Kátia Cristina Cruz Capel

UNIVERSIDADE FEDERAL DO RIO DE JANEIRO Instituto de Biologia

Sistemática do gênero Tubastraea (Scleractinia: Dendrophylliidae) e estrutura genética das espécies invasoras do Atlântico Sul Ocidental

Rio de Janeiro 2018 Kátia Cristina Cruz Capel

Sistemática do gênero Tubastraea (Scleractinia: Dendrophylliidae) e estrutura genética das espécies invasoras do Atlântico Sul Ocidental

Tese de doutorado apresentada ao Programa de Pós-graduação em Ciências Biológicas (Biodiversidade e Biologia Evolutiva), Instituto de Biologia, Universidade Federal do Rio de Janeiro, como requisito parcial à obtenção do título de Doutor em Ciências Biológicas (Biodiversidade e Biologia Evolutiva).

Orientadora: Dra. Carla Zilberberg Coorientador: Dr. Marcelo Kitahara

Rio de Janeiro Janeiro de 2018

Kátia Cristina Cruz Capel

Sistemática do gênero Tubastraea (Scleractinia: Dendrophylliidae) e estrutura genética das espécies invasoras do Atlântico Sul Ocidental / Kátia Cristina Cruz Capel. Rio de Janeiro: UFRJ/IB – 2018 Xi + 222 fls

Tese (Doutorado) – Universidade Federal do Rio de Janeiro, Instituto de Biologia, Programa de Pós-graduação em CIências Biológicas (Biodiversidade e Biologia Evolutiva), 2018.

Orientadora: Dra. Carla Zilberberg Coorientador: Dr. Marcelo Kitahara

Referências: f. 210-222

1. taxonomia, 2. espécies invasoras, 3. clonalidade, 4. Tubastraea coccinea, 5. Tubastraea sp. cf. T. diapahana. - Tese

I. Zilberberg, Carla (Orient.). II. Universidade Federal do Rio de Janeiro. Programa de Pós-graduação em Ciências Biológicas (Biodiversidade e Biologia Evolutiva). III. Sistemática do gênero Tubastraea (Scleractinia: Dendrophylliidae) e estrutura genética das espécies invasoras do Atlântico Sul Ocidental

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iii

Nothing is constant but change! All existence is a perpetual flux of "being and becoming!" That is the broad lesson of the evolution of the world.

Ernst Haeckel

Á minha família, Tárcia, Armanado e Kelly.

Agradecimentos

Meu maior agradimento é aos meus orientadores, Carla Zilberberg e Marcelo Kitahara. Sou grata à vocês por terem sido maravilhosos em todos os momentos, por me fornecerem toda a ajuda necessária nesses quatro anos, por todo o apoio e todo o conhecimento que vocês me passaram. Vocês sempre foram mais que orientadores pra mim, são exemplos de carreira, de caráter e acima de tudo grandes amigos. Espero um dia ter pelo menos parte do conhecimento, talento e dedicação que vocês tem com tudo o que fazem. Nada disso teria sido possível sem vocês. Obrigada por sempre acreditarem em mim! Não posso deixar de dar um toque especial no meu agradecimento à Carla. Orientadora, amiga e atual rommate. Você é a maior responsável por eu estar onde estou hoje e é difícil colocar em palavras o quão importante você é pra mim. Você me acolheu no seu laboratório quando vim bater na sua porta e desde então nossa relação só ficou mais próxima. No meu último ano você me acolheu na sua casa, dividindo seu espaço comigo e proporcionando inúmeras noites de alegria, diversão e discussões científicas. Você nunca duvidou da minha capacidade e isso foi imprescindível pra mim. Agradeço aos meus orientadores no exterior, Rob Toonen e Zac Forsman, por terem proporcionado uma das melhores experiências da minha vida. Foi expecional para o meu crescimento profisional trabalhar em um laboratório fora do Brasil. É incrível o quanto é possível aprender em um ano quando se convive em um ambiente de trabalho tão harmonioso e repleto de pessoas extremamente competentes. Esse agradecimento se extende aos professores e pesquisadores Brian Bowen e Steve Karl, que sempre acescentaram em cada comentário e sugestão. Obrigada à todos por me proporcionarem a oportunidade de fazer um Comprehensive Exam, foi sem dúvida a época de maior aprendizado da minha vida. À minha família linda, Tárcia, Armando, Kelly, Rafael, e mihas avós Terezinha e Beatriz. Vocês são meu alicerce, minha razão de continuar. Obrigada por sempre estarem ao meu lado e por me apoiar em cada decisão. Não é fácil ficar longe de vocês, mas vocês nunca restringiram a minha vontade de voar. Eu não seria quem eu sou sem vocês. Um mais que obrigada ao Bananal, minha família carioca, Renatinha, Bruno, Jiló, Alex, Ric, Fabi e o mais recente membro, Larinha. Eu não sei o que seria da minha vida no Rio sem vocês! O Bananal sempre foi mais que uma república, vocês são família. Obrigada por me receberem nessa casa e tornar minha vida no Rio de Janeiro mais animada, feliz e cheia de momentos únicos. Um obrigada especial ao Jiló (o moço que mora comigo) por ser a companhia mais divertida que eu poderia ter nesses últimos meses e por sempre me alimentar, fazendo comidas deliciosas com ou sem amendoim. Um agradecimento também à saudosa Grade, Fernanda Caju, Mariana Geada, Paula Pola, maravilhosas! Vocês são todos mais que especiais! vi

Obrigada ao LaBiCni, por ser o laboratório mais florido e unido do Brasil! Mari, Amana e Lívia, suas lindas, obrigada por todo o apoio, pela amizade e pelo companheirismo de sempre. Obrigada aos melhores estagiários do mundo, Lígia, Alê e Márcio, vocês são demais! Esse doutorado não seria possível sem vocês. Um obrigada especial à todos os professores e pesquisadores que me ajudaram durante o processo, Cristiano Lazoski, Paulo Paiva, Michele Klautau, Andrea Junqueira, Joel Creed, Simone Oigman Pszczol, Guilherme Longo, Álvaro Migoto, Augusto Flores, Sérgio Floeter, Carlos Eduardo Ferreira (Cadu), entre outros. Aos grandes amigos que tive a sorte de conciver nesses últimos anos. Nico, Jorge, Linda, Chuck, Jemili, vocês ajudaram a tornar minha vida no Rio ainda mais feliz. Um obrigada especial pra Bianca del Bianco, minha parça, a melhor amiga que o Rio de Janeiro podia me dar. Aos amigos de laboratório do HIMB, ToBofriends Annick, Ingrid, Molly, Eillen, Erika, Alea, Derek, Mykle, Matt, Garett, Evan, Richard, e aos tantos outros amigos que o Hawaii me trouxe, Leon, Michelle, Adam, Sam, Greg, Henry, Howard, Ryan, Kyle. Meu ano no Hawaii foi ótimo graças à vocês! À todos que já me ajudaram de alguma forma, seja em campo ou coletando pra mim quando eu não pude ir: Bruno Masi, Larissa Marques, Edson Chuck, Mariana Frias, Fernanda Gianini, Serginho, Marcelo Mantellato, Carla Menegola, Gisele Gregório, Bruna Luz, Flora Sarti, Júlia Nunes, Mardia Eduarda Alves (Duda), Gilberto Mourão, Carlos Eduardo Ferreira (Cadu), Allen Chen e outros. À equipe do ICMBio de Alcatrazes e ao Ignacio Santos, do IBAMA, pelo apoio em campo. Por último agradeço ao programa de pós-graduação em Biodiversidade e Biologia Evolutiva (UFRJ), á CAPES pela bolsa concedida, ao programa Ciência sem Fronteiras pela bolsa de doutorado sanduiche e à PADI Foundation pelo financiamento concedido.

Mahalo!

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RESUMO

Apesar dos corais azooxantelados representarem a metade da diversidade das espécies de Scleractinia, de forma geral, permanecem pouco estudados. A taxonomia tradicional da ordem é baseada unicamente em caracteres morfológicos; no entanto, a alta variabilidade morfológica, a plasticidade fenotípica e a convergência de caracteres frequentemente desafiam a identificação das espécies. Tubastraea (Scleractinia, Dendrophylliidae) é um gênero de coral azooxantelado de águas rasas que compreende seis espécies recentes, sendo todas originárias do Indo-Pacífico e Pacífico. Duas espécies, Tubastraea coccinea e T. sp. cf. T. diaphana foram introduzidas no Oceano Atlântico Sudoeste e, desde então, vêm aumentando sua distribuição drasticamente competindo com espécies nativas. No entanto, identificações erradas não são incomuns para o gênero e existem poucas informações sobre a estrutura genética, histórico da invasão e estratégia reprodutiva das espécies invasoras. Neste contexto, a presente tese teve como objetivo (1) apresentar uma extensa revisão morfológica do gênero e reconstruções filogenéticas baseadas no genoma mitocondrial, (2) reavaliar a espécie T. caboverdiana, (3) descrever o genoma mitocondrial completo das espécies invasoras no Atlântico Sudoeste, (4) descrever um novo conjunto de microssatélites para estudos genéticos populacionais do gênero Tubastraea, (5) analisar a clonalidade, a diversidade genética e a estrutura das populações invasoras e vetores de introdução e (6) avaliar a produção assexuada de larvas em populações invasoras. Resultados da parte taxonômica indicam que uma das espécies invasoras do Atlântico previamente identificada como T. tagusensis possui morfologia e genética mais próxima à T. diaphana, sendo desta forma considerada neste trabalho como Tubastraea sp. cf. T. diaphana. Em adição, saliento que a espécie T. aurea, previamente sinonimizada como T. coccinea, pode ser uma espécie válida e, por fim, nesta tese é proposto um novo gênero Laborelia para acomodar espécimes encontrados em Cabo Verde que foram previamente identificados como T. caboverdiana. Outra evidência que apoia a hipótese de que identificações equivocadas de espécimes do gênero não são incomuns é apontada pela análise de genomas mitocondriais completos, os quais indicam que a sequencia de "T. coccinea" depositada no GenBank pertence provavelmente a uma espécie do gênero Dendrophyllia demonstrando que o genoma mt pode ser usado como marcador para identificação de gênero ou mesmo espécie. Para avaliar a clonalidade, a diversidade genética e a estrutura populacional das espécies invasoras de Tubastraea, foi desenvolvido um novo conjunto de marcadores microssatélites, os quais demonstram uma alta proporção de clones, com poucos genótipos espalhados por locais distantes até 2.000 km na costa brasileira. Esta ampla distribuição é provavelmente resultado de introduções secundárias através do transporte de vetores contaminados. Todos os sítios analisados mostraram um excesso de heterozigotos, o que é possivelmente explicado por uma combinação de fatores como estratégia reprodutiva, alta taxa de crescimento e alta pressão de propágulos. Os resultados também indicam predominância de produção assexuada de larvas em populações invasoras de Tubastraea spp., o que é provavelmente uma das principais razões para o sucesso da invasão no Atlântico Sudoeste. Em geral, os resultados apresentados nesta tese corroboram o status de invasor para o gênero Tubastraea no Atlântico, recomendam uma revisão do gênero em toda a sua distribuição natural e apoiam a hipótese de introdução através de bioincrustação em vetores como plataformas de petróleo e monoboias. Palavras-chave: taxonomia, espécies invasoras, clonalidade, Tubastraea coccinea, Tubastraea sp. cf. T. diapahana

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ABSTRACT

Although comprising half of the diversity of Scleractinian corals, azooxanthelate corals remain poorly studied. Traditional taxonomy relies solely on morphological characters for species delimitation; however, high morphological variability, phenotypic plasticity and convergence of characters frequently challenge species identification. Tubastraea (Scleractinia, Dendrophylliidae) is a shallow water genus with six extant species originally from the Indo-Pacific and Pacific Oceans. Two species, Tubastraea coccinea and T. sp. cf. T. diaphana were introduced in the Southwestern Atlantic Ocean and are spreading fast outcompeting native species. However, misidentifications are not uncommon within the genus and there is little information regarding the genetic structure, invasion history and reproductive strategies of the invasive species. The study aimed to (1) present an extensive morphological review of the genus and phylogenetic reconstructions based on mitochondrial genome, (2) re-evaluate the species T. caboverdiana, (3) describe the complete mitochondrial genome of the invasive species in the Southwestern Atlantic, (4) describe a new set of microsatellite loci for population genetic studies of the genus Tubastraea, (5) analyze the clonality, genetic diversity and structure of invasive populations and the role vectors of introduction and (6) evaluate the asexual production of larvae in invasive populations. The taxonomic results suggests that one of the Atlantic invasive species previously identified as T. tagusensis is morphologically and genetically similar to T. diaphana and it was here considered as Tubastraea sp. cf. T. diaphana. Aditionally, we suggest that T. aurea, previously synonymized to T. coccinea, may be a valid species and finally we propose the new genus Laborelia to accommodate specimens found at Cape Verde previously identified as T. caboverdiana. Another evidence supporting the assumption that misidentification within the genus is not uncommon was revealed by analyzing the complete mitochondrial genome, showing that the “T. coccinea” sequence deposited on GenBank is probably a species of the genus Dendrophyllia showing that the mt genome can be used as marker for genus or species identification. In order to explore the clonality, genetic diversity and population structure of Tubastraea we described a new set of microsatellite markers, which revealed a high proportions of clones, with few genotypes spread over sites distant up to ~2,000 km within the Brazilian coast. This broad distribution is probably result of secondary introductions through the transport of contaminated vectors. All analyzed sites showed an excess of heterozygotes, which is probably explained by a combination of factors such as reproductive strategy, high growth rate and high propagule pressure. Results also showed a predominance of asexual produced of larvae in the invasive range of Tubastraea spp., which is likely one of the main reasons for their invasion success in Southwestern Atlantic. In general, our results support the status of invasive for the genus Tubastraea in the Atlantic, recommend a review of the genus throughout their natural range of distribution and support the hypothesis of introduction though bioincrustation in vectors such as oil platforms and monobouys.

Key-words: taxonomy, invasive species, clonality, Tubastraea coccinea, Tubastraea sp. cf. T. diapahana

LISTA DE FIGURAS

Introdução

Figura 1. Quadro de invasão, mostrando a terminologia, estágios de invasão, barreiras a serem superadas e as medidas de manejo aplicáveis à cada estágio Fonte: Modificado de Blackburn et al. (2011)...... 4

Figura 2. Mapa da distribuição das espécies Tubastraea coccinea (em vermelho), T. sp. cf. T. diaphana (em laranja), ambas as espécies (em verde) e espécies do gênero Tubastraea não identificadas (em preto) (a) em costões rochoso ao longo da costa brasileira e (b) em vetores de introdução. Fonte: Creed et al. (2016)...... 5

SUMÁRIO

1. Introdução ...... 1 1.1 Bioinvasão ...... 2 1.2 A invasão de Tubastraea spp. no Atlântico ...... 4 1.3 Sistemática do gênero Tubastraea ...... 7 2. Objetivos ...... 10 2.1 Objetivo geral ...... 11 2.2 Objetivos específicos, apresentados sob a forma de capítulos ...... 11 3. Capítulos ...... 12 Capítulo I ...... 13 On the taxonomy of the widely distributed shallow water coral Tubastraea Lesson, 1829 (Cnidaria: Anthozoa: Dendrophylliidae) with morphological and molecular data: avoiding misidentification Capítulo II ...... 94 Laborelia, a new genus of Dendrophylliidae (Cnidaria: Scleractinia) from Eastern Atlantic Capítulo III ...... 117 Complete mitochondrial genome sequences of Atlantic representatives of the invasive Paciﬁc coral species Tubastraea coccinea and T. tagusensis (Scleractinia, Dendrophylliidae): Implications for species identiﬁcation Capítulo IV ...... 127 Clone wars: asexual reproduction dominates in the invasive range of Tubastraea spp. (Anthozoa: Scleractinia) in the South-Atlantic Ocean Capítulo V ...... 155 Marine hitchhikers: multiple introductions of Tubastraea spp. in the Southwestern Atlantic and the role of vectors on dispersion Capítulo VI ...... 188 Aliens do not make sex 4. Discussão ...... 202 4.1 Sistemática e taxonomia ...... 203 4.2 Clonalidade ...... 204 4.3 Panorama geral sobre a invasão Atlântico ...... 205 5. Conclusões ...... 208 6. Referências bibliográgicas ...... 210

1. Introdução

1 1.1 Bioinvasão

Espécie exótica invasora é aquela introduzida por atividades antrópicas em locais fora da sua área de distribuição natural, onde elas causam impactos negativos ao ambiente e espécies nele encontradas (IUCN). A invasão biológica é atualmente uma das principais ameaças à biodiversidade global (Bax et al. 2003; Bellard et al. 2016). Milhares de espécies são acidentalmente transportadas anualmente no ambiente marinho através de diversos meios de introdução, como a liberação deliberada de espécies por aquariofilistas, abertura de canais, aquacultura, água de lastro e bioincrustações (Bax et al. 2003; Hewitt et al. 2009; Padilla & Williams 2004; Ruiz et al. 2000). Estima-se que embarcações de madeira do século XVIII transportavam até 120 organismos incrustados ou perfuradores nos cascos (Bax et al. 2003) e o aumento do transporte aquaviário observado nas últimas décadas tem sido um dos principais responsáveis pelo transporte de milhares de espécies no ambiente marinho (Carlton & Geller 1993; Ferreira et al. 2006; Roberts & Tsamenyi 2008; Wanless et al. 2010; Wonham & Carlton 2005).

Grande parte das espécies transportadas não sobrevive à viagem ou não consegue se estabelecer no novo ambiente (Blackburn et al. 2011, 2015). Blackburn et al. (2011) propõe um quadro de bioinvasão, onde quatro estágios são reconhecidos: transporte, introdução, estabelecimento e dispersão, cada um associado a uma ou mais barreiras a serem superadas para o estabelecimento de uma espécie exótica (Figura 1). Quando uma espécie tem a habilidade de se dispersar além da área inicial de introdução, a invasão pode ser considerada como bem-sucedida (Catford et al. 2009). De forma geral, uma invasão de sucesso depende de uma combinação dos fatores bióticos (como a presença de predadores e competidores), e abióticos, que envolvem características ambientais do local (Catford et al. 2009), bem como a pressão de propágulos (definida como o número de indivíduos introduzidos multiplicado pela frequência de eventos de invasão) (Eppstein & Molofsky 2007). Além de potencializar as chances de estabelecimento de uma espécie exótica, a pressão de propágulos pode ainda levar a um aumento da diversidade genética da população invasora, o que, consequentemente, aumenta suas chances de sobrevivência (Blackburn et al. 2015; Lockwood et al. 2005, 2009). Um processo de invasão é geralmente seguido por uma perda de diversidade genética em decorrência de deriva genética e fatores como o efeito gargalo e/ou endocruzamento (Ellstrand & Elam 1993; Geller et al. 2008; Johnson & Woollacott 2015; Roman & Darling 2007; Sakai et al. 2001; Wrange et al. 2016). No entanto, múltiplos eventos de invasão podem amenizar esses efeitos e até levar a uma maior diversidade genética nas populações invasoras

2 (Blackburn et al. 2015; Bock et al. 2015; Carlton 1996; Dlugosch & Parker 2008; Lockwood et al. 2005, 2009; Simberloff 2009). As características biológicas das espécies também influenciam no sucesso de invasão, e espécies exóticas invasoras frequentemente compartilham uma série de características que facilitam a dispersão e o rápido estabelecimento em novos locais, como crescimento rápido, maturidade reprodutiva precoce e a ocorrência de estratégias diferenciadas para reprodução e sobrevivência (Lockwood et al. 2005; Sakai et al. 2001; Sax et al. 2007; Sax & Brown 2000).

Uma vez estabelecida, espécies exóticas têm o potencial de alterar a comunidade (Lages et al. 2011; Vitousek et al. 1997), podendo provocar um desequilíbrio na dinâmica local, seja por predação (Cure et al. 2012) ou competição com espécies nativas (Santos et al. 2013). Inúmeros exemplos podem ser citados para o ambiente marinho, destacando-se a alga Caulerpa taxifolia, que se alastrou pelo Mediterrâneo, excluindo espécies nativas (Meinesz et al. 2001), o ctenóforo Mnemiopsis leidyi, responsabilizado por colapsar o estoque pesqueiro do Mar Negro (Shiganova 1998), e o peixe leão Pterois spp., que levou a uma redução no recrutamento de peixes nativos no Caribe (Albins & Hixon 2008). Em casos extremos, espécies exóticas invasoras podem levar à extinção de espécies nativas (Clavero et al. 2009).

O manejo de espécies exóticas invasoras depende do conhecimento do histórico da invasão, incluindo a identificação dos principais meios e rotas de introdução (Hewitt et al. 2009), sendo o controle dos vetores, ou seja, o meio físico pelo qual uma espécie é transportada, a forma mais eficiente de prevenção (Davidson et al. 2008). No entanto, apesar da bioincrustação em embarcações ser considerada o principal vetor de introdução de espécies exóticas invasoras atualmente, o manejo dos vetores ainda é incipiente em nível internacional (Creed et al. 2016).

Atualmente, ferramentas moleculares têm sido amplamente usadas em estudos de bioinvasão para detectar diversidade genética, populações de origem, vetores de introdução, padrões de dispersão, dentre outros (Concepcion et al. 2010; Hellberg et al. 2002; Sammarco et al. 2012; Viard & Comtet 2015). Descobertos na década de 80 (Ellegren 2004), microssatélites são regiões de repetições simples do genoma, considerados como marcadores moleculares de alta resolução empregados em estudos populacionais, principalmente devido ao seu caráter altamente polimórfico (Bruford & Wayne 1993; Ellegren 2004; Hale et al. 2012; Kitahara et al. 2016; Underwood et al. 2006). Em estudos com espécies invasoras, marcadores microssatélites têm sido utilizados para compreender o processo de invasão, incluindo a detecção de populações de origem (Andreakis et al. 2009; Blakeslee et al. 2017;

3 Reusch et al. 2010) e a possível ocorrência de múltiplas invasões (Darling et al. 2008; Roman 2006).

<]l------1I Exótica 1 ----~ I> Term inologia ~<] 1 Estabelecido 1 ~----, I>

Estágio Transporte Introdução Estabelecimento Dispersão

Barreira

Fracasso da introdução Expansão

Manejo <]-Prevenção,> <]--Mitigação -C>

Figura 1. Quadro de invasão, mostrando a terminologia, estágios de invasão, tipos de barreiras a serem superadas e as medidas de manejo aplicáveis à cada estágio Fonte: Modificado de Blackburn et al. (2011).

1.2 A invasão de Tubastraea spp. no Atlântico

Atualmente, quatro registros de corais escleractíneos exóticos são conhecidos no Atlântico Ocidental: Fungia scutaria na Jamaica (Bush et al. 2004); Tubastraea coccinea no Caribe (Cairns 2000), Golfo do México, Flórida (Fenner & Banks 2004) e Brasil (de Paula & Creed 2004); T. sp. cf. T. diaphana (previamente identificada como T. tagusensis, ver Capítulo 1) no Brasil (de Paula & Creed 2004); e T. micranthus no Golfo do México (Sammarco et al. 2010). A espécie Fungia scutaria, originária do Indo-Pacífico, foi introduzida propositalmente na Jamaica na década de 1960 para fins de pesquisa, e apesar de diversas tentativas de retirada a espécie persistiu por mais de 35 anos, tendo o último registro ocorrido em 2004 (Bush et al. 2004). Já o gênero Tubastraea, foi acidentalmente introduzido em eventos múltiplos por incrustações em cascos de navios e plataformas de petróleo (Cairns 2000; Castro & Pires 2001; Fenner & Banks 2004; Sammarco et al. 2010). Dentre estas, T. micranthus é reportada no Golfo do México somente em plataformas de petróleo e, assim como F. scutaria, não causa impactos visíveis à biodiversidade local e, portanto, ambas não são consideras invasoras.

4 O primeiro registro do gênero Tubastraea no Atlântico foi da espécie T. coccinea em Curaçao e Porto Rico na década de 1940 (Vaughan & Wells 1943), locais de onde a espécie se dispersou por todo o Caribe (Cairns 2000). Cerca de 30 anos depois, as espécies T. coccinea e T. sp. cf. T. diaphana foram acidentalmente introduzidas na costa brasileira (Castro & Pires 2001), onde atualmente estão distribuídas descontinuamente em costões rochosos ao longo de mais de 3.500 km (Figura 2), incluindo Unidades de Conservação (Creed et al. 2016; Soares et al. 2016) e áreas adjacentes ao mais diverso e importante banco de corais da costa brasileira, o Arquipélago dos Abrolhos (Costa et al. 2014). Em 2001 a espécie T. coccinea foi registrada pela primeira vez em plataformas de petróleo no Golfo do México (Fenner 2001) e três anos depois invadiu a costa da Flórida (Fenner & Banks 2004). Acredita-se que a introdução no Atlântico Sul Ocidental tenha ocorrido por transporte de espécimes incrustados em plataformas de Petróleo (Creed et al. 2016). De fato, o primeiro registro das espécies foi em plataformas de petróleo na Bacia de Campos, litoral do Estado do Rio de Janeiro (Castro & Pires 2001), sendo que a distribuição de ambas as espécies está intimamente associada à presença de vetores e/ou terminais aquaviários.

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De forma geral, T. coccinea e T. sp. cf. T. diaphana são espécies oportunistas de crescimento rápido e maturidade reprodutiva precoce (Glynn et al. 2008; de Paula et al. 2014), possuem uma elevada capacidade regenerativa (Luz et al. 2016) e apresentam uma série de estratégias reprodutivas, incluindo brotamento extratentacular (Cairns 1991) e intratentacular (observação pessoal), “polyp bail-out” (Capel et al. 2014) e incubação de larvas sexuadas e assexuadas (Ayre & Resing 1986; Glynn et al. 2008). Sob o ponto de vista evolutivo a reprodução sexuada é vantajosa por permitir a recombinação e fluxo gênico entre indivíduos (Muller 1932), enquanto a reprodução assexuada propaga os genótipos melhor adaptados (Holman 1987; Radtkey et al. 1995; Weider et al. 1999). A ocorrência de reprodução assexuada com reprodução sexuada ocasional se beneficia das vantagens dos dois tipos de reprodução, evitando um acúmulo de mutações deletérias e a perda da diversidade genética, ambas associadas a predominância da reprodução assexuada (Hurst & Peck 1996; Peck & Waxman 2000). Além disso, em condições ambientais desfavoráveis os organismos tem a capacidade de aumentar a taxa de reprodução sexuada, criando novos genótipos e aumentando a probabilidade de sobrevivência sob as novas condições (Combosch & Vollmer 2013). A liberação de larvas assexuadas parece ser a forma predominante de reprodução da espécie T. coccinea e indícios de reprodução sexuada ainda são escassos (Ayre & Resing 1986; de Paula et al. 2014). Glynn et al. (2008) observaram a liberação de espermatozoides no Panamá e Galápagos, no entanto, existem observações pontuais de liberação de gametas masculinos e estudos acerca da reprodução das espécies T. coccinea e T. sp. cf. T. diaphana indicam um número escasso de gametas masculinos nas colônias examinadas (Glynn et al. 2008; de Paula et al. 2014).

O sucesso da invasão e a ampla distribuição do gênero Tubastraea spp. no Atlântico Sul Ocidental é possivelmente resultado de uma combinação entre as características biológicas das espécies, as condições propícias para o estabelecimento das espécies no Atlântico Sul e a elevada pressão de propágulos. O gênero já foi encontrado em pelo menos 23 vetores, incluindo monoboias e plataformas de petróleo, sendo que vetores “contaminados” são frequentemente transportados ao longo da costa, potencializando a dispersão e chance de estabelecimento em novas áreas (Creed et al. 2016).

As espécies T. coccinea e T. sp. cf. T. diaphana vêm causando alterações significantes na comunidade bentônica do Atlântico Sul, chegando a ocupar mais de 90% do substrato rochoso (Mantelatto et al. 2011) e competindo diretamente com espécies nativas (Creed 2006;

6 Santos et al. 2013). Estudos têm demonstrado relações antagonistas entre as duas espécies de Tubastraea e a espécie endêmica Mussismilia hispida quando em contato direto (Creed 2006; Santos et al. 2013), além da produção de compostos químicos com potenciais efeitos negativos à fauna nativa (Lages et al. 2010b). Interações alelopáticas já foram observadas entre a congênere T. faulkneri e larvas de pelo menos 11 espécies zooxanteladas (Koh & Sweatman 2000).

Apesar dos diversos estudos ecológicos acerca das espécies T. coccinea e T. sp. cf. T. diaphana no Atlântico Sul (Capel et al. 2014; Carlos-Júnior et al. 2015; Creed 2006; Creed & de Paula 2007; Glynn et al. 2008; Lages et al. 2010a, 2012; Moreira & Creed 2012; Riul et al. 2013; Santos et al. 2013), não existem informações sobre a diversidade genética das populações, assim como se as espécies foram introduzidas uma ou múltiplas vezes no Atlântico Sul e quais teriam sido as rotas de entrada. Tais informações são essenciais para o desenvolvimento de estratégias eficazes de manejo e prevenção de novas invasões (Sax et al. 2007).

1.3 Sistemática do gênero Tubastraea

A ordem Scleractinia Bourne, 1900 compreende mais de 1500 espécies exclusivamente marinhas, diferenciadas pela deposição de esqueletos calcários contínuos (Cairns 1999, 2009; Huang & Roy 2015). A ordem pode ser subdividida em dois grupos ecológicos em função da presença de algas simbiontes dinoflageladas (zooxantelas) na gastroderme do animal, fornecendo oxigênio e produtos fotossintéticos ao coral em troca de abrigo e nutrientes. Corais zooxantelados são restritos à zona fótica de regiões tropicais e subtropicais e são os principais constituintes dos recifes de coral de água rasa. Em contraste, os corais azooxantelados ocorrem em regiões tropicais, temperadas e polares em profundidades de até 6000 metros (Wells 1956; Cairns 1982; Huston 1985; Dawson 2002).

A família Dendrophylliidae é a terceira mais diversa dentro da ordem Scleractinia, contendo 22 gêneros, incluindo Tubastraea Lesson, 1829 (Cairns & Hoeksema 2010). O gênero é conhecido desde o Eoceno (aproximadamente 55 milhões de anos atrás) e compreende espécies azooxanteladas de águas rasas (Cairns 2001). Atualmente são reconhecidas quatro espécies fósseis e sete espécies viventes, das quais seis são originárias do Oceano Pacífico (T. coccinea Lesson, 1829, T. tagusensis Wells, 1982, T. diaphana (Dana, 1846), T. floreana Wells, 1982, T. faulkneri Wells, 1982 e T. micranthus (Ehrenberg, 1834)) e

7 uma recentemente descrita para o Oceano Atlântico Oriental (T. caboverdiana Ocaña & Brito 2015) (Cairns & Zibrowius 1997; Ocaña et al. 2015; Wells 1982).

Devido às cores vibrantes e por serem encontrados em águas rasas, inúmeros espécimes foram coletados e descritos entre o século XVII e início do século XVIII, muitos dos quais foram sinonimizados por Boschma (1953) e Wells (1983) (Cairns 2001). A taxonomia do grupo é baseada exclusivamente em caracteres macromorfológicos, como forma da colônia, tamanho e proximidade de coralitos e arranjo dos septos (Cairns & Zibrowius 1997; Wells 1982). No entanto, corais escleractíneos frequentemente apresentam uma elevada variabilidade morfológica entre indivíduos da mesma espécie, dificultando assim suas identificações (Todd 2008). A espécie recém descrita T. caboverdiana permaneceu sem uma identidade definida por mais de 40 anos, e mesmo a descrição atual permanece questionável (ver Capítulo 2). Descrições das espécies viventes podem ser encontradas nos trabalhos de Wells (1982, 1983), Cairns and Zibrowius (1997) e Cairns (2000, 2001). No entanto, espécies não identificadas ainda são encontradas na literatura (Arrigoni et al. 2014; Fenner 2005), assim como erros de identificação (Capel et al. 2016, Capítulo 2), ressaltando a necessidade de revisões atualizadas do gênero.

Nas últimas décadas, a implementação e o barateamento de técnicas moleculares revolucionou a sistemática tradicional, e tem gerado uma grande quantidade de dados, utilizados para esclarecer a relação entre espécies e evolução de grupos taxonômicos em diversas escalas (Kitahara et al. 2016). Uma das maiores mudanças dos últimos anos foi a separação da ordem Scleractinia em três grandes clados: “complexos”, “robustos” e “basais”, discordantes da classificação original de cinco subordens, baseada exclusivamente em caracteres morfológicos (Kitahara et al. 2010; Romano & Palumbi 1996, 1997; Stolarski et al. 2011). Tais estudos tem se baseado principalmente em genes mitocondriais para investigar relações filogenéticas e evolutivas em Scleractinia (Kitahara et al. 2014, 2016; Lin et al. 2014; Park et al. 2012). Apesar de exibir uma baixa taxa evolutiva (Huang et al. 2009; van Oppen et al. 1999; Shearer et al. 2002), regiões de evolução rápida no genoma mitocondrial de Anthozoa também têm sido utilizadas em estudos com espécies próximas (Hsu et al. 2014; Keshavmurthy et al. 2013). Concomitantemente, diversos estudos têm usado uma abordagem conjunta de dados morfológicos e moleculares para resolver a evolução e sistemática do grupo (Benzoni et al. 2010, 2012; Budd et al. 2012; Fukami et al. 2008; Kitahara et al. 2013; Stefani et al. 2007; Wallace et al. 2007) e descrever novas espécies (Arrigoni et al. 2017; Forsman et al. 2017; Johnston et al. 2017). Os corais azooxantelados possuem uma

8 diversidade semelhante à encontrada entre os zooxantelados, abrangendo cerca de 706 espécies, no entanto, estudos moleculares e taxonômicos acerca desses corais ainda se encontram defasados na literatura (Kitahara et al. 2016).

A identificação precisa e rápida em nível de espécie, facilitada pela utilização de ferramentas moleculares, é essencial para detectar, manejar e prevenir invasões biológicas (Ereshefsky 2008; Pyšek et al. 2013). Além disso, espécies apresentam histórias de vida particulares (p.ex. taxa de crescimento, estratégias reprodutivas, capacidade competitiva, etc.) e respondem de forma diferente ao ambiente, tornando planos de manejo impraticáveis sem uma identificação correta.

Espécies invasoras são consideradas uma ameaça à biodiversidade global, e a presença dos corais invasores Tubastraea coccinea e T. sp. cf. T. diaphana tem causado alterações significativas na dinâmica de comunidades ao longo da costa brasileira, o que tem se tornado cada vez mais preocupante devido à rápida expansão na distribuição de ambas as espécies (Costa et al. 2014; Miranda et al. 2016; Silva et al. 2014). Nesse contexto, o presente trabalho fornece dados inéditos abordando sistemática e estrutura genética das duas espécies que invadiram e tem se espalhado rapidamente ao longo do Atlântico Sul Ocidental. O gênero Tubastraea apresenta uma taxonomia complicada e descrições morfológicas atualizadas das espécies são indispensáveis para uma identificação precisa, prevenindo possíveis erros de identificação. Da mesma forma, as análises da estrutura genética são essenciais para esclarecer como se deu o processo de invasão ao longo da costa e quais os principais meios de dispersão. Os dados apresentados no presente trabalho fornecem uma perspectiva mais clara sobre o cenário da invasão no Brasil, apresentando informações essenciais para o desenvolvimento de estratégicas realmente eficazes no manejo e prevenção de novas invasões.

2. Objetivos

10 2.1 Objetivo geral

Apresentar uma revisão taxonômica do gênero Tubastraea e examinar a estrutura genética das duas espécies que invadiram o Atlântico Sul, T. coccinea e T. sp. cf. T. diaphana.

2.2 Objetivos específicos, apresentados sob a forma de capítulos

Capítulo 1 – Apresentar uma revisão do gênero Tubastraea, baseada em caracteres macromorfológicos e moleculares, incluindo um histórico taxonômico do grupo, descrições detalhadas das espécies atuais e uma chave de identificação.

Capítulo 2 – Reavaliar a espécie recém descrita Tubastrara caboverdiana, apresentando a descrição de um novo gênero para acomodar a espécie.

Capítulo 3 – Determinar, descrever e comparar os genomas mitocondriais das espécies T. coccinea e T. tagusensis, avaliando a possibilidade de identificação genética destas espécies.

Capítulo 4 – Descrever 12 loci de microssatélites para as espécies T. coccinea e T. tagusensis, apresentando uma análise preliminar da diversidade genética de duas populações da costa brasileira.

Capítulo 5 – Avaliar a diversidade genética dos corais invasores T. coccinea e T. sp. cf. diaphana na costa brasileira e em vetores de introdução, investigar a estrutura populacional e a ocorrência de múltiplas invasões, e discutir a importância dos vetores na dispersão das espécies.

Capítulo 6 – Examinar a produção larvas sexuadas e assexuadas nas espécies T. coccinea e T. sp. cf. diaphana em uma população invasora.

OBS: Os capítulos 3 e 4 foram publicados antes da finalização da revisão apresentada no capítulo 1 e a consequente alteração do nome T. tagusensis para T. sp. cf. T. diaphana, e, portanto, o primeiro nome foi mantido nos dois capítulos.

3. Capítulos

Capítulo I

On the taxonomy of the widely distributed shallow water coral Tubastraea Lesson, 1829 (Cnidaria: Anthozoa: Dendrophylliidae) with morphological and molecular data: avoiding misidentification

Kátia Cristina Cruz Capel, Carla Zilberberg, Stephen Cairns, Ingrid Knapp, Zac Forsman, Robert Toonen, Joel Creed & Marcelo Kitahara

Revista alvo: Invertebrate Systematics.

13 On the taxonomy of the widely distributed shallow water coral Tubastraea Lesson, 1829 (Cnidaria: Anthozoa: Dendrophylliidae) with morphological and molecular data: avoiding misidentification

KCC Capel1,2*, C Zilberberg1,2, SD Cairns3, Knapp I4, Z Forsman4, RJ Toonen4, JC Creed2,5, MV Kitahara6,7

1 Departamento de Zoologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil 2 Associate Researcher, Coral-Sol Research, Technological Development and Innovation Network 3 Department of Invertebrate Zoology, Smithsonian National Museum of Natural 4 School of Ocean & Earth Science & Technology, Hawaiʻi Institute of Marine Biology, University of Hawaiʻi at Mānoa, Kāneʻohe, HawaiʻI, United States 5 Departamento de Ecologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil 6 Departamento de Ciências do Mar, Universidade Federal de São Paulo, Santos, Brazil 7 Centro de Biologia Marinha, Universidade de São Paulo, São Sebastião, Brazil

* Corresponding author Kátia Capel e-mail: [email protected] Laboratório de Biodiversidade de Cnidária. Departamento de Zoologia Universidade Federal do Rio de Janeiro.

14 Abstract

Azooxanthelate corals comprise half of the diversity of Scleractinian corals but remain poorly studied. Traditional taxonomy relies solely on morphological characters for species delimitation; however, high morphological variability, phenotypic plasticity and convergence of characters frequently challenge species identification. Tubastraea (Scleractinia, Dendrophylliidae) is a shallow water genus with six extant species originally from the Indo- Pacific and Pacific Oceans. Due to their vibrant color, some species have been described multiple times resulting in a large list of synonyms, some of which remain controversial. A lack of a comprehensive taxonomic review has led to misidentifications within the genus and even in the identity of invasive populations, which have been introduced in the Atlantic during the past decades, and are in question. The present study provides the first thoroughly morphological review of the genus with a phylogenetic reconstruction based on mitochondrial genome. Museum collections and new material from the invasive range were examined for morphological analyses and twelve specimens representing four species and three unidentified morphotypes were included in the phylogenetic analyses. A protocol of restriction site associated DNA sequencing was used to recover the mitochondrial genome. For all valid species a full description with a list of synonyms, distribution and images is presented. Results corroborate the proposed transfer of T. caboverdiana to a new genus, identify the Atlantic invasive species previously considered to be T. tagusensis as Tubastraea sp. cf. T. diaphana and suggest that T. aurea may be a valid species. Reliable taxonomic identities are crucial for conservation purposes and this is the first step towards a worldwide revision of Tubastraea.

Key words: Azooxanthelate, invasive species, taxonomy, Atlantic Ocean

15 Introduction

The order Scleractinia Bourne, 1900 comprises over 1500 species exclusively found in marine habitats (Cairns 1999; Cairns 2009; Huang and Roy 2015). Within other Anthozoa, the group is discriminated by their ability to build a continuous calcareous skeleton, which has been used as the foundation for the taxonomy since the first classifications by Milne- Edwards and Haime (1857) and Ogilvie (1897). Although significant and extensively used, shallow-water coral classification based on skeleton morphology can be problematic (Kitahara et al. 2016) and even lead to misidentifications in the absence of comprehensive/detailed descriptions. Scleractinian corals display high intraspecific variability and phenotypic plasticity (Foster 1979; Todd 2008), which frequently challenges species identification/delimitation (Kitahara et al. 2016).

The advance of molecular techniques during the past decades has significantly improved the study of systematics with new insights into the evolutionary relationships within Scleratinia (Kitahara et al. 2016). The most relevant finding of the “DNA era” is the separation of the order in three main clades (“complex”, “robust” and “basal”) (Romano and Palumbi 1996; 1997; Kitahara et al. 2010; Stolarski et al. 2011) instead of the five accepted suborders based on morphology (Wells 1956). Notably, these findings also report discordances with morphological data at the suborder level (Romano and Palumbi 1996; 1997). During the past 10 years an increasing number of studies using morphological and molecular data improved our understanding not only on the evolution, but also on the systematics at family (Fukami et al. 2008; Benzoni et al. 2012; Budd et al. 2012; Kitahara et al. 2013), genera (Wallace et al. 2007; Benzoni et al. 2007; Benzoni et al. 2010) and species level (Arrigoni et al. 2017; Forsman et al. 2017; Johnston et al. 2017). However, azooxanthelate corals remain poorly studied with several ambiguities to be solved (Kitahara et al. 2016). Despite the significance of molecular data, traditional taxonomy based on morphology should not be neglected and a comprehensive and clear description of the morphology is crucial to assist in species identification.

Taxonomic history

Dendrophylliidae is the third most diverse family within the order Scleractinia, with solitary and colonial forms found worldwide at depths up to 2165 m (Cairns 2001). The

16 family is known from the Early Cretaceous (about 120 million years ago) and currently includes 364 valid species (198 being exclusively fossil) (Cairns 2001) divided into 22 genera, mostly of which are exclusively azooxanthellate (Cairns and Hoeksema 2010). Morphologically, the family is distinguished by having a porous synapticulotheca, septa composed by one fan system, and the occurrence of Pourtalès plan, although the latter is missing in some genera (Cairns 2001).

The dendrophylliid genus Tubastraea Lesson, 1829 embraces azooxanthellate species, which first appeared in the fossil record in the Eocene (about 55 million year ago). Due to their vibrant colors and occurrence in shallow waters, several species were collected and described between the XVII and early XVIII centuries, many of which have been later synonymized (Cairns 2001). Currently there are five synonyms accepted for the genus, all described between 1848 and 1884 (Coenopsammia Milne Edwards and Haime, 1848; Pachypsammia Verril, 1866; Astropsammia Verril, 1869; Placopsammia Duncan, 1876; and Dendrophyllia (Coenopsammia) Duncan, 1884). The most recent and comprehensive diagnosis of the genus is given by Cairns (2001), however, the occurrence of septal arrangement according to the Pourtalès plan remains unclear. It is considered to be absent in this group (Cairns 2001), although Arrigoni et al. (2014) reports it for one morphotype identified as Tubastraea sp. 1 (pg. 18, Fig. 11R).

Tubastraea divergence is estimated to have occurred around 55 million years ago (Cairns 2001), and four fossils (T. sp. sensu Stolarski, 1996; ?T. nomlandi Durham, 1942; T. puyricardensis (Chevalier, 1961); and T. puyricardensis var. grandiflora Chevalier and Demarcq, 1964) and six living species (T. coccinea Lesson, 1829; T. micranthus (Ehrenberg, 1834); T. diaphana (Dana, 1846); T. faulkneri Wells, 1982; T. tagusensis Wells, 1982; and T. floreana Wells, 1982) are currently accepted and found from 0 to 110 meters deep (Wells 1982; Cairns and Zibrowius 1997; Cairns 2001). Species delimitation relies on macromorphological characters such as colony form, corallite size and septal arrangement (Wells 1982; Cairns and Zibrowius 1997). However, due to pervasive morphological plasticity and lack of a comprehensive taxonomic review, the identification of some morphotypes remains challenging (e.g. Fenner 2005; Arrigoni et al. 2014) and misidentifications are not uncommon (see Capel et al. 2016).

