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UNIVERSIDADE ESTADUAL DE CAMPINAS Instituto de Biologia

RENATA APARECIDA DOS SANTOS ALITTO

RECONHECIMENTO E DESCRIÇÃO DE ESPÉCIES DE OPHIUROIDEA (ECHINODERMATA) POR MEIO DA TAXONOMIA INTEGRATIVA

RECOGNITION AND DESCRIPTION OF OPHIUROIDEA (ECHINODERMATA) THROUGH INTEGRATIVE

CAMPINAS 2019 2

RENATA APARECIDA DOS SANTOS ALITTO

RECONHECIMENTO E DESCRIÇÃO DE ESPÉCIES DE OPHIUROIDEA (ECHINODERMATA) POR MEIO DA TAXONOMIA INTEGRATIVA

RECOGNITION AND DESCRIPTION OF OPHIUROIDEA SPECIES (ECHINODERMATA) THROUGH INTEGRATIVE TAXONOMY

Tese apresentada ao Instituto de Biologia da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Doutora em BIOLOGIA , na Área de BIODIVERSIDADE ANIMAL.

Thesis presented to the Institute of Biology of the University of Campinas in partial fulfillment of the requirements for the degree of Doctor, in the area of ANIMAL BIOLOGY.

ESTE ARQUIVO DIGITAL CORRESPONDE À VERSÃO FINAL DA TESE DEFENDIDA PELA ALUNA RENATA APARECIDA DOS SANTOS ALITTO E ORIENTADA PELA MICHELA BORGES.

Supervisor/Orientador: DRA. MICHELA BORGES Co-supervisor/Coorientador: DR. MAIKON DI DOMENICO

CAMPINAS 2019 3

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COMISSÃO EXAMINADORA

Prof. Dra. Michela Borges (Orientadora)

Prof. Dra. Luciana Ribeiro Martins

Prof. Dr. Leonardo Querobim Yokoyama

Prof. Dra. Fosca Pedini Pereira Leite

Prof. Dr. Fabrizio Marcondes Machado

Os membros da Comissão Examinadora acima assinaram a Ata de Defesa, que se encontra no processo de vida acadêmica do aluno.

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DEDICATÓRIA

Se Deus colocou um sonho em seu coração, Ele tem toda a intenção de fazê-lo acontecer.

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AGRADECIMENTOS

O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001 e Programa de Doutorado Sanduíche no Exterior (PDSE) 88881.131940/2016-01. Agradeço também as agências de fomento FAPESP (2011/50317-5 e 2011/50724-0) e FAEPEX (2101/16) que possibilitaram a realização desse trabalho e pelo financiamento ao projeto.

Primeiramente quero agradecer a Deus por ter tido o privilégio de viver tantas experiências. Agradecer por Ele ter me dado coragem para seguir confiante atrás dos meus sonhos, e fazer planos para alcançá-los. Agradecer por ter colocado o Thiago em meu caminho e por me permitir partilhar com ele tantas experiências e descobrir o real sentido da vida e da felicidade. Sem ele, nada disso faria sentido. Só tenho a agradecer. Meu Deus, muito obrigada! Também quero agradecer... Aos meus pais, João Dimas e Maria Helena, por me amarem tanto e por pensarem em mim em todas as decisões que tomam em suas vidas. Obrigada por me ensinarem a amar. Obrigada por me incentivarem nos estudos e por nunca me deixarem desistir mesmo quando as situações foram muito difíceis. Ao meu marido Thiago, por embarcar nesse sonho comigo que hoje é realidade. Não foi só eu que cresci, vejo que você também cresceu e amadureceu. Hoje entendo como foi importante essa etapa ter acontecido em nossas vidas. Eu faria tudo de novo com você. À Nina, muito mais que um bichinho de estimação! É meu despertador, minha companheira de passeios e refeições, meu depósito de amor incondicional. Te abraçar me faz tão bem! Com você meus dias são mais leves e doces!! As minhas melhores amigas Erica e Ana Lucia. Obrigada pela amizade de vocês que é tão importante para mim. Vocês são uma benção em minha vida. À Michela, mais que orientadora, uma incentivadora. Obrigada por me apoiar em todas as decisões, não só científicas, mas também pessoais. Obrigada por toda a paciência, carinho e amor em ensinar. Lembro-me da primeira vez que você me mostrou um ofiuróide – seus olhos brilharam! E isso me marcou profundamente! É por isso que você é uma grande inspiração para mim, não só como pesquisadora, mas também como mãe – presente em todos os momentos! Tenho certeza que tudo isso fez e faz toda a diferença em minha formação profissional e pessoal. Ao Maikon, meu co-orientador. Obrigada por me desafiar a sempre dar o melhor de mim. Com você aprendi a argumentar, questionar, defender minhas opiniões. Obrigada por me mostrar como é maravilhoso aprender e descobrir habilidades que eu nem imaginava. À Karin, minha “co-orientadora” e amiga. Obrigada por me explicar todas as técnicas moleculares com tanto carinho e com exemplos que só você para pensar. Imagina se eu estivesse dentro de um ependorfe? Eu imaginei, entendi e jamais esquecerei. 7

À Maris e ao Pablo, grandes amigos e incentivadores. Obrigada pela disponibilidade, paciência e carinho em sempre me ajudar! À Ana Christensen por me ensinar sobre biologia marinha com um amor incondicional. Obrigada por ter me desafiado todos os dias e me incentivar dizendo: “You are set”. Sim! Você tem razão, eu estou pronta! Eu estou pronta para aparecer, aparecer para o mundo. Obrigada por me mostrar isso e me ensinar a me autoconhecer. A todos do Museu: Adriana, Artur, Karina, Simone e Jean, por proporcionarem um ambiente ideal tanto para trabalhar como para descontrair e dar risada. A todos da equipe Equi.Lab: Gabriela, Letícia, Helena, Gisella e Cecília. Vocês foram muito importantes no desenvolvimento desta tese. Sem vocês, não teríamos resultados tão impactantes. Afinal, sozinhos vamos mais rápido, mas juntos vamos mais longe. Muito obrigada!! A todos do LabEsc: Karin, Kaleb, Alessandra, Profª Luciana e Rê Tenório por todo apoio nos experimentos moleculares. À Capes pela bolsa de estudos que pude usufruir no Brasil e nos Estados Unidos. Essa experiência foi imensamente importante para mim, não só profissionalmente, mas pessoalmente. Aos professores Danilo C. Miguel e Silmara M. Allegretti, coordenadores da Pós- graduação em Biologia Animal da UNICAMP. Obrigada por todo o apoio e ajuda. À professora Cecília Amaral pela dedicação intensiva na coordenação do projeto BIOTA/Araçá e pela disponibilização do material utilizado. Aos professores Silvana Siqueira, André Garraffoni, Danilo C. Miguel, Leonardo Yokoyama, Luciana Martins, Fosca Pedini e Fabrizio Marcondes pelos excelentes comentários, correções e sugestões durante a qualificação, pré-banca e banca. À UNICAMP e ao curso de pós-graduação em Biologia Animal do IB/UNICAMP pela formação proporcionada. Aos meus professores do curso de graduação em Ciências Biológicas da Pontíficia Universidade Católica de Campinas (PUC/Campinas). Em especial, o Prof. Ed Gonçalves e a Profa. Monica Oliveira por me despertaram o amor pela Genética e Zoologia. Ao Museu de Zoologia da UNICAMP, Laboratório de Microscopia Eletrônica da UNICAMP e Laboratório de Estudos Cromossômicos da UNICAMP pela infraestrutura para realização deste trabalho. À Lamar University. Aqui incluo todos os professores e alunos que me receberam com tanto carinho e tiveram tanta paciência comigo durante minha experiência nos Estados Unidos. Enfim, obrigada a todos que direta ou indiretamente, fazem parte da minha história. 8

RESUMO

Os Ophiuroidea estão entre os invertebrados marinhos mais abundantes na fauna de fundo oceânico e representam um táxon modelo ideal na análise de padrões globais de diversidade e distribuição. Diante de toda essa notabilidade, a taxonomia dos Ophiuroidea tem recebido mais atenção nos últimos anos. Isto porque trabalhos taxonômicos não acurados podem prejudicar outras disciplinas que, direta ou indiretamente, dependem da taxonomia. Nesta tese, reconhecemos e descrevemos espécies de Ophiuroidea por meio da taxonomia integrativa (TI). A TI visa delimitar as unidades de biodiversidade a partir de perspectivas múltiplas e complementares, como por exemplo, caracteres morfológicos, genéticos, ecológicos, comportamentais e filogeográficos. Nesta tese, as espécies candidatas (hipóteses) foram testadas por meio de caracteres morfológicos (morfologia externa, morfologia das microestruturas braquiais e bucais; morfometria de estruturas diagnósticas) e caracteres moleculares (fragmentos dos genes mitocondriais 16S e COI). O conteúdo desta tese encontra-se em três capítulos. No primeiro capítulo foi descrita e validada a espécie trindadensis Tommasi, 1970. Ophiothrix trindadensis é uma espécie nativa do Brasil, até então, sem série-tipo depositada em museus e, por isso, foi proposta a designação de um neótipo coletado na Ilha Trindade (localidade tipo). No segundo capítulo foi feito um estudo comparativo entre Ophiothela sp. do Brasil (BR), Ophiothela mirabilis Verrill, 1867 e Ophiothela danae Verrill, 1869. Estas espécies são fissíparas, e isso dificulta o seu reconhecimento com base somente em caracteres morfológicos externos. Ophiothela sp. BR, O. mirabilis, e O. danae estão intimamente relacionadas. No entanto, Ophiothela sp. BR tem um diferente conjunto de haplótipos e Ophiothela sp. BR está mais próxima de O. danae do que de O. mirabilis nas análises morfométricas. Ophiothela sp. BR é diferente das demais espécies analisadas, porém o conjunto de dados não permitiu verificar se é uma linhagem geográfica ou uma espécie nova para a Ciência. A descrição de Ophiothela sp. BR revelou características interessantes, especialmente com relação a morfologia das microestruturas. Durante os estudos morfométricos, ocorrências morfológicas não usuais de Ophiothela sp. BR foram observadas e descritas no terceiro capítulo. Ophiothela sp. BR tem uma alta incidência de fissiparidade evidenciada por um alto número de configurações do disco. Nossas redescrições serão úteis em novas identificações de espécimes e os resultados gerados poderão ser usados em análises de biodiversidade, filogenia, genética de populações, evolução e ecologia. Palavras-chave: equinodermos, bentos marinho, biodiversidade, ofiuróides 9

ABSTRACT

Ophiuroids (brittle stars) are an ideal model taxon to analyze global patterns of species diversity as they are a major component of the benthic fauna. Due to their apparent significance, the taxonomy of Ophiuroidea has received more attention lately. However, taxonomy without an accurate delimitation can impair other disciplines that, directly or indirectly, depend on it. In this thesis, we recognize and describe Ophiuroidea species through the integrative taxonomy (IT). IT is defined as the science that aims to delimit the units of life’s diversity from multiple and complementary perspectives, such as comparative morphology, population genetics, ecology, behavior, and phylogeography. Species boundaries were examined using morphological data (external morphology, morphometry of diagnostic structures, arm microstructures) and molecular data (16S and COI). Research presented in this thesis is divided in three chapters. In the first chapter, I re-describe and confirm the validity of Ophiothrix trindadensis Tommasi, 1970 (Echinodermata: Ophiuroidea). This is a native species from Brazil, however it lacked a type series deposited in scientific collections. I proposed a neotype designation based on topotype specimen from Trindade Island. In the second chapter, a comparative study between Ophiothela sp. BR, Ophiothela mirabilis Verrill, 1867 and Ophiothela danae Verrill, 1869 was developed. They are fissiparous species, which made the morphological studies and the delimitation of Ophiothela species even more difficult. Ophiothela sp. BR, O. mirabilis, and O. danae are closely related. However, Brazilian specimens have a different set of haplotypes and Ophiothela sp. BR is closer to O. danae than to O. mirabilis on the morphometric analysis. The Brazilian specimens are distinct, but current evidence is not enough to work out if they are geographic lineage or species. The detailed description of Ophiothela sp. BR revealed interesting features, particularly regarding its microstructural morphology. Unusual morphological occurrences were observed on Ophiothela sp. BR and were presented in the third chapter. Ophiothela sp. BR has high incidence of fissiparity evidenced by a great number of disc configuration categories. Our redescriptions will be useful in future specimens’ identifications and the results can be used in analyses concerning biodiversity, phylogeny, population genetics, evolution and ecology. Keywords: , benthos, biodiversity, brittle stars

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SUMÁRIO

INTRODUÇÃO ...... 11

CAPÍTULO 1 ...... 40 Atlantic West Ophiothrix spp. in the scope of integrative taxonomy: confirming the existence of Ophiothrix trindadensis Tommasi, 1970 ...... 40

CAPÍTULO 2 ...... 110 Unraveling the taxonomic identity of Ophiothela Verrill, 1867 (Ophiuroidea) along the Brazilian coast ...... 110

CAPÍTULO 3 ...... 159 Unusual morphological occurrences in the small six-rayed Ophiothela sp. BR (Echinodermata: Ophiuroidea) ...... 159

ANEXO 1 ...... 194 ANEXO 2 ...... 195 ANEXO 3 ...... 196 ANEXO 4 ...... 197 ANEXO 5 ...... 198 ANEXO 6 ...... 199 ANEXO 7 ...... 200

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INTRODUÇÃO

Os Ophiuroidea são diversos e abundantes em comunidades bentônicas e são considerados um táxon modelo ideal para análises de padrões globais de diversidade, em estudos filogeográficos, evolutivos e ecológicos (O’Hara & Tittensor 2010; Stöhr et al. 2012; Woolley et al. 2016; O’Hara et al. 2019). Eles ocorrem desde regiões equatoriais as polares e desde o entremarés à zona hadal e apresentam uma variedade de estratégias de dispersão que afetam a conectividade das populações, como larvas planctotróficas, larvas lecitotróficas, viviparidade e fissiparidade (Byrne & Selvakumaraswamy 2002; Stöhr et al. 2012). São conhecidos por possuírem muitos registros fósseis, especialmente na forma de ossículos isolados, os quais tem recebido destaque em estudos filogenéticos recentes (O’Hara et al. 2014; Thuy & Stöhr 2016). Devido a sua abundância, hábitos alimentares e altos níveis de atividade, os ofiuróides têm um significativo impacto no balanço energético e na ecologia das comunidades de fundos não-consolidados pela utilização, processamento e redistribuição da matéria orgânica (Summers & Nybakken 2000). Além disso, eles são considerados o principal elo entre as cadeias alimentares locais envolvendo peixes e outros macroinvertebrados de significativo valor comercial (Fell 1966; Manso & Farias 1999). Os Ophiuroidea também são usados como bioindicadores de poluição, pois por serem residentes permanentes refletem as condições ambientais locais (Harmelin et al. 1981; Manso & Absalão 1988). Em um estudo recente, Barboza et al. (2015a) investigaram o padrão de distribuição dos ofiuróides ao longo de um gradiente de contaminação na Baía de Paranaguá, sul do Brasil. Os autores concluíram que a abundância de indivíduos, especialmente Amphipholis januarii, foi significativamente menor em locais com altas concentrações de esteróides fecais e hidrocarbonetos alifáticos. Por outro lado, Alitto (2015) destacou a abundância de Hemipholis cordifera próximo ao emissário de esgoto sanitário na Baía do Araçá, corroborando a tolerância da espécie em sedimentos poucos oxigenados (Christensen & Colacino 2000). Apesar de toda esta notabilidade, estudos sobre a taxonomia e evolução dos ofiuróides são ainda bastante restritos (Stöhr 2012; O'Hara et al. 2017). De acordo com O'Hara et al. (2017) a riqueza de espécies conhecida (aproximadamente 2100 espécies) é certamente uma grande subestimativa, já que estudos recentes identificaram linhagens crípticas para muitas morfo-espécies examinadas (Stöhr et al. 2009; O’Hara et al. 2019). 12

No Brasil, o último levantamento apontou 133 espécies de Ophiuroidea (Barboza & Borges 2012). É inquestionável que o conhecimento do grupo aumentou, especialmente na região sudeste, devido aos inventários de diversidade elaborados nos últimos anos e que foram financiados no âmbito do Programa BIOTA/FAPESP (Borges & Amaral 2005; Borges et al. 2011; Amaral et al. 2016; Alitto et al. 2018). Porém, segundo Stöhr et al. (2012) a América do Sul é uma das regiões menos diversas para a fauna de Ophiuroidea, o que está relacionado à escassez de taxonomistas e estudos com o grupo para esta área. Sendo assim, o reconhecimento desta biodiversidade depende de um trabalho acurado de taxonomia, com formação de recursos humanos e integração de diferentes metodologias corroborando ou não as identificações propostas. Neste contexto taxonômico, algumas espécies de Ophiuroidea, classe mais diversa e abundante dos Echinodermata (Stöhr et al. 2012), foram estudadas sob um ponto de vista mais integrativo, no intuito de seguramente reconhecê-las como unidade biológica. A seguir apresentamos algumas informações sobre: (1) Características gerais e filogenia dos Echinodermata; (2) Morfologia, taxonomia e filogenia dos Ophiuroidea; (3) Conceito de espécie e taxonomia integrativa; (4) Objetivos e principais resultados.

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1. Características gerais e filogenia dos Echinodermata

Características gerais

O filo Echinodermata é exclusivamente marinho com representantes encontrados em costões rochosos, praias, baías, entremarés e mar profundo (Hendler et al. 1995; Pawson 2007). Dentre as cinco classes viventes, Ophiuroidea (serpentes-do-mar, ofiuróides) contém o maior número de espécies descritas (2087), seguido por Asteroidea (estrelas-do-mar, 1800), Holothuroidea (pepinos-do-mar, 1700), Echinoidea (ouriços-do-mar e bolachas-da-praia, 1000) e a menos estudada Crinoidea (lírios-do-mar, 700) (Alvarado & Solís-Marín 2013; Kroh & Mooi 2018; Mah 2018; Stöhr et al. 2018). Outras 13.000 espécies são conhecidas (Pawson 2007) devido aos ricos registros fósseis, que datam desde o Cambriano inferior (Zamora et al. 2013).

O primeiro trabalho com Echinodermata do Brasil foi feito por J. Marcgrave, em 1648, o qual cita quatro espécies (dois asteróides, um equinóide e um ofiuróide) (Marcgrave 1942). Descrições de Rathbun (1879) compuseram o primeiro catálogo de Echinodermata da costa brasileira e em 1882, Ludwig apresentou um estudo de 30 espécies (Ludwig 1882). A partir do século XX, estudos taxonômicos começaram a se desenvolver devido aos esforços do Professor Dr. Luiz Roberto Tommasi, do Instituto Oceanográfico da Universidade de São Paulo. Em seus trabalhos, Prof. Tommasi estudou a morfologia das diferentes classes de Echinodermata e forneceu as primeiras chaves de identificação para as espécies brasileiras (Tommasi 1965, 1966, 1969, 1970a, b). Atualmente, há 329 espécies registradas no litoral brasileiro. Ophiuroidea é a classe mais diversa com 143 espécies, seguida por Asteroidea (75), Holothuroidea (51), Echinoidea (45) e Crinoidea (17) (Borges et al. em preparação).

O filo inclui membros com papéis ecológicos fundamentais na cadeia alimentar dos ecossistemas costeiros. Alguns exemplos: predadores vorazes como as estrelas-do-mar que controlam a densidade de outros invertebrados (Lawrence 2013); consumidores primários como os ouriços-do-mar (Jangoux & Lawrence 1982); responsáveis pela ciclagem de matéria orgânica como os pepinos-do-mar (Uthicke 1999; Ginger et al. 2001); responsáveis pela transferência da produção primária para o sedimento, enriquecendo o ecossistema bentônico como os ofiuróides (Josefson & Conley 1997).

Algumas espécies são consideradas modelo para estudos de embriologia, ecotoxicologia e biotecnologia (Petzelt 2005; Pawson 2007; Benavides-Serrato et al. 2011). Isto porque equinodermos possuem diversos mecanismos de desenvolvimento, fácil 14

manutenção em laboratório (especialmente ouriços-do-mar), presença de hemoglobina no sistema hidrovascular de algumas espécies e posição filogenética próxima aos cordados (Christensen et al. 2015; Reich et al. 2015).

Filogenia

O grupo Echinodermata é monofilético e possui sinapomorfias como sistema hidrovascular de canais celômicos, simetria pentarradial, tecido conjuntivo mutável e esqueleto interno constituído por ossículos calcários (Hendler et al. 1995; Hickman 1998; Benavides-Serrato et al. 2011; Reich et al. 2015). Estes atributos os tornam ainda mais interessantes e intrigantes (Hyman 1955), especialmente para estudos zoológicos e filogenéticos.

Com relação à árvore da vida, há um consenso de que os Echinodermata pertencem ao clado Ambulacraria juntamente com os Hemichordata (Edgecombe et al. 2011). Ambulacraria é suportado por vários padrões exclusivos de genes não compartilhados com os Chordata (Brown et al. 2008). No entanto, as relações filogenéticas dentro do grupo são controvérsas (Smith 1984; Janies 2001; Pisani et al. 2012; Schlegel et al. 2014).

O ancestral comum mais recente dos Echinodermata data de 500 milhões de anos, e logo após houve um período de especiação que durou cerca de 10 a 15 milhões de anos (Smith et al. 2013). Este período curto, porém, intenso, teve uma alta taxa de divergência morfológica, e isto dificulta os estudos filogenéticos dentro do grupo. Atualmente, duas hipóteses são mais discutidas entre os equinodermatólogos, a Asterozoa-Echinozoa e a Cryptosyringida. Em ambas hipóteses, Crinoidea é o ancestral comum e Echinoidea e Holothuroidea formam um clado denominado Echinozoa. A diferença entre as duas hipóteses é com relação a posição dos Ophiuroidea.

A hipótese Asterozoa-Echinozoa foi primeiramente proposta por Bather (1900). Nesta hipótese o clado Asterozoa é formado por Ophiuroidea e Asteroidea (Fig. 1A) e é suportado por análises moleculares e morfológicas (Mooi & David 2000; Janies 2001; O’Hara et al. 2014; Telford et al. 2014; Reich et al. 2015). A sinapomorfia morfológica de Asterozoa é o plano corporal do adulto, que inclui a forma estrelada e a construção do quadro oral e ossículos ambulacrais (Mooi & David 2000). 15

A hipótese Cryptosyringida foi primeiramente descrita por Smith (1984). Nesta hipótese, o clado Cryptosyringida é composto por Echinoidea, Holothuroidea e Ophiuroidea (Fig. 1B) e é suportado por análises morfológicas e embriológicas (Janies 2001; Raff & Byrne 2006). Uma das sinapomorfias morfológicas que suporta esta hipótese é a posição do nervo radial: dentro de um canal epineural fechado nos Cryptosyringida, e na epiderme dos Asteroidea (Nichols 1969). A sinapomorfia embriológica está relacionada à nutrição da larva, que não se alimenta no caso dos Crinoidea e se alimenta no caso dos Cryptosyringida (Raff & Byrne 2006).

Figura 1. Hipóteses das relações filogenéticas dentro do grupo dos Echinodermata viventes. A) Hipótese Asterozoa. B) Hipótese Cryptosyringida. Modificado de Reich et al. (2015).

A análise filogenética mais recente utilizou, pela primeira vez, dados do táxon Xyloplax, o qual é considerado um Asteroidea enigmático devido ao seu tamanho pequeno e morfologia estranha (circular) quando comparado as outras estrelas-do-mar (Janies & Mooi 1998). A análise foi feita com base em transcriptomas (RNAs mensageiros, RNAs ribossômicos, RNAs transportadores e os microRNAs) e suporta a hipótese Asterozoa (Linchangco et al. 2017). 16

É imprescindível que novas análises sejam feitas com a inclusão de mais representantes de diferentes famílias e integração de dados morfológicos. É inegável que novas técnicas e análises moleculares trouxeram um maior número de caracteres para serem analisados em estudos filogenéticos. Porém, caracteres morfológicos devem ser integrados às análises, pois caso contrário, perdemos uma chance valiosa de se aprender e entender a evolução do grupo. Compreender essas relações é essencial para inferir estados plesiomórficos dos caracteres dos Echinodermata e Hemichordata, assim como entender as relações entre Ambulacraria e Deuterostomia (Cannon et al. 2014).

Dentre as cinco classes de Echinodermata, os Ophiuroidea: i) são os que mais intrigam com relação aos estudos filogenéticos; ii) são os mais bem-sucedidos devido à sua mobilidade por meio da articulação das vértebras braquiais; iii) ocupam uma variedade de nichos tróficos em diferentes profundidades, e iv) exibem uma gama de modos de alimentação e reprodução (O’Hara & Tittensor 2010; Benavides-Serrato et al. 2011; Stöhr et al. 2012; Woolley et al. 2016). Em seguida, apresentamos uma descrição da classe e um resumo sobre a morfologia, taxonomia e filogenia do grupo.

2. Morfologia, taxonomia e filogenia dos Ophiuroidea

Morfologia

Os Ophiuroidea são semelhantes aos Asteroidea, porém algumas características únicas os colocam em um grupo a parte. Uma das sinapomorfias são os canais radiais do sistema ambulacral encontrados na parte inferior dos braços. Nos ofiuróides, estes canais são completamente fechados por partes rígidas do esqueleto braquial (placas dorsais, ventrais e laterais), enquanto nos asteróides os sulcos são abertos ao longo dos braços (Hendler et al. 1995; Stöhr et al. 2012).

Os ofiuróides possuem um disco bem definido com formato de circular a pentagonal. Dorsalmente coberto por escamas, espinhos, grânulos, placas ou até mesmo tegumento. Na superfície dorsal (contrária à boca) há escudos radiais, localizados próximos à base dos braços, placas primárias e placa centrodorsal, primeiras a se desenvolverem no disco do indivíduo jovem (Fig. 2A). Estas placas podem ser distinguíveis ou não no espécime adulto (Borges & Amaral 2005; Bueno et al. 2018). 17

A superfície ventral (oral) pode ser coberta por um revestimento igual ou diferente da superfície dorsal. A boca está localizada no centro da superfície ventral e ao seu redor há cinco mandíbulas, constituídas por várias peças calcárias (Fig. 2B). Cada conjunto mandibular é composto por: i) dentes (série vertical de papilas localizadas na extremidade interna da mandíbula); ii) escudos orais e adorais situados na parte distal da mandíbula; iii) papilas orais laterais; e iv) papilas infradentais ou apicais situadas sob os dentes (Fig. 2C). O madreporito é constituído por um escudo oral modificado. Em cada margem interradial ventral observa-se uma ou duas fendas bursais, que representam a conexão entre as bursas e o meio externo (Fig. 2B) (Borges & Amaral 2005; Bueno et al. 2018).

Figura 2. Morfologia externa dos Ophiuroidea. A) Disco - vista dorsal. B) Disco - vista ventral. C) Detalhe da mandíbula.

Tipicamente, os ofiuróides apresentam cinco braços longos e afilados, que podem ser simples ou ramificados, constituídos por vários segmentos articulados entre si (Fig. 3A). Cada segmento braquial é formado por: uma vértebra, uma placa dorsal, uma placa ventral e duas placas laterais (Fig. 3B). Em cada segmento há um par de pés ambulacrais os quais se 18

comunicam com o exterior através dos poros tentaculares que, na maioria das espécies, são protegidos por escamas tentaculares. As placas braquiais dorsais, ventrais e laterais variam em forma e tamanho e representam caracteres taxonômicos importantes. Cada vértebra possui seis faces: proximal (Fig. 3C), distal (Fig. 3D), dorsal (Fig. 3E), ventral (Fig. 3F) e duas laterais.

Figura 3. Ossículos braquiais de Ophiuroidea. A) Disco dorsal - modificado de Hendler et al. (1995). B) Esquema do corte transversal do braço. Vértebra: C) vista proximal, D) vista distal, E) vista dorsal e F) vista ventral. Abreviações: d: dorsal; di: distal; p: proximal; v: ventral. B, C e D modificado de LeClair & LaBarbera (1997). E e F modificado de Irimura & Fujita (2003).

As superfícies articulares dos ossículos vertebrais são classificadas em cinco categorias: i) articulação com botões – provavelmente mais primitiva, flexível em todas as direções, porém os botões de articulação são ocos e por isso os movimentos mais bruscos não são estáveis; ii) articulação em forma de sela – permite maior flexibilidade ao braço, com movimentos em todas as direções, inclusive espiral; iii) articulação universal – flexível mas sem movimento espiral; iv) articulação retangular – mais alongada no sentido dorso ventral e 19

movimentação mais restrita com curva para baixo e v) articulação aberrante – flexível verticalmente e restrito horizontalmente (Litvinova 1994).

Taxonomia e filogenia

Os caracteres utilizados na taxonomia morfológica dos Ophiuroidea sempre foram considerados inconsistentes (Martynov 2010). Alguns autores, como por exemplo, Smith et al. (1995) e Chen & McNamara (2006) acreditam que estas inconsistências estão relacionadas com a rápida especiação da classe, que levou a escassez de sinapomorfias morfológicas e moleculares (Perseke et al. 2010). Entretanto, Stöhr (2012) discorda deste ponto de vista e sugere que a falta de sinapomorfias reconhecidas para o grupo é consequência do nosso parco conhecimento da morfologia e evolução dos ofiuróides.

Este cenário justifica uma ampla revisão taxonômica e filogenética, que deve ser feita a partir de estudos morfológicos e moleculares, com a descrição e ilustração de novos caracteres. Desta forma, será possível compreender a diversidade e as relações de parentesco entre os táxons de Ophiuroidea. Isto já vem acontecendo em níveis taxonômicos superiores, porém o mesmo não ocorre para os níveis taxonômicos mais baixos como gêneros e espécies (O'Hara et al. 2017; O'Hara et al. 2018).

Alguns pesquisadores começaram a buscar novos caracteres, especialmente nas placas e ossículos braquiais e peristomiais, para resolver questões taxonômicas complexas (Martynov 2010; Thuy & Stöhr 2011; Wilkie & Brogger 2018). A morfologia da placa braquial lateral (PBL) e a articulação do espinho braquial foram considerados caracteres conservados dentro do nível de família e importantes para elucidar a posição sistemática de gêneros e espécies (Thuy & Stöhr 2011). Esta classificação com base na PBL foi a mais parecida com a filogenia realizada em nível molecular, feita a partir de um conjunto de dados com 425 genes (O’Hara et al. 2014). Assim, pela primeira vez, discutiu-se a importância destas placas como sinal filogenético.

Por conta destes últimos trabalhos (Martynov 2010; Thuy & Stöhr 2011; O’Hara et al. 2014), os estudos que envolvem a sistemática dos Ophiuroidea iniciaram uma nova fase com foco em caracteres moleculares e morfológicos microestruturais, como placas orais, peristomiais e braquiais. Um exemplo é a análise filogenética feita com 130 caracteres morfológicos (Thuy & Stöhr 2016) que foi comparada àquela realizada com dados 20

moleculares (O’Hara et al. 2014). Mesmo com conjuntos de dados diferentes, os resultados foram parecidos. A partir disso, Thuy & Stöhr (2016) sugeriram a utilização de dados morfológicos e moleculares em uma matriz combinada, uma vez que a morfologia pode influenciar positivamente a árvore de dados combinados, apoiando sua utilidade em análises multigênicas (Nylander et al. 2004; Wortley & Scotland 2006). No entanto, apesar de ser uma boa alternativa para análises, a matriz combinada ainda não foi testada para os ofiuróides.

Caminhando a passos largos, a biologia molecular veio como uma ferramenta poderosa no estudo dos ofiuróides. Isso porque sequências de DNA fornecem muitos caracteres que são facilmente comparáveis entre os organismos (Vogler & Monaghan 2007; Cardoso et al. 2009). Recentemente, uma análise filogenética feita a partir de 1484 éxons de 576 espécies de ofiuróides sugeriu a existência de cinco superfamílias e 33 famílias, das quais 10 são novas para a Ciência, resultado absolutamente inovador para os especialistas (Hugall et al. 2016; O'Hara et al. 2017). Estas mesmas famílias foram posteriormente descritas e redescritas com base em caracteres morfológicos tradicionalmente aceitos e com a adição dos microestruturais nas diagnoses (O'Hara et al. 2018).

No entanto, muito trabalho ainda precisa ser feito porque vários gêneros são suspeitos de serem não-monofiléticos em sua composição atual de espécies, mostrando a necessidade de mais estudos com o grupo (O'Hara et al. 2017; O'Hara et al. 2018).

Neste contexto, nos deparamos com outro desafio: conceituar espécie e encontrar ferramentas seguras para que possamos delimitá-las com a maior exatidão possível. A seguir, discursamos sobre o conceito de espécie e como a taxonomia integrativa pode ajudar a resolver alguns problemas taxonômicos específicos.

3. Conceito de espécie e taxonomia integrativa

O que é espécie?

Espécie é uma das unidades fundamentais da biologia (Mayr 1982; de Queiroz 2005) e sua conceituação e delimitação são centrais para o desenvolvimento da sistemática, biogeografia, ecologia, evolução e especialmente para pesquisas na biologia da conservação e em biodiversidade (Caldecott et al. 1996; Agapow et al. 2004; Padial et al. 2006).

A questão de como melhor definir o termo "espécie" tem ocupado os biólogos há séculos. Darwin publicou há quase 160 anos uma frase que acreditamos valer até os dias de 21

hoje: “não há definição que satisfaça todos os naturalistas” (Darwin 1859, p. 44). Muitos trabalhos com diversas revisões e opiniões foram publicados nos últimos 20 anos (Luckow 1995; Mayden 1997; de Queiroz 1998; Wilkins 2009; Freudenstein et al. 2017). Um dos mais notáveis listou 22 definições (Mayden 1997), enquanto uma revisão mais recente listou 34 (Zachos 2018). Diante de tantas variações, concordamos com a fórmula proposta por Wilkins (2011): n + 1, onde n é o número de biólogos discutindo sobre conceito de espécie. Isto significa que o número de conceitos será maior a medida que o número de biólogos aumenta.

