this taxon from ouranalysis and suggest that its placement in the Bugula sensustricto clade was likely due misidentification of a specimenin a previous work (Lee et al. 2011).Finally, that same preliminary BI phylogenetic analysis (not

shown) placed Bugula umbelliformis incertae sedis (GenBankHQ896148–HQ8961489; Table S1) in a clade with the cte-nostome Flustrellidra hispida (Fabricius, 1780) (GenBankNC_008192). The analysis by Lee et al. (2011) similarlyshowed this species as the sister group to Flustrellidra armataGrischenko, Seo and Min, 2010. These results suggest thatthe COI-5P sequences for Bugula umbelliformis incertae sedisrepresent contaminant sequences originating from ctenosto-mes. Misidentified species and contaminant sequences of bry-ozoans have been previously detected in another study (seeWaeschenbach et al. 2012), and their continued identificationwill allow more accurate systematic revisions.

Final considerationsAs a direct result of the increasing number of studies thathave presented molecular phylogenies for bryozoans atdistinct taxonomic scales (e.g. Hao et al. 2005; Lim-Fong

et al. 2008; Fuchs et al. 2009; Tsyganov-Bodounov et al.2009; Knight et al. 2011; Lee et al. 2011; Waeschenbachet al. 2012), many useable COI and 16S sequences for spe-cies in Buguloidea have become available in GenBank. Thisavailability, combined with our newly generated sequences,allowed us to evaluate the current classification of Bugulafrom a phylogenetic perspective.In accordance with the BI molecular phylogeny, we rede-

fined the genus Bugula Oken to include 30 species, charac-terized by biserial colonies, bifurcation modified type 3 (withuniserial appearance) or type 4, spherical ooecia attached atthe inner distal angle of the autozooids, and zooids withoutarticulated spines. The 24 species reassigned to Bugulina aredistinguished from Bugula by having multiserial colonies andooecia with proximal and lateral calcified ectooecium and adistal membranous area. Twenty-three species of the ‘Bugulaturrita’ group (see Vieira et al. 2012) were reassigned toCrisularia; they are characterized by bifurcation pattern type3 and rounded cap-shaped ooecia, with distally calcifiedectooecium. Finally, one species was assigned to Virididentulagen. n., characterized by biserial colonies with branchingpattern type 4 (Harmer 1923), jointed outer distal spines andooecium with ectooecium reduced at the lateral proximaledge, with a membranous frontal area and smooth entooe-cium. Ten species could not be assigned to any of the abovegenera, remaining as incertae sedis.The taxonomic value of the red or green pigment of bugu-

lids remains unclear. The only species whose red pigmenthas been studied in detail is Bugula neritina (see Winston &Woollacott 2008 for references). Other red-pigmented spe-cies studied by Winston &Woollacott (2008) are placed herein Bugula, for example Bugula robusta and Bugula minima, butred pigment is also found in Crisularia (e.g., Crisulariapupurotincta). The dark-green-pigmented Antarctic speciesBugula longissima is placed not with Virididentula, but withthose species still considered incertae sedis.

AcknowledgementsThis is a contribution of the Center for Marine Studies, Fed-eral University of Paran�a (CEM/UFPR), the Center forMarine Biology of the University of S~ao Paulo (CEBIMar/USP) and the Center for Marine Biodiversity Research(NP-BioMar/USP). This research was partially supported byThe National Council for Scientific and TechnologicalDevelopment (CNPq) (151221/2013-8, granted to Paulo C.Lana and Karin H. Fehlauer-Ale; 305608/2006-1, 474605/2013-2, granted to Leandro M. Vieira; 564945/2010-2 – ‘Br-Bol’, granted to Mariana C de Oliveira) and the S~ao PauloResearch Foundation (FAPESP) (2009/08940-7, granted toKarin H Fehlauer-Ale; 2009/08941-3 to Alvaro E Migotto;2012/24285-1, granted to Leandro M Vieira). Contributionno. 979 from the Smithsonian Marine Station, Fort Pierce,



http://www.ncbi.nlm.nih.gov/nuccore/KF714804

http://www.ncbi.nlm.nih.gov/nuccore/AY633480







http://www.ncbi.nlm.nih.gov/nuccore/AF061433


http://www.ncbi.nlm.nih.gov/nuccore/HQ896150




http://www.ncbi.nlm.nih.gov/nuccore/NC_008192

Florida. We wish to thank Alvaro E. Migotto (Center forMarine Biology of the University of S~ao Paulo), Paulo C.Lana (Center for Marine Studies of the Federal Universityof Paran�a) and Tim Littlewood (Natural History Museum ofLondon) for providing laboratory and office facilities. Sev-eral collaborators donated exemplars and/or helped withfieldwork, and we are very grateful to all of them: AlbertoLindner (Federal University of Santa Catarina), AndreaWaeschenbach (Natural History Museum of London),Arnaud Czaja (Imperial College of London), Dennis Gordon(National Institute of Water & Atmospheric Research),Javier Souto (University of Santiago de Compostela), JudithFuchs (Marine Biological Association UK), Oscar Reverter-Gil (University of Santiago de Compostela) and Rosana M.da Rocha (Federal University of Paran�a). Andrew Ostrovskyprovided helpful information on brood chamber structuresincluded in this study. Three anonymous reviewers providedvery helpful comments and suggestions on the manuscript.

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Supporting InformationAdditional Supporting Information may be found in theonline version of this article:Fig. S1. Morphological features of Bugula sensu stricto.Fig. S2. Morphological features of Bugulina.Fig. S3. Morphological features of Crisularia.Fig. S4. Morphological features of Virididentula gen. n.Table S1. Bryozoan species included in the Bayesian

phylogenetic analysis, with information on genes and sam-pling sites.Table S2. Primers used, PCR product sizes in bp,

cycling conditions and primers reference.



identifying monophyletic groups within bugula sensu lato … · 2017. 6. 18. · parana, avenida...

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