The natural distribution of all living species is the Indo-Pacific region, while populations found in the Atlantic Ocean are either classified as invasive (Cairns 2000; Fenner

17 2001; Castro and Pires 2001; De Paula and Creed 2004) or cryptogenic (Creed et al. 2016). In the Western Atlantic three species (T. coccinea, T. tagusensis and T. micranthus) were introduced through biofouling on ship hulls and oil platforms (Cairns 2000; Sammarco et al. 2004; 2010; Creed et al. 2016) between 1940 and 2010. The absence of these species on previous surveys and a rapid increase in their distribution and abundance support the invasion hypothesis. Nevertheless, the status of the East Atlantic populations remains under discussion (Creed et al. 2016). Laborel (1974) recorded two morphotypes of Tubastraea sp. for Gabon, Gulf of Guinea, Sierra Leone and Cape Verde, and highlighted the need of a broad taxonomic review of the genus. Recently, Ocaña et al. (2015) described some of the specimens from Cape Verde as new (Tubastraea caboverdiana Ocaña and Britto 2015). However, their description relied solely on morphology, and molecular analyses revealed that this species does not belong to the genus Tubastraea. In addition, Boekschoten and Borel Best (1988) identified fossil and living specimens from Cape Verde as T. coccinea but did not provide a description or images of the specimens. Therefore, the taxonomy and, consequently, the evolutionary history of genus remain ambiguous.

Phylogenetic studies

Cairns (2001) provided the first phylogeny of the family Dendrophylliidae, based on 10 morphological characters, including corallum morphology, thecal structure, calicular elements numbers, size, position, etc, and presence of zooxanthellae. The family was recovered as monophyletic and the genus Tubastraea was recovered as a sister clade of Turbinaria, being close related to Astroides, Paleoastroides, Cladopsammia and Rhizopsammia (Cairns 2001). However, recent phylogenetic analyses of the family based on three molecular markers and including 30 species from 11 genera suggested that the morphological characters used were not informative for the recovery of evolutionary relationships within the group (Arrigoni et al. 2014). In their study, Tubastraea was recovered as a monophyletic clade within a monophyletic lineage that included Astroides calycularis, Dendrophyllia cornigera and Leptopsammia pruvoti.

Although easily found in shallow waters, the taxonomy of the genus Tubastraea remains unclear and ambiguous. The broad distribution of some species may enhance the phenotypic plasticity, which may add to an overlap of morphological characters within

18 species. As such, the present study aims to provide the first thoroughly morphological review of the accepted species of the genus with a phylogenetic reconstruction of the group based on mitochondrial genomes. Results presented herein might represent the baseline for further studies on the diversity, evolution and phylogeography of the genus.

Methods

Sampling

For the morphological analyses, specimens of T. coccinea and T. sp. cf. diaphana (previously identified as T. tagusensis, see discussion) were sampled from three localities in their invasive range along the Brazilian coast: Búzios Island (23º48’S/45º06’W); Archipelago of Alcatrazes (24°05′S/45°41′W); and an artificial substrate (monobuoy IMODCO-IV) located in the São Sebastião Channel (23°48′55″S/45°24′01″W). After collection of tissue samples, colonies were immersed in a sodium hypochlorite solution during 48 hours to remove the tissue. For phylogenetic analyses, twelve specimens representing four species and three unidentified morphotypes of Tubastraea were collected by SCUBA diving and snorkeling in Japan, Taiwan, Palmyra Atoll, Hawaii and Brazil (Table 1). Tissue samples were preserved in 96% ethanol, CHAOS solution (Fukami et al. 2004) or salt-satured DMSO (dimethyl sulfoxide) buffer (Gaither et al. 2011).

DNA extraction, library preparation and Illumina sequencing

DNA was extracted using DNeasy Tissue and Blood Kit (QIAGEN) and EZNA Tissue DNA Kit (OMEGA) following manufactures’ instruction and using HPLC grade water for elution. Extraction was checked in 1% agarose gel and quantified using AccuBlue High Sensitivity dsDNA quantification kit (Biotium, Inc.) measured in a SpectraMax M2 microplate reader. A protocol of restriction site associated DNA sequencing (ezRAD; see Toonen et al. 2013) was used for sequencing. This method uses a combination of restriction enzymes to digest high quality genomic DNA in fragments of appropriate size for sequencing, followed by size-selection and the standard Illumina TruSeq library preparation (Toonen et al. 2013). DNA was digested using the restriction enzymes DPNII or a combination of MboI and Sau3AI in 50 µl reactions (18-19 µl HPLC grade water, 1 µl of each restriction enzyme - 10

19 units), 5 µl Cutsmart Buffer and 25 µl of DNA, followed by 3 hours incubation at 37°C and 20 minutes at 65°C. Samples were cleaned with Ampure XP beads in a proportion of 1:1.8 DNA:Beads and libraries were generated using KAPA library preparation kit (Roche). All libraries were sequenced as 300 bp single-end on the Illumina GAIIx at EPSCoR Genetics Core Facility at the Hawai’i Institute of Marine Biology.

Phylogenetic analyses

Sequences obtained were trimmed for quality and adaptors and assembled with a reference mitochondrial genome (Tubastraea coccinea KX024566) using Geneious 7.1.9 (Kearse et al. 2012). Sequences were annotated and all protein coding, ribosomal and transfer RNAs genes were concatenated. Sequences were aligned using MUSCLE algorithm implemented in Geneious 7.1.9 (Kearse et al. 2012), and final alignment was 15,322 bp in length. Phylogenetic analyses were performed using RAxML (Stamatakis 2014) and MrBayes 3.2.6 (Ronquist and Huelsenbeck 2003) available on Geneious with specific evolutionary models for each locus and codon position as suggested by PartitionFinder2 (Lanfear et al. 2012). Bayesian analyses were run for 1 million generations with sampling every 500 with a burn-in of 100,000.

Morphological analyses

Species descriptions were based on type specimens, museum collections and/or new collected material. The holotypes of four species of Tubastraea are held at the National Museum of Natural History (NMNH) at the Smithsonian Institution (T. tagusensis – USMN 46977; T. floreana – USNM 46974; T. faulkneri – USNM 47145 and T. diaphana – USNM 180). For T. micranthus, whose type has been lost, the material from the NMNH was considered. For T. coccinea, whose type is deposited at the Muséum National d’Histoire Naturelle (MNHN) (Paris, France) (no collection number) (Wells 1936), images of the type were considered additionally to the material from the NMNH. Specimens were photographed and the macromorphology (e.g. shape of corallum, corallite diameter, septa arrangement, columella) was examined. Identification followed Wells (1982; 1983), Cairns and Zibrowius (1997), Cairns (1994; 2000; 2001) and Fenner (2005). For each species a list of synonyms,

20 type species, taxonomic history, distribution, description, illustration, and discussion are provided. All analyzed material will deposited at Museu Nacional do Rio de Janeiro (MNRJ).

Table 1. List of specimens included in the phylogenetic analyses with GenBank accession number and percentage of mitochondrial genome recovered. An asterisk (*) indicates sequences obtained in the present study. Two asterisks (**) indicate specimens previously identified as T. tagusensis (Capel et al. 2016) and herein after identified as Tubastraea sp. cf. T. diaphana.

Species – ID Accession Locality Reference or Mitogenome number collector coverage Dendophyllia cribrosa NC026026 South Korea Kwak et al. 100% Unpublished Dendrophyllia arbuscula NC027590 Japan Luz et al. 2015 100% Porites porites NC008166 USA - Florida Medina et al. 100% 2006 Laborelia caboverdiana – CVL-1 * Cape Verde J Creed 81,1% Tubastraea coccinea Mba-3 * Brazil - Buoy KCC Capel 99,8% Tubastraea coccinea Mbv-31 * Brazil - Buoy KCC Capel 100% Tubastraea coccinea Taiw-1 * Taiwan A Chen 97,5% Tubastraea coccinea BIG-126 * Brazil - Coast M Mantellato 84,7% Tubastraea coccinea KX024566 Brazil - Buoy MV Kitahara 100% Tubastraea diaphana Taiw-6 * Taiwan A Chen 100% Tubastraea diaphana DAR * Hawaii Z Forsman 94,6% Tubastraea sp. cf. T. diaphana ** KX024567 Brazil - Buoy MV Kitahara 100% Tubastraea sp. cf. T. diaphana BIG-144 ** * Brazil - Coast M Mantellato 100% Tubastraea sp. cf. T. diaphana Mb-52 ** * Brazil - Buoy KCC Capel 100% Tubastraea micranthus Tm-JP3 * Japan ME Santos 99,1% Tubastraea sp. 02-Tub-HI * Hawaii KCC Capel 64,8% Tubastraea sp. 01-Tub-HI * Hawaii KCC Capel 82,3% Tubastraea sp. 106-PA * Palmyra Atol JE Maragos 94,6% Turbinaria peltata KJ725201 - Shi et al. 2014 100%

Results

Phylogenetic analyses

Of the fifteen analyzed specimens, 64.8 to 100% of the mitochondrial genome was recovered, all comprising 13 protein-coding genes, 2rRNAs and 2tRNA, following the canonical gene organization. To better understand the relationships among Tubastraea representatives, mitochondrial protein-coding genes, rRNA and tRNA from four valid species and three unidentified morphotypes were used to reconstruct the evolutionary history of the group. Notwithstanding the applied methodology, Tubastraea was always recovered as two main clades with strong statistical support (Figure 1 and Supplemental File 2: posterior probability ≥0.99; bootstrap ≥98). In order to avoid any influence of unequal proportion of

21 missing data among specimens (ranging from 0 to 32.7%) a second phylogeny was reconstructed removing all sites with missing data, resulting in a final alignment of 9,064 bp (Supplemental File 2). The second analysis resulted in no major differences, except for the position of Tubastraea sp. 01-Tub-HI. Nevertheless, this terminal had low support in both topologies.

Jr------Porites porites OI l ~------Turbinaria peltata e ..e u ~------7_,,/,,______. Dendrophyllia cribrosa e ,.._cu Tubastraea caboverdiana CVLl .._co Dendrophyllia arbuscula "O "õ Tubastraea micranthus Tm-JP3 ã:i u cu Tubastraea diapahana Taiw-6 ..e a.. Tubastraea diaphana DAR

Tubastraea sp. cf. T. dlaphana KX024567

Tubastraea sp. cf. T. diaphana BIG-144

Tubastraea sp.

Tubastraea coccinea Mba-3 "O o Tubastraea coccinea Mbv-31 u o Tubastraea sp. 106-PA a..

Tubastraea sp. 02-Tub-HI

Tubastraea coccinea Taiw-1

Tubastraea coccinea BIG-126

Tubastraea coccinea KX024566

0.005

Figure 1. Phylogenetic analyses based on Bayesian inference and Maximum likelihood of all 13 protein-coding genes, two ribosomal RNA (rRNA) and two transfer RNA (tRNA) from 18 dendrophylliid corals and Porites porites as external group. Red dots indicate node statistical support (Posterior probability and bootstrap) of 1 and ≥98 respectively, while the blue dot indicates Posterior probability of 1 and bootstrap support value of 82.

Systematics

Class Anthozoa Ehrenberg, 1834

Subclass Hexacorallia Haeckel, 1896

22 Order Scleractinia Bourne, 1900

Family Dendrophylliidae Gray, 1847

Genus Tubastraea Lesson, 1829

Tubastraea Lesson, 1829:93. —Wells, 1936b:132 [nomenclatural note]. —Vaughan and Wells, 1943:238-239, pl. 50, fig. 5. —Alloiteau, 1952:681. —Boschma, 1953:109-118. — Wells, 1956:F436; 1982:216; 1983:243 [nomenclatural note]. —Ogawa and Takahashi, 1993:95-109. —Cairns, 1994:93. —Cairns, 2000: 178. —Cairns, 2001:28. —de Paula and Creed, 2004:177.

Coenopsammia Milne Edwards and Haime, 1848:106-107; 1850:liii [type species designated]; 1851:138-139. —Milne Edwards, 1860:125. —Chevalier and Beauvais, 1987:694.

Astropsammia Verrill, 1869:392. —Duncan, 1884:179. —Vaughan and Wells, 1943:239. — Chevalier and Beauvais, 1987:694.

Pachypsammia Verrill, 1866:30. —Duncan, 1884:179.

Dendrophyllia (Coenopsammia). —Duncan, 1884:178.

Placopsammia Duncan, 1876:441.

Type species

Tubastraea coccinea Lesson, 1829 (also designated as type species of Coenopsammia by Milne Edwards and Haime, 1848), by monotypy.

Type locality

Bora Bora, Society Islands (depth unknown).

Diagnosis

Colony plocoid, phaceloid or dendroid. Extratentacular budding from a basal coenosteum predominant but intratentacular budding also observed. Corallites cylindrical with a spongy columella. Septa normally arranged.

Distribution

Holocene: circumtropical. Depth: 0-110 meters.

Tubastraea coccinea Lesson, 1829

Figs. 2 A-L

Tubastraea coccinea Lesson, 1829: 93. –Wells, 1936: 132. –Scatterday, 1974: 86. –Scheer and Pillai, 1974: 10, 64-65, pl. 30. –Maragos, 1977: 197, 199-200. –Cairns, 1979: 207. –Zlatarski, 1982: 320-321, 323-324, 341-342, figs. 70-71, 149-152. –Wells, 1982: 216. –Wells, 1983: 243-244, pl. 18, figs. 1-2. –Wood, 1983: 66. –Veron, 1986: 580-581. – Prahl, 1987: 230-231, fig. 8. –Wilson, 1990: 137-138, fig. 1. –Cairns et al., 1991: 48. – Cairns, 1991a: 26-27, pl. 12, figs c-e. –Humann, 1993: 164-165. –Ogawa and Takahashi, 1993: 98, pl. 1, figs. 1-8, pl. 2, figs. 1-4, pl. 5, figs. 1-5. –Cairns and Keller, 1993: 282-284. –Cairns, 1994: 93-94, pl. 39, figs. g-i. –Cairns and Zibrowius, 1997: 197. –Cairns, 1998: 409. –Cairns, 2000: 178-180, figs. 212-215. –Song, 2000: 286-288. –Cairns, 2001: 29, pl. 10, figs. i-l.–Cairns, 2004a: 318. –de Paula and Creed, 2004: 175- 183. –Fenner, 2005: 26, 82. –Tachikawa, 2005: 20, pl. 13, figs. A-C.. –Kitahara, 2007: 504-505, 515, fig. 5K. –Lam et al., 2009: 736, figs. 2A-B.

Lobopsammia aurea Quoy and Gaimard, 1833: 195, pl. 15, figs. 7-11.

Dendrophyllia aurantiaca (?) Quoy and Gaimard, 1833: 195. –Dana, 1846: 388.

Coenopsammia coccinea. –Milne Edwards and Haime, 1848b: 107-108.

Coenopsammia ehrenbergiana Milne Edwards and Haime, 1848b: 109, pl. 1, fig. 12. – Milne Edwards and Haime,1860:liii. –Marenzeller 1907:74. –Harrison and Poole, 1909:909.

Coenopsammia gaimardi Milne Edwards and Haime, 1848b: 109.

Coenopsammia tenuilamellosa Milne Edwards and Haime, 1848b: 110, pl. 1, fig. 11.

Coenopsammia urvillii Milne Edwards and Haime, 1848b: 109.

Coenopsammia radiata Verrill, 1864: 44.

Coenopsammia manni Verrill, 1866: 30-31. –Verrill, 1869: 101. –Harrison and Poole, 1909:907-909.

Pachypsammia valida Verrill, 1866: 30.

Astropsammia pederseni Verrill, 1869: 392.

24 Dendrophyllia surcularis Verrill, 1869: 393.

?Placopsammia darwini Duncan, 1876: 441, pl. XL, fig. 4.

Dendrophyllia manni. –Quelch, 1886: 30, 196. –Vaughan, 1907: 156, pl. 46, figs. 6, 6a, 7, 7a. –Harrison and Poole, 1909:907-909. –Vaughan, 1918: 144. –Hoffmeister, 1925: 48.

Dendrophyllia (Coenopsammia) affinis Duncan, 1889: 18-19, pl. 1, figs. 29-30.

Dendrophyllia aurea. –Whitelegge, 1889:191. –Eguchi, 1934: 367. –Macnae and Kalk, 1958: 123. –Rosen, 1971:83.

Dendrophyllia willeyi. –Vaughan, 1918: 143-144, pl. 60, figs. 4, 4a.

Tubastraea tenuilamellosa. –Durham and Barnard, 1952: 105-106, pl. 12, fig. 50d. – Boschma, 1953: 109-117, pl. 9, figs. 1-4, pl. 10, figs. 1, 3-5, pl. 11, figs. 1, 3. –Roos, 1971: 84, pl. 53.

Tubastrea tenuilamellosa. –Durham, 1947: 38, pl. 11, figs. 1, 2, 4, 9, pl. 12, figs. 6, 7. Boschma, 1951: 44-46. –Durham, 1962: 42, 44-46. –Ross, 1964: 17, 48. –Olivares, 1971: 75-77, pl. 2, figs. a-b. –Smith, 1971: 95. –Erhardt, 1974: 407. –Erhardt and Meinel, 1975: 246.

Lobopsammia darwini. –Durham and Barnard, 1952:2. –Durham, 1966:125.

Tubastraea aurea. –Boschma, 1953: 111-118 (in part: pl. 10, figs. 2, 6, pl. 11, figs. 4-6, pl. 12, figs. 1-6). –Searles, 1956: 24, pl. 38B. –Stephenson and Wells, 1956: 59. –Squires, 1959b: 427-428. –Pichon, 1964: 191. –Eguchi, 1965: 295. –Squires, 1966: 169. –Pfaff, 1969: 23. –Eguchi, 1968: C68-70, pl. C16, figs. 5-6, pl. 17, fig. 17, pl. C26, figs. 2-3. – Utinomi, 1971: 220-221. –Wells and Lang, 1973: 58. –Eguchi and Miyawaki, 1975: 54, pl. 7, fig. 3. –Pichon, 1978: 441. –Best et al., 1980: 621. –Betterton, 1981: 242-243, fig. 201. –Castañares and Soto, 1982. –Scheer and Pillai, 1983: 173-174, pl. 40, fig. 8. – Wood, 1983: 121, 124. –Schuhmacher, 1984: 94-95. –Veron, 1986: 584-585, fig. 1. – Latypov, 1990: 65-66, pl. 27, fig. 4, pl. 32, fig. 5.

Tubastrea aurea. –Zans, 1959: 29, 35. –Almy and Carrión-Torres, 1963: 161, pl. 21, fig. b. – Wells, 1964: 109. –Colin, 1978: 291, 293. –Prahl and Erhardt, 1985: 181-182, figs. 108a-b, 109.

?Dendrophyllia turbinata Nemenzo, 1960: 18-19, pl. IX, fig. 2.

Dendrophyllia coccinea. –Eguchi, 1965: 296.

Dendrophyllia ? coccinea. Milne Edwards and Haime, 1848b: 107-108.

Tubastrea coccinea. –Latypov, 1990: 66-67, pl. 27, fig. 1, pl. 32, fig. 3. –Steiner, 2003: 5, 10.

Material examined

25 Types

Holotype of T. coccinea (MNHN – analyzed in picture). Type locality: Bora Bora, Society Islands. Depth unknown. The same specimen is the designated holotype of Coenopsammia coccinea.

Syntype of A. pederseni (USNM 38354). Type locality: La Paz, Gulf of California. Depth unknown.

Other examined material

Galápagos Islands, 8 colonies (USMN 77258, 77270, 77262, 77263, 77264, 77267). Panama, 3 colonies (USMN 83658, 83668). Virgin Islands, 3 colonies (USMN 61849). Jamaica, 6 colonies (USMN 47030, 61848, 83686, 83698, 83699, 94415). Florida, 1 colony (USMN 1016054). Cocos Islands, Costa Rica, 1 colony (USNM 83647). Lesser Antilles, 3 colonies (USNM 1113133). Bonaire, 1 colony (USNM 61845). Curacao, 1 colony (USNM 83692). Ecuador, 1 colony (USMN 77260). Kiribati, 2 colonies (USNM 83639, 83695). Society Islands, 1 colony (USNM 83642). Borneo Island, 1 colony (USNM 83684). Singapore, 1 colony (USNM 83662). Archiphelago of Alcatrazes, Brazil, 13 colonies. Búzios Islands, Brazil, 5 colonies. Underneath a monobuoy (IMODCO 4), Brazil, 15 colonies.

Taxonomic history

The species was described by Lesson (1829) based on specimens collected at Bora Bora, Pacific Ocean. During the next 60 years T. coccinea was placed in different genera and described under several different names, mostly of which were synonymized by Boschma (1953) and Wells (1983). Further descriptions were based mainly on characters that are now considered to be highly variable intraspecifically or even within a colony, such as size of the columella and septa width. T. coccinea was redescribed by Cairns (1991; 1994; 2000) and Cairns and Zibrowius (1997).

Distribution

26 Cosmopolitan in tropical shallow waters of the Pacific and Indian Oceans. Cryptogenic in the Tropical Eastern Pacific (from Gulf of California to Galápagos) and Eastern Atlantic (Cape Verde, Gulf of Guinea and Canary Islands) (Creed et al., 2016; Brito et al. 2017). Invasive in the Western Atlantic: Florida (Fenner and Banks, 2004), Gulf of Mexico (Fenner, 2001), Caribbean (Vaughan and Wells, 1943), Brazil (Castro and Pires, 2001; de Paula and Creed, 2004) (Figure 5). Depth: 1-110 meters.

Description

Corallum plocoid to subphaceloid, forming nearly spherical colonies firmly attached to substratum. Extratentacular budding from basal coenosteum, rarely occurring from theca of parent corallite. Intratentacular budding less frequent. Corallites cylindrical, up to 20 mm in calicular diameter, closely spaced, and rarely projecting more than 10 mm from coenosteum. Synapticulotheca thin and porous with fine granular costae. Coenosarc reddish or orange, tentacles orange or yellow.

Septa hexamerally arranged in four nonexsert cycles with a narrow rudimentary fifth cycle according to formula: S1≥S2>S3>S4>S5. S1 axial edge entire, straight and obliquely oriented (usually narrower at calicular edge). Deep in fossa S1 merges to columella. S2 equal in size to slightly smaller than S1, with smooth or occasionally dentate axial edge. S1-2 upper septal faces covered with rounded granules. S3 narrow and bearing laciniate axial edge, but eventually similar to S1-2 and also merge to columella deep in fossa. Half-systems bearing developed S3 bear narrow rudimentary S5. S4 rudimentary with lacinate axial edges. Fossa shallow to moderately deep. Columella spongy.

Discussion

T. coccinea is most similar to T. faulkneri and T. diaphana, but can be distinguished based on the distance between adjacent corallites and colony growth form. Among the cited synonyms, the status of T. aurea - originally described as Lobopsammia aurea by Quoy and Gaimard (1833) - remains controversial, as this species continues to be cited in several scientific studies (e.g.: Cheng et al., 2011; Latypov, 2011; Won et al., 2011; Arrigoni et al., 2014). On a phylogenetic reconstruction of the family Dendrophylliidae, Arrigoni et al. (2014) recognize

27 T. aurea as a separate species (based on samples from the Indian Ocean), which seems to be supported by molecular data. The low support retrieved in the phylogenetic analyses (Figure 1) suggests that the Hawaiian specimen Tubastraea sp. 01-Tub-HI is actually T. aurea and it might indeed represent a sister species to T. coccinea. The latter seems to have a high morphological variability, which justifies the several synonyms; nevertheless, the occurrence of cryptic species is not ruled out here. Originally described from the Pacific Ocean, T. coccinea is considered the most widespread shallow-water coral species known to date. However, a natural cosmopolitan distribution is uncommon and frequently associated to anthropogenic transport (Pérez-Portela et al., 2013). T. coccinea is an opportunistic species found incrusted on ship hulls and oil platforms worldwide (Sammarco et al., 2004; Friedlander et al., 2014; Creed et al., 2016). A thorough phylogeographic study is needed for a full understanding of the invasion process and verification of the native status of some populations. In an extensive review of its Atlantic invasion, Creed et al. (2016) classified populations from the Tropical Eastern Pacific and the Eastern Atlantic as cryptogenic. Indeed, the Eastern Pacific Barrier (EPB) is a strong obstacle that prevents dispersion between West and East Pacific for several marine species (Quintanilla et al., 2017) and given the Pacific origin of this species, a natural occurrence on the Eastern Atlantic is challenging to explain. In contrast, in the Western Atlantic, the status of invasive species seems to be well established. It has been suggested that the introduction occurred around the 1940’s in the Caribbean, by incrustation on ship hulls (Vaughan and Wells, 1943; Cairns, 2000) and in the late 1980’s in Southwestern Atlantic by incrustation on oil platforms (Castro and Pires, 2001). For both the Caribbean and Southwestern Atlantic the distributional range and abundance in the following years have increased quite rapidly, supporting the occurrence of an invasion process. Currently, established populations of this species can be found from Florida (26°42’N, 77°09’W) to Southern Brazil (27°17’S, 48°22’W) (Fenner and Banks, 2004; Capel, 2012), outcompeting native and endemic species in the Southwestern Atlantic (Creed, 2006; Santos et al., 2013; Miranda et al., 2016).

Tubastraea tagusensis Wells, 1982

Figs. 3 A-D

28 Tubastraea tagusensis Wells, 1982: 216-218, pl. 4: figs. 1-4; –Wells, 1893:244-245, pl. 20: figs. 1-6.

Tubastraea floreana Wells, 1982:218 [in part: specimen from Pinzon].

Material examined

Type

Holotype of T. tagusensis (USNM 46977). Type locality: Isabela Island, Galápagos. Depth 4.5 meters.

Other examined material

Galápagos Islands, 14 colonies (USNM 19151, 46978, 46979, 77256, 77268, 78877, 78881, 78882, 78883, 78886, 84851, 93274).

Taxonomic history

This species was described by Wells (1982) and re-described by Cairns (1991).

Distribution

Natural from Galápagos (Isabela, Santiago; Pinzón, Daphne Minor). Cryptogenic at Kuwait, Nicobar Island, Palau and Mexico (Creed et al., 2016) (Figure 5). Depth: 1-43 meters.

Description

Corallum plocoid. Corallites nearly spaced. Extratentacular budding from basal coenosteum. Intratentacular budding absent. Corallites cylindrical, ranging between 4 and 6 mm in calicular diameter and projecting 1-4 mm above coenosteum. Coenosteum quite porous. Costae smooth to slightly granular, separated by thin furrows.

29 Septa hexamerally arranged in four nonexsert cycles according to the formula: S1=S2≥S3>S4. S1-2 thin, entire, and merge deep in fossa to columella. Upper S1-2 axial edges vertical in orientation. Lower S1-2 edges almost horizontal. S3 dimorphic in development: some are 1 only /3 width of S1-2 and bear lacinate axial edges, whereas large corallites usually have S3 as developed as S1-2. S1-3 merge to columella. If present, S4 rudimentary and bearing lacinate axial edge and S3 well developed. Septal faces smooth or only slightly granular. Fossa of moderate depth containing a spongy and usually small columella. However, some corallites have a prominent columella augmented by septal extensions.

Discussion

T. tagusensis was described from the Galápagos and has only five records outside the archipelago, one of which is an invasive population and four are currently classified as cryptogenic (Creed et al., 2016). Indeed, a careful analysis of these records reveal misidentifications, such as the Kuwait specimen described by Hodgson and Carpenter (1995). They described a reptoid colony with oval corallites and a well-developed fourth cycle, features absent in T. tagusensis. Another ambiguous record of T. tagusensis is the invasive populations established in the Southwestern Atlantic Ocean. Specimens found in the Atlantic have notorious differences when compared to the type specimens and will hereafter be referred as Tubastraea sp. cf. T. diaphana (see discussion below). Therefore, it is possible that T. tagusensis might be endemic to Galápagos. Due to these morphological disparities, the description provided above was based exclusively on specimens from the type locality. Compared to other Tubastraea, T. tagusensis (type species) can be distinguished by having 12-24 thin septa that are vertical on upper axial edges and almost horizontal deep in fossa.

Tubastraea sp. cf. T. diaphana

Fig. 3 E-L

Material examined

30 Archipelago of Alcatrazes, Brazil, 14 colonies. Búzios Islands, Brazil, 6 colonies. Artificial substrate, underneath a monobuoy (IMODCO 4), Brazil, 2 colonies.

Taxonomic history

This species was previously identified as T. tagusensis (de Paula and Creed 2004 – and all subsequent publications) and is here tentatively identified as Tubastraea sp. cf. T. diaphana until the true identity can be defined.

Distribution

Introduced in the Southwestern Atlantic, occurring in natural and artificial substrate along the Brazilian coast, from Fortaleza (2º36’S, 39º42’W) to São Paulo (24º07’S, 45º42’W) (Sampaio et al., 2012; de Paula and Creed, 2004; Mantelatto et al., 2011; Creed et al., 2016) (Figure 5). Depth: 1-40 meters. Natural distribution currently unknown.

Description

Corallum phaceloid. Corallites closely spaced. Extratentacular budding from basal coenosteum and occasionally from the edge of parent corallites. Intratentacular budding frequent. Corallites cylindrical and ranging between 6-15 mm in calicular diameter. Polyps undergoing intratentacular division might reach 18 mm in calicular diameter. Corallites project up to 50 mm above coenosteum. Coenosteum porous. Costae granular and separated by thin furrows. Coenosarc yellow or orange.

Septa hexamerally arranged in four nonexsert cycles according to the formula: S1≥S2>>S3>S4>S5. S1-2 thin, entire, vertical and merge deep in fossa to columella. Upper S1-2 axial edges narrower than lower edges. S2 equal to only slightly smaller than S1. S3 1 around /3 width of S2 and bearing entire or slightly dentate axial edge. On larger corallites 2 (~15 mm in diameter) S3 reaches /3 width of S2 and merge deep in fossa to columella. S4 always rudimentary and bearing lacinate axial edges. If present, S5 rudimentary. Septal faces granular. Fossa deep containing a poor developed spongy columella.

Discussion

The former description was based on specimens sampled from the invasive populations along the Southwestern Atlantic. Previously identified as T. tagusensis, it is here considered a separated species due to several morphological disparities. Tubastraea sp. cf. T. diaphana has a phaceloid growth form, bearing corallites projecting up to 40 mm above the coenosteum with occasional intratentacular budding. The phaceloid growth form resembles T. diaphana and small colonies can be quite similar. However, those species differ on the size of colonies (smaller bushy colonies in T. diaphana), height of projecting corallites, development of S3-4 and pigmentation of the coenosarc. Some colonies resemble T. coccinea but with taller corallites, deeper fossa, and less developed collumela. An extensive sampling from the Indo- Pacific and Eastern Pacific regions is necessary to evaluate the identity and origin of this invasive species.

Tubastraea faulkneri Wells, 1982

Figs. 4 A-B

Tubastraea faulkneri Wells, 1982: 216, pl. 3, figs. 1-3. –Wells, 1983: 244, pl. 19, figs. 1-4. – Veron, 1986: 578, 582, 584, 585. –Cairns and Zibrowius, 1997: 64. –de Paula and Creed, 2004: 176, 181.

Dendrophyllia aurea van der Horst, 1926: 46, pl. 2, fig. 1.

Tubastraea aurea Boschma, 1953: 112, pl. 9, figs. 5, 6.

Tubastraea aurea Nemenzo, 1971: 182, pl. 12, fig. 3.

Tubastraea (new species) Faulkner and Chesher, 1979: 307, pl. 192.

Material examined

Type

32 Holotype of T. faulkneri (USNM 47145). Type locality: North Pacific Ocean, Palau Islands, Bailechesngel Island. Depth 7.6 meters.

Other examined material

Indonesia, Moluccas, 1 colony (USNM 62570). Madagascar, Radama Island, 1 colony (USNM 91841). Mauritius Island, 1 colony (USNM 91842). Philippines, 1 colony (USNM 92580).

Taxonomic history

Tubastraea faulkneri was described by Wells (1982) based on specimens from Palau and re- described by Cairns (1991).

Distribution

Indo-Pacific Ocean at Galápagos, Indonesia, Madagascar, Mauritius (Figure 5). Depth 1.5-15 meters.

Description

Corallum plocoid. Budding extratentacular from a porous coenosteum. Corallites widely spaced (5-15 mm apart), cylindrical, up to 15 mm in diameter and up to 8 mm in height. Costae thin, granular and separated by deep ridges, somewhat continuous to coenosteum.

Septa hexamerally arranged in four cycles accordingly to the formula: S1>S2>S3>S4. S1 vertical, entire, slightly exsert and thicker than higher septal cycles. S1 frequently extending all distance to the columella. S2 usually smaller and thinner than S1. S2 merge to columella deeper in fossa than S1. S1-2 axial edges smooth. S3 axial edge smooth or lacinate, occasionally fusing to S2 before columella (not Pourtalès plan). S4 rarely complete, with lacinate axial edges, frequently fusing to S3 half way to columella. Septal faces granular. Fossa shallow containing a spongy columella better developed in larger corallites.

Discussion

T. faulkneri is most similar to T. coccinea, differing in having: i) well-spaced corallites (5-15 mm apart); ii) S1 more exsert and thicker than remaining septa; and iii) S4 merging to S3. There are only few records of T. faulkneri resulting in a poorly understood species. According to Cairns (1991), the occurrence in Galápagos is doubtful and no new collections have been done to date.

Tubastraea micranthus (Ehrenberg, 1834)

Figs. 5 A-C

Oculina micranthus Ehrenberg, 1834: 304.

Dendrophyllia nigrescens Dana, 1846: 387. –Vaughan, 1918: 143-144, pl. 60, figs. 1, 1a. – Searles, 1956: 24, pl. 39A. –Stephenson and Wells, 1956: 55. –Wells, 1964: 108.

Coenopsammia viridis Milne Edwards and Haime, 1848b: 110.

Coenopsammia aequiserialis Milne Edwards and Haime, 1848b: 110-111. –Semper, 1872: 267.

Dendrophyllia micranthus. –Van der Horst, 1922: 49-51 (in part: Siboga stn. 277). –Van der Horst, 1926: 43-44, pl. 2, figs. 6-7. –Faustino, 1927: 218-220, pl. 72, figs. 1-2. – Crossland, 1952: 171-172. –Stephenson and Wells, 1956: 55. –Nemenzo, 1960: 16-17, pl. 8, fig. 2. –Scheer and Pillai, 1974: 63, pl. 29, fig. 3. –Pillai and Scheer, 1976: 16. – Betterton, 1981: 242, figs. 199-200.

Dendrophyllia micranthus var. grandis Crossland, 1952: 173, pl. 55, fig. 1, pl. 56, fig. 1.

Tubastrea micrantha. –Wells, 1964: 108. –Ogawa and Takahashi, 1993: 99-100, pl. 4, figs. 1- 6, pl. 6, figs. 5-6.

Dendrophyllia cf. micrantha. –Best et al., 1980: 621.

Tubastraea micranthus. –Macnae and Kalk, 1958: 123. –Scheer and Pillai, 1983: 175-176, pl. 41, figs. 7-8. –Schuhmacher, 1984: 94, figs. 1a-b, 4. –Cairns and Zibrowius, 1997: 195- 196. –Cairns, 1998: 410. –Cairns et al, 1999: 28. –Paula and Creed, 2004: 176, 181. – Cairns, 2004a: 267, 318. –Tachikawa, 2005: 20-21, pl. 13, figs. G-K.

Tubastraea micrantha. –Pichon, 1978: 441. –Wells, 1983. –Veron, 1986: 583, fig. 3, 585, figs. 3, 7. –Cairns and Keller, 1993: 282.

34 Tubastrea micranthus. –Latypov, 1990: 68, pl. 26, figs. 1-2.

Material examined

Type

Type specimen not traced, probably lost (Cairns and Zibrowius, 1997). Type locality unknown.

Other examined material

Rodrigues Island, 1 colony (USNM 22018). Palau, 2 colonies (USNM 78552, 83680). Madagascarar, 6 colonies (USNM 91833). Indonesia, 5 colonies (USNM 78551).

Taxonomic history

T. micranthus was described as Oculina micranthus, later transferred to Dendrophyllia (Van der Horst, 1922) and, more recently to Tubastraea. The species was redescribed by Cairns and Zibrowius (1997).

Distribution

Indo-West Pacific, from Madagascar to the Fiji Islands (Figure 5). Depth: 0-50 meters.

Description

Corallum dendroid up to one-meter heigh. Massive basal plate with at least two axial branches. Extratentacular budding from all branch faces. Corallites small (up to 8 mm in calicular diameter), cylindrical, and projecting up to 10 mm above branch coenosteum. Costae granular and separated by deep ridges. Coenosarc dark green.

Septa hexamerally arranged in four nonexsert cycles according to the formula: S1≥S2>>S3>>S4. S1-2 entire, narrow at upper axial edge and merge to columella deep in

35 fossa. S2 usually ¾ width of S1. S3 dimorphic: usually rudimentary with lacinate axial edges, but occasionally similar in profile to S2. S4 absent, but if present S4 poorly developed and some fuse to S3 half way to columella. Septal faces granular. Fossa shallow bearing a poorly developed, spongy columella.

Discussion

T. micranthus is considered to be a primary reef-builder (Schuhmacher, 1984), and the only true branching species within the genus. The branching nature of this species is different from all other congeners. The species was introduced in the Gulf of Mexico around 2005 encrusted on oil platforms, where it can be found up to 183 meters deep (Sammarco et al., 2010; Sammarco et al., 2017). Although restricted to oil platforms, T. micranthus seems to be competitive superior against local fauna with the potential to become a strong invader in the Atlantic (Sammarco et al., 2015).

Tubastraea floreana Wells, 1982

Figs. 5 D-E

Tubastraea floreana Wells, 1882: 218, pl. 4, figs. 5, 6. –Wells, 1983: 245, pl. 18: figs. 3-6. – Cairns, 1991: 28, pl. 12, fig. h, i.

Material examined

Type

Holotype of T. floreana (USNM 46974). Type locality: Galápagos - Playa Prieta, West side of Floreana Island). Depth: specimen sampled was washed up on the beach.

Other examined material

Galápagos, 7 colonies (USNM 46975, 78879, 77261, 78879, 78885, 93275).

36 Taxonomic history

T. floreana was described by Wells (1982) from the Galápagos Islands and re-described by Cairns (1991).

Distribution

Endemic from Galápagos, found at the Islands Floreana, Isabela, San Salvador, Espanola and Pinzon (Figure 5). Depth: 2-5 m.

Description

Corallum plocoid to subphaceloid forming small encrusting colonies. Corallites slightly spaced. Budding extratentacular from basal coenosteum. Corallites cylindrical, fragile (theca thin), projecting up to 4 mm above coenosteum, and bearing a circular to slightly elliptical calice measuring 4 to 6 mm in diameter. Coenosteum porous or eventually compact. Costae granular, separated by porous ridges discontinuous to that from coenosteum. Coenosarc bright pink.

Septa hexamerally arranged in three nonexsert cycles accordingly to the formula: S1≥S2>S3. S1-2 merge to columella with vertical, and entire to slightly lacinate axial edges. S3

1 rudimentary and bearing lacinate axial edge. Upper edge of S3 about /3 width of S1-2. Septal faces smooth or slightly granular. Fossa moderate depth containing a poorly developed spongy columella.

Discussion

Distinguished from congeners by having smaller corallite diameter with only three septal cycles, T. floreana is endemic to Galápagos and currently classified as critically endangered by the IUCN Red List after suffering a severe decline associated with an El Niño event on 1982-1983 (Hickman et al., 2017).

37 Tubastraea diaphana (Dana, 1846)

Fig. 5 F-K

Dendrophyllia diaphana Dana, 1846: 389, pl. 27, fig. 3. –Vaughan, 1918: 144-145, pl. 60, figs. 2-3.

Dendrophyllia aequiserialis. –Quelch, 1886: 147.

Dendrophyllia sibogae van der Horst, 1922: 56-57, pl. 8, figs. 18-19.

Dendrophyllia micranthus var. fruticosa Nemenzo, 1960: 17-18, pl. 9, fig. 1.

Tubastrea diaphana. –Stephenson and Wells, 1956: 59. –Wells, 1964: 108.

Tubastraea diaphana. –Wells, 1964: 108. –Scheer and Pillai, 1983: 174, pl. 41, figs. 1-4. – Veron, 1986: 580-582, fig. 2. –Cairns and Keller, 1993: 284, pl. 13, fig. H. –Ogawa and Takahashi, 1993: 99, pl. 2, figs. 9-10, pl. 5, fig. 7. –Cairns and Zibrowius, 1997: 196– 197. –Cairns, 1998: 409–410.

Material examined

Type

Holotype of T. diaphana (USMN 180). Type locality: Singapore. Depth unknown.

Other material

Australia, 3 colonies (USNM 45586, 83676, 1014923). Malaysia, 3 colonies (USNM 48039, 48049, 83670). Philippines, 4 colonies (USNM 77162, 78522, 83653, 83675). Singapure, 3 colonies (USNM 78521, 78525, 78526). Tonga, 1 colony (USNM 78528). Phoenix Islands, 1 colony (USNM 78529). Palau, 2 colonies (USNM 83674, 83677). Tanzania, 1 colony (USNM 83697). Madagascar, 2 colonies (USNM 91838, 91839). Solomon Island, 1 colony (USNM 97776). Sumatra, 1 colony (USNM 1121443).

Taxonomic history

38 T. diaphana was first described as Dendrophyllia diaphana by Dana (1846) based on specimens from Singapore. Detailed descriptions are provided by Vaughan (1918), Nemenzo (1960) and Cairns and Zibrowius (1997).

Distribution

Widespread in the tropical Indo-Pacific and Indian Ocean (Figure 5). Depth: 1-15 meters.

Description

Corallum phaceloid forming small corallites clusters. Corallites cilindrical, projecting up to 20 mm above a narrow but thick coenosteum. Budding extratentacular from basal coenosteum and occasionally from theca of a parent corallite. Costae granular, separated by deep ridges. Coenosarc dark brown to black.

Septa hexamerally arranged in four nonexsert cycles according to the formula: S1>S2>>S3≥S4. S1 vertical, occasionally narrower at upper edge. S1 bear smooth axial edge 1 extending /2 to the entire distance to columella. S2 only slightly smaller than S1, but otherwise similar. Deep in fossa S1-2 merge to columella. S3 usually poorly developed, up to 1 /3 width of S1, and bear slightly lacinate axial edge. Septal faces covered with rounded granules. If present, S4 rudimentary. Fossa deep containing a poorly developed columella.

Discussion

T. diaphana is most similar to T. coccinea and Tubastraea sp. cf. T. diaphana, but can be distinguished by the branching growth form, height of corallites, development of S3-4 and dark brown/black pigmented tissue.

Identification key for all extant Tubastraea species

1a. Corallum ceroid or plocoid …………….………...…………………...….…..……...(2)

39 1b. Corallum phaceloid …………….………...………………….……....….…………..(3)

1c. Corallum dendroid ……………...…………………..…...…………...…T. micranthus

2a. Adult corallite diameter ≥ 7 mm ……………....………..…….…….………..…...... (4)

2b. Adult corallite diameter ≤ 6 mm .…………....………………….….….…..T. floreana

3a. Corallites up to 20 mm in height and coenosarc black ...... T. diaphana

3b. Corallites up to 40 mm in height and coenosarc yellow/orange ..T. sp. cf. T. diaphana

4a. Corallites closely spaced (< 5mm apart) ……………………….………...…………(5)

4b. Corallites widely spaced (>5 mm apart) …..……………………………..T. faulkneri

5a. Lower axial edges of S1-2 and eventually S3 nearly horizontal ….…..…T. tagusensis

5b. Lower axial edges of S1-2 and eventually S3 always vertical….…...……..T. coccinea

Figure 2. Skeleton images of colonies and corallites of Tubastraea coccinea. (A-B) Holotype, Bora Bora. (C-D) Taiw-1, Taiwan. (E-F) Alcatrazes Archipelago, Brazil. (G-H) Mba-3, Artificial substrate, Brazil. (I-J) Mbv-31, Artificial substrate, Brazil. (K-L) Alcatrazes Archipelago, Brazil.

Figure 3. Skeleton images of colonies and corallites of Tubastraea. (A-B) T. tagusensis, holotype, Galápagos (USNM 46977). (C-D) T. tagusensis, paratype, Galápagos (USNM 46979). (E-F) Tubastraea sp. cf. T. diaphana, artificial substrate, Brazil (MB 50). (G-H) Tubastraea sp. cf. T. diaphana, Alcatrazes Archipelago Brazil, showing intratentacular budding. (I-J) Tubastraea sp. cf. T. diaphana, Alcatrazes Archipelago, Brazil (EAL 1). (K-L) Tubastraea sp. cf. T. diaphana, artificial substrate, Brazil.