Estas controvérsias geraram inúmeras crises na taxonomia e motivaram constantes questionamentos sobre qual a melhor maneira de delimitar espécies (Gewin 2002; Godfray 2002; Mallet & Willmott 2003; Wilson 2003; Wheeler et al. 2004). A discussão mais recente enfatizou a utilização de espécies como hipóteses explanatórias (Fitzhugh 2005, 2009). Pesquisas com base explanatória tem o objetivo de explorar os fenômenos estudados, ao invés de simplesmente descrevê-los (Maxwell & Mittapalli 2012). Desta forma, tratar espécie como hipótese é ter uma resposta provisória a uma pergunta, mas que ainda não foi testada. Alguns pesquisadores chamam este primeiro passo de Hipótese Primária de Espécie (HPS) (Puillandre et al. 2012). Em seguida a HPS é testada por meio de ferramentas disponíveis a critério do pesquisador.

Nesta tese, as espécies são tratadas como hipóteses e testadas a partir de perspectivas múltiplas e complementares. Esta perspectiva é chamada de taxonomia integrativa (Dayrat 2005).

Taxonomia integrativa

A taxonomia integrativa (TI) visa delimitar as unidades de biodiversidade a partir de perspectivas múltiplas e complementares, como por exemplo, caracteres morfológicos, genéticos, ecológicos, comportamentais e filogeográficos (Dayrat 2005; Padial et al. 2010). É uma nova forma de taxonomia, com novos conceitos e métodos (Dayrat 2005; Valdecasas et al. 2008; Schlick-Steiner et al. 2010), o que certamente mudará a percepção da biodiversidade global e será de grande valor prático para outras disciplinas que dependem de uma taxonomia mais utilizável (Riedel et al. 2013), confiável e realizada em tempo aceitável.

A TI já foi repercutida com sucesso em diversos trabalhos, especialmente para explorar níveis taxonômicos mais baixos como espécies ou complexo de espécies de vários 22

metazoários (Fonseca et al. 2008; Cardoso et al. 2009; Glaw et al. 2010; Arribas et al. 2013). Porém, a implementação da TI pode ser problemática para níveis taxonômicos mais altos, como famílias e gêneros. Neste sentido, Korshunova et al. (2017) listaram várias regras operacionais enraizadas em fatores biológicos, e que posteriormente foram aplicadas a um grupo de moluscos. Os autores acreditam que as mesmas regras possam ser aplicadas a outros grupos de metazoários.

A novidade desta tese é a utilização de diferentes fontes de dados na delimitação de algumas espécies de Ophiuroidea. Desta forma, a TI será utilizada a partir de caracteres morfológicos e genéticos. Ainda, a utilização e comparação de espécies e espécimes com ocorrência em áreas continentais e insulares.

A tendência em estudar caracteres moleculares a partir de dados gerados com sequenciamentos de DNA trouxe aos equinodermatólogos uma nova ferramenta para detectar e diferenciar espécies morfologicamente muito semelhantes (Boissin et al. 2008; Hoareau & Boissin 2010; Pérez‐Portela et al. 2013). Recentemente, alguns trabalhos definiram diferentes linhagens genéticas de ofiuróides a partir da análise de DNA mitocondrial, como CO1 e 16S, para Amphipholis, família Amphiuridae (Boissin et al. 2015), Ophiothrix, (Baric & Sturmbauer 1999; Muths et al. 2009; Pérez‐Portela et al. 2013; Taboada & Pérez- Portela 2016) e para Ophiactis, Ophiactidae (Roy & Sponer 2002; Christensen et al. 2008; Barboza et al. 2015b).

No entanto, estudos recentes revelaram algumas deficiências ao se utilizar exclusivamente caracteres moleculares para a taxonomia (Wheeler et al. 2004; Bond & Stockman 2008; Sauer & Hausdorf 2012), como por exemplo, a incapacidade de se relacionar e comparar espécies viventes e fósseis (Wheeler et al. 2004). Sauer & Hausdorf (2012), em um trabalho que analisa métodos baseados em DNA para a delimitação de espécies, reforçam a importante combinação de abordagens morfológicas e moleculares, uma vez que somente a TI pode desvendar todas as facetas da Biodiversidade da Terra e torna tal delimitação mais segura.

4. Família Ophiotrichidae Ljungman, 1867

Ophiotrichidae tem um registro fóssil relativamente recente, especificamente do Eoceno (O’Hara et al. 2014). Ela foi erigida por Ljungman em 1867 para agrupar os gêneros 23

Ophiothrix, Ophiocnemis e Ophiogyma (Ljungman 1867). Tem como diagnose: disco com escamas que sustentam espinhos e/ou grânulos espinulosos, opacos ou hialinos; espinhos do disco podem ser curtos ou longos e podem ou não ocorrer sobre os escudos radiais; estes espinhos apresentam dentículos hialinos e podem ser bífidos e/ou trífidos; um feixe de papilas apicais e não há papilas orais (Clark 1966; Albuquerque 1986; Hendler et al. 1995; Borges & Amaral 2005).

Apesar de ser considerada bem estabelecida (O'Hara et al. 2017), Ophiotrichidae é problemática no que se refere as distinções entre os gêneros, consequência da ampla variabilidade fenotípica interespecífica (Clark 1966; Hendler 2005). A maioria dos integrantes desta família é encontrada em uma variedade de substratos, incluindo conchas, rochas, cascalho, e associados a esponjas, algas e corais (Hendler et al. 1995; Borges & Amaral 2005). Ophiotrichidae é composta por 177 espécies distribuídas em 13 gêneros (O'Hara et al. 2017), dos quais 10 espécies e dois gêneros (Ophiothrix e Ophiothela) são registrados para o Brasil (Barboza & Borges 2012).

Ophiothrix Müller & Troschel, 1840 é um dos gêneros de Ophiotrichidae que mais chama a atenção, pois possui cores brilhantes e associações encantadoras com corais e esponjas (Hendler 2005). Quanto a diagnose, Ophiothrix é reconhecido por possuir disco dorsalmente coberto por espinhos bífidos e/ou trífidos; espinhos dorsais do braço são tipicamente espinhosos e vítreos (Clark 1966; Tommasi 1970b; Albuquerque 1986; Alitto et al. 2018). É também o mais estudado em análises moleculares, por se tratar de um táxon extremamente problemático com variação intraespecífica na morfologia, coloração, ecologia e distribuição geográfica (Baric & Sturmbauer 1999). Recentemente, alguns trabalhos definiram diferentes linhagens genéticas a partir da análise de DNA mitocondrial, como CO1 e 16S para Ophiothrix fragilis e O. quinquemaculata (Muths et al. 2009; Pérez‐Portela et al. 2013; Taboada & Pérez-Portela 2016).

Ophiothela tem como diagnose: escudos radiais grandes (cerca de 2/3 do disco); disco e braços dorsalmente cobertos por grânulos e ventralmente cobertos por um tegumento, que pode obscurecer as escamas e placas braquiais (Verrill 1867; Alitto et al. 2018). Ophiothela Verrill, 1867 era um gênero restrito ao Oceano Pacífico (Clark 1976). Porém, recentemente, alguns espécimes foram identificados em populações emergentes do Atlântico (Hendler et al. 2012). Desde então, Ophiothela tem sido comumente encontrada associada a substratos biológicos, como corais e esponjas e as implicações dessa associação ainda são desconhecidas (Mantelatto et al. 2016; Lawley et al. 2018). Araújo et al. (2018) observaram 24

espécimes de Ophiothela perto dos pólipos de octocorais Leptogorgia punicea. Segundo os autores, a alta concentração de Ophiothela pode comprometer a habilidade destes octocorais em obter alimento. No entanto, mais estudos sobre a ecologia trófica, especialmente usando técnicas modernas (por exemplo, isótopos estáveis), são necessários para testar esta hipótese.

5. Objetivo e principais resultados

Objetivo

O objetivo da tese foi delimitar algumas espécies de Ophiotrichidae utilizando-se dos princípios que regem a taxonomia integrativa. As espécies candidatas (hipóteses) foram testadas por meio de caracteres morfológicos (morfologia externa, morfologia das microestruturas braquiais e bucais, morfometria de estruturas diagnósticas) e caracteres dados moleculares (fragmentos dos genes mitocondriais 16S e COI). As espécies candidatas foram classificadas de acordo com protocolo de cumulação de Padial et al. (2010). Enfatizamos que os dados moleculares tiveram o mesmo peso dos dados morfológicos em nossas análises.

Principais resultados

O conteúdo encontra-se em três capítulos. No primeiro capítulo foi redescrita e validada Ophiothrix trindadensis Tommasi, 1970. Ophiothrix trindadensis é uma espécie nativa do Brasil, e até então, sem série-tipo depositada em museus. A partir da investigação integrativa, foi proposta a designação de um neótipo coletado na Ilha Trindade (localidade tipo). A espécie foi redescrita com base em caracteres morfológicos externos e microestruturais como placas braquiais, orais e dentais. Os resultados contribuem para uma identificação confiável não só para O. trindadensis, mas também para Ophiothrix angulata – espécie morfologicamente semelhante. Este capítulo foi publicado pela revista PLoSOne (Alitto et al. 2019).

No segundo capítulo foi realizado um estudo comparativo entre Ophiothela sp. do Brasil (BR), Ophiothela mirabilis Verrill, 1867 e Ophiothela danae Verrill, 1869. Estas espécies são fissíparas, e isso dificulta o seu reconhecimento com base somente em caracteres morfológicos. Ophiothela sp. BR, O. mirabilis e O. danae estão intimamente relacionadas. No entanto, Ophiothela sp. BR tem um diferente conjunto de haplótipos. Com relação as análises morfométricas, Ophiothela sp. BR está mais próxima de O. danae do que de O. mirabilis. 25

Apesar de Ophiothela sp. BR ser diferente das demais espécies analisadas, o conjunto de dados não permitiu verificar se é uma linhagem geográfica ou uma espécie nova para a Ciência. A descrição detalhada de Ophiothela sp. BR revelou características interessantes, especialmente com relação as microestruturas: i) articulação da vértebra do tipo estreptospôndila (pela primeira vez na família Ophiotrichidae); ii) oito botões na superfície dorsal da vértebra (até o momento presente apenas em Ophiothela); iii) quilha distal dividida em duas pontas (primeira vez na literatura); iv) orifício nos lados abradial e adradial da placa oral (assim como outras espécies fissíparas Ophiactis savignyi e Ophiocomella ophiactoides). Durante os estudos morfométricos, ocorrências morfológicas não usuais de Ophiothela sp. BR foram observadas e descritas em um capítulo separado. O segundo capítulo está em fase final de preparação e será submetido à revista Zoologica Scripta.

No terceiro capítulo é apresentado um estudo morfológico de Ophiothela sp. BR. Concluiu-se que a espécie tem uma alta incidência de fissiparidade evidenciada pelas ocorrências morfológicas não usuais e um alto número de configurações do disco (DC). É importante destacar dois eventos. Primeiro, o registro de um espécime com 1 + 1 DC com apenas um par de escudos radiais, um braço longo e um braço curto - resultado de uma fissão recente e desigual. Segundo, foram registradas 12 DC diferentes, sugerindo que os espécimes sofrem fissão antes mesmo de terminar a regeneração. Neste capítulo, foram discutidos alguns fatores que podem estar relacionados a alta incidência de fissiparidade de Ophiothela sp. BR. O gênero Ophiothela é considerado invasor no litoral brasileiro e sua alta taxa de regeneração e fissiparidade tem facilitado sua expansão em diferentes ambientes e substratos biológicos. Este capítulo está em fase final de preparação e será submetido à revista Marine Biodiversity.

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CONCLUSÕES

Na presente tese mostramos como reconhecer e descrever espécies de Ophiuroidea por meio da taxonomia integrativa. Duas espécies da família Ophiotrichidae (Ophiothrix trindadensis e Ophiothela sp. BR) foram descritas em detalhe por meio de caracteres morfológicos e moleculares. Nossos resultados contribuem para novos estudos em, ao menos, cinco ramos da biologia:

1º) Biodiversidade e conservação. As redescrições de Ophiothrix trindadensis e a descrição de Ophiothela sp. BR serão úteis em novas identificações de espécimes, especialmente porque comparamos estas espécies com as mais próximas de cada gênero. Os resultados gerados poderão ser usados em análises de biodiversidade e gerarão subsídios para futuras propostas de manejo e conservação.

2º) Filogenia. Os novos caracteres taxonômicos, tanto morfológicos como moleculares, poderão ser usados para estudar as relações filogenéticas entre gêneros e espécies, extrapolando os resultados para as famílias.

3º) Genética de populações. Os dados moleculares gerados podem ser integrados a novas amostras. Assim, estes resultados poderão ser usados em estudos sobre genética de populações, fluxo gênico e história demográfica.

4º) Evolução. Ophiothrix trindadensis e Ophiothrix angulata têm potencial para serem usados em estudos sobre processos de especiação ao longo da plataforma continental brasileira. Isso porque O. trindadensis ocorre em uma ilha oceânica (Ilha Trindade) e O. angulata na costa sudeste e sul brasileira. Por outro lado, Ophiothela sp. BR pode ser usada para testar teorias sobre as vantagens evolutivas de fissiparidade.

5º) Ecologia. Espécimes de Ophiothrix trindadensis e Ophiothrix angulata foram amostrados em diferentes substratos biológicos e diferentes localidades. Por isso, reiteramos a necessidade de novos estudos para testar se as diferenças morfológicas encontradas podem estar relacionadas ao substrato biológico, diferentes salinidades e/ou profundidades, bem como outras variáveis. Para Ophiothela sp. BR, enfatizamos a realização de testes em laboratório a fim de se identificar possíveis iniciadores de fissão, como temperatura e salinidade.

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40

CAPÍTULO 1

Atlantic West Ophiothrix spp. in the scope of integrative taxonomy: confirming the existence of Ophiothrix trindadensis Tommasi, 1970

Renata Aparecida dos Santos Alitto, Antonia Cecília Zagagnini Amaral, Letícia Dias de

Oliveira, Helena Serrano, Karin Regina Seger, Pablo Damian Borges Guilherme, Maikon Di

Domenico, Ana Beardsley Christensen, Luciana Bolsoni Lourenço, Marcos Tavares, Michela

Borges

Capítulo publicado pela revista PlosOne (Anexo 1)

Cadastro de Acesso ao Patrimônio Genético AF4BF9C (Anexo 2) e AC6C194 (Anexo 3). 41

Atlantic West Ophiothrix spp. in the scope of integrative

taxonomy: confirming the existence of Ophiothrix

trindadensis Tommasi, 1970

Renata Aparecida dos Santos Alitto1,2*¶, Antonia Cecília Zacagnini Amaral2&, Letícia Dias de

Oliveira1,2¶, Helena Serrano1,2¶, Karin Regina Seger1,3¶, Pablo Damian Borges Guilherme4¶,

Maikon Di Domenico5&, Ana Beardsley Christensen6¶, Luciana Bolsoni Lourenço3&, Marcos

Tavares7&, Michela Borges1,2¶

1Departamento de Biologia Animal, Universidade Estadual de Campinas, Campinas, São Paulo, Brasil

2Museu de Zoologia “Adão José Cardoso”, Universidade Estadual de Campinas, Campinas, São Paulo, Brasil

3Departamento de Biologia Celular, Universidade Estadual de Campinas, Campinas, São Paulo, Brasil

4Laboratório de Biologia Marinha, Universidade Estadual do Paraná, Paranaguá, Paraná, Brasil

5Centro de Estudos do Mar, Universidade Federal do Paraná, Pontal do Paraná, Paraná, Brasil

6Department of Biology, Lamar University, Beaumont, Texas, USA

7Museu de Zoologia da Universidade de São Paulo, São Paulo, Brasil.

* Corresponding author

E-mail: [email protected] (RASA)

¶These authors contributed equally to this work.

&These authors also contributed equally to this work. 42

Abstract

We re-describe and confirm the validity of Ophiothrix trindadensis Tommasi, 1970 (Echinodermata: Ophiuroidea). This is a native species from Brazil, however it lacked a type series deposited in scientific collections. The recognition of O. trindadensis was made possible using integrative taxonomy applied to many specimens from the type locality (Trindade Island) as well as from different locations along the Brazilian coast (Araçá Bay and Estuarine Complex of Paranaguá). Initially, 835 specimens were studied and divided into four candidate species (CS) inferred from external morphological characters. Afterwards, the CSs were compared using integrative taxonomy based on external morphology, arm microstructures morphology (arm ossicle), morphometry, and molecular studies (fragments of the mitochondrial genes 16S and COI). Analyses indicated CS1 and CS2 as O. trindadensis, and CS3 as O. angulata, both valid species. CS4 remains O. cf. angulata as more data, including their ecology and physiology, are needed to be definitively clarified. Our integrative investigation using specimens from the type locality overcame the lack of type specimens and increased the reliable identification of O. trindadensis and O. angulata.

Keywords: DNA barcoding, morphometric, arm vertebrae, zygospondylous joint, lateral arm plate.

Introduction

The Family Ophiotrichidae is one of the most interesting among Ophiuroidea, due to the brilliant colors of some species and their association with corals and . This family has been revised multiple times, however, it continues to be problematic owing to the intraspecific variability [1, 2]. Currently, Ophiotrichidae has 169 species distributed across 16 genera [3], with ten species and two genera reported from Brazil [4].

The genus Ophiothrix Müller & Troschel, 1840 is considered taxonomically difficult due to the high degree of variability in morphological characters. Therefore, molecular characters have been used as a tool to detect and distinguish species that are morphologically similar [5-7]. A successful example applied to ophiotrichids is a study conducted across the Atlantic-Mediterranean area, which demonstrated the existence of three 43

genetic and morphologically distinct lineages of Ophiothrix, although they have yet to be formally described [8, 9].

A total of 93 Ophiothrix species are recognized at present [3], and six are recorded from Brazil [4]: O. ailsae Tommasi, 1970; O. angulata (Say, 1825), O. brachyactis H. L. Clark, 1915, O. rathbuni Ludwig, 1882, O. suensoni Lütken, 1856, and O. trindadensis Tommasi, 1970. Of these, three are endemic: O. ailsae described from the coastal island Vitoria, State of São Paulo, O. trindadensis described from the remote oceanic island Trindade [10] and O. rathbuni, for which there is no type locality information [11]. Our attempts to locate the type series of the endemic Brazilian species O. ailsae, O. trindadensis and O. rathbuni at Oceanographic Institute of University of São Paulo, Museum of Zoology of University of São Paulo and Museum of Zoology of University of Campinas have been in vain.

The present study used an integrative approach to investigate Ophiothrix specimens and to clarify their taxonomic status and distribution in Brazil. External morphology was initially employed to propose candidate species. Then, the species boundaries among populations were evaluated using four independent character sets related to: i) external morphology, ii) arm microstructures morphology (arm ossicles), iii) morphometry, and iv) molecular data (nucleotide sequences of the mitochondrial genes 16S and COI).

One species of particular interest, Ophiothrix trindadensis, is redescribed here in detail. Since its type specimens were lost, we propose a neotype designation based on topotype specimen from Trindade Island following Article 75 of the International Code for Zoological Nomenclature [12].

Material and methods

1) Study site and data collection

Brittle stars were collected from three sites in Brazil: i) Trindade (type locality of Ophiothrix trindadensis) and Martin Vaz Oceanic Archipelago, ii) Araçá Bay, and iii) Estuarine Complex of Paranaguá. These samples were then compared to those from three additional sites: i) São Pedro and São Paulo Archipelago – Brazil (molecular data), ii) Port 44

Aransas, Texas, United States (morphological and molecular data), and iii) South Carolina, United States (type locality of O. angulata, morphological data) (S1 Fig). The geographic coordinates, substrate type, salinity, and main references for each locality sampled are shown in S1 Table. All specimens collected in the present study were fixed and preserved in 70% or 90% ethanol and deposited in the Museum of Zoology of the University of Campinas (ZUEC) labeled as ZUEC OPH (identification number) and Museum of Zoology of University of São Paulo labeled as MZUSP (identification number). The following is a brief description of each location as well as the sampling strategies that were used.

Trindade and Martin Vaz Oceanic Archipelago, Brazil (TMV) TMV is a group of one large (Trindade) and several smaller islands (Martin Vaz) located in the southwestern Atlantic approximately 1200 km east of Vitória, the capital of the Brazilian State of Espírito Santo. Trindade Island and the much smaller Martin Vaz Islands are only 49 km apart from each other. Trindade Island is 13.5 km2 in area and is almost totally composed of volcanic and subvolcanic rocks formed between the end of the Pliocene and the Holocene [13, 14]. During the project ProTrindade/CNPq, five trips to the TMV were conducted between 2012 and 2015. Most of the material reported was collected from scuba diving operations between 4–30 m, which resulted in 605 lots of shallow-water echinoderms. More information can be found in Anker et al. [15].

Araçá Bay, Brazil (AB) AB is located on the northeastern coast of the State of São Paulo, Brazil. It encompasses three beaches, two shores islets, three main stands of mangrove, rocky shores, and an extensive muddy sand flat extending to the subtidal zone. The latter comprises a vast intertidal flat up to 300 meters wide [16, 17]. Brittle stars were sampled during the project BIOTA/FAPESP – Araçá conducted between 2012 and 2016 from rubble bottom with a dredge and by hand associated with the Amphimedon viridis.

Estuarine Complex of Paranaguá, Brazil (ECP) ECP is located on the southern coast of the State of Paraná, Brazil. The estuary measures 612 km2 and it is part of the largest remaining sectors of the Atlantic Rain Forest 45

along the Brazilian coast. ECP is part of a large interconnected subtropical estuarine system comprised of two main water bodies, extensive sandy beaches, and rocky shores [18]. Brittle stars were sampled in 2014 in the intertidal zone of Banana Island and Perigo tidal flat by hand. All the specimens were associated with the sponge Mycale (Zygomycale).

São Pedro and São Paulo Archipelago, Brazil (SPSPA) SPSPA lies on the mid-Atlantic ridge, some 1000 km away from the coast of the State of Rio Grande do Norte, northeastern Brazil, and approximately 1960 km from the African coast [19]. SPSPA, the only Brazilian oceanic islands above the equator, is comprised of four larger islets plus several minor rocks, rising 4000 m from the ocean depths. The emerged area of the archipelago covers approximately 13000 m2 and is 420 m across at the greatest width [19, 20]. Two brittle stars identified as Ophiothrix angulata by Barboza et al. [21] were sampled by scuba diving in 2013. Logistics were supported by the PROARQUIPÉLAGO Program. The specimens were found associated with Chaetopterus polychaete tubes [21].

Port Aransas South Jetty, Texas, United States (TX-US) The South Jetty is at the north end of Mustang Island. The jetty, constructed in the early 1900's of large granite blocks quarried from Marble Falls, Texas, extends approximately 1 mile from the northern tip of Mustang Island into the Gulf of Mexico, stabilizing the entrance of the Corpus Christi Ship Channel [22, 23]. Brittle stars were collected between 2011 and 2012 on South Jetty by hand. The specimens were found in colonies of the sandy lobed tunicate Eudistoma carolinense.

South Carolina, United States (SC-US) A total of 18 specimens were borrowed from Smithsonian United States National Museum (USNM). Voucher E24138 contained nine specimens (dried) collected from South Creek, near the entrance in N. Edisto River, South Carolina in May 31, 1966. Voucher E28587 (in alcohol) with five specimens was sampled off South Carolina in September 12, 1980. Voucher 33710 (in alcohol) with four specimens was sampled in Calibogue Sound, SC in January 16, 1891.

46

2) Delimitation of the candidate species (CS) inferred from morphological characters As a starting point for species validation, morphological hypotheses about the identity of CSs were proposed (step A, Fig 1A). They were based on characters usually reported as diagnostic in morphological overviews and keys [10, 24-30].

Fig 1. Schematic diagram of integrative taxonomy applied to Ophiothrix spp. (A) A priori classification inferred from morphological characters. (B) Morphological characters (B1) external morphology and (B2) arm microstructures morphology. (C) Morphometry: measurements and linear discriminant analysis (LDA). (D) Molecular characters: (D1) phylogenetic analyses (Maximum Parsimony, Bayesian Inference, and Maximum Likelihood) and (D2) genetic diversity and species delimitation test (genetic distances, AMOVA, haplotype network, bPTP). (E) Congruence framework of integrative taxonomy.

47

3) Morphological characters

External morphology

Comparisons of external characters of all CSs were made against descriptions and museum specimens of Ophiothrix angulata and O. trindadensis. In relation to O. angulata, the CSs were compared to its original description [24], redescription [29] and samples from type locality (South Carolina, United States) available from USNM (vouchers E24138 – 9 spms, E28587 – 5 spms, 33710 – 4 spms). They were also compared with the original description of O. trindadensis [10] (Step B1, Fig 1B).

Arm microstructures morphology

The best-preserved specimens of each CS were selected to study the arm ossicles, which were extracted from a small part of the proximal arm, between the fifth and the tenth segment. The arm segment was immersed in regular household bleach (NaClO) until the soft tissues were removed [31]. The ossicles were then washed with distilled water, air-dried, and prepared for examination with a scanning electron microscope (SEM) model JEOL JSM5800LV dx.doi.org/10.17504/protocols.io.tz6ep9e [PROTOCOL DOI]. The terms applied to the arm ossicles are based on literature [32-38] and are illustrated in Alitto et al. [30]. Arm vertebra joints were classified into types as proposed by Litvinova [39] (Step B2, Fig 1B).

4) Morphometry

Measurements were taken using an ocular micrometer and through the AxioVision VS program 40.4.8.20 (Carl Zeiss Microscopy, Germany) attached to a ZEISS Discovery V20 stereomicroscope for specimens less than 10 mm disc diameter and with a digital Mitutoyo CD-6 CS caliper for the larger specimens. A detailed list of measurements is presented in S2 Table and illustrated in S2 Fig.

The linear discriminant analysis (LDA) was applied using the R environment [40]. To avoid multicollinearity among morphological characters a correlation matrix was constructed and the variables that were significantly correlated were removed with a threshold value of 0.9. To investigate the differences in morphological characters, lda function (package 48

MASS) [40, 41] was used to distinguish the four Ophiothrix CS. The classification rate of each CS was assessed by the “lda” function.

The predict function (package STATS) and table function (package BASE) were used to assess the classification based on the linear discriminants and to verify the model error. Visualization was performed using the package ggplot2 [42] (Step C, Fig 1C).

A permutational multivariate analysis of variance (PERMANOVA) [43] was conducted (function adonis) and homogeneity of group dispersions (functions betadisper and permutest) were then used to formally test whether morphological characters and dispersion differed between CSs (package vegan) [44] dx.doi.org/10.17504/protocols.io.tz5ep86 [PROTOCOL DOI].

Ophiothrix angulata specimens from the type locality (USNM) were predicted by the model to verify the CS to which they were most related.

5) Molecular characters

DNA extraction, amplification, and sequencing

DNA sequences used in phylogenetic analyses were obtained as follows. Samples of tube feet from the arms or gonads were soaked in two changes of Tris-EDTA (TE) buffer for 30 minutes each. The samples were then macerated in 500 μl of TE buffer with a pestle. A volume of 300 μl of a 10% Chelex 100 solution (BioRad) was added. The samples were then incubated at 55°C for 60 minutes, boiled for 8 minutes, cooled to room temperature, vortexed for 20 seconds followed by 60 seconds of centrifugation at 14,000 xg. The supernatant was decanted and kept refrigerated until use in polymerase chain reactions (PCR).

A fragment of the mitochondrial 16S gene was PCR-amplified using the forward primer 16S Sofi F (5′-CAGTACTCTGACTGTGCAA-3′) and the reverse primer 16S Sofi R (5′-GGAAACTATGATCCAACATC-3′) [8]. A fragment of the mitochondrial cytochrome oxidase subunit 1 (COI) gene was amplified using the forward primer COIf-L (5′- CCTGCAGGAGGGGGAGAYCC-3′) and the reverse primer COIa-H (5′- TGTATAGGCGTCTGGATAGTC-3′) [45].

PCR reactions were performed using PuReTaq Ready-To-Go™ PCR Beads for 25

μl reactions (GE Heathcare), complemented with 0.5 to 1 μl of 25mM MgCl2. Alternatively, 49

PCR reactions were composed of 10 mM Tris-HCl, pH 8/1 mM KCl buffer, 1.5 mM MgCl2, and 200 μM each dNTP (Invitrogen). In both cases, 0.4 μM of each primer was used and template DNA concentration (ranging from 25.7 to 917.9 ng/μl) was tested.

Thermal cycling consisted of a single step at 94°C for 5 minutes, which was followed by 39 cycles (denaturation at 94°C for 45 seconds, annealing at 45.7°C to 51.3°C for 45 seconds, and extension at 72°C for 60 seconds) and a final extension at 72°C for 3 minutes on a thermal cycler (Eppendorf Mastercycler®).

PCR products were separated from excess primers and dNTP using a purification kit (Promega). Purified product was then used as a template for DNA sequencing reactions using BigDye Terminator (Applied Biosystems) and sequenced by Serviço de Sequenciamento de DNA – SSDNA IQUSP with the same primers used in the amplification reaction. A 372 bp fragment from 28 individuals was obtained for 16S and 466 bp fragment from 17 individuals for COI. All the sequences are deposited in GenBank (S3 Table). The nucleotide sequences were edited using BIOEDIT Sequence Alignment Editor v. 7.0.1 [46] dx.doi.org/10.17504/protocols.io.tz7ep9n [PROTOCOL DOI].

Phylogenetic analyses

All 16S and COI sequences were aligned with the MUSCLE algorithm [47], providing a unified matrix. All the sequences from Ophiothrix angulata (16S) and additional sequences from others Ophiotrichidae (16S, COI) available on GenBank were added to the matrix for comparison. Amphipholis squamata (Amphiuridae) was used as outgroup. All samples used are listed in S3 Table.

Three mitochondrial datasets were considered: i) 16S, ii) COI and iii) concatenated (COI+16S). Phylogenetic reconstructions were made on the basis of three different optimality criteria, Maximum Parsimony (MP), Bayesian Inference (BI), and Maximum Likelihood (ML) to assess whether there were any differences in the trees recovered with regard to the method used (Step D, Fig 1D).

The MP was performed with the TNT 1.5 program [48]. Most parsimonious trees were obtained by a heuristic search (best length was hit 100 times), using the new technology search option, which included sectorial searches, ratchet, tree drifting and tree fusing. The 50

gaps were considered as fifth state. The cladograms had their nodes evaluated by the Bootstrap resampling test [49], based on 1000 pseudoreplicates using the Traditional Search.

For BI, the best-fitting model of molecular evolution was GTR+G, chosen based on the AIC and Hierarchical Likelihood Ratio Tests according to the estimation by MR.MODELTEST v.2.3 [50]. The BI analyses used one cold and three incrementally heated Monte Carlo Markov chains (MCMC) on two simultaneous runs. The standard deviation of the split frequencies between the two runs reached a value lower than c. 0.005 at two million generations, with one tree sampled every 100th generation, each using a random tree as a starting point and a temperature parameter value of 0.2 (the default in MrBayes). The first 25% of the total sampled trees were discarded as ‘burnin’ to achieve the MCMC log- likelihoods that had become stationary and converged. The analyses resulted in similar likelihood scores, with ESS > 200, as verified using TRACER.

The ML was performed with the MEGA v.7.0 program [51] using the Kimura 2- parameter model [52]. The statistical support was obtained with a bootstrap function using 1000 replicates.

The phylogenetic trees were visualized and edited in FIGTREE v.1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/) and Adobe Illustrator dx.doi.org/10.17504/protocols.io.wimfcc6 [PROTOCOL DOI].

Analyses of the genetic diversity

The genetic distances between and within the different groups were estimated by p-distance using MEGA v. 6.0 [53], ignoring the alignment gaps in pairwise comparisons. Following Hart and Podolsky [6] and Pérez-Portela et al. [8], pairwise genetic p-distances >3% for 16S and >15% for COI were considered interspecific variations since most of the species already described have been differentiated by this minimum (Step D2, Fig 1D).

The concatenated matrix was submitted to the AMOVA (Analysis of Molecular Variance) test [54] to evaluate the genetic difference between and among the studied groups formed by the phylogenetic analyses. The overall fixation index (FST) [55] was calculated to verify the gene flow between the groups. The test was simulated with 1000 permutations in Arlequin software v. 3.1 [56] (Step D2, Fig 1D). 51

The haplotype network was constructed based on the concatenated matrix by the median-joining network [57], implemented in Network software v. 5.0.0.1 (http://www.fluxus-engineering.com/) (Step D2, Fig 1D) 10.17504/protocols.io.t2peqdn [PROTOCOL DOI].

Species delimitation using bPTP

The phylogenetic tree generated by the Bayesian Inference was submitted to the Bayesian Poisson Tree Processes - bPTP [58] to test species boundaries. This test adds Bayesian support (BS) values to the nodes of the input tree and delimit species based on the Phylogenetic Species Concept (Step D2, Fig 1D).

The analyses were conducted on the web server (available at http://species.h- its.org/ptp/). The parameters for the run were 500000 MCMC generations, thinning of 100, and Burn-in of 0.25 dx.doi.org/10.17504/protocols.io.t2jeqcn [PROTOCOL DOI].

6) Integrative taxonomy

Species boundaries among populations of Ophiothrix were evaluated using four independent character sets in order to test: i) external morphology (Step B1, Fig 1B), ii) arm microstructures morphology (Step B2, Fig 1B), iii) morphometry (Step C, Fig 1C), and iv) molecular data (16S and COI fragments) (Step D, Fig 1D). The CSs were classified according to the congruence framework of Padial et al. [59] when there was concordance of, at least, three data sets analyzed (Step E, Fig 1E). Therefore, we highlight that molecular data was not weighted more heavily than morphological features dx.doi.org/10.17504/protocols.io.t2meqc6 [PROTOCOL DOI].