Figure 4. Skeleton images of colonies and corallites of Tubastraea. (A-B) T. faulkneri, holotype, Palau (USNM 47145). (C-D) Tubastraea sp. 02-Tub-HI, Hawaii. (E-F) Tubastraea sp. 01-Tub-HI, Hawaii. (G-H) Tubastraea sp. 106-PA, Palmyra Atoll.

Figure 5. Skeleton images of colonies and corallites of Tubastraea. (A-C) T. micranthus, Rodrigues Island (USNM 22018). (D-E) T. floreana, holotype, Galápagos (USNM 46974). (F-G) T. diaphana, holotype, Singapure (USNM 180). (H-I) T. diaphaha, Madagascar (USNM 91839). (J-K) T. diaphana, Taiwan (Taiw-6).

44 A B

e D ,,.. - ~ -· -;;:..;-·......

·· ,. ~, ··- ~'::

E F

Figure 6. Worldwide distribution of native (blue), introduced (red) and crytogenic (green) records of: (A) T. coccinea; (B) T. tagusensis; (C) Tubastraea sp. cf. T. diaphana; (D) T. faulkneri; (E) T. micranthus; (F) T. floreana; and (G) T. diaphana. List of all records in Supplemental File 1. A, B and E modified from Creed et al. (2016).

Discussion

Although comprising nearly half of the extant corals, azooxanthellate species remain understudied when compared to zooxanthelate hermatipic corals. Tubastraea representatives are found worldwide in shallow waters, but several ambiguities are unresolved such as several unidentified species (e.g. Fenner 2005; Arrigoni et al. 2014). This is the first taxonomic

45 review of the genus, with morphological descriptions and mitophylogenomic analyses, discussing the identity of native and invasive specimens. Corroborating with Arrigoni et al. (2014), notwithstanding the evolutionary model or inference methodology (i.e. Bayesian inference or Maximum Likelihood) applied, the genus Tubastraea is recovered as monophyletic. Results presented herein challenge the validity of T. caboverdiana (see chapter 2) (Figure 1), and we propose a different identification of the Atlantic invasive specimens Tubastraea tagusensis, here identified as Tubastraea sp. cf. T. diaphana, and suggest that T. aurea might be a valid species.

Within Tubastraea, the mitogenomic analyses reveals two main clades correlated with the main corallite arrangement of the colony: (1) T. micranthus, T. diaphana and T. tagusensis – Brazil; and (2) T. coccinea, Tubastraea sp. 01-Tub-HI, Tubastraea sp. 106-PA and Tubastraea sp. pink. Species from the first clade have dendroid and phaceloid corallum with new buds arising occasionally from the upper theca of a parent corallite. Conversely, plocoid growth form is predominant within species from the second clade (except for Tubastraea sp. pink, which is more phaceloid). Dendrophylliidae corals display a wide variety of growth forms (Cairns 2001; Arrigoni et al. 2014) and intrageneric variability in corallite arrangement is observed for other coral genera (e.g.: Turbinaria [Arrigoni et al. 2014]; and Blastomussa [Benzoni et al. 2014]). Within the first clade, T. micranthus is separated with high support values, while the invasive species Tubastraea sp. cf. T. diaphana clusters with T. diaphana. Despite this molecular similarity, these species differ in colony gross morphology (size and growth form), corallite height, S3-4 development, and coenosarc pigmentation (see Figures 3 and 5).

The phylogenetic position of Tubastraea sp. 01-Tub-HI (Hawaii) has low statistical support as a result of its “unstable” position when comparing the topologies recovered using all data and the one retrieved when missing data were filtered (Figure 1, Supplemental File 2). As discussed in the taxonomic section, it is likely that Tubastraea sp. 01-Tub-HI represents T. aurea, a species previously synonymized as T. coccinea, but still commonly cited in the literature (e.g.: Won et al. 2001; Cheng et al. 2011; Latypov 2011; Arrigoni et al. 2014). Although a clear description of T. aurea is still lacking, a few characters traditionally used to distinguish it from T. coccinea, such as width and smoothness of septa are observed in Tubastraea sp. 01-Tub-HI (Figures 4 E-F) (Squires 1959; Song 1982). However, these characters are highly variable both inter- and intraspecifically and a new description of T.

46 aurea is needed. Currently, four Tubastraea species are recorded from Hawaii: T. coccinea (Orange Cup Coral), T. diaphana (Black Cup Coral), Tubastraea sp. (Pink Cup Coral) and Tubastraea sp. (Deep Pink Cup Coral) (Fenner 2005). Nevertheless, all but T. diaphana used to be recognized as T. aurea before its synonimization with T. coccinea (see Harris 1968; 1973). Tubastraea sp. 01-Tub-HI matches the Orange Cup Coral description but, as mentioned above, molecular data suggests that this species is not T. coccinea and might represent the true T. aurea. An exhaustive study including both the type specimen and new collected material is necessary to corroborate this hypothesis.

Interestingly, the cosmopolitan species T. coccinea split into two subclades: (1) T. coccinea Mba-3, T. coccinea Mbv-31 and Tubastraea sp. 106-PA; and (2) Tubastraea sp. 02- Tub-HI, T. coccinea Taiw-1, T. coccinea BIG-126 and T. coccinea KX024566 (Figure 6). Tubastraea sp. 106-PA is a small colony from Palmyra Atoll (South Pacific Ocean) and a precise identification based on morphology was unreliable due to the small colony size. Although morphologically different from T. coccinea (smaller corallites and S1>>S2), the recovered phylogeny suggests that they might represent the same species, and such differences might be the result of ontogenetic development. Similarly, Tubastraea sp. 02-Tub- HI was here recovered within the second subclade of T. coccinea (Figure 6) despite exhibiting morphological differences such as corallite size and arrangement (plocoid versus subphaceloid), septal size, and coenosarc and tentacle pigmentation. Tubastraea sp. 02-Tub- HI matches the Pink Cup Coral described by Fenner (2005) as “Pink Cup Coral forms pink colonies that when mature are round lumps with projecting long corallites…”. Fenner (2005) also discussed that although similar to T. coccinea, he classified it as a separate species based on biological features observed by Harris (1973). Analyzing association between nudibranch and corals Harris (1973) found that the nudibranch Phestilla melanobranchia died after a few days of feeding on a pink morphotype of Tubastraea from Hawaii and Singapore, suggesting that it might be a different species. If the observed cluster with T. coccinea, Tubastraea sp. 106-PA and Tubastraea sp. 02-Tub-HI holds true, an updated description of T. coccinea will be necessary to accommodate such variability. However, hybridization, the occurrence of cryptic or recent speciation might also explain the observed topology. Indeed, cryptic species and hybridization have already been reported for other scleractinian corals with wide distributional ranges (e.g. Richards and van Oppen 2012; Keshavmurthy et al. 2013; Warner et al. 2015; Richards and Hobbs 2015).

47 The use of molecular tools has revolutionized taxonomic studies in Scleractinia and, together with morphological data, new species have been described during the past years (Benzoni et al. 2012; Benzoni et al. 2014; Arrigoni et al. 2015; Arrigoni et al. 2016; Arrigoni et al. 2017). Nevertheless, azooxanthellate corals remain understudied when compared to zooxanthellate reef building species. Many of the morphological characters used to delimit species within the genus Tubastraea are highly variable both intra- and interspecifically (Boschma 1953; Squires 1959; Laborel 1974), which complicates taxonomy. This review is the first step towards a necessary worldwide revision of Tubastraea, combining morphological and molecular data of all valid species and unidentified morphotypes, in order to clarify the ambiguities within the genus, avoid future misidentifications and better comprehend their evolutionary relationships. Studies including nuclear data might provide new insights on the evolution of the genus and are higly recommended. Reliable taxonomic identities are crucial and the primary step for conservation purposes, especially dealing with a genus with high potential invasive species.

Acknowledgments

We are grateful to Maria Eduarda Santos, Marcelo Mantellato, and Estação Ecológica Tupinambás - ICMBio for sample collections. This research was supported by São Paulo Research Foundation (FAPESP), granted to A.E. Migotto (grant # 2012/21583-1), M.V. Kitahara (grant # 2014/01332-0) and K.C.C. Capel (grant # 2013/02696-2). K.C.C.Capel also thanks the PADI Foundation for the grant # 21882. J.C.Creed acknowledges the support of Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (#E- 26/010.003031/2014 PENSA Rio and #E26/201.286/2014 CNE) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (#307117/2014-6).This is Scientific Contribution No. 33 of the Projeto Coral-Sol. ZHF would like to acknowlege the support of the Seaver Institute.

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58 Supplemental File 1 Global records of Tubastraea, substrate occupied, introduced status, date reported and information sources. Modified from Creed et al. (2016).

Tubastraea tagusensis Wells, 1982 Decimal Decimal Country Region Substrate Category Date Information source Latitude* Longitude* reported** 29,623 47,638 Kuwait - Natural Cryptogenic 1988-1994 Hodgson and Carpenter (1995) 18,790 -110,930 Mexico Socorro Island Natural Cryptogenic 1995 Online database 7,513 134,585 Palau - Natural Cryptogenic 1975 Wells (1982) 6,861 93,660 India Nicobar Island Natural Cryptogenic 1975 Wells (1982) -0,260 -91,380 Ecuador Pinzon Island, Galápagos Natural Native 1966 Online database -0,274 -90,703 Ecuador Galápagos Natural Native 1872 Cairns (1991) -0,275 -90,706 Ecuador Cousin's Rock, Santiago Island, Natural Native 1975 Wells (1982) Galápagos -0,397 -90,352 Ecuador South Daphne Minor Natural Native 1975 Wells (1982) Island,Galápagos -0,600 -90,670 Ecuador Isabela Island Natural Native 1888 Online database -0,868 -91,170 Ecuador Tagus Cove, Isabela Island, Galápagos Natural Native 1975 Wells (1982) -2,492 -39,863 Brazil Acaraú Artifical, shipwreck Non-native 2016 M. Braga, pers. comm. -10,983 -36,932 Brazil Off Aracajú Artifical, oil platform Non-native 2013 G.S. do Nascimento, pers. comm. -11,098 -36,959 Brazil Off Aracajú Artifical, oil platform Non-native 2013 G.S. do Nascimento, pers. comm. -12,485 -38,341 Brazil Todos-os-Santos Bay, near Maré Artificial, nautical signs Non-native 2012 Miranda et al. (2012) island -12,502 -38,474 Brazil Paraguaçu estuary Artificial, pier Non-native 2012 Miranda et al. (2012) -12,502 -38,473 Brazil Paraguaçu estuary Natural Non-native 2012 Miranda et al. (2016) -12,532 -38,413 Brazil Todos-os-Santos Bay, north Itaparica Artificial, floating pier Non-native 2011 Sampaio et al. (2012) island -12,574 -38,305 Brazil Todos os Santos Bay, Salvador Artificial, seawall Non-native 2015 Miranda et al. (2016)

-12,855 -38,838 Brazil Canteiro de São Roque, Paraguaçu Artifical, oil platform Non-native 2014 J.C. Creed, pers. obs. -12,894 -38,578 Brazil Todos-os-Santos Bay Natural Non-native 2011 Sampaio et al. (2012) -13,055 -38,526 Brazil Todos-os-Santos Bay Artifical, shipwreck Non-native 2008 Sampaio et al. (2012) -14,783 -38,967 Brazil Ilheus Artifical, drilling Non-native 2009 M.D. Correia, pers. comm. platform -19,570 -39,258 Brazil Peroá-Cangoá Field, Espírito Santo Artifical, gas platform Non-native 2012 Costa et al. (2014) Basin -20,298 -40,263 Brazil Vitória Artifical, port structures Non-native 2008 Costa et al. (2014) -22,489 -40,378 Brazil Namorado field, Campos Basin Artifical, oil platform Non-native 2005 C.E.L. Ferreira, pers. comm. -22,775 -41,828 Brazil Armação de Búzios Natural Non-native 2011 Projeto Coral-Sol database -22,877 -43,122 Brazil Niterói Artifical, oil platform Non-native 2002 De Paula and Creed (2004) -22,891 -42,007 Brazil Cabo Frio Natural Non-native 2013 Projeto Coral-Sol database -22,973 -42,014 Brazil Arraial de Cabo Artificial, monobuoys Non-native 2007 Mizrahi (2008) -22,984 -42,002 Brazil Arraial do Cabo Natural Non-native 2011 Projeto Coral-Sol database -22,999 -43,966 Brazil Sepetiba Bay Natural Non-native 2011 Projeto Coral-Sol database -23,035 -43,200 Brazil Cagarras Archipelago Natural Non-native 2004-2011 Projeto Coral-Sol database -23,111 -44,267 Brazil Ilha Grande Bay Natural Non-native 1990's De Paula and Creed (2004) -23,117 -44,283 Brazil Ilha Grande Bay Artifical, shipwreck Non-native 2015 J. C. Gomes, pers. comm. -23,117 -44,283 Brazil Ilha Grande Bay Artifical, piers, docks Non-native 2012 Mangelli and Creed (2012) and decks -23,140 -44,165 Brazil Ilha Grande, Vila do Abraão Artificial, small pleasure Non-native 2014 M.C. Mantelatto, pers. obs. boat -23,797 -45,157 Brazil Ilhabela Natural Non-native 2008 Mantelatto et al. (2011) -23,813 -45,403 Brazil São Sebastião Artificial, monobuoys Non-native 2012 J.C. Creed, pers. obs. -23,813 -45,403 Brazil São Sebastião Artificial, monobuoys Non-native 2012 J.C. Creed, pers. obs. -24,098 -45,687 Brazil Alcatrazes Archipelago Natural Non-native 2011 Projeto Coral-Sol database -24,104 -45,691 Brazil Alcatrazes Archipelago Natural Non-native 2011 Projeto Coral-Sol database

Tubastraea micranthus Ehrenberg, 1834 Decimal Decimal Country Region Substrate Category Date Information source Latitude* Longitude* reported** 29,001 34,755 Egypt, Israel Gulf of Aqaba Natural Cryptogenic 1984 Schuhmacher (1984) 60

, Jordan, and Saudi Arabia 28,860 -88,930 USA Gulf of Mexico Artifical, oil platform Non-native 2013 Sammarco et al. (2013); Sammarco et al. (2014) 28,720 -89,430 USA Gulf of Mexico Artifical, oil platform Non-native 2014 Sammarco et al. (2014) 28,680 -89,390 USA Gulf of Mexico Artifical, oil platform Non-native 2014 Sammarco et al. (2014) 28,640 -89,790 USA Gulf of Mexico Artifical, oil platform Non-native 2014 Sammarco et al. (2014) 28,580 -90,070 USA Gulf of Mexico Artifical, oil platform Non-native 2014 Sammarco et al. (2014) 28,550 -90,670 USA Gulf of Mexico Artifical, oil platform Non-native 2006 Sammarco et al. (2010); Sammarco et al. (2013); Sammarco et al. (2014) 28,450 -90,380 USA Gulf of Mexico Artifical, oil platform Non-native 2014 Sammarco et al. (2014) 28,310 -90,070 USA Gulf of Mexico Artifical, oil platform Non-native 2013 Sammarco et al. (2013); Sammarco et al. (2014) 28,310 -90,020 USA Gulf of Mexico Artifical, oil platform Non-native 2014 Sammarco et al. (2014) 20,921 107,394 Vietnam Bai Tu Long Archipelago Natural Native 2011 Latypov (2011) 20,238 39,588 Saudi Arabia Red Sea Natural Cryptogenic 1991 Shepard and Shepard (1991) 19,634 37,289 Sudan Port Sudan area, Red Sea Natural Cryptogenic 1984 Schuhmacher (1984) 17,903 106,679 Vietnam Ze Island Natural Native 2011 Latypov (2011) 16,819 96,933 Myanmar Natural Native 1886 Duncan (1886) 16,136 108,274 Vietnam Cape Danang, Cham and Son Tra Natural Native 2011 Latypov (2011) islands 15,377 109,121 Vietnam Re Island Natural Native 2011 Latypov (2011) 13,532 121,101 Phillipines Verde Island Natural Native 1984 Schuhmacher (1984) 13,504 120,953 Phillipines Near Puerto Galera Bay Natural Native 1984 Schuhmacher (1984) 12,401 92,646 India Andaman Islands Natural Native 2001 D. Fenner, pers. obs. 12,207 109,233 Vietnam Khanh Hoa Province Natural Native 2011 Latypov (2011) 10,520 108,963 Vietnam Thu Island Natural Native 2011 Latypov (2011) 10,290 123,922 Phillipines off Cebu Natural Native 1984 Schuhmacher (1984) 10,074 114,357 Vietnam Spratly Islands Natural Native 2011 Latypov (2011) 9,996 109,075 Vietnam Ca Thuik Islands Natural Native 2011 Latypov (2011)

9,964 104,961 Vietnam Rach Gia bay Natural Native 2011 Latypov (2011) 9,934 104,008 Vietnam An Thoi Archipelago and Namsu Natural Native 2011 Latypov (2011) Islands 9,755 118,647 Phillipines Palawan Natural Native 1998 D. Fenner, pers. obs. 9,698 108,163 Vietnam Royal Bishop and Astrolab shoals Natural Native 2011 Latypov (2011) 9,429 123,388 Phillipines Sumilon Island Natural Native 1984 Schuhmacher (1984) 9,306 103,485 Vietnam Tho Chu Island Natural Native 2011 Latypov (2011) 8,711 106,583 Vietnam Con Dao Islands Natural Native 2011 Latypov (2011) 8,450 123,140 Phillipines Natural Native 1991 Online database 8,380 98,390 Thailand Natural Native 1991 Online database 7,540 98,310 Thailand Natural Native 1990 Online database 7,538 134,473 Palau Western Channel Natural Native 2003 Faucci et al. (2007) 7,330 134,500 Palau Caroline Island Natural Native 1983 Wellington and Trench (1985) 7,020 93,920 India Great Nicobar Natural Native 2009-2013 Mondal et al. (2015) 6,891 158,203 Federated Pohnpei Natural Native 2005 Turak and DeVantier (2005) States of Micronesia 5,949 116,081 Malasya Sabah Natural Native 2001 D. Fenner, pers. obs. 5,870 120,870 Phillipines Parang Natural Native 1997 Online database 5,791 103,019 Malaysia Redang Island Natural Native 2000 D. Fenner, pers. obs. 2,821 104,141 Malaysia Tioman Island Natural Native 2000 D. Fenner, pers. obs. 2,318 104,103 Malaysia Tinggi Island Natural Native 2000 D. Fenner, pers. obs. 1,680 127,540 Indonesia North Malaku Natural Native 1997 Online database 1,552 110,355 Malasya Sarawak Natural Native 2001 D. Fenner, pers. obs. -0,170 130,230 Indonesia Alyui Bay, Waigeo Natural Native 1998 Fenner (2002) -0,200 130,130 Indonesia Between Waigeo and Kawe Islands Natural Native 1998 Fenner (2002) -0,250 130,290 Indonesia Wofah Island Natural Native 1998 Fenner (2002) -0,320 130,130 Indonesia Ju Island, Batang Pele Natural Native 1998 Fenner (2002) -0,410 130,270 Indonesia Between Fowoyo and Yefnab Islands Natural Native 1998 Fenner (2002) -0,430 130,560 Indonesia Gam-Waigeo Passage Natural Native 1998 Fenner (2002)

-0,500 130,730 Indonesia Mios Kon Island Natural Native 1998 Fenner (2002) -0,520 130,670 Indonesia Mike`s Reef Natural Native 1998 Fenner (2002) -0,540 130,720 Indonesia Sardine Reef Natural Native 1998 Fenner (2002) -0,550 130,250 Indonesia Fam Island Natural Native 1998 Fenner (2002) -0,558 130,690 Indonesia Kri Island Natural Native 1998 Fenner (2002) -0,560 130,680 Indonesia Kri Island dive camp Natural Native 1998 Fenner (2002) -0,590 130,320 Indonesia Melissa`s Garden Natural Native 1998 Fenner (2002) -0,610 130,560 Indonesia W. Mansuar Island Natural Native 1998 Fenner (2002) -0,690 130,710 Indonesia Pulau Dua, Wai Reefs Natural Native 1998 Fenner (2002) -0,700 130,650 Indonesia Wai Reef complex Natural Native 1998 Fenner (2002) -1,940 120,565 Indonesia Sulawesi Natural Native 1998 D. Fenner, pers. obs. -4,530 129,880 Indonesia Banda Island Natural Native 1975 Online database -5,530 132,430 Indonesia off Pualu Tajondo Natural Native 1997 Online database -7,336 72,424 British Chagos Archipelago Natural Native 2014 D. Fenner, pers. obs. Indian Ocean Territory -7,336 72,424 British Chagos Archipelago Natural Native 1999 Shepard (1999) Indian Ocean Territory -9,400 46,330 Seychelles Aldabra atoll Natural Native NA Online database -9,466 159,950 Solomon Natural Native NA Online database Islands -9,569 150,671 Papua New Milne Bay Natural Native 2001 Fenner (2003) Guinea -9,900 144,100 Australia Murray Island Natural Native 2004 Online database -11,270 152,320 Papua New Guinea Natural Native 1990 Online database -12,170 44,361 Comoro Natural Native 1984 Schuhmacher (1984) Islands -12,170 122,970 Australia Ashmore Reef Natural Native 1998 Online database -12,250 143,250 Australia Piper Islands Natural Native 2002 Online database -13,320 48,250 Madagascar Natural Native NA Online database 63

-13,910 144,350 Australia Queensland Natural Native 2003 Online database -14,470 145,530 Australia Queensland Natural Native NA Online database -14,496 144,975 Australia Howick Island Natural Native 1990 Online database -14,660 145,470 Australia Queensland Natural Native 2010 Online database -14,710 145,530 Australia Queensland Natural Native 2010 Online database -16,335 179,216 Fiji North Vanua Natural Native 2004 Lovell and McLardy (2008) -16,370 145,550 Australia Queensland Natural Native 1997 Online database -16,500 147,830 Australia Queensland Natural Native 2002 Online database -16,510 174,850 Australia Queensland Natural Native 1997 Online database -16,750 145,960 Australia Queensland Natural Native 1997 Online database -16,760 145,000 Australia Queensland Natural Native 1997 Online database -16,838 178,292 Fiji Yadua and Yadua Taba Island Natural Native 2002 Lovell and McLardy (2008) -17,610 146,440 Australia Queensland Natural Native 2004 Online database -17,677 177,081 Fiji Mamanuca Island and Coral Coast Natural Native 2005 Lovell and McLardy (2008) -18,217 177,707 Fiji Votua Village Natural Native 2006 Lovell and McLardy (2008) -18,570 147,420 Australia Queensland Natural Native 1987 Online database -18,641 178,529 Fiji Astrolabe Reef Natural Native 2003 Lovell and McLardy (2008) -18,874 178,518 Fiji Great Astrolab Reefs Natural Native 1997 Lovell and McLardy (2008) -19,710 63,420 Mauritius Natural Native NA Online database -20,205 164,355 New Northwestern Grande-Terre Natural Native 2007 Fenner and Muir (2009) Caledonia -20,340 148,930 Australia Queensland Natural Native 1995 Online database -20,416 115,555 Australia Montebello Islands Natural Native 1993 Marsh (2000) -20,745 165,285 New Touho-Ponérihouen Natural Native 2009 Fenner (2011) Caledonia -21,000 194,540 Australia Queensland Natural Native 1988 Online database -21,117 -175,190 Tonga Natural Native 1997 Lovell and McLardy (2008) -21,250 165,289 New Natural Native 2006 Pichon (2006) Caledonia -21,551 49,108 Madagascar Natural Native 1984 Schuhmacher (1984) -23,260 151,930 Australia Queensland Natural Native 2010 Online database 64

-25,360 153,010 Australia Queensland Natural Native 2009 Online database -26,620 153,170 Australia Queensland Natural Native 2008 Online database 33,578 126,573 Korea Off Jeju-do Natural Native 2017 Choi and Song (2017) 26,681 128,138 Japan Manza Natural Native 2016 M.E. Santos, pers. comm. 27,095 124,716 Tawian Natural Native 2017 Choi and Song (2017)

Tubastraea coccinea Lesson, 1829 Decimal Decimal Country Region Substrate Category Date Information source Latitude* Longitude* reported** 35,138 139,618 Japan Misaki, Sagami Bay Natural Native 1914 Cairns (1994) 33,728 135,378 Japan off Tanabe, Kii Strait Natural Native 1888-1909 Cairns (1994) 33,678 135,348 Japan off Shirahama Natural Native 1888-1909 Cairns (1994) 33,356 126,564 South Korea off Cheju Do, East China Sea Natural Native 1994 Cairns (1994) 33,303 131,992 Japan Bungo Strait Natural Native 1994 Cairns (1994) 32,342 130,227 Japan Amakusa Islands Natural Native 1994 Cairns (1994) 31,293 -80,930 USA Greys Reef National Marine Artificial, weather buoy Non-native 2006 S. Fangman pers. comun. Sanctuary 30,832 130,325 Japan Osumi Shoto Natural Native 1994 Cairns (1994) 30,240 130,770 Japan Yakushima Natural Native 1992 Cairns (1994) 29,623 47,638 Kuwait Natural Native 1988-1994 Hodgson and Carpenter (1995) 29,405 -88,584 USA Gulf of Mexico Artificial, platform Non-native 2003-2006 Sammarco et al. (2012) 29,347 -88,282 USA Gulf of Mexico Artificial, platform Non-native 2003-2006 Sammarco et al. (2012) 29,347 -88,216 USA Louisiana Artificial, platform Non-native 2001 Fenner (2001) 29,292 -88,669 USA Gulf of Mexico Artificial, platform Non-native 2003-2006 Sammarco et al. (2012) 29,259 -88,442 USA Gulf of Mexico Artificial, platform Non-native 2003-2006 Sammarco et al. (2012) 29,240 -88,410 USA Gulf of Mexico Artificial, platform Non-native 2003-2006 Sammarco et al. (2012) 28,865 -88,931 USA Gulf of Mexico Artificial, platform Non-native 2014 Sammarco et al. (2014) 28,787 -90,427 USA Gulf of Mexico Artificial, platform Non-native 2014 Sammarco et al. (2014) 28,720 -89,431 USA Gulf of Mexico Artificial, platform Non-native 2014 Sammarco et al. (2014) 28,680 -89,388 USA Gulf of Mexico Artificial, platform Non-native 2014 Sammarco et al. (2014) 65

28,668 -93,886 USA Gulf of Mexico Artificial, platform Non-native 2003-2006 Sammarco et al. (2012) 28,658 -89,155 USA Louisiana Artificial, platform Non-native 1994 Fenner (2001) 28,643 -89,794 USA Gulf of Mexico Artificial, platform Non-native 2013 Sammarco et al. (2013); Sammarco et al. (2014) 28,575 -90,072 USA Gulf of Mexico Artificial, platform Non-native 2014 Sammarco et al. (2014) 28,575 -90,072 USA Gulf of Mexico Artificial, platform Non-native 2014 Sammarco et al. (2014) 28,552 -93,602 USA Texas Artificial, platform Non-native 2000 Fenner (2001) 28,549 -90,069 USA Gulf of Mexico Artificial, platform Non-native 2014 Sammarco et al. (2014) 28,540 -90,275 USA Gulf of Mexico Artificial, platform Non-native 2013 Sammarco et al. (2013) 28,539 -90,092 USA Louisiana Artificial, platform Non-native 2001 Fenner (2001) 28,501 -90,381 USA Gulf of Mexico Artificial, platform Non-native 2003-2006 Sammarco et al. (2012) 28,500 -178,330 USA Kure Natural Native 2000-2002 Maragos et al. (2004) 28,496 -90,203 USA Gulf of Mexico Artificial, platform Non-native 2013 Sammarco et al. (2013); Sammarco et al. (2014) 28,475 -90,236 USA Gulf of Mexico Artificial, platform Non-native 2013 Sammarco et al. (2013); Sammarco et al. (2014) 28,466 -90,446 USA Gulf of Mexico Artificial, platform Non-native 2003-2006 Sammarco et al. (2012) 28,454 -90,383 USA Gulf of Mexico Artificial, platform Non-native 2013 Sammarco et al. (2013); Sammarco et al. (2014) 28,376 -93,491 USA Gulf of Mexico Artificial, platform Non-native 2003-2006 Sammarco et al. (2012) 28,361 -93,769 USA Gulf of Mexico Artificial, platform Non-native 2003-2006 Sammarco et al. (2012) 28,339 -92,452 USA Sonnier Bank Natural Non-native 2007 Schmahl et al. (2008) 28,309 -90,071 USA Gulf of Mexico Artificial, platform Non-native 2013 Sammarco et al. (2013); Sammarco et al. (2014) 28,308 -90,022 USA Gulf of Mexico Artificial, platform Non-native 2014 Sammarco et al. (2014) 28,308 -96,228 USA Texas Artificial, platform Non-native 1999 Fenner (2001) 28,299 -91,088 USA Gulf of Mexico Artificial, platform Non-native 2003-2006 Sammarco et al. (2012) 28,231 -16,427 Spain Canary Islands Artificial, platform Non-native 2017 Brito et al. (2017) 28,214 -90,420 USA Gulf of Mexico Artificial, platform Non-native 2003-2006 Sammarco et al. (2012) 28,210 -177,363 USA Midway Atoll Natural Native 2000-2002 Maragos et al. (2004) 28,208 -92,952 USA Gulf of Mexico Artificial, platform Non-native 2001-2002 Sammarco et al. (2004) 28,196 -90,541 USA Gulf of Mexico Artificial, platform Non-native 2003-2006 Sammarco et al. (2012) 66

28,165 -94,296 USA Stetson Bank Natural Non-native 2012 Precht et al. (2014) 28,096 -93,478 USA Gulf of Mexico Artificial, platform Non-native 2001-2002 Sammarco et al. (2004) 28,070 -93,469 USA Gulf of Mexico Artificial, platform Non-native 2001-2002 Sammarco et al. (2004) 28,041 -93,759 USA Texas Artificial, platform Non-native 1994 Fenner (2001) 28,037 -93,231 USA Gulf of Mexico Artificial, platform Non-native 2001-2002 Sammarco et al. (2004) 28,003 -93,294 USA Gulf of Mexico Artificial, platform Non-native 2001-2002 Sammarco et al. (2004) 27,985 -93,458 USA Gulf of Mexico Artificial, platform Non-native 2001-2002 Sammarco et al. (2004) 27,981 -93,034 USA Gulf of Mexico Artificial, platform Non-native 2001-2002 Sammarco et al. (2004) 27,976 -94,144 USA Gulf of Mexico Artificial, platform Non-native 2001-2002 Sammarco et al. (2004) 27,972 -93,518 USA Gulf of Mexico Artificial, platform Non-native 2001-2002 Sammarco et al. (2004) 27,962 -93,671 USA Gulf of Mexico Artificial, platform Non-native 2001-2002 Sammarco et al. (2004) 27,962 -93,671 USA Texas Artificial, platform Non-native 1994 Fenner (2001) 27,956 -94,027 USA Gulf of Mexico Artificial, platform Non-native 2001-2002 Sammarco et al. (2004) 27,941 -91,038 USA Louisiana Artificial, platform Non-native 2001 Fenner (2001) 27,917 -93,917 USA Gulf of Mexico Artificial, platform Non-native 2001-2002 Sammarco et al. (2004) 27,913 -93,935 USA Gulf of Mexico Artificial, platform Non-native 2001-2002 Sammarco et al. (2004) 27,904 -96,089 USA Gulf of Mexico Artificial, platform Non-native 2003-2006 Sammarco et al. (2012) 27,901 -93,577 USA Texas Artificial, platform Non-native 1991 Fenner (2001) 27,855 -96,036 USA Gulf of Mexico Artificial, platform Non-native 2003-2006 Sammarco et al. (2012) 27,827 -93,830 USA West Flower Garden Bank Natural Non-native 2011 Hickerson et al. (2012) 27,819 -94,323 USA Texas Artificial, platform Non-native 1994 Fenner (2001) 27,808 -93,067 USA Geyer Bank Natural Non-native 2004 Precht et al. (2014) 27,779 -93,310 USA Gulf of Mexico Artificial, platform Non-native 2003-2006 Sammarco et al. (2012) 27,761 -93,138 USA Gulf of Mexico Artificial, platform Non-native 2003-2006 Sammarco et al. (2012) 27,442 -111,856 Mexico Baja California Natural Cryptogenic 2004 Online Database 27,430 -111,880 Mexico Baja California Natural Cryptogenic 2004 Online Database 27,260 -112,090 Mexico Baja California Natural Cryptogenic 2004 Online Database 27,240 -112,110 Mexico Baja California Natural Cryptogenic 2004 Online Database 27,218 -78,401 Bahamas SW Walker's Cay Natural Non-native 2001 Fenner (2001)

27,117 -94,045 USA Texas Artificial, platform Non-native 2000 Fenner (2001) 26,794 -80,045 USA Florida Artificial, floating dock Non-native 2004 Fenner and Banks (2004) 26,793 -80,004 USA Florida Articial, shipwreck Non-native 2004 Fenner and Banks (2004) 26,704 -77,153 Bahamas Guana Cay, Abaco Natural Non-native 2003 Fenner and Banks (2004) 26,637 -77,039 Bahamas Man of War Cay, Abaco Natural Non-native 2000 Fenner and Banks (2004) 26,500 128,002 Japan Ryukyu Islands Natural Native 1994 Cairns (1994) 26,418 -78,532 Bahamas Grand Bahamas Natural Non-native 1994 Frink (1994); Fenner (2001) 26,302 -80,062 USA Florida Articial, shipwreck Non-native 2004 Fenner and Banks (2004) 26,231 -80,064 USA Florida Articial, shipwreck Non-native 2004 Fenner and Banks (2004) 26,231 -80,066 USA Florida Articial, shipwreck Non-native 2004 Fenner and Banks (2004) 26,227 -80,065 USA Florida Articial, shipwreck Non-native 2004 Fenner and Banks (2004) 26,159 -80,079 USA Florida Articial, shipwreck Non-native 2004 Fenner and Banks (2004) 26,060 -111,180 Mexico Baja California Natural Cryptogenic 2004 Online Database 26,009 -80,094 USA Florida Articial, shipwreck Non-native 2001 Fenner and Banks (2004) 26,000 -173,920 USA Lisianski/Neva Shoal Natural Native 2000-2002 Maragos et al. (2004) 25,983 -80,085 USA Florida Articial, sunken oil rig Non-native 2004 Fenner and Banks (2004) jackets 25,887 -111,219 Mexico Baja California Natural Cryptogenic 2004 Online Database 25,821 -80,085 USA Florida Articial, shipwreck Non-native 2004 Fenner and Banks (2004) 25,790 -111,250 Mexico Baja California Natural Cryptogenic 2004 Online Database 25,780 -111,250 Mexico Baja California Natural Cryptogenic 2004 Online Database 25,770 -171,750 USA Laysan Natural Native 2000-2002 Maragos et al. (2004) 25,748 -80,095 USA Port of Miami, Mitigation Reef Artificial, breakwater Non-native 2004 Fenner and Banks (2004) 25,651 -79,294 Bahamas Bimini Artifical, shipwreck Non-native 1986-1987 Szmant et al (1990) 25,628 34,588 Egypt, Israel, Gulf of Aqaba Natural Native 1967-1970 Scheer and Pillai (1983) Jordan, and Saudi Arabia 25,420 -170,830 USA Maro Natural Native 2000-2002 Maragos et al. (2004) 25,124 121,866 Taiwan Yin-Yan Sea Natural Native 2014 Chan et al. (2014) 25,000 -168,000 USA Gardener Pinnacles Natural Native 2000-2002 Maragos et al. (2004) 68

24,990 -80,381 USA Florida Artifical, shipwreck Non-native 1999 Fenner and Banks (2004) 24,961 -80,453 USA Florida Keys, Conch Reef Artificial, vessels sunk Non-native 2014 Precht et al. (2014) as artificial reef 24,952 -80,462 USA Florida Keys, Conch Reef Artificial, Aquarius Non-native 2014 Precht et al. (2014) underwater habitat 24,952 -80,462 USA Florida Keys, Conch Reef Natural Non-native 2014 Precht et al. (2014) 24,935 -80,550 USA Key Largo/Chicken and Hen Reef Natural Non-native 2001 Fenner (2001) 24,830 121,970 Taiwan Kueishan Islet Natural Native 2014 Chan et al. (2014) 24,623 -81,982 USA Florida Artifical, shipwreck Non-native 2000 Fenner and Banks (2004) 24,479 -110,345 Mexico off Partida Island, Gulf of California Natural Cryptogenic 1888-1909 Cairns (1994) 24,479 -110,393 Mexico Espiritu Santo Islands, Gulf of Natural Cryptogenic 1888-1909 Cairns (1994) California 24,172 -110,365 Mexico La Paz, Gulf of California Natural Cryptogenic 1994 Cairns (1994) 23,870 -166,270 USA French Frigate Shoals Natural Native 2000-2002 Maragos et al. (2004) 23,766 -76,087 Bahamas Lee Stocking/ Perry Reef Natural Non-native 2001 Fenner (2001) 23,570 -164,700 USA Necker Natural Native 2000-2002 Maragos et al. (2004) 23,398 59,133 Oman Gulf of Oman Natural Native 1988 Sheppard and Salm (1988) 23,110 117,270 China Zhao’an Bay Natural Native 2010 Yang et al. (2013) 23,050 -161,920 USA Nihoa Natural Native 2000-2002 Maragos et al. (2004) 22,650 121,430 Taiwan Green Island Natural Native 2014 Chan et al. (2014) 22,600 69,500 India Gulf of Kutch Natural Native NA Online Database 22,479 -74,154 Bahamas Long Cay Artifical, sunken Non-native 2001 Fenner (2001) airplanes 22,273 114,175 China Hong Kong Natural Native 1984 Scott (1984) 21,850 -105,880 Mexico Baja California Natural Cryptogenic 2005 Online Database 21,777 -80,038 Cuba La Boca Artifical, shipwreck Non-native 2001 Fenner (2001) 21,750 -106,680 Mexico Maria Madre Island Natural Cryptogenic 1997 Online Database 21,701 -82,830 Cuba Isle of Youth Natural Non-native 2001 Fenner (2001) 21,620 -106,580 Mexico Maria Madre Island Natural Cryptogenic 1997 Online Database 21,570 -159,050 USA Hawaii Natural Native 1853-1856 Verril (1866) 21,484 -71,520 British South Caicos Artifical, sunken Non-native 2001 Fenner (2001) Overseas airplanes 69

Territory 21,431 -157,980 USA Oahu, Hawaii Natural Native 2002 Faucci et al. (2007) 21,404 -71,819 British Turks and Caicos Artifical, sunken Non-native 2001 Fenner (2001) Overseas airplanes Territory 21,404 -71,819 British Turks and Caicos Natural Non-native 1994-1996 Steiner (1999); Fenner (2001) Overseas Territory 21,156 -75,939 Cuba Santa Lucia Artifical, shipwreck Non-native 2001 Fenner (2001) 21,000 -75,580 Cuba Eastern Natural Non-native 1982 Zlatarski and Estallela (1982); Fenner (2001) 20,921 107,394 Vietnam Bai Tu Long Archipelago Natural Native 2011 Latypov (2011) 20,540 -105,300 Mexico Bahía de Banderas Natural Cryptogenic 1997 Online Database 20,482 -69,756 Dominican Silver Bank Natural Non-native 1965 Cairns (2000); Fenner (2001) Republic 20,333 -87,033 Mexico Cozumel Artifical, sunken Non-native 1999 Fenner (1999); Fenner (2001) airplanes 20,238 39,588 Saudi Arabia Red Sea Natural Native 1991 Shepard and Shepard (1991) 19,895 -155,587 USA Hawaii Natural Native 1981 Jokiel et al. (1985) 19,320 -110,820 Mexico off Socorro Island Natural Cryptogenic NA Online Database 19,291 -81,241 British Grand Cayman Natural Non-native 1994 Roberts (1994); Fenner Overseas (2001) Territory 18,691 -64,341 British Virgin Anagada Natural Non-native 1975 Dunne and Brown (1979); Islands Fenner (2001) 18,573 -87,316 Mexico Chinchoro Bank Natural Non-native 2001 Fenner (2001) 18,573 -87,316 Mexico Chinchoro Bank Artifical, shipwreck Non-native 2001 Fenner (2001) 18,464 -77,407 Jamaica Discovery Bay Artificial, undersides of Non-native 2014 Precht et al. (2014) buoys 18,443 -67,271 Puerto Rico Natural Non-native 1930´s Boschma (1953); Almy and Carrion-Torres (1963); Fenner (2001) 18,432 -64,629 British Virgin Tortola Island Artifical, shipwreck Non-native 2001 Fenner (2001) Islands 18,416 -77,100 Jamaica Natural Non-native 1955-1958 Goreau (1959); Goreau and

Wells (1967); Wells (1973); Wells and Lang (1973); Cairns (2000); Fenner (2001) 18,311 -64,935 USA St.Thomas Natural Non-native 2001 Fenner (2001) 18,291 -64,736 U.S. Virgin Steven´s Reef, St. John Natural Non-native 1968 Cairns (2000); Fenner (2001) Islands 18,218 -67,444 Puerto Rico Mono Island Artifical, pier pilings Non-native 2001 Fenner (2001) 18,203 -63,068 British Anguilla Natural Non-native 1986 Bouchon and Laborel (1986); Overseas Fenner (2001) Territory 18,048 -63,057 France/ St. Martin Natural Non-native 1975 Bak (1975); Bouchon and Netherlands Laborel (1990); Fenner (2001) 18,048 -63,057 Netherlands St. Eustatius Natural Non-native 1975 Bak (1975); Fenner (2001) 17,903 106,679 Vietnam Ze Island Natural Native 2011 Latypov (2011) 17,880 -62,833 Martinique St. Barthelemy Natural Non-native 1986 Bouchon and Laborel (1986); (France) Fenner (2001) 17,793 -70,737 Dominican Republic Natural Non-native 2001 Fenner (2001) 17,713 -64,885 U.S. Virgin Fredrickstad, St. Croix Artifical, pier pilings Non-native 2001 Fenner (2001) Islands 17,677 -64,836 U.S. Virgin St. Croix Natural Non-native 1977 Adey et al. (1977); Cairns Islands (2000); Fenner (2001) 17,650 -63,246 U.S. Virgin Saba Natural Non-native 1971 Roos (1971); Edmunds et al. Islands (1990) 17,329 -87,918 Belize Calabash Cay, Turneffe Atoll Natural Non-native 1994 Fenner (1999); Bengtsson et al. (2015) 16,955 -25,313 Cape Verde Santo Antao Natural Cryptogenic 1988 Boekschoten and Best (1988) 16,819 96,933 Myanmar Mergui Archipelago Natural Native 1886 Duncan (1886) 16,781 -22,792 Cape Verde Sal Natural Cryptogenic 1970 Laborel (1974); Boekschoten and Best (1988) 16,745 -24,555 Cape Verde Sao Vicente Natural Cryptogenic 1970 Laborel (1974); Boekschoten and Best (1988) 16,500 151,800 Philippines Philippines Sea Natural Native 1830 Online Database 16,583 -24,300 Cape Verde Sao Nicolau Natural Cryptogenic 1988 Boekschoten and Best (1988) 16,237 -61,552 France Guadeloupe Natural Non-native 1986 Bouchon and Laborel (1986); Fenner (2001) 71

16,136 108,274 Vietnam Cape Danang, Cham and Son Tra Natural Native 2011 Latypov (2011) islands 16,086 -22,611 Cape Verde Boa Vista Natural Cryptogenic 1970 Laborel (1974); Boekschoten and Best (1988) 16,046 -86,522 Honduras Roatan Natural Non-native 2001 Fenner (2001) 15,377 109,121 Vietnam Re Island Natural Native 2011 Latypov (2011) 14,916 -23,429 Cape Verde Sao Tiago Natural Cryptogenic 1970 Laborel (1974); Boekschoten and Best (1988) 14,852 -24,248 Cape Verde Fogo Natural Cryptogenic 1970 Laborel (1974) 14,842 -24,858 Cape Verde Brava Natural Cryptogenic 1970 Laborel (1974); Boekschoten and Best (1988) 14,274 -80,391 Colombia Serrana Natural Non-native 2001 Fenner (2001) 13,618 -80,166 Colombia Roncador Natural Non-native 2001 Fenner (2001) 13,520 120,950 Philippines Mindoro Natural Native NA Online Database 13,504 120,953 Phillipines near Puerto Galera Bay Natural Native 1984 Schuhmacher (1984) 13,482 144,744 Micronesia Guam Natural Native 1983 Randall and Myers (1983); Randall (2003); Faucci et al. (2007) 13,320 -81,371 Colombia Isla Providencia Natural Non-native 1970 Geister (1975,1992); Prahl and Erhardt (1985,1988); Fenner (2001) 12,577 -81,694 Colombia Isla San Andrés Natural Non-native 2001 Fenner (2001) 12,502 -69,969 Aruba Natural Non-native 1950 Roos (1971); Boschma (1953); Bak (1975,1977); Fenner (2001) 12,207 109,233 Vietnam Khanh Hoa Province Natural Native 2011 Latypov (2011) 12,166 -68,238 Bonaire Natural Non-native 1950 Boschma (1953); Roos (1971); Scatterday (1974); Van Veghel (1997); Scatterday (1874); Fenner (2001) 12,154 -68,990 Curaçao Natural Non-native 1930´s Boschma (1953); Roos (1964, 1971); Bak (1975, 1977); Cairns (2000); Fenner (2001) 11,960 121,480 Philippines Caluya Natural Native NA Online Database

11,802 -66,755 Venezuela Los Roques Islands Natural Non-native 2001 Fenner (2001) 10,708 -68,252 Venezuela P.N. Morrocoy Natural Non-native 2001 Fenner (2001) 10,520 108,963 Vietnam Thu Island Natural Native 2011 Latypov (2011) 10,300 -63,972 Venezuela Gulf of Cariaco Natural Non-native 1968-1971 Olivares (1971); Antonius (1980); Fenner (2001) 10,486 -67,965 Venezuela P.N. San Esteban Artifical, shipwreck Non-native 2001 Fenner (2001) 10,293 -64,479 Venezuela Baía de Mochima Natural Non-native 1982 Pauls (1982); Fenner (2001) 10,167 -75,750 Colombia Natural Non-native 1965-1968 Pfaff (1969); Erhardt and Meinel (1975); Werding (1979); Cairns (2000) apud Fenner(2001); Prahl (1985) 10,130 -109,220 France Clipperton Island Natural Cryptogenic 1997 Carricart-Ganivet and Reyes- Bonilla (1999) 10,074 114,357 Vietnam Spratly Islands Natural Native 2011 Latypov (2011) 9,996 108,963 Vietnam Ca Thuik Islands Natural Native 2011 Latypov (2011) 9,964 104,961 Vietnam Rach Gia bay Natural Native 2011 Latypov (2011) 9,934 104,008 Vietnam An Thoi Archipelago and Namsu Natural Native 2011 Latypov (2011) Islands 9,755 118,647 Phillipines Palawan Natural Native 1998 D. Fenner, pers. obs. 9,698 108,163 Vietnam Royal Bishop and Astrolab shoals Natural Native 2011 Latypov (2011) 9,306 103,485 Vietnam Tho Chu Island Natural Native 2011 Latypov (2011) 9,115 78,621 India Gulf of Mannar Natural Native 1988-1996 Venkataraman et al. (2002) 9,070 123,270 Philippines Apo Island Natural Native 1979 Online Database 8,787 -79,557 Panama Taboga Island Natural Cryptogenic 1990 Glynn et al., (2008) 8,711 106,583 Vietnam Con Dao Islands Natural Native 2011 Latypov (2011) 8,705 -83,879 Costa Rica Caño Island Natural Cryptogenic 1985 Glynn et al., (2008) 8,676 -77,405 Panama Natural Non-native 1972 Porter (1972); Robertson and Glynn (1977); Fenner (2001) 8,626 -79,055 Panama Saboga Island Natural Cryptogenic 1989 Glynn et al., (2008) 8,500 79,000 Sri Lanka Palk Strait Natural Native NA Online Database 8,460 93,620 India Andaman and Nicobar Islands Natural Native 1957-1958 Scheer and Pillai (1974) 7,965 -82,034 Panama Secas Island Natural Cryptogenic 2002 Glynn et al., (2008)

7,809 -81,761 Panama Uva Island Natural Cryptogenic 1990 Glynn et al., (2008) 7,797 98,537 Thailand Koh Kai Natural Native 1990 Online Database 7,480 98,320 Thailand Ko Racha Noi Natural Native 1990 Online Database 7,430 134,540 Palau Natural Native 1992 Online Database 7,340 134,480 Palau Natural Native 1993 Online Database 7,210 93,760 India Great Nicobar Natural Native 1957-1958 Scheer and Pillai (1974) 7,020 93,920 India Great Nicobar Natural Native 2009-2013 Mondal et al. (2015) 6,891 158,203 Federated Pohnpei Natural Native 2005 Turak and DeVantier (2005) States of Micronesia 6,860 122,060 Philippines Great Santa Cruz Island Natural Native NA Online Database 6,063 171,995 Marshall Natural Native 1926 Van der Horst (1926) Islands 5,800 -162,400 USA Palmyra Atoll Natural Native 2000-2002 Online Database 5,791 103,019 Malaysia Redang Island Natural Native 2000 D. Fenner, pers. obs. 5,529 -87,059 Costa Rica Chatham Bay, Cocos Island Natural Cryptogenic 1986-1991 Cairns (1991) 4,629 118,758 Malasya Bodgaya Islands, Sabah Natural Native 1987 Wood and Tan (1987) 4,250 111,170 Malasya Natural Native 1879 Online Database 2,821 104,141 Malaysia Tioman Island Natural Native 2000 D. Fenner, pers. obs. 2,810 171,710 Kiribati Taburao Natural Native 1972 Online Database 2,460 104,530 Malasya Pulau Aur Natural Native NA Online Database 2,318 104,103 Malaysia Tinggi Island Natural Native 2000 D. Fenner, pers. obs. 1,968 73,541 Maldives Natural Native 1926 Van der Horst (1926) 1,678 -92,003 Ecuador Darwin Island, Galápagos Natural Cryptogenic 1986-1991 Cairns (1991) 1,552 110,355 Malaysia Sarawak Natural Native 2001 D. Fenner, pers. obs. 1,343 103,819 Singapore Natural Native 1881 Studer (1881) 0,190 -176,480 USA Baker Island Natural Native 2004 Online Database -0,170 130,230 Indonesia Alyui Bay, Waigeo Natural Native 1998 Fenner (2002) -0,170 130,010 Indonesia Wayag Islands Natural Native 1998 Fenner (2002) -0,200 130,130 Indonesia Between Waigeo and Kawe Islands Natural Native 1998 Fenner (2002)

-0,268 -90,700 Ecuador Sullivan Bay, Santiago, Galápagos Natural Cryptogenic 1986-1991 Cairns (1991) -0,270 -91,380 Ecuador Isabella Island, Galápagos Natural Cryptogenic 1986-1991 Cairns (1991) -0,274 -90,703 Ecuador Santiago Island, Galápagos Natural Cryptogenic 1986-1991 Cairns (1991) -0,275 -90,706 Ecuador Cousin's Rock, Bartolomé, Natural Cryptogenic 1986-1991 Cairns (1991) Galápagos -0,320 130,130 Indonesia Ju Island, Batang Pele Natural Native 1998 Fenner (2002) -0,350 -90,450 Ecuador Galápagos Natural Cryptogenic 1931-1952 Durham and Barnard (1952) -0,380 -160,020 USA Jarvis Island Natural Native 2000-2004 Online Database -0,397 -90,352 Ecuador Daphne Menor Island, Galápagos Natural Cryptogenic 1986-1991 Cairns (1991) -0,410 130,270 Indonesia Between Fowoyo and Yefnab Natural Native 1998 Fenner (2002) Islands -0,430 130,560 Indonesia Gam-Waigeo Passage Natural Native 1998 Fenner (2002) -0,485 -90,267 Ecuador Itabaca Canal/Galápagos Natural Cryptogenic 1985-1993 Glynn et al., (2008) -0,520 130,670 Indonesia Mike`s Reef Natural Native 1998 Fenner (2002) -0,540 130,720 Indonesia Sardine Reef Natural Native 1998 Fenner (2002) -0,558 130,690 Indonesia Kri Island Natural Native 1998 Fenner (2002) -0,590 130,320 Indonesia Melissa`s Garden Natural Native 1998 Fenner (2002) -0,590 130,260 Indonesia SE Miosba Island Natural Native 1998 Fenner (2002) -0,610 130,560 Indonesia W. Mansuar Island Natural Native 1998 Fenner (2002) -0,610 130,160 Indonesia Equator Islands Natural Native 1998 Fenner (2002) -0,631 -90,376 Ecuador Santa Cruz Island, Galápagos Natural Cryptogenic 1986-1991 Cairns (1991) -0,690 130,710 Indonesia Pulau Dua, Wai Reefs Natural Native 1998 Fenner (2002) -0,750 -90,310 Ecuador Academy Bay, Santa Cruz, Natural Cryptogenic 1986-1991 Cairns (1991) Galápagos -0,754 -90,309 Ecuador Punta Estrada/Galápagos Natural Cryptogenic 1985-1993 Glynn et al., (2008) -0,820 -90,060 Ecuador off Santa Fe Island, Galápagos Natural Cryptogenic 1986-1991 Cairns (1991) -0,868 -91,170 Ecuador Tagus Cove, Galápagos Natural Cryptogenic 1986-1991 Cairns (1991) -1,294 -90,435 Ecuador Floreana Island Natural Cryptogenic 1986-1991 Cairns (1991) -1,300 -90,436 Ecuador Floreana Island Natural Cryptogenic 1980's to Glynn et al., (2008) 1998 -1,378 -89,674 Ecuador Española Island, Galápagos Natural Cryptogenic 1986-1991 Cairns (1991)

-1,940 120,565 Indonesia Sulawesi Natural Native 1998 D. Fenner, pers. obs. -3,167 40,167 Kenia Watamu Marine Reserve Natural Native 1973-1979 Lemmens (1993) -4,980 119,220 Indonesia Makassar Natural Native 1980 Online Database -5,104 119,285 Indonesia Gusung Kodingarengkeke Natural Native 1979 Online Database -5,123 119,340 Indonesia Pulau Samalona Natural Native 1979 Online Database -6,714 39,277 Tanzania SW of Bongoyo Natural Native 1974 Hamilton and Brakel (1984) -7,336 72,424 British Indian Chagos Archipelago Natural Native 2014 D. Fenner, pers. obs. Ocean Territory -8,677 115,488 Indonesia Pulua Penida Natural Native 2013 M.C. Mantelatto, pers. obs. -9,400 46,330 Seychelles Aldabra Atoll Natural Native 1993 Cairns and Keller (1993) -9,569 150,671 Papua New Milne Bay Natural Native 2001 Fenner (2003) Guinea -10,492 105,625 Australia Christmas Island Natural Native 1900 Bernard (1900) -2,492 -39,863 Brazil Acaraú Artifical, shipwreck Non-native 2016 K. Capel, pers. obs. -10,983 -36,932 Brazil Off Aracajú Artificial, platform Non-native 2013 G.S. do Nascimento, pers. comm. -12,485 -38,341 Brazil Todos-os-Santos Bay, near Maré Artificial, nautical Non-native 2012 Miranda et al. (2012) island signage -12,502 -38,474 Brazil Paraguaçu estuary Artificial, pier Non-native 2012 Miranda et al. (2012) -12,502 -38,473 Brazil Paraguaçu estuary Natural Non-native 2012 Miranda et al. (2016) -12,532 -38,413 Brazil Todos-os-Santos Bay, north Itaparica Artificial, floating pier Non-native 2011 Sampaio et al. (2012) island -12,574 -38,305 Brazil Todos os Santos Bay, Salvador Artificial, seawall Non-native 2015 Miranda et al. (2016) -12,854 -38,840 Brazil Canteiro de São Roque, Paraguaçu Artificial, platform Non-native 2014 J.C. Creed, pers. obs. -12,855 -38,838 Brazil Canteiro de São Roque, Paraguaçu Artificial, platform Non-native 2014 J.C. Creed, pers. obs. -12,894 -38,578 Brazil Todos-os-Santos Bay Natural Non-native 2015 Miranda et al. (2016) -13,055 -38,526 Brazil Todos-os-Santos Bay Artifical, shipwreck Non-native 2008 Sampaio et al. (2012) -13,320 48,250 Madagascar Ambaro Natural Native 1993 Cairns and Keller (1993) -13,630 125,620 Australia Cassini Island Natural Native 1991 Cairns (1998) -14,267 -170,650 American Aua Reef Natural Native 1973-1979 Lamberts (1983) Samoa

-14,272 -170,612 American Faga'itua pass Natural Native 1973-1979 Lamberts (1983) Samoa -14,306 -170,696 American Samoa Natural Native 2008 Lovell and McLardy (2008) -14,626 -145,199 French Takapoto Atoll Natural Native 1985 Vasseur (1985) Polynesia -14,670 145,450 Australia Lizard Island Natural Native 2004 Cairns (2004) -14,783 -38,967 Brazil Ilheus Artificial, drilling Non-native 2009 M.D.Correia, pers. comm platform -15,350 123,530 Australia Western Australia Natural Native 1991 Cairns (1998) -16,540 -151,760 French Society Islands, Bora Bora Natural Native 1958 Cairns (1994) Polynesia -17,538 -149,838 French Moorea Natural Native 1985 Vasseur (1985) Polynesia -17,908 178,009 Fiji Natural Native 2008 Lovell and McLardy (2008) -18,407 147,626 Australia Central region of the Great Barrier Natural Native 1984 Ayre and Resing (1986) Reef -19,045 35,848 Mozambique Natural Native 1988-1989 Cairns and Keller (1993) -19,670 63,436 Mauritius Rodrigues/Port Mathurin Natural Native NA Online Database -20,100 149,040 Australia off Hook Island Natural Native 1987 Online Database -20,160 149,075 Australia Deloraine Island Natural Native 1987 Online Database -20,205 164,355 New Northwestern Grande-Terre Natural Native 2007 Fenner and Muir (2009) Caledonia -20,431 115,556 Australia Montebello Islands Natural Native 2000 Marsh (2000) -20,470 149,230 Australia off Shaw Island Natural Native 1987 Online Database -20,480 116,530 Australia Kendrew Island Natural Native 1974 Online Database -20,700 -40,408 Brazil Escalvada Island, Espírito Santo Natural Non-native 2011 I. Caldas, pers. comm. state -20,745 165,285 New Touho-Ponérihouen Natural Native 2009 Fenner (2011) Caledonia -20,780 149,390 Australia Cockermouth Reef Natural Native 1987 Online Database -21,250 165,289 New Natural Native 2006 Pichon (2006) Caledonia -21,963 -39,675 Brazil Roncador Field, Campos basin Artificial, oil platform Non-native 2013 G.B. Almada, pers. comm. -22,320 113,800 Australia Cape Range National Park Natural Native 1977 Online Database

-22,367 -40,400 Brazil Voador Field, Campos Basin Artificial, oil platform Non-native 2013 R.G. dos Santos, pers. comm. -22,489 -40,378 Brazil Namorado field, Campos Basin Artificial, oil platform Non-native 2005 C.E.L. Ferreira, pers. comm. -22,775 -41,828 Brazil Armação de Búzios Natural Non-native 2005 Projeto Coral-Sol database -22,877 -43,122 Brazil Niterói Artificial, oil platform Non-native 2002 De Paula and Creed (2004) -22,891 -42,007 Brazil Cabo Frio Natural Non-native 2013 Projeto Coral-Sol database -22,900 -40,983 Brazil Pampo field, Campos Basin Artificial, oil platform Non-native 1997 Nicolau (1997) -22,969 -41,999 Brazil Arraial de Cabo Artificial, drill ship Non-native 2005 Ferreira et al. (2006) -22,969 -41,999 Brazil Arraial de Cabo Artificial, drilling Non-native 2005 Ferreira et al. (2006) platform -22,969 -41,999 Brazil Arraial de Cabo Artificial, drilling Non-native 2005 Ferreira et al. (2006) platform -22,969 -41,999 Brazil Arraial de Cabo Artificial, oil platform Non-native 2005 Ferreira et al. (2006) -22,972 -41,999 Brazil Arraial de Cabo Artificial, drill ship Non-native 2005 Ferreira et al. (2006) -22,973 -42,014 Brazil Arraial de Cabo Artificial, monobuoys Non-native 2007 Mizrahi (2008) -22,984 -42,002 Brazil Arraial do Cabo Natural Non-native 1999 Projeto Coral-Sol database -22,999 -43,966 Brazil Sepetiba Bay Natural Non-native 2011 Projeto Coral-Sol database -23,015 -44,317 Brazil Angra dos Reis Artificial, oil platform Non-native 2007 J.C. Creed, pers. obs. -23,111 -44,267 Brazil Ilha Grande Bay Natural Non-native 1990's De Paula and Creed (2004) -23,117 -44,283 Brazil Ilha Grande Bay Artifical, shipwreck Non-native 2015 J. C. Gomes, pers. comm. -23,117 -44,283 Brazil Ilha Grande Bay Artifical, piers, docks Non-native 2012 Mangelli and Creed (2012) and decks -23,140 -44,165 Brazil Ilha Grande, Vila do Abraão Artificial, small pleasure Non-native 2014 M.C. Mantelatto, pers. obs. boat -23,230 151,820 Australia North West Island Natural Native 1972 Online Database -23,797 -45,157 Brazil Ilhabela Natural Non-native 2008 Mantelatto et al. (2011) -23,813 -45,403 Brazil São Sebastião Artificial, monobuoys Non-native 2012 J.C. Creed, pers. obs. -23,813 -45,403 Brazil São Sebastião Artificial, monobuoys Non-native 2012 J.C. Creed, pers. obs. -23,920 152,400 Australia Lady Musgrave Reef Natural Native 1971 Online Database -24,104 -45,691 Brazil Alcatrazes Archipelago Natural Non-native 2014 K. Capel, pers. obs. -24,321 -46,179 Brazil Laje de Santos Natural Non-native 2012 A. Costa, pers. comm. -25,500 113,030 Australia Dirk Hartog Island Natural Native NA Online Database

-26,767 -46,784 Brazil Caravelas field, Itajaí Artificial, oil platform Non-native 2000 Barreiros et al. (2000) -27,284 -48,366 Brazil Arvoredo Natural Non-native 2012 K. Capel, pers. obs. -28,470 113,700 Australia West Wallabi Island Natural Native 1978 Online Database -28,730 113,770 Ausralia Houtman Abrolhos Natural Native NA Online Database -29,273 -177,921 New Zealand Kermadec Islands, Raoul and Natural Native 1985-1995 Brook (1999) adjacent islets -31,530 159,070 Australia Lord Howe Island Natural Native NA Online Database -32,020 115,500 Australia Rottnest Island Natural Native 1972 Online Database -33,820 151,275 Australia Port du Roi George and Port Jackson Natural Native 1833 Cairns (1994) -43,485 171,265 New Zealand New Zealand Natural Native 2009 Cairns et al. (2009)

Tubastraea floreana Wells, 1982 Decimal Decimal Country Region Substrate Category Date Information source Latitude* Longitude* reported** -1,268 -90,434 Galápagos Floreana Island, Playa Preta Natural Native 1975 Cairns (1991) -0,201 -90,830 Galápagos Buccaneer Cove Natural Native 1978 Cairns (1991) -0,792 -91,196 Galápagos Isabela Island, Caleta Iguana Natural Native 1975 Cairns (1991) -1,333 -90,294 Galápagos Espanola Island, Gardiner Island Natural Native 1977 Cairns (1991) -0,688 -90,541 Galápagos Pinzon Island Natural Native 1991 Cairns (1991) -0,034 -91,495 Galápagos Isabela Island, Tagus Cove, North Natural Native 1934 Online Database Shore Of Cove

Tubastraea faulkneri Wells, 1982 Decimal Decimal Country Region Substrate Category Date Information source Latitude* Longitude* reported** 7,097 134,284 Palau Bailechesengel Island Natural Native 1973 Online Database -4,529 129,865 Indonesia Moluccas, Banda Island, Goeneng Natural Native 1975 Online Database Api -14,028 47,964 Madagascar Radama Islands, Berafia Island Natural Native NA Online Database -20,300 57,694 Mauritius Flic-En-Flac Point, Lagoon Natural Native NA Online Database 79

13,658 120,897 Philippines Luzon Island, Batangas Bay, Se Natural Native 1994 Online Database Corner of Mouth of Bay, Off Pulangbuli -18,824 147,650 Australia Davies Reef Natural Native 2000 Koh and Sweatman (2000) 22,300 114,369 Hong Kong Steep Isandls Caves Natural Native 2008 Lam et al. (2008) 33,578 126,573 Korea Off Jeju-do Natural Native 2017 Choi and Song (2017)

Tubastraea diaphana (Dana, 1846) Decimal Decimal Country Region Substrate Category Date Information source Latitude* Longitude* reported** -29,683 32,097 South Africa Off Natal Natural Native 1988 Cairns e Keller 1993 -12,169 96,908 Australia Cocos Keelings Islands Natural Native NA Online Database 5,780 103,000 Malaysia Pulau Redang, Kerengga Besar Natural Native 1977 Online Database 9,421 123,303 Philippines Cebu Island, Liloan Point Natural Native 1978 Online Database 1,160 103,741 Singapure Raffles Lighthouse Natural Native NA Online Database 6,975 122,330 Philippines Zamboanga Del Sur, Santa Cruz Natural Native 1967 Online Database Island -20,725 -175,046 Tonga - Natural Native 1969 Online Database -2,804 -171,663 Kiribati Canton Island Natural Native 1954 Online Database 6,889 122,070 Philippines Zamboanga Del Sur, Santa Cruz Natural Native 1967 Online Database Island 4,109 116,520 Malaysia Sabah, Borneu Island Natural Native NA Online Database -23,439 151,920 Australia Heron Island, Lee Reef, NW Side Of Natural Native 1954 Online Database Island -5,806 39,181 Tanzania Zanzibar Natural Native 1959 Cairns and Keller (1993); Online Database -13,330 48,250 Madagascar Nosy Be, Tankley Natural Native NA Cairns and Keller (1993); Online Database -12,790 160,036 Solomon Indispensable reef, , Leeward Side Natural Native 1994 Online Database Island -0,029 98,521 Sumatra Pulo Bai Natural Native 1963 Online Database 21,252 -157,611 USA Hawaii Natural Native 2005 Fenner (2005); Online Database 80

12,900 121,225 Philippines West coast of Mindoro Natural Native 1997 Cairns e Zibrowius 1997 9,217 121,762 Philippines Sulu Sea Natural Native 1997 Cairns e Zibrowius 1997 9,217 121,762 Philippines Negros Natural Native 1997 Cairns e Zibrowius 1997 9,600 121,754 Indonesia Savu Sea Natural Native 1997 Cairns e Zibrowius 1997 22,366 114,391 Hong Kong Conic Islands, Steep Isandls Caves Natural Native 2008 Lam et al. (2008) -20,569 116,795 Dampier Legendre Island Natural Native 2004 Griffith 2004 Archipelago -21,206 165,844 New northeast Natural Native 2009 Fenner and Muir (2013), Caledonia Online Database 27,095 124,716 Taiwan Taiwan Natural Native 2016 A. Chen, pers. comm. 28,580 -90,070 USA Gulf of Mexico Artifical, oil platform Non-native 2010 Online Database 10,564 119,737 Philippines Palawan Island, Small Island NE Of, Natural Native 1985 Online Database In Dumuran Passage 7,276 134,591 Palau Auluptagel Island, Ngell Channel Natural Native 1983 Online Database -21,025 149,901 Australia Queensland, Great Barrier Reef, Natural Native NA Online Database Penrith Island -20,782 149,390 Australia Queensland, Great Barrier Reef, Natural Native 1987 Online Database Cockermouth Island -32,005 115,560 Australia Western Australia, Rottnest Island Natural Native 1956 Online Database 7,352 135,024 Palau Great Reef Natural Native 1973 Online Database -14,300 -170,680 American Tutuila Island, Pago Pago Harbor Natural Native NA Online Database Samoa 20,698 116,733 Dongsha Islan Dongsha Natural Native 2016 M. E. Santos, pers. Comm. ds -29,306 -177,858 New Zeland Kermadec Islands Natural Native 1985-1988 Brook (1999)

*Coordinates in gray are estimated coordinates based on the name/description of the geographical region in order to build distribution maps

**Dates in gray are those of the publication from which the record was taken when the original date of observation was not

81 presented/available NA = not available

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Online databases: Global Biodiversity Information Facility: http://www.gbif.org/ Species Link: http://splink.cria.org.br/ Encyclopedia of Life: http://www.eol.org Hexacorallians of the World: http://www.hercules.kgs.ku.edu WoRMS: http://www.marinespecies.org/ USNM Invertebrate Zoology Cnidaria Collection: http://collections.nmnh.si.edu/search/iz/

Supplemental File 2 Phylogenetic analyses based on Maximum likelihood 13 mitochondrial protein-coding genes, two ribosomal RNA (rRNA) and two transfer RNA (tRNA), after removing all sites with missing data (final alignment 9,064 bp long) from 18 Dendrophylliidae corals and Porites porites as external group.

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Capítulo II

Laborelia, a new genus of Dendrophylliidae (Cnidaria: Scleractinia) from Eastern Atlantic

Kátia Cristina Cruz Capel, Carla Zilberberg, Joel Creed, Ingrid Knapp, Zac Forsman, Robert Toonen & Marcelo Kitahara

Revista alvo: Zootaxa.

Laborelia, a new genus of Dendrophylliidae (Cnidaria, Scleractinia) from Eastern Atlantic

KCC Capel1,2*, C Zilberberg1,2, JC Creed2,3, Knapp I4, Z Forsman4, RJ Toonen4, O Ocaaña,5, MV Kitahara6,7

1 Departamento de Zoologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil 2 Associate Researcher, Coral-Sol Research, Technological Development and Innovation Network 3 Departamento de Ecologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil 4 School of Ocean & Earth Science & Technology, Hawaiʻi Institute of Marine Biology, University of Hawaiʻi at Mānoa, Kāneʻohe, HawaiʻI, United States 5 Museo del Mar, Ceuta, Spain 6 Departamento de Ciências do Mar, Universidade Federal de São Paulo, Santos, Brazil 7 Centro de Biologia Marinha, Universidade de São Paulo, São Sebastião, Brazil

* Corresponding author Kátia Capel e-mail: [email protected] Laboratório de Biodiversidade de Cnidária. Departamento de Zoologia Universidade Federal do Rio de Janeiro.

Abstract

Laborelia is a new genus of the Dendrophylliidae family found at Cape Verde, Eastern Atlantic. Specimens were first recognized as Enallopsammia micranthus and further described as a new species Tubastraea caboverdiana, changing the status of this genus as native for the Atlantic Ocean. Here we describe a new genus to accommodate this species based on morphological and molecular analyses, comparing to other Dendrophylliidae genus. The new genus has normally arranged septa (not Portualès Plan), poor developed columella and a shallow water distribution, all supporting the new classification and validated by molecular data. Our results support the status of invasive for the genus Tubastraea and recommend an extensive review of shallow water dendrophylliids in the Eastern Atlantic.

Key words: Cape verde; azooxanthellate corals; Tubastraea

Introduction

The Dendrophylliidae is the third most speciose family of the order Scleractinia, comprising 21 genera that embrace 364 valid species of which 166 are extant (Cairns 2001; Pascual-Sánchez 2013). Such diversity is followed by a wide variety of growth forms (e.g. solitary and colonial), life styles (i.e. zooxanthellate, azooxanthellate and facultative), and an extensive distribution range, occurring from the tropics to polar regions at depths up to 2165 m (Cairns 2001). Although recovered as monophyletic (Arrigoni et al. 2014; Kitahara et al. 2010; Nunes et al. 2008), the generic evolutionary relationships of the family remains unclear, with several poly/paraphyletic genera (Arrigoni et al. 2014; Kitahara et al. 2016).

Scleractinian classical taxonomy relies on morphological characters of the skeleton (Milne-Edwards & Haime 1857), but high intraspecific variation, convergence and homoplasy frequently challenges species identification, especially in shallow water species. Within dendrophylliids, morphological characters used to reconstruct the evolutionary history of the group (i.e. corallum morphology, theca structure, calicular elements, and presence of zooxanthellae) does not seem to be informative (Arrigoni et al. 2014). In fact, not all evolutionary changes that result in speciation such as changes in reproduction and ecology are accompanied by morphological changes (Gélin et al. 2017; Paz-García et al. 2015).

The genus Tubastraea Lesson, 1829 comprises six extant species and several unidentified morphotypes, all of which are azooxanthellate and native from the Eastern Pacific or Indo-Pacific Oceans (Arrigoni et al. 2014; Cairns 2001; Fenner 2005). In the Eastern Atlantic (EA) the status of the genus is unclear and has been under discussion for more than four decades (Creed et al. 2016; Laborel 1974). Laborel (1974) considers the occurrence of the genus Tubastraea in the EA as a recent invasion from the Indo-Pacific or Caribbean waters, but fossils of T. coccinea from the Pleistocene and also living specimens have been mentioned from Cape Verde (Boekschoten & Best 1988). However, description/figures have not been provided for these fossil specimens. The genus was first recorded in the EA with two morphotypes from Gulf of Guinea, Gabon, Sierra Leone and Cape Verde (Laborel 1974). At the first three sites two distinct morphotypes differing by colony growth form and tissue pigmentation were mentioned, one with orange subplocoid colonies and the other branching with yellow coenosarc. However, two morphologically undistinguishable forms displaying both orange and yellow tissue pigmentation and branching colony were observed at Cape Verde (Laborel 1974). Due to its dendroid colony growth form, 97 the species from Cape Verde was firstly identified as Enallopsammia micranthus by Chevalier (1966); however, in a revision of the genus Zibrowius (1973) considered it a different species: “Or, il s'agit apparemment d'une espèce de Coenopsammia différente des formes indo-pacifiques précédemment désignées sous ce même nom spécifique (micranthus Ehrenberg).” In 1974, Laborel identified this species as Tubastraea sp. and highlighted the need of a taxonomical revision of the genus. More recently, Ocaña et al. (2015) re-examined specimens from Cape Verde and described a new species, Tubastraea caboverdiana, based on morphological differences to other Tubastraea, especially T. coccinea.

Following the examination of new samples from the type locality of T. caboverdiana we observed several morphological characters that are inconsistent to those from Tubastraea. Such morphological divergence is mirrored at molecular level and together, suggests that T. caboverdiana has more affinity to other dendrophylliid genera than to Tubastraea. Here we describe a new genus to accommodate this species based on morphological and molecular data and discuss main morphological divergences with other Dendrophylliidae genera.

Material and methods

Sampling

A total of 21 samples of the two color morphotypes (ten orange and eleven yellow) were collected by SCUBA from T. caboverdiana type locality (Tarrafal, Sao Tiago Island, Cape Verde - 15º10’N, 23º47’W). Tissue samples of each colony were preserved in CHAOS solution (4 M guanidine thiocyanate, 0.1% N-lauroyl sarcosine sodium, 10 mM Tris pH 8, 0.1 M 2-mercaptoethanol) (Fukami et al. 2004) for molecular analyses and the skeleton was bleached in a sodium hypochlorite solution for morphological analyses. All dry specimens will be deposited at the Museu Nacional do Rio de Janeiro (MNRJ).

Morphological analyses

Species re-description was based on the new collected material from the type locality and also from pictures of the holotype of T. caboverdiana, deposited at the Museo del mar de Ceuta (MMC) (Spain) (MMC-26) (Ocaña et al. 2015). Identification and comparison to other

Dendrophylliidae followed Chevalier (1966), Zibrowius (1980), Cairns (2001) and Cairns & Kitahara (2012).

DNA extraction and sequencing

One individual of each color morph was used for molecular analyses. DNA was extracted using DNAeasy Tissue and Blood Kit (Quiagen Inc., Valencia, CA, USA) following the manufactures instruction. Extraction was checked in a 1% agarose gel and quantified using AccuClear UltraHigh Sensitivity dsDNA quantification kit (Biotium, Inc.) measured in a SpectraMax M2 microplate reader. A protocol of restriction site associated DNA sequencing (ezRAD; see Knapp et al. 2016; Toonen et al. 2013) was used for sequencing using the GATC cut site restriction enzyme DPNII in 50 µl reactions (18-19 µl HPLC grade water, 1 µl of the restriction enzyme - 10 units), 5 µl Cutsmart Buffer and 25 µl of DNA, followed by 3 hours incubation at 37ºC and 20 minutes at 65ºC. Samples were cleaned with Ampure XP beads in 1:1.8 ratio of DNA:Beads and libraries were generated using KAPA library preparation kit (Roche) including the size-selection (350-700bp) and PCR steps. All libraries were sequenced as 300 bp single-end on the Illumina MiSeq at Genetics Core Facility at the Hawai’i Institute of Marine Biology.

Phylogenetic analyses

Sequences obtained were trimmed for quality and adaptors and assembled with a reference species (AQ2 Tubastraea coccinea HG965344, HG965278 and HG965410) to recover two mitochondrial and one nuclear marker using Geneious 7.1.9 (Kearse et al. 2012). The three regions used was (1) COI, (2) an intragenic region between COI and trnM, trnM and a portion of large ribosomal subunit (herein after called IGR), and (3) ITS1, 5.8S, ITS2 2 and a portion of 18S and 28S (herein called rDNA). Additional sequences of 67 Dendrophylliidae and one Poritidae (to be used as external group) were downloaded from GenBank for phylogenetic analyses (Supplemental File 1). Sequences were aligned using MUSCLE algorithm implemented in Geneious 7.1.9 (Kearse et al. 2012) and concatenated in a final alignment of 1,931 bp in length. Maximum Likelihood (ML) and Bayesian inference (BI) phylogenetic reconstruction analyses were performed with PhyML (Guindon et al. 2010) 99 and MrBayes 3.2.6 (Ronquist & Huelsenbeck 2003) available on Geneious. For the ML analyses the evolutionary model HKI+I was used, as suggested by jModel Test (Darriba et al. 2015) for the concatenated sequences. For the BI, specific evolutionary models were used for each locus and codon position for COI as suggested by PartitionFinder 2 (Lanfear et al. 2012). PartitionFinder 2 suggested HKY+G for COI_pos1, IGR and COI_pos2, F81+I for COI_pos3 and K80+I+G for rDNA. Bayesian analyses were run for 1 million generations with sampling every 200 generations and a burn-in of 100.0000.

Results

Systematics

Class Anthozoa Ehrenberg, 1834

Subclass Hexacorallia Haeckel, 1896

Order Scleractinia Bourne, 1900

Family Dendrophylliidae Gray, 1847

Type genus. Dendrophyllia de Blainville, 1830.

Diagnosis (from Cairns 2001). Wall synapticulothecate, formed of irregularly arranged synapticulae, resulting in an irregularly porous theca through which extratentacular mesenterial extensions protrude. Septa consisting of one laminar fan system of simple trabeculae, but trabeculae irregularly developed and not closely united, resulting in thickened and usually perforate septa; upper and axial margins of lower-cycle septa smooth. Septa usually arranged in Pourtalès plan at some stage of skeletal ontogeny.

Laborelia new genus

Type species. Laborelia caboverdiana (Ocaña & Brito, 2015), by monotype.

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Diagnosis. Colonies bushy, phaceloid to dendroid, all achieved by extratentacular budding (frequently from theca of a parent corallite). No epitheca. Septa normally arranged and covered with granules. Columella poorly or moderately developed.

Remarks. Laborelia caboverdiana is morphologically similar to Cladopsammia, Leptopsammia, Astroides, Enallopsammia and Dendrophyllia. The new genus differs from existing genera by having colonial forms with normally arranged septa (not Portualès Plan), poor developed columella and shallow water distribution. Morphological differences were supported by molecular data.

Distribution. Archipelago of Cape Verde, Eastern Atlantic.

Etymology. The name is dedicated to Laborel, who provided an extensive contribution to the knowledge on the Atlantic coral fauna.

Laborelia caboverdiana (Ocaña & Brito, 2015), new combination

Figs. 1-4

Enallopsammia micranthus. –Chevalier, 1966: 1387-1390.

Tubastraea sp. –Laborel, 1974: 434-435.

Tubastraea caboverdiana Ocaña & Brito, 2015: 48-52.

Type material. MMC-26 (Holotype) (Ocaña et al. 2015).

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Type locality. Sao Tiago Island, Cape Verde, 10 meters.

Material examined. Yellow morphotype: Tarrafal, Cape Verde, 11 colonies. Orange morphotype: Tarrafal, Cape Verde, 10 colonies.

Taxonomic history. This species was first identified as Enallopsammia micranthus by Chevalier (1966) and later moved to Tubastraea by Laborel (1974). A species level identification was given only recently by Ocaña et al. (2015) who described it as T. caboverdiana. The species is herein re-described as Laborelia caboverdiana.

Distribution. Currently known only from the Archipelago of Cape Verde but possibly occurring on Gulf of Guinea (based on descriptions from Laborel [1974]).

Description. Corallum colonial, phaceloid to dendroid forming bushy colonies. Budding extratentacular from commom corallum base and frequently from theca of a parent corallite. Largest colony examined (number) bear 89 corallites. Corallite cylindrical, calice circular to slightly elliptical ranging between 3 and 11 mm in diameter (GCD). Most examined colonies bear a few main corallites projecting up to 56 mm above common base, from which new buds arise. Calicular edge slightly thinner than remaining theca. Theca porous especially near calicular edge. Costae granular, separated by deep narrow ridges. Coenosarc orange or yellow.

Septa hexamerally arranged in four nonexsert cycles according to the formula: S1>S2>S3>S4. All septa thin. S1 extend about at least 2/3 distance to columella with entire and vertical axial edges. S2 slightly smaller than S1. S1-2 always merge with the columella. S3 about ½ width of S2. In each system, a pair of S3 fuse to common S2 near columella. S3 bear laciniate axial edge in smaller corallites but entire in larger corallites, when it merges with S2 deep in fossa. S4 rudimentary, entire or with slightly laciniate axial edge. Septal faces

102 covered with pointed granules. Fossa deep containing a poorly or sometimes moderately developed columella.

Discussion. Laborelia caboverdiana differs from Tubastraea representatives by having a phaceloid to dendroid corallum forming a bushy colony with new buds often arising from the theca of a parent corallite in an acute angle. Septa width also differentiates Laborelia from Tubastraea species, since in the former septa are wider and project further into the calice. Two morphologically undistinguishable color forms of Laborelia caboverdiana are found in Cape Verde, a yellow and a red morph, both undistinguishable morphologically. According to Laborel (1974), these specimens from Cape Verde resemble the yellow morph found at the Gulf of Guinea.

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Figure 1. In situ images of Laborelia caboverdiana at Cape Verde. (A) orange color-morph; (B) yellow color- morph; and (C) Both color-morphs growing together.

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Figure 2. Skeleton images of colonies and corallites of the holotype of Laborelia caboverdiana (MMC-26), deposited at Museo del mar de Ceuta (MMC) (Spain). Images courtesy from Juan Antonio Rosa Montes.

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Figure 3. Skeleton images of colonies and corallites of Laborelia caboverdiana species. Specimens CVl-1 (A), CVl-l3 (B-C), CVa-10 (D) and CVa-21 (E-F).

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Figure 4. Microscopy electron scanning images Laborelia caboverdiana species. Specimens CVl-13. (A-C) and CVa-10 (D-F).

Phylogenetic analysis

Phylogenetic analyses were performed based on two mitochondrial (COI and IGR) and one nuclear (rDNA) gene, having 601, 450 and 888 pb, respectively. Sequences were concatenated in a final alignment of 1,931 bp for a total of 70 specimens. Within these partial sequences the rDNA holds the highest phylogenetic signal with 257 informative sites out of 365 variable sites, contrasting to 42 out of 70 and 36 out of 63 for COI and IGR, respectively. Both methods BI and ML recovered similar topologies (Figure 5; Supplemental File 2),

107 corroborating the position of Laborelia caboverdiana outside the clade including seven Tubastraea species. In all evolutionary reconstructions Laborelia caboverdiana was recovered close to a clade containing Dendrophyllia conigera and Leptopsammia pruvoti (Figure 5).

~------Goniopora c-ol,inma ~ ---- AQ J Cludop.w;mm/a :-p. l 1I S:! I ] 4 l(gudtip,thntmfa ,te,:1w..m [11t1 HS3185 Egm.:l:ipscw:mia s<:rpe,:iifu, MQ002 Ctadopsammilr eguci:ii MQ071 CladopMmmiaegw: hff MQ ISS Claa'opsa,,;mia c.e,ffcMi AS3 Heterop.vanm:ú;• cucli!eu \!C.:688 l i(W!.ropsammlt: Córhl1;a KC/76 Ht:ü:rop.:;c,-mmiu r.:uc:Mt·u AK22 7irrhl,wdu mewmt?:rina AK4 'forb/Jrar/11 n!llijormls 14 3 B.\ 124 Turbi,wria mest.~11frri11a HS 1747 Turhi,:aria sr. 2 HS 1775 Turbinarh1 sp. 2 ._____ BAL261iu-bin

AS6 Dt111cono,{)j'(mm1ia axi_j.i,ga 0.1!.'60 ,------e KTj6 Dum.:w.upwmmw a.rüügu 1'02 Turófr,ariu peita:., HS:205& 1i,rbi,:al'ia pd,ata l lS28t;7 Dalpl;vlJl11 rmgna MYl06 Ri:frop:.ummiu w<:!ISü..' ilii SR 11 Deudropl~f•lNu ar!m.-. c;rtla UJ2.t? Rhizops1Jm111fa weUstéiJtf , .MQ 180 Rhiw psammüt v... -- n·,Wi o.9~®HS2888 Rluzop.mmmfa verrUli ~ I) l ~ AO 147 1 Rliiw psammill cf. veni!U ~-----·' · SR26 Cladopsammia gracfh's AO 105 C:!adop.wmmw g,.m:i!i.,; M().0]5 lli:iMt>famm/h vn,·Uii M765 Rhizovsammia »w1s11..·i,,i º~'i'°f"jm~1ii,;ª~"~'•~'";1e1~·~m·c_ ____ n.v:,... rj---;:::=::::::::::::::S0:::=02:::9:::R:-'hMY~ú~· 105 1i1bo.s1rac-a sp. 1 7 S0119 T11bast1cwa aurca M762 Tllba.'i1raea mm"'.. MY070 Tuh

0.06 Figure 5. Phylogenetic analyses based on Bayesian inference of the concatenated genes COI, IGR and rDNA from 70 Dendrophylliidae corals and Goniopora columna as external group. Black dots indicate branches with Posterior probability ≥ 98 and bootstrap support value ≥ 98.

108

Discussion

Although all studies using molecular data have recovered Dendrophylliidae as monophyletic, several genera within the family have been shown to be poly or paraphyletic (Arrigoni et al. 2014; Kitahara et al. 2010). Dendrophylliidae is the third most diverse family within the order and has an intricate and challenging taxonomy. Morphological plasticity, intraspecific variability, convergence and homoplasy are some of the factors that frequently challenge traditional scleractinian taxonomy, especially for shallow waters species (Kitahara et al. 2010). Here we described a new Dendrophylliidae genus to accommodate a recently described species from Cape Verde, L. caboverdiana.

The species was previously described as Tubastraea sp., however morphological and molecular evidences support the new combination proposed herein. Despite some similarities to Cladopsammia, Astroides, Enallopsammia and Dendrophyllia, the analyzed material does not fit within any Dendrophylliidae genera. According to the diagnosis, Cladopsammia build “small bushy colonies formed by extratentacular budding from common basal coenosteum and occasionally from edge zone of larger corallites…Pourtalès plan well developed” (Cairns 2001). However, although budding from thecal edge of larger corallites is frequent, Pourtalès Plan is absent in L. caboverdiana. On the recovered phylogeny, Cladopsammia is polyphyletic and none of the three species included are closely related to L. caboverdiana (Figure 5). Astroides is a monospecific genus with variable morphology (cerioid, plocoid and phaceloid), characterized by having a massive columella, a shallow fossa and septa with dentate axial edges (Cairns 2001), none observed in L. caboverdiana. Furthermore, no closely relationship was recovered by molecular data (Figure 5).