7) Ethics Statements

Field work was authorized by the Chico Mendes Institute for Biodiversity Conservation – ICMBio, under the license number: 19887-1 to Cecília Amaral (AB) 38463-3 to Maristela Bueno (ECP) and 53376-1 to Marcos Tavares (TMV). Genetics data were registered at the National System for the Management of Genetic Heritage and Associated 52

Traditional Knowledge – SisGen, according to Brazilian legislation Law number 13.123/2015 and Decree 8772/2016. Approval ID for this study was AC6C194.

Results

1) Candidate Species (CS) inferred from morphological

characters

A total of 835 Ophiothrix specimens were analyzed and then classified into four CS: CS1 and CS2 from TMV and CS3 and CS4 from ECP and AB (Step A, Fig 1A). Table 1 and Fig 2 summarizes the main characteristics identified as diagnostic among the CS.

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Fig 2. Candidate Species of Ophiothrix. (A, B) CS1 – MZUSP 1426, Trindade Island. (C, D) CS2 – MZUSP 1425, Trindade Island. (E, F) CS3 – ZUEC OPH 2811, Araçá Bay. (G, H) CS4 – ZUEC OPH 2783, Estuarine Complex of Paranaguá. as: arm spine; dap: dorsal arm plate; ds: disc spine; lds: longer disc spine; sds: small disc spine; vap: ventral arm plate; vap1: first ventral arm plate; vap2: second ventral arm plate. Scale bars: 0.5 mm. 54

Table 1. Characters identified as diagnostic in the separation of Ophiothrix CSs.

Characters Ophiothrix sp. CS1 Ophiothrix sp. CS2 Ophiothrix sp. CS3 Ophiothrix sp. CS4 Figs 2A and 2B Figs 2C and 2D Figs 2E and F Figs 2G and 2H Locality TMV TMV ECP and AB ECP and AB Disc Covered by hyaline spines up Covered by hyaline spines up Mostly covered by small Mostly covered by spines to 0.10 mm in length to 0.17 mm in length hyaline spines of similar to those of the arms approximately 0.15 mm in (denticulate) up to 2 mm in length and some longer ones length and small hyaline of approximately 0.8 mm in spines of approximately 0.2 length scattered on the disc mm in length

Dorsal arm With a prominent ridge at With a prominent ridge at Without a prominent ridge at Without a prominent ridge at plates median portion similar to median portion similar to median portion median portion CS2 CS1 prominent ridge at median portion of the dorsal arm plates (dap). This structure was previously called “carena” (in Portuguese) by 55

Tommasi Ventral arm Distal edge concave, as long Distal edge concave, as long Distal edge concave, slightly Distal edge concave, slightly plates as wide, except the first as wide, except the first and as long as wide as long as wide which did not have a distal the second which did not concavity have a distal concavity and are twice as long as wide (similar to a paddle shape)

Length of the Two to three times greater Two to three times greater Two to three times greater More than three times larger longest arm than the width of one segment than the width of one segment than the width of one segment than the width of one segment spines AB, Araçá Bay, São Paulo, Brazil; ECP, Estuarine Complex of Paranaguá, Paraná, Brazil; TMV, Trindade and Martin Vaz Oceanic Archipelago,

Brazil. 56

A detailed comparison of the CSs and six Brazilian Ophiothrix species listed by Barboza and Borges [4] were perfomed. The possibility that CSs belong to some of the species was rejected as follows: O. rathbuni has triangular and naked radial shields; O. suensoni possesses naked radial shields; O. ailsae has red bands on the arms and a disc covered only by trifid spines; and O. brachyactis possesses granules on dorsal disc.

The most similar species to CSs were Ophiothrix angulata and O. trindadensis as they have subpentagonal disc and the dorsal disc and radial shields are covered by bifid and/or trifid spines. However, some differences were noted among the specimens, particularly the length of the spines covering the dorsal disc, the dorsal and ventral arm plates, and number of arm spines.

2) Morphological characters

Taxonomic review

Three groups of Ophiothrix were formed according to their main morphological characters (Table 2). Group 1 included O. trindadensis from original description [10], CS1 and CS2. Their major characteristics are the absence of longer hyaline spines scattered on the disc, absence of denticulate spines on the disc, presence of a prominent ridge at median portion, termed carena by Tommasi, on dorsal arm plate, and five to 14 arm spines.

57

Table 2. Main morphological characters of all CSs compared with Ophiothrix angulata and O. trindadensis.

Group 1 Group 2 Group 3 O. angulata O. angulata Characters O. trindadensis O. angulata CS1 CS2 Santana et al. Type locality CS3 CS4 Tommasi [10] Say [24] [29] USNM Locality TMV TMV TMV SC-US SC-US SC-US ECP, AB ECP, AB Disc (sub) pentagonal ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Small hyaline spines and some longer ones scattered X X X ND X X ✓ X on the disc

Denticulate spines on the X X X ND X X X ✓ disc

Carena on dorsal arm plate ✓ ✓ ✓ ND X X X X

Ventral arm plates heart- ✓ ✓ ✓ ND ✓ ✓ ✓ ✓ shaped

Number of arm spines 9 5 – 13 7 – 14 7 5 – 9 5 – 7 5 – 8 4 – 9 Main morphological characters of all CSs with Ophiothrix angulata – original description [24], redescription [29] and specimens from type locality (USMN); O. trindadensis – original description [10]. AB, Araçá Bay, São Paulo, Brazil; ECP, Estuarine Complex of Paranaguá, Paraná, Brazil; ND, not described; SC-US, South Carolina, United States; TMV, Trindade and Martin Vaz Oceanic Archipelago, Brazil; USNM, Smithsonian Museum of Natural History; ✓ present; X absent. 58

Group 2 included all the O. angulata from original description [24], the redescription [29], samples from type locality (USNM) and CS3. Their main characteristics are the absence of longer hyaline spines scattered on the disc, absence of denticulate spines on the disc, absence of the carena on dorsal arm plate, and five to nine arm spines.

Group 3 included the CS4. Despite the similarities between Ophiothrix angulata and CS4, they were kept separate due to the denticulate disc spines on the latter.

Arm ossicles

A total of 10 specimens were analyzed: a) CS1 = 2 (samples MZUSP 1426, ZUEC OPH 2883); b) CS2 = 1 (MZUSP 1425); c) CS3 = 2 (MZUSP 1695, ZUEC OPH 2811); and d) CS4 = 5 (ZUEC OPH 2803, ZUEC OPH 2798, ZUEC OPH 2783, ZUEC OPH 2455, ZUEC OPH 2149). The dorsal, ventral, and lateral arm plates (S3 Fig) and arm vertebrae (S4 Fig) were examined.

The primary differences between the CSs are listed in S4 Table. All the differences are related to the dorsal and lateral arm plates. The dorsal arm plates of CS1 and CS2 were one and a half times as wide as long, distal region three to four times wider than the proximal one, with a carena at the median portion. The dorsal arm plates of CS3 and CS4 were as wide as long, distal and proximal regions at the same size, without a carena at median position. CS1 and CS2 have eight or more spine articulations on the lateral arm plates, while CS3 and CS4 have at most seven.

There were no differences in the vertebral ossicles. A true keel was observed in all CS. This was evident due the presence of a dorsal distal keel, dorsal projections, and depressions (large groove) connected by dorsal accessory muscles. These were also evident at the dorsal vertebral surface.

3) Morphometry

A total of 75 specimens were measured: CS1 = 11; CS2 = 10; CS3 = 25; CS4 = 19 and Ophiothrix angulata from type locality (USNM) = 10. 59

The LDA using all 17 morphological characters was effective in discriminating between the four Ophiothrix CSs (n=65), supported by the results for within group dispersion BETADISPER (F3,61 = 7.1677, p<0.001), and PERMANOVA

(F3,61 = 7.5725, p<0.001) analysis which confirmed significant multivariate differences in at least one of the CS.

The first and second linear discriminant axes described 72.94% and 24.54% of the among CSs variation in morphological characters, respectively (Fig 3). The classification success of LDA among the four CSs was 86.79%, showing CS3 and CS4 as the candidates classified correctly most often (100%), while CS1 was the most commonly misclassified (54%).

Fig 3. Axes 1 and 2 from Linear Discriminant Analysis (LDA) based on 17 brittle stars’ characters. See S2 Table and S2 Fig for definitions of the morphological characters used for the morphometric analysis of Ophiothrix CSs. a: specimens from AB; g: specimen from TX-US; p: specimens from ECP; s: specimens from SC-US; t: specimens from TMV; X: Ophiothrix angulata from type locality (USNM).

60

The width of the first ventral arm plate and width of the oral shield (vap1_w and os_w) were the morphological characters with highest positive coefficients in the first discriminant vector (LD1) indicating a strong contribution of these two characters in the multivariate separation model (Fig 3), separating mainly CS4 from the other CS. The highest negative coefficients were length of second ventral arm plate (vap2_l) and width of dorsal arm plate (dap_w), separating in two groups, one from CS1 and CS3 (TMV) and another from CS3 (AB, ECP) and Ophiothrix angulata from type locality.

Alternatively, the second discriminant vector (LD2) has length of first ventral arm plate (vap1_l) and width of dorsal arm plate (dap_w) as a positive coefficient and length of adoral shield (ads_l) and width of second ventral arm plate (vap2_w) as a negative coefficient, separating CS3 from the other CS.

4) Molecular characters

Phylogenetic analyses

The phylogenetic cladograms obtained either in the Bayesian analyses or in the Maximum Parsimony (concatenated matrix, Fig 4; 16S and COI individual matrices, S5 Fig) and Maximum Likelihood analyses (concatenated matrix, S6 Fig) of all datasets show two main clades with high support values, which were referred to as Clade A and Clade B. Clade A is formed by CS1, CS2 from TMZ and specimens from SPSPA, while Clade B is formed by CS3, CS4 from ECP and AB, and the single specimen from TX-US. 61

Fig 4. Cladogram inferred from the Bayesian analysis of 16S and COI sequences (concatenated). The numbers above the branches represent posterior probabilities. For the clades also inferred in the MP analysis, bootstrap values (%) are provided (below the branches). Branches are identified by individual codes (S3 Table) and their localities: AB: Araçá Bay, São Paulo, Brazil; ECP: Estuarine Complex of Paranaguá, Paraná, Brazil; EUR: Europe; SPSPA: São Pedro and São Paulo Archipelago, Brazil; TMV: Trindade and Martin Vaz Oceanic Archipelago; TX-US: Texas, United States. The scale bar represents the average nucleotide substitutions per site. Asterisks (*) indicate that the support value was lower than 0.7 (Bayesian Inference) or 70% (Maximum Parsimony), and a dash (-) indicates that the branch was not recovered in MP analysis. 62

Five clades within Clade A were only found in Bayesian Inference, three of them with high support value (more than 0.8). Within Clade B, only one clade was found in Bayesian Inference and Maximum Parsimony, and two clades in Maximum Likelihood, but all with low support values.

Analyses of the genetic diversity

Genetic distances. The genetic distances observed in 16S and COI genes between CS1 and CS2 were low (1.4% for 16S, 3.5% for COI), as well as those observed between CS3 and CS4 (0.4% for 16S, 1.5% for COI). In contrast, high genetic distances were observed between specimens included in Clade A and those of Clade B which range from 11.4% to 12.1% for 16S gene and from 16.8% to 17.2% for COI gene (S5 Table).

AMOVA. AMOVA test revealed that 93.49% of total genetic variability occurred among the Clades A and B that were inferred in the phylogenetic analyses. Only 6.63% of total genetic diversity refers to variability within these groups. The overall fixation index (FST) was extremely high (0.934) (S6 Table). These results indicate that the two groups are genetically distinct and well structured, indicating low or null gene flow between them.

Haplotype Network. The haplotype network indicated the existence of two sharply divergent lineages (1: CS1+CS2 and 2: CS3+CS4+TX-US) lacking intermediate haplotypes. Divergence among clades was caused by 34 mutations. The first lineage (Fig 5, right) revealed a star-like pattern with H_7 as the ancestral haplotype surrounded by low-frequency (mostly private) haplotypes, separated by a few mutational steps. The second lineage (Fig 5, left) is composed of three dominant haplotypes (H_11, H_15 and H_14) surrounded by private haplotypes (Fig 5). 63

Fig 5. Median-joining haplotype network based on the concatenated matrix of 16S and COI gene sequences. Each circle represents one haplotype and their size is proportional to the number of individuals possessing it. SPSPA: Saint Peter and Saint Paul Archipelago, Brazil; TX- US: Texas, United States.

Species delimitation using bPTP

Clades A (CS1, CS2, SPSPA) and B (CS3, CS4, TX-US) found in the phylogenetic analyses were supported as distinct species by the PTP test. High values of posterior delimitation probabilities supported each of these clades (0.76 and 0.9 for Clade A and Clade B, respectively) (S7 Fig).

5) Integrative taxonomy

To integrate the results reported for the different operational criteria, a congruence framework was followed which considered that concordant divergence patterns among several taxonomic characters indicated that full lineage separation, as it was highly improbable that a coherent concordance pattern would emerge by chance [59].

Concordance was found between the analyses based on external morphology, arm microstructures morphology (dorsal arm plates and lateral arm plates), morphometry, and molecular data (phylogenetic inferences, genetic distances, AMOVA, haplotype network, and bPTP) pointing to the existence of at least two species: (1) composed of CS1 and CS2 from TMV, and (2) composed of CS3 from ECP and AB. CS4 remains as O. cf. angulata as more data is needed to be definitively clarified (Fig 6). 64

Fig 6. Synthetic representation of the integrative taxonomy approach within the Ophiothrix CSs. dap: dorsal arm plates; lap: lateral arm plates.

In the following, we present the redescription of Ophiothrix trindadensis Tommasi, 1970, which is represented by CS1 and CS2, and Ophiothrix angulata (Say, 1825) as CS3. We propose CS4 as O. cf. angulata due to its different morphology, particularly the presence of spines on the disc similar to those of the arms (denticulate). The redescription of O. trindadensis contains the amended diagnosis, the description of an adult specimen (5.8 mm of dd) and juvenile specimen (less than 4 mm of dd) due to the importance of considering both in taxonomic practice [60-62]. We also added the variation in adult specimens, comparisons with O. angulata, taxonomic comments, remarks, and distribution.

6) Morphological description

Ophiothrix trindadensis Tommasi, 1970

CS1 and CS2

Neotype MZUSP 1425 (sample 14).

65

Type locality. Trindade Island.

Maximum size. dd up to 8.5 mm (present study)

Material examined. 541 specimens. See S7 Table.

Amended diagnosis. Pentagonal disc covered by small hyaline spines. Dorsal arm plates fan-triangular, distal region three to four times greater than the proximal, and with a prominent carena at median portion. Eight to eleven long arm spines (longest about three arm joints), vitreous and denticulate (Fig 7).

Description of the adult neotype. Disc (dd: 5.8 mm). Pentagonal, covered by small hyaline spines. Radial shields triangular, three times as long as wide, one-third of dd, united distally and separated proximally (Fig 7A). Ventral interradius covered by scales with small spines, the same as the dorsal (Fig 7B).

Mouth plating. Oral shields almost twice as wide as long, tapered proximally and with a slight projection at the distal edge. Madreporite larger than other oral shields, but with a similar shape. Adoral shields broadened distally and united proximally. Depression between two oral plates. A cluster of dental papillae on the apex of the jaw. Infradental papillae and lateral oral papillae absent. Oral tentacle pore visible (Fig 7B). Dental plate with equal width all over, an outer column of small holes at each edge on ventral half, dorsal half with fenestrations and a septum (Fig 7C). Abradial view of oral plate with rib-like branching structures on muscle attachment area (Fig 7D). Adradial view of oral plate with a large, dorsal, spoon-shaped depression on muscle attachment area (Fig 7E).

Arms. Dorsal arm plates fan-triangular, one and a half times as wide as long, and with a prominent carena at median portion (Fig 7F). Ventral arm plates as long as wide, with a slightly notch at distal edge, proximal edge with a small tip (Fig 7G). One tentacle scale. Eight to eleven long arm spines (longest about three arm joints), vitreous and denticulate, the second to ventral-most being the smallest and the ventral-most modified into a hook with hyaline teeth facing proximal side (Fig 7G).

Lateral arm plates (Figs 7H and 7I): general outline: arched (wrapped around the arm); without constriction; projecting ventro-proximalwards; ventro-distal tip not projecting ventralwards. Outer surface ornamentation: trabecular intersections protruding to form knobs approximately the same size as stereom pores. Outer proximal edge: surface lined by discernible band of different stereom structure, restricted to central part; without 66 spurs; central part protruding; surface without horizontal striation. Arm spine articulations: nine, on elevated portion not bordered proximally by ridge; directly adjacent to the distal edge; arranged over entire distal edge; middle spine articulation(s) larger; distance between spine articulations equidistant. Lobes merged at their proximal tips by smooth connection; lobes parallel, equal-sized, bent, with tilted orientation; stereom with perforations; sigmoidal fold absent. Inner side: dominated by two separate central knobs with a ridge; without additional dorsal structure; single large perforation. Vertebrae: zygospondylous of universal type and keeled. Large groove on proximal side of vertebrae dorsally corresponding to distalwards projecting dorso-distal muscular fossae of distal side (Fig 7J). Zygocondyles dorsalwards converging and zygosphene fused with pair of zygocondyles (Fig 7K). Narrow dorsal keel protruding distalwards far beyond vertebra edge, matching large dorsal groove proximally (Figs 7L and 7M).

Variation in the adult specimens. Disc covering: with larger and/or smaller spines. Shape of oral shield: with straight and/or rounded edges. Adoral shields: separated or united proximally. Ventralmost arm spine modified into a hook with hyaline teeth facing the disc: often starting at 8th or 9th segment but occasionally from the 5th segment. Spine on the first dorsal arm plate: sometimes present and easily observable. Number of arm spines: eight to eleven, but one was identified with 14.

Color patterns in preserved adult specimens. Most common are (S8 Fig): purple disc, gray and purple arms; purple disc, pink and purple arms; purple with white – disc and arms. Some others are: red disc and arms; orange disc and purple arms; and brown with white – disc and arms.

Juvenile specimens - less than 4 mm of dd

Material examined. 4 specimens. See S7 Table.

Disc (dd = 2.1 mm): subpentagonal, covered by small hyaline spines. Radial shields triangular, two times as long as wide, one-fourth of dd, united distally and separated proximally (Fig 8A). Ventral interradius covered by scales with small spines, the same as the dorsal (Fig 8B). Oral shields almost three times as wide as long, tapered proximally and without a slight projection at the distal edge. Madreporite not distinguishable. Adoral shields broadened distally and united proximally. Depression 67 between two oral plates. A cluster of dental papillae on the apex of the jaw and without lateral oral papilla. Oral tentacle pore visible (Fig 8C).

Arms. Dorsal arm plates fan-triangular, one and a half times as wide as long, and with a prominent carena at median portion only in specimens greater than 2.9 mm of dd (Fig 8D). Ventral arm plates twice as long as wide, and the notch at distal edge is smaller than the notch in adult specimens (Fig 8E). One tentacle scale. Four to eight long arm spines (longest about three arm joints), vitreous and denticulate, the second to ventral- most being the smallest and the ventral-most modified into a hook with hyaline teeth facing the disc (Fig 8E).

Color patterns in preserved juvenile specimens. Most common are purple radial shields; purple and white on disc and arms; and with a bright whitish circle in the center of the disc.

Taxonomic comments. The main differences between O. trindadensis and O. angulata are: i) prominent carena at median portion of the dorsal arm plates of O. trindadensis, which is absent in O. angulata; and ii) eight to eleven long arm spines in O. trindadensis, while O. angulata is frequently characterized by at most seven arm spines. Barboza et al. [21] described the dorsal arm plates of the Ophiothrix angulata from SPSPA with the distal edge slightly lobed, which we assume to be the carena found on O. trindadensis. The specimens from SPSPA were deposited in the collection of the National Museum of Rio de Janeiro, Brazil, but unfortunately, they were not accessible. In addition to the carena on dorsal arm plates, the low genetic differences between specimens from TMV and SPSPA shows the existence of just one species in TMV and SPSPA, O. trindadensis.

Remarks. Ophiothrix trindadensis was collected from rubble bottom or associated with Lithothamnion. It has also been sampled in corals [10].

Distribution. Tropical Atlantic (realm), Tropical Southwestern Atlantic (province): São Pedro and São Paulo Islands [21], Northeastern Brazil [63], Eastern Brazil [64], Trindade and Martin Vaz Islands [10]. The present study samples occurred at depths ranging from 7 to 25 m. 68

Fig 7. Ophiothrix trindadensis sample MZUSP 1425 (5.8 mm dd). (A) Dorsal view. (B) Ventral view. (C) Dental plate. (D) Abradial view of oral plate. (E) Adradial view of oral plate. (F) Detail of dorsal arm. (G) Detail of ventral arm. (H) Lateral arm plate – external side. (I) Lateral arm plate – internal side. (J-M) Vertebrae ossicle: (J) proximal surface. (K) distal surface. (L) dorsal surface. (M) ventral surface. ads: adoral shields; as: arm spine; cdp: cluster of dental papillae; dap: dorsal arm plate; d: dorsal; ddi: dorso-distal; ddmf: dorso-distal muscular fossae; di: distal; dk: distal keel; ds: disc spine; dp: dorso-proximal; kn: knob; lg: large groove; ma: madreporite; os: oral shields; otp: oral tentacle pore; p: proximal; pe: perforation; ribs: rib-like branching structures; ret: regular teeth; rs: radial shields; sh: small hole; v: ventral; vap: ventral arm plate; vdi: ventro-distal; vi: ventral interradius; vp: ventro-proximal; zd: zygocondyle; zp: zygosphene. Stereomicroscope photos (A, B, F, G), scale bar equal to 0.5 mm. SEM photos (C-E, H-M), scale bar equal to 100 μm. 69

Fig 8. Ophiothrix trindadensis – juvenile with 2.1 mm of dd (MZUSP 1687). (A) Dorsal view. (B) Ventral view. (C) Detail of the oral view. (D) Detail of dorsal arm. (E) Detail of ventral arm. ads: adoral shields; as: arm spine; cdp: cluster of dental papillae; dap: dorsal arm plate; di: distal; os: oral shields; otp: oral tentacle pore; p: proximal; rs: radial shields; vap: ventral arm plate; vi: ventral interradius. Stereomicroscope photos, scale bar equal to 0.5 mm.

Ophiothrix angulata (Say, 1825)

Type locality. Charleston Harbour, South Carolina (United States).

CS3. Araçá Bay and Estuarine Complex of Paranaguá, Brazil; South Carolina and Texas, United States.

Maximum size. dd up to 10 mm [27].

Material examined. 181 specimens. See S7 Table.

Amended diagnosis. Pentagonal disc, covered by small and hyaline spines and some longer ones scattered on the disc. Dorsal arm plates fan-triangular, as wide as long. Five to eight long arm spines (longest about three arm joints), vitreous and denticulate. 70

Description. Original description in Say [24], neotype description in Santana et al. [29], microstructures description in Alitto et al. [30]. Taxonomic comments. Specimens from type-locality (USNM, voucher E24138) were compared with our samples. The only difference observed was the shape of the ventral arm plates: slightly concave distally in our specimens while it was cordiform in specimens from type-locality. Two characters were not observed in the specimens from USNM and the present study: i) carena on dorsal arm plates and ii) disc covered by spines similar to those of the arms (denticulate). Remarks. In Araçá Bay, it was collected from rubble bottom with a dredge or associated with the sponge Amphimedon viridis. In Estuarine Complex of Paranaguá, it was sampled with the sponge Mycale (Zygomycale). Distribution. See Alitto et al. [30]. Specimens from type locality occurred at depths ranging from 13 to 66 m. The present study samples occurred at depths ranging from intertidal to 20 m.

Ophiothrix cf. angulata

CS4. Araçá Bay and Estuarine Complex of Paranaguá, Brazil.

Maximum size. dd up to 7.8 mm (present study).

Material examined. 110 specimens. See S7 Table.

Amended diagnosis. Pentagonal disc, covered by small and hyaline spines, mostly covered by spines similar to those of the arms (denticulate). Dorsal arm plates fan- triangular, as wide as long. Five to eight long arm spines (longest about three arm joints), vitreous and denticulate.

Description of the adult. Disc (dd: 6.4 mm) pentagonal, covered by small hyaline, bifid and/or trifid spines and some longer ones scattered on the disc. Radial shields triangular, twice as long as wide, one-third of dd, united distally and separated proximally, covered by bifid and/or trifid spines (Fig 9A). Ventral interradius covered by scales, except near the oral shields and bursal slits (Fig 9B).

Mouth plating. Oral shields as long as wide, tapered proximally and with a slight projection at the distal edge. Madreporite larger than other oral shields, but with a similar shape. Adoral shields broadened distally and separated proximally. Depression 71 between two oral plates. A cluster of dental papillae on the dental plate. Infradental papillae and lateral oral papillae absent. Oral tentacle pore visible (Fig 9B). Dental plate with equal width all over, an outer column of small holes at each edge on ventral half, dorsal half with fenestrations and a septum (Fig 9C). Abradial view of oral plate with rib-like branching structures on muscle attachment area (Fig 9D). Adradial view of oral plate with a large, dorsal, spoon-shaped depression on muscle attachment area (Fig 9E).

Arms: dorsal arm plates fan-triangular, as long as wide and contiguous (Fig 9F). Ventral arm plates straight or slightly concave on distal edge, proximal edge straight, lateral edges convex and slightly as long as wide (Fig 9G). One tentacle scale. Five to eight long arm spines (longest about four arm joints), vitreous and denticulate, the second to ventral-most being the smallest and the ventral-most modified into a hook with hyaline teeth facing the disc (Fig 9E).

Lateral arm plates (Figs 9H and 9I): general outline: arched (wrapped around the arm); without constriction; ventral portion projecting ventro-proximalwards; ventro-distal tip not projecting ventralwards. Outer surface ornamentation: trabecular intersections protruding to form knobs approximately the same size as stereom pores. Outer proximal edge: surface lined by discernible band of different stereom structure, restricted to central part; without spurs; central part protruding; surface without horizontal striation. Arm spine articulations: seven, on elevated portion not bordered proximally by ridge; directly adjacent to the distal edge; arranged over entire distal edge; all similar in size; distance between spine articulations dorsalwards increasing. Lobes merged at their proximal tips by smooth connection; lobes parallel, equal-sized, bent, with tilted orientation; stereom with perforations; sigmoidal fold absent. Inner side: dominated by two separate central knobs with a ridge; without additional dorsal structure; single large perforation.

Vertebrae: zygospondylous of universal type and keeled. Large groove on proximal side of vertebrae dorsally corresponding to distalwards projecting dorso-distal muscular fossae of distal side (Fig 9J). Zygocondyles dorsalwards converging and zygosphene fused with pair of zygocondyles (Fig 9K). Narrow dorsal keel protruding distalwards far beyond vertebra edge, matching large dorsal groove proximally (Figs 9L and 9M).

Taxonomic comments. A total of five specimens were considered juvenile (less than 4 mm of dd). Spines similar to those of the arms (denticulate) were observed on the disc 72 even in the smallest samples. The number of arm spines of most adults was 6 to 7, however three specimens had 8 arm spines.

Remarks. In Araçá Bay, specimens were collected from rubble bottom with a dredge or associated with the sponge Amphimedon viridis. In the Estuarine Complex of Paranaguá, it was sampled with the sponge Mycale (Zygomycale).

Distribution. Tropical Atlantic (realm), Tropical Southwestern Atlantic (province): Southeastern Brazil (present study). The present study samples occurred at depths ranging from intertidal to 20 m. 73

Fig 9. Ophiothrix cf. angulata ZUEC OPH 2783 (6.4 mm dd). (A) Dorsal view. (B) Ventral view. (C) Dental plate. (D) Oral plate - abradial view. (E) Oral plate - adradial view. (F) Detail of dorsal arm. (G) Detail of ventral arm. (H) Lateral arm plate – external side. (I) Lateral arm plate – internal side. (J-M) Vertebrae ossicle: (J) proximal surface. (K) distal surface. (L) dorsal surface. (M) ventral surface. ads: adoral shields; as: arm spine; cdp: cluster of dental papillae; d: dorsal; dap: dorsal arm plate; ddi: dorso-distal; di: distal; dk: distal keel; ddmf: dorso-distal muscular fossae; dp: dorso-proximal; kn: knob; lds: longer disc spine; lg: large groove; ma: madreporite; os: oral shields; otp: oral tentacle pore; p: proximal; pe: perforation; ribs: rib-like branching structures; ret: regular teeth; rs: radial shields; sh: small holes; v: ventral; vap: ventral arm plate; vdi: ventro-distal; vi: ventral interradius; vp: ventro- proximal; zd: zygocondyle; zp: zygosphene. Stereomicroscope photos (A, B, F, G), scale bar equal to 0.5 mm. SEM photos (C-E, H-M), scale bar equal to 100 μm. 74

Discussion

The combined integrative taxonomy based on external morphology, arm microstructures morphology, morphometry, and molecular data has confirmed the identity of two species of Ophiothrix. The candidate species that were previously identified as CS1, CS2, CS3, and CS4 are now classified as: 1) Ophiothrix trindadensis Tommasi, 1970, formed by CS1 and CS2, and redescribed here in detail, 2) Ophiothrix angulata as CS3. The CS4 was described as O. cf. angulata as more data are needed to be definitively clarified.

The conclusion that CS1 and CS2 are Ophiothrix trindadensis is due to the four distinct analyses (external morphology, arm microstructures morphology, morphometry, and molecular) that distinguished it from CS3 and CS4, and because they are from the type locality of O. trindadensis, Trindade Island. As the analyses failed to separate CS1 from CS2, they have been combined as a single species with intraspecific morphological variation, such as larger and/or smaller spines on the disc.

The main character observed in the arm ossicles of Ophiothrix trindadensis was a prominent ridge at median portion of the dorsal arm plates (DAP). This structure was previously called “carena” (in Portuguese) by Tommasi [10] and is rediscribed here as a diagnosis. Another diagnostic feature related to DAP was that the distal region was three to four times wider than the proximal one, unlike O. angulata, which is the same width. And the last feature was the number of the spine articulations on the arm ossicles, and consequently, the arm spines on the lateral arm plates. O. trindadensis can have up to 14 arm spines, unlike O. angulata, which has at most nine.

The morphometric analysis of the Ophiothrix trindadensis yielded two characters that separated it from the others CS: i) length of the second ventral arm plate, and ii) width of dorsal arm plate. For these two characters, we made initial comments and predictions, which aided in the separation of the CSs before the tests. We did not know if these characters would be significant a priori. This demonstrates the power of morphometry in the separation of species for taxonomic studies. Measures of ventral and dorsal arm plates are highly indicative of separation of Ophiothrix species and should be considered in diagnosis. 75

The specimens from Trindade and Martin Vaz Archipelago (TMV) formed a clade, here named Clade A, with strong support in both phylogenetic analyses. The level of genetic divergence between Clades A and B was high (11.4–12% and 16.8– 17.2% for 16S and COI, respectively). These levels of 16S and COI divergence are similar to genetic distances found between Ophiothrix fragilis and O. quinquemaculata (about 9.5% for 16S and 16.5% for COI) in Europe [8, 9].

The hypothesis that the TMV specimens were Ophiothrix angulata was discarded for three reasons. First because the original description of O. angulata [24] depicts seven arm spines. Second because the genetic divergence of 16S gene between TMV specimens and O. angulata (KU672428) was high (11.7–12.1%). And third due to the absence of the carena on dorsal arm plates in the O. angulata from type locality (USNM).

Another feature of interest was the similarity between the specimens from TMV and São Pedro and São Paulo Archipelago (SPSPA). The genetic divergence of 16S gene between these two localities was low (0.6–1.2%), which is indicative of intraspecific variations. According to Barboza et al. [21], the specimens from SPSPA were deposited in the collection of the National Museum of Brazil (NMRJ), but unfortunately, they were not found. Despite this, Barboza et al. [21] described the dorsal arm plates of the specimens with distal edge slightly lobed, which we interpret to be the carena present on Ophiothrix trindadensis.

The diagnosis that CS3 from Clade B is Ophiothrix angulata was made for three reasons. First is because of the similarities between CS3 with the original description of Say [24] and redescription of Santana et al. [29]. Second is due to the low genetic divergence of 16S gene between CS3 and O. angulata (KU672428) (0.8%). The sample KU672428 is from Port Aransas, Texas, USA, which is the nearest place of the O. angulata type locality (Charleston Harbour, South Carolina, USA). And finally, due to the morphological similarities between CS3 and O. angulata from type locality (USNM).

CS4 was redescribed here as Ophiothrix cf. angulata. This conclusion was due to the low genetic divergence (0.9% and 1.5% for 16S and COI, respectively) between CS4 and Ophiothrix angulata (KU672428) and the great similarity between CS4 and O. angulata from type locality (USNM). Despite this, we believe that it is 76 necessary to continue the studies of CS4 for the following reasons: i) the morphometric analysis separated the CS4 from other CSs; ii) the presence of disc spines similar to those of the arms (long and denticulate); and iii) necessity of comparison with other Ophiothrix species.

CS3 was frequently found in Araçá Bay (AB), while CS4 was often in Estuarine Complex of Paranaguá (ECP). This must be addressed in future studies, particularly when considering the environments in which they live. The AB is a more saline region with oceanographic features distinct from ECP. Furthermore, the specimens were sampled in different sponges, CS3 in Amphimedon viridis while CS4 in Mycale (Zygomycale). This observation has prompted several questions concerning their ecology and physiology as well. What degree can the differences observed on disc coverage (presence or absence of denticulate spines) could be related to their i) biological substrate? ii) the different salinities and/or depths sampled? In any case, we emphasize the need for further studies, including ones concerning their ecological traits.