Dendrophyllia, another comparable genus, is also polyphyletic (Arrigoni et al. 2014) and morphologically divided into three groups according to the colony growth form: monopodial; sympodial; and bushy (Cairns 2009). All Dendrophyllia have septa arranged in Pourtalès Plan, which as stated above is absent in L. caboverdiana. Phylogenetic analyses comprising 11 of the 21 recognized genera within the family recovered L. caboverdiana more closely related to Dendrophyllia cornigera and Leptopsammia pruvoti, both found in the Northeastern Atlantic (Zibrowius 1980). D. cornigera have a ramose growth form, somewhat similar to L. caboverdiana, but differing by having septa arranged in Pourtalès Plan and a

109 deeper distributional range (98-600 meters deep) (Cairns 2009). Leptopsammia pruvoti is similar to L. caboverdiana in having normally arranged septa (not Portualès Plan) and the first cycles of septa (S1 and S2) with smooth axial edges (Cairns 2001). However, Leptopsammia is composed only by solitary species. Due to these morphological disparities, we opted to not synonymize D. cornigera and L. pruvoti. We recommend the inclusion of more specimens, as well as more species from both genera to better understand their evolutionary relationship.

Although lacking half of the extant genus diversity within the family, the evolutionary reconstruction includes representatives of almost all colonial genera, except for Dichopsammia Song, 1994 and Enallopsammia Michelotti, 1871. Dichopsammia is a monospecific genus found in the North Pacific, of which colonies are formed exclusively by intratentacular budding, contrasting to Laborelia. Enallopsammia, on the other hand, is morphologically similar to L. caboverdiana. Those specimens from Cape Verde were first identified as Enallopsammia micranthus but later transferred to Tubastraea (see Zibrowius 1973; Laborel 1974). Enallopsammia comprises three extant species with arborescent growth form, a papillose columella and are distributed in the Atlantic and Pacific Oceans at depths from 110 to 2165 meters (Cairns 2001), all contrasting to L. caboverdiana.

Azooxanthellate corals remains understudied compared to their symbiotic counterparts (Kitahara et al. 2016) and the status of the genus Tubastraea in the EA remained under discussion for several decades (Creed et al. 2016; Laborel 1974). Here we described a new genus to accommodate L. caboverdiana, previously described as Tubastraea from Cape Verde, EA. Thus, we suggest that Tubastraea remains classified as invasive in the EA and indicates that a reevaluation of the EA shallow water dendrophylliids, mainly those currently identified as belonging to the genus Tubastraea is urgently needed to confirm species identity and clarify the current distribution of both Tubastraea and Laborelia.

Acknowledgments

We are grateful to Inácio Domingos Silva Neto for the support with Microscopy electron scanning and Oscar Ocaña and Juan Antonio Rosa Montes for holotype pictures. This research was supported by the PADI Foundation granted to K.C.C.Capel thanks (grant # 21882).

110

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Supplemental File 1 List of specimens of Dendrophylliidae included in phylogenetic analyses with corresponding identification, locality and accession numbers. * indicates new sequences obtained by the present study. Remaining sequences (except for Goniopora columna) are from Arrigoni et al. (2014).

Species Identification Locality Accession numbers COI IGR rDNA Astroides calycularis MED842 Mediterranean HG965307 HG965239 HG965371 Sea Astroides calycularis MED843 Mediterranean HG965308 HG965240 HG965372 Sea Balanophyllia SOL1 Mediterranean HG965309 HG965241 HG965373 (Balanophyllia) europaea Sea Balanophyllia SOL2 Mediterranean HG965310 HG965242 HG965374 (Balanophyllia) europaea Sea Balanophyllia SOL4 Mediterranean HG965311 HG965243 HG965375 (Balanophyllia) europaea Sea Balanophyllia HS3312 New HG965312 HG965244 HG965376 (Eupsammia) imperialis Caledonia Balanophyllia HS2887 New HG965313 HG965245 HG965377 (Eupsammia) imperialis Caledonia Balanophyllia SOL3 Mediterranean HG965314 HG965246 HG965378 (Balanophyllia) regia Sea Balanophyllia SOL7 Mediterranean HG965315 HG965247 HG965379 (Balanophyllia) regia Sea Cladopsammia sp. 1 AQ1 Japan HG965316 HG965248 HG965380 Cladopsammia eguchii MQ002 Marquesas, HG965317 HG965249 HG965381 French Polynesia Cladopsammia eguchii MQ071 Marquesas, HG965318 HG965250 HG965382 French Polynesia Cladopsammia eguchii MQ155 Marquesas, HG965319 HG965251 HG965383 French Polynesia Cladopsammia gracilis AO105 Japan - HG965252 HG965384 Cladopsammia gracilis SR26 Japan HG965320 HG965253 HG965385 Dendrophyllia arbuscula SR11 Japan HG965321 HG965254 HG965386 Dendrophyllia cornigera CA01 Mediterranean HG965322 HG965255 HG965387 Sea Dendrophyllia cornigera MI01 Mediterranean HG965323 HG965256 HG965388 Sea Duncanopsammia axifuga AS6 Australia HG965325 HG965258 HG965390 Duncanopsammia axifuga KT56 Australia HG965326 HG965259 HG965391 Eguchipsammia serpentina HS3134 New HG965327 HG965260 HG965392 Caledonia Eguchipsammia serpentina HS3185 New HG965328 HG965261 HG965393 Caledonia Heteropsammia cochlea AS3 Australia HG965329 HG965262 HG965394 Heteropsammia cochlea NC688 New HG965330 HG965263 HG965395 Caledonia Heteropsammia cochlea NC776 New HG965331 HG965264 HG965396 Caledonia Leptopsammia pruvoti MD02 Mediterranean HG965332 HG965265 HG965397 Sea Leptopsammia pruvoti MD03 Mediterranean HG965333 HG965266 HG965398 Sea Rhizopsammia cf verrilli AO147 Japan - HG965267 HG965399 113

Rhizopsammia verrilli MQ035 Marquesas, HG965334 HG965268 HG965400 French Polynesia Rhizopsammia verrilli MQ180 Marquesas, HG965335 HG965269 HG965401 French Polynesia Rhizopsammia verrilli HS2888 New HG965336 HG965270 HG965402 Caledonia Rhizopsammia wettsteini DJ247 Djibouti HG965337 HG965271 HG965403 Rhizopsammia wettsteini M765 Maldives HG965338 HG965272 HG965404 Rhizopsammia wettsteini MY106 Mayotte HG965339 HG965273 HG965405 Island Rhizopsammia wettsteini SO029 Socotra HG965340 HG965274 HG965406 Island, Yemen Tubastraea cf aurea M762 Maldives HG965341 HG965275 HG965407 Tubastraea cf aurea MY070 Mayotte HG965342 HG965276 HG965408 Island Tubastraea cf aurea SO119 Socotra HG965343 HG965277 HG965409 Island, Yemen

Tubastraea coccinea AQ2 Japan HG965344 HG965278 HG965410 Tubastraea coccinea SR144 Japan HG965345 HG965279 HG965411 Tubastraea coccinea SR28 Japan HG965346 HG965280 HG965412 Tubastraea diaphana AO101 Japan - HG965281 HG965413 Tubastraea micranthus AO100 Japan - HG965282 HG965414 Tubastraea micranthus HS3129 New HG965347 HG965283 HG965415 Caledonia Tubastraea micranthus M768 Maldives HG965348 HG965284 HG965416 Tubastraea micranthus MY072 Mayotte HG965349 HG965285 HG965417 Island Tubastraea micranthus Y756 Yemen HG965350 HG965286 HG965418 Tubastraea sp. 1 MY105 Mayotte HG965351 HG965287 HG965419 Island Tubastraea sp. 2 HS2883 New HG965352 HG965288 HG965420 Caledonia Tubastraea sp. 2 HS2884 New HG965353 HG965289 HG965421 Caledonia Tubastraea sp. 2 HS2890 New HG965354 HG965290 HG965422 Caledonia Tubastraea sp. 3 KI2 Japan HG965355 HG965291 HG965423 Tubastraea sp. 3 KI3 Japan HG965356 HG965292 HG965424 Turbinaria heronensis HS1986 New HG965357 HG965293 HG965425 Caledonia Turbinaria heronensis HS2178 New HG965358 HG965294 HG965426 Caledonia Turbinaria mesenterina AK22 Japan HG965359 HG965295 HG965427 Turbinaria mesenterina BA124 Yemen HG965360 HG965296 HG965428 Turbinaria patula HS2283 New HG965361 HG965297 HG965429 Caledonia Turbinaria patula HS1835 New HG965362 HG965298 HG965430 Caledonia Turbinaria peltata HS2058 New HG965363 HG965299 HG965431 Caledonia Turbinaria peltata NG2 Japan HG965364 HG965300 HG965432 Turbinaria reniformis AK4 Japan HG965365 HG965301 HG965433 Turbinaria reniformis BA126 Yemen HG965366 HG965302 HG965434 Turbinaria sp. 1 HS1752 New HG965367 HG965303 HG965435 Caledonia Turbinaria sp. 1 HS1793 New HG965368 HG965304 HG965436

114

Caledonia Turbinaria sp. 2 HS1747 New HG965369 HG965305 HG965437 Caledonia Turbinaria sp. 2 HS1775 New HG965370 HG965306 HG965438 Caledonia Goniopora columna Y698 Yemen JF825141 JF825141 AB906954 Laborelia caboverdiana CVL-1 Cape Verde * * * Laborelia caboverdiana CVa-6 Cape Verde * * *

115

Supplemental File 2 Phylogenetic analyses based on Maximum likelihood of the concatenated genes COI, IGR and rDNA from 70 Dendrophylliidae corals and Goniopora columna as external group.

~------GoniopOfa

., 72 l__- ---""'[==- HHS:,838:2 o MEOSO ,1,1,10842

T_u.bowrdiana._am,1ret.i ----=, ---,____ T_ClbMtd.ana,_l'-rJnj.J

116

Capítulo III

Complete mitochondrial genome sequences of Atlantic representatives of the invasive Paciﬁc coral species Tubastraea coccinea and T. tagusensis (Scleractinia, Dendrophylliidae): Implications for species identiﬁcation

Kátia Cristina Cruz Capel, Alvaro Esteves Migotto, Carla Zilberberg, Mei Fang Lin, Zac Forsman, David Miller & Marcelo Kitahara

Publicado na revista Gene em 2016.

117

Cene 590 (2016) 270-277

Contents lists available at ScienceDirect Gene

j ournal homepage: www.elsevi er.com/locate/ gene

Research paper

Complete mitochondrial genome sequences of Atlantic representa tives of (1) CrossMark the invasive Pacific coral species Tubastraea coccinea and T. tagusensis (Scleractinia, Dendrophylliidae): Implications for species identification

K.C.C. Capei ª. A.E. Migotto b, C. Zilberberg ª. M.F. Lin e.d.e, Z. Forsman r, D.J. Miller e.e. M.V. Kitahara b.g.* ' Deparramenco de Zoclogia, Universidade Federal do Rio de Janeiro. Rio deJ aneiro. Brazil • Centro de Biologia Marinha. Universidade de São /'auto. São Sebasriãa. São Paulo, Brazil e Comparadve Cenomics Centre and Department ofMolecularand Cell Biology.James Cook University. Townsville, Queensland, Au,;tralia d Biod.iversiry Researc.t, Centre, Academia Sinica. Taipel raiwan ' ARC Cmrre ofExc ellencefor Coral ReefStudies,James Cook Universiry. Townsville, Qµeensland. Ausrro/ia ' flawon lnsrirure of Marine Biology. Universiry of Hawai'i. USA • Departamento de Cifocins do Mar. Universidade Federal de São Pau/o, Sa1110s, São Pau/o, Brazil

ARTICLE IN FO A 8 S T R A C T

Ardcle hisro,y: Members or lhe a20ox.1nthellate coral genus Tubasrraea are invasive species with particular conce 111 beca use they Received 8 April 2016 have become established and are fierce compecitors in the invaded areas in rnany pares or 1he world. Pacific Receivecl in revisecl form 17 May 2016 Tubasrmea species are spreading fasr rhroughour che Arlanric Ocean, occupying over 95% or che available substrate Accepted 23 May 2016 in some areas and out-competing native endemic specics. Approximaccly half or ali known coral species are Available online 25 May 2016 azooxamhellace but chesc are seriously undcr-rcpresented compared to zooxanthcllate corais in terms of the availability of mitochondrial (mt) genome data. ln the present study, the complete mt DNA sequences of Atlantic Keywords: Sun-coral individuais of the invasive scleractinian species Tubascraea coecinea and Tubastmea tagusemis werc determined Exoric speóes and comparcd to thc GenBank rcfercncc sequence available for a Padfic ·r. coccinea· individual. At 19.094 bp Sourh Atlancic (compareci to 19,070 bp for the GenBank specimen), the mt genomes assemblcd for the Allantic T. coccinea and mrgenome T. cagusensis were among the longest sequence determined to date for "Complex" sclerattinians. Comparisons of genomes data showed 1hat the "T. coccineo· sequence deposited on GenBank was more closely related to thar from DendrophylliCI arbuscula than 10 the Atlantic n,bas1raea spp .. in terms of genome length and base pair similarilies. Thís w,1s confirmed by phylogenetic analysis. suggesting that the former was misidenrified and might actually be a member from the genus Dendrop/1yllia. ln addition. although ín general rhe COX1 locus has a slow evolutíonary rate in Scleractinia, ít was rhe most variable region or the Tubasrmea mt genome and can be used as markers for genus or species ídentification. Given the limited data available for azooxanthellate corais, the results presented herc represent an impo,tant contribution to our undcrstanding or phylogenetic relationships and the evolutionary history ofthe Scleractínia. © 2016 Elsevier 8.V. AII rights reserved.

1. lntroductioo

Human acrivity has been responsible for unprecedented connectivity in the marine environment, particularly by the accidental ttansport of many species of crustaceans, mollusks. fishes. algae, cnidarians Abbreviacions: A adenine: aa. aminoadd(s); ATI'6,ATP synthase FO subunit6: ATP8. ATP and ctenophores (Moinar et ai.. 2008; Ghabooli et ai .. 2013). When synthase FO subunir 8: bp, base pair(s): C, cytosine: COB, cytochrome b: COX 1-3.cytochrome es tablished. exotic species may cause dramatic changes in the new oxidase subunir 1-3: G, guaninc: ICS. incergenic spacer: indel, insertion or delerion: rrs. internai rransa'ibcd spaccr: Kb, kilobase; m, mecer: min, minute: mt. mirochondrial: NDl- environment by altering community structure and displacing native 5. NADH dchydrogenase subunirs 1-5: ND4L NIIDH dehydrogcnase subunit 4~: nr. species (Moinar et ai., 2008). Tubasrraea Lesson, 1829 is an nucleocide(s); mi, 165 ribosomal RNA: ms, 12S ribosomal RNA: rRNA. ribosomal RNA: s. azooxanthellate scleractinian genus originally described in the Pacific second(s): T. thymine: tRNA transíer RNA: trnM, tRNA•Met (merhionine): trnW, tRNA-Trp Ocean inhabiting tropical shallow waters (Cairns. 2000). but has recently (tryptophan). attracted intense public and media concem dueto the highly competitive • Corresponding author .-:tt: Oep,1rtamento de Ciências do Mar. Universidade Federal de São Paulo. Santos:, Brazil. anel invasive properties of the members in this genus (Costa et ai., E-mail address: [email protected],· (M.V. Kitahara). 2014; Silva et ai .. 2014; Sammarco et ai .. 2015 ). With fast growth,

http://dx.doi.o1'g/10. 1O16/j.gene.2016.05.034 0378·1 l 19/() 2016 Elsevier 8.V. Ali rights reserved.

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K.CC Capei ec ai. / Gene 590 (2016) 270-277 271 early reproductive maturity and absence of natural predators in the differs substantially (Lin et ai., 2014). A shared characteristic of the Atlantic Ocean. Tubastraea species are able to cover nearly 95% of azooxanthellate corais and corallimorphs that differ in mt genome tl1e available surface, out-competing native endemic species (Creed, organization to the respective canonical pattems is that they inhabit 2006: Mantellato et ai., 2011 : Santos et ai., 2013: Hennessey and temperate and/or deep-water environments (Cairns, 2007; Fautin Sammarco, 2014: Sammarco et ai., 2015: Miranda et ai., 2016). et ai., 2009). By contrast. Tubastraea species inhabit tropical shallow Among its represemarives, Tubasrraea coccínea Lesson. 1829 and waters: mt genome organization in representa tives of this genus is Tubastraea tagusensis Wells. 1982 ( described from Bora Bora of French therefore of particular interest in terms of the apparent correlation Polynesia and Galapagos of Ecuador. respectively) are spreading fast between the mt genome structure and tl1e presence/absence of symbiotic throughout the Atlantic, where they have been first reported in Puerto clinoílagellates. Rico and Curaçao (Vaughan and Wells. 1943: Boschma, 1953 ). ln This study provides the complete mt genome sequences of Atlantic Brazilian waters, the presence of this genus had been documented since specimens of the invasive coral species Tubastraea coccinea and 1980's (Castro and Pires, 2001 ), although they were first identified to spe T. ragusensis. Together with all of the scleractinian data available in cies levei in 2004 ( de Paula and Creed, 2004 ). Since then, the genus has GenBank. these novel sequences were subjected to phylogenetic spread over 3000 km along the Brazilian coast (e.g.: de Paula and Creed, analysis, providing new perspectives on relationships within and 2004: Mantellato et ai .. 2011: Capei, 2012; Sampaio et ai.. 2012; Costa between dendrophylliids and the evolution of mt genomes within et ai., 2014) and predictions indicate that there is a high risk that the Scleractinia. Comparisons across the range of species suggest T. cocci11ea could colonize the entire coast of Brazil (Riul et ai .. 2013). that the COX1 locus may provide markers useful for identification Tubastraea belongs to the order Scleractinia, app roximately half of Tubastraea at the genus or species leve!. (-706 species) of the representatives of which do not l1ost the sym biotic dinoílagellate Symbiodinium (zooxanthellae) (Cairns, 2007). 2. Materiais and methods Despite being highly diverse. azooxanthellate scleractinians are under-represented in terms of available molecular data. Complete 2.1. DNA exrraction and sequencing mitochondrial (mt) genome sequence data are available for 55 scleractinians (see Tseng et ai.. 2005: Medina et ai., 2006: Chen Specimens of Tubastraea coccinea and T. tagusensis (specimen # et ai., 2008: Li n et al. , 201 1; Arrigoni et al., 2014; Kitahara et ai., MVK-CEBIMar 6 and # MVK-CEBIMar 43. respectively) were collected 2014: Zeng et ai., 2014) and only nine of these are azooxanthellate on May 2nd. 2013 from underneath a monobuoy (IMODCO 4) around species. A complete mt genome sequence for Tubascraea coccinea 5 m depth in the São Sebastião channel (23º48'55"S/45º24'01 •w). has been lodged in GenBank ( NCBI accession number NC026025 ), Brazil. Upon collection. total genomic DNA was extracted from the but phylogenetic analyses indicate that this sequence has a higher specimens and skeleton vouchers dried and deposited in the Cnidaria similarity with that from Dendrophyllia arbuscula van der Horst, collection of the Center for Marine Biology (CEBIMar-USP). Species 1922 than with other Tubastraea species ( Luz et ai. . 2015 ). ra ising identification followed Wells (1982) and Cairns ( 1991. 2000). the possibility of misiclentification. Although widely dispersed. Whole mesenteries were dissected from each species and total Tubasrraea has poorly defined taxonomic characters with severa! un genomic DNA extracted using the DNeasy Tissue Kit (Qiagen. Seoul, identified morphotypes (e.g.: Fenner. 2005: Arrigoni et ai., 2014), Korea). fo llowing the manufacturer's instructions. Portions of all mt which highlights che challenges of species identification in this genus. protein-coding and rRNA genes were amplified using the he DNeasy lndeed, many shallow-water scleractinians exh ibit high intraspecific Tissue Kit (Qiagen. Seoul, Korea ). following the manufacturer's instruc morphological variation (Todd, 2008) that frequently challenges tions. Portions of all mt protein-coding and rRNA genes were amplified taxonomy based exclusively on morphology. using the "Complex" scleractinian universal primers CS-1 to CS-21 Anthozoa mt genomes are atypical in te1ms of the presence ofonly 2 under the polymerase chain reaction (PCR) conditions describecl by Lin tRNAs compareci to > 20 in Bilateria ( Beagley et ai.. 1998: Boore. 1999; et ai. (2011 ). using the TopTaq polymerase master mix kit (Qiagen. see also Chen et ai.. 2008 that reported a o-nw duplication in Seriatopora Seoul, Korea ). To obtain sequences from regions not covered by the spp. Lamarck, 1916). ln addition. they have relatively loose gene universal primers. 26 specific primers were developed based on packing. especially those species that belongs to the "Basal" and T. cocdnea and T. ragusensis sequences (Supplementa1y Material, "Complex" clades (van Oppen et ai.. 2002; Kitahara et ai.. 2014; Lin Table S1) . For the specific primers, PCR were carried out using the same et ai., 2011). Despite an extremely low rate of evolution (van mix as fo r the universal primers and the following cycling conditions: Oppen et ai.. 1999: Shearer et ai.. 2002; Huang et al. , 2008), mt One cycle at 95 •e for 3 min. followed by 30 cycles of 30 s at 94 •c. 45 s genome data have been exrensively exploired to investigate phylogenetic at 50 to 52 •e (depending on the primers annealing temperature) and and evolutionaiy relationships within the Scleractinia and related groups 90 s at 72 •e. and ending with 4 min at 72 •e. Amplicons ranged in size be (Park et al.,2012 ; Kitahara et ai.. 2014; Lin et ai., 2014). Furthermore, DNA tween - 500 and 1500 bp, and were subjected to direct (Sanger) sec1uenc barcoding methods based on mt genes. such as Cytochrome Oxidase ing at Macrogen (South Korea). subunit 1 (COXl ). have recently been developed for coral genus (Hsu et ai .. 2014) or species (Keshavmurthy et ai.. 2013) identification. 2.2. Sequence ana/yses and armotation of the complete mirochondrial Despite the limited data available for azoox.anthellate corais, there are genomes some intriguing differences between zooxanthellate and awoxanthellate taxa in terms of mt genome characteristics. For instance. although mt Sequences were verified, assembled and analyzed using Geneious gene order is highly conserved among zooxanthellate corais (Medina v.6.1 .6 (Biomatters) and Sequencher 5.1 (Gene Codes). Sequences et ai .. 2006; Chen et ai., 2008: Kitahara et ai., 2014). two gene rearrange were aligned to previously published data in MEGA 6 using a weighted ment events have occurred across the nine azooxanthellate corais that matrix of Clustal W (TI1ompson et al., 1994) in order to identify protein have so far been examined (Embelm et ai., 201 1; Lln et ai.. 2012). fntrigu coding and ribosomal RNA genes. Examination of open reading frames ingly. a similar trend has also been observed in corallimorpha1ians, che (ORFs) and codon usage. as well as other DNA statistics. were pe1fonned anthozoan order most closely related to Scleractinia (Lln et ai .. 2014). ln using Dual Organelle Genome Annotator {Wyman et ai., 2004), brief. ali of the zooxanthellate corallimorphs for which data are available Sequence Manipulation Suite v.2 (Stothard. 2000). and MEGA 6 (10 species) have the same mt gene organization (which differs from (Tamura et ai., 2013). tRNAs were predicted using tRNAscan -SE the scleractinian norm). while in Corynactis ca/ífornica Carlgren, 1936 search server vl.21 (Lowe and Eddy. 1997). Tandem repeat sections and Corallimorpfms proftmdus Moseley. 1877, the two azooxanthellate were searched in the five largest intergenic spacers ( IGS-1. IGS-3, corallimorphs for which data are avai lable. mt gene organization IGS-6, lGS-8 and JGS-18) using Tandem Repeat Finder (Benson.

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1999). Mitochondrial genome sequences are available on CenBank NOS (51 (NCBI accession numbers KX024566 and KX024567. for T. coccineo and mi T. tagusellsis. respectively).

2.3. P/Jylogenetic onalyses

Nucleotide sequences of ali protein-coding and ribosomal genes from 57 scleractinians representing the "Basal" (1 ). "Complex" (39). and "Robust" ( 17) clades. in addition to sequences from Corallimorpharia COX1 Tubastraea coccinea ( 12), Actiniaria (2), Antipatharia (1), Zoantharia (1 ). and Octocorallia 19,094 bp ND2 ( 1) were used to reconstruct anthozoan evolutionary history. The octocoral Antillogorgio bipinnora (Verrill. 1864) was used as outgroup. Tubastraea tagusensis The final alignment comprised a total of 14,953 bp. of which 9714 19,094 bp ATP8 - positions were phylogenetically informative. Maximum likelihood analyses were conducted using PhyM L (Cuindon et ai .. 2010) llllder tmW the GTR + I + G nucleotide evolutionary model, selected as the ATP6 most appropriate for the complete dataset using MECA 6. Phylogenetic analysis of the dendrophyllids was also conducted based on the COXl gene using maximum likelihood analyses under the CfR + I + G nucleo tide evolutionary model. The final alignment comprised a total of 1577 bp. of which n1 positions were phylogenetically info,mative. ms COX3 3. Results and discussion Fig. t. Mitocliondrial gene map of die scleractinians T11bas1T<100 cocdnea and T. ragusensis. Scaling is approxima1e only. Protein-coding. tRNA. and rRNA genes were abbreviated as in 3.1. Orgonization ond gene content the rext. Blank regions between genes represem incergenic spacers. The NOS intron is indkated by the inner green line. At 19,094 bp, the mt genomes ofT. coccineo and r. cagusensis were at the high end ofthe size range of"Complex" scleractinians (see Kitahara et ai .. 2014). ln general. Tubastroea mt genomes were intermediate in size between those of"Basal" (-19.5 kb) and "Robust" scleractinians 32. Codon usage (typically-17 kb ). but were among the longest "Complex" scleractinian mt genomes determined to date (see Medina et ai .. 2006: Lin et ai .. The 13 mt protein-coding genes of T. coccinea and T. tagusensis 2011 ; Chuang and Chen. 2015 ). The inrermediate size of Tubascraea comprise 3945 codons. with all 62 amino acid codons but one ( cysteine mt genomes is consistem with a relatively early divergence ofthe fami ly TCC) being used. Leucine ( 15.1 %) and cysteine (0.9%) were the most Dendrophylliidae. implied by molecular phylogenetic reconstructions and least frequent amino acids, respectively. while the most and least (Romano and Cairns, 2000; Fukami et ai .. 2008; Kitahara et ai .. 2010; frequently used codons were phenylalanine (ID) and arginine (CCC) Stolarski et ai.. 2011 ). (Table 2). ln general. there was a strong bias towards codons end ing Whereas in two other azooxanthellate scleractinians (Embelm et ai. . with thymine ( 44%). a trend also observed in other scleracrinians, 20 11 : Lin et ai .. 2012). mt gene organization differs, Tuboscraea spp. especially those belonging to the "Robust'' clade (Kitahara et ai., 2014). follow the canonical scleractinian pattern (van Oppen et ai .. 2002; As with the majority of anthozoans (van Oppen et ai., 2002; Chen Fukami and Knowlton. 2005: Tseng et ai.. 2005: Medina et ai.. 2006: Flor et ai., 2008). nine of the 13 me protein-coding genes ofT. coccinea and and Tillier, 2007; Chen et ai.. 2008). As most hexacorallians (see Chen r. tagusensis use methionine (ATC) as start codon. while ND3- NDS et ai., 2008). T. coccinea and T. ragusensis mt genomes comprised each and ND4L use valine (CTC ) and ND6 isoleucine (ATA). TI1e usage of 13 protein-coding genes, 2 rRNAs. and 2 tRNAs, ali transcribed from the CTC and ATA as start codons has already been documented in otl1er sarne strand (Fig. 1). However, unlike most scleractinians, there were no scleractinians (e.g.: Acropora tenuis (Dana. 1846): Pod/lopará damicomis; overlaps between genes (Table 1) . Tubascraea mt genome also has a Seriaropara hystrix Dana 1846 - van Oppen et ai .. 2002: Chen et ai .. COXl group I intron. obseived in some "Complex" corais and ali 2008). Additionally. as in other Anthozoa ( e.g.: Flor and Tillier, 2007). Corallimorpharia. but absent in "Robust'' scleractinians (Lin et ai., 20 14). for both Tubascraea species, ali protein-coding genes have complete Moreover, the COXl group I intron. as well as the rRNAs and lGSs. were (TAC or TAA) stop codons. with TAA being more frequently obse1ved. slightly larger in Tubascraea than those previously published for other scleractinians. The ND5 gene was also interrupted by another group I in 3.3. RNA genes and non-coding regions tron that contains 11 genes (Fig. 1 ). The sense strand of the mt genomes from r. coccinea and The boundaries of T. coccinea and T. tagusensis rRNA genes were T. ragusensis were composed of 25.3%A. 13.6%e. 23.7%G, and 37.4%T. deduced by comparison with data from other anthozoans. As observed and 25.4%A. 13.6%C, 23.6%C and 37.5% T, respectively. Within different in other cora is, the rns and mi were located almost opposite to each regions. the (A+ T)-content ranges from 45.1 % in rrnM to 69.7% in other (Fig. 1). Also, as reporteei in most scleractinians, only two tRNAs ND4L This (A+ T)-bias is common for hexacorallians (Kitahara et al.. have been found in Tubastraea: trnM (methionine) and rrnW(trypto 2014) as for other metazoans (e.g.: Lavrov et ai., 2008; Perseke et ai.. phan) (Fig. 2). which differs from the two species of Seriacopora 2010). Over<1ll, the (A + T)-content in Tubastraea was -62.8%. which is (S. caliendrum Eh renberg. 1834 anel S. /Jystrix) that possess three mt similar to the mean value observed from ocher "Complex" scleractinians. rRNAs (Chen et ai .. 2008). but significantly lower ifcompared to "Robust" coral representatives ( e.g.: The T. coccinea and T. tagusensis mt genomes have each 18 IGSs, Pocillopora damicomis (Linnaeus. 1858) (- 70.2% - Chen et ai.. 2008 (. totaling 2404 and 2406 bp respectively, or 12.6%ofthe total mtgenome Astrangia sp. Milne Edwards & Haime, 1848 168.3% - Medina et ai.. size. Four ofthe 18 ICSs (ICS-1. -3, -6, and -8) account for >60%ofthe 2006]). The higher values observed in "Robusr· corais has been related non-coding regions. van Oppen et ai. (2002) argued that the ICS with a recluced efficiency of a putative DNA repair systems (Kitahara between rns and COX3 of Acropora tenuis has severa! features charac et ai .. 2014 ). teristic of control regions of higher animais (i.e.: repetitive sequences.

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Table 1 Mt geno,nc organization in n,bosrraea coccinea and T. cagusensis.

Posit:ion Lengrh (bp) AT% Start•Stop codon IGS Rcgion T. coccinea T. tagusensis T. cocci11earr. raguse11sis T. cocci1100/T, cagusensis T. cocci11earr. raguse11sis T. coccinea/T, cagusensis NOS (5') 168-887 168-887 720020 62.4%,162.4% GTG-/GTG- 430/430 (IGS 18) Croup I intron 888-12,2!0 888- 12,213 11,323/ 11.324 63.2%,163.1% 0/0 NOl 1220-2203 1220-2203 984/984 62.0%,162.0% ATC-TAA/ATC-TM 332/332 ( ICS 1) COB 2331 -3494 2331 -3494 1164/ 1164 61.1%/61.0% ATG-TAA/ATC-TM 127/ 127 (ICS 2) ND2 4133-5230 4133-5230 1098/ 1098 62.5%162.7% ATC-TANATC-TM 638/638 (ICS 3) N06 5264-5857 5264-5857 594/594 64.8%/64.8% ATA-TM/ATA-TM 33/33 (IGS 4) ATP6 5940-6638 5940-6638 699/699 64.2%/64.2% ATC-TAC/A1'C-TAC 82/82 (ICS 5) N04 6940-8415 6940-8415 1476/1476 63.6%,163.7% ATC-TANATC-TM 301/301 (IGS 6) ms 8529-9593 8529-9593 1065/1065 58.9%158.9% 113/113 (IGS7) COX3 9813- 10,601 9814- 10.602 789/789 61.9%,162.1% ATC-TAC/ATG-TAC 219/220 (ICS 8) COX2 10.639-11,382 10,640-11,383 744/744 63.2%/63.2% ATC-TAG/ATG-TAC 37/37 (IGS 9) N04L 11,436- 11,735 11.437- 11.736 300/300 69.7%,169.7% GTC-TM/CTC-TM 53/53 (ICS 10) N03 11,754-12, 110 11,756-12.112 357/357 65.0%/65.0% CTG-TAG/CTC-TAC 18/19 (ICS 11 ) NOS (3') 12.210-13.325 12.212-13.327 1116/1116 62.5%/62.8% TAC/-TAG 99/99 (ICS 12) tmW 13,365- 13.434 13,367- 13.436 70/70 50.0%,150.0% 39/39 (ICS 13) ATP8 13,468-13.683 13.470-13.685 216/216 68.5%168.5% ATC-TAA/ATC-TM 33/33 (IGS 14} COXI (5') 13.807- 14.700 13.809-14.702 894/894 60.3%160.3% ATC-/ATC- 123/123 (ICS 15) COXl-intron 14,701- 15,664 14,703- 15,666 964/964 63.9%,164.4% COXI (3') 15.665-16.348 15.667-16.350 684/684 63.6%163.3% TM/-TM rmM 16.470-16.540 16,472-16.542 71/71 45.1%/45.1% 121/ 121 (IGS 16) mi 16.577-18,83 1 16.579-18.831 2255/2253 61.3%/61.3% 36/36 (IGS 17)

conserved sequence blocks and seconda,y structure porentially associat protein-coding genes. rnl and the IGS regions, of which 34 were ed with the initiation of heavy-strand replication). To idemify candidate transicions. 16 transversions. and 4 indels (Fig. J) (sequence diversity control regions in the Tubastaea mt genomes, searches were conducted [p-distanceJ =0.0026 ± 0.0005 ). For 6 ofthe protein coding genes. nucle for tandem sequence repeats in IGS-1. -3. -6. and -8 but none were otide divergence resulted in changes at the amino acid leve!. Toe COX1 detected. lnterestingly, among rhe IGS checked, the smallest one anel mi loci were rhe mosr variable. with 8 and 6 variable sites (0.33% (IGS-8). also founcl between rns and COX3, had a high degree of and 0.27% of differences) respectively (Table 3). The average difference similarity to the putative mt control region of some other scleractinians between genomes was 0.28%. similar to what has been observed between (Enallopsammia rostraca (Pou1talmm. 1878) and Porites spp. Link. 1807) other scleractinians congeners ( e.g.: 0.18% berween Podllopora damicomis (dara not shown), indicating thar this region in Tubasrraea may, in fact. and Pocillopora eydouxi Milne Eclwards. 1860 (Flot and Tillier. 2007 1; and correspond to the mt control region. ln other coral species. different IGS 0.48% between 5eriatapora caliendrum and S. hysrrix [Chen et ai., 20081). regions may function as control regions; rhat between ATP6 and ND4 in At 19,070 bp, the database Tubastraea coccinea mt genome sequence pocilloporids (Flot and Tillier, 2007: Chen et ai., 2008) and the IGS (GenBank accession number NC026025) is 24 bp smaller than the between COB and ND2 for the Dendrophylliid Turbinaria peltata (Esper, T. coccinea mr genome presented herein, the elifference being accounted 1794) (Sh i et ai .. 2014). For the sponge Amphimedon queenslandica for by a 23 bp iodei in ms (Fig. 4) anel a 1 bp inelel in mi. Additionally, Hooper & van Soest. 2006, the longest IGS is thought to contain the 117 nr differences were found between the genomes, which is control region on the basis that it contains repeatecl sequences and abour two rimes higher than the difference berween T. coccinea anel resembles the control region of higher Metazoa (Erpenbeck et ai .. 2006). T. tag11se11sis presented here (54 nt). Previous studies have already indicared the anomalously high similarity between the GenBank darabase 3.4. Comparison among Tubas1raea genomes T. coccinea anel Dendrophyllia arbuscu/a (accession number KR82493 7) sequences relative to other Tubascraea species (Luz er ai.. 2015). The ln total, there were 54 nt differences between the T. coccinea and sizes of rhe T. coccinea NC026025 and D. arbuscula KR824937 mt genomes T. ragusensis mt genomes reporteei here. distributed across 9 of the 13 were vecy similar ( 19.070 and 19,069 bp respectively) and differ only at

Table2 Number of occurrcnccs of eaeh codon in lhe 13 protein-coding genes from Tubasrraeo coccinea (Te) and T. ragusensis (Tr) mt genomes.

Te/Tt Te/TI Te/TI Tc/Tt Phe uuu 312/314 Ser ucu 113/113 Tyr UAU 148/ 148 Cys UGU 37/37 uuc 23/22 ucc 20/20 UAC 11 / 11 UGC 0/0 UUA 278/278 UCA 33/33 End UM 8/8 Trp UCA 39/40 uuc 161/ 160 ucc 37/37 UAC 5/5 UGG 59/58 Leu cuu 92/91 Pro ccu 68/67 His CAU 68/69 Arg ccu 12/ 13 cuc 18/ 17 CCC 25/26 CAC 11/10 CCC 5/4 CUA 33/35 CCA 31/31 Clu CM 57/57 CCA 20/20 cuc 13/ 13 CCC 28/28 CAC 17/ 17 CCC 12/ 12 lle AUU 180/ 181 Thr ACU 79/79 Asn MU 75/75 Ser ACU 70/71 AUC 36/37 ACC 18/18 AAC 22/22 ACC 11/ 11 AUA 117/116 ACA 38/38 Lys AM 63/62 Arg ACA 41/41 Met AUC 118/118 ACC 28/28 AAC 38/39 ACC 12/ 12 Vai cuu 198/ 196 Ala ccu 128/127 Asp GAU 64/65 Cly ccu 90/91 cuc 30/31 CCC 47/49 CAC 22/22 GGC 34/32 CUA 71/71 CCA 43/43 Clu CM 55/55 GCA 58/58 cuc 89/88 CCC 61/61 GAC 65/64 GCC 150/ 150

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274 K.C.C. Capei ,rol/ c,n, 590(20 16) 270-277 a b

• u • • u . ,. ,. ,,. ••o ••e • •e

e u • ç • A

Fig. 2. Predicred rRNA secondary srmcrures fmm the mr genomes ofTubastraea cocdnea and T. ragusensis: (a) nnW(nyprophan): (b) rmM (merhianine).

48 nt positions. Furthermore, D. arbuscu/a KR824937 and T. coccínea NC026025 share a 23 bp gap in the rns that is not present in the Tubasrraea sequences presented here or in other dendrophyllids. Table3 including Dendrophyl/ia cribrosa Milne Edwards & Haime, 1851 (Fig. 4). Nudeotide (nt) and aminoacid (aa) sequence identities (%) and numbersofvariable sites Given the extremely low rate of mt genome evolution in Scleractinia (Vn) between Tuba.çn·aea cocdnoo anel T. rctgusensis mr genomes. relative to other Metazoa (van Oppen et ai.. 1999; Shearer et ai.. 2002: nt Huang et ai., 2008), the levei of sequence divergence between the ªª Locus ldentity Vn ldentity Vn database (NC026025) and novel (reported here) T. cocdnea sequences is unprecedented and highly unlikely. Altogether. rhese similarities Protein-coding suggest that T. coccinea NC026025 might be a nüsidentified member NOS (5') 100.0 o 100.0 o N01 99.7 3 99.1 3 of the genus Dendrophyllia. Severa! Dendrophyllia species that inhabit COB 99.7 4 99.5 2 tropical shallow-waters are deceptively similar to Tubastraea in tenns of N02 99.8 2 100.0 o colony morphology and have a Tubasrroea-like reddish-orange coenosarc, N06 99.8 99.5 so the potential for mis-identifkation is high. ATP6 100.0 o 100.0 o N04 99.9 2 99.8 1 COX3 99.7 2 100.0 o COX2 99.6 3 99.2 2 35. Phylogenetic analyses N04L 100.0 o 100.0 o N03 100.0 o 100.0 o Data from mt genomes from 57 anthozoans, representing two NOS (3') 99.7 3 100.0 o rrn-Trp 100.0 o 100.0 o subclasses and six orders, were used to reconstruct the phylogenetic ATP8 100.0 o 100.0 o relationship using maximum likelihood methocls (Fig. 5). The recovered COXl (5') 99.6 4 99.3 2 topology was largely consistent with those previously published COX 1 - intron 99.4 6 ( Fukami er ai., 2008: Kitahara et ai.. 201O : Kirahara er ai., 2014 ). ln general, COXl (3') 99.4 4 99.1 2 trn-Met 100.0 o 100.0 o the phylogenetic reconstruction presented herein recovered the three main scleractinian clades. "Complex". "Robust" anel "Basal", supports rRNA rhe monophyly of the family Dendrophylliidae. and places the rns 100.0 o mi 99.7 6 dendrophylliids as sister group of Poritidae ( Medina et ai., 2006; Fukami et ai.. 2008; Ki tahara et ai.. 2010; Arrigoni et ai., 2014). The rRNA rmM 100.0 o rn,w 100.0 o ln1roge11ic sp11cer lCS 1 99.7 1 1CS2 100.0 o i 7 e6 +------"'Transvmiont 1CS3 99.4 4 ill IGS4 100.0 o ~ s ■ Transirions lGSS 100.0 o b 4 +---~------· ---o,-.. ■ lndcls IGSG 100.0 o 11+-~ ~ ------~~- ..-~- --1i---..rw-1 ~ ---~ 1CS7 99.1 1 ;,< 2 lGSS 98.6 3 1CS9 100.0 o lCS 10 100.0 o IGS 11 94.4 1 IGS 12 100.0 o lCS 13 100.0 o lGS 14 100.0 o lGS 15 99.2 1 lCS 16 99.2 1 fig. 3. Repart.ition of the sequence differen

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K.CC Capei et a[ /Cene 590 (2016) 270-277 275

1 ~ • l!J 40 •p 'P Constnsus ■TIIIG TI'r ■T G U GG TI ~ TD G u _lTGD GI'r ciê T c■c GG TGl!'l;G O.ndrophylla a ibrosa NC026026 CTAGTATCTAAAGATTGGTCTTCAGACTTAGACGATAGAGTGCGAACAGGTGAGGAAACCC Oendrophyllla arbuscul,o NC027590 CTAGTAT T TAAA ------TAGAGTGCGAACAGGTGAGGAAACCC Tubastrau cocdnea NC026025 CTAGTAT T TAAA ------TAGAGTGCGAACAGGTGAGGAAACCC Tuba.sua.ta cocclnu CTAGTATCTAAAGATTGGTCTTCAGACTTAGACGATAGAGTGCGAACAGGTGAGGAAACCC Tuba,straea tagusensts CTAGTATCTAAAGATTGGTCTTCAGACTTAGACGATAGAGTGCGAACAGGTGAGGAAACCC Turblnarla pehua NC02 ◄ 671 CTAGTATCTAAAGATTGGTCTTCAGACTTAGACGATAGAGTGCGAACAGGTGAGGAAACCC Porltes po(ites NC008166 CTAGTATCTAAAGATTGGTCTTCA■ACTT ------TAGAGTGCGAACAGGTGAGGAAA■CC

Fig. 4, lnirial fragment of rns showing lhe 23 bp indel in lhe CenBank mi genome sequences for Tubastraea cocdnea (NC026025) and Dendrophyllia orbuscula (NC027590). recovered topology also supports the monophyly ofScleractinia and 3.6. Potenâa/ for species idenâficaâon Corallimorpharia, placing them as sister groups. The r. coccinea sequence retrieved from GenBank was included in The issue of molecular markers for coral identification is a challenging analyses. Dendrophyllía and Tubastraea were the only scleractinian genera subject since COX1. the near-universal marker for DNA barcoding of that were not recovered as monophyletic (Fig. 5 ). According to an exten metazoan species (Hebert et ai., 2003). is highly conserved in Scleractinia sive molecular phylogeny of the family, Dendrophyl/ia is a polyphyletic (<2%, Shearer and Coffroth, 2008), as in other anthozoans (Hellberg. genus. while Tubastraea is monophyletic with low intrageneric distances 2006). Additionally, the most variable regions often differ among coral (Anigoni et ai., 2014). The phylogenetic results presented here were genera. making it unlikely that a universal mt marker can be developed consistent with the idea that T. coccinea NC026025 is more closely related for coral identification. For example. in Seriaropora spp. NOS (3' ). ATP6 to D. arbuscula KR824937 than to other Tubastraea species (Fig. 5). and IGS-9 (between trnW anda putative ATP8) were the most variable

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=~rslenuls Ar:ropor, yonge, Sderaclinia

Fig. 5. ML phylogcny of scleracônian corais based on ali 13 mitochondrial protein-coding genes. rns and mi with ML boorstrap (upper) and Sh-Uke (lower) nodc suppor1 values. Nodcs wi1hout supporr numbcrs indicace boamrap and Sh-Ukc support ovcr ~98.

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276 K. C.C. Capei etal / Gene 590 (2016) 270-277

Table4 Matri.x of mean difference between sped es based on COX 1 sequences (subs.titutions: per site and stalldard errors. caJ culated by the Kimura ( 1980) method ). An asrerisk (•) indicates the sequences obtained by 1he present worl<.

TubasCTaea Tubastraea rubasrraea cocdnea Dendrophyllia arbusrula Dendrophyllia cribrosa Turbinaria peltata ragusen.sis .. coccinea· NC026025 NC027590 NC026026 NC024671 Tubastroea coccinca· 0.005 1 ± 0.001 8 n,bastraea coccineo NC026025 0.0083 ± 0.0022 0.01 08 ± 0.0025 Dendrophyllia arbuscula NC027590 0.0076 ± 0.0023 0.0102 ± 0.0026 0.0019 ± 0.001 1 Dendrophyllia cribrosa NC026026 0.0114 ± 0.0027 0.0127 ± 0.0027 O.O1 33 ± 0.0028 o.o1 27 ± 0.0028 Turbinaria peitara NC02467 1 0.0273 ± 0.0041 0.0298 ± 0.0043 0.0317 ± 0.0044 0.03 11 ± 0.0045 0.0337 ± 0.0047 Parires porites NC008166 0.0565 ± 0.0060 0.0590 ± 0.0059 0.0603 ± 0.0062 0.0610 ± 0.0062 0.061O ± 0.0060 0.0578 ± 0.0062

regions {Chen et ai .. 2008). while in l'oci/lopora spp. (Flot and Tillier. 2007) 4. Conclusion the co1Tesponding regions were ND3. ATP6 and IGS-11 (between COXl and ATP8). Among these regions, only the NOS (3') and che IGS between The complete mt genome sequences (19,094 bp) oftwo shallow ATP8 anel COXl (IGS-15) differed between T. cocdnea and T. cagusensis water invasive coral species. Tubastraea coccinea anel T. tagusensiS have (Table 3). been determined. adding data for ecologically important species to the Severa! regions of the mitochondrial (l

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K.CC Capei er ai. / Gene 590 (2016) 270-277 277

an invasive coral spccics ovcr Abmlhos Bank. Southwestcm Atlanric Mar. Pollut. Buli. Lowc. T.M.. Eddy. S.R.. 1997. tRNAscan-SE: a program for improvcd derection of transfc r 85. 252-253. RNA genes ln genomic sequence. Nucleic Acids Res. 25. 955-964. Creed. J.C.. 2006. Two invasive .:1 1ien azooxanrhellare corais. Tubasrraeo cocdnea and Luz. B.LP .. Capei. K.C.C.. Stampar. S.N.. Kirahara. M.V.. 2015. Description of thc mirochondrial Tu bastroeo ragusensis, dominate the naóve zooxanthellate Mussismilia llispida in gcoomc ofthe rrcc coral Dendropllyl/ioorlJusailo (Amhoz0<1. Sclel'actinia). Mitochondrial Brazil Coral Reefs 25, 350. DNA 29. 1-2. de Paula. A.F.. Creed.J.C. 2004. Two species of the coral l'uoostmea (Cnidaria. Scleractinia) Mantellato, M.C.. Mourão, e .e .. Migotto. A.E.. Lindner. A.. 2011. Range expansion of the in Brazil: a case of accidenral introduction. Buli. Mar. Sei. 74, 175-183. invasive corais Tubasrraea cocdnea and Tubastraea tQg,,sensis in the Southwcst Atlantic. Embelm. A.. Karlsen. 8.0.. EVercscn.J.. Johanscn. S.D.. 201 1. Mitogenome rearrangement in Coral Rcds 30. 397. the cold-water scleractinian coral Lophelia pertusa (Cnidaria. Anthozoa) involves a Medina. M.. Collins, A.e .. Takaoka. T.L. Kuehl.J.V.. Boore. J.L. 2006. Naked corais: skeleton long-term evolving group 1 intron. Mo!. Phylogenet. Evol. 61. 495-503. loss in Sclcractinia. Proc. Narl. Acad. Sei. U. S. A. 103. 96-100. Erpenbeck. o.. Hooper.J.N.A.. Wõrhcide, e.. 2006. co l phylogcnics in diploblasts and me Miranda. RJ. Cniz. I.C.S .. Barros. F.. 2016. EJfeccs of the alicn coral Tubasrroea r~•serisis on ·sarcoding of Life' -are we sequcncing a suboptimal parrition? Mol. Ecol. No1es 6. native coral assemblagcs in a soudiwesrem Atlanric coral ,-cer. Mar. Biodivcrs. 163. 45. 550-553. Molnar.j.M.. earnbo.1, R.L, Revenga, C.. Spalding. M.D.. 2008. Assessing the global threat of Fautin. o.e .. Guinonc. J.M.. Orr. J.C 2009. Comparative dcpth distribution or invasivo spccies to marine biodivcrsity. Front. Eco!. Environ. 6. 485-492. corallimorpharians and scleractinians (Cnldal'ia: Anthozoa). Mar. Ecol. Prog. Ser. 397. Park. E.. Hwang. O.S. • Lec. J.S.. Song.J.1 .. Seo. T.K., Won, Y.J.. 2012. Estimation of divergence 63-70. times in cnidarian evolution ba.sed on mitochondrial protein-

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Additional file 1. Primer names and sequences designed in the present study for the amplification/sequence of the mitochondrial genome of Tubastraea coccinea and T. tagusensis. The position (bp) and amplicon length (bp) of primers are provided.

Primer name Sequence (5' to 3') Position (bp) Length (bp) Tubastraea coccinea Tc_MVK1_F forward TTTGGGGGCGATTCGGGCAG 1,645-1,980 368 Tc_MVK1_R reverse CCAAACCACCCGGCTCCACC 1,695-2,012 Tc_MVK2_F forward CCACTGCGCAAAGAGAATCCGC 2,393-3,268 905 Tc_MVK2_R reverse CGCCCAAATGGACGAAAGGGCA 2,364-3,231 Tc_MVK3_F forward TGCCCTTTCGTCCATTTGGGCG 3,329-4,230 932 Tc_MVK3_R reverse GGCACTTTGCCCAGACCCCC 3,299-4,190 Tc_MVK4_F forward TGCTTTATTGTGTGGATTTACGGGGG 4,343-4,689 354 Tc_MVK4_R reverse ATGCCCCACCCCACTGTAGGC 4,336-4,641 Tc_MVK5_F forward AGCCTGGCTTTGTTTGGGGGT 5,947-6,335 418 Tc_MVK5_R reverse AGGGGCCCCACTCGGCATAA 5,918-6,292 Tc_MVK6_F forward TTATGCCGAGTGGGGCCCCT 6,397-6,908 562 Tc_MVK6_R reverse CGCCCCTGCCACAGCAACAA 6,347-6,854 Tc_MVK7_F forward TGCTTTGGATGGGGCCTCTTTG 7,248-7,656 450 Tc_MVK7_R reverse CCAGAAACGGGGGCCTCCAC 7,207-7,620 Tc_MVK8_F forward TTCGCTGGGGGTTTTCTACTAACCA 13,888-14,329 486 Tc_MVK8_R reverse TTACCCCGGGAGCCCGCATA 13,844-14,299 Tc_MVK9_F forward GGATTAACTCCGGCGTGGGGC 15,683-15,931 298 Tc_MVK9_R reverse ACCGCCCCCATAGAAAGGACA 15,634-15,875 Tc_MVK10_F forward TGGACAGACAGACAGGGGGCG 17,362-17,968 650 Tc_MVK10_R reverse GGTCAGTGTTACCGCGGCCATT 17,318-17,909

Tubastraea tagusensis Tc_MVK2_R reverse CGCCCAAATGGACGAAAGGGCA 2,364-3,232 869 Tc_MVK3_F forward TGCCCTTTCGTCCATTTGGGCG 3,327-4,097 807 Tt_MVK3_R reverse GGCAAGTGCGCATTCGCTTGA 3,291-4,053 Tc_MVK5_F forward AGCCTGGCTTTGTTTGGGGGT 5,963-6,336 422 Tc_MVK5_R reverse AGGGGCCCCACTCGGCATAA 5,915-6,287 Tc_MVK6_F forward TTATGCCGAGTGGGGCCCCT 6,400-6,908 564 Tc_MVK6_R reverse CGCCCCTGCCACAGCAACAA 6,345-6,862 Tt_MVK5_F forward TGTGGGCCCATCATATGTTTACGGT 14,754-15,734 981 Tt_MVK5_R reverse ACAAACCCCATTGCCCAAAGCA 14,754-15,527 Tt_MVK6_F forward CCGGGCTCATGCCCCGAAGA 16,579-17,090 576 Tt_MVK6_R reverse CCCCTAACCACAGGTCATCCGAGG 16,515-17,039 Tc_MVK10_F forward TGGACAGACAGACAGGGGGCG 17,369-17,969 653 Tc_MVK10_R reverse GGTCAGTGTTACCGCGGCCATT 17,317-17,915 Tt_F20.5 forward 5' - TAGATAAGTGGGACAGTTTG 18,364-363 1093

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Capítulo IV

Clone wars: asexual reproduction dominates in the invasive range of Tubastraea spp. (Anthozoa: Scleractinia) in the South-Atlantic Ocean

Kátia Cristina Cruz Capel, Robert Toonen, Caio Rachid, Joel Creed, Marcelo Kitahara, Zac Forsman & Carla Zilberberg

Publicado na revista PeerJ em 2017.

127

Clone wars: asexual reproduction dominates in the invasive range of Tubastraea spp. (Anthozoa: Scleractinia) in the South-Atlantic Ocean

1 2 3 2 Katia Cristina Cruz Capel • • , Robert J. Toonen , Caio T.C.C. Rachid ', 3 5 3 6 7 2 1 3 Joel C. Creed • , Marcelo V. Kitahara · • , Zac Forsman and Carla Zilberberg •

1 Departamento de Zoologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazi l 2 School of Ocean & Earth Science & Technology, Hawai'i Jnstitute of Marine Biology, University of Hawai'i at Maooa, Kaoe'ohe, Hawai'i, United States ofAmerica 1 Coral-Sol Research, Tech nological Development and !nnovation Network, Brazi l ◄ Jnstituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil ' Departamento de Ecologia, Unive rsidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil 6 Departamento de Ciências do Mar, Universidade Federal de São Paulo, Santos, Brazil 7 Centro de Biologia Ma ri nha, Universidade de São Paulo, São Sebastião, Brazil

ABSTRACT Although the invasive azooxanthellate corals Tubastraea coccinea and T. tagusensis are spreadiug quickly and outcornpeting native species in the Atlantic Ocean, there is little information regarding the genetic structure and path of i.ntroduction for these species. Here we present the first data on genetic diversity and clonai structure from these two species using a new set of microsatellite markers. High proportions of clones were observed, indicating that asexual reproduction has a major role in the local population dynamics and, therefore, represents one of the main reasons for the invasion success. Although no significant population structure was ÍOLu1d, results suggest the occurrence of multiple invasions for T. coccinea and also that both species are being transported along the coast by vectors such as oil platforms and monobouys, spreading these invasive species. fn addition to the description of novel microsatellite markers, this Submitted 20 July 2017 study sheds new üght into the invasive process of Tubastraea. Accepted 9 September 2017 Published 5 October 2017

Corresponding author Subjects Biodiversity, Conservation Bioloi,ry, Ecology, Genetics, Marine Biology Katia Cristina Cruz Capei, katiacapel7@grna il .com Keywords Sun-coral, Clone structure, Mjcrosatellites, Population genetics, T. coccinea, T. tagusensis Academia editor James Reimer Additional lnformation and INTRODUCTION Declarations can be found on page 13 The marine environment is continuously subjected to multiple stressors, many of which are associated with human activities (e.g., over-exploitation of resources, pollution, clirnate 00110.7717/peerj.3873 change and invasive species) (Halpem et ai., 2014; Gal/ardo etal., 2016). Among these @ Copyright stressors, invasive species are considered to be a major threat to biodiversity (Moinar 2017 Capei et ai. et ai., 2008) with the poteutial to quickly trigger changes in native comrnunities and the Distributed under ecosystem services and functions, which can have wide-ranging negative impacts. There are Creative Commons CC-BY 4.0 numerous examples of marine invasions which impact humans or native biota, such as in C-1RMl-!Biâ-i-i the Mediterranean Sea with the invasion of the ctenophore Mnemiopsis leidyi, which caused

How to cite this artfcle Capei et ai. (2017), Clone wars: asexual reproduction dominate.s in the invasive range of 'fobastmea spp. (Antho• zoa: Sdcmctini:.t) in thcSo1.tlh•Ad,.uuicOc~111. PeerJ 5:e3873; DOI 10.7717/pcerj.3873 128

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the collapse of the fishing industry (Sliiga11ova, 1998), the algae Womersleyella setacea, that negatively affected sponge reproduction ( Caralt & Cebrian, 2013) and the lionfish Pterois spp., responsible for a reduction in the native fish recruitment in the Atlantic (Albí11s & Hixo11, 2008). Scleractinian corais are known to play a key role in the marine environment by building structurally complex and highly diverse ecosystems (Reaka-Kudla, 1997). As ecosystem engineers that are under threat globally (Hoegh-Guldberg, 1999; Pandolfi et ai., 2003), scleractinian corals are rarely seen as an environmental risk. However, three scleractinian species from the genus Tubastraea were introduced and are spreading rapidly throughout the Western Atlantic Ocean (De Paula & Creed, 2004; Fe1111er, 2001 ; Fenner & Banks, 2004; Samnwrco, Atchison & Boland, 2004; Smnmarco, Porter & Caims, 20/0; Capei, 2012; Sampaio et ai., 2012; Co,tn et ai. , 2014; Silva et ai., 2014), threatening na tive and endemic species (Mantellnto et ai., 2011 ; Santos, Ribeiro & Creed, 2013; Creed, 2006) and fouling man-made structures and vessels. Tubastraea is an azooxanthellate dendrophyllid genus from the Pacific and Indian Oceans that was first reported in the Caribbean in 1943 (Vaughan & We/1s, 1943). Since then, three species have been identified in the Western Atlantic Ocean: (1) T. coccinea, now reported along 9,000 km of coastline of the Western Atlantic Ocean from Florida (26°47'N, 80º 02'W) (Fe1111er & Ba11ks, 2004) to Southern Brazil (27°17'$, 48°22'W) (Capei, 2012); (2) T. tagusensis, along the Brazilian coast (De Paula & Creed, 2004); and (3) T. micranthus in the Gulf of Mexico (Sammarco, Porter & Caims, 201 O). All three are considered opportunistic species most likely associated with transport on ships and/or oi! platforms in the Caribbean, Gulf of Mexico and Brazilian coast (Caims, 2000; Castro & Pires, 2001 ; Sammarco, Porter & Cairns, 2010). Once established, invasive species can alter the structure oflocal communities, displacing and outcompeting native species (\fito11sek, 1990; Mooney & Cleln11d, 2001 ; Lnges, Flewy & Me11egoln, 2011 ; Cure etal., 2012; Santos, Ribeiro d,- Creed, 2013; Miranda, Cruz & Barros, 2016). ln contrast to the native range, where Tubastraea is largely restricted to shaded or marginal habitats, studies on oi! rigs in the Gulf of Mexico have shown that both T. coccinea and T. micranthus are excellent competitors and can overgrow other species (Hen11essey & Snmmarco, 2014; Sammarco et ai., 2015). Similarly, in Brazil, T. coccinea a.nd T. tagusensis can cover up to 100% of the available surface in some areas (Ma111ellato e/ ai., 2011 ), killing native and endemic coral species upon direct contact (Creed, 2006; Sa1110s, Ribeiro dr Creed, 2013; Ma11tellato & Creed, 2014; Miranda, Cruz & Barros, 2016). Fast growth rate, rapid range expansion, early reproductive age, propagule pressure and a wide variety of reproductive and survival strategies are biological characteristics usually associated with invasion success (Snx & Brown, 2000; Sakai et nl, 2001 ; Lockwood, Cassey é- Black/Jurn, 2005; Sax et ai., 2007). Tubastraea species possess ali of these characteristics (Cairns, 1991 ; Ayre & Resing, 1986; Glym1 et ai., 2008; H11rriso11, 2011 ; Capei el nl., 2014; De Paula, Pires & Creed, 2014), which are enhanced by the fact that within the invaded areas they generally Jack natural predators and dorn i11ant competitors. ln addition, a large number of infested vectors (e.g., oi! plalforms and monobuoys) have been recorded

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transporting Tubastraea spp. along the Brazilian coast, leading to rapid range expansion throughout the Southwestern Atlantic Ocean (Creed et nl., 2016). Asexual reproduction improves coral ability to reach high abundance (Ayre & Miller, 2004) and may be an important trait of many invasive species, mainly in the first stage of invasion (Taylor & Ilastings, 2005). When associated with early reproductive age and high propagule pressure it can rapidly increase abundance. Asexual production of brooded planulae has been reported in several anthozoans, including actinarians ( Ottaway & Kirby, 1975; Blnck & Johnso11, 1979), octocorals (Brnzea11 & Lasker, 1989) and scleractinians (Stoddart, 1983; Ayre & Resing, 1986). Although T. coccinea and T. diaphana appear to reproduce mainly by asexually produced larvae (Ayre & Resing, 1986) , there is no inforrnation for their congeners, and the proportion of sexual versus asexual reproduction remains unknown within the genus. Furthermore, Ayre & Resi11g (1986) were able to score only two allozyme loci to infer asexual production ofbrooded Jarvae of Tubastraea spp. and the use of a larger number of more polymorphic loci, such as microsatellites, is desirable to corroborate their findings. Although Tubastraea species are spreading rapidly and changing local benthic communities throughout the tropical Western Atlantic, information about their genetic diversity and reproductive strategies are stül scarce. The study of reproductive strategies of invasive species is fundamental to understanding the invasion process, preventing new invasions, development of effective management strategies, and resolving the ecological and evolutionary processes involved in their invasion success (Sakní et ai., 200 I; Sax et ai., 2007). However, to date there was no molecular marker developed to perform such studies with Tubastraea. Here, we report 12 novel microsatellite loci specifically developed for T. coccinea and cross-amplified in T. tagusensis and investigate the clonai structure and genetic diversity of populations of these alien invasive corais iJ1 the Southwestern Atlautic Ocean.

MATERIAL$ ANO METHODS Sampling and DNA extraction Microsatellite development was performed using samples of T. coccinea coUected from Búzios Island (23º47'S, 45°08'W, 6 m in depth) and also from a monobuoy (IMODCO 4) at the São Sebastião channel (23°48'S, 45°24' W, 5 m of depth), Brazil. Additional samples of T. coccinea and T. tagusensis, collected from Todos-os-Santos Bay (TSB), northeastern Brazil (12º 49'5, 38º46' W), and ilha Grande Bay (1GB ) (23º06'S, 44°151 W), southeastern Brazil ( ~24 colonies/species/loca]ity), were used to test the markers and evaluate their genetic diversity (Fig. 1). Samples were preserved in 96% ethanol or CHAOS buffer (Fuknmi et ai., 2004) prior to extraction. Total DNA was extracted using the Qiagen DNeasy tissue and blood kit following the manufacturer's instructions or using the Phenol:Chloroform method described by F11kami et ai. (2004).

Microsatellite development and primer testing Two genomic libraries were constructed at the National Laboratory for Scientific Computing (LNCC, Petrópolis, Brazil) using the 454 Genome Sequencer FLX platform

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40°W

Brazil

20°5

AtlanticOcean 30º

600km

figure I Distributional range and samp)e localities on Southwestern Atlantic. Map showing the dis tributional range ofT11b11stmet1 spp. on Soutl1western Atlantic with the northern (NL) and southern (SL) limits oftbe distribution and sampled localities: Todos-os-Santos Bay (TSB) and !lha Grande Bay (1GB) are showed by dark-gray stars; light-gray star represent Bú1Jos lsland and São Sebastião channel where ini tial collections to isolate microsatellite loci were performed. Map layout from hnp://d-map~.com/carte. php?num_car-152J&lan!\ cn.

(Fernandez-Silva et ai., 2013). Reads were trimmed fo r adapters and quality using the FASTX-Toolkit. The software Newbler 2.3 (Roche, Basel, Switzerland) was used to perform the de novo assembly. The programs MSATCOMMANDER 0.8 (Fnirc/01'1, 2008) and SSRfinder were used to search for di-, tri-, tetra-, penta-, and hexa-nucleotide repetitions. Thirty-nine pairs of primers flanking the microsatellite regions were designed using Primer3 (http://bioínfo.ut.cc/primcr3-0.4.0/) a.nd p rimer characteristics were checked using OligoAnalyzer 3. 1 (hllps://www.idtdna.com/calc/analyzer/). Forward primers were designed with a MI3 taiJ at their 51 end (TGT AAA ACG ACG GCC AGT) for dye labeled (6-FAM, VIC, NED, or PET) prin1ers anneaJing to the replicated strand during PCR reactions (Schuelke, 2000).

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A total of 47 specimens of T. coccinea and 48 T. tagusensis were amplified by Polymerase Chain Reactions (PCRs) . PCRs were perforrned in 10 µI reactions including 0.2 µM of forward primer with M13 tail, 0.4 µM of labeled prirner (Ml3 with VIC, NED, PET, or 6-FAM fluorescent dyes), 0.8 µ.M of reverse primer, lU GoTaq (Promega, Fitchburg, WI, USA), lx PCR Buffer (Promega), 0.20 mM dNTPs (Jnvitrogen, Carlsbad, CA, USA), between 1.5 and 2.5 mM MgCli (Table 1), 10 µ.g BSA (Invitrogen), and 5-10 ng of DNA. Cycling conditions were: 95 ºC for 3 rnin fo llowed by 5 cycles at 95 º C, 30 s; 52- 62 º C (Table 1), 30 s; 72 ºC, 45 s; and 30 cycles at 92 ºC, 30 s; 52-62 º C, 30 s; 72 º C, 55 s; with a final extension at 72 ºC for 30 rnin (Toone11, 1997). Amplification was verified in 2% agarose gel. PCR products were pooled with GS600-LIZ size standard (Applied Biosystems, Waltham, MA, USA) and genotyped in the ABI 3500 geneticAnalyzer (Applied Biosystems). Genotypes were deterrnined using the prograrn Geneious 7. l.9.

Statistical analyses Clonal structure of each species was assessed using the 'GenClone' on R 3.2.3 package (R Core Team, 2015). Samples with the sarne alleles at all loci (ramets) were assigned to the sarne multilocus genotype (MLG, or genets) and considered to be a product of asexual reproduction. To check if individuais with the sarne MLG were truly clones, the probability of finding identical MLGs, resulting from distinct sexual reproductive events (Psex), was calculated following Ar111rnd-J-Iaond et ai. (2007). When Psex < 0.001, sarnples are considered ramets belonging to the sarne genet. ln order to avoid the overestirnation of genotype nurnbers due to scoring errors or sornatic mutations (Douhovnikojf6 Dodd, 2003), a second analysis calculating the genetic distance among all pairs of genets was performed. Based on the genetic distances, MLGs that differed at only one allele were assigned to the sarne multi-locus Lineage (MLL) (Arnaud-Haond et ai., 2007). For the genetic diversity and population structure analyses, only unique MLLs were considered. To assess the clonai structure ofeach popuiation, two indexes were calculated as proposed by Ama11d-Hao11d et ai. (2007): (1) clonal richness, to evaluate the proportions of clones in each population (R = G-1/N - l), where G represents distinct multilocus lineages (MLL) and N is the total number of individuais sampled. The index ranges from zero (when ali individuals are clones) to one (when aU sarnples analyzed correspond to a different MLL); and (2) the genotypic evenness, to evaluate the equitability in the distribution of the MLL, calculated by the Simpson's complement evenness index (V = (D-Dm;n)/(Dmax - Dm;n)), where D represents the observed diversity, Dmax the value assumed if all genets have the sarne number of ramets, and Dmin the diversity value when all but one genet has one individual (Hurlbert, 1971). This índex ranges from zero (when one genet dominates the population) to one (when genets each have the sarne number of ramets). Quality control of loci folJowed Selkoe & Toone11 (2006). To assess each population's genetic diversity, the number of alleles (Na), observed (Ho) and expected heterozygosities (He) were calculated using the 'diveRsity' in R 3.2.3 package (R Core Tenm, 2015). Significant deviations from Hardy- Weinberg equilibrium (HWE) and linkage equilibrium were tested with the FSTAT program (Goudet; 1995). The occurrence of null alleles was investigated using the Micro-Checker program ( V n11 Oosterlwut et nl., 2004). To measure

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C') ,::,"' ~ ~ ~ -,_, g Table 1 Description of Tubastraea cocci11ea and Tubastraea taguse11sis m.icrosatellite loci with their respective GeneBank Accession number ;J elude an M 13 sequence (S' -TGTAMACGACGGCCAGT-3').

Primt-rtitQWntt Ratpeat Sr«~ TA(ºC)I ..... TSB{N•ll,.Tcll4~TI) l Ac\'.t$$'=""1 0fl I\IIJ'l~ mollf IIMgO, (bp) ~ ( mM ) o Q Na fio "' P1s N ~ F:TCTAAMCCACCCCCACTACITCCCTCATOOCAOC.AC•Prl' T.rocil "612 0.12 0.18 0.00 :1 F": TCTJ.AMCCACGGCCAGT(;TGGAÇAGTCAATMGCITGCC..NEI> T. tvN'Ír'N'n 61)12 1.00 0.50 - 1.00 ~ Ttô4/ "-.) 1~ _,._. tTC,\)-4 2.5'-l.~ ~ R!GCCTGATOGlTfCTI'CACGTC T •~uiJ >812 1),40 .,, - 0.14 ~ . ..::l. F: TCTMMCCACCCCCAC1TC1,CGAccx;c,.ffMTAccrc~A~I 1. <0i.""WMI' W2 o.,o 0.76 O,J9 T"°Y K) (CMA)S 368.,.,jJ? O> "'..... R!TCTGCACTCAAí'CTCCTCAAC T. ,~c;b W 2.S 0.60 0.42 - 0,.JJ "' F: TCTMMCCACCCCCACTúCTCc"..AGTGTAAATT(',CrrCC•PET T.i«dl'N'n .. ,, 1.00 0.50 - t.00 Too81 ► Y111 .. CGGA)& J4J.. .},lj lt; G,\C\ACTCG.,\AACCÇCACG T. rngAA"t1# $?/2 1.00 0.,0 - 1.00

F: TCTAAMCCACCC:CCAC111'GACC'.i\CGTACTGCCAAG•\'1C T,<41((,lJMT 6011 Tco9H ll't . ('Tl,)10 3H -J51 lt; TCTCTTCAGACAGCTCCCC T. r~ 11if 6012 0.20 O. IS 0.00

F": 'rCTMMCCACGCCCAC1'CTCCCCTACCTCC\TCCff'f-VIC T.('l)(d,r,n1 6211.S 0.70 O.SI -0.JI Tc.l029f t-\1~74 (ATA}?O '?11-2.'U R: CCCGCITCTATATACGCITCC T. r~csi, ..,, 0.20 0.46 o... F: TCTMMCCACCGC'.0.GTCCG,,V,TIC CCA TCCMlTAT4'PAM T•'"""" 6(111,S ,... MI -o.OJ T«>JO/ K'íl 1 (ACA'll6 2S2-l6-f R: crcrc·rcc,.Arc1iCCT'CCAA T r~i;-is 60ll.2~ 1.00 0,,0 -1.00

F': r CTAMACCACCCCCACTCCCTCC1'CTCCTCTrn'CAT-6F'AM T.t~i;-b ..,, 1.00 0.50 -1.00 T00Ji.t 1,., 1~74\ (ATA)JJ 2◄0--2.W, R: ACééACTlT(;Aé:CTG·mcc

Tc,o.l2b/ ~, 1 T mgtUrllfiF l10-2U 1.00 0.,0 - 1.00

F: TCTAAAACCACCCCCACl'CCCCCTACTJ\CCACACCMT-PET T.rom11(n ..,, O.ló O,:H - 0,lO Tc,oJ,4/ ~"'""•V• (1TA)l9 139-217 lt: TccrrrcrACACCCCACClT T.t,l;flói,11d ..,, 0.80 0.53 -0.28

f: TCl'MAACCACGCCéACTCC.V.'rCACMO.GCC\CAAC-\'IC T.tMmn, S,81 1.S Too36/ f.: ,. (ATA)l5 l.}8~150 • R: TITCGTCl'GCCACATJ'C1TC

F: TCTMAACGAOOGCCACTAAACATTa;AlTCCÇACTCÇ,NED T. rofalM.t 62/1.S 1.00 0.1~ - 0.,2 T«,)7/ 1,;'íl~ 1~ (CTA}Z◄ 2◄2-261 ": ACCCCCCC\CTAATA TITCC T r~lfh 61/1.S 1.00 0.62 -0.50

F': r CTAMACCACCCCCACTrn'CACTn'CACifTAil'CACTOCiJ'•NFJ> T.cr of lndividt1als gcno1yp,cd; N:,1 , numbcr of alicies; Hc, cxpecl<.'d hc1cn erOZ)'gosity; f 1s, inbreeJingcoefficient (nega1ive value.s indicate an excess o( he1eroz)'go1es). •Loci wilh ._,...., idcnce of link"

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population structure two indexes were calculated using the programs Genetix (Belkhir et ai., 2004) and GenoDive (Meim1a11s & Van Tienderen, 2004). (1) Wright's fi.xation index FsT, ranging from zero, when different populations have identical alteies frequencies, to one, when each population has different fixed aUeles (Wriglzt, J965 ). However, when applied to highly polymorphic markers, such as microsateUites, this index never reaches one and can underestimate genetic differentiation (Hedrick, 1999; Meinnans & Hedrick, 2011 ; Bird et ai., 2011 ). The second measure, (2) Meirmans and Hedrick's differentiation índex G;r, is a standardized measure rescaled from zero to one based on the maximum value of G;T which simplifi.es interpretation of the degree of genetic differentiation among populations when using highly polymorphic microsatellite markers (Meirmans cf Hedrick, 201 /; Bird etal., 2011 ). A Bayesian analysis was performed to estimate the number of genetic clusters in the dataset using STRUCTURE v. 2.3.4 software (Pritchard, Stephens & Do1111elly, 2000) with the admixture ancestry model and correlated alicie frequency. The analysis was performed with an initial bum-in of 500,000 cycles followed by 500,000 additional cycles and the number of clusters (K) tested varied from one to three with 15 iterations for each K-value. A higher range in the number of dusters (K ranging from one to five) was also tested to verify possible substructure within the populations. The most likely K-value was estimated by estimating the "log probabili ty of data" for each value of K (mean LnP(K)) (Pritchard, Stephe11s & Donnelly, 2000) using STRUCTURE HARVESTER (Ear/ & Von f-loldt, 2012). The ó.K criterion, frequently used in population genetic studies, is applied for datasets with more than two populations and as one of the hypotheses here is that the two localities are one panmitic population, this criterion was not used in the present work (Evamzo, Reg11aut & Goudet, 2005).

RESULTS Characterization of microsatellite markers The two 454 runs resulted in a total of 329,832 reads with an average size of ±708.5 bp. A total of 1,077 regions with 2- 6 bp microsatellite repeats with at least four units were found. Among these regions, 39 were selected for pri.mer design, based on the size and position of the repeat within the sequence, and the primer characteristics (e.g., lacking primer-dimer formation). Within these, 11 and 10 were successfolly amplified and genotyped for Tubastraea coccinea and T. tagusensis respectively (Accession numbers: KYl 98738- KY198749). While two loci failed to amplify for T. tagusensis (Tco36 and Tco38), this species also exhibited two loci ata single locus with no evidence of linkage disequilibrium between them (Tco32a and Tco32b), so both were included in these analyses. Evidence for null alicies for T. coccinea TSB population was observed in the sarne two loci (Tco36 and Tco38) that failed to amplify for T. tagusensis. Since both loci had only homozygote genotypes at the two analyzed localities, these loci were removed from the genetic diversity analyses. The loci Tcol and Tco9 showed evidence of linkage disequilibrium with other loci and were also removed from the remaining analyses. The

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Table 2 Genetic diversity of Tubastraea coccinea and T. tagusensis in two localities on tbe Soutbwestern Atlantic Oceau, Todos os Santos Bay (TSB) aod Uba Grande Bay (1GB), Brazil.

Specie Location N MLG MLL R V A AR Ap Ho He F,s TSB 23 13 13 0.55 0.845 21 2.74 4 0.80 0.56 -0.380 r cocci11e11 1GB 24 6 6 0.2 1 l.13e-16 17 2.18 o 0.77 0.45 - 0.65 1 TSB 24 7 5 0.17 0.54 25 1.98 4 0.67 0.43 -0.468 T. tagusensis 1GB 24 6 3 0.09 ].04e- 16 22 1.87 1 0.64 0.37 -0.615

Notes. N, Number of individualssampled; MLG, multilocus genot)'l>e; Ml.L, multilocus. lineagesi R, clonai rkhne.-.s.i V. genotypic cvenness; ~ pareteo distribution; A, alleles number; AR, allele richness: Ap, number of private alleles: Ho, observed hererozigosities; He, expccted heterozigosities; F1s, inbreeding coeffident.

number of aUeles per locus ranged from one to five in T. coccinea and one to four in T. tagusensis. Between localities, Ho ranged from 0.38 to 1 (TSB) and 0.17 to 1 (1GB) for T. coccinea and from 0.2 to 1 (TSB) and O to 1 (1GB) for T. tagusensis. He ranged from 0.31 to 0.76 (TSB) and 0.15 to 0.58 (1GB) for T. coccinea and from 0.18 to 0.62 (TSB) and O to 0.61 (1GB) for T. tagusensis (Table 1). ln general, the observed heterozygosity was higher than expected for most loci in both populations ofboth species, with up to 100% of individuais being heterozygous at some loci (Tablc l ), although no sign ificant deviation from HWE was observed.

Clonality Psex values observed were highly significant ( <0.001) for ali but two and seven individuais of T. coccinea and T. tagusensis respectively. Thus, these data do not support the hypothesis of severa! individuais with the sarne MLG having originated by chance from distinct sexual reproduction events. A high proportion of clones were observed at both localities for both species (Tablc 2). For T. coccinea, at TSB of the 23 colonies sampled l3 MLLs were fou nd, whil e at 1GB only six MLLs out of the 24 colonies sampled were found. T. tagusensis had five (at TSB) and three (at 1GB) unique MLLs among the 24 sampled colonies at each locality (Table 2). Missing values were considered as different aUeles by the program, and although only specimens with missing information at no more than one locus were kept, it is important to note that the final number of MLL might be overestimated slightly. Clonal richness observed for T. coccinea indicates that 1GB is mostly composed of clones (R = 0.22), with only six MLLs out of 24 individuais, while TSB has nearly half of the individuais comprised of clones (13 MLL in 23 individuals sampled; R = 0.55) (Table 2). ln addition to the low MLL diversity at 1GB, 19 individuais had the sarne predominant 16 MLL, which was observed by the evenness indexes (V= 1,13- ). Conversely, the TSB population of T. coccinea had more equaUy distributed MLLs, with the most common one beingshared among on]y 4 individuals (V= 0.85). For T. tagusensis, both populations were composed mainly of clones, with very low clonai richness (IGB: R = 0.09; TSB: R = 0.17). Similarly to what was observed for T. coccinea, MLLs were more equaUy distributed at TSB, with 14 individuals belonging to the sarne MLL (V =0.54), while in 1GB the most common one was shared among 22 individuais (V =-l.04P- l6).

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Genetic diversity and population structure Only unique MLLs were used to assess genetic diversity and population structure in each species. For both species, TSB had higher number of alleles, allelic richness and number of private alleles compared to 1GB, with T. coccinea presenting the more accentuated differences (1 ablc 2). There were no significant deficits of heterozygosity; both observed (Ho) and expected (He) heterozygosity were similar when comparing between localities for both T. coccínea (TSB: 0.80 and 0.56; 1GB: 0.77 and 0.45) and T. tagusensis (TSB: 0.67 and 0.43; 1GB: 0.64 and 0.37). The inbreeding coefficient (F1s), although not significant, was negative for both localities and in both species, indicating an excess of heterozygotes (Table 2). FsT and c;Tvalues were 0.06 (p = 0.08) and 0.13 (p = 0.07) for T. coccinea and indistinguishable from zero (p = 0.69 and p = 0.69) for T. tagusensis. The lack of significant population structure among the sampled localities indicates similar allele frequencies for both species across these sites. Although Bayesian analysis recovered two genetic clusters for T. coccinea for both ranges of K tested, these groups are nota function of population structure between localities (Fig. 2), but instead, reflect the presence of population strucrure within each locality. Furthermore, there is no evidence of interbreeding betv.reen the two clusters, and the FsT values between these sites is likely a result of the strikingly different proportion of these two groups in each site. ln contrast, no clustering was observed between or within localities for T. tagusensis, with the most likely K val ue being one for both ranges of K tested (Fig. 2).

DISCUSSION The novel microsatellite markers reported herein will enable further studies regarding the genetic diversity and population structure of Tubastraea spp. corais in the Atlantic and na tive ranges of these invasive populations. Using these microsatellites, this study shows that both invasive coral species (T. coccinea and T. tagusensis) have high proportions of clones at both localities on the Brazilian coast with identical multilocus lineages (MLLs) found i11 sites separated by more than 1,500 km. The results indicate that asexua] reproduction dominates in tlle invasive range of Tubastraea spp. in the Southwestern Atlantic and despi te the large distance between localities, no significant population structure couJd be found. ln contrast, there are clear signs of popuJation structure across this sarne region in an endemic spawning coral species (Mussismilia hispida, Azevedo, 2015). Our results support previous work reporting reproduction via asexual larvae in T. coccinea (Ayre & Resing, 1986). Likewise, the high proportion of clones foUJ1d at both sampled localities for T. tagusensis indicares likely reproduction by asexual larvae for this species also, a reproductive mode previously recorded for onJy three scleractinian species: Pocillopora damicornis (Stoddart, 1983), Tubastraea diaphana and T. coccinea (Ayre & Resi11g, 1986). Indeed, a study on the reproductive strategies of T. coccinea and T. tagusensis in the Southwestern Atlantic observed a small number of spermaries and the presence of embryos and planula at different times of the year, concluding that asexual reproduction could be important fo r both species (De Paula, Pires & Creed, 2014). For most corais,

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a L(K) (mean +- SD) b L(K) (mean +• SD) -190 J!l • J!l. 121.5 ..,"' ..,"' õ-200 .oe ~- 122.0 a. ea. e· 210 j _, 5.122.5 i -;;;., õ-220 Ô - 123.0 e + ..., .,e ::. ::;"' - 230 • - 123.5 1.0 1.5 2.0 2.5 3.0 1.0 1.5 2.0 2.5 3.0 K K

Tubastraea coccinea __J TSB 1GB

d Tubastraea tagusensis

TSB 1GB

Figure 2 Bayesian clustering analyses for T11bastraea cocci,rea and T. tag11se11sis. (A) and (B) shows the most likely K-value estimated by the mean ofestimated " log probability of data" for each value ofK for T. cocci11ea (K = 2) and T. 111gusensis (K = 1), respectively; (C) and (D) shows the genetic clusters, where each individual is represented by a vertical bar with different colors indicating the relative proportion of each genetic duster. TSB, Todos os Santos Ba)'; 1GB, Ilha Grande Bay.

clonality ís a result of mechanícal fragmentation due to physical dísturbances (Poster et a/., 2013 ; Nakajima ct ai., 2015). T. coccinea and T. tagusensis, however, are JJot prone to fragmentation, so the high number of clones observed for both species in this study seems more líkely to result from asexually produced larvae. Nevertheless, ít is desírable to confirm the production of asexual larvae for both T. coccinea and T. tagusensis by performíng paternity studies in the future. For invasive species, asexual reproduction can be crucial in the first stage of invasion, when sexual partners are scarce or absent, because it significantly enhances the chances of survival for the colonists (Taylor & llastings, 2005). Successful invasions originating from a few clonai genotypes have been prevíously recorded for plants (Ren, Zlumg & Zlia11d, 2005; Li11 e/ ai., 2006) and otl1er cnidarians (Rcitzel et ai., 2008). Asexual reproduction is dominant in the invasive range and it may have contributed to the invasive success of Tubastraea in the Southwestern Atlantic, where the rocky shores provide a suitable habitat and release from enemies (Enemy Release Hypothesis, Kea11e & Crawley, 2002). At 1GB

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both studied coral species have high percentage of clones and an extremely low genotypic eve1mess, indicating that most colonies are clones belonging to the same genet. Sampling more areas surrounding each coUection site is needed to thoroughly examine clonai diversity for these regions, but particularly in TSB where samples were more widely spaced, this observation supports the role of asexual reproduction in increasing local abundance. Gregarious settlement has been previously observed for both T. coccinea (Glynn et ai., 2008; De Paula, Pires & Creed, 2014), and T. tagusensís (De Pa11ln, Pires & Crced, 2014), although these studies did not determine if the aggregated larvae were sexually or asexually derived. It is noteworthy that T. coccinea has higher numbers of MLLs, clonai richness and genotypic evenness at TSB than at 1GB, suggesting increased occurrence of sexual reproduction or a greater number of successful colonists at the former site. Rates of sexual and asexual reproduction can be highly variable among geographic regions in other corals (Bawns, Miller & Hellberg, 2006; Noree 11, Harriso11 & Vnn Oppen, 2009; Combosch & Vollmer, 2011; Gorospe e!,~ Karl, 2013), but it remains unknown what governs the difference in the proportion of sexual and asexual reproduction at different localities. Severa! factors can influence both genotypic and genetic diversity in invasive species, induding the number of invasions, the genetic diversity of the source population(s) and a variety of biological factors, such as the main reproductive strategy adopted by the species ( Dl11gosch & Pnrker, 2008). Although sexual reproduction might also occur in Tubastraea, the results obtained for T. coccinea might be an effect of the occurrence of recent multiple introductions from different native populations (Ro111a11 ó- Darli11g, 2007). Another hypothesis would be the presence of cryptic species, which has been found in other scleractinian corais (Pi11zó11 & Weil, 2011 ; Wamer, Van Oppen & Willis, 2015; Nakajima et ai., 2017). Morphological analyses combined with molecular data including native populations are necessary to corroborate this hypothesis. A decrease in genetic diversity as a result of a small founding population has been previously recorded for severa] invasive populations (Roman & Darling, 2007; Geller et nl., 2008; Jo/111so11 & Woollacott, 2015; Wrange et ai., 2016; but see Gaither et ai., 2010; Gaither, Too11e11 & Bowen, 2012 for counter-examples). Here, we report excess ofheterozygosity for both populations ofboth species and the presence of up to 100% heterozygous individuaJs at some loci (Table l ). High leveis of heterozygosity can resu.lt from an isolate-breaking effect, when multiple introductions mix previously separated native populations (Holla11d, 2000; Ha111ilto11, 2010). However, in this case, there is no evidence of mixing between the two genetic clusters (Fig. 2), indicating that they are not interbreeding. Thus, it seems more likely that TSB and IGB were colonized by different native populations followed by recent transport between localities without sufficient time for them to interbreed, although the possibility of cryptic species that are incapable of ü1terbreeding should also be considered. If the first scenario of introduction by different native populations proves true, the high heterozygosity could be either a result of a founder effect in which the new area was, by chance, colonized by a higher number of heterozygote genotypes, or due to a higher fitness of the heterozygote genotypes, either of which could be propagated by asexual reproduction (De Meeus & Ballo11x, 2005). Alternatively, Gaither, Toone11 & Bowe11 (2012) showed that introduced fishes in Hawai'i with a known history actually had higher and

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more even genetic diversity than was observed in the na tive range, and such an effect could also explain the observed pattern here. ln contrast to what is observed with T. coccinea, we recover only a single genetic cluster for T. tagusensis between both populations. This single cluster could result from either invasion ofboth localities frorn the sarne source population, or a secondary invasion along the Brazilian coast from the original locality being spread to another. UnJike T. coccinea, which is now considered cosmopolitan (Caims, 2000), T. tagusensis has a restricted distribution (Cairns, 1991 ) and may have naturaUy low genetic diversity. The distinction between these species is reminiscent of the pattern reported by Caither, Bowen & Toonen (2013) in which population structure of species in their native range predicts the diversity and rate of spread in the invasive range. Considering that (i) both T. coccinea and T. tagusensis brood larvae competent for only ~18 days (in aguaria) that typically display gregarious setdement ( Glynn et ai., 2008; De Paula, Pires & Creed, 2014) and (ii) the absence of Tubastraea in extensive areas between the two localities, it is highly unJikely that they are connected through larval dispersai. On the other hand, oil platforms are known to be moved between these regions (Sampaio et ai., 2012), and are considered the main vector for the introduction of Tubastraea into the southwestern Atlantic (Castro 6· Pires, 2001; Creed et al., 2016). Thus, our data showing a lack of structure between localities, and the occurrence of shared MLLs for each species among these distant sites, indicate that anthropogenic vectors, such as oil platforms, monobuoys, or other vessels have played an important role in dispersing these alien invasive species, and possibly assisting other species to spread along the coast (Al111eida et ai., 2015; Creed et ai., 2016).