A true keel was observed in all CS. Unfortunately, knowledge about the vertebrae is still scarce, with only a few studies describing possible correlations between vertebral morphologies and ecological specialization [65-68]. The need for further studies such as behavioral observation and mechanical testing of individual arm segments is necessary to elucidate the function of these structures.

Our results demonstrate the utility of applying integrative taxonomic approaches for brittle stars specimens, particularly at the species level according to Padial et al. [59]. This method has demonstrated great efficiency and accuracy in the taxonomic delimitation of several groups, for instance beetles, as shown by Arribas et al. [69]. However, implementation of integrative taxonomy may be problematic for hidden species and/or higher taxonomic levels, such as family and genera. To assist in this issue, Korshunova et al. [70] developed several operational rules rooted in biological facts, which were successfully applied to a group of mollusks. These certainly may be applied for other metazoan groups.

We highlight the importance of depositing samples in museums, as they document historical and current patterns of biological diversity, which cannot be replaced. Additionally, future studies should consider the use of morphometry 77 combined with scanning electron microscopy in order to detect patterns undetected by the naked eye.

Acknowledgments

We are grateful to Camila Silva, Guilherme Corte, Helio Checon, Nathalia Padovanni, Angélica Godoy, Renato Luchetti, Gustavo Dias, and Felipe Oricchio for helping collect and sieve all the Araçá Bay samples and Maristela Bueno for collecting the Paranaguá specimens. We thank Joel Braga de Mendonça Junior and Andre Pol for conducting fieldwork in Trindade. We are in debit with Adriane Sprogis and Stella de Ferraz for their great help with the scanning electron microscopy at UNICAMP. We also thank to Smithsonian United States National Museum for specimens’ loan. We gratefully acknowledge the Brazilian Navy (1st District) and SECIRM (Interministerial Secretaria for Marine Resources) in the person of Commanders R. Otoch and S. da Costa Abrantes for all the support provided in Trindade and Martin Vaz, including laboratory facilities and diving operations. This is contribution number 5 of the ProTrindade Marine Invertebrate Project. Finally, many thanks to the reviewers whose valuable comments were greatly appreciated.

78

Supporting information

S1 Fig. Study sites and data collection. Triangles represent the samples that were collected during the present study: Trindade and Martin Vaz Oceanic Archipelago, Araçá Bay, and the Estuarine Complex of Paranaguá. Circles represent the location of samples used in our comparisons: São Pedro and São Paulo Archipelago (molecular data), South Carolina (morphological data), and Texas (morphological and molecular data). The molecular data were obtained from GenBank. The shapefile was imported from the Natural Earth project (the 1:50m resolution version).

79

S2 Fig. Illustrations of the 17 morphological characters used for the morphometric analysis. The abbreviations and definitions are described in Table S2.

80

S3 Fig. Arm ossicles of Ophiothrix CSs. dorsal (dap), ventral (vap), and lateral arm plates (lap). CS1 MZUSP 1426: (A) dap; (B) vap; (C,D) lap. CS2 MZUSP 1425: (E) dap; (F) vap; (G, H) lap. CS3 ZUEC OPH 2811: (I) dap; (J) vap; (K,L) lap. CS4 ZUEC OPH 2783: (M) dap; (N) vap; (O, P) lap. d: dorsal; ddi: dorso-distal; di: distal; dp: dorso-proximal; p: proximal; pe: perforation; v: ventral; vdi: ventro-distal; vp: ventro-proximal. Scale bars: 100 μm. 81

S4 Fig. Four main sides of the vertebrae ossicle of Ophiothrix. Proximal view (pv), distal view (div), dorsal view (dov), ventral view (vv). CS1 MZUSP 1426: (A) pv; (B) div; (C) dov; (D) vv. CS2 MZUSP 1425: (E) pv; (F) div; (G) dov; (H) vv. CS3 ZUEC OPH 2811: (I) pv; (J) div; (K) dov; (L) vv. CS4 ZUEC OPH 2783: (M) pv; (N) div; (O) dov; (P) vv. d: dorsal; di: distal; dk: distal keel; lg: large groove; p: proximal; v: ventral. Scale bars: 100 μm. 82

S5 Fig. Phylogenetic relationships inferred from the 16S (left) and COI (right) matrices by Bayesian analysis (MrBayes analysis). Numbers above and below branches represent the support values for BI (posterior probabilities) and MP (bootstrap values in %), respectively. The MP tree was not represented. Branches are identified by individual codes and their localities (AB: Araçá Bay, São Paulo, Brazil; ECP: Estuarine Complex of Paranaguá, Paraná, Brazil; EUR: Europe; SPSPA: São Pedro and São Paulo Archipelago, Brazil; TMV: Trindade and Martin Vaz Oceanic Archipelago). The scale bar represents the average nucleotide substitutions per site. Asterisks (*) indicates that the support value was lower than 70% (MP) or 0.7 (BI), and a dash (-) indicates that the branch was not recovered. 83

S6 Fig. Cladogram inferred from the Maximum Likelihood of 16S and COI sequences (concatenated). The numbers in nodes represent the support values (bootstrap). Branches are identified by individual codes (S3 Table) and their localities: AB: Araçá Bay, São Paulo, Brazil; ECP: Estuarine Complex of Paranaguá, Paraná, Brazil; EUR: Europe; SPSPA: São Pedro and São Paulo Archipelago, Brazil; TMV: Trindade and Martin Vaz Oceanic Archipelago; TX-US: Texas, United States. The scale bar represents the average nucleotide substitutions per site. Asterisks (*) indicate that the support value was lower than 70%.

84

S7 Fig. Species delimitation hypothesis obtained in the maximum likelihood solution of the Bayesian Poisson Tree Processes (bPTP) analysis. Branches in red connect haplotypes included in same species, in blue distinct species. Branches are identified by individual codes (Table S3) and their localities: AB: Araçá Bay, São Paulo, Brazil; ECP: Estuarine Complex of Paranaguá, Paraná, Brazil; EUR: Europe; SPSPA: Saint Peter and Saint Paul Archipelago, Brazil; TMV: Trindade and Martin Vaz Oceanic Archipelago; TX-US: Texas, United States.

85

S8 Fig. Ophiothrix trindadensis – color patterns in some preserved specimens. (A) ZUEC OPH 2907, dd = 5.8 mm. (B) MZUSP 1661, dd = 5.3 mm. (C) MZUSP 1687, dd = 3,1 mm. (D) MZUSP 1450, dd = 6.1 mm. (E) MZUSP 1686 dd = 4.2 mm. (F) ZUEC OPH 2883, dd = 8.5 mm. Stereomicroscope photos, scale bar equal to 1 mm. 86

S1 Table. Study sites that brittle stars were collected (TMV, AB, ECP) and compared (SPSPA, TX-US, SC-US). AB, Araçá Bay; ECP, Estuarine Complex of Paranaguá; SC-US, South Carolina, United States; SPSPA, São Pedro and São Paulo Islands; TMV, Trindade and Martin Vaz Oceanic Archipelago; TX-US, Texas, United States; USNM, United States National Museum; – absent.

Marine Trindade and Martin Southeastern Brazil São Pedro and São Northern Gulf of Carolinian Ecoregions Vaz Islands Paulo Islands Mexico Geographic TMV AB ECP SPSPA TX-US SC-US location Project ProTrindade/CNPq BIOTA/FAPESP – – PROARQUIPÉLAGO – From USNM Araçá Program

Environment Subtidal (7 – 25 m) Intertidal and Intertidal Subtidal (5 m) Intertidal Subtidal (13 m – type subtidal (0 – 20 m) 66 m) Sampling Scuba diving Dredge and by hand By hand Scuba diving By hand Not informed, but strategies probably by dredge. Geographic -20.500, -29.333 -23.814, -45.404 -25.422, -48.407 0.917, -29.345 27.843, -97.061 32.486, -80.353 coordinates (Trindade Island) (Edisto River) -20.500, -28.850 32.097, -80.837 (Martin Vaz Islands) (Calibogue Sound) Substrate type Rubble bottom and Rubble bottom and Biological Biological substrate Biological Not informed. biological substrate biological substrate substrate (sponge) (polychaete tubes) substrate (sandy (algae) (sponge) lobed tunicate colonies) Salinity ~36 ppt (present study) ~34 ppt (present ~29 ppt (present ~35 ppt (Becker 2002) ~30 ppt Not informed. study) study) Water 25 ºC (present study) 21 ºC – 24 ºC 24 ºC (present 20 ºC – 26 ºC (Becker 25 ºC Not informed temperature (present study) study) 2002) References Almeida [1, 2]; Anker Amaral et al. [4,5]; Lessa et al. [7], Edwards & Lubbock The University of – et al. [3] Siegle et al. [6] Lana et al. [8] [9,10]; Barboza et al. Texas at Austin [11]; Viana et al. [12] [13,14] 87

References: 1. Almeida FFM. Ilha de Trindade – registro de vulcanismo cenozóico no Atlântico Sul. In: Schobbenhaus C, Campos DA, Queiroz ET, Winge M, Berbert-Born MLC, editors. Sítios geológicos e paleontológicos do Brasil. Brasília: DNPM/CPRM, Comissão Brasileira de Sítios Geológicos e Paleobiológicos (SIGEP); 2002. pp. 369–377. 2. Almeida FFM. Ilhas oceânicas brasileiras e suas relações com a tectônica atlântica. Terrae Didat. 2006;2: 3–18. 3. Anker A, Tavares M, Mendonça JB. Alpheid shrimps (Decapoda: Caridea) of the Trindade & Martin Vaz Archipelago, off Brazil, with new records, description of a new species of Synalpheus and remarks on zoogeographical patterns in the oceanic islands of the tropical southern Atlantic. Zootaxa. 2016;4138: 1–58. doi: 10.11646/zootaxa.4138.1.1 4. Amaral ACZ, Migotto AE, Turra A, Schaeffer-Novelli Y. Araçá: biodiversidade, impactos e ameaças. Biota Neotrop. 2010;10: 219–264. doi: 10.1590/s1676-06032010000100022 5. Amaral ACZ, Turra A, Ciotti AM, Rossi-Wongstschowski CLDB, Schaeffer-Novelli Y. Life in Araçá Bay: diversity and importance. 3rd ed. São Paulo: Lume; 2016. http://www.bibliotecadigital.unicamp.br/document/?code=73819&opt=1 6. Siegle E, Dottori M, Villamarin BC. Hydrodynamics of a subtropical tidal flat: Araçá Bay, Brazil. Ocean & Coastal Management. 2017;in press: doi: 10.1016/j.ocecoaman.2017.11.003 7. Lessa GC, Meyers SR, Marone E. Holocene stratigraphy in the Paranagua Bay estuary, southern Brazil. Journal of Sedimentary Research. 1998;68: 1060–1076. 8. Lana PC, Marone E, Lopes RM, Machado EC. The Subtropical Estuarine Complex of Paranaguá Bay, Brazil. In: Seeliger U, Kjerfve B, editors. Coastal Marine Ecosystems of Latin America. Berlin, Heidelberg: Springer Berlin Heidelberg; 2001. pp. 131–145. 9. Edwards A, Lubbock R. Marine Zoogeography of St Paul's Rocks. Journal of Biogeography. 1983a;10: 65–72. doi: 10.2307/2844583 10. Edwards A, Lubbock R. The ecology of Saint Paul's Rocks (Equatorial Atlantic). J Zool. 1983b;200: 51–69. doi: 10.1111/j.1469- 7998.1983.tb06108.x 11. Barboza CAM, Mattos G, Paiva PC. Brittle stars from the Saint Peter and Saint Paul Archipelago: morphological and molecular data. Mar Biodivers Rec. 2015;8: 1–9. doi: 10.1017/S1755267214001511 12. Viana DL, Hazin FHV, Oliveira JEL, Souza MAC. Saint Peter and Saint Paul archipelago: Brazil in the mid atlantic. Recife: Vedas Edições; 2017. 13. The University of Texas at Austin. The Texas High School Coastal Monitoring Program 2006. Available from: http://www.beg.utexas.edu/coastal/thscmp/fg_mustang_6.htm. 14. The University of Texas at Austin. Marine Science Institute College of Natural Sciences 2018. Available from: https://utmsi.utexas.edu/. 88

S2 Table. Abbreviations and definitions of the 17 morphological characters used for the morphometric analysis.

Abbreviation Character name Definition dd Disc diameter Length from the distal edge of the radial shield to the interradial edge of the disc (Fig. S2 B) rs_l Radial shield length Length of one radial shield (Fig. S2 A) rs_w Radial shield width Width of one radial shield (Fig. S2 A) disc_spine Disc spine Length of the largest denticulate spine (as those of the arms) near to the disc center as Arm spine Number of arm spines at the third free arm segment (Fig. S2 B) as2 Second arm spine Length of the second arm spine at the third free arm segment (Fig. S2 B) od Oral diameter Length from the proximal edge of adoral shield to distal edge of the first ventral arm plate (Fig. S2 E) os_l Oral shield length Length of one oral shield (Fig. S2 D) os_w Oral shield width Width of one oral shield (Fig. S2 D) ads_l Adoral shield Length of one adoral shield (Fig. S2 D) length ads_w Adoral shield width Width of one adoral shield (Fig. S2 D) dap_l Dorsal arm plate Length of the third dorsal arm plate at the free length arm segment (Fig. S2 C) dap_w Dorsal arm plate Width of the third dorsal arm plate at the free arm width segment (Fig. S2 C) vap1_l 1st ventral arm plate Length of the first ventral arm plate (Fig. S2 F) length vap1_w 1st Ventral arm Width of the first ventral arm plate (Fig. S2 F) plate width vap2_l 2nd ventral arm Length of the second ventral arm plate (Fig. S2 F) plate length vap2_w 2nd ventral arm Width of the second ventral arm plate (Fig. S2 F) plate width

89

S3 Table. Specimens used in the phylogenetic analyses with their locality, museums vouchers, and GenBank accession numbers for DNA sequences. AB, Araçá Bay, Brazil; AM, Australian Museum; ECP, Estuarine Complex of Paranaguá, Brazil; EUR, Europe; FMC, French Mediterranean Coast; IC, individual codes in the present study; NMRJ, National Museum of Rio de Janeiro, Brazil; ROS, Roscoff/France; SPSPA, São Pedro and São Paulo Archipelago; TMV, Trindade and Martin Vaz Oceanic Archipelago, Brazil; TX-US, Texas, United States; ZUEC OPH, Scientific Collection of Ophiuroidea in Museum of Zoology of the University of Campinas.

Museum voucher IC Species Locality GenBank (16S) GenBank (COI) Reference MZUSP 1425 14 Ophiothrix trindadensis TMV MH281576 MH281604 Neotype – present study MZUSP 1426 15 Ophiothrix trindadensis TMV MH281577 Present study MZUSP 1427 16 Ophiothrix trindadensis TMV MH281578 MH281605 Present study MZUSP 1428 17 Ophiothrix trindadensis TMV MH281579 MH281606 Present study MZUSP 1429 27 Ophiothrix trindadensis TMV MH281580 Present study MZUSP 1430 30 Ophiothrix trindadensis TMV MH281581 MH281607 Present study ZUEC OPH 2813 32 Ophiothrix trindadensis TMV MH281582 MH281608 Present study ZUEC OPH 2880 33 Ophiothrix trindadensis TMV MH281583 MH281609 Present study ZUEC OPH 2881 34 Ophiothrix trindadensis TMV MH281584 Present study ZUEC OPH 2882 35 Ophiothrix trindadensis TMV MH281585 MH281610 Present study ZUEC OPH 2883 97 Ophiothrix trindadensis TMV MH281586 Present study ZUEC OPH 2803 10 Ophiothrix cf. angulata ECP MH281587 MH281613 Present study ZUEC OPH 2149 100 Ophiothrix angulata AB MH281603 Present study ZUEC OPH 2802 99 Ophiothrix angulata AB MH281600 Present study ZUEC OPH 2811 95 Ophiothrix angulata AB MH281599 Present study ZUEC OPH 2785 91 Ophiothrix cf. angulata ECP MH281597 MH281620 Present study ZUEC OPH 2455 90 Ophiothrix cf. angulata AB MH281598 Present study ZUEC OPH 2796 89 Ophiothrix angulata AB MH281601 Present study MZUSP 1696 88 Ophiothrix angulata AB MH281602 Present study ZUEC OPH 2783 68 Ophiothrix cf. angulata ECP MH281596 MH281619 Present study ZUEC OPH 2808 65 Ophiothrix angulata ECP MH281595 MH281618 Present study 90

ZUEC OPH 2807 64 Ophiothrix angulata ECP MH281594 Present study MZUSP 1695 62 Ophiothrix angulata ECP MH281593 MH281617 Present study ZUEC OPH 2799 61 Ophiothrix cf. angulata ECP MH281592 MH281616 Present study ZUEC OPH 2798 60 Ophiothrix cf. angulata ECP MH281591 MH281615 Present study ZUEC OPH 2797 59 Ophiothrix cf. angulata ECP MH281590 MH281614 Present study ZUEC OPH 2792 58 Ophiothrix cf. angulata ECP MH281589 MH281612 Present study ZUEC OPH 2805 56 Ophiothrix angulata ECP MH281588 MH281611 Present study NMRJ Ophiothrix angulata SPSPA KM234228 1 NMRJ Ophiothrix angulata SPSPA KP128041 1 Absent Ophiothrix angulata TX-US KU672428 2 Absent Ophiothrix fragilis EUR JX947928 3 Absent Ophiothrix fragilis EUR JX947929 3 Absent Ophiothrix quinquemaculata EUR AJ002795 4 AM Macrophiothrix caenosa EUR AY365147 5 AM Macrophiothrix longipeda EUR AY365160 5 Absent Amphipholis squamata FMC KT780340 6 Absent Amphipholis squamata FMC KT780339 6 Absent Amphipholis squamata ROS NC013876 7

References

1. Barboza CAM, Mattos G, Paiva PC. Brittle stars from the Saint Peter and Saint Paul Archipelago: morphological and molecular data. Mar Biodivers Rec. 2015;8: 1–9. doi: 10.1017/S1755267214001511 2. Hunter RL, Brown LM, Alexander Hill C, Kroeger ZA, Rose SE. Additional insights into phylogenetic relationships of the Class Ophiuroidea (Echinodermata) from rRNA gene sequences. Journal of Zoological Systematics and Evolutionary Research. 2016;1–7. doi: 10.1111/jzs.12135 91

3. Pérez‐Portela R, Almada V, Turon X. Cryptic speciation and genetic structure of widely distributed brittle stars (Ophiuroidea) in Europe. Zool Scripta. 2013;42: 151–169. doi: 10.1111/j.1463-6409.2012.00573.x 4. Baric S, Sturmbauer C. Ecological Parallelism and Cryptic Species in the Genus Ophiothrix Derived from Mitochondrial DNA Sequences. Mol Phylogenet Evol. 1999;11: 157–162. doi: 10.1006/mpev.1998.0551 5. Hart MW, Podolsky RD. Mitochondrial DNA phylogeny and rates of larval evolution in Macrophiothrix brittlestars. Mol Phylogenet Evol. 2005;34: 438–447. doi: 10.1016/j.ympev.2004.09.011 6. Boissin E, Egea E, Féral J, Chenuil A. Contrasting population genetic structures in Amphipholis squamata, a complex of brooding, self- reproducing sister species sharing life history traits. Marine Ecology Progress Series. 2015;539: 165–177. doi: 10.3354/meps11480 7. Perseke M, Bernhard D, Fritzsch G, Brümmer F, Stadler PF, Schlegel M. Mitochondrial genome evolution in Ophiuroidea, Echinoidea, and Holothuroidea: insights in phylogenetic relationships of Echinodermata. Mol Phylogenet Evol. 2010;56: 201–211. doi: 10.1016/j.ympev.2010.01.035 92

S4 Table. Comparative table of the differences in dorsal and lateral arm plates between the CS.

Character CS1, CS2 CS3, CS4 Dorsal arm One and a half times as wide as As wide as long plates long

Distal region three to four times Distal and proximal regions at greater than the proximal one the same width

With a prominent keel at median Without a prominent keel at portion median portion Lateral arm Eight or more spine articulations At most seven spine plates articulations

S5 Table. Genetic distance (%) between and within the CS. Genetic distance between the CSs estimated from the mitochondrial gene fragments 16S (downward triangle) and COI (top triangle). In the gray diagonal line, average genetic distance (%) of both genes within the groups of sequences. AB, Araçá Bay, São Paulo, Brazil; CS, Candidate Species inferred from morphological characters; ECP, Estuarine Complex of Paranaguá, Paraná, Brazil; EUR, Europe; Nc, Value not calculated because of the inexistence of groups’ sequences for the considered gene; SPSPA, Saint Peter and Saint Paul Archipelago, Brazil; TMV, Trindade and Martin Vaz Oceanic Archipelago; TX-US, Texas, United States. # value of genetic distance within the group not calculated because only one sequence was considered in the analysis.

Taxa groups 1 2 3 4 5 6 7 8 9 1 Ophiothrix CS1 – TMV 1.6 3.5 17.0 16.8 Nc Nc Nc Nc 24.9 2 Ophiothrix CS2 – TMV 1.4 0.9 17.2 17.0 Nc Nc Nc Nc 25.1 3 Ophiothrix CS3 – ECP, AB 12.0 11.6 0.4 1.5 Nc Nc Nc Nc 24.7 4 Ophiothrix CS4 – ECP, AB 11.9 11.4 0.4 0.4 Nc Nc Nc Nc 24.8 5 Ophiothrix angulata SPSPA 1.2 0.6 11.3 11.2 0 Nc Nc Nc Nc 6 Ophiothrix angulata TX-US 12.1 11.7 0.8 0.9 11.4 # Nc Nc Nc 7 Ophiothrix spp. EUR 23.2 22.8 20.5 20.8 22.9 20.3 7.3 Nc Nc 8 Macrophiothrix spp. EUR 19.2 19.1 19.3 19.2 18.8 18.9 22.6 9.9 Nc 9 Amphipholis squamata 31.2 31.1 32.6 32.4 30.4 32.5 35.0 34.7 4.6

93

S6 Table. Analysis of Molecular Variance (AMOVA) for the population structuring scenario given by the groups I and II. Group I is composed of the specimens from Trindade and Martin Vaz Oceanic Archipelago, Saint Peter and Saint Paul Archipelago – which corresponds to Clade A inferred in the phylogenetic analysis. Group II is composed of the specimens from Estuarine Complex of Paranaguá, Araçá Bay, and Texas – which corresponds to Clade B inferred in the phylogenetic analysis. Source of variation d.f. Sum of Variance Percentage squares components of variation Among groups 1 268.700 17.72975 Va 93.49 Among populations within groups 3 3.474 -0.02370 Vb -0.12 Within populations 26 32.697 1.25758 Vc 6.63 Total 30 304.871 18.96363

94

S7 Table. Material used in this study. MZUSP = Museum of Zoology of University of São Paulo. ZUEC OPH = Museum of Zoology of the University of Campinas. * = juvenile specimens (less than 4 mm of dd). GenBank GenBank Nº Accession Accession Number of Observatio Bio Mol Species Study sites Date Station Latitude Longitude Coletor Substrate Depth numbers numbers specimens ns Sample (16S) (COI) MH281576 MH281604 14 Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 15.III.2013 Sample 3 20S 29'52.3" 29W 19'15.6" Projeto Protrindade 1 12m MH281577 15 Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 15.III.2013 Sample 3 20S 29'52.3" 29W 19'15.6" Projeto Protrindade 1 12m MH281578 MH281605 16 Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 15.III.2013 Sample 3 20S 29'52.3" 29W 19'15.6" Projeto Protrindade 1 12m MH281579 MH281606 17 Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 15.III.2013 Sample 3 20S 29'52.3" 29W 19'15.6" Projeto Protrindade 1 12m MH281580 27 Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 15.III.2013 Sample 3 20S 29'52.3" 29W 19'15.6" Projeto Protrindade 1 12m MH281581 MH281607 30 Ophiothrix trindadensis * Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 15.III.2013 Sample 3 20S 29'52.3" 29W 19'15.6" Projeto Protrindade 1 12m MH281582 MH281608 32 Ophiothrix trindadensis * Ilha da Trindade, Laje da Ponta Noroeste, ES, Brasil 16.VII.2012 Sample 2A 20S 29'57.8" 29W 20'39.2" Projeto Protrindade 1 15.3m MH281583 MH281609 33 Ophiothrix trindadensis Ilha da Trindade, Laje da Ponta Noroeste, ES, Brasil 16.VII.2012 Sample 2A 20S 29'57.8" 29W 20'39.2" Projeto Protrindade 1 15.3m MH281584 34 Ophiothrix trindadensis Ilha da Trindade, Laje da Ponta Noroeste, ES, Brasil 16.VII.2012 Sample 2A 20S 29'57.8" 29W 20'39.2" Projeto Protrindade 1 15.3m MH281585 MH281610 35 Ophiothrix trindadensis Ilha da Trindade, Laje da Ponta Noroeste, ES, Brasil 16.VII.2012 Sample 2A 20S 29'57.8" 29W 20'39.2" Projeto Protrindade 1 15.3m Sample MH281586 97 Ophiothrix trindadensis Ilha da Trindade, Ponta da Calheta, ES, Brasil 26.X.2014 20S 30'18.72" 29W 18'31.67" J.B.Mendonça - Projeto Protrindade 1 9.9m 79A MH281587 MH281613 10 Ophiothrix cf. angulata Paranaguá, Ilha da Banana, PR, Brasil 20.VI.2014 Sample 171 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 MH281588 MH281611 56 Ophiothrix angulata Paranaguá, Ilha da Banana, PR, Brasil 20.VI.2014 Sample 171 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 MH281589 MH281612 58 Ophiothrix cf. angulata * Paranaguá, Ilha da Banana, PR, Brasil 20.VI.2014 Sample 171 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 MH281590 MH281614 59 Ophiothrix cf. angulata Paranaguá, Baixio do Perigo, PR, Brasil 20.VII.2014 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 MH281591 MH281615 60 Ophiothrix cf. angulata * Paranaguá, Baixio do Perigo, PR, Brasil 20.VII.2014 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 MH281592 MH281616 61 Ophiothrix cf. angulata * Paranaguá, Baixio do Perigo, PR, Brasil 20.VII.2014 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 MH281593 MH281617 62 Ophiothrix angulata * Paranaguá, Baixio do Perigo, PR, Brasil 20.VII.2014 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 MH281594 64 Ophiothrix angulata * Paranaguá, Baixio do Perigo, PR, Brasil 20.VII.2014 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 MH281595 MH281618 65 Ophiothrix angulata * Paranaguá, Baixio do Perigo, PR, Brasil 20.VII.2014 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 MH281596 MH281619 68 Ophiothrix cf. angulata Paranaguá, Baixio do Perigo, PR, Brasil 20.VII.2014 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 MH281597 MH281620 91 Ophiothrix cf. angulata Paranaguá, Baixio do Perigo, PR, Brasil 20.VII.2014 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 Sponge MH281598 90 Ophiothrix cf. angulata * Baía do Araçá, São Sebastião, SP, Brasil 27.VI.2014 OFI 021 23S 48'60'' 45W 24'24'' Gustavo Dias 1 Sponge MH281599 95 Ophiothrix angulata Baía do Araçá, São Sebastião, SP, Brasil 21.II.2013 Est 11 23S 49'20'' 45W 24'10'' Maristela Bueno 1 21.6m MH281600 99 Ophiothrix angulata Baía do Araçá, São Sebastião, SP, Brasil 01.X.2012 OFI 004 23S 45'36" 45W 24'35.64" Gustavo Dias 1 Hard bottom MH281601 89 Ophiothrix angulata * Baía do Araçá, São Sebastião, SP, Brasil 27.VI.2014 OFI 021 23S 48'60'' 45W 24'24'' Gustavo Dias 1 Sponge MH281602 88 Ophiothrix angulata * Baía do Araçá, São Sebastião, SP, Brasil 27.VI.2014 OFI 021 23S 48'60'' 45W 24'24'' Gustavo Dias 1 Sponge MH281603 100 Ophiothrix angulata Baía do Araçá, São Sebastião, SP, Brasil 01.X.2012 OFI 004 23S 45'36" 45W 24'35.64" Gustavo Dias 1 Hard bottom Ophiothrix angulata Paranaguá, Baixio do Perigo, PR, Brasil 20.VII.2014 25S 31’12'' 48W 30’33.12" Maristela Bueno 30 Sponge 57 Ophiothrix angulata Paranaguá, Ilha da Banana, PR, Brasil 20.VI.2014 Sample 171 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 Ophiothrix angulata Paranaguá, Baixio do Perigo, PR, Brasil 20.VII.2014 25S 31’12'' 48W 30’33.12" Maristela Bueno 112 Sponge 55 Ophiothrix angulata Paranaguá, Ilha da Banana, PR, Brasil 20.VI.2014 Sample 171 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 92 Ophiothrix angulata Paranaguá, Baixio do Perigo, PR, Brasil 20.VII.2014 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 Sponge 93 Ophiothrix angulata Paranaguá, Baixio do Perigo, PR, Brasil 20.VII.2014 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 Sponge

95

S7 Table. Continued.

GenBank GenBank Nº Accession Accession Number of Observatio Bio Mol Species Study sites Date Station Latitude Longitude Coletor Substrate Depth numbers numbers specimens ns Sample (16S) (COI) 94 Ophiothrix angulata Paranaguá, Baixio do Perigo, PR, Brasil 20.VII.2014 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 Sponge 53 Ophiothrix angulata Paranaguá, Ilha da Banana, PR, Brasil 20.VI.2014 Sample 171 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 54 Ophiothrix angulata Paranaguá, Ilha da Banana, PR, Brasil 20.VI.2014 Sample 171 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 66 Ophiothrix angulata Paranaguá, Baixio do Perigo, PR, Brasil 20.VII.2014 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 67 Ophiothrix angulata Paranaguá, Baixio do Perigo, PR, Brasil 20.VII.2014 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 52 Ophiothrix angulata Paranaguá, Ilha da Banana, PR, Brasil 20.VI.2014 Sample 171 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 Ophiothrix angulata Paranaguá, Baixio do Perigo, PR, Brasil 20.VII.2014 25S 31’12'' 48W 30’33.12" Maristela Bueno 15 Sponge 87 Ophiothrix angulata * Baía do Araçá, São Sebastião, SP, Brasil 22.II.2013 23S 48'53.9'' 45W 24'24.4'' Felipe Oricchio 1 Hard bottom 63 Ophiothrix angulata * Paranaguá, Baixio do Perigo, PR, Brasil 20.VII.2014 25S 31’12'' 48W 30’33.12" Maristela Bueno 1 98 Ophiothrix angulata * Baía do Araçá, São Sebastião, SP, Brasil 01.X.2012 OFI 004 23S 45'36" 45W 24'35.64" Gustavo Dias 1 Hard bottom 86 Ophiothrix angulata * Baía do Araçá, São Sebastião, SP, Brasil 21.II.2013 Est 11 23S 49'20'' 45W 24'10'' Maristela Bueno 1 Hard bottom 21.6m 96 Ophiothrix angulata * Baía do Araçá, São Sebastião, SP, Brasil 21.II.2013 Est 11 23S 49'20'' 45W 24'10'' Maristela Bueno 1 Hard bottom 21.6m Ophiothrix cf. angulata Paranaguá, Baixio do Perigo, PR, Brasil 20.VII.2014 25S 31’12'' 48W 30’33.12" Maristela Bueno 30 Sponge Ophiothrix cf. angulata Paranaguá, Baixio do Perigo, PR, Brasil 20.VII.2014 25S 31’12'' 48W 30’33.12" Maristela Bueno 72 Sponge Water Sample Ophiothrix trindadensis Arquipélago Martin Vaz, Ilhote principal Martin Vaz, ES, Brasil 22.I.2012 20S 28'26.98'' 28W 51'20.98'' C. H. Guimarães - Projeto Protrindade 2 Corals temperatur 19.2m 44A e: 25ºC Ophiothrix trindadensis Arquipélago Martin Vaz, Ilha Martin Vaz, ES, Brasil 24.VII.2013 Sample 32B 20S 30'45.7'' 29W 18'21.9'' J.B.Mendonça - Projeto Protrindade 3 12.3m

Ophiothrix trindadensis Arquipélago Martin Vaz, Ilha Martin Vaz, ES, Brasil 23.VII.2013 Sample 42C 20S 30'45.7'' 29W 18'21.9'' J.B.Mendonça - Projeto Protrindade 2 13m

Ophiothrix trindadensis Arquipélago Martin Vaz, Ilha Martin Vaz, ES, Brasil 23.VII.2013 Sample 64B 20S 30'45.7'' 29W 18'21.9'' J.B.Mendonça - Projeto Protrindade 6 13m 18 Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 15.III.2013 Sample 3 20S 29'52.3" 29W 19'15.6" Projeto Protrindade 1 12m 19 Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 15.III.2013 Sample 3 20S 29'52.3" 29W 19'15.6" Projeto Protrindade 1 12m 20 Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 15.III.2013 Sample 3 20S 29'52.3" 29W 19'15.6" Projeto Protrindade 1 12m 21 Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 15.III.2013 Sample 3 20S 29'52.3" 29W 19'15.6" Projeto Protrindade 1 12m 22 Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 15.III.2013 Sample 3 20S 29'52.3" 29W 19'15.6" Projeto Protrindade 1 12m 23 Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 15.III.2013 Sample 3 20S 29'52.3" 29W 19'15.6" Projeto Protrindade 1 12m 24 Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 15.III.2013 Sample 3 20S 29'52.3" 29W 19'15.6" Projeto Protrindade 1 12m 25 Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 15.III.2013 Sample 3 20S 29'52.3" 29W 19'15.6" Projeto Protrindade 1 12m 36 Ophiothrix trindadensis Ilha da Trindade, Laje da Ponta Noroeste, ES, Brasil 16.VII.2012 Sample 2A 20S 29'57.8" 29W 20'39.2" Projeto Protrindade 1 15.3m Sample Ophiothrix trindadensis Ilha da Trindade, SECON/ECIT (Enseada dos Portugueses), ES,14.V.2014 Brasil 20S 30'20.9'' 29W 18'43.7'' J.B.Mendonça - Projeto Protrindade 2 10m 91A

96

S7 Table. Continued.