CONCLUSIONS Tnvasive Tubastraea spp. are spreading quickly tluoughout the Atlantic, in some areas covering up to 100% of the available surface (Mantellato et ai., 2011 ) and outcompeting na tive and endemic species (Ma11tellato et ai., 201 /; Sm1tos, Ribeiro & Creed, 2013; Creed, 2006). Despite this documented impact and concern, Little is known about the genetic diversity and reproductive strategies of Tubastraea species globally. This study provides the first survey of genetic diversity and likely reproductive strategies along the southwestern Atlantic coast, demonstrating that asexual reproduction has an important role in the popuJation dynamics of both T. coccinea and T. tagusensis and is probably a relevant feature leading to their invasive success. Results also indicate that there were likely at least two different populations ofT. coccinea introduced into the southwestern Atlantic. A molecular systematic examination of the genus is highly recommended in order to check for the occurrence of cryptic species. Future studies shouJd focus on the identification of potential source populations and the global phylogeograpy of Tubastraea with the goaJ of tracking and limiting future invasions, as well as the establishment of effective management and prevention strategies.

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ACKNOWLEDGEMENTS We are grateful to Antonio Solé-Cava for helping editing the first set ofNext-Generation Sequencing data and to Marcelo Mantellato and Projeto Coral-Sol for samples. We are also thankful to Diane Bailleul for aJJ the support on the clonai analyses. This is Scientific Contribution No. 30 of the Projeto Coral-Sol.

ADDITIONAL INFORMATION ANO DECLARATIONS

Funding This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Joel C. Creed, Ciências do Mar 1137/2010); Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (Katia Cristi11a Cruz Capei, Joel C. Creed and Carla Zilberberg, FAPERJ-E-26/010.003031/2014 PensaRio; Joel C. Creed E26/201.286/2014); Conselho Nacional de Desenvolvimento Científico e Tecnológico (Joel C. Creed, CNPq-305330/2010-1) and Fundação de Amparo à Pesquisa do Estado de São Paulo (Marcelo V. Kitahara, FAPESP 2014/01332-0); and Award NSF-OA#l4-16889 (National Science Foundation). The funders had no role in study design, data collection and anaJysis, decision to publish, or preparation of the manuscript. Grant Disclosures The following grant information was disclosed by the authors: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior: 1137/2010. Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro: FAPERJ-E-26/010.003031/2014, E26/201.286/2014. Conselho Nacional de Desenvolvimento Cientifico e Tecnológico: CNPq-305330/2010-1. Fundação de Amparo à Pesquisa do Estado de São Paulo: FAPESP 2014/01332-0. National Science Foundation: NSF-OA#l4-l6889. Competing lnterests Robert J. Toonen is an Academic Editor for PeerJ. Author Contributions • Katia Cristina Cruz Capei conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. • Robert J. Toonen wrote the paper, reviewed drafts of the paper. • Caio T.C.C. Rachid analyzed the data, reviewed drafts of the paper. • Joel C. Creed contributed reagents/materiaJs/analysis tools, wrote the paper, reviewed drafts of the paper. • Marcelo V. Kitahara contributed reagents/materials/analysis tools, wrote the paper, reviewed drafts of the paper. • Zac Forsman wrote the paper, reviewed drafts of the paper. • Carla Zilberberg conceived and designed the experiments, contributed reagents/materi als/analysis tools, wrote the paper, reviewed drafts of the paper.

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Field Study Permissions The following information was supplied relating to field study approvals (i.e., approving body and any reference numbers): The sarnples used in the study were collected under the permit No 003/2014 frorn Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis - Ministério do Meio Ambiente, Brazil.

DNA Deposition The following information was supplied regarding the deposition of DNA sequences: Ali microsatellite developed will be available via GenBank accession numbers KY 198738 to KYl 98749. Sequences were uploaded as File S 1.

Data Availability The following information was supplied regarding data availability: The raw data has been uploaded as a Supplemental File.

Supplemental lnformation Supplemental information for this article can be found online at http:/ /dx.doi.org/10.771 7/ peerj.3873#supplcmental-infonnation.

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Supplemental File 1. Microsatellites accession numbers and sequences.

Tco1 – ACCESSION KY198738 acccggtcatgtaaggctgtacactgatctgtttaatcgttcgcnacgtaaaaantagggtgttttaactctacagatcagcttctgttttgtt atattgtctaacctttattattttcaattatattacttttctctgcctcgaatagcgtttttgctaattcgcngggnaactgtacttaagttgatatgt ctaataaagttttgtttgtttgttgttgttgttgttgtttgtagaatgttataataaggttcgtgaactcgcaaacgttgacgtcagagagtcgtct taaacggcctcgtttccatgtaacgggtcttccaattaagaactcgtacagatagcagacaagagtgtctgaaatgaaatttgatgatgtct gtaagtacagactacgcctagtcaaagcaagtacccgtgct

Tco4 – ACCESSION KY198739 gtggagagtgaataagcttggggtgagtgaataaaagggggtaagtgaataagagggggnagtgaatattataaagagggggagtc gagtgaataagagggggntgaaaatgttagagacaagataccataaagttttgataatatcatcancatcatttattnattagccttttttttn aantaacagatttaacaagttacagcagnttgttaggcgaggagacctcaagaaaccatcaggc

Tco5 – ACCESSION KY198740 tcaggagccgattaatacctgattatttttctgtaagattaaggagactctacttattcatgaactcagacctacccttaacgataatgtctgat tatttttgttattttaacgagtgtttngacataatgtagacaagataaaccattggaaaaataaaatattaatagcagcctttgttaaatcattga aaaaagaacgttaacagcaatgcaatatcttaattattgggctcaatatataaccacagaagcttatcacgtatgagcttctgataaccata actcaaagttccagtttcagtgaaactggaaagaaagaangaaagaangaaatatttctttctttcttacgaatgaacgaatggacggac ggacgaacggacgaacgaacgaacttgagcacattcactgcaca

Tco8 – ACCESSION KY198741 tgctgccgcgatacgagcatcgtttcatcacccccgagtattggcaagaggaagaggaggaggaggaggaggaagagtcgatgga gtgaactgacacaaaacacgcgttttgttttacaagtggttgatttttattaaattttcaactcaacgttggattttacaataaagtgttcttttac aatttaaacgtttcttgactgcatgattttttcacaatcttctcngncngggtatnttttcccgncaggtngntgatcccncggancnttttttc gtccgctttccacttgtc

Tco9 – ACCESSION KY198742 ttgaccacgtactgccaagttcatctgccagtttttaatattatgatcgacagaactcgtaattttaaattaaaaactccaaacgttatcgtatt aatttgttcctgactctttctgtgatagcgtcaccagcggccagtgtttattttcttgcttagagcctatctttcatatagaattgttatcttagtaa tttactctgttattagttttctatttgaaaggcttgtcgagctgcattcaaaaccctatattaagtctcgtcaaactccaggcacattccacaata tatatatatatatatataggctgagccaaacagcggagctctctgaacaga

Tco29 – ACCESSION KY198743

149 gtgccctaggtccatggtttggacccggcccccatggagaacacataattatatagagaacacataataataataataataataataataa taataataataataataataataataataatacaatgcactagaaatttataataacttaaaagacaatagactacgaaatgattaaggggta gtgacaaggaagcctatatagaagccgg

Tco30 – ACCESSION KY198744 gggaattcggatgcaattataacatacatacatacatacatacatactttattggctcgtccccatggggtttttcagagtcaatttaaaatta cataactaagtacaagacaataaaagtaaaaagtaaaacttgtggattaacttaaaaagtcattaagaagtttttgcctaagtaagcgctta aacaccatcacggaagggctaagtctgagcgagggttgcagctcattccacagag

Tco32 – ACCESSION KY198745 gcgtggtctggtcttttcattaaaaaagaatcagggtatcgttaccgttcaccagcttactgacttagtcattgctgtttggaacctctccggt aaaatataataataataataataataataataataataataataacgaacttattctttcgttttctctgcagttaatgatcagtttgagaagctat atcaaacactcgaaagagtgtttcattaggtatccaaacacctcaaagtgggt

Tco34 – ACCESSION KY198746 gcgcctactaccacacgaatggtttattattattattattattattattattattattattattattattattattattatggtacagttactttaattgcaa atggtttctcactctcaacctccgagtccaaaccatggacttagggatgcaaagaggatttctacttgatggaaagcacccgcctaagaa ggtgcgctgtagaaagga

Tco36 – ACCESSION KY198747 gcaatgacaacagccagaacatgatgttaaacaaagataataataataataataataataataataataataataataattatcatcaagaa caacaaggacaagatatgcacacttatcgatgtagccattccctctgacaaaaacatctccaccaaagtctctgagaaaacatccaaata taaagacctagaacttgagattgcaagaatgtggcagacgaaa

Tco37 – ACCESSION KY198748 aaacattcgattcccactcggcaatcttgtaaatcagtttttatagttattttaatttttataaatgattgtaattaaaatactattagtgtaactgaa gatttttaaccgagtttaaataaagatctactactactactactactactactactactactactactactactactactactactactactacta ctacggatgcggtatataatttgaacggaaatattagtggccgggt

Tco38 – ACCESSION KY198749 tttgagtttgagtttattgactccttagctacatacatacatacatacatacatactttattgaccacttccccaaaggggcttttcagtgccaat tacaaagaaaaagctaaaaataaaatatatacaaattatttaaagttacaattacaaattattaggatatcattcctccctcttaaatttgtctaa aagcacccctctaagcttactcc

150

Supplemental File 2. Allele sizes for each genotyped individual.

Species: Tubastraea coccinea Ind Pop Microsatellite loci Tco1 Tco4 Tco5 Tco8 Tco9 Tco29 Tco30 Tco34 Tco37 Tco36 Tco38 BTS104 TSB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BTS105 TSB 567 573 253 259 380 440 343 349 347 347 213 222 252 256 186 189 260 263 238 238 227 227 BTS106 TSB 567 600 253 259 0 0 343 349 347 351 222 222 252 256 189 189 242 245 250 250 227 227 BTS107 TSB 567 573 253 259 380 440 343 349 347 347 213 222 252 256 186 189 260 263 238 238 227 227 BTS108 TSB 567 600 253 259 368 368 0 0 347 351 222 222 252 256 189 189 242 245 250 250 227 227 BTS109 TSB 567 573 253 259 380 440 343 349 347 347 213 222 252 256 186 189 260 263 238 238 227 227 BTS110 TSB 573 573 253 259 0 0 343 349 357 357 216 222 252 264 189 189 260 263 238 238 227 235 BTS111 TSB 567 573 253 259 384 440 343 349 0 0 213 222 252 256 186 189 260 263 238 238 227 227 BTS112 TSB 567 573 253 259 0 0 343 349 347 347 213 222 252 256 186 189 260 263 238 238 227 227 BTS113 TSB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BTS114 TSB 567 573 253 259 380 440 343 349 347 347 213 222 252 256 186 189 260 263 238 238 227 227 BTS115 TSB 567 573 253 259 380 440 0 0 347 347 213 222 252 256 186 189 242 245 238 238 227 227 BTS116 TSB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235

BTS117 TSB 567 600 253 259 368 1 380 343 349 347 351 222 222 252 256 189 189 242 245 250 250 227 227 BTS118 TSB 567 600 253 259 368 368 343 349 347 351 0 0 252 256 189 189 242 245 250 250 227 227 BTS119 TSB 567 573 253 259 380 440 343 349 347 347 0 0 252 256 186 189 260 263 238 238 227 227 BTS120 TSB 567 600 253 259 368 368 343 349 347 351 0 0 252 256 189 189 242 245 250 250 227 227 BTS121 TSB 567 600 253 259 368 380 343 349 347 351 222 222 252 256 189 189 242 245 250 250 227 227 BTS122 TSB 567 573 253 259 380 440 343 349 347 347 0 0 252 256 186 189 260 263 238 238 227 227 BTS123 TSB 567 600 253 259 368 368 343 349 347 351 0 0 252 256 189 189 242 245 250 250 227 227 BTS124 TSB 573 573 253 259 432 432 343 349 355 357 216 222 252 264 189 189 260 263 232 232 227 227 BTS125 TSB 567 600 253 259 368 380 343 349 347 351 222 222 252 256 189 189 242 245 250 250 227 227 BTS127 TSB 573 573 253 259 432 432 343 349 355 357 0 0 252 264 189 189 260 263 238 238 235 235

151

BIG95 IGB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BIG96 IGB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BIG97 IGB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BIG98 IGB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BIG99 IGB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BIG100 IGB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BIG101 IGB 573 573 253 259 432 432 0 0 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BIG102 IGB 573 573 0 0 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BIG103 IGB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BIG104 IGB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BIG105 IGB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BIG106 IGB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BIG107 IGB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BIG108 IGB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BIG109 IGB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BIG111 IGB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BIG112 IGB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BIG113 IGB 573 573 253 259 432 432 343 349 357 357 216 222 0 0 189 189 260 263 238 238 235 235 BIG114 IGB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BIG115 IGB 573 573 253 259 0 0 343 349 347 357 216 222 252 264 189 189 260 263 238 238 235 235 BIG116 IGB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BIG117 IGB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235 BIG118 IGB 567 573 253 259 380 440 343 349 347 347 213 222 252 256 186 189 260 263 238 238 227 227 BIG119 IGB 573 573 253 259 432 432 343 349 357 357 216 222 252 264 189 189 260 263 238 238 235 235

TSB= Todos os Santos Bay IGB= Ilha Grande Bay

152

Species: Tubastraea tagusensis Ind Pop Microsatellite loci Tco1 Tco4 Tco5 Tco8 Tco9 Tco29 Tco30 Tco34 Tco37 Tco32 Tco32_b BTS73 TSB 573 573 253 253 380 424 343 349 347 347 213 225 252 256 213 219 245 248 240 246 270 276 BTS74 TSB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BTS75 TSB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BTS76 TSB 573 573 253 259 424 424 343 349 347 347 213 213 252 256 189 213 245 260 240 246 270 276 BTS77 TSB 573 573 253 259 424 424 343 349 347 347 213 213 252 256 189 213 245 260 240 246 270 276 BTS78 TSB 567 573 253 259 424 424 343 349 347 351 222 222 252 256 189 213 0 0 240 246 270 276 BTS79 TSB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BTS81 TSB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BTS82 TSB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 189 213 245 260 240 246 270 276 BTS83 TSB 573 573 253 259 424 424 343 349 347 347 213 213 252 256 189 213 245 260 240 246 270 276 BTS84 TSB 573 573 253 259 424 424 343 349 347 347 213 213 252 256 189 213 245 260 240 246 270 276 BTS85 TSB 573 573 253 259 424 424 343 349 347 347 213 213 252 256 189 213 245 260 240 246 270 276 BTS86 TSB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276

BTS87 TSB 573 573 253 253 380 1 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BTS88 TSB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BTS89 TSB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BTS90 TSB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BTS91 TSB 573 573 253 253 380 424 343 349 347 347 213 213 0 0 189 189 245 248 240 246 270 276 BTS92 TSB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BTS93 TSB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BTS94 TSB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BTS95 TSB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BTS96 TSB 573 573 253 259 424 424 343 349 347 347 213 213 252 256 189 189 245 260 240 246 270 276 BTS97 TSB 573 573 253 259 424 424 343 349 347 347 213 213 252 256 189 189 245 260 240 246 270 276

153

BIG65 IGB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BIG66 IGB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BIG67 IGB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 273 276 BIG68 IGB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 242 245 240 246 270 276 BIG69 IGB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 242 245 240 246 270 276 BIG70 IGB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BIG71 IGB 573 573 253 253 380 424 343 349 347 347 213 225 252 256 213 219 245 248 240 246 270 276 BIG72 IGB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BIG73 IGB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BIG74 IGB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BIG75 IGB 573 573 253 259 424 424 343 349 347 347 213 213 252 256 189 213 245 260 240 246 270 276 BIG76 IGB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BIG77 IGB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BIG78 IGB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 242 245 240 246 270 276 BIG79 IGB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BIG80 IGB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BIG81 IGB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 242 245 240 246 270 276 BIG82 IGB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 242 245 240 246 270 276 BIG83 IGB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 242 245 240 246 270 276 BIG84 IGB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BIG85 IGB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 242 245 240 246 270 276 BIG86 IGB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 246 246 270 276 BIG87 IGB 573 573 253 253 380 424 343 349 347 347 213 213 252 256 213 219 245 248 240 246 270 276 BIG88 IGB 573 573 253 253 380 424 343 349 347 347 213 225 252 256 213 219 245 248 240 246 270 276

TSB= Todos os Santos Bay IGB= Ilha Grande Bay

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Capítulo V

Marine hitchhikers: multiple introductions of Tubastraea spp. in the Southwestern Atlantic and the role of vectors on dispersion

Kátia Cristina Cruz Capel, Joel Creed, Marcelo Kitahara & Carla Zilberberg

Revista alvo: Molecular Ecology.

155

Marine hitchhikers: multiple introductions of Tubastraea spp. in the Southwestern Atlantic and the role of vectors on dispersion

KCC Capel1,2*, Creed J2,3, MV Kitahara4,5, C Zilberberg1,2

1 Departamento de Zoologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil 2 Associate Researcher, Coral-Sol Research, Technological Development and Innovation Network 3 Departamento de Ecologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil 4 Departamento de Ciências do Mar, Universidade Federal de São Paulo, Santos, Brazil 5 Centro de Biologia Marinha, Universidade de São Paulo, São Sebastião, Brazil

* Corresponding author Kátia Capel e-mail: [email protected] Laboratório de Biodiversidade de Cnidária. Departamento de Zoologia Universidade Federal do Rio de Janeiro.

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Abstract

Marine traffic had increased exponentially in the past decades and accidental introduction through ballast water and biofouling are currently the main responsible for spreading non- indigenous species in the marine realm. In the Southwester Atlantic Ocean, two species of scleractinian corals, Tubastraea coccinea and T. sp. cf. T. diaphana, had been introduced by opportunistic colonization of oil platforms in 1980. After 40 years, the species are widespread over more than 3,500 km, from, outcompeting native and endemic species. In order to better understand the invasion process and the role of vectors on spreading these species we sampled and genotyped 306 and 173 colonies of T. coccinea and T. sp. cf. T. diaphana from invaded sites, possible vectors and one native population. Analyses revealed a high proportion of clones for both species, with only 84 and 30 unique multilocus genotypes within all sampled sites. In general, invaded sites were dominated by few genotypes, spread over sites distant around ~1,500 and ~2,000 km. The observed broad distribution of clones is likely a result from secondary introductions through the transport of contaminated vectors throughout the coast. An excess of heterozygous was observed, mainly for invaded sites, which disagree to the general expected loss of genetic diversity following an invasive process. We suggest that the observed genetic diversity for Tubastraea spp. in Southwestern Atlantic may be explained by a combination factors such as reproductive strategy, high growth rate and high propagule, through the occurrence of multiple invasions. Results presented here show that clonality and secondary introductions are the main reasons for the broad spread and invasive success of Tubastraea spp. in the Southwestern Atlantic. Consequently, the correct control of vectors is the most effective approach for an effective management and prevention of new invasions.

Key words: invasive species, clonality, Tubastraea coccinea, T. sp. cf. T. diaphana

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Introduction

Marine bioinvasion is reshaping the distribution and biogeographic patterns worldwide, and is reaching unprecedented levels with hundreds of species being transported to new environments every year (Molnar, Gamboa, Revenga, & Spalding, 2008; Ricciardi, 2007; Seebens, Gastner, & Blasius, 2013; Wilson, Dormontt, Prentis, Lowe, & Richardson, 2009). Accidental introductions can occur through many ways such as aquaculture, trade of ornamental species, canal linking unconnected waters, ballast water and biofouling, the last two being the main responsible for spreading non-indigenous species in the marine realm (Carlton, 1996; Minchin, Gollasch, Cohen, Hewitt, & Olenin, 2009; Molnar et al., 2008; Williams et al., 2013). As a consequence of the increasing marine traffic, vessels (e.g. cargo ships, oil platforms, floating docks, buoys; herein on called vectors) transport a large number of species (either by ballast water or biofouling), some of which will be able to establish and disperse, becoming invasive in the new environment (Ricciardi, 2016; Williams et al., 2013).

Recently, 15 non-indigenous species including two azooxanthellate corals (Tubastraea coccinea and T. tagusensis, the later re-identified as T. sp. cf. T. diaphana by Capel et al. in prep., Chapter 1) are causing major negative impacts in the South Atlantic (Castro, Fileman, & Hall-Spencer, 2017). T. coccinea was first reported in the Atlantic during the 1940’s in Curaçao and Puerto Rico, probably introduced from Indo-Pacific waters encrusted in ship hulls (Vaughan and Wells 1943; Cairns, Häussermann, & Försterra, 2005; Creed et al., 2016). Further records of the genus were reported in the Northwestern Atlantic in 2004 (T. coccinea) (Fenner & Banks, 2004), Southwestern Atlantic in the 1980’s (T. coccinea and T. sp. cf. T. diaphana) (Castro & Pires, 2001; de Paula & Creed, 2004) and Gulf of Mexico in 2010 (T. coccinea and T. micranthus) (Sammarco, Porter, & Cairns, 2010). Given the pattern of sea surface currents and previous examples from other invasive species, the Gulf of Mexico and Florida could have been naturally invaded by Caribbean populations (Creed et al., 2016). However, the occurrence of T. micranthus, a species not yet found in the Caribbean, on oil platforms at Gulf of Mexico indicates that human vectors had also been responsible for introduction at this region (Sammarco, Porter, Sinclair, & Genazzio, 2013). In the Southwestern Atlantic, Tubastraea spp. were first introduced through encrustation on offshore oil platforms in the Rio de Janeiro State (Castro & Pires, 2001; Creed et al., 2016). The genus is now widespread on rocky shores and artificial substrate (oil platforms, buoys and drill ships) along more than 3,500 km, from Ceará (02º29′S, 39º51’W) (Soares, Davis, & de

158

Macêdo Carneiro, 2016) to Santa Catarina (27º17’S, 48º22’W) (Kátia Cristina Cruz Capel, 2012) and both species have been shown to be outcompeting native and endemic species (Creed, 2006; Mantelatto, Creed, Mourão, Migotto, & Lindner, 2011; Mantelatto & Creed, 2015; Miranda, Cruz, & Barros, 2016; Santos, Ribeiro, & Creed, 2013). Although there is no doubt that the introduction in the Southwestern Atlantic was through biofouling (Creed et al., 2016), the invasion history is unclear and there are no studies elucidating why these two species are so successful in the Brazilian coast and what is the role of vectors on spreading them.

Successful invasive species frequently share a set of life history and ecological traits that facilitate their establishment, such as rapid growth, large number of offspring (r-select species), sexual and asexual reproduction, early maturity and phenotypic plasticity (Sakai et al., 2001). When combined with a high propagule pressure, a measure of the number of individuals released and the number of releasing events, it considerably enhances the chances of survival in a new environment (Lockwood, Cassey, & Blackburn, 2005, 2009). Tubastraea spp. possesses all cited traits typical of successful invasive species (Ayre & Resing, 1986; Cairns, 1991; Capel, Migotto, Zilberberg, & Kitahara, 2014; de Paula, de Oliveira Pires, & Creed, 2014; Glynn et al., 2008; Harrison, 2011), which may have facilitated their establishment and dispersion in the Southwestern Atlantic. Furthermore, considering the reproductive biology of these species and the rapid expansion throughout more than 3,500 km in less than 40 years it is likely that vectors have been playing a key role in these species spread along the Southwestern Atlantic (Capel et al., 2017; Chapter 4). Indeed, Tubastraea spp. have been recorded on at least 23 vectors, some of which have been towed along the Brazilian coast without any biofouling control (Creed et al., 2016).

The occurrence of multiple introductions has also been correlated with invasion success (Bock et al., 2015; Dlugosch & Parker, 2008; Keller & Taylor, 2010; Kolbe et al., 2004; Rius & Darling, 2014; Verhoeven, Macel, Wolfe, & Biere, 2011). When founded by a small number of individuals, recently established populations can suffer a drastic reduction on genetic diversity as a consequence of genetic drift. Ultimately, the genetic drift can have negative consequences such as the fixation of deleterious alleles and/or decrease of the species resilience (Ellstrand & Elam, 1993; Geller, Sotka, Kado, Palumbi, & Schwindt, 2008; Johnson & Woollacott, 2015; Joe Roman & Darling, 2007; Sakai et al., 2001; Wrange et al., 2016). On the other hand, multiple introduction events of non-indigenous species from more

159 than one native population can lead to an increase in genetic diversity by mixing previously separated populations and increasing the propagule pressure, consequently reducing negative outcomes of an invasion process and enhancing the possibility of a successful invasion (Blackburn, Lockwood, & Cassey, 2015; Bock et al., 2015; Carlton, 1996; Dlugosch & Parker, 2008; Lockwood et al., 2005, 2009; Simberloff, 2009).

A better understanding of the invasion process and the ways by which invasive species are spreading in new environments is essential for improve management effectiveness and control plans in order to reduce and/or prevent future invasions (Hewitt, Gollasch, & Minchin, 2009; Sakai et al., 2001; Williams et al., 2013). Here we use a set of microsatellite markers recently developed for Tubastraea spp. (Capel et al., 2017; Chapter 4) to (1) investigate genetic diversity and clonality on Brazilian invaded sites and vectors, (2) provide insights on the role of vectors at spreading these invasive corals in the Southwestern Atlantic and (3) evaluate the population structure on invaded sites and the possibility for the occurrence of multiple introductions.

Methods

Sampling

A total of 306 and 173 colonies of T. coccinea and T. sp. cf. T. diaphana were sampled by SCUBA diving from 15 and nine sites, respectively, between 2012 and 2017 (Figure 1, Supplemental File 1). Samples of T. coccinea and T. sp. cf. T. diaphana were taken from eight and six invaded sites along the Southwestern Atlantic, covering the entire range of distribution of each species in the Brazilian coast, and from six and three possible vectors, respectively. The vectors include two monobouys (IMODCO-IV, SBM-V), two oil platforms (P14 and P27) and one drill ship (FPSO Marlim Sul). Additionally, a native population of T. coccinea (Taiwan) was sampled for comparison (Figure 1). On each site, 11-27 colonies of T. coccinea and 6-24 of T. sp. cf. T. diaphana were sampled and preserved in 96% ethanol or CHAOS buffer (Fukami et al., 2004) prior to extraction.

DNA Extraction and microsatellite amplification

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Total DNA was extracted using the Phenol:Chloroform method described by Fukami et al. (2004). Eight and ten microsatellite markers developed by Capel et al. (2017) (Chapter 4) were amplified by Polymerase Chain Reactions (PCRs) for all individuals of T. coccinea and T. sp. cf. T. diaphana, respectively. PCRs were performed in 10 µl reactions including 0.2 µM of forward primer with M13 tail at their 5′ end (TGT AAA ACG ACG GCC AGT), 0.4 µM of labeled primer (M13 with VIC, NED, PET, or 6-FAM fluorescent dyes) (Schuelke, 2000), 0.8 µM of reverse primer, 1U GoTaq (Promega), 1X PCR Buffer (Promega), 0.20 mM dNTPs

(Invitrogen), between 1.5 and 2.5 mM MgCl2 (following Capel et al. 2017), 10 µg BSA (Invitrogen), and 5-10 ng of DNA. Cycling conditions were: 95°C for 3 min followed by 5 cycles at 95°C, 30 s; 52–62°C, 30 s; 72°C, 45 s; and 30 cycles at 92°C, 30 s; 52–62°C, 30 s; 72°C, 55 s; with a final extension at 72°C for 30 min (Toonen, 1997). Final concentration of

MgCl2 and annealing temperature followed Capel et al. (2017). Amplification was verified in 2% agarose gel. PCR products were pooled with GS600-LIZ size standard (Applied Biosystems) and genotyped in the ABI 3500 genetic Analyzer (Applied Biosystems). Genotypes were determined using the program Geneious 7.1.9 (Kearse et al., 2012).

Clonal analyses

The package ‘GenClone’ on R 3.2.3 (R Development Core Team 2015) was used to assess the clonal structure of each species on all sites. A total of eight samples from T. coccinea (3%) and six samples from T. sp. cf. T. diaphana (4%) failed to amplify for more than one locus and were excluded from the analyses. Of the remaining samples (298 T. coccinea and 166 T. sp. cf. T. diaphana), individuals with identical alleles at all loci (ramets) were assigned to the same multilocus genotype (MLG, or genets). To check if individuals with the same MLG are truly clones, the probability of finding identical MLGs resulting from distinct sexual reproductive events (Psex) was calculated for each population (Arnaud-Haond,

Duarte, Alberto, & Serrão, 2007). When Psex >0.001, samples were considered product of distinct sexual reproduction events (not truly clones) and included in analyses of genetic diversity. Two indexes were used to describe the clonal diversity on each population, the clonal richness (R), taking into account the number of individuals sampled (R = (MLG – 1)/(N – 1), ranging from 0 to 1, when all samples analyzed correspond to a different MLG); and the genotypic evenness (V), calculated by the Simpson’s complement evenness index to

161 evaluate equitability in the distribution of the MLG (ranging from 0 to 1, when genets each have the same number of ramets) (Arnaud-Haond et al., 2007; Hurlbert, 1971). For genetic diversity analyses, only unique MLGs per population were considered.

Genetic diversity

FSTAT program (Goudet, 1995) was used to test linkage disequilibrium among all pair of loci. Subsequent analyses were done removing loci in linkage disequilibrium with more than one other locus. The software INEst was used to evaluate the occurrence of null alleles using the individual inbreeding model (IIM) and taking into account intrapopulation inbreeding (Chybicki & Burczyk, 2009). The presence of null alleles can bias several parameters usually measured in population analyses such as the inbreeding coefficient, the observed heterozigosity and fixation indexes (Chybicki & Burczyk, 2009). To assess each population’s genetic diversity, the number of alleles (A), private alleles (Ap), allelic richness (Ar), observed (Ho) and expected heterozygosities (He) and were calculated using the package ‘diveRsity’ in R 3.2.3 (R Development Core Team 2015). The inbreeding coefficient

(FIS) and deviations from Hardy-Weinberg equilibrium were calculated with the software FSTAT (Goudet, 1995).

Populations structure

To explore the topology of phylogeographic relationships among sampling sites, a neighbor-joining (NJ) tree based on Cavalli-Sforza’s and Edwards chord distance, suitable for microsatellite data (Takezaki & Nei, 1996), was constructed using the software Populations 1.2.32 (Langella 1999) and the package ‘ape’ in R 3.2.3 (R Development Core Team 2015).

To estimate the number of genetic clusters in the dataset, clones within each population were removed from the data set, remaining 84 and 30 individuals for T. coccinea and T. sp. cf. T. diaphana, respectively. A Bayesian analysis was performed in the new data set using the software STRUCTURE v. 2.3.4 (Pritchard, Stephens, & Donnelly, 2000) with the admixture ancestry model, correlated allele frequency and no sampling locations as prior. The analysis was performed with an initial burn-in of 500,000 cycles followed by 500,000 additional

162 cycles and the number of clusters (K) tested varied from 1 to 14 for T. coccinea and 1 to 9 for T. sp. cf. T. diaphana with 15 iterations for each K-value. The most likely K-value was estimated by estimating the “log probability of data” for each value of K (mean LnP(K)) and ΔK criterion (Pritchard et al., 2000) using STRUCTURE HARVESTER (Earl & vonHoldt, 2012).

Table 1. Summary of statistics per samples site for the species Tubastraea coccinea (N = 298) and T. sp. cf. T. diaphana (N = 166). * indicates significant deviations from Hardy-Weinberg equilibrium.

Status Site N MLG Psex A Ar Ae Ho He FIS Tubastraea coccinea Invaded Todos os Santos Bay (TSB) 25 10 0 22 2.2 3 0.58 0.55 -0.01 Âncora Island (AI) 21 8 0 20 2.2 2 0.50 0.54 0.13 Ilha Grande Bay (IGB) 24 3 1 13 1.8 0 0.50 0.39 -0.16 Búzios Island (BI) 12 2 1 14 2.2 0 0.54 0.42 -0.15 Alcatrazes (Alc) 21 6 0 20 2.4 1 0.57 0.52 0.02 Laje de Santos (LS) 24 1 2 11 1.8 0 0.83 0.42 -1 Queimada Grande (QG) 17 2 1 11 1.8 0 0.83 0.42 -1 Arvoredo Island (ArI) 24 2 2 11 1.8 0 0.83 0.42 -1 Vectors IMODCO-IV 39 21 0 41 2.8 8 0.54 0.66 0.21* SBM-V 25 16 0 23 2.2 0 0.41 0.48 0.18 P14 11 3 1 11 1.7 0 0.42 0.32 -0.18 P27 14 8 0 23 2.5 0 0.54 0.57 0.12 FPSO Marlim Sul 21 15 0 27 2.3 3 0.42 0.61 0.34* Native Taiwan 20 11 0 22 2.2 4 0.39 0.49 0.25 Tubastraea sp. cf. T. diaphana Invaded Petroleiro do Acaraú (PA) 22 4 2 14 1.8 11 0.67 0.35 -0.91 Todos os Santos Bay (TSB) 24 6 2 17 1.9 0 0.53 0.35 -0.49 Âncora Island (AI) 22 5 0 19 2.0 4 0.50 0.42 -0.07 Ilha Grande Bay (IGB) 24 5 3 14 1.7 0 0.58 0.32 -0.81 Búzios Island (BI) 24 1 2 13 1.6 0 0.62 0.31 -1 Alcatrazes (Alc) 19 7 2 16 1.9 2 0.62 0.36 -0.69 Vectors IMODCO-IV 14 5 1 18 1.9 2 0.65 0.41 -0.51 SBM-V 6 5 0 20 2.2 5 0.46 0.45 0.09 P14 11 6 0 20 2.1 4 0.57 0.41 -0.30 N = number of sampled individuals, MLG = number of unique multilocus genotypes per site, Psex = number of individuals with Psex ≥ 0.01, A = number of alleles, Ar = allelic richness, Ae = number of exclusive alleles, Ho = observed heterozygosity, He = expected heterozygosity.

Results

Clonality

Analyses revealed a high proportion of clones for T. coccinea and T. sp. cf. T. diaphana with only 84 (N=298) and 30 (N=166) MLGs within all sampled sites. It is 163 necessary to highlight that missing values are considered as a different genotype, which may lead to a slight overestimation of MLG. A total of 24 and 10 individuals of T. coccinea and T. sp. cf. T. diaphana, respectively, had missing data at one locus. For T. coccinea, invaded sites had in general lower numbers of MLGs when compared to vectors and the native population, with five sites holding three or less MLGs (Table 1, Figure 1). The same was not observed for T. sp. cf. T. diaphana, for which the highest number of MLGs observed was on an invaded site (Alcatrazes: MLG = 7, Table 1, Figure 2). The existence of two MLGs for T. coccinea at the invaded sites Queimada Grande and Santa Catarina is probably a consequence of missing data in one locus and these two populations are likely dominated by only one MLG. For T. coccinea, clonal richness was lower on invaded sites, ranging from 0 to 0.4, which contrast to vectors and native population that ranged from 0.5 to 0.7 (Figure 3A), The genotypic evenness did not show a clear pattern, with five invaded sites, one vector and the native site being dominated by one genotype. However, the remaining sites (two invaded sites and three vectors) had more equitable distribution of ramets among the observed MLGs (genotypic evenness (V) close to 1, Figure 3A). Interestingly, the same pattern for MLGs and clonal richness was not as clear for T. sp. cf. T. diaphana. Invaded sites and vectors had similar numbers of MLGs (except for Búzios Island, with one MLG) and clonal richness was slightly lower on vectors, reaching 0.8 in the vector SBM-V (Figure 3B). The genotypic evenness did not change between invaded sites and vectors for this species (Figure 3B).

164

40º B

,' r Taiwan

-30º

e Pl4~ ~õ'''~' P27 0

Alcatrazes IMODCO-íV -30º

~redoo lsland Queimada Grande .....

Figure 1. Sampling sites for T. coccinea (A) along the invasive range in Southwestern Atlantic, (B) at a native population in Taiwan and (C) at five vectors located on the Brazilian coast. Pie diagrams show the number of multilocus genotypes (MLGs) (inner circle) and the allele frequency of the locus Tco 29 (with a total of 13 alleles, outer circle) per population. Colors indicate MLGs or alleles that are shared among two or more sites and gray scale indicate MLGs and alleles that are exclusive for the correspondent site (not observed on any other analyzed site). The locus Tco 29 is the second most diverse for T. coccinea and was chosen to exemplify allele sharing among invaded sites, vectors and Taiwan.

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-60° -30º

Petroleiro do Acaraú Búzios Island B '" fj

IMODCO-IV - -30° (1) Âncora lsJ,od SBM-V e Ilha Grande Bay

Figure 2. Sampling sites for T. sp. cf. T. diaphana (A) along the invasive range in Southwestern Atlantic and (B) at three vectors located on the Brazilian coast. Pie diagrams show the number of multilocus genotypes (MLGs) (inner circle) and the allele frequency of the locus Tco 34 (with a total of six alleles, outer circle) per population. Colors indicate MLGs or alleles that are shared among two or more sites and gray scale indicate MLGs and alleles that are exclusive for the correspondent site (not observed on any other analyzed site). The locus Tco 34 is the second most diverse for T. sp. cf. T. diaphana and was chosen to exemplify allele sharing among invaded sites and vectors.

A B 0.8 0.8 • • • 0.7 • 0.7 • 0.6 • 0.6 • • • • 0.5 • • • 0.5 • • • • • 0.4 • 0.4 • • • • • 0.3 0.3 • • 0.2 • 0.2 • • • • 0 .1 • • 0. 1 • • • • • o • •

Figure 3. Clonal indicess for (A) T. coccinea and (B) T. sp. cf. T. diaphana. Black dots indicate the clonal richness (R), ranging from 0 to 1, when all samples analyzed correspond to a different MLG and gray dots indicate the genotypic evenness (V), ranging from 0 to 1, when genets each have the same number of ramets. Purple and green areas indicate invaded sites and vectors, respectively.

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Of the observed MLGs, ten (T. coccinea) and five (T. sp. cf. T. diaphana) are shared with one or more sampled sites (Figures 1 and 2). T. coccinea had five MLGs shared among invaded sites and vectors, while the remaining five are shared exclusively among vectors. Vectors also showed a higher number of exclusive MLGs, not shared with any other sampled site (Table 1; Figure 1). Neither the invaded sites nor the vectors shared any MLGs with the native population from Taiwan. One MLG was found on three vectors and all invaded sites (except for Alcatrazes), indicating the occurrence of clones separated by over 1,900 km along the Brazilian coast (Figure 1). Although for T. coccinea no MLG was shared between Alcatrazes and other invaded sites, the predominant MLG found at this site differed by only one allele to the predominant MLG found at other invaded sites (green MLG showed at the inner circle in Figure 1). Similarly, for T. sp. cf. T. diaphana, the predominant MLG found at Alcatrazes also differed by only one allele to the predominant MLG found at other invaded sites (red MLG showed at the inner circle in Figure 2). Of the five MLGs observed for T. sp. cf. T. diaphana, four were shared between at least one vector and one invaded site and one was found exclusively on invaded sites. The northernmost sampled site, Petroleiro do Acaraú, did not shred MLGs with any other invaded site or vectors.

For analyses of genetic diversity, all clones found within population were removed and only one representative of each MLG was included, additionally to those individuals with

Psex value higher than 0.001 (Table 1). Within all data set, a total of eight and eight individuals of T. coccinea and 12 T. sp. cf. T. diaphana had Psex value higher than 0.001, being considered product of distinct sexual reproduction events.

Genetic diversity and population structure

Two loci from T. coccinea (Tco1 and 30) and two from T. sp. cf. T. diaphana (Tco4 and 37) showed evidence of linkage disequilibrium with at least two other loci and were excluded from genetic diversity analyses. There was no evidence of null alleles for T. coccinea, while for T. sp. cf. T. diaphana three loci showed evidence of null alleles for one population each (Supplemental File 2).

For T. coccinea, the number of alleles and allelic richness ranged from 11 to 41 and 1.7 to 2.8, respectively. For this species, exclusive alleles were found at three invaded sites

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(Todos os Santos Bay, Âncora Island and Alcatrazes), two vectors (SBM-V Araça and FPSO Marlim Sul) and in the native population (Taiwan) (Table 1). The frequency distribution of alleles for one locus (Tco 29, with 13 alleles – Figure 1, outer circle) shows that invaded sites, vector and the native population share four alleles and that the number of exclusive alleles is higher on vectors and the native population. All but one analyzed locus (Tco 5) has at least one allele shared among all sites and Taiwan shares at least one allele per locus with one or more site (invaded or vector) (Supplemental File 3). Observed (Ho) and expected (He) heterozygosity ranged from 0.39 to 0.83 and 0.32 to 0.66, respectively, with the smallest values found in one vector (SBM-V) and the native population (Table 1). Only two vectors (IMODCO-IV and FPSO Marlim Sul) had significant deficits of heterozygosity (Table 1). Although not significant, many sites (e.g., LS, QG and ArI; Table 1) had Ho values twice higher than He, indicating heterozygote excesses. These values need to be interpreted with caution, since all sites that displayed Ho excess had very low number of individuals. The inbreeding coefficient (Fis), was negative for most invaded sites (except for AI and Alc) and P14, indicating an excess of heterozygotes (Table 1). The remaining vectors and the native population had inbreeding coefficients ranging from 0.12 (i.e. P27) to 0.34 (i.e. FPSO Marlim Sul).

For T. sp. cf. T. diaphana, the number of alleles and allelic richness were similar among sampled sites, ranging from 13 to 20 and 1.6 to 2.2, respectively. Exclusive alleles were found at all but three invaded sites (TSB, IGB and BI), with the highest number observed at the northernmost site, Petroleiro do Acaraú (Ae = 11; Table 1). Figure 2 shows the frequency distribution of alleles for the locus Tco 34 (outer circle), with all sites but Petroleiro do Acaraú sharing at least one allele. Five out of eight loci had shared alleles among Petroleiro do Acaraú and at least one other site and only three loci has shared alleles among all samples sites (Supplemental File 3). Observed heterozygosity (Ho) (ranging from 0.46 to 0.67) was higher than the expected (He) (ranging from 0.31 to 0.45) in all sites (Table 1), with no significant deficits of heterozygosity. The inbreeding coefficient (Fis) was negative for all sites (but SBM-V), indicating an excess of heterozygotes (Table 1).

For T. coccinea, the NJ recovered three groups: (1) five southern invaded sites, (2) three northern invaded sites and one vector (P14), (3) the remaining four vectors and (4) the native population, separated from all groups (Figure 4A). Although with low support values, the observed topology corroborates the overall observed MLGs distribution (Figure 1).

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Bayesian clustering analyses did not recover any clear genetic cluster for any invaded site, vector or the native population of T. coccinea (Figure 5). The two methodologies used to estimate K gave similar results of five, eight or nine possible genetic clusters (Figure 5). All sites with more than one individual had evidence of more than two genetic clusters, including the native population of Taiwan.

For T. sp. cf. T. diaphana, all invaded sites and three vectors were recovered at the same cluster by NJ analysis, while Petroleiro do Acaraú was the most divergent site (Figure 4B), corroborating to the low number of shared alleles between this site and the remaining invaded sites and vectors (Figure 2). Bayesian analysis recovered three genetic clusters (Figure 6), suggesting that the Southwestern Atlantic was colonized by more than one native population, as observed for T. coccinea. Except for Petroleiro do Acaraú, the observed genetic clusters were not a function of population structure between localities (Figure 6). No substructure was observed when analyzing higher K (data not show). Evidence of interbreeding between two clusters could be observed at Todos os Santos Bay, Alcatrazes and IMODCO-IV (Figure 6).