GenBank GenBank Nº Accession Accession Number of Observatio Bio Mol Species Study sites Date Station Latitude Longitude Coletor Substrate Depth numbers numbers specimens ns Sample (16S) (COI) Sample Ophiothrix trindadensis Ilha da Trindade, Rampa Nova (Praia dos Portugueses), ES, Brasil23.IV.2014 20S 30'17.7" 29W 18'56.7" J.B.Mendonça - Projeto Protrindade 7 Hard bottom 10.7m 81A Sample Ophiothrix trindadensis Ilha da Trindade, Rampa Nova (Praia dos Portugueses), ES, Brasil23.IV.2014 20S 30'17.7" 29W 18'56.7" J.B.Mendonça - Projeto Protrindade 1 Hard bottom 10.7m 85A Sample Ophiothrix trindadensis Ilha da Trindade, Rampa Nova (Praia dos Portugueses), ES, Brasil23.IV.2014 20S 30'17.7" 29W 18'56.7" J.B.Mendonça - Projeto Protrindade 1 Hard bottom 10.7m 86A Sample Ophiothrix trindadensis Ilha da Trindade, Rampa Nova (Praia dos Portugueses), ES, Brasil23.IV.2014 20S 30'17.7" 29W 18'56.7" J.B.Mendonça - Projeto Protrindade 1 Hard bottom 10.7m 88A Sample Ophiothrix trindadensis Ilha da Trindade, Rampa Nova (Praia dos Portugueses), ES, Brasil23.IV.2014 20S 30'17.7" 29W 18'56.7" J.B.Mendonça - Projeto Protrindade 1 Hard bottom 10.7m 90A Sample Ophiothrix trindadensis Ilha da Trindade, Rampa Nova (Praia dos Portugueses), ES, Brasil23.IV.2014 20S 30'17.7" 29W 18'56.7" J.B.Mendonça - Projeto Protrindade 1 Hard bottom 10.7m 92A Sample Ophiothrix trindadensis Ilha da Trindade, Rampa Nova (Praia dos Portugueses), ES, Brasil23.IV.2014 20S 30'17.7" 29W 18'56.7" J.B.Mendonça - Projeto Protrindade 1 Hard bottom 10.7m 93A Sample Ophiothrix trindadensis Ilha da Trindade, Rampa Nova (Praia dos Portugueses), ES, Brasil18.IV.2014 20S 30'17.7" 29W 18'56.7" J.B.Mendonça - Projeto Protrindade 1 10.2m 107A Sample Ophiothrix trindadensis Ilha da Trindade, Rampa Nova (Praia dos Portugueses), ES, Brasil23.IV.2014 20S 30'17.7" 29W 18'56.7" J.B.Mendonça - Projeto Protrindade 1 Hard bottom 10.7m 116A Sample Ophiothrix trindadensis Ilha da Trindade, Rampa Nova (Praia dos Portugueses), ES, Brasil23.IV.2014 20S 30'17.7" 29W 18'56.7" J.B.Mendonça - Projeto Protrindade 8 10.7m 118A Sample Ophiothrix trindadensis Ilha da Trindade, Rampa Nova (Praia dos Portugueses), ES, Brasil23.IV.2014 20S 30'17.7" 29W 18'56.7" J.B.Mendonça - Projeto Protrindade 5 10.7m 122A Sample Ophiothrix trindadensis Ilha da Trindade, Rampa Nova (Praia dos Portugueses), ES, Brasil23.IV.2014 20S 30'17.7" 29W 18'56.7" J.B.Mendonça - Projeto Protrindade 1 Hard bottom 10.7m 99A Ophiothrix trindadensis Ilha da Trindade, Praia/Ilha da Rocha, ES, Brasil 16.VII.2013 Sample 62B 20S 30'26.5'' 29W 20'48.0'' J.B.Mendonça - Projeto Protrindade 1 20.8m Sample Ophiothrix trindadensis Ilha da Trindade, Praia/Ilha da Rocha, ES, Brasil 01.VII.2016 20S 30'26.5'' 29W 20'48.0'' J.B.Mendonça - Projeto Protrindade 4 21.4m 124B Ophiothrix trindadensis Ilha da Trindade, Praia do Andrada, ES, Brasil 17.VII.2013 Sample 40B 20S 30'71.8'' 29W 18'24.7'' J.B.Mendonça - Projeto Protrindade 2 9.9m

Ophiothrix trindadensis Ilha da Trindade, Praia das Tartarugas/Parcel, ES, Brasil 11.VII.2012 Sample 50B 20S 31'01.3'' 29W 17'56.9'' J.B.Mendonça - Projeto Protrindade 2 12.3m Sample Ophiothrix trindadensis Ilha da Trindade, Praia das Tartarugas/Parcel, ES, Brasil 10.VII.2015 20S 31'01.3'' 29W 17'56.9'' J.B.Mendonça - Projeto Protrindade 4 13.9m 80A

97

S7 Table. Continued.

GenBank GenBank Nº Accession Accession Number of Observatio Bio Mol Species Study sites Date Station Latitude Longitude Coletor Substrate Depth numbers numbers specimens ns Sample (16S) (COI) Sample Ophiothrix trindadensis Ilha da Trindade, Praia das Tartarugas/Parcel, ES, Brasil 30.IV.2014 20S 31'01.3'' 29W 17'56.9'' J.B.Mendonça - Projeto Protrindade 5 9.8m 98A Sample Ophiothrix trindadensis Ilha da Trindade, Praia das Cabritas, ES, Brasil 28.IV.2014 20S 29'32.0" 29W 19'46.5" J.B.Mendonça - Projeto Protrindade 6 9.2m 95A Sample Ophiothrix trindadensis Ilha da Trindade, Praia das Cabritas, ES, Brasil 28.IV.2014 20S 29'32.0" 29W 19'46.5" J.B.Mendonça - Projeto Protrindade 7 9.2m 97A Sample Ophiothrix trindadensis Ilha da Trindade, Praia da Calheta, ES, Brasil 21.VI.2016 20S 30'29.5" 29W 18'37.0" J.B.Mendonça - Projeto Protrindade 26 12.5m 110A Sample Ophiothrix trindadensis Ilha da Trindade, Praia da Calheta (SECON), ES, Brasil 18.VI.2012 20S 30'20.9'' 29W 18'43.7'' J.B.Mendonça - Projeto Protrindade 6 12m 34C, D Ophiothrix trindadensis Ilha da Trindade, Praia da Calheta (SECON), ES, Brasil 03.VII.2012 Sample 48C 20S 30'20.9'' 29W 18'43.7'' J.B.Mendonça - Projeto Protrindade 6 12.3m

Ophiothrix trindadensis Ilha da Trindade, Praia da Calheta (SECON), ES, Brasil 18.VI.2012 Sample 60B 20S 30'20.9'' 29W 18'43.7'' J.B.Mendonça - Projeto Protrindade 10 12m

Ophiothrix trindadensis Ilha da Trindade, Praia da Calheta (SECON), ES, Brasil 20.VI.2016 Sample 64B 20S 30'20.9'' 29W 18'43.7'' J.B.Mendonça - Projeto Protrindade 13 7m Sample Ophiothrix trindadensis Ilha da Trindade, Praia da Calheta (SECON), ES, Brasil 11.XI. 2014 20S 30'20.9'' 29W 18'43.7'' J.B.Mendonça - Projeto Protrindade 6 11m 74A Ophiothrix trindadensis Ilha da Trindade, Ponta Norte, ES, Brasil 03.VII.2012 Sample 1A 20S 29'18.7'' 29W 20'18.3'' J.B.Mendonça - Projeto Protrindade 6 15.6m Sample Ophiothrix trindadensis Ilha da Trindade, Ponta Norte, ES, Brasil 02.VII.2015 20S 29'18.7" 29W 20'18.3" J.B.Mendonça - Projeto Protrindade 5 14.2m 114A Sample Ophiothrix trindadensis Ilha da Trindade, Ponta Norte, ES, Brasil 23.X.2014 20S 29'40.2'' 29W 20'32.9'' J.B.Mendonça - Projeto Protrindade 4 14m 87A Sample Ophiothrix trindadensis Ilha da Trindade, Ponta Norte, ES, Brasil 01.IV.2014 20S 29'40.2'' 29W 20'32.9'' J.B.Mendonça - Projeto Protrindade 11 11.5m 111C Ophiothrix trindadensis Ilha da Trindade, Ponta Noroeste, ES, Brasil 04.VII.2012 Sample 15B 20S 29'46.4'' 29W 20'35.4'' J.B.Mendonça - Projeto Protrindade 1 11.6m Sample Ophiothrix trindadensis Ilha da Trindade, Ponta da Calheta, ES, Brasil 28.IV.2014 20S 30'18.72'' 29W 18'31.67" J.B.Mendonça - Projeto Protrindade 3 16.7m 68A Sample Ophiothrix trindadensis Ilha da Trindade, Ponta da Calheta, ES, Brasil 12.V.2014 20S 30'18.72" 29W 18'31.67" J.B.Mendonça - Projeto Protrindade 1 15.9m 67A Sample Ophiothrix trindadensis Ilha da Trindade, Ponta da Calheta, ES, Brasil 30.IV.2014 20S 30'18.72" 29W 18'31.67" J.B.Mendonça - Projeto Protrindade 18 15.6m 70A Sample Ophiothrix trindadensis Ilha da Trindade, Ponta da Calheta, ES, Brasil 29.IV. 2014 20S 30'18.72" 29W 18'31.67" J.B.Mendonça - Projeto Protrindade 2 15.9m 72A

98

S7 Table. Continued.

GenBank GenBank Nº Accession Accession Number of Observatio Bio Mol Species Study sites Date Station Latitude Longitude Coletor Substrate Depth numbers numbers specimens ns Sample (16S) (COI) Sample Ophiothrix trindadensis Ilha da Trindade, Ponta da Calheta, ES, Brasil 26.X.2014 20S 30'18.72" 29W 18'31.67" J.B.Mendonça - Projeto Protrindade 1 9.9m 84A Sample Ophiothrix trindadensis Ilha da Trindade, Ponta da Calheta, ES, Brasil 25.VI.2015 20S 30'18.72" 29W 18'31.67" J.B.Mendonça - Projeto Protrindade 1 16.9m 89A Sample Ophiothrix trindadensis Ilha da Trindade, Ponta da Calheta, ES, Brasil 26.X.2014 20S 30'18.72" 29W 18'31.67" J.B.Mendonça - Projeto Protrindade 18 9.9m 109A Sample Ophiothrix trindadensis Ilha da Trindade, Ponta da Calheta, ES, Brasil 03.VIII.2015 20S 30'18.72" 29W 18'37.67" J.B.Mendonça - Projeto Protrindade 8 17.7m 112A Sample Ophiothrix trindadensis Ilha da Trindade, Ponta da Calheta, ES, Brasil 20.V.2014 20S 30'18.72" 29W 18'37.67" J.B.Mendonça - Projeto Protrindade 1 16.3m 115A Sample Ophiothrix trindadensis Ilha da Trindade, Ponta da Calheta, ES, Brasil 03.VIII.2015 20S 30'18.72" 29W 18'37.67" J.B.Mendonça - Projeto Protrindade 4 17.7m 128A Sample Ophiothrix trindadensis Ilha da Trindade, Ponta da Calheta, ES, Brasil 08.V.2014 20S 30'18.72" 29W 18'37.67" J.B.Mendonça - Projeto Protrindade 2 16.6m 129A Sample Ophiothrix trindadensis Ilha da Trindade, Ponta Crista do Galo, ES, Brasil 21.V.2014 20S 29'14.84" 29W 20'13.94" J.B.Mendonça - Projeto Protrindade 13 15m 71A Ophiothrix trindadensis Ilha da Trindade, Paredão, ES, Brasil 19.VI.2012 Sample 36B 20S 31'36.9'' 29W 18'14.3'' J.B.Mendonça - Projeto Protrindade 7 19m Sample Ophiothrix trindadensis Ilha da Trindade, Paredão, ES, Brasil 20.VI.2012 20S 31'36.9'' 29W 18'14.3'' J.B.Mendonça - Projeto Protrindade 2 20m 52A Ophiothrix trindadensis Ilha da Trindade, Laje na ponta do monumento, ES, Brasil 30.VI.2013 Sample 7A 20S 30'10.3'' 29W 20'36.1'' J.B.Mendonça - Projeto Protrindade 1 19.5m Sample Ophiothrix trindadensis Ilha da Trindade, Laje da Ponta Noroeste, ES, Brasil 09.VII.2012 20S 29'57.8" 29W 20'39.2" J.B.Mendonça - Projeto Protrindade 7 17.7m 33C, D Sample Ophiothrix trindadensis Ilha da Trindade, Ilha Sul, ES, Brasil 06.XI.2014 20S 31'34.32" 29W 19'27.98" J.B.Mendonça - Projeto Protrindade 7 17.6m 69A Sample Ophiothrix trindadensis Ilha da Trindade, Ilha Sul, ES, Brasil 21.X.2014 20S 31'34.32" 29W 19'27.98" J.B.Mendonça - Projeto Protrindade 11 17.8m 83A Sample Ophiothrix trindadensis Ilha da Trindade, Ilha Sul, ES, Brasil 06.XI.2014 20S 31'34.32" 29W 19'27.98" J.B.Mendonça - Projeto Protrindade 4 17.6m 94A Sample Ophiothrix trindadensis Ilha da Trindade, Ilha da Rocha, ES, Brasil 16.VII.2013 20S 30'26.5'' 29W 20'48.0'' J.B.Mendonça - Projeto Protrindade 2 20.8m 53A Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 15.VII.2013 Sample 9A 20S 29'52.3" 29W 19'15.6" J.B.Mendonça - Projeto Protrindade 2 12m Sample Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 10.VII.2012 20S 29'52.3" 29W 19'15.6" J.B.Mendonça - Projeto Protrindade 8 14.6m 11A

99

S7 Table. Continued.

GenBank GenBank Nº Accession Accession Number of Observatio Bio Mol Species Study sites Date Station Latitude Longitude Coletor Substrate Depth numbers numbers specimens ns Sample (16S) (COI) Sample Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 15.VII.2013 20S 29'52.3" 29W 19'15.6" J.B.Mendonça - Projeto Protrindade 1 12m 54A Sample Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 22.IV.2014 20S 29'52.3" 29W 19'15.6" J.B.Mendonça - Projeto Protrindade 28 13.7m 82A Sample Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 24.VI.2016 20S 29'52.3" 29W 19'15.6" J.B.Mendonça - Projeto Protrindade 2 11.3m 96A Sample Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 22.IV.2014 20S 29'52.3" 29W 19'15.6" J.B.Mendonça - Projeto Protrindade 40 13.7m 100A Sample Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 17.IV.2014 20S 29'52.3" 29W 19'15.6" J.B.Mendonça - Projeto Protrindade 1 13.3m 113B Sample Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 17.IV.2014 20S 29'52.3" 29W 19'15.6" J.B.Mendonça - Projeto Protrindade 2 12.9m 117A Sample Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 17.IV.2014 20S 29'52.3" 29W 19'15.6" J.B.Mendonça - Projeto Protrindade 7 13.3m 120A Sample Ophiothrix trindadensis Ilha da Trindade, Estação Orelhas, ES, Brasil 01.XI.2014 20S 29'40.2'' 29W 20'32.9'' J.B.Mendonça - Projeto Protrindade 2 12m 106A Ophiothrix trindadensis Ilha da Trindade, Enseada dos Portugueses (SECON), ES, Brasil18.VII.2013 Sample 5F 20S 30'20.9'' 29W 18'43.7'' J.B.Mendonça - Projeto Protrindade 5 12.2m Ophiothrix trindadensis Ilha da Trindade, Enseada do Príncipe (Pedra da Garoupa), ES,16.VII.2013 Brasil Sample 37C 20S 31'35.5'' 29W 18'94.0'' J.B.Mendonça - Projeto Protrindade 3 10.4m Sample Ophiothrix trindadensis Ilha da Trindade, Enseada do Príncipe (Paredão), ES, Brasil 09.VII.2013 20S 31'22.4'' 29W 19'52.0'' J.B.Mendonça - Projeto Protrindade 7 19.5m 57A, B, C Sample 8A, Ophiothrix trindadensis Ilha da Trindade, Enseada do lixo, ES, Brasil 15.VII.2013 20S 31'29.8'' 29W 19'43.9'' J.B.Mendonça - Projeto Protrindade 4 17m B, D Sample Ophiothrix trindadensis Ilha da Trindade, Enseada do lixo, ES, Brasil 02.VII.2012 20S 31'29.8'' 29W 19'43.9'' J.B.Mendonça - Projeto Protrindade 4 25m 43A, B Sample Ophiothrix trindadensis Ilha da Trindade, Enseada do lixo, ES, Brasil 15.VII.2013 20S 31'29.8'' 29W 19'43.9'' J.B.Mendonça - Projeto Protrindade 7 17m 58A Sample Ophiothrix trindadensis Ilha da Trindade, Enseada do lixo, ES, Brasil 19.VI.2016 20S 31'29.8'' 29W 19'43.9'' J.B.Mendonça - Projeto Protrindade 11 14.5m 105A Water Sample Ophiothrix trindadensis Ilha da Trindade, Enseada do Lixo, ES, Brasil 23.II.2012 20S 31'43.5'' 29W 19'28.1'' C. H. Guimarães - Projeto Protrindade 2 Hard bottom temperatur 20m 18A e: 26ºC Water Sample Ophiothrix trindadensis Ilha da Trindade, Enseada do Lixo, ES, Brasil 17.II.2012 20S 31'43.5'' 29W 19'28.1'' C. H. Guimarães - Projeto Protrindade 1 Hard bottom temperatur 23m 63A e: 26ºC

100

S7 Table. Continued.

GenBank GenBank Nº Accession Accession Number of Observatio Bio Mol Species Study sites Date Station Latitude Longitude Coletor Substrate Depth numbers numbers specimens ns Sample (16S) (COI) Ophiothrix trindadensis Ilha da Trindade, Enseada das Orelhas, ES, Brasil 06.VII.2013 Sample 13B 20S 29'40.2'' 29W 20'32.9'' J.B.Mendonça - Projeto Protrindade 2 14m

Ophiothrix trindadensis Ilha da Trindade, Enseada das Orelhas, ES, Brasil 30.VI.2012 Sample 41C 20S 29'40.2'' 29W 20'32.9'' J.B.Mendonça - Projeto Protrindade 13 13.9m Sample Ophiothrix trindadensis Ilha da Trindade, Enseada das Orelhas, ES, Brasil 24.X.2014 20S 29'40.2'' 29W 20'32.9'' J.B.Mendonça - Projeto Protrindade 2 15.4m 76A Sample Ophiothrix trindadensis Ilha da Trindade, Enseada da Cachoeira, ES, Brasil 12.VII.2012 20S 30'55.6'' 29W 20'21.7'' J.B.Mendonça - Projeto Protrindade 6 12m 10A, B, D, Sample 4A, Ophiothrix trindadensis Ilha da Trindade, Enseada da Cachoeira, ES, Brasil 09.VII.2012 20S 30'57.1'' 29W 20'15.2'' J.B.Mendonça - Projeto Protrindade 3 13.8m B, F Sample Ophiothrix trindadensis Ilha da Trindade, Enseada da Cachoeira (Praia do M), ES, Brasil08.VII.2013 20S 30'53.8'' 29W 20'19.2'' J.B.Mendonça - Projeto Protrindade 12 15m 39D Ophiothrix trindadensis Ilha da Trindade, Enseada da Cachoeira (Farrilhões), ES, Brasil08.VII.2013 Sample 19B 20S 31'22.4'' 29W 19'52.0'' J.B.Mendonça - Projeto Protrindade 1 9.5m

Ophiothrix trindadensis Ilha da Trindade, Enseada da Cachoeira (Farrilhões), ES, Brasil20.VI.2012 Sample 45B 20S 31'22.4'' 29W 19'52.0'' J.B.Mendonça - Projeto Protrindade 5 11.8m Sample Ophiothrix trindadensis Ilha da Trindade, Enseada da Cachoeira (Farrilhões), ES, Brasil16.VI.2012 20S 31'22.4'' 29W 19'52.0'' J.B.Mendonça - Projeto Protrindade 2 11.9m 49B, D Ophiothrix trindadensis Ilha da Trindade, Enseada da Cachoeira (Farrilhões), ES, Brasil16.VI.2012 Sample 55B 20S 31'22.4'' 29W 19'52.0'' J.B.Mendonça - Projeto Protrindade 2 11.9m

Ophiothrix trindadensis Ilha da Trindade, Enseada da Cachoeira (Farrilhões), ES, Brasil16.VI.2012 Sample 59B 20S 31'22.4'' 29W 19'52.0'' J.B.Mendonça - Projeto Protrindade 1 11.9m Sample Ophiothrix trindadensis Ilha da Trindade, Enseada da Cachoeira (Farrilhões), ES, Brasil05.V.2014 20S 31'22.4'' 29W 19'52.0'' J.B.Mendonça - Projeto Protrindade 14 14.4m 119A Sample Ophiothrix trindadensis Ilha da Trindade, SECON/ECIT (Enseada dos Portugueses), ES,04.VII.2016 Brasil 20S 30'20.9'' 29W 18'43.7'' J.B.Mendonça - Projeto Protrindade 1 11.4m 75A Sample Ophiothrix trindadensis Ilha da Trindade, Rampa Nova (Praia dos Portugueses), ES, Brasil23.IV.2014 20S 30'17.7" 29W 18'56.7" J.B.Mendonça - Projeto Protrindade 1 Hard bottom 10.7m 73A Sample Ophiothrix trindadensis Ilha da Trindade, Rampa Nova (Praia dos Portugueses), ES, Brasil23.IV.2014 20S 30'17.7" 29W 18'56.7" J.B.Mendonça - Projeto Protrindade 2 10.7m 118A Sample Ophiothrix trindadensis Ilha da Trindade, Praia/Ilha da Rocha, ES, Brasil 01.VII.2016 20S 30'26.5'' 29W 20'48.0'' J.B.Mendonça - Projeto Protrindade 1 21.4m 124B Ophiothrix trindadensis Ilha da Trindade, Praia das Tartarugas/Parcel, ES, Brasil 12.VII.2013 Sample 12C 20S 31'01.3'' 29W 17'56.9'' J.B.Mendonça - Projeto Protrindade 1 10.8m

101

S7 Table. Continued.

GenBank GenBank Nº Accession Accession Number of Observatio Bio Mol Species Study sites Date Station Latitude Longitude Coletor Substrate Depth numbers numbers specimens ns Sample (16S) (COI) Ophiothrix trindadensis Ilha da Trindade, Praia das Tartarugas/Parcel, ES, Brasil 26.VI.2012 Sample 56C 20S 31'01.3'' 29W 17'56.9'' J.B.Mendonça - Projeto Protrindade 1 9.5m Sample Ophiothrix trindadensis Ilha da Trindade, Praia das Tartarugas/Parcel, ES, Brasil 10.VII.2015 20S 31'01.3'' 29W 17'56.9'' J.B.Mendonça - Projeto Protrindade 1 13.9m 80A Sample Ophiothrix trindadensis Ilha da Trindade, Praia das Tartarugas/Parcel, ES, Brasil 30.IV.2014 20S 31'01.3'' 29W 17'56.9'' J.B.Mendonça - Projeto Protrindade 1 9.8m 98A Sample Ophiothrix trindadensis Ilha da Trindade, Praia das Cabritas, ES, Brasil 28.IV.2014 20S 29'32.0" 29W 19'46.5" J.B.Mendonça - Projeto Protrindade 1 9.2m 95A Sample Ophiothrix trindadensis Ilha da Trindade, Praia da Calheta, ES, Brasil 21.VI.2016 20S 30'29.5" 29W 18'37.0" J.B.Mendonça - Projeto Protrindade 3 12.5m 110A Ophiothrix trindadensis Ilha da Trindade, Praia da Calheta (SECON), ES, Brasil 18.VI.2012 Sample 60B 20S 30'20.9'' 29W 18'43.7'' J.B.Mendonça - Projeto Protrindade 2 12m Sample Ophiothrix trindadensis Ilha da Trindade, Praia da Calheta (SECON), ES, Brasil 20.VI.2016 20S 30'20.9'' 29W 18'43.7'' J.B.Mendonça - Projeto Protrindade 2 7m 66A Ophiothrix trindadensis Ilha da Trindade, Ponta Norte, ES, Brasil 03.VII.2012 Sample 1A 20S 29'18.7'' 29W 20'18.3'' J.B.Mendonça - Projeto Protrindade 1 15.6m Sample Ophiothrix trindadensis Ilha da Trindade, Ponta Norte, ES, Brasil 01.IV.2014 20S 29'40.2'' 29W 20'32.9'' J.B.Mendonça - Projeto Protrindade 1 11.5m 111C Sample Ophiothrix trindadensis Ilha da Trindade, Ponta da Calheta, ES, Brasil 30.IV.2014 20S 30'18.72" 29W 18'31.67" J.B.Mendonça - Projeto Protrindade 1 15.6m 70A Sample Ophiothrix trindadensis Ilha da Trindade, Ponta da Calheta, ES, Brasil 26.X.2014 20S 30'18.72" 29W 18'31.67" J.B.Mendonça - Projeto Protrindade 1 9.9m 109A Sample Ophiothrix trindadensis Ilha da Trindade, Ponta da Calheta, ES, Brasil 03.VIII.2015 20S 30'18.72" 29W 18'37.67" J.B.Mendonça - Projeto Protrindade 1 17.7m 112A Sample Ophiothrix trindadensis Ilha da Trindade, Ponta Crista do Galo, ES, Brasil 21.V.2014 20S 29'14.84" 29W 20'13.94" J.B.Mendonça - Projeto Protrindade 1 15m 71A Sample Ophiothrix trindadensis Ilha da Trindade, Ilha Sul, ES, Brasil 06.XI.2014 20S 31'34.32" 29W 19'27.98" J.B.Mendonça - Projeto Protrindade 1 17.6m 69A Sample Ophiothrix trindadensis Ilha da Trindade, Ilha Sul, ES, Brasil 21.X.2014 20S 31'34.32" 29W 19'27.98" J.B.Mendonça - Projeto Protrindade 1 17.8m 83A Sample Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 10.VII.2012 20S 29'52.3" 29W 19'15.6" J.B.Mendonça - Projeto Protrindade 1 14.6m 11A Sample Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 22.IV.2014 20S 29'52.3" 29W 19'15.6" J.B.Mendonça - Projeto Protrindade 2 13.7m 82A Sample Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 22.IV.2014 20S 29'52.3" 29W 19'15.6" J.B.Mendonça - Projeto Protrindade 2 13.7m 100A

102

S7 Table. Continued.

GenBank GenBank Nº Accession Accession Number of Observatio Bio Mol Species Study sites Date Station Latitude Longitude Coletor Substrate Depth numbers numbers specimens ns Sample (16S) (COI) Sample Ophiothrix trindadensis Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 17.IV.2014 20S 29'52.3" 29W 19'15.6" J.B.Mendonça - Projeto Protrindade 1 13.3m 120A Sample Ophiothrix trindadensis Ilha da Trindade, Enseada do Príncipe (Paredão), ES, Brasil 09.VII.2013 20S 31'22.4'' 29W 19'52.0'' J.B.Mendonça - Projeto Protrindade 1 19.5m 57A, B, C Sample Ophiothrix trindadensis Ilha da Trindade, Enseada do lixo, ES, Brasil 15.VII.2013 20S 31'29.8'' 29W 19'43.9'' J.B.Mendonça - Projeto Protrindade 1 17m 58A Sample Ophiothrix trindadensis Ilha da Trindade, Enseada do lixo, ES, Brasil 19.VI.2016 20S 31'29.8'' 29W 19'43.9'' J.B.Mendonça - Projeto Protrindade 1 14.5m 105A Ophiothrix trindadensis Ilha da Trindade, Enseada das Orelhas, ES, Brasil 30.VI.2012 Sample 41C 20S 29'40.2'' 29W 20'32.9'' J.B.Mendonça - Projeto Protrindade 1 13.9m

Sample Ophiothrix trindadensis Ilha da Trindade, Enseada da Cachoeira, ES, Brasil 12.VII.2012 20S 30'55.6'' 29W 20'21.7'' J.B.Mendonça - Projeto Protrindade 1 12m 10A, B, D, Sample Ophiothrix trindadensis Ilha da Trindade, Enseada da Cachoeira (Praia do M), ES, Brasil08.VII.2013 20S 30'53.8'' 29W 20'19.2'' J.B.Mendonça - Projeto Protrindade 2 15m 39D Sample Ophiothrix trindadensis Ilha da Trindade, Enseada da Cachoeira (Farrilhões), ES, Brasil05.V.2014 20S 31'22.4'' 29W 19'52.0'' J.B.Mendonça - Projeto Protrindade 2 14.4m 119A Ophiothrix trindadensis Arquipélago Martin Vaz, Ilha Martin Vaz, ES, Brasil 23.VII.2013 Sample 64B 20S 30'45.7'' 29W 18'21.9'' J.B.Mendonça - Projeto Protrindade 1 13m 26 Ophiothrix trindadensis * Ilha da Trindade, Farol Enseada dos Portugueses, ES, Brasil 15.III.2013 Sample 3 20S 29'52.3" 29W 19'15.6" Projeto Protrindade 1 12m

103

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CAPÍTULO 2

Unraveling the taxonomic identity of Ophiothela Verrill, 1867 (Ophiuroidea) along the Brazilian coast

Renata Aparecida dos Santos Alitto, Gabriela Granadier, Ana Beardsley Christensen, Timothy O’Hara, Maikon Di Domenico, Michela Borges

Capítulo formatado para a revista-alvo Zoologica Scripta.

Cadastro de Acesso ao Patrimônio Genético AF4BF9C (Anexo 2), AB7038A (Anexo 4) e AACEF44 (Anexo 5).

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Corresponding author Name: Renata A. S. Alitto Address: Charles Darwin Street, Campinas, São Paulo, Brazil, ZIP Code 6109 Telephone number: 55 (19) 3521-6385 Email address: [email protected]

Unraveling the taxonomic identity of Ophiothela Verrill, 1867 (Ophiuroidea) along the Brazilian coast RENATA A. S. ALITTO, GABRIELA GRANADIER, ANA B. CHRISTENSEN, TIMOTHY O’HARA, MAIKON DI DOMENICO, MICHELA BORGES

Ophiothela along the Brazilian coast R. A. S. Alitto, G. Granadier et al.

Abstract Delimiting species boundaries remains an important challenge in systematics and becomes urgent when unresolved taxonomy complicates other scientific disciplines that depend on it. We examined species boundaries of the small six-rayed brittle star Ophiothela using three independent character sets utilizing morphology (external morphology and morphometry), and molecular data (16S and COI). Concordance was found between the analyses pointing that Ophiothela sp. from Brazil (BR), Ophiothela mirabilis, and Ophiothela danae are closely related. However, Brazilian specimens have a different set of haplotypes and Ophiothela sp. BR is closer to O. danae than to O. mirabilis on the morphometric analysis. We conclude that the Brazilian specimens are distinct, but current evidence is not enough to work out if they are geographic lineage or species. Ophiothela sp. BR was rigorously described and documented with high resolution photos through scanning electron microscopy, providing new features and augment its description.

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Introduction

The genus Ophiothela was first described by Verrill (1867) as a subgenus of Ophiothrix. According to the author, Ophiothela agrees well with the typical species of Ophiothrix, but differs in having the arms distinctly covered with a membranous skin with scattered granules. Due to these distinct characters, Ophiothela is currently accepted as a genus (Stöhr, O’Hara, & Thuy, 2018).

Ophiothela mirabilis was the first species described in the genus Ophiothela, followed by O. danae described by Verrill (1869). Although the descriptions of these species were made by the same author and time period, he made no comparison between them. In the following years, many species were described into Ophiothela. To complicate matters, the small size (at most 7 mm of dd) and fissiparous nature, made the morphological studies and delimitation of Ophiothela species even more difficult.

In the Ophiothela genus, the most synonymized species is O. danae with a total of seven synonymous names (Stöhr et al., 2018). The main argument used by the authors for synonymization was the lack of differences between the species. An example is O. danae, O. isidicola, and O. verrilli which have overlapping features in their original descriptions with no differences between them (Koehler, 1905; Matsumoto, 1917). All three species are accepted as O. danae (Stöhr et al., 2018).

Ophiothela was confined to Pacific waters (A. M. Clark, 1976), but recently introduced populations have been described in the Atlantic (Araújo, Soares, Matthews- Cascon, & Monteiro, 2018; Hendler, Migotto, Ventura, & Wilk, 2012; Lawley, Fonseca, Júnior, & Lindner, 2018; Mantelatto et al., 2016). Since then, there is no consensus on the identification of Ophiothela species due to its fissiparity ability, which increases the difficulty to delimit taxa based on external morphology. The accurate identification of Ophiothela species, particularly from Brazil, is a mandatory requirement because its expansion has changing the appearance of coral reefs, sponges, algae and other echinoderms. The implications of these associations are still unclear. However, the identification and description of the species of Ophiothela in Brazil is the first and essential step to proceed ecological studies or even environmental monitoring. 113

In order to feel these gaps, an integrative approach was used to investigate Ophiothela specimens and to clarify their taxonomic status and distribution in Brazil. Our study was evaluated using morphology (external morphology and morphometry) and molecular data (nucleotide sequences of the mitochondrial genes 16S and COI).

Material and Methods

Institutional abbreviations: MCZ, Museum of Comparative Zoology; USNM, United States National Museum, Smithsonian Institution; ZUEC OPH, Ophiuroidea Scientific Collection from Museum of Zoology of the University of Campinas.

Abbreviations: dd, disc diameter; BR, Brazil; FG, French Guyana; MA, Madagascar; MP, Mexico Pacific; NZ, New Zealand; PE, Peru; SEM, scanning electron microscope; spm, specimen(s); sq – sequence.

Delimitation of the candidate species inferred from morphological characters

As a starting point, we performed a morphological comparison of our specimens of Ophiothela sp. from Brazil (BR) to an additional six Ophiothela species listed by Stöhr et al. (2018). This comparison was based on characters usually reported as diagnostic in morphological overviews and keys (Alitto et al., 2018; Verrill, 1867, 1869). The species most similar to our specimens are Ophiothela mirabilis Verrill, 1867 and O. danae Verrill, 1869 as they have six arms and five arm spines (step A, Fig 1A).

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Figure 1. Schematic diagram of the integrative taxonomy applied to Ophiothela spp. A) A priori classification inferred from morphological characters based on literature. B) Morphological characters: B1) external morphology, B2) microstructural morphology, B3) morphometry. C) Molecular characters: C1) phylogenetic analyses (Maximum Parsimony and Bayesian Inference), and C2) genetic diversity (genetic distances, AMOVA, haplotype network). D) Congruence framework of integrative taxonomy.

Morphological characters

Data collection. A total of 132 spms were studied from four sites from Brazil: i) Todos os Santos Bay, Bahia; ii) Araçá Bay, São Paulo; iii) Praia Preta, São Paulo, and iv) Estuarine Complex of Paranaguá, Paraná. These samples were then compared to those from scientific collections:

1) MCZ: i) Ophiothela mirabilis from Panama (1 dried syntype spm, OPH- 2495); ii) Ophiothela danae from Fiji Islands (1 dried syntype spm, OPH-2492; 1 115 syntype spm in ethanol, OPH-2631) and from South Africa Cape Colony (1 dried syntype spm, OPH-2493).

2) USNM: Ophiothela mirabilis from Panama (4 dried spms, E27240 and E31768) and Ophiothela danae from Philippines (2 dried spms, 41274 and 41275).

The geographic coordinates, substrate type, and main references for each specimen studied are shown in Supplementary Table 1.

External morphology. Comparisons of external characters of Ophiothela sp. BR were made against original descriptions of Ophiothela mirabilis and O. danae (Verrill, 1867, 1869) and against museum specimens from MCZ (syntypes) and USNM (Fig. 1B, Step B1). The terms applied are based on the literature (O'Hara, Stöhr, Hugall, Thuy, & Martynov, 2018; Stöhr, O'Hara, & Thuy, 2012; Thuy & Stöhr, 2016).

Microstructural morphology. The best-preserved specimens of Ophiothela sp. BR were selected to study the arm ossicles, which were extracted from a small part of the proximal arm, between the fifth and the tenth segment. The arm segment was immersed in regular household bleach (NaClO) until the soft tissues were removed (Stöhr, Conand, & Boissin, 2008). The ossicles were then washed with distilled water, air- dried, and prepared for examination with a SEM model JEOL JSM5800LV. The terms applied to the microstructures are based on the literature (Hotchkiss & Glass, 2012; Hotchkiss, Prokop, & Petr, 2007; Martynov, 2010; O'Hara, Stöhr, et al., 2018; Stöhr et al., 2012; Thuy & Stöhr, 2011, 2016). The arm ossicles are illustrated in Alitto et al. (2018). Arm vertebra joints were classified into types as proposed by Litvinova (1994) (Fig. 1B, Step B2).

Morphometry. Measurements were taken using an ocular micrometer and through the AxioVision VS program 40.4.8.20 (Carl Zeiss Microscopy, Germany) attached to a ZEISS Discovery V20 stereomicroscope or by using digital measurement (Adobe Photoshop®). A detailed list of measurements is presented in Table 1.

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Table 1. Abbreviations and definitions of the 13 morphological characters used for the morphometric analysis.

Abbreviation Character name Definition dd Disc diameter Length from the distal edge of the radial shield to the interradial edge of the disc rs_l Radial shield Length of one radial shield. Subdivided into: length rs_l1, rs_l2, rs_l3, rs_l4, rs_l5, and rs_l6 representing the first, second, third, fourth, fifth, and sixth respectively rs_w Radial shield width Width of one radial shield. Subdivided into: rs_w1, rs_w2, rs_w3, rs_w4, rs_w5, and rs_w6 representing the first, second, third, fourth, fifth, and sixth respectively as Arm spine Number of arm spines at the third free arm segment od Oral diameter Length from the proximal edge of adoral shield to distal edge of the first ventral arm plate os_l Oral shield length Length of one oral shield os_w Oral shield width Width of one oral shield ads_l Adoral shield Length of one adoral shield length ads_w Adoral shield width Width of one adoral shield vap2_l 1st ventral arm Length of the first ventral arm plate plate length vap2_w 1st Ventral arm Width of the first ventral arm plate plate width vap3_l 2nd ventral arm Length of the second ventral arm plate plate length vap3_w 2nd ventral arm Width of the second ventral arm plate plate width

The linear discriminant analysis (LDA) was applied using the R environment (R Development Core Team, 2018). To avoid multicollinearity among morphological characters a correlation matrix was constructed and the variables that 117 were significantly correlated were removed with a threshold value of 0.9. To investigate the differences in morphological characters, lda function (package MASS) (R Development Core Team, 2018; Venables & Ripley, 2002) was used to distinguish the Ophiothela spp. The classification rate of each Ophiothela species was assessed by the “lda” function.

The predict function (package STATS) and table function (package BASE) were used to assess the classification based on the linear discriminants and to verify the model error. Visualization was performed using the package ggplot2 (Wickham, 2009) (Fig. 1B, Step B3).

Molecular characters

Data collection. A total of nine COI sequences of Ophiothela spp. were used (Supplementary Table 1 and Table 2):

1) Ophiothela danae from New Zealand (NZ) and Australia (AUS) (2 sq).

2) Ophiothela vincula from AUS (2 sq).

3) Ophiothela venusta from Madagascar (MA) (1 sq).

4) Ophiothela tigris from MA (1 sq).

5) Ophiothela mirabilis from Peru (PE), Mexico Pacific (MP), French Guyana (FG) (3 sq).

DNA extraction, amplification, and sequencing. DNA sequences used in phylogenetic analyses were obtained as follows. Samples of tube feet from the arms were soaked in two changes of Tris-EDTA (TE) buffer for 30 minutes each. The samples were then macerated in 500 μl of TE buffer with a pestle. A volume of 300 μl of a 10% Chelex 100 solution (BioRad) was added. The samples were then incubated at 55°C for 60 minutes, boiled for 8 minutes, cooled to room temperature, vortexed for 20 seconds, followed by 60 seconds of centrifugation at 14,000 xg. The supernatant was decanted and kept refrigerated until use in polymerase chain reactions (PCR). 118

A fragment of the mitochondrial 16S gene was PCR-amplified using the forward primer 16S Sofi F (5′-CAG TAC TCT GAC TGT GCA A-3′) and the reverse primers 16S Sofi R (5′-GGA AAC TAT GAT CCA ACA TC-3′) (Pérez‐Portela, Almada, & Turon, 2013) and 16S Sbr-H (5′-CCG GTC TGA ACT CAG ATC ACG T- 3′) (Palumbi et al., 1991). A fragment of the mitochondrial cytochrome oxidase subunit 1 (COI) gene was amplified using the forward primer COIceF (5′-ACT GCC CAC GCC CTA GTA ATG ATA TTT TTT ATG GTN ATG CC-3′) and the reverse primer COIceR (5′- TCG TGT GTC TAC GTC CAT TCC TAC TGT RAA CAT RTG-3′) (Hoareau & Boissin, 2010).

PCR reactions were performed using PuReTaq Ready-To-Go™ PCR Beads for 25 μl reactions (GE Heathcare), complemented with 0.5 to 1 μl of 25mM MgCl2. Alternatively, PCR reactions were composed of 10 mM Tris-HCl, pH 8/1 mM KCl buffer, 1.5 mM MgCl2, and 200 μM each dNTP (Invitrogen). In both cases, 0.4 μM of each primer was used and template DNA concentration (ranging from 2.7 to 11 ng/μl) was tested.

Thermal cycling consisted of a single step at 94°C for 4 minutes, which was followed by 40 cycles (denaturation at 94°C for 60 seconds, annealing at 45.7°C to 55.7°C for 70 seconds, and extension at 72°C for 80 seconds) and a final extension at 72°C for 5 minutes on a thermal cycler (Eppendorf Mastercycler®).

PCR products were separated from excess primers and dNTP using a purification kit (Promega). Purified product was then used as a template for DNA sequencing reactions using BigDye Terminator (Applied Biosystems) and sequenced by Serviço de Sequenciamento de DNA – SSDNA IQUSP with the same primers used in the amplification reaction. A 320 bp fragment from 17 individuals was obtained for 16S and 704 bp fragment from five individuals for COI. All the sequences will be deposited in GenBank (Table 2). The nucleotide sequences were edited using BIOEDIT Sequence Alignment Editor v. 7.0.1 (Hall, 1999) and GeneStudio 2.2.0.0 (Genestudio, Suwanee, GA, USA).

Phylogenetic analyses. Two mitochondrial datasets were considered: i) 16S, and ii) COI. All sequences were aligned with GeneStudio 2.2.0.0 via Clustal W 1.83 (Genestudio, Suwanee, GA, USA) providing a unified matrix. All the additional 119 sequences from other Ophiotrichidae (16S, COI) available on GenBank were added to the matrix for comparison. Amphipholis squamata (Amphiuridae) was used as outgroup. All samples used are listed in Table 2.

Phylogenetic reconstructions were made on the basis of two different optimality criteria, Maximum Parsimony (MP) and Bayesian Inference (BI), to assess whether there were any differences in the trees recovered with regard to the method used (Fig. 1C, Step C1).

The MP was performed with the TNT 1.5 program (Goloboff & Catalano, 2016). Most parsimonious trees were obtained by a heuristic search (best length was hit 100 times), using the new technology search option, which included sectorial searches, ratchet, tree drifting and tree fusing. The gaps were considered as fifth state. The cladograms had their nodes evaluated by the Bootstrap resampling test (Felsenstein, 1985), based on 1000 pseudoreplicates using the Traditional Search.

The BI was performed with MrBayes version 3.2 (Ronquist et al., 2012), the best-fitting model of molecular evolution for 16S and COI datasets were SYM+G chosen based on the Akaike Information Criterion (AIC) and GTR chosen based on the Hierarchical Likelihood Ratio Tests (hLRTs) according to the estimation by MR.MODELTEST v.2.3 (Nylander, 2004). The BI analyses used one cold and three incrementally heated Monte Carlo Markov chains (MCMC) on two simultaneous runs. The standard deviation of the split frequencies between the two runs reached a value lower than c. 0.001 at one million generations, with one tree sampled every 100th generation, each using a random tree as a starting point and a temperature parameter value of 0.2 (the default in MrBayes). The first 25% of the total sampled trees were discarded as ‘burnin’ to achieve the MCMC log-likelihoods that had become stationary and converged. The analyses resulted in similar likelihood scores, with ESS > 200, as verified using TRACER. The phylogenetic trees were visualized and edited in FIGTREE v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).

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Table 2. Specimens used in the phylogenetic analyses with their locality, museums vouchers/catalogue, and GenBank accession numbers for DNA sequences. Abbreviations: ACUNHC, Abilene Christian University Natural History Collection; AM, Australian Museum; IC, individual codes in the present study; MNHN, National Museum of Natural History, Paris; MV, Museums Victoria; MZUSP, Museum of Zoology of the University of São Paulo; TMV-BR, Trindade and Martin Vaz Oceanic Archipelago, Brazil; UF, Florida Natural History Museum; UNAM, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México.

Museum voucher or GenBank GenBank IC Species Locality Reference catalogue (16S) (COI) ZUEC OPH 2814 11 Ophiothela sp. BR X X Present study

ZUEC OPH 2819 145 Ophiothela sp. BR X Present study

ZUEC OPH 2817 143 Ophiothela sp. BR X Present study

ZUEC OPH 2822 148 Ophiothela sp. BR X Present study

ZUEC OPH 2818 144 Ophiothela sp. BR X Present study

ZUEC OPH 2825 151 Ophiothela sp. BR X Present study

ZUEC OPH 2826 152 Ophiothela sp. BR X Present study

ZUEC OPH 2877 82 Ophiothela sp. BR X X Present study

ZUEC OPH 2876 80 Ophiothela sp. BR X Present study

ZUEC OPH 2862 81 Ophiothela sp. BR X Present study 121

ZUEC OPH 2828 154 Ophiothela sp. BR X Present study

ZUEC OPH 2851 178 Ophiothela sp. BR X Present study

ZUEC OPH 2847 174 Ophiothela sp. BR X Present study

ZUEC OPH 2848 175 Ophiothela sp. BR X X Present study

ZUEC OPH 2849 176 Ophiothela sp. BR X Present study

ZUEC OPH 2850 177 Ophiothela sp. BR X X Present study

ZUEC OPH 2852 179 Ophiothela sp. BR X X Present study

CZA E094 TANIA304 Ophiothela mirabilis PE X Present study

UNAM 3.138.36 TANIA610 Ophiothela mirabilis MP X Present study

UF 16885 UF16885 Ophiothela mirabilis FG X Present study Hugall, O’Hara, Hunjan, AM J24839 J24839 Ophiothela danae NZ KU895425 Nilsen, and Moussalli (2016) MV F168055 TOH_796 Ophiothela danae AUS X Present study AUS: King George River MV F193491 KGR28232 Ophiothela vincula KU895424 Hugall et al. (2016) region AUS: King George River WAM KGR23129 Ophiothela vincula KU895423 Hugall et al. (2016) region 122

MNHN IE.2007.4207 07.4207 Ophiothela venusta MA KU895426 Hugall et al. (2016)

UF 7611 UF 7611 Ophiothela tigris MA KU895427 Hugall et al. (2016) (Pérez‐Portela et al., Absent Ophiothrix fragilis Atlantic-Mediterranean JX948073 2013) (Pérez‐Portela et al., Absent Ophiothrix fragilis Atlantic-Mediterranean JX948074 2013) (Pérez‐Portela et al., Absent Ophiothrix fragilis Atlantic-Mediterranean JX948075 2013) (Hunter, Brown, Absent Ophiothrix angulata Texas, United States KU672428 Alexander Hill, Kroeger, & Rose, 2016) ZUEC OPH 2803 Ophiothrix cf. angulata ECP-BR MH281587 (Alitto et al., 2019)

ZUEC OPH 2811 Ophiothrix angulata AB-BR MH281599 (Alitto et al., 2019)

MZUSP 1426 Ophiothrix trindadensis TMV-BR MH281577 (Alitto et al., 2019)

MZUSP 1425 Ophiothrix trindadensis TMV-BR MH281576 (Alitto et al., 2019)

ACUNHC 02008 Amphipholis squamata Texas, United States KU672403 (Hunter et al., 2016)

MV F211339 Amphipholis squamata Australia KU895012 (Hugall et al., 2016)

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Analyses of the genetic diversity. The genetic distances between and within the different groups were estimated by p-distance using MEGA v. 7.0 (Kumar, Stecher, & Tamura, 2016), ignoring the alignment gaps in pairwise comparisons. Following Hart and Podolsky (2005) and Pérez‐Portela et al. (2013), pairwise genetic p-distances >3% for 16S and >15% for COI were considered interspecific variations since most of the species already described have been differentiated by this minimum (Fig. 1C, Step C2).

The COI matrix was submitted to the AMOVA (Analysis of Molecular Variance) test (Excoffier, Smouse, & Quattro, 1992) to evaluate the genetic difference between and among the studied groups formed by the phylogenetic analyses. The overall fixation index (FST) (Wright, 1978) was calculated to verify the gene flow between the groups. The test was simulated with 10000 permutations in Arlequin software v. 3.1 (Excoffier, Laval, & Schneider, 2005) (Fig. 1C, Step C2).

Haplotype networks were constructed using the TCS method in PopART v1.7 (Leigh & Bryant, 2015).

Integrative taxonomy Specimens of Ophiothela sp. BR were evaluated using three independent character sets in order to test: i) external morphology, ii) morphometry, and iii) molecular data (16S and COI fragments). The species were classified according to the cumulative framework described in Padial, Miralles, De la Riva, and Vences (2010) when there was concordance of at least two data sets analyzed (Fig. 1D). Therefore, we highlight that molecular data was not weighted more heavily than morphological features.

Results

Morphological characters The possibility that our specimens belong to some species was rejected as follows: O. tigris Lyman, 1871 has five arms and dorsal arm plates represented by a double row of irregular elongated warts, which only at the base of the arm are increased in number; O. venusta (de Loriol, 1900) has five arms and triangular ventral arm plates; O. gracilis Nielsen, 1932 and O. vincula Mortensen, 1913 have five arms.

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External morphology. A detailed comparison between Ophiothela sp. from Brazil (BR), O. danae, and O. mirabilis is presented according to main characteristics observed in their original descriptions, syntypes from MCZ, and additional material from USNM.

Dorsal disc

Verrill (1867, 1869) described Ophiothela danae and O. mirabilis with rounded granules scattered on dorsal disc as we observed on our BR specimens and on the syntypes from MCZ. However, some of analyzed specimens have more granules than others, which may be related to their size (Figs. 2A,C,E). One specimen of O. danae had three larger and rounded granules in the interradial recesses (USNM 41275) (Fig. 2A). Although uncommon, some specimens of O. danae without granules on dorsal surface were described (Matsumoto, 1917), but this did not occur for our specimens.

Clusters of sharp conical spines in the interradial recesses and in the center of the disc were described by Verrill (1867) for Ophiothela mirabilis. These spines were not observed in any specimen examined.

Dorsal disc with central area depressed

Verrill (1869) described the central area depressed only for Ophiothela danae. We observed this feature in most of our BR specimens and in the both syntypes from MCZ (Figs. 2C,E). We do not consider this character appropriate for a species diagnosis since it may be related to the preservation method or even possibly to food items ingested.

Radial shields

Verrill (1867, 1869) described prominent radial shields occupying almost the entire disc, covered by rounded granules in Ophiothela mirabilis and O. danae. We also observed this feature in most of our BR specimens and in all syntypes from MCZ. The number of granules on the radial shields was variable and possibly related to their size or regeneration state, even in the same individual. One specimen of O. mirabilis had granules only around the radial shields, not covering them (USNM E27240) (Fig. 2B).

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Continuous ring around the mouth

Plates united around the mouth forming a continuous ring were described for the genus Ophiothela as a diagnosis (Verrill, 1867). Some of our BR specimens did not have skin covering the ventral disc surface, exposing ventral arm plates between the adoral shields radially, contradicting the original description (Alitto et al., 2018). The oral and adoral shields of the syntype of O. mirabilis (MCZ OPH-2495) were not observed due to the poor condition of the ventral side. The same plates were observed in the syntype of O. danae (MCZ OPH-2631) with ventral arm plates between the adoral shields radially (Fig. 2C).

Dorsal arm plates

Verrill (1867) described the dorsal arm plates (DAP) covered by rounded granules for Ophiothela mirabilis but did not describe the DAP for O. danae. A higher concentration of granules was observed proximally to the disc in both species, suggesting that older segments have more granules (Fig. 2D). A lower concentration of granules was observed on regenerating arms both BR and syntypes specimens. A central series of the largest granules (CGR) on DAP was described only for O. danae (Verrill, 1869), while numerous and unequal granules on DAP were described for O. mirabilis (Verrill, 1867). The CGR was not observed on syntypes specimens due to their poor condition. However, we observed that some specimens of O. mirabilis (USNM E31768) had a central large granule for each arm segment on the more developed arms (Fig. 2E). We assume that the DAP is very tiny as it was not discernible under the stereomicroscope and the household bleach dissolved it quickly (Alitto et al., 2018).

Ventral arm plates

Verrill (1867) described the VAP in Ophiothela mirabilis about as long as wide, widest outwardly, outer edge convex, and sides converging to the rounded inner angle. We agree with Nielsen (1932) that the Verrill's description appears to be the combination of the VAP and the vertebra as seen through the thick skin (Fig. 2F). The naked skin was observed in most of BR specimens, obscuring or making difficult to see the ventral arm plates (VAP). The VAP is very tiny as it was not observed under the stereomicroscope, but through SEM it is three times as long as wide, distal region wider than the proximal, and with a notch in distal edge.

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Number of arm spines

Ophiothela mirabilis was described with five to six arm spines in specimens with 4 to 5.5 mm of dd (Verrill, 1867), while O. danae was described with five arm spines (Verrill, 1869). The number of arm spines ranged from three to five, with the ventral shortest and bearing sharp teeth in all our specimens. Due to the poor condition of the arms of the syntype of O. mirabilis (MCZ OPH-2495), the arm spines were not observed. Four arm spines were observed in O. danae (MCZ OPH-2492). We believe that this feature is variable and should not be used for species identification.

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Figure 2. Ophiothela spp. variations. A) O. danae USNM 41275; B) O. mirabilis USNM E27240; C) O. danae MCZ OPH-2631 (Available from http://mczbase.mcz.harvard.edu/guid/MCZ:IZ:OPH-2631); D) O. danae USNM 41275; E) O. mirabilis USNM E31768; F) O. mirabilis ZUEC OPH 1020. Abbreviations: ads, adorsal shield; as, arm spine; cgr, central granule; gr, granule; os, oral shield; rs, radial shield; ts, thick skin; ve, vertebra. Stereomicroscope photos, scale bar equal to 0.5 mm.

Ophiothela sp. BR and the syntypes of Ophiothela mirabilis and O. danae are represented in Fig. 3. Table 3 summarizes the main characteristics observed between them.

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Figure 3. Ophiothela spp. including syntypes. A, B) Ophiothela sp. BR ZUEC OPH 1020; C, D) O. mirabilis MCZ OPH-2495 (Available from http://mczbase.mcz.harvard.edu/guid/MCZ:IZ:OPH-2495); E, F) O. danae MCZ OPH- 2492 (Available from http://mczbase.mcz.harvard.edu/guid/MCZ:IZ:OPH-2492). Abbreviations: as, arm spine; cad, central area depressed; gr, granule; rs, radial shield. Stereomicroscope photos, scale bar equal to 0.5 mm.

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Table 3. Main morphological characters of Ophiothela sp. from Brazil compared with Ophiothela danae and Ophiothela mirabilis from original description and observations on syntypes from MCZ. Abbreviations: NDI, not discernible; NDE, not described. ✓ present; X absent.

Observation Original description Observation O. mirabilis O. danae Characters Ophiothela sp. BR O. mirabilis O. danae Syntype MCZ OPH-2495 Syntype MCZ OPH-2492 Figs. 3A,B Verrill, 1867 Verrill, 1869 Figs. 3C,D Figs. 3E,F Locality Brazil Panama Fiji Islands Panama Fiji Islands Disc diameter 1 to 7.7 mm 4 to 5.5 mm 4.5 to 5 mm 2.6 mm 4.5 mm Dorsal disc with rounded ✓ ✓ ✓ ✓ ✓ granules scattered Dorsal disc with central ✓ NDE ✓ ✓ ✓ area depressed Prominent radial shields covered by rounded ✓ ✓ ✓ ✓ ✓ granules Plates united around the mouth to form a X ✓ ✓ NDI NDI continuous ring Dorsal arm with the ✓ X ✓ NDI NDI central granules largest Ventral arm plates covered ✓ ✓ NDE ✓ ✓ with skin Number of arm spines 3 – 5 5 – 6 5 NDI 4

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All analyzed specimens have the following features: i) rounded granules scattered on the disc and on the arms dorsally; ii) prominent radial shields (two-thirds of dd); iii) ventral surface covered by skin, vi) six arms; v) dorsal arm plates very tiny and not discernible under the stereomicroscope; vi) ventral arm plates between the adoral shields radially; vii) three to six arm spines.

The following features were considered as variations: i) higher or lower quantity of granules on the dorsal disc and on the radial shields; ii) dorsal disc with or without central area depressed; iii) presence or absence of rounded granules in the interradial recesses; iv) granules only around the radial shields, not over them; v) presence or absence of a central series of largest granules on dorsal arm plates.

We could not distinguish Ophiothela mirabilis from O. danae based on the descriptions of morphological characters by Verrill (1867, 1869) and on our observations of syntypes from MCZ and specimens from USNM. This indicates that they cannot be separated morphologically.

Morphometry. The LDA performed shows that specimens identified as Ophiothela sp. BR overlapped on some specimens identified as O. danae, particularly from Phillippines. One specimen of O. danae from Fiji Islands and the specimens of O. mirabilis were isolated from other specimens (Fig. 4). The first and second linear discriminant axes described 76.93% and 23.07% of the among species variation in morphological characters, respectively. The classification success of LDA among the three species was 92%, showing Ophiothela sp. BR and O. mirabilis as the species classified correctly most often (96%, 60% respectively), while O. danae was the most commonly misclassified (0%).

The width of the third radial shield (rs_w3) and the length of the fifth radial shield (rs_l5) were the morphological characters with highest positive coefficients in the first discriminant vector (LD1). The highest negative coefficients were width of the first radial shield (rs_w1) and length of oral shield (os_l). Alternatively, the second discriminant vector (LD2) has length of oral shield (os_l) and width of the third radial shield (rs_w3) as a positive coefficient and width of the third ventral arm plate (vap3_w) and length of the fourth radial shield (rs_l4) as a negative coefficient.

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In summary, the LDA result shows a well-defined lineage of Ophiothela sp. in Brazil which is closer to Ophiothela danae than to Ophiothela mirabilis.

Figure 4. Axes 1 and 2 from Linear Discriminant Analysis (LDA) based on 13 brittle stars’ characters. See Table 1 for definitions of the morphological characters used for the morphometric analysis of Ophiothela spp. a: Araçá Bay, Brazil; b: Fiji Islands; c: South Africa Cape Colony; d: Panama; e: Estuarine Complex of Paranaguá, Brazil; f: Praia Preta, Brazil; g: Todos os Santos Bay, Brazil; h: Phillippines.

Molecular characters Phylogenetic analyses. The phylogenetic cladograms obtained either in the Bayesian (BI) analyses or in the Maximum Parsimony (MP) analyses of the two datasets (16S, Fig. 5; COI, Fig. 6) show a clade containing all Ophiothela sp. BR with high support values (Figs. 5,6).

Using the 16S dataset, two clades were noted in BI and MP with high support values (1, 100%, respectively) (Fig. 5): i) Ophiothela clade which comprises all Ophiothela sp. BR; ii) Ophiothrix clade which contains all Ophiothrix samples.

An Ophiothela subclade was noted in BI and MP using the COI dataset (Fig. 6) with high support values (1, 100%, respectively). Ophiothela subclade is formed by Ophiothela sp. BR, O. mirabilis, O. danae, and O. vincula. Two main clades were

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Figure 5. Cladogram inferred from the Bayesian analysis of 16S sequences. The numbers above the branches represent posterior probabilities. For the clades also inferred in the MP analysis, bootstrap values (%) are provided (below the branches). Branches are identified by individual codes (Table 2) and their localities: BR, Brazil.

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Figure 6. Cladogram inferred from the Bayesian analysis of COI sequences. The numbers above the branches represent posterior probabilities. For the clades also inferred in the MP analysis, bootstrap values (%) are provided (below the branches). Branches are identified by individual codes (Table 2) and their localities: AUS, Australia; BR, Brazil; FG, French Guyana; MA, Madagascar; MP, Mexico Pacific; NZ, New Zealand; PE, Peru. The dash (-) indicates that the branch was not recovered in MP analysis.

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Analyses of the genetic diversity Genetic distances

The genetic distances observed in 16S and COI between the Ophiothela sp. BR were low (from 0.3% to 0.7% for 16S and from 0.1% to 0.2% for COI).

Utilizing the COI dataset, the Ophiothela subclade, which is formed by Ophiothela sp. BR, O. mirabilis, O. danae, and O. vincula has a mean of 1.8% of genetic distance within it. The lowest genetic distances were between Ophiothela mirabilis and O. vincula (1.4%), O. mirabilis and O. danae (1.7%), and O. vincula and O. danae (1.8%). The highest genetic distances were between O. tigris and O. danae (20.6%), and O. tigris and Ophiothela sp. BR (20.3%) (Table 4).

Table 4. Genetic distance (%) between Ophiothela spp. estimated from the mitochondrial gene fragments COI. Abbreviation: Nc, Value not calculated because of the lack of groups’ sequences for the gene; WGMD, Compute Within Group Mean Distance.

Taxon 1 2 3 4 5 6 7 WGMD

1 Ophiothela sp. BR 0.2

Ophiothela mirabilis PE, 2 2.1 0.9 MP, FG Ophiothela danae NZ, 3 2.5 1.7 1.8 AUS

4 Ophiothela vincula AUS 2.7 1.4 1.8 0.0

5 Ophiothela venusta MA 16.2 15.4 15.2 14.7 Nc

6 Ophiothela tigris MA 20.3 20.0 20.6 19.5 18.8 Nc

7 Ophiothrix group 20.7 20.1 21.3 20.1 19.5 20.8 1.0

8 Amphipholis squamata 23.8 23.8 23.5 24.0 23.9 24.2 24.8 Nc

AMOVA AMOVA test revealed that 44.36% of total genetic variability of COI sequences occurred among Ophiothela subclade (inferred in the phylogenetics analysis) and the group formed by Ophiothela venusta and O. tigris (Table 5). On the other hand, 34.48% of total genetic diversity refers to variability within these groups. The overall

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fixation index (FST) was 0.65. These results indicate that the two groups are genetically distinct and well structured, indicating low or null gene flow between them.

Table 5. Analysis of Molecular Variance (AMOVA) of COI sequences for the population structuring scenario given by group I which is the Ophiothela subclade inferred in the phylogenetics analysis (Ophiothela sp. BR, O. mirabilis, O. danae, and O. vincula) and II (Ophiothela tigris and O. venusta).

Source of variation d.f. Sum of Variance Percentage of squares components variation Among groups 1 59.994 9.28675 Va 44.36 Among populations within groups 5 75.830 4.42992 21.16 Within populations 7 50.533 4.42992 Vc 34.48 Total 13 186.357 20.93572

Haplotype Network The haplotype network indicated the existence of two divergent lineages (1: Ophiothela sp. BR and 2: O. mirabilis, O. danae, and O. vincula) with intermediate haplotypes. Divergence among lineages was caused by five mutations. The first lineage (Fig. 7, left) is composed by low-frequency (mostly private) haplotypes, separated by a few mutational steps. The second lineage (Fig. 7, right) is composed of five haplotypes sharply separated, but connected with the intermediate haplotypes (Fig. 7, black circles).

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Figure 7. Median-joining haplotype network based on COI gene sequences. Each circle represents one haplotype and their size is proportional to the number of individuals possessing it. The black circles represent possible intermediate haplotypes.

Our studies based on molecular characters showed two main results: i) analyses with 16S and COI sequences indicate only one lineage in Brazil, and ii) comparisons based on COI sequences revealed that the haplotypes from Brazilian specimens are different from the east Pacific and West Pacific, suggesting some geographic structure.

Integrative taxonomy To integrate the results reported for the different operational criteria, a cumulative framework was followed (Padial et al., 2010). Concordance was found between the analyses based on external morphology, morphometry, and molecular (16S, genetic distances, haplotype network) pointing that Ophiothela sp. BR, O. mirabilis, and O. danae are closely related (Fig. 8). However, Brazilian specimens have a different set of haplotypes and Ophiothela sp. BR is closer to O. danae than to O. mirabilis on the morphometric analysis. We suggest further studies to better define the species into Ophiothela.

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Figure 8. Synthetic representation of the integrative taxonomy approach within the Ophiothela spp.

In the following, we present the description of Ophiothela sp. BR based on specimens from Brazil. The description contains the amended diagnosis, taxonomic comments, remarks, and distribution. The classification is in accordance with O'Hara, Hugall, Thuy, Stöhr, and Martynov (2017) and O'Hara, Stöhr, et al. (2018).

Morphological description Order AMPHILEPIDIDA (O'Hara et al., 2017) Family OPHIOTRICHIDAE Ljungman, 1867 Genus Ophiothela Verrill, 1867 Type species. Ophiothela mirabilis Verrill, 1867 Amended diagnosis. Dorsal disc with scattered rounded granules. Radial shields very large, covering most of the disc. Ventral side of arms and disc covered by skin, usually obscuring the arm plates and scales. Dorsal arm plates very tiny or covered by granules. Ventral arm plates between the adoral shields radially. Arm spines mostly turned downward, armed with thorns or hooks. Streptospondylous vertebrae with hourglass- shaped articulation and keeled. Dorsal groove of vertebrae lined by smooth knobs (Alitto et al., 2018; Verrill, 1867).

Ophiothela sp. BR Figures 9 – 11

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Maximum size. dd up to 7 mm. Material examined. 132 specimens (dd 0.7–7.7mm). See Supplementary Table 1. Amended diagnosis. Dorsal disc with scattered rounded granules. Radial shields covering about two-thirds of dd. Ventral side of arms and disc covered by skin. Typically hexamerous, occasionally pentamerous. Dorsal arm plates very tiny and not discernible under the stereomicroscope. Ventral arm plates between the adoral shields radially. Three to six arm spines. Streptospondylous vertebrae with hourglass-shaped articulation and keeled. Dorsal groove of the vertebrae lined by a series of four smooth knobs to either side.

Description. Disc: (dd: 2.5 mm). Star-shaped, with interradial recesses, rounded granules scattered over the entire dorsal surface, larger between radial shields (Fig. 9A). Radial shields elevated, triangular (Fig. 9B), approximately two-thirds of dd, united distally and separated proximally by one scale. Radial shield pairs separated by a narrow row of plates bearing granules (Fig. 9A). Abradial genital plate shorter than half the adradial length; adradio-distal tip shape concave (Fig. 9C). Ventral side of arms and disc covered by skin (Fig. 9D).