A B Âncora lsland Todos os Santos Oay P14 11 23 Alcoum.cs Vectors Ak:01rozes 21 48 ------SBM·~' ' , IMODCO-IV 4 56 83 ' ~h~ Grau

Taiwan

AJlCOt'.i ls1:and ~ Todo$ os Santos 0.1y., , Petroleiro do Acarnú Pl4 Northem sites

Figure 4. Neighbor-joining (NJ) tree based on Cavalli-Sforza’s and Edwards chord distance for (A) T. coccinea and (B) T. sp. cf. T. diaphana.

169

A Della K - rll

o -1400 2 4 6 8 LO 12 14 o 2 4 6 8 10 12 14 e K K K c 5

Figure 5. Bayesian clustering analyses for Tubastraea coccinea. (A) shows the most likely K-value estimated by delta K and (B) estimated by the mean of estimated “log probability of data” for each value of K (both K = 3); and (C) shows the genetic clusters for K =5, K=8 and K = 9, where each individual is represented by a vertical bar with different colors indicating the relative proportion of each genetic cluster.

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A lkhn K = mcan (IL"(K )l)/sd(L(K)) B L(K}(mcan +- SD) 100 350 . • . s . . .g + + 80 ~ o 400 . 60 l "'" 5 8 40 ij 450 ~o ~ 20 !) ::;: o 500 .

2 3 4 ; 6 7 8 2 4 6 R e K K K = 3

t, QJ~ ;;, .:. .:. ~ -~ (/j ~ !V .... ~ e:,~ ~ ,,,-. ~ & ~ ~ o '<" ;; -'" ,::iº ~ ~ çf ~ e, ;;; (5~ "' '<""" ,f .,f & ''f' ~ :::,~ " ,;,º f.l& ~" Figure 6. Bayesian clustering analyses for Tubastraea sp. cf. T. diaphana. (A) Shows the most likely K-value estimated by delta K and (B) estimated by the mean of estimated “log probability of data” for each value of K (both K = 3); and (C) shows the genetic cluster for K =3, where each individual is represented by a vertical bar with different colors indicating the relative proportion of each genetic cluster.

Discussion

Forty years after its first record, the genus Tubastraea have spread over more than 3,500 km along the Brazilian coast with increasing densities (Mantelatto et al., 2011; Silva, Paula, Fleury, & Creed, 2014). Here we show evidence that T. coccinea and T. sp. cf. T. diaphana were likely introduced in the Southwestern Atlantic by more than one event and suggest that the transport of oil platforms and buoys have contributed to the spread of these species along the Brazilian coast.

Clonality

Supporting previous results (see Capel et al. 2017), for both T. coccinea and T. sp. cf. T. diaphana, the Southwestern Atlantic invaded sites have a higher proportion of clones, with sites dominated by one genotype (Figures 1 and 2). Thus, their ability to reproduce asexually is probably one of the main factors responsible for their success as invaders. Such ability enables high population densities from a small starting number of individuals. Interestingly, for T. coccinea, although Taiwan had less clones when compared to the majority of invaded 171 sites, half of the population was composed by clones of the same genet. This demonstrates that asexual reproduction is also important in its native range of distribution. As discussed by previous studies, asexual reproduction in Tubastraea occurs through the asexual production of larvae (Ayre & Resing, 1986; Capel et al., 2017; also see Chapter 6), a strategy broadly used by anthozoans (Black & Johnson, 1979; Brazeau & Lasker, 1989; Ottaway & Kirby, 1975), but to date only observed in three scleractinian species, T. coccinea, T. diaphana and Pocillopora damicornis (D. J. Ayre & Resing, 1986; Combosch & Vollmer, 2013; Stoddart, 1983).

Along the Southwestern Atlantic, both T. coccinea and T. sp. cf. T. diaphana had one dominant genotype (shared by 36% and 46% of all analyzed individuals, respectively), showing an over-representation of few genotypes. This may be a consequence of the invasive process, where only one or a few genotypes better fitted to the local environment were able to establish and disperse (Caron, Ede, & Sunnucks, 2014; Gorospe & Karl, 2013). A similar pattern was previously observed for Pocillopora damicornis in Hawaii (Gorospe & Karl, 2013), Reunion Islands (Gélin et al., 2017) and Acropora palmata in the French Antilles (Japaud, Fauvelot, & Bouchon, 2014). Nevertheless, those studies analyzed samples collected on small geographic scales (less than 20 km), while the dominant genotype of T. coccinea and T. sp. cf. T. diaphana was spread over invaded sites distant around ~1,500 and ~2,000 km, respectively (Figures 1 and 2). Three non-exclusive hypotheses could explain such broad distribution of clones: (1) long-distance dispersal of asexual larvae; (2) different events of introduction from the same native population; or (3) secondary introduction from one invaded site to another.

Although possible for close localities, larval dispersal itself does not explain the broad distribution of clones at sites separated by more than 1,500 km. Tubastraea have a gregarious settlement behavior and most larvae settle within 1 to 3 days (Glynn et al., 2008), even though experiments in aquaria have shown that larvae are competent for ∼18 days (Alline Figueira de Paula et al., 2014; Glynn et al., 2008) and one study mentions T. coccinea larvae being competent after 100 days (Richmond, pers. comm. on Fenner, 2001). The gregarious settlement with high local clonality observed for Tubastraea follows the “strawberry-coral” model (Willians 1975), when organisms use sexual reproduction to disperse genotypically diverse individuals, while asexual reproduction helps to spread locally adapted genotypes. Interestingly, an opposite hypothesis of dispersal capability was proposed for P. damicornis,

172 where the asexual larvae would be responsible for long distance dispersal (Schmidt-Roach, Miller, Woolsey, Gerlach, & Baird, 2012). Indeed, studies showing evidence of local recruitment from sexual larvae with low clonality corroborate the hypothesis that asexual produced larvae of P. damicornis may travel further (Ayre & Miller, 2004; Sherman, Ayre, & Miller, 2006). Future studies comparing size and duration of sexual and asexual larvae of Tubastraea spp. in situ are recommended to test if they follow this pattern.

Dispersion through vectors

The second and third hypotheses are the most likely explanations for the observed clonal distribution. The first record of Tubastraea spp. in the Southwestern Atlantic was on offshore oil platforms from Campos Basin, north of Rio de Janeiro State, in the late 1980s. The transport through vectors, such as oil platforms and buoys are the most probable means of introduction of these species along the Brazilian rocky shores (Creed et al., 2016). Indeed, the distributional range of Tubastraea spp. in the Southwestern Atlantic appears to be directly associated to sites with intense ship traffic and waterway terminals. de Paula & Creed (2005) analyzed the distribution and expansion of Tubastraea spp. at Ilha Grande Bay and found that, at this locality, the Petrobras oil terminal and Verolme shipyard are likely points of introduction of these invasive species. A number of vessels used by oil companies in Brazil (e.g. oil platforms and buoys) were built by foreign companies and may have been colonized with the local fauna that were further transported as biofouling. Ferreira, Gonçalves, & Coutinho (2006) found 22 non-indigenous species when analyzing drill-ships, platforms and cargo ships in Arraial do Cabo, including T. coccinea. Furthermore, previous studies had demonstrated that artificial substrates indeed facilitate invasion (Dafforn, Johnston, & Glasby, 2009; Glasby, Connell, Holloway, & Hewitt, 2007; Ruiz, Freestone, Fofonoff, & Simkanin, 2009). Tubastraea spp. seems to be opportunistic species and have been reported on artificial substrates at both invaded (Castro & Pires, 2001; Creed et al., 2016; Fenner & Banks, 2004; Ferreira et al., 2006) and native localities (Ho, Hsu, & Chen, 2016; Sunken City, Hawaii, personal observation). To confirm the hypothesis of different events of introduction from the same native population it is essential to know the origin of the vessels and when they first arrived in the Southwestern Atlantic. However, this information is not easily traceable and could not be verified up to now. Of the five vectors included in the study we were able to recover information of two: P27 came from Singapore to Arraial do Cabo, Brazil, in 1998 and 173

P14 was built in France in 1983, with no information of where and when it has first arrived in Brazilian waters. Singapore is within the natural distribution of Tubastraea spp. (Cairns, 2001) and it is highly possible that the platform P27 was infested before its arrival in the Atlantic. Evidence supporting this assumption is the large size of colonies sampled at the platform and the first records of Tubastraea spp. in Arraial do Cabo only one-year after the arrival of P27 in this region (Creed et al., 2016). Regarding the platform P14, it has probably arrived free of Tubastraea, once there are no records of this genus in Europe. However, it was probably contaminated in Brazilian or Caribbean waters and then became a vector. Although we cannot confirm the origin of all vectors, the i) occurrence of unique MLGs, ii) exclusive alleles not found within invaded sites (Figures 1 and 2), and iii) the higher number of alleles and clonal richness observed at all analyzed vectors (except for P14) compared to invaded sites, suggest that those vectors were already contaminated with Tubastraea spp. prior to their arrival in the Southwestern Atlantic. Determining the exact origin of the Atlantic populations is out of the scope of this work, nevertheless results presented here give us one more clue that the Indo-Pacific is the likely source region for the Atlantic populations.

The occurrence of secondary introduction from one invaded site to another is supported by information of vessels being transported along the Brazilian coast (Table 2) (Creed et al., 2016) and the occurrence of clones on both invaded sites and vectors. The platforms P14 and P27 and the monobuoy IMODCO-IV were transported at least once along the Brazilian coast after being contaminated (Table 2) (Creed et al., 2016). Although Tubastraea spp. have been previously recorded at all cited sites prior to the arrival of the analyzed vectors, the transport of contaminated vectors further support the spread of genotypes that may have not been at an invaded location. Tubastraea spp. were recorded on at least 23 vessels related to oil production (e.g. platforms, drill ships, monobuoys) (Creed et al., 2016) of which we have analyzed samples from only five. It is highly possible that vectors not included in this study were the primary responsible for introductions along several localities along the Southwestern Atlantic.

Table 2. Locations where the five analyzed vectors where recorded in the Southwestern Atlantic. Tc = T. coccinea; Tsp = T. sp. cf. T. diaphana. Vector Location (coordinates) Year Species Source P14 Caravelas field, Itajaí 2000 Tc Identified by J.C. Creed (26º46’2’’S, 46º47’2.15’’W) from photographic register of Barreiros et al. (2000). 174

Angra dos Reis, Ilha Grande Bay 2007 Tc In port, J.C. Creed (pers. (23º00’53’’S, 44º18’59’’W) obs.) Canteiro de São Roque, Todos os 2014 Tc / Tsp In port, J.C. Creed (pers. Santos Bay (12º51’16’’S, obs.) 38º50’17’’W) P27 Voador field, Campos Basin 2013 Tc Identified by J.C. Creed (22º22’S, 40º24’W) from photographic register communicated by Ricardo Guedes dos Santos (pers. comm.). Canteiro de São Roque, Todos os 2014 Tc In port, J.C. Creed (pers. Santos Bay (12º51’16’’S, obs.) 38º50’17’’W) IMODCO Arraial do Cabo (22º58’21’’S, 2007 Tc / Tsp Mizrahi (2008) IV 42º0’49’’W) São Sebastião (23º48’48’’S, 2014 Tc / Tsp In port, J.C. Creed (pers. 45º24’11’’W) obs.) SBM-5 São Sebastião (23º48’48’’S, 2012 Tc / Tsp In port, J.C. Creed (pers. Araça 45º24’11’’W) obs.) FPSO Bacia de Campos (22º32’38’’S, 2016 Tc Identified by C. Zilberberg Marlim Sul 40º01’15’’W) from samples provided by the company SBM-Off- shore

Genetic diversity

Supporting preliminary analyses showed by Capel et al. (2017), we observed an excess of heterozygotes for T. coccinea, which was higher at most invaded sites when compared to vectors and is conflicting with the expected decrease in genetic diversity following an invasive process (Geller et al., 2008; Johnson & Woollacott, 2015; Joe Roman & Darling, 2007; Wrange et al., 2016). Nonetheless, counter examples have been observed for marine organisms, where invasive populations harbor higher genetic diversity than native populations, showing that the genetic structure of invasive populations is an intricate function of several factors, such as genetic drift, the lag time before population expansion, propagule pressure and the occurrence of multiple invasions (Gaither, Toonen, & Bowen, 2012; Kolbe et al., 2004; Lockwood et al., 2005, 2009; J. Roman, 2006). It also has been shown that populations with high growth rates usually experience a smaller decrease in heterozygosity after a bottleneck process and are less prompt to have genetic diversity reduced by genetic drift (Dlugosch & Parker, 2008; Nei, Maruyama, & Chakraborty, 1975). Gaither et al. (2012) found high genetic diversity and no change in haplotype frequency for invasive populations of the reef fish Lutjanus kasmira in Hawaii, which had also a rapid population growth. Furthermore, lineages where asexual reproduction predominates tend to have high levels of

175 heterozygosity and negative FIS as a consequence of an independent evolution of loci (e.g. “Meselson effect”) accumulating divergence within alleles (Balloux, Lehmann, & De Meeûs, 2003; Birky, 1996; Halkett, Simon, & Balloux, 2005; Judson & Normark, 1996). Tubastraea spp. reproduce mainly through asexual larvae (Ayre & Resing, 1986; Chapter 6), is able to reproduce after only three months of settlement (de Paula et al., 2014) and a single colony is able to release ~2,000 larvae in one reproductive event (Bruna Luz, personal comm.), evidencing their “premature” maturity and high propagule pressure, which can result in a fast range expansion. Thus, the observed genetic diversity for Tubastraea spp. in the Southwestern Atlantic may be explained by a combination of factors, such as reproductive strategy, high growth rate, high propagule pressure and through the occurrence of multiple invasions.

Population structure and multiple introductions

Our results corroborate the hypothesis of multiple introductions events (Capel et al., 2017), with at least five genetic clusters with no geographic pattern for T. coccinea. The lack of geographic pattern is likely a result from the transport of infested vectors previously discussed. However, STRUCTURE is not appropriate for organisms that reproduce mainly asexually (Halkett et al., 2005) and results should be interpreted with caution. Sammarco, Brazeau, McKoin, & Strychar (2017) found similar results when analyzing invasive populations of T. micranthus at two oil platforms in the Gulf of Mexico, with four distinct genetic clusters observed in one single platform, likely resulting from multiple introductions from distinct source populations. Besides increasing the propagule pressure, multiple introductions can lead to an increase in genetic diversity via isolate breaking effect by creating new genotypes, potentially benefiting invasive populations and enhancing their chance of survival (Bock et al., 2015; Dlugosch & Parker, 2008; Keller & Taylor, 2010; Kolbe et al., 2004; Rius & Darling, 2014; Verhoeven et al., 2011). Patterns of high genetic diversity of invaded populations associated with multiple introductions have been observed for marine organisms, such as the green crab Carcinus maenas in Canada (Roman, 2006), the nassariid gastropod Cyclope neritea, the caprellid Caprella scaura at the Iberian Peninsula (Cabezas, Xavier, Branco, Santos, & Guerra-García, 2014; Simon-Bouhet, Garcia-Meunier, & Viard, 2006) and others (Ashton et al., 2008; Facon, Pointier, Jarne, Sarda, & David, 2008). Interestingly, three groups were recovered by NJ analyses segregating vectors, northern 176

(including P14) and southern invaded sites. Despite the low support values, results agree with the observed genotypic distribution. Northern sites were likely invaded more than once and had higher genotypic diversity when comparing to the southern sites, which were invaded less than ten years ago, mostly by the same genotypes, suggesting single invasion events. Except for P14, vectors had higher genotypic diversities, with genotypes not found at invaded sites, and were grouped together.

For T. sp. cf. T. diaphana, both STRUCTURE and NJ results suggest that the northernmost invaded site (Petroleiro do Acaraú) was colonized by a different population, not present in any other invaded site or vector analyzed and most probably resulted from a single introduction event. This site is a shipwreck 1,500 km distant from Todos os Santos Bay, the closest invaded site on the coast, and the direction of sea surface currents are westward, which possibly prevents any gene flow between Petroleiro do Acaraú and the remaining invaded sites. Nevertheless, Mucuripe waterway terminal is ~200 km east of the shipwreck and it is possible that this invasion was also through biofouling in vessels sailing around this area. The remaining invaded and vectors are likely derived from two different native populations. The native distribution of T. sp. cf. T. diaphana is currently unknown until an extensive revision of the genus is undertook (Capel et al. in prep; Chapter 1), hindering any assumptions of the origin of the Southwestern Atlantic populations.

Understand the invasion process and identify the vectors responsible for the transport of invasive species is the primordial steps for improving management and control. Our results show that clonality and transport through vectors by more than one event are the main reasons for the fast spread and invasive success of Tubastraea spp. in the Southwestern Atlantic. High clonality capability is a common feature among successful invasive species, enabling invaders with low number of individuals/low genetic diversity to reach high densities and successfully dominate the invaded region (Caron et al., 2014; Roman & Darling, 2007). We also suggest that the Indo-Pacific is a possible source of the Southwestern Atlantic populations, although a more extensive sampling of native populations and other invaded sites, such as the Caribbean, are recommended to track the exact origin of the Atlantic invaders. We observed that vectors still hide most of the genetic and genotypic diversity and new invasions can worsen the situation by enhancing the diversity and, consequently, increase the resilience of the populations along the coast. Local strategies have been taken to control the expansion of Tubastraea spp. in the Southwestern Atlantic coast (Creed, Junqueira, Fleury, Mantelatto, &

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Oigman-Pszczol, 2017); however, controlling the vectors responsible for introduction and dispersion is a key procedure to turn management more effective and to prevent further invasions/population expansions (Reaser et al., 2007; Ruiz and Carlton 2003).

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Supplemental File 1 Map of the sampling sites of T. coccinea (red), Tubastraea sp. cf. T. diaphana (green) or both (blue) at (A) invaded sites along the Brazilian coast and (B) vectors along the Brazilian coast (i.e. the location of each vector when samples were taken).

A B -30'

Todos os Santos Bay P27 • P14

.J()'

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Supplemental File 2 Tubastraea coccinea and T. sp. cf. T. diaphana null allele frequencies for invaded, vectors and native sites. Only three loci for three different sites showed frequency of null alleles ≥ 14%. One asterisk (*) indicates locus with linkage disequilibrium and were excluded from analyses. Two asterisks (**) indicate the locus that did not amplify for T. coccinea.

Status Site Mean Null Allele Frequency Tco1 Tco4 Tco5 Tco8 Tco9 Tco29 Tco30 Tco34 Tco32 Tubastraea coccinea Invaded Todos os Santos Bay * 0,01 0,04 0,01 0,13 0,04 * 0,01 ** Âncora Island 0,01 0,01 0,01 0,02 0,01 0,01 Ilha Grande Bay 0,02 0,01 0,02 0,02 0,01 0,01 Búzios Island 0,03 0,03 0,03 - 0,03 0,03 Alcatrazes 0,01 0,01 0,01 - 0,01 0,01 Laje de Santos 0,03 0,03 0,03 - 0,03 0,03 Queimada Grande 0,03 0,03 0,03 - 0,03 0,03 Santa Catarina 0,02 0,02 0,02 - 0,02 0,02 Vector IMODCO-IV 0,00 0,00 0,00 0,00 0,00 0,00 SBM-V Araça 0,00 0,01 0,00 0,02 0,01 0,00 P14 0,02 0,02 0,02 0,02 - - P27 0,02 0,02 0,01 0,01 0,01 0,01 FPSO Marlim Sul 0,02 0,02 0,01 0,01 0,01 0,01 Native Taiwan 0,01 0,01 0,01 0,07 0,01 - Tubastraea tagusensis Invaded Petroleiro do Acaraú 0,03 * 0,03 0,03 - - 0,15 0,02 0,14 Todos os Santos Bay 0,01 0,01 0,01 0,01 0,01 0,01 0,01 0,01 Âncora Island - 0,01 0,01 0,01 0,01 0,01 0,01 0,01 Ilha Grande Bay - 0,01 0,01 - - 0,01 0,01 0,01 Búzios Island - 0,04 0,03 - - 0,03 0,03 0,03 Alcatrazes - 0,02 0,02 - 0,01 0,02 0,21 0,01 Vector IMODCO-IV 0,01 0,01 0,01 - 0,01 0,01 0,01 0,01 SBM-V Araça 0,02 0,02 0,02 - 0,02 0,02 0,08 0,02 P14 0,02 0,01 0,02 - 0,01 0,02 0,08 0,01

186

Supplemental File 3 Allelic information showing number of alleles per loci (1) within all sampled sites, (2) shared within all sampled sites and (C) shared between at least one site (invaded or vector) and the most divergent site, Taiwan for T. coccinea and Patroleiro do Acarú (PA) for T. sp. cf. T. diaphana. One asterisk (*) indicates locus with linkage disequilibrium and were excluded from analyses. Two asterisks (**) indicate the locus that did not amplify for T. coccinea.

Tco1 Tco 4 Tco 5 Tco 8 Tco 9 Tco 29 Tco30 Tco 34 Tco32 Tubastraea coccinea (1) Total * 4 22 3 7 13 * 7 ** (2) Shared with all 2 0 2 1 1 1 (3) Shared with Taiwan 4 6 2 2 3 1 T. sp. cf. T. diaphana (1) Total 5 * 7 3 3 4 5 6 3 (2) Shared with all 0 0 1 0 0 1 0 1 (3) Shared with PA 1 0 2 1 0 2 0 2

187

Capítulo VI

Aliens do not make sex

Kátia Cristina Cruz Capel, Marcelo Kitahara & Carla Zilberberg

Revista alvo: Coral Reefs.

188

Aliens do not make sex

KCC Capel1*, MV Kitahara2,3, C Zilberberg1

1 Departamento de Zoologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil 2 Departamento de Ciências do Mar, Universidade Federal de São Paulo, Santos, Brazil 3 Centro de Biologia Marinha, Universidade de São Paulo, São Sebastião, Brazil

* Corresponding author Kátia Capel e-mail: [email protected] Laboratório de Biodiversidade de Cnidária. Departamento de Zoologia Universidade Federal do Rio de Janeiro.

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Abstract

Asexual reproduction allows the spread of well adapted genotypes and can strong influence genetic structure and colonization patterns, mainly for invasive species. However, on the long run, predominance of asexual reproduction has several disadvantages, and a mixed strategy with both sexual and asexual reproduction seems to overcome these negative effects. The genus Tubastraea comprises two species that had invaded and are spreading fast throughout the Southwestern Atlantic and the occurrence of asexual reproduction may be favoring their invasion success. Although production of asexual larvae has been previously reported for Tubastraea, there is a lack of information regarding the occurrence of sexual reproduction and the contribution of both strategies. Using a set of microsatellites loci we show that in the Southwestern Atlantic both T. coccinea and T. sp. cf. T. diaphana reproduce mainly through asexual production of larvae. A small percentage of the analyzed larvae (0,5% for T. coccinea and 2,2% for T. sp. cf. T. diaphana) differed by one or more alleles and were considered result of sexual reproduction. The observed high clonality and low genotypic distances suggest that larvae are formed through pseudogamy, which occasionally allows the incorporation of portions of sperm’s genome. Results presented here corroborate the predominance of asexual larvae production for both T. coccinea and T. sp. cf. T. diaphana throughout their invasive range, although the occurrence and proportion of sexual reproduction remains controversial. Nonetheless, the formation of asexual larvae is likely one of the main reasons for their invasion success of these species in the Southwestern Atlantic.

Key words: invasive species; Southwestern Atlantic; T. coccinea; T. sp. cf. T. diaphana

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Introduction

Asexual reproduction has been observed in several taxa including unicellular organisms, fungi, plants, rotifers, cladocerans, cnidarians, porifera, insects, among others (Stoddart 1983; Ayre and Resing 1986; Halkett et al. 2005; Zilberberg et al. 2006). In general, it can strongly influence species’ genetic structure and colonization patterns (McFadden et al. 1997; Brown and Eckert 2005; Halkett et al. 2005). Advantages of asexual reproduction include the spread of well adapted genotypes, avoidance of the costs of meiosis and the possibility of population expansion when mate partners are scarce (Shick et al. 1979; Holman 1987; Ayre and Willis 1988; Radtkey et al. 1995; Hurst and Peck 1996; Weider et al. 1999; Peck and Waxman 2000; Taylor and Hastings 2005). However, on the long run, the predominance of asexual reproduction can have several evolutionary disadvantages for the species, such as the accumulation of deleterious alleles due to Muller’s ratchet (Muller 1964; Felsenstein 1974) and a decrease in evolutionary potential achieved through recombination (Peck 1994). Nevertheless, asexual reproduction remains a commonly used strategy among animals, occurring in several ways, such as binary fission (Bunting and Wenrich 1929), regeneration (Carnevali 2006), fragmentation (Highsmith 1982), budding (Fautin 2002), gemmule formation (Manconi and Pronzato 2016), asexual larvae (Stoddart 1983; Ayre and Resing 1986) and parthenogenesis (Tucker 1958; Bogart et al. 2007).

Scleractinian corals have a diversified set of both sexual (i.e. broadcast spawning and broadcasting) and asexual (i.e. budding, fission, fragmentation and broadcasting asexual larvae) reproduction strategies (Highsmith 1982; Fautin 2002; Harrison 2011), frequently combined. Among these, the asexual formation of larvae is extremely rare, recorded to date for only three species (Pocillopora damicornis, Tubastraea coccinea and T. diaphana) (Stoddart 1983; Ayre and Resing 1986). Analyses on the genetic diversity of these species suggest that they also reproduce sexually (Ayre and Miller 2004, also see Chapter 5); however, studies using allozyme loci failed to find evidence of sexual reproduction in all three of them (Stoddart 1983; Ayre and Resing 1986). Nevertheless, studies with Tubastraea spp. used only two loci in a relatively conserved marker and, thus, the prevalence of asexual larvae still needed to be further examined. If in fact these species with facultative asexual reproduction prioritize asexual larvae production, the low frequency of sexual reproduction could confound the detection of sex (Schurko et al. 2009). Recent studies using hyper- variable microsatellite markers have demonstrated the occurrence of a combination of sexual

191 and asexual larvae for P. damicornis (Miller and Ayre 2004; Yeoh and Dai 2010; Combosch and Vollmer 2013), but the existence of a mixed reproduction strategy in Tubastraea remains unclear. Likewise, nothing is known about the process that regulates the formation of sexual and asexual larvae and even though parthenogenesis is the most likely explanation for the formation of asexual larvae, the exact biological process remains uncertain (Diah-Permata et al. 2000; Combosch and Vollmer 2013).

Mixed reproduction, combining sexual and asexual larvae, allow species to have the best of both strategies, acquiring genetic diversity through recombination and spreading local adapted genotypes through clone production (Hurst and Peck 1996; Peck and Waxman 2000). The genus Tubastraea comprises azooxanthellate species originally from the Indo-Pacific region (Cairns 2001) of which three have successfully invaded the Western Atlantic (T. coccinea, T. sp. cf. T. diaphana and T. micranthus) (Castro and Pires 2001; de Paula and Creed 2004; Sammarco et al. 2010) and are spreading fast throughout natural substrates (Mantelatto et al. 2011; Sampaio et al. 2012; Silva et al. 2014). For invasive species, the ability to reproduce asexually can be a key feature for success and, indeed, can be observed among successful invaders within plants (Brown and Eckert 2005) and animals (Ting and Geller 2000).

It has been shown that asexual reproduction predominates in the invasive range of T. coccinea and T. sp. cf. T. diaphana, and it may also be important on native populations (see Chapter 5). However, there is a lack of information regarding the occurrence of sexual reproduction within Tubastraea. Molecular analyses using hyper-variable markers, such as microsatellites, can provide strong evidence of clonal and sexual reproduction (Halkett et al. 2005; Schurko et al. 2009). Here we use a set of recently developed microsatellites loci to investigate and quantify the contribution of sexual and asexual larvae formation in colonies of the two species T. coccinea and T. sp. cf. T. diaphana within their invasive distributional range.

Material and methods

Sampling

192

A total of 14 colonies of each species, T. coccinea and T. sp. cf. diaphana, were sampled from Búzios Island (23º47′S, 45º08′W, 6 m depth) in March 2015 and six additional colonies of T. coccinea were sampled from São Sebastião channel (23º48′S, 45º24′W, 5 m of depth) in December 2016. Only five colonies of each species (the ones that released the highest number of larvae) were analyzed. For larvae collection, colonies were kept separated in open-water system containers with filtered seawater (Figure 1) and plankton-fed every other day. During 13 days the aquaria were monitored four times a day for the presence of larvae, which was sampled with a Pasteur pipette and preserved individually in 100% ethanol. Experiments were performed in March, following previous observations of the highest period of larval release in the laboratory (de Paula et al. 2014). Tissue samples of parental colonies were collected at the end of larvae sampling and preserved in CHAOS solution.

Jê, Jê,Jê__, Jê, ~ Jê, Jê, Jê, Jê,

F ilt er t------c.,,.__

• • • • • t.J • --• • • •

Figure 1. Diagram of the open-water system aquariums with filtered seawater were colonies were kept for larvae collection.

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DNA Extraction and microsatellite amplification

For DNA extraction all larvae were transferred to a 96-well plate (1 larvae per well) with a 22 µl solution including 0.5% of SDS, 10% of proteinase K and 20 µl of AE buffer (10 mM Tris-Cl and 0.5 mM EDTA; pH 9.0) and incubated at 55ºC︎ for 3 hr and at 95ºC for 30 min (protocol modified from Hubert and Hedgecock 2004). DNA from parental colonies were extracted using the Phenol:Chloroform method (Fukami et al. 2004).

Microsatellite markers developed by Capel et al. (2017) (Chapter 4) were amplified by Polymerase Chain Reactions (PCRs) for all parental colonies and 184 and 183 larvae of T. coccinea and T. sp. cf. T. diaphana, respectively. Six loci were selected based on their levels of polymorphism and differed between species (T. coccinea: Tco1, Tco4, Tco5, Tco29, Tco30 and Tco34; T. sp. cf. T. diaphana: Tco5, Tco8, Tco30, Tco34, Tco37 and Tco32). PCRs were performed in 10 µl reactions including 0.2 µM of forward primer with M13 tail at their 5′ end (TGT AAA ACG ACG GCC AGT), 0.4 µM of labeled primer (M13 with VIC, NED, PET, or 6-FAM fluorescent dyes) (Schuelke 2000), 0.8 µM of reverse primer, 1U GoTaq (Promega),

1X PCR Buffer (Promega), 0.20 mM dNTPs (Invitrogen), between 1.5 and 2.5 mM MgCl2 (following Capel et al. 2017), 10 µg BSA (Invitrogen), and 5-10 ng of DNA. Cycling conditions were: 95°C for 3 min followed by 5 cycles at 95°C, 30 s; 52–62°C, 30 s; 72°C, 45 s; and 30 cycles at 92°C, 30 s; 52–62°C, 30 s; 72°C, 55 s; with a final extension at 72°C for

30 min (Toonen 1997). Final concentration of MgCl2 and annealing temperature followed Capel et al. (2017). Amplification was verified in 2% agarose gel. PCR products were pooled with GS600-LIZ size standard (Applied Biosystems) and genotyped in the ABI 3500 genetic Analyzer (Applied Biosystems). Genotypes were determined using the program Geneious 7.1.9 (Kearse et al. 2012).

Data analyses

The locus Tco29 failed to amplify in 18% (N = 33) of all T. coccinea larvae and, thus, was excluded from analyses. Within each of the five analyzed families (each family being composed by a parental colony and all sampled larvae) a range of 0 to 2 (0-8%) and 0 to 6 larvae (0-33%) failed to amplify at one or more loci for T. coccinea and T. sp. cf. T. diaphana, respectively (Table 1). To avoid any bias in the estimative of clones, those individuals that

194 failed to amplify for one or more loci were excluded from analyses. The remaining larvae (182 T. coccinea and 170 T. sp. cf. T. diaphana) were compared and assigned to the same multilocus genotype (MLG) when exhibiting identical alleles at all loci using the package ‘GenClone’ on R 3.2.3 (R Development Core Team 2015). Total number of alleles per loci, inbreeding coefficient (FIS) and deviations from Hardy-Weinberg Equilibrium (HWE) were calculated with GenALEx 6.5 (Peakall and Smouse 2006).

Results and discussion

On March 2015 only two out of 14 colonies of T. coccinea released larvae, while all 14 colonies of T. sp. cf. T. diaphana released at least one larva. On February 2016, when only T. coccinea was sampled, all six colonies released at least 50 larvae each. Our results demonstrate that in the Southwestern Atlantic both T. coccinea and T. sp. cf. T. diaphana reproduce mainly through asexual production of larvae with 181 out of 182 (99,5%) and 166 out of 170 (97,6%) larvae of each species having multilocus genotype identical to the parental colonies (Table 1). The predominance of clonal reproduction is also evidenced by the observed negative values of FIS, showing high levels of heterozygosity and deviations from random mating (Table 1) (Balloux et al. 2003; Halkett et al. 2005). Clonal lineages enable alleles to accumulate divergence indefinitely due to an independent evolution of loci (e.g. “Meselson effect”), which leads to high allelic diversity and heterozygosity (Birky 1996; Judson and Normark 1996; Balloux et al. 2003; Halkett et al. 2005). Furthermore, it is highly unlikely that high levels of identical heterozygous genotypes result from sexual reproduction (Ayre and Resing 1986).

Ayre and Resing (1986) were the first to document asexual production of larvae for 11 colonies of T. coccinea and T. diaphana in their natural range of distribution using two allozyme loci. Even though they used only two loci of a conserved marker, all 91 larvae analyzed were identical to the parental colonies with no evidence of sexual reproduction. Here we found a small percentage of sexually derived larvae ranging from 2,1% to 4,4%, with a higher proportion being observed for T. sp. cf. T. diaphana (Table 1). Only one larvae of T. coccinea differed from the parental colony by two alleles at two different loci. For T. sp. cf. diaphana, three colonies released a total of four larvae differing from the parental colony by only one allele, all at the same locus. The two larvae released from the same parental colony

195 were identical siblings. Interestingly, at all cases the parental colony was a heterozygote while the sexually produced larvae were homozygous for one of the alleles. To avoid the overestimation of asexual reproduction, larvae with one or more alleles difference was considered a product of sexual reproduction. However, single allele differences could be a result from several processes including genotyping errors (e.g. allele dropout) (Hoffman and Amos 2005), somatic mutation or chimerism (van Oppen et al. 2011). Some asexual reproduction strategies also allow a certain level of recombination or even exchange of genetic material, which could explain the observed difference (Uecker 2017).

Table 1. Summary of results for Tubastraea coccinea and T. sp. cf. T. diaphana parental colonies showing the number of polyps (No. polyps), number of larvae sampled (No. larvae), number of individuals with missing data (No. missing) and number and percentage of sexually produced larvae per parent (No. sex and % sex).

Parent No. larvae No. missing No. sex % sex FIS Tubastraea coccinea Tc1 48 0 0 0 -1 Tc2-5 42 0 0 0 -1 TcG-20 48 0 1 2.1 -0.98 Tc2 21 0 0 0 -1 Tc2-11 25 2 0 0 -1 Total 184 2 1 0.5 Tubastraea sp. cf. T. diaphana Tt1 45 4 2 4.4 -0.96 Tt5 45 1 0 0 -1 Tt4 48 2 1 2.1 -0.99 Tt12 18 6 0 0 -1 Tt2 27 0 1 3.7 -0.99 Total 183 13 4 2.2 Colonies with identical multilocus genotype are shown by gray shadows.

Both native and invasive populations of T. coccinea and T. sp. cf. diaphana, display some genetic diversity suggesting the occurrence of recombination (Capel et al. in prep., Chapter 5). Nonetheless, sexual reproduction remains poorly documented for Tubastraea spp.. Only two studies have evaluated the reproductive biology of Tubastraea spp. in their natural (Glynn et al. 2008) and invasive range (de Paula et al. 2014) and both found year round reproductive activity despite a rare observation of spermaries. Furthermore, there are only a few observations of spawning of spermatozoa in Panamá, Galápagos and the Great Barrier Reef (Glynn et al. 2008), but never in the invasive range. The rarity of male gametes may indicate a low frequency of sexual reproduction or the occurrence of sexual reproduction restricted to specific environmental conditions, season or be limited by low population 196 densities (cyclical parthenogenesis) (Gómez and Carvalho 2000; Simon et al. 2002; Decaestecker et al. 2009), since simultaneous mixed reproduction is rare in animals (Combosch and Vollmer 2013). Nevertheless, our results indicate the occurrence of simultaneous mixed reproduction within the same colony for Tubastraea spp., a strategy also observed for Pocillopora damicornis (Combosch and Vollmer 2013). Mix reproduction with facultative parthenogenesis incorporates positive features of both sexual and asexual reproduction (Shick et al. 1979; Holman 1987; Ayre and Willis 1988; Radtkey et al. 1995; Hurst and Peck 1996; Weider et al. 1999; Peck and Waxman 2000) and is found on a variety of organisms including crayfish (Buřič et al. 2011), sharks (Dudgeon et al. 2017), and Komodo dragon (Watts et al. 2006).

Two hypotheses could explain the formation of asexual larvae within Tubastraea spp., apomictic parthenogenesis and pseudogamy. Apomitic parthenogenesis occur through mitotic oogenesis, generating identical clones, while pseudogamy depends on the presence of sperm to stimulate the development of parthenogenic larvae (sperm-dependent parthenogenesis) and allows the incorporation of portions of the sperm’s genome (Beukeboom et al. 1998; D’Souza and Michiels 2010). Although the biological process of formation of asexual larvae could not be confirmed, our observation of a high number of larvae with genotype identical to their parental colonies and genotypic distances of only one or two alleles suggests pseudogamy as the most likely explanation. This strategy is assumed to explain asexual larvae on Pocillopora damicornis (Combosch and Vollmer 2013) and it is recorded for several organisms among plants and animals (Stenseth et al. 2014).

Thus, herein we show evidence that invasive populations of T. coccinea and T. sp. cf. T. diaphana reproduce mainly through asexually produced larvae, with controversial signs of sexual reproduction. Due to the low variation of the observed sexually derived larvae, an extensive sampling on different seasons and environmental conditions is recommend to verify the real proportion of sexual versus asexual reproduction within Tubastraea spp.. Previous studies with Pocillopora damicornis found that different populations invest differently on asexual reproduction, although they failed to find any correlation with environmental conditions to explain such pattern (Sherman et al. 2006). For Tubastraea spp., asexual reproduction have been now recorded for both native (Ayre and Resing 1986) and invasive populations, and it is likely one of the main reasons for their invasion success in the Southwestern Atlantic.

197

Acknowledgements

We are grateful to Bruna Luz for helping collecting the larvae and to Alvaro Migotto and CEBIMar (Center for Marine Biology, University of São Paulo) for providing the laboratory structure.

References

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4. Discussão

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4.1 Sistemática e taxonomia

A família Dendrophyllidae é a terceira mais diversa da ordem Scleractinia e compreende uma grande diversidade de organismos solitários e coloniais, zooxantelados e azooxantelados, ocorrendo em águas rasas e profundas (Cairns 2001). A taxonomia de corais azooxantelados ainda é incipiente quando comparada aos corais zooxantelados (Kitahara et al. 2016). Entre as espécies que ocorrem em águas rasas, muitas possuem uma coloração alaranjada e morfologia similar, o que frequentemente dificulta a identificação até mesmo em nível de gênero. Os resultados apresentados aqui, com base em dados morfológicos e moleculares, sugerem: (1) uma nova identificação para uma das espécies introduzidas no Atlântico, incialmente identificada como T. tagusensis e aqui identificada por T. sp. cf. T. diaphana; (2) a possível revalidação da espécie T. aurea, anteriormente sinonimizada à T. coccinea; e (3) a transferência da espécie recém descrita T. caboverdiana para um novo gênero aqui descrito, Laborelia.

A espécie T. tagusensis foi descrita para o Arquipélago de Galápagos e apresenta quatro registros questionáveis distribuídos entre o Atlântico Sul, Indo-Pacífico e Pacífico leste (Creed et al. 2016). Análises morfológicas demonstram que os espécimes do Atlântico Sul não se encaixam na descrição da espécie tipo, e a análise de mais exemplares incluindo espécimes de Galápagos é recomendado para uma identificação precisa (Capítulo 1). Corais escleractíneos tendem a apresentar uma elevada variabilidade morfológica intraespecífica (Todd 2008), no entanto, por vezes essa variabilidade pode esconder a ocorrência de espécies crípticas, hibridização ou especiação recente (Keshavmurthy et al. 2013; Richards & Hobbs 2015; Richards & van Oppen 2012; Warner et al. 2015). No caso da espécie T. coccinea, existem cerca de 14 sinonímias reconhecidas, e os resultados aqui apresentados sugerem que a sinonímia T. aurea é uma espécie válida e é provável que ambas façam parte de um complexo de espécies ainda não esclarecido (Capítulo 1). A inclusão de um maior número de espécimes é indispensável para esclarecer essas ambiguidades.

Outra problemática envolvendo o gênero Tubastraea são os espécimes encontrados em Cabo Verde, no Atlântico Leste. Indivíduos dessa localidade foram inicialmente identificados como pertencentes ao gênero Enallopsammia (Chevalier 1966) e posteriormente transferidos para o gênero Tubastraea (Laborel 1974). No entanto, comparações morfológicas com os demais gêneros da família Dendrophylliidae e análises moleculares utilizando genes mitocondriais e nucleares demonstram que a espécie de Cabo Verde não pertence ao gênero 203

Tubastraea, corroborando a transferência para o novo gênero aqui descrito, Laborelia (Capítulo 2). A avaliação do genoma mitocondrial sugere também que um espécime depositado no GenBank e identificado como T. coccinea (número de acesso: NC026025) é uma identificação errônea, sendo mais próximo à espécie Dendrophyllia arbuscula (Capel et al., 2016; Capítulo 3). Embora o genoma mitocondrial de antozoários tenha uma baixa taxa evolutiva, os resultados mostram que ele apresenta resolução suficiente ao nível de gênero, podendo ser usado para confirmar identificações ambíguas (Capel et al., 2016; Capítulo 3).

Além de resolver questões taxonômicas do grupo, o trabalho apresenta a primeira chave de identificação específica para as espécies válidas de Tubastraea. De forma geral, foi observado que o gênero Tubastraea apresenta uma elevada variabilidade morfológica intraespecífica, e uma revisão incluindo um número maior de espécimes e uma abordagem integrativa com dados morfológicos e moleculares é essencial para esclarecer a diversidade do grupo e suas relações evolutivas.

4.2 Clonalidade

Os corais escleractíneos apresentam uma série de estratégias reprodutivas assexuadas, incluindo brotamento, fissão, fragmentação e liberação de larvas assexuadas (Ayre & Resing 1986; Fautin 2002; Harrison 2011; Highsmith 1982). Os resultados apresentados mostram que as espécies T. coccinea e T. sp. cf. T. diaphana se reproduzem predominantemente pela liberação de larvas assexuadas, uma estratégia pouco difundida entre corais escleractíneos e até então encontrada em apenas três espécies (Ayre & Resing 1986; Stoddart 1983). Diversas vantagens podem ser atribuídas à predominância de reprodução assexuada, incluindo um baixo custo energético, dispersão de genótipos bem adaptados e possibilidade de reprodução quando não existem parceiros reprodutivos próximos (Ayre & Willis 1988; Holman 1987; Hurst & Peck 1996; Peck & Waxman 2000; Radtkey et al. 1995; Shick et al. 1979; Taylor & Hastings 2005; Weider et al. 1999), no entanto, a sobrevivência de uma linha exclusivamente clonal é prejudicial a longo prazo e extremamente rara na natureza (Muller 1964; Peck 1994). Dessa forma, muitos organismos apresentam as duas estratégias, se beneficiando de ambas e evitando os custos e malefícios associados à uma única estratégia reprodutiva. A diversidade genética observada em Tubastraea spp., particularmente em vetores e na população nativa avaliada, sugere que as espécies avaliadas utilizam ambas as estratégias, produzindo tanto

204 larvas sexuadas quanto assexuadas. A análise de cerca 184 e 183 larvas de T. coccinea e T. sp. cf. T. diaphana, respectivamente, demonstrou que a produção de larvas assexuadas é predominante, enquanto a produção de larvas sexuadas ficou pouco evidente (Capítulo 6). Embora 0,5% e 2,2% das larvas analisadas tenham sido atribuídas à reprodução sexuada, a diferença de dois ou menos alelos com relação a colônia parental pode ser resultante de outros fatores, como erros de genotipagem, mutações somáticas ou quimerismo (van Oppen et al. 2011). Uma outra hipótese é a ocorrência de pseudogamia, um tipo de partenogênese que depende da presença de gametas masculinos e permite, eventualmente, a passagem de conteúdo genético paterno (em parte ou completo) (Beukeboom et al. 1998). É possível ainda que a reprodução sexuada seja restrita para determinadas