Mouth plating. Oral shields slightly broadened proximally. Adoral shields broadened distally and united proximally. Depression between two oral plates. A cluster of dental papillae on the dental plate and without lateral oral papilla. Oral tentacle pore large and opening at the mouth angle (Figs. 9D,E). Dental plate entire; ventral half widest; dorsal half with fenestrations and a septum (Fig. 9F). Oral plates longer than wide (approximately 1.5 times) (Figs. 9G,H); abradial and adradial surface with an opening smaller than the opening of the first tube foot; abradial muscle attachment area with normal stereom (Fig. 9H); adradial muscle attachment area in ventral position of the articulation area (Fig. 9G).

Arms. Six arms, dorsoventrally coiled and covered with scattered granules, dorsal arm plates very tiny and not discernible under the stereomicroscope (Fig. 9A). Ventral arm plates three times as long as wide, distal region wider than the proximal, notch in distal edge, lateral edges with clear incisions for tentacle openings, the first three plates contiguous, the others separated by lateral arm plates (Fig. 10A). Three to five arm spines, ventral shortest and bearing sharp teeth (Fig. 10B).

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Lateral arm plates (Figs. 10C,D). General outline: ventral portion projecting ventro- proximalwards; ventro-distal tip not projecting ventralwards; tentacle notch pointing ventralwards. Outer surface ornamentation: trabecular intersections protruding to form knobs larger than stereom pores on most of outer surface. Outer proximal edge: surface without discernible band of different stereom structure; without spurs; central part protruding; surface without horizontal striation. Spine articulations: on same level as remaining outer surface; dorsalwards increasing in size; distance between spine articulations equidistant. Lobes simply separated, equal-sized; lobes parallel, straight, oriented nearly horizontal; stereom massive; sigmoidal fold absent. Inner side, ridges and knobs: inner side with two separate knobs; without additional dorsal structure on inner side; single large perforation on inner side.

Vertebrae. Streptospondylous with hourglass-shaped articulation and keeled (although it is not well developed due to the high rate of fission). Two large grooves on proximal side of vertebrae dorsally corresponding to distalwards projecting dorso-distal muscular fossae of distal side (Fig. 10E). Hourglass-shaped articulation (Fig. 10F). Zygosphene absent; tiny spines on the end of the distal keel seen from the distal aspect (Figs. 10G,H). Dorso-distal muscular fossae transformed distalwards clearly projecting beyond the zygocondyles (true keel) and divided into two separate end processes matching the two large dorsal depressions proximally. Dorsal groove lined by a series of four smooth knobs to either side (Fig. 10I). Distinct suture line in ventral view (Fig. 10J).

Color patterns. The most common color pattern in Brazil has been the yellow-orange (Hendler et al., 2012; Lawley et al., 2018; Mantelatto et al., 2016), but purple was also described in specimens from Araçá Bay (Alitto et al., 2018). In the present study, must common are orange and purple in living specimens and reddish purple, brown, cream, or white for those fixed in alcohol. Other patterns observed: purple with white and pink with purple (Fig. 11). A few studies reported that the variety of color observed for Ophiothela was correlated with the color of their hosts (A. M. Clark, 1976; Granja- Fernández et al., 2014). In our study, these patterns do not appear to have any correlation with the color of the hosts used by O. mirabilis, which was the same as reported by Mantelatto et al. (2016). These observations suggest that color is not important to the Brazilian specimens, possibly because it possesses an innate predator deterrent such as chemical defense (Mantelatto et al., 2016).

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Taxonomic comments. The following features were considered as variations: i) number of granules on the dorsal disc and on the radial shields; ii) dorsal disc with central area depressed; iii) presence of rounded granules in the interradial recesses; and iv) granules only around the radial shields, not over them. Two characters of the Ophiotrichidae diagnosis were not observed in Ophiothela mirabilis (O'Hara, Stöhr, et al., 2018): i) small holes at each edge on ventral half of the dental plate, and ii) three knobs on the inner side of lateral arm plates – only one knob was observed. An opening on the both sides of the oral plate was observed. This opening is often present in self- dividing (fissiparous) species as Ophiocomella spp. and Ophiactis savignyi and may suggest a relationship to the phenomenon of fissiparity (Devaney, 1970). Only two specimens with seven arms was observed. This is considered an unusual morphological occurrence and it was described by Matsumoto (1917) for O. danae. Two features contradict the description of Ophiothela sp. made by Alitto et al. (2018): i) one, not none, knob on the inner side of lateral arm plate, and ii) vertebrae streptospondylous (not zygospondylous). The distal keel divided into two separate end processes and with tiny spines was not reported in the literature. The series of knobs on the dorsal groove were only described for Ophiothela tigris (Mortensen, 1913). Although the vertebra was not well developed in our specimens due to the high rate of fission in O. mirabilis, we conclude that its articulation is hourglass-shaped which is very similar to the saddle- shaped described by Litvinova (1994).

Remarks. Ophiothela mirabilis is commonly found on biological substrates, particularly gorgonians. In the present study, it was associated with sponges Mycale (Zygomycale) and Callyspongia sp., and the coral Carijoa riisei. In Brazil, O. mirabilis is reported to use a total of 29 novel host taxa (i.e. sponges, corals, echinoderms, algae, bryozoan, seahorse) (Mantelatto et al., 2016).

Distribution. According to marine ecoregions (Spalding et al., 2007). Tropical Atlantic (realm), North Brazil Shelf (province): Guianan (Hendler & Brugneaux, 2013); Tropical Southwestern Atlantic (province): Northeastern and Eastern Brazil (Araújo et al., 2018; Hendler et al., 2012). Temperate South America (realm), Warm Temperate Southwestern Atlantic (province): Southeastern Brazil (Bumbeer & Rocha, 2016; Hendler et al., 2012; Mantelatto et al., 2016).

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Figure 9. Ophiothela sp. BR ZUEC OPH 1020 (2.5 mm dd). A) Dorsal view. B) Radial shield. C) Genital plate. D) Ventral view. E) Detail of the oral view. F) Dental plate. G) Oral plate in adradial view. H) Oral plate in abradial view. Abbreviations: ab, abradial; ad, adradial; ads, adoral shield; abmaa, abradial muscle attachment area; adt, adradial- distal tip; admaa, adradial muscle attachment area; as, arm spine; cd, central depression; cdp, cluster of dental plate; d, dorsal; di, distal; fe, fenestration; ff, first foot; gr, granule; jd, jaw depression; op, oral plate; ope, opening; os, oral shield; otp, oral tentacular pore; p, proximal; rs, radial shield; v, ventral; vap, ventral arm plate; sp, septum; sk, skin. Stereomicroscope photos: (A,D,E), scale bar equal to 0.5 mm. SEM photos: (B,C,F–H), scale bar equal to 100 μm.

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Figure 10. Ophiothela sp. BR ZUEC OPH 1020 (2.5 mm dd). A) Ventral arm plate. B) Detail of a hooked arm spine. C) Lateral arm plate – external view. D) Lateral arm plate – internal view. E, F) Vertebra – proximal face. G, H) Vertebra – distal face. I) Vertebra – dorsal face. J) Vertebra – ventral face. Abbreviations: as, arm spine; d, dorsal; ddi, dorso-distal; ddmf, dorsodistal muscular fossae; de, depression; di, distal; dk, distal keel; dp, dorso-proximal; hj, hourglass joint; kn, knob; no, notch; p, proximal; pe, perforation; sa, spine articulation; sl, suture line; sp, spine; tn, tentacle notch; v, ventral; vdi, ventro-distal; vp, ventro-proximal; zd, zygocondyle. Stereomicroscope photo: (B), scale bar equal to 0.1 mm. SEM photos: (A,C–J), scale bar equal to 100 μm.

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Figure 11. Ophiothela mirabilis – color patterns in some living (A,B) and preserved (C,D) specimens. A) Orange ZUEC OPH 1020. B) Purple with white ZUEC OPH 2814. C) Reddish purple ZUEC OPH 2876. D) Brown USNM 41275. Stereomicroscope photos, scale bar equal to 0.5 mm.

Discussion

The combined integrative taxonomy based on external morphology, morphometry, and molecular data shows that Ophiothela sp. BR, Ophiothela mirabilis, and Ophiothela danae are closely related. Despite this, two points are important to highlight. First, Ophiothela sp. BR has a different set of haplotypes from O. mirabilis and O. danae. Second, Ophiothela sp. BR is closer to O. danae than to O. mirabilis on the morphometric analysis. We conclude that the Brazilian specimens are distinct, but current evidence is not enough to work out if they are a new geographic lineage or species, suggesting further studies with this group. Ophiothela sp. BR was rigorously described using external morphology and microstructural elements, providing additional

145 diagnostic characters for future studies of the group's taxonomy, phylogeny, and ecological traits.

The main character observed in nonregenerating arms of Ophiothela sp. BR was a central and larger granule for each arm segment, which may be a remnant of the dorsal arm plate (DAP). The DAP is very tiny as it was not discernible under the stereomicroscope in most of the specimens, particularly the ones from Brazilian coast. Mortensen (1913) described lateral granules on the dorsal side of arms as parts of the vertebra for O. tigris. We assume that these granules could be the same smooth knobs observed on the dorsal face of the vertebra in our specimens under the SEM. These knobs were observed in series of four to either side on the vertebra of Ophiothela sp. BR. This quantity of knobs was not observed on the dorsal arm surface when analyzed under the stereomicroscope. We concluded that Brazilian specimens have granules on the dorsal arm and smooth knobs on the dorsal face of its vertebra.

A true vertebral keel was observed in Ophiothela sp. BR. This keel is different from the keel described for Ophiothrix angulata (Alitto et al., 2018), which also belongs to the family Ophiotrichidae. In Ophiothela sp. BR the keel is divided into two separate end processes matching the two large dorsal grooves proximally, while in O. angulata the keel is not divided, and its end process matches a single dorsal groove proximally. An ancestral state reconstruction of the evolutionary history of ophiuroid vertebral morphologies was recently proposed by E. G. Clark et al. (2018). The authors suggested that the non-keeled vertebral morphology was present in the most recent common ancestors of Ophiuroidea and the keeled morphology appears to have evolved convergently at least twice within the Amphilepidida to which Ophiotrichidae belongs.

Ophiothela sp. BR has streptospondylous joint with an hourglass-shaped articulation. This kind of articulation is found in several taxa with epizoic life style, whereas their relatives have zygospondylous articulations (O'Hara, Stöhr, et al., 2018). The hourglass-shaped articulation is similar to the saddle-shaped articulation which provides the arm the most flexibility, being able to flex in every direction and to bend spirally (Litvinova, 1994).

Due to the streptospondylous joint described here for Ophiothela sp. BR, we recognize the family Ophiotrichidae as the third non-euryalid family with this joint. The others two non-euryalid families are Ophiacanthidae and Hemieuryalidae (LeClair,

146

1996; Stöhr, 2012). In addition to these families, this joint is also present in the order Euryalida, and is a trait that exhibits homoplasy (E. G. Clark et al., 2018). Since the arm vertebrae have been subject to ecological adaptation (O'Hara, Stöhr, et al., 2018), the need for further studies, such as behavioral observation and mechanical testing, are necessary to elucidate the function of the different joints present in ophiuroids.

Another feature of interest is the opening on the both sides of the oral plate of Ophiothela sp. BR. We agree with Devaney (1970) that this feature may suggest a relationship to the phenomenon of fissiparity, since it has only been described in self- dividing (fissiparous) species, such as Ophiocomella spp. and Ophiactis savignyi. This opening is thought to lead to the water vascular system (Devaney, 1970).

Peristomial plates, such as oral and dental plates, have been largely neglected as a taxonomic resource. However, a recent study suggested that the description of these plates can improve our understanding of the evolutionary processes responsible for the diversity and distribution of the extant ophiuroid fauna (Wilkie & Brogger, 2018). We hope that our detailed description of the dental and oral plates of Ophiothela sp. BR could integrate future studies of comparative morphology and systematics.

The morphometric analysis of the Ophiothela sp. BR, O. mirabilis, and O. danae shows that these species did not totally intermingle on the LDA. Brazilian specimens are closer to Ophiothela danae than to Ophiothela mirabilis. Morphometry has been used as a powerful tool for delimiting species, particularly when applied with an integrative approach (Alitto et al., 2019; Arribas, Andújar, Sánchez-Fernández, Abellán, & Millán, 2013; Di Camillo et al., 2018; Marinho, Cônsoli, Penteado-Dias, & Zucchi, 2017). In the case of O. mirabilis, the morphometry was relevant detecting patterns related to its morphological variation. We highlight the use of morphometry in several areas of biology as delimiting species and ontogenetic series in order to detect patterns undetected by the eye.

The specimens of Ophiothela mirabilis, O. danae and O. vincula form a subclade with strong support in both phylogenetic analyses based on COI fragments. The level of genetic divergence between these species was low (0.3–0.7% and 1.4–2.7% for 16S and COI, respectively). The COI levels are slightly higher than those found

147 within Ophiothrix trindadensis (about 1.5%), but they continue to be indicative of intraspecific variations (Alitto et al., 2019; Pérez‐Portela et al., 2013).

Although Ophiothela vincula Mortensen, 1913 has been considered as an accepted species (Stöhr et al., 2018), attention should be given to it for three main reasons. First is because of the low genetic divergence of COI gene between our specimens of O. mirabilis and O. vincula (2.7%). Second because O. vincula was described from a few specimens sampled from Philippines Islands, same locality as some of the O. mirabilis observed from USNM. Thirdly because the only morphological difference observed from the description of Mortensen (1913) is that O. vincula has five arms while O. mirabilis has six arms.

Due to the high number of similar species, low levels of genetic distance, and the high degree of fissiparity, Ophiothela may represent a hybrid swarm similar to Ophiocomella (O'Hara, Hugall, et al., 2018). Ophiothela mirabilis, O. vincula, and probably O. gracilis may be better thought of as samples from a larger facultative asexual complex (Birky & Barraclough, 2009). Hybridization has been evidenced in ophiuroids (Taboada & Pérez-Portela, 2016; Weber, Stöhr, & Chenuil, 2019) and we believe that the improvement of new technologies, such as next-gene sequence-capture, will provide new possibilities for testing the hybridization in Ophiothela spp.

Our findings have important taxonomic implications, particularly at the species level. But we also emphasize the necessity for an integrative approach at a family level since considerable morphological differences were found in Ophiothela. Further studies should include nuclear genes, such as 28S, in order to understand the phylogenetic relationships of the Ophiotrichidae family and its relatives.

The genus Ophiothela has been continuously expanding. The North Atlantic Ocean as well the Pacific Ocean (particularly at the coasts of North and South America) are the only sites where Ophiothela was not reported. This places Ophiothela in the same group as other fissiparous and widespread brittle stars, Ophiactis lymani and Ophiactis savignyi, which have circum-tropical and circum-subtropical distribution. We propose the use of Ophiothela sp. BR, Ophiothela mirabilis, and Ophiothela danae as a model organism to: i) explain the importance of asexual species in marine realms; ii) for testing theories concerning the evolutionary advantages of fissiparity; and iii) for testing its regeneration ability in applied biology and gene expression.

148

Acknowledgements

We are grateful to Professor Cecília Amaral (IB/UNICAMP) for coordinating Project BIOTA-Araçá and to Marine Biology Center in University of São Paulo (CEBIMar-USP). We are grateful to Camila Silva, Guilherme Corte, Helio Checon, Nathalia Padovanni, Angélica Godoy, Renato Luchetti, Fabrizio Marcondes, and Ariane Campos for helping collect and sieve all the Araçá Bay samples; Maristela Bueno for collecting the Paranaguá specimens and Alisson Santana for collecting the Salvador specimens. We are in debit to Adriane Sprogis and Stella de Ferraz for their great help with the scanning electron microscopy at UNICAMP and Karin R. Seger and Luciana B. Lourenço for all support provided with the genetic analysis at UNICAMP. Many thanks to the reviewers Luciana Martins, Leonardo Yokoyama, Fabrizio Marcondes and Fosca Pedini whose valuable comments were greatly appreciated. We also thank the Smithsonian United States National Museum for loan of specimens and Museum of Comparative Zoology, particularly Adam Baldinger, for the excellent photos provided. Finally, many thanks to the reviewers whose valuable comments were greatly appreciated.

Financial Support

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001, grant 2011/50317-5, São Paulo Research Foundation (FAPESP), grant 2018/10313-0 (FAPESP), and 2017/09987-3 (FAPESP).

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Supplementary Table 1

Sample Number of Species Study sites Observation Date Latitude Longitude Coletor Substrate Depth number specimens

Ophiothela mirabilis Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 11 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m Ophiothela mirabilis Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m Ophiothela mirabilis Porto da Barra, Salvador, BA, Brasil XI.2013 18 -13.003 -38.533 Karline Rebello, Renato Guimarães 1 sponge Callyspongia sp. 1 to 3 m Ophiothela mirabilis Porto da Barra, Salvador, BA, Brasil XI.2013 18 -13.003 -38.533 Karline Rebello, Renato Guimarães 1 sponge Callyspongia sp. 1 to 3 m Ophiothela mirabilis Porto da Barra, Salvador, BA, Brasil XI.2013 18 -13.003 -38.533 Karline Rebello, Renato Guimarães 1 sponge Callyspongia sp. 1 to 3 m Ophiothela mirabilis Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 3 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m Ophiothela mirabilis Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 10 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m Ophiothela mirabilis Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 23 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m Ophiothela mirabilis Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 24 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m Ophiothela mirabilis Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 48 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m Ophiothela mirabilis Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 50 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m Ophiothela mirabilis Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 53 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m Ophiothela mirabilis Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 57 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m Ophiothela mirabilis Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 59 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m Ophiothela mirabilis Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 67 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m Ophiothela mirabilis Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 68 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m Ophiothela mirabilis Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 74 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m Ophiothela mirabilis Porto da Barra, Salvador, BA, Brasil XI.2013 18 -13.003 -38.533 Karline Rebello, Renato Guimarães 1 sponge Callyspongia sp. 1 to 3 m Ophiothela mirabilis Ilha de Itaparica, Salvador, BA, Brasil VI.2015 12 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m Ophiothela mirabilis Ilha de Itaparica, Salvador, BA, Brasil VI.2015 12 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m Ophiothela mirabilis Ilha de Itaparica, Salvador, BA, Brasil VI.2015 12 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m Ophiothela mirabilis Ilha de Itaparica, Salvador, BA, Brasil VI.2015 12 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m Ophiothela mirabilis Ilha de Itaparica, Salvador, BA, Brasil VI.2015 12 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m Ophiothela mirabilis Ilha de Itaparica, Salvador, BA, Brasil VI.2015 12 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m Ophiothela mirabilis Ilha de Itaparica, Salvador, BA, Brasil VI.2015 12 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m Ophiothela mirabilis Ilha de Itaparica, Salvador, BA, Brasil VI.2015 12 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m Ophiothela mirabilis Ilha de Itaparica, Salvador, BA, Brasil VI.2015 12 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m Ophiothela mirabilis Porto da Barra, Salvador, BA, Brasil XI.2013 18 -13.003 -38.533 Karline Rebello, Renato Guimarães 1 sponge Callyspongia sp. 1 to 3 m Ophiothela mirabilis Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 172 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) sp. 1 m Ophiothela mirabilis Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 172 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) sp. 1 m Ophiothela mirabilis Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 172 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) sp. 1 m Ophiothela mirabilis Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 172 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) sp. 1 m Ophiothela mirabilis Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 172 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) sp. 1 m Ophiothela mirabilis Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 172 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) sp. 1 m Ophiothela mirabilis Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 172 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) sp. 1 m Ophiothela mirabilis Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 172 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) sp. 1 m Ophiothela mirabilis Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 172 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) sp. 1 m Ophiothela mirabilis Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 172 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) sp. 1 m Ophiothela mirabilis Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 172 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) sp. 1 m Ophiothela mirabilis Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 172 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) sp. 1 m Ophiothela mirabilis Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 172 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) sp. 1 m Ophiothela mirabilis Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 172 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) sp. 1 m Ophiothela mirabilis Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 172 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) sp. 1 m Ophiothela mirabilis Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 172 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) sp. 1 m Ophiothela mirabilis Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 172 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) sp. 1 m

150

Supplementary Table 1. Continued

Nº Museum Sample Number of Type of data Bio Mol Species Study sites Observation Date Latitude Longitude Coletor Substrate Depth Voucher nº number specimens Sample ZUEC OPH 2854 Morphological 181 Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 172 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) sp. 1 m ZUEC OPH 2855 Morphological 182 Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 172 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) sp. 1 m ZUEC OPH 2856 Morphological 183 Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 172 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) sp. 1 m ZUEC OPH 2837 Morphological 184 Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 12 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2838 Morphological 185 Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 12 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2839 Morphological 186 Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 12 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2840 Morphological 187 Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 12 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2841 Morphological 188 Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 12 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2842 Morphological 189 Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 12 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2843 Morphological 190 Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 12 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2844 Morphological 191 Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 12 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2845 Morphological 192 Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 12 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2846 Morphological 193 Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 12 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2857 Morphological 194 Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 56 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2858 Morphological 195 Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 58 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2859 Morphological 196 Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 63 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2860 Morphological 197 Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 64 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2861 Morphological 198 Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 65 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2871 Morphological 199 Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 66 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2872 Morphological 200 Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 69 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2873 Morphological 201 Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 60 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2874 Morphological 202 Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 60 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2875 Morphological 203 Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 60 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 1020 Morphological Ophiothela sp. BR Praia Preta, São Sebastião, SP, Brasil 13.X.2006 -23.760 -45.409 Alessandra Pereira Majer 1 2-3m ZUEC OPH 2377 Morphological, molecular Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 172 -25.423 -48.406 G. J. Silvério 3 sponge Mycale (Zygomycale ) sp. 1 m MCZ OPH-2495 Morphological - Syntype Ophiothela mirabilis Archipelago Las Perlas, Golfo de Panama, Panama 1866 8.333 -79.116 Frank H. Bradley 1 - 1838-1842 U.S. Exploring Expedition 1838-1842 MCZ OPH-2492 Morphological - Syntype Ophiothela danae Fiji Islands Pacific Ocean: Pacific, Western Central -17.02 178.06 1 - (approximate) James Dwight Dana 1838-1842 U.S. Exploring Expedition 1838-1842 MCZ OPH-2631 Morphological - Syntype Ophiothela danae Fiji Islands Pacific Ocean: Pacific, Western Central -17.02 178.06 1 - (approximate) James Dwight Dana MCZ OPH-2493 Morphological - Syntype Ophiothela danae South Africa Cape Colony: Algoa Bay Atlantic Ocean No collection date -33.888 259.644 SS Pieter Faure 1 - USNM E27240 Morphological Ophiothela mirabilis Panama Bay 26.VIII.1974 8.187 -79.521 2 - USNM E31768 Morphological Ophiothela mirabilis Panama Bay 13.IV.1972 8.187 -79.521 2 - USNM 41274 Morphological Ophiothela danae Jolo Island, Jolo, Daingapic Point, Sulu, Philippines 14.II.1908 6.16 121.364 1 37 m USNM 41275 Morphological Ophiothela danae Tapul Group, Tapul Island, NW of, Sulu, Philippines 16.II.1908 6.16 121.364 1 38 m AM J24839 Molecular J24839 Ophiothela danae New Zealand; Kermadec Islands, north west corner of North Meyer Island -29.241667 177.877 1 3-15 m WAM Molecular KGR23129 Ophiothela vincula Australia; King George River region -13.828850 127.261 1 22-29m MNHN IE.2007.4207Molecular 74.207 Ophiothela venusta Madagascar; Sud-Ouest Pointe Barrow -25.050000 44.000000 1 132-153m UF 7611 Molecular UF 7611 Ophiothela tigris Madagascar; Nosy Be, across bay from CNRO complex, off Lokobe Reserve -13.413900 48.305600 1 0-12m MV F193491 Molecular KGR28232 Ophiothela vincula Australia; King George River region -13.828850 127.261 1 - CZA E094 Molecular TANIA304 Ophiothela mirabilis Peru UNAM 3.138.36 Molecular TANIA610 Ophiothela mirabilis Mexico Pacific Chamela07_E.5_Mexico_Pacific UF 16885 Molecular UF16885 Ophiothela mirabilis Franch Guyana GUYANE_French_Guyana_CP4383 MV F168055 Molecular TOH_796 Ophiothela danae NW Australia NW_Australia_RVS_4545_D104 ZUEC OPH 2980 Morphological Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2981 Morphological Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2982 Morphological Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2983 Morphological Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2984 Morphological Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m

151

Supplementary Table 1. Continued

Nº Museum Sample Number of Type of data Bio Mol Species Study sites Observation Date Latitude Longitude Coletor Substrate Depth Voucher nº number specimens Sample ZUEC OPH 2985 Morphological Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2986 Morphological Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2987 Morphological Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2988 Morphological Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2989 Morphological Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2990 Morphological Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2991 Morphological Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2992 Morphological Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2993 Morphological Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2994 Morphological Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2995 Morphological Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2996 Morphological Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2997 Morphological Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2998 Morphological Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2999 Morphological Ophiothela sp. BR Baía do Araçá, São Sebastião, SP, Brasil 16.II.2016 -23.820 -45.394 Renata Alitto, Fabrizio Marcondes, Ariane Campos 1 coral Carijoa riisei 17 m ZUEC OPH 2940 Morphological Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) 1 m ZUEC OPH 2941 Morphological Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) 1 m ZUEC OPH 2942 Morphological Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) 1 m ZUEC OPH 2943 Morphological Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) 1 m ZUEC OPH 2944 Morphological Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) 1 m ZUEC OPH 2945 Morphological Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) 1 m ZUEC OPH 2946 Morphological Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) 1 m ZUEC OPH 2947 Morphological Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) 1 m ZUEC OPH 2948 Morphological Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) 1 m ZUEC OPH 2949 Morphological Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) 1 m ZUEC OPH 2950 Morphological Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) 1 m ZUEC OPH 2951 Morphological Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) 1 m ZUEC OPH 2952 Morphological Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) 1 m ZUEC OPH 2953 Morphological Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) 1 m ZUEC OPH 2954 Morphological Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) 1 m ZUEC OPH 2955 Morphological Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) 1 m ZUEC OPH 2956 Morphological Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) 1 m ZUEC OPH 2957 Morphological Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) 1 m ZUEC OPH 2958 Morphological Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) 1 m ZUEC OPH 2959 Morphological Ophiothela sp. BR Ilha da Banana, Paranaguá, PR, Brasil 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale (Zygomycale ) 1 m ZUEC OPH 2960 Morphological Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2961 Morphological Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2962 Morphological Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2963 Morphological Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2964 Morphological Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2965 Morphological Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2966 Morphological Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2967 Morphological Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2968 Morphological Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2969 Morphological Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2970 Morphological Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2971 Morphological Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2972 Morphological Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2973 Morphological Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2974 Morphological Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2975 Morphological Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2976 Morphological Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2977 Morphological Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2978 Morphological Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m ZUEC OPH 2979 Morphological Ophiothela sp. BR Ilha de Itaparica, Salvador, BA, Brasil VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 m

152

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CAPÍTULO 3

Unusual morphological occurrences in the small six-rayed brittle star Ophiothela sp. BR (Echinodermata: Ophiuroidea)

Renata Aparecida dos Santos Alitto, Gabriela Granadier, Ana Beardsley Christensen, Maikon Di Domenico, Michela Borges

Capítulo formatado para a revista-alvo Marine Biodiversity.

Cadastro de Acesso ao Patrimônio Genético AF4BF9C (Anexo 2), AB7038A (Anexo 4) e AACEF44 (Anexo 5).

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Unusual morphological occurrences in the small six-rayed brittle star Ophiothela sp. BR (Echinodermata: Ophiuroidea)

Renata Aparecida dos Santos Alitto*1,2 ORCID 0000-0001-9732-4877, Gabriela Granadier1,2, Ana B. Christensen3, Maikon Di Domenico4, Michela Borges2

1Departamento de Biologia Animal, Universidade Estadual de Campinas, Campinas, São Paulo, Brasil 2Museu de Zoologia “Adão José Cardoso”, Universidade Estadual de Campinas, Campinas, São Paulo, Brasil 3Department of Biology, Lamar University, Beaumont, Texas, USA 4Centro de Estudos do Mar, Universidade Federal do Paraná, Pontal do Paraná, Paraná, Brasil *corresponding author: [email protected]

Abstract Ophiothela sp. BR is considered an invasive species in Brazil appearing in 2000. It has been recorded from different localities with a high degree of fission and regeneration. However, the level of fission in Ophiothela sp. BR, as well as the role of the morphological and biological characters related to fission play in its success in marine invasions, were never discussed. A detailed morphological study with specimens from the West Atlantic Ocean is presented. The study was developed through the measure of radial shields, a first in studying the incidence of fission. All samples were divided into disc configuration (dc) designated by an x + y system (x for the number of longer older radial shields, y for the number of shorter regenerating radial shields). Two events are important to highlight, which are considered unusual for brittle stars: i) a specimen with 1 + 1 dc with only two pairs of radial shields, one long arm and one shorter arm resulting from a recent unequal fission; ii) a total of 12 dc categories comprised of different sizes of regenerated parts in the same individual. The high incidence of fission recorded in specimens from Atlantic Ocean allows us to discuss four main factors that may be correlated: disturbance in the environment, epizoic habit, absence of mates, and hybridization. Our results show the diversification of Ophiothela sp. BR, highlighting its fissiparous ability involved in the marine invasions, which is changing the appearance of coral reefs, sponges, algae and other echinoderms. Keywords: fissiparous, regeneration, echinoderms, disc diameter, disc configuration

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General information Funding: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001; grant 2011/50317-5, São Paulo Research Foundation (FAPESP), grant 2018/10313-0 (FAPESP), and 2017/09987-3 (FAPESP).

Conflict of Interest: The authors declare that they have no conflict of interest.

Ethical approval: All applicable international, national, and/or institutional guidelines for animal testing, animal care and use of were followed by the authors.

Sampling and field studies: All necessary permits for sampling and observational field studies have been obtained by the authors from the competent authorities and are mentioned in the acknowledgements, if applicable. The study is compliant with CBD and Nagoya protocols.

1) Introduction Echinoderms, particularly asteroids, ophiuroids, and holothuroids, are capable of asexual reproduction by fissiparity, a process wherein the central disc divides in two and each half forms a complete animal by regeneration (Emson and Wilkie 1980, 1984; Mladenov 1996). Fissiparity confers many advantages, such as enhanced survival of the progeny and maintaining the size of the population (Mladenov et al. 1983). It also may be an effective strategy for replicating a successful genotype, or like viviparity, for founding a population with a limited number of propagules (Hendler 1991).

There are currently 73 echinoderm species known to be capable of reproducing asexually by fission, of these 44 are ophiuroids (Mladenov 1996). These species are commonly six-rayed possessing a set of three long and three shorter arms. An example is Ophiothela sp. BR, which is small, epizoic, and occurs in a great variety of colors (Clark 1976; Alitto et al. 2018).

Ophiothela sp. BR belongs to the family Ophiotrichidae, which is considered a problem for taxonomists due to its poor morphological delineation with many overlapping features (Clark 1966; Hendler 2005). The genus Ophiothela presently includes five species (Stöhr et al. 2018).

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It was originally thought that Ophiothela was confined to Pacific waters (Clark 1976). However, since 2000, it has been recorded in the Atlantic Ocean (Hendler et al. 2012) and is considered an invasive species (Hendler and Brugneaux 2013). Two main hypotheses have been used to explain the presence of Ophiothela sp. BR in the Atlantic Ocean (Hendler and Brugneaux 2013): i) spreading in ballast water or in the fouling communities on marine vessels; or ii) planktonic larvae and/or rafting adults carried by oceanic currents. Since then, the rapid expansion of Ophiothela sp. BR has been changing the appearance of several invertebrates (i.e. sponges, corals, echinoderms), algae, and seahorses in Brazil which are densely covered by this species (Mantelatto et al. 2016).

Many studies have reported the occurrence of Ophiothela in different localities from the Atlantic Ocean with a high degree of fission and regeneration (Hendler et al. 2012; Mantelatto et al. 2016; Alitto et al. 2018; Araújo et al. 2018; Lawley et al. 2018). However, the level of fission in Ophiothela sp. BR, as well as the role of the morphological and biological characters related to fission play in its success in marine invasions, were never discussed. Our purpose was to address these issues by presenting a detailed morphological and morphometrical study with specimens from the West Atlantic Ocean (Northeast and Southeast of Brazil). Our report describes unusual morphological occurrences and discusses the main factors that may be correlated with the high incidence of fission events recorded in the samples from the West Atlantic Ocean.

2) Material and methods Institutional abbreviations: MCZ, Museum of Comparative Zoology; USNM, United States National Museum, Smithsonian Institution; ZUEC OPH, scientific collection of Ophiuroidea from the Museum of Zoology of the University of Campinas.

Abbreviations: dc, disc configuration; AB, Araçá Bay, Brazil; ECP, Estuarine Complex of Paranaguá, Brazil; TBS, Todos os Santos Bay, Brazil; spm, specimen(s).

2.1) Study sites and data collection

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A total of 131 specimens of Ophiothela sp. BR were sampled from three sites in Brazil: i) Todos os Santos Bay (44 spms), ii) Araçá Bay (44 spms), and iii) Estuarine Complex of Paranaguá (43 spms) (Fig. 1). All specimens were fixed and preserved in 70% or 90% ethanol and deposited in ZUEC OPH.

Figure 1. Study sites and data collection. Triangles represent the localities where samples were collected during the present study. The shapefile was imported from the Natural Earth project (the 1:50m resolution version).

The following is a brief description of each location as well as the sampling strategies that were used.

Todos os Santos Bay (TSB) TSB is located on the coast of the State of Bahia, Brazil. TSB has an approximate area of 1223 km2 (Cirano and Lessa 2007) and has approximately 200 km of coastline and encompasses a series of beaches, peninsulas, coves, and several islands, such as Itaparica located at its entrance (Manso et al. 2008).

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Brittle stars were collected from two sites by hand from 1 to 3 m deep in November 2013 and June 2015: i) at Itaparica Island on the coral Carijoa riisei; ii) at Porto da Barra on the sponges Callyspongia sp. and Desmapsamma sp.

Araçá Bay (AB) AB is located on the northeastern coast of the State of São Paulo, Brazil. It encompasses three beaches, two small islets, three main stands of mangrove, rocky shores, and an extensive muddy sand flat extending to the subtidal zone. The latter comprises a vast intertidal flat up to 300 m wide (Amaral et al. 2010; Amaral et al. 2016).

Brittle stars were collected during the project BIOTA/FAPESP – Araçá, in February 2016 from rubble bottom by dredge at 17 m deep. All the specimens were associated with the coral Carijoa riisei.

Estuarine Complex of Paranaguá (ECP) ECP is located on the southern coast of the State of Paraná, Brazil. The estuary measures 612 km2 and it is part of the largest remaining sectors of the Atlantic Rain Forest along the Brazilian coast. ECP is part of a large interconnected subtropical estuarine system comprised of two main water bodies, extensive sandy beaches, and rocky shores (Lana et al. 2001).

Brittle stars were collected in July 2014 in the intertidal zone of Banana Island by hand at 1 m deep. All the specimens were associated with the sponge Mycale (Zygomycale).

A detailed list of all specimens used is presented in Supplementary Table 1.

2.2) Taxonomic study The specimens were identified based on descriptions of the most comprehensive studies (Verrill 1867, 1869; Clark 1976; Cherbonnier and Guille 1978; Alitto et al. 2019, 2nd chapter). Each individual was studied under a stereomicroscope (Stemi 2000 ZEISS). The specimens of interest were chosen for photography with a video camera (ZEISS TK 1270U) mounted on a stereomicroscope. The photos were organized into plates using Adobe Photoshop.

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2.3) Morphometry Measurements of all the radial shields of each specimen were taken using an ocular micrometer and through the AxioVision VS program 40.4.8.20 (Carl Zeiss Microscopy, Germany) attached to a ZEISS Discovery V20 stereomicroscope or by using digital measurement (Adobe Photoshop®). The length of the radial shield was measured from the distal edge to the proximal edge.

All samples were divided into disc configuration (dc). Each dc category was designated by an x + y system, with x for the number of longer older radial shields and y for the number of shorter regenerating radial shields. Shorter radial shields were defined as those with < 50% length of the longest radial shields.

It was not possible to measure the arms of Ophiothela sp. BR due to the dorsoventral coiling of the arms. Therefore, the methodology used here is a first in studying the incidence of fission through measures of the disc, particularly radial shields. The designation x + y was adapted from Clark (1967) and Mladenov et al. (1983) where the authors used the same system, but applied to arm configuration. The definitions of longest and shortest was adapted from Rubilar et al. (2005) where the authors used them to study the arm configuration in asteroids.

3) Results Ophiothela sp. BR exhibits a great variety of dc which were classified into 12 categories:

1) (1 + 1): 1 pair of radial shields with > 50% length of the 1 shortest pair of radial shields; 1 long arm and 1 shorter arm resulting from a recent unequal fission (Fig. 2A)

2) (1 + 5): 1 pair of radial shields with > 50% length of the 5 shortest pairs of radial shields; 1 long arm and 5 shorter arms resulting from an unequal fission (Fig. 2B)

3) (1 + 6): 1 pair of radial shields with > 50% length of the 6 shortest pairs of radial shields; 1 long arm and 5 shorter arms resulting from an unequal fission (Fig. 2C)

4) (2 + 4): 2 pairs of radial shields with > 50% length of the 4 shortest pairs of radial shields; 2 long arms and 4 shorter arms resulting from an unequal fission (Fig. 2D)

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5) (2 + 5): 2 pairs of radial shields with > 50% length of the 5 shortest pairs of radial shields; 2 long arms and 5 shorter arms resulting from an unequal fission (Fig. 2E)

6) (3 + 0): 3 pairs of radial shields with similar lengths; 3 long arms resulting from a recent equal fission (Fig. 2F)

7) (3 + 3): 3 pairs of radial shields with > 50% length of the 3 shorter pairs of radial shields; 3 long arms and 3 shorter arms (Fig. 2G)

8) (3 + 4): 3 pairs of radial shields with > 50% length of the 4 shorter pairs of radial shields; 3 long arms and 4 shorter arms (Fig. 2H)

9) (4 + 2): 4 pairs of radial shields with > 50% length of the 2 shortest pairs of radial shields; 4 long arms and 2 shorter arms resulting from an unequal fission (Fig. 3A)

10) (4 + 3): 4 pairs of radial shields with > 50% length of the 3 shortest pairs of radial shields; 4 long arms and 3 shorter arms resulting from an unequal fission (Fig. 3B)

11) (5 + 1): 5 pairs of radial shields with > 50% length of the 1 shortest pair of radial shields; 5 long arms and 1 shorter arm resulting from an unequal fission (Fig. 3C)

12) (6 + 0): 6 pairs of radial shields with similar lengths; 6 long arms (Fig. 3D)

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Figure 2. Disc configurations exhibited by Ophiothela sp. BR: A) 1 + 1 (ZUEC OPH 2882), B) 1 + 5 (ZUEC OPH 2855), C) 1 + 6 (ZUEC OPH 2959), D) 2 + 4 (ZUEC OPH 2850), E) 2 + 5 (ZUEC OPH 2377), F) 3 + 0 (ZUEC OPH 2877), G) 3 + 3 (ZUEC OPH 2876), H) 3 + 4 (ZUEC OPH 2944). Each type of disc configuration is designated as described in the text. Scale bar = 0.5 mm.

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Figure 3. Disc configurations exhibited by Ophiothela sp. BR: A) 4 + 2 (ZUEC OPH 2878), B) 4 + 3 (ZUEC OPH 2951), C) 5 + 1 (ZUEC OPH 2985), D) 6 + 0 (ZUEC OPH 2822). Each type of disc configuration is designated as described in the text. Scale bar = 0.5 mm.

Specimens 6 + 0 dc (Fig. 3D), easily recognized by having 6 long arms with almost the same length, were less frequent (35% of spms). The majority of specimens of Ophiothela sp. BR were in some stage of regeneration (65% of spms) and they were divided into three groups.

Group I is composed of specimens with different sizes of regenerated parts in the same individual (24% of spms) here called as unusual events. In this group, 9 dc categories were observed: 1 + 1 (Fig. 2A), 1 + 5 (Fig. 2B), 1 + 6 (Fig. 2C), 2 + 4 (Fig. 2D), 2 + 5 (Fig. 2E), 3 + 4 (Fig. 2H), 4 + 2 (Fig. 3A), 4 + 3 (Fig. 3B), and 5 + 1 (Fig. 3C). These may indicate fission events taking place before animals fully regenerate from the previous event and splitting along different planes with each fission.

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Group II is comprised of specimens 3 + 0 dc (22% of spms, Fig. 2F). This group recently experienced fission and has yet to regenerate the disc and arms. We highlight one specimen with three different lengths of radial shields suggesting three stages of recovery following fission.

Group III is composed of specimens 3 + 3 dc (18% of spms, Fig. 2G). This group appears to have recently undergone fission as one half of the disc and the new arms are distinctly shorter than the other half and arms.

4) Discussion Ophiothela sp. BR from the West Atlantic Ocean has large capacity for fission as evidenced by the large number of dc categories. Moreover, we observed unusual morphological occurrences and different sizes of regenerated parts in the same specimen, indicating the ability of Ophiothela sp. BR to split itself before fully regenerating. Since it is considered an invasive species in Brazil (original distribution: Pacific Ocean), our results demonstrate the imperative need to identify the initiators of fission, highlighting this ability of Ophiothela sp. BR as key to its success in marine invasions.

We suggest that specimens from West Atlantic have prioritized asexual reproduction through fissiparity for two reasons. First, because individuals with ripe gonads were not observed, indicating that sexual reproduction is very infrequent or occurs at a constant, but very low and hence undetectable rate. Second, because of the great number of specimens with various stages of recovery following fission, suggesting the ability of Ophiothela sp. BR to split itself before fully regenerating.

Unusual morphological occurrences

Two unusual morphological occurrences in Ophiothela sp. BR were described, both related to its fission capacity:

i) A specimen with 1 + 1 dc with only two pairs of radial shields, one long arm and one shorter arm. This is possibly a consequence of a recent unequal fission or the result of a predatory event, in which the regeneration of the new arm probably began from a single portion of the disc and its arm. This event has only been reported for Ophiocomella ophiactoides in one experiment involving attempts to initiate fission by

170 means of small surgical incisions in the disc (Mladenov et al. 1983). The 1 + 1 dc is considered unusual for ophiuroids, and so far, only described for some asteroids, where new arms can grow if a portion of the central disc remains attached (Ducati et al. 2004; Candia Carnevali 2006). However, we cannot discard the hypothesis of another mechanism only described for some asteroids species where a single arm can regenerate a whole animal, called a comet stage (Ben Khadra et al. 2017). Laboratory testing with living specimens are needed to verify these premises.

ii) A total of 11 dc categories composed of different sizes of regenerated parts in the same individual. These dc categories suggest the ability of Ophiothela sp. BR to split itself before fully regenerating. This may be one of its keys to being successful in the West Atlantic Ocean or any other new area, where the population is in various stages of regeneration following fission. This strategy minimizes the interval between successive fissions and maximizes the increase in specimen numbers. Similar events were reported in Ophiactis virens (Simroth 1877) and Ophiocomella ophiactoides (Mladenov et al. 1983) and are suggested for Ophiothela mirabilis, Ophiactis savignyi, Ophiactis modesta, and Astroceras annulatum (Yamazi 1950).

Morphometric analyses

Morphometric analyses have been used as a powerful tool for delimiting species, particularly when applied with an integrative approach (Arribas et al. 2013; Marinho et al. 2017; Di Camillo et al. 2018). However, morphometry may also have other uses as to compare sizes of intraspecific specimens between different localities, i.e. Sanches et al. (2016), or assist in the description of ontogenetic series (Stöhr 2005; Borges et al. 2015; Martynov et al. 2015).

Our morphometric analysis of Ophiothela sp. BR allowed us to compare the lengths of the radial shields in the same specimen revealing different dc categories and suggesting that Ophiothela sp. BR splits itself before fully regenerating. Therefore, we highlight the use of morphometry as a powerful tool to detect patterns undetected by the eye.

Factors influencing asexual reproductive processes

Ophiothela sp. BR has demonstrated its great ability to colonize new habitats through the production of multiple clones well adapted to local conditions.

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Hence, due to its evident adaptive and strategic significance/value, regeneration certainly contributes to its fitness and success throughout the marine ecosystem (Ben Khadra et al. 2018).

Environmental stress of various kinds may mediate fission through stimulation of the nervous system (Mladenov 1996). The most significant study that has investigated the possible initiators of fission was developed by Mladenov et al. (1983). The authors tested several factors, including salinity, temperature, hypoxia, mechanical deformation, and incisions in the disc on the brittle star Ophiocomella ophiactoides. They concluded that the external factors tested did not play an important role in the induction of fission in this species and they suggested that internal factors of some sort are involved in the process. In contrast, for another fissiparous species Ophiactis lymani, the fission may be related to the low salinity (Lima et al. 2013). For Ophiactis savignyi, McGovern (2002) compared the seasonal peaks in asexual reproduction of specimens from Taiwan and Florida Keys. The author concluded that the peaks occur earlier in Taiwan than in Florida Keys, possibly as a result of the many environmental differences that likely exist between these two localities.

Temperature may be a limiting factor for the development of marine invertebrates. For instance, Ophiothela sp. BR is absent in temperatures below 16°C, characteristic of waters from the South of Brazil (Lawley et al. 2018). On the other hand, the higher temperatures may be favoring it as well as other invasive species like the sun corals. While high temperatures lead to extensive bleaching in native Brazilian corals, the sun corals Tubastraea spp., continue to regenerate (Luz et al. 2018). These studies demonstrate the importance of investigating the initiators of fission and the factor that is favoring the regeneration of Ophiothela sp. BR in Brazil. Therefore, all this information needs to be considered during management efforts of invasive species.

There appears to be a correlation between fissiparity and epizoic habit in ophiuroids (Clark 1967, 1976). However, the converse is not the case since there is a larger number of non-fissiparous species that have an epizoic habit (Clark 1976). Heretofore, this premise has not been tested. Beyond fission, Ophiothela sp. BR has another set of characters that assist it in an epizoic habit: i) sharp teeth on the arm spines which serve as hooks for adhesion (Verrill 1867); ii) streptospondylous joint with a hourglass-shaped articulation (Alitto et al. 2019, 2nd chapter) allowing it to flex its arms vertically (Litvinova 1994); iii) the reduction and absence of dorsal arm plates (Alitto et

172 al. 2018; Alitto et al. 2019, 2nd chapter) enabling greater movement of its arm vertically; iv) rounded granules scattered over the entire dorsal surface as disc and arms (Verrill 1867; Alitto et al. 2019, 2nd chapter) which we believe cause more friction with the water reducing the chances of its being carried away.

These features may help explain the extreme ability of Ophiothela sp. BR to be opportunistic and a generalist in relation to its host species. The most recent study reported 29 novel host groups, including sponges, cnidarians, ascidians, echinoderms (sea cucumber and sea urchin), algae, bryozoans, and seahorses used by Ophiothela in Brazil (Mantelatto et al. 2016). Since the implications of this association are still unclear, we emphasize the need for further studies.

Sexual reproduction in Ophiothela sp. BR has not been investigated, so we do not know if it is monoecious or dioecious. Therefore, the hypothesis of Ophiothela sp. BR having high incidence of fission due to the absence of mates cannot be discarded. This is called plastic reproductive allocation and it was reported in Ophiactis savignyi (McGovern 2003), an epizoic, fissiparous and widely distributed brittle star, like Ophiothela sp. BR. The plasticity in O. savignyi is likely adaptive because: i) it maximizes sexual-reproductive potential when fertilization is more probable; and ii) maximizes survival of the clone when mates are absent, and gametes are unlikely to be converted to offspring (McGovern 2003).

The high incidence of fission in Ophiothela sp. BR may also be associated with the presence of hybrid and sterile specimens. This is because hybrids between closely related species are often inviable or, if they live, they are sterile, and their presence can reduce the exchange of genetic variants between species (Johnson 2008). Despite the complexity in being detected, hybridization has been evidenced as a frequent phenomenon (Weber et al. 2019), widespread, diverse in form, with a potential to contribute to individual speciation events (Abbott et al. 2013). In ophiuroids, the most recent studies that have suggested hybridization are: Stöhr and Muths (2010) for Acrocnida spp., Hugall et al. (2016) for Amphistigma minuta, Taboada and Pérez- Portela (2016) for Ophiothrix fragilis, and Weber et al. (2019) for Ophioderma spp. We believe that the improvement of new technologies, such as the next-generation sequence-capture methodology, will provide new possibilities for testing the possibility of hybridization in Ophiothela spp.

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5) Acknowledgments We are grateful to Professor Cecília Amaral (IB/UNICAMP) for coordinating Project BIOTA-Araçá and to Marine Biology Center in University of São Paulo (CEBIMar-USP). Many thanks to Alisson Santana, Karline Rebello, and Renato Guimarães for collecting the Salvador samples, Gilson Silvério and Maristela Bueno for collecting the Paranaguá specimens, and Camila Silva, Guilherme Corte, Helio Checon, Nathalia Padovanni, Angélica Godoy, Renato Luchetti, Fabrizio Marcondes, and Ariane Campos for helping collect and sieve all the Araçá Bay samples. Finally, many thanks to the reviewers Luciana Martins, Leonardo Yokoyama, Fabrizio Marcondes and Fosca Pedini whose valuable comments were greatly appreciated. Field work was authorized by the Chico Mendes Institute for Biodiversity Conservation – ICMBio, under the license number: 19887-1 to Cecília Amaral and 38463-3 to Maristela Bueno. All data were registered at the National System for the Management of Genetic Heritage and Associated Traditional Knowledge – SisGen, according to Brazilian legislation Law number 13.123/2015 and Decree 8772/2016. Approval ID for this study were AF4BF9C, AB7038A, and AACEF44.

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6) Supplementary Table 1 Specimens of Ophiothela sp. BR used in the present study. NS, number of specimens.

Museum Study sites Date Latitude Longitude Colector Number of Substrate Depth Voucher nº specimens

ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2863 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2864 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2865 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2920 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2866 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2867 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2868 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2869 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2879 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2870 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2847 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2848 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2849 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2850 Brasil (Zygomycale)

175

ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2851 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2852 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2853 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2854 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2855 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2856 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 3 sponge Mycale 1 m 2377 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2940 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2941 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2942 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2943 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2944 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2945 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2946 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2947 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2948 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2949 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2950 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2951 Brasil (Zygomycale)

176

ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2952 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2953 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2954 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2955 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2956 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2957 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2958 Brasil (Zygomycale) ZUEC OPH Ilha da Banana, Paranaguá, PR, 20.VII.2014 -25.423 -48.406 G. J. Silvério 1 sponges Mycale 1 m 2959 Brasil (Zygomycale) ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2814 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2883 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2815 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2816 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2817 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2818 SP, Brasil Fabrizio

177

Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2819 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2820 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2821 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2822 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2823 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2824 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2825 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2826 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2857 SP, Brasil Fabrizio Marcondes, Ariane Campos

178

ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2858 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2859 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2860 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2861 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2871 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2872 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2873 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2874 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2875 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2980 SP, Brasil Fabrizio

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Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2981 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2982 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2983 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2984 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2985 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2986 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2987 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2988 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2989 SP, Brasil Fabrizio Marcondes, Ariane Campos

180

ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2990 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2991 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2992 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2993 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2994 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2995 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2996 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2997 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2998 SP, Brasil Fabrizio Marcondes, Ariane Campos ZUEC OPH Baía do Araçá, São Sebastião, 16.II.2016 -23.820 -45.394 Renata Alitto, 1 coral Carijoa riisei 17 m 2999 SP, Brasil Fabrizio

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Marcondes, Ariane Campos ZUEC OPH Porto da Barra, Salvador, BA, XI.2013 -13.003 -38.533 Karline Rebello, 1 sponge Callyspongia 1 to 3 2876 Brasil Renato Guimarães sp. m ZUEC OPH Porto da Barra, Salvador, BA, XI.2013 -13.003 -38.533 Karline Rebello, 1 sponge Callyspongia 1 to 3 2862 Brasil Renato Guimarães sp. m ZUEC OPH Porto da Barra, Salvador, BA, XI.2013 -13.003 -38.533 Karline Rebello, 1 sponge Callyspongia 1 to 3 2877 Brasil Renato Guimarães sp. m ZUEC OPH Porto da Barra, Salvador, BA, XI.2013 -13.003 -38.533 Karline Rebello, 1 sponge Callyspongia 1 to 3 2827 Brasil Renato Guimarães sp. m ZUEC OPH Porto da Barra, Salvador, BA, XI.2013 -13.003 -38.533 Karline Rebello, 1 sponge Callyspongia 1 to 3 2836 Brasil Renato Guimarães sp. m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2828 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2829 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2830 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2831 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2832 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2833 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2834 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2835 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2878 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2837 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2838 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2839 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2840 Brasil m

182

ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2841 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2842 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2843 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2844 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2845 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2846 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2960 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2961 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2962 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2963 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2964 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2965 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2966 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2967 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2968 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2969 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2970 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2971 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2972 Brasil m

183

ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2973 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2974 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2975 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2976 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2977 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2978 Brasil m ZUEC OPH Ilha de Itaparica, Salvador, BA, VI.2015 -12.999 -38.577 Alisson Santana 1 coral Carijoa riisei 1 to 3 2979 Brasil m

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7) Supplementary Table 2 Measurements of all specimens (mm). See Table 1 for definitions of the morphological characters used for the morphometric analysis. Bold measure represents the longest radial shields; * represents shorter radial shields defined as those with < 50% length of the longest radial shields.

Locality Voucher number Disc configuration dd rs_l1 rs_l2 rs_l3 rs_l4 rs_l5 rs_l6 rs_l7 AB ZUEC OPH 2883 1+1 0.90 0.90 0.32* ECP ZUEC OPH 2853 1+5 1.50 0.80 0.38* 0.30* 0.20* 0.29* 0.39* ECP ZUEC OPH 2855 1+5 1.60 1.10 0.49* 0.45* 0.26* 0.30* 0.35* ECP ZUEC OPH 2959 1+6 2.20 0.90 0.39* 0.42* 0.41* 0.38* 0.38* 0.39* ECP ZUEC OPH 2952 2+4 1.90 0.30* 0.70 0.60 0.20* 0.20* 0.20* ECP ZUEC OPH 2850 2+4 2.00 0.97 0.48* 0.47* 0.46* 0.47* (0.94) ECP ZUEC OPH 2377 2+5 1.34 0.76 0.74 0.35* 0.28* 0.32* 0.37* 0.33*

ECP ZUEC OPH 2940 3+0 4.70 2.50 2.10 2.20

ECP ZUEC OPH 2950 3+0 1.70 0.80 0.60 1.10

ECP ZUEC OPH 2958 3+0 2.90 2.30 1.00 0.70

TSB ZUEC OPH 2961 3+0 2.20 0.80 0.90 1.10

TSB ZUEC OPH 2962 3+0 2.70 1.30 1.20 1.20

TSB ZUEC OPH 2964 3+0 1.80 0.80 0.70 0.70

TSB ZUEC OPH 2966 3+0 2.70 1.50 1.20 1.00

TSB ZUEC OPH 2967 3+0 2.50 1.10 1.40 1.20

TSB ZUEC OPH 2969 3+0 1.70 0.80 0.60 0.60

TSB ZUEC OPH 2970 3+0 2.20 1.10 0.90 0.60

TSB ZUEC OPH 2971 3+0 2.70 0.70 1.30 1.00

TSB ZUEC OPH 2972 3+0 2.20 0.90 0.90 0.80

TSB ZUEC OPH 2973 3+0 4.10 2.10 1.90 1.40

TSB ZUEC OPH 2974 3+0 3.10 1.80 1.00 1.20

TSB ZUEC OPH 2978 3+0 3.60 1.80 1.60 0.80

AB ZUEC OPH 2988 3+0 3.20 1.50 2.00 1.50

185

AB ZUEC OPH 2993 3+0 4.80 2.40 2.80 2.10

ECP ZUEC OPH 2377 3+0 1.73 1.26 0.84 0.77

AB ZUEC OPH 2824 3+0 1.30 0.80 0.66 0.52

TSB ZUEC OPH 2827 3+0 1.30 0.80 0.62 0.36

TSB ZUEC OPH 2836 3+0 1.30 0.50 0.44 0.64

TSB ZUEC OPH 2840 3+0 2.50 1.20 1.27 0.96

TSB ZUEC OPH 2842 3+0 1.40 0.90 0.97 0.68

TSB ZUEC OPH 2844 3+0 1.91 1.00 0.68 0.77

TSB ZUEC OPH 2845 3+0 1.22 0.70 0.46 0.35

TSB ZUEC OPH 2846 3+0 1.41 0.60 0.66 0.72

AB ZUEC OPH 2860 3+0 1.60 0.90 0.65 0.70

AB ZUEC OPH 2861 3+0 1.00 0.60 0.62 0.48

TSB ZUEC OPH 2877 3+0 0.70 0.60 0.69 0.63 ECP ZUEC OPH 2945 3+3 3.00 1.80 0.70* 0.60* 0.80* 1.20 1.00 ECP ZUEC OPH 2954 3+3 3.20 1.30 1.80 1.20 0.60* 0.70* 0.80* ECP ZUEC OPH 2955 3+3 2.50 1.40 0.90 1.10 0.30* 0.50* 0.60* ECP ZUEC OPH 2957 3+3 2.40 1.10 1.10 1.30 0.60* 0.60* 0.60* TSB ZUEC OPH 2977 3+3 6.00 3.20 2.30 2.40 1.20* 1.20* 1.20* ECP ZUEC OPH 2377 3+3 1.60 0.93 0.64 0.58 0.21* 0.27* 0.30* AB ZUEC OPH 2816 3+3 1.20 0.70 0.35* 0.28* 0.34* 0.62 0.48 TSB ZUEC OPH 2833 3+3 1.50 0.95 0.53 0.30* 0.41* 0.21* 0.64 TSB ZUEC OPH 2835 3+3 1.50 0.84 0.63 0.36* 0.42 0.33* 0.41* TSB ZUEC OPH 2843 3+3 1.50 0.80 0.64 0.58 0.36* 0.23* 0.30* ECP ZUEC OPH 2851 3+3 1.40 0.80 0.36* 0.32* 0.39* 0.61 0.65 ECP ZUEC OPH 2852 3+3 1.80 0.85 0.40* 0.42* 0.42* 0.55 0.61 ECP ZUEC OPH 2856 3+3 1.50 0.80 0.60 0.46 0.32* 0.39* 0.36* AB ZUEC OPH 2859 3+3 1.30 0.80 0.57 0.32* 0.25* 0.24* 0.45 TSB ZUEC OPH 2862 3+3 1.60 0.60 0.47 0.33* 0.33* 0.23* 0.84

186

ECP ZUEC OPH 2864 3+3 1.70 0.90 0.33* 0.40* 0.65 0.47 0.40* ECP ZUEC OPH 2865 3+3 1.80 0.90 0.53 0.43* 0.38* 0.45* 0.59 ECP ZUEC OPH 2866 3+3 2.10 1.30 1.28 0.59* 0.33* 0.44* 0.87 ECP ZUEC OPH 2867 3+3 1.70 0.90 0.81 0.42* 0.44* 0.31* 0.51 ECP ZUEC OPH 2868 3+3 1.80 1.10 0.65 0.33* 0.44* 0.49* 1.01 ECP ZUEC OPH 2870 3+3 1.30 0.80 0.78 0.55 0.19* 0.32* 0.37* AB ZUEC OPH 2871 3+3 1.50 0.70 0.27* 0.27* 0.28* 0.56 0.74 AB ZUEC OPH 2872 3+3 1.30 0.70 0.31* 0.57 0.35* 0.35* 0.42 TSB ZUEC OPH 2876 3+3 1.40 0.75 0.69 0.48* 0.38* 0.28* 0.60 ECP ZUEC OPH 2944 3+4 3.50 0.70 0.60 0.46 0.20* 0.29* 0.29* ECP ZUEC OPH 2948 4+2 3.40 1.90 1.50 1.40 0.80* 1.00 0.80* ECP ZUEC OPH 2949 4+2 2.70 1.40 1.30 1.00 0.60* 0.40* 1.00 TSB ZUEC OPH 2960 4+2 3.30 1.60 1.20 1.10 1.00 0.60* 0.50* TSB ZUEC OPH 2963 4+2 2.70 1.20 1.40 1.00 0.80 0.40* 0.60* TSB ZUEC OPH 2965 4+2 2.30 1.20 1.00 0.60* 0.90 0.60* 0.70 TSB ZUEC OPH 2975 4+2 4.30 2.10 2.30 2.10 1.20 0.80* 1.10* AB ZUEC OPH 2982 4+2 4.30 2.20 2.50 0.90* 1.30 0.90* 2.20 AB ZUEC OPH 2992 4+2 4.60 2.50 1.00* 1.10* 1.40 1.80 2.00 TSB ZUEC OPH 2830 4+2 1.80 0.90 0.79 0.63 0.43* 0.39* 0.62 TSB ZUEC OPH 2831 4+2 1.70 0.80 0.43 0.35* 0.33* 0.63 0.81 TSB ZUEC OPH 2839 4+2 2.50 1.55 1.22 0.77* 0.69* 0.81 1.04 TSB ZUEC OPH 2841 4+2 2.10 1.09 0.53* 0.46* 0.63 0.93 0.62 ECP ZUEC OPH 2854 4+2 2.00 1.00 0.73 0.56 0.47* 0.50* 0.70 TSB ZUEC OPH 2878 4+2 2.50 1.30 1.22 0.84 0.87 0.59* 0.60* ECP ZUEC OPH 2951 4+3 2.30 1.10 0.80 1.30 0.50* 0.40* 0.40* 0.55 ECP ZUEC OPH 2941 5+1 3.60 1.30 1.20 1.30 0.70 0.70 0.60* ECP ZUEC OPH 2943 5+1 5.50 2.30 1.10* 1.40 1.90 2.80 2.20 ECP ZUEC OPH 2956 5+1 3.20 1.40 1.00 1.10 0.90 1.00 0.60*

187

TSB ZUEC OPH 2968 5+1 1.80 1.00 0.90 0.80 0.40* 0.60 0.70 TSB ZUEC OPH 2976 5+1 3.90 1.80 2.20 1.30 1.00* 1.40 1.70 TSB ZUEC OPH 2979 5+1 3.60 1.80 1.70 1.60 0.90 0.80* 1.10 AB ZUEC OPH 2989 5+1 7.30 3.80 3.00 3.20 2.50 1.50* 2.50 AB ZUEC OPH 2990 5+1 5.30 3.40 2.70 2.60 2.40 1.60* 2.30 AB ZUEC OPH 2997 5+1 4.90 3.00 1.80 1.30* 1.80 2.20 2.40 ECP ZUEC OPH 2942 6+0 5.10 1.70 1.30 1.60 2.60 1.50 1.90 ECP ZUEC OPH 2946 6+0 3.40 1.40 1.40 1.30 1.10 1.00 1.90 ECP ZUEC OPH 2947 6+0 3.10 0.90 1.20 0.90 1.10 1.20 1.50 ECP ZUEC OPH 2953 6+0 2.90 1.30 1.50 0.90 0.90 1.00 0.80 AB ZUEC OPH 2980 6+0 0.70 0.40 0.30 0.20 0.30 0.40 0.40 AB ZUEC OPH 2981 6+0 6.10 2.90 2.90 1.90 2.00 2.40 2.60 AB ZUEC OPH 2983 6+0 6.00 2.90 2.90 2.70 1.80 2.50 2.50 AB ZUEC OPH 2984 6+0 6.50 3.30 3.50 3.00 1.80 2.50 2.40 AB ZUEC OPH 2985 6+0 7.70 3.40 3.60 3.50 3.00 2.40 2.30 AB ZUEC OPH 2986 6+0 4.80 2.60 2.40 1.50 1.70 1.90 2.30 AB ZUEC OPH 2987 6+0 6.40 3.10 2.60 2.90 2.00 2.00 2.00 AB ZUEC OPH 2991 6+0 5.40 2.80 2.30 2.00 2.10 2.40 2.30 AB ZUEC OPH 2994 6+0 4.90 2.80 2.30 1.40 1.70 2.10 2.20 AB ZUEC OPH 2995 6+0 6.80 3.90 3.30 2.40 2.10 2.90 3.20 AB ZUEC OPH 2996 6+0 7.20 3.10 2.70 2.70 3.40 2.40 3.10 AB ZUEC OPH 2998 6+0 1.30 0.70 0.50 0.40 0.50 0.60 0.60 AB ZUEC OPH 2999 6+0 1.20 0.50 0.50 0.50 0.50 0.50 0.50 AB ZUEC OPH 2814 6+0 1.50 0.70 0.67 0.57 0.59 0.36 0.42 AB ZUEC OPH 2815 6+0 1.40 0.70 0.75 0.57 0.45 0.44 0.51 AB ZUEC OPH 2817 6+0 1.60 0.60 0.49 0.50 0.90 0.76 0.69 AB ZUEC OPH 2818 6+0 1.30 0.76 0.68 0.72 0.52 0.53 0.43 AB ZUEC OPH 2819 6+0 1.50 0.70 0.63 0.46 0.39 0.49 0.76

188

AB ZUEC OPH 2820 6+0 1.90 0.90 0.81 0.66 0.57 0.68 0.75 AB ZUEC OPH 2821 6+0 1.50 0.80 0.66 0.49 0.56 0.71 0.75 AB ZUEC OPH 2822 6+0 1.44 0.70 0.60 0.69 0.45 0.61 0.58 AB ZUEC OPH 2823 6+0 1.50 0.60 0.72 0.77 0.51 0.41 0.52 AB ZUEC OPH 2825 6+0 1.50 0.60 0.63 0.55 0.59 0.61 0.51 AB ZUEC OPH 2826 6+0 1.30 0.50 0.45 0.41 0.40 0.74 0.68 TSB ZUEC OPH 2828 6+0 2.00 1.20 0.68 0.66 0.71 0.98 1.00 TSB ZUEC OPH 2829 6+0 2.00 1.00 1.03 0.83 0.58 0.61 0.85 TSB ZUEC OPH 2832 6+0 1.70 0.70 0.86 0.49 0.88 0.61 0.66 TSB ZUEC OPH 2834 6+0 2.10 1.00 0.84 0.57 0.59 0.57 0.78 TSB ZUEC OPH 2837 6+0 1.70 0.72 0.67 0.58 0.42 0.60 0.70 TSB ZUEC OPH 2838 6+0 1.50 0.70 0.43 0.39 0.44 0.41 0.65 ECP ZUEC OPH 2847 6+0 1.80 0.90 0.58 0.56 0.55 0.51 0.72 ECP ZUEC OPH 2848 6+0 1.40 0.70 0.65 0.58 0.50 0.48 0.56 ECP ZUEC OPH 2849 6+0 1.70 0.80 0.65 0.48 0.53 0.58 0.73 AB ZUEC OPH 2857 6+0 1.77 0.90 0.71 0.73 0.63 0.43 0.46 AB ZUEC OPH 2858 6+0 1.30 0.70 0.54 0.48 0.41 0.70 0.59 ECP ZUEC OPH 2863 6+0 1.50 0.97 0.74 0.50 0.64 0.62 0.56 ECP ZUEC OPH 2869 6+0 1.70 0.90 0.57 0.51 0.52 0.59 0.59 AB ZUEC OPH 2873 6+0 1.50 0.80 0.66 0.59 0.60 0.45 0.60 AB ZUEC OPH 2874 6+0 1.30 0.70 0.67 0.65 0.38 0.46 0.53 AB ZUEC OPH 2875 6+0 1.60 0.70 0.72 0.67 0.53 0.59 0.46 ECP ZUEC OPH 2879 6+0 1.50 0.70 0.70 0.40 0.38 0.38 0.58 ECP ZUEC OPH 2920 6+0 1.40 0.70 0.46 0.50 0.51 0.59 0.84

189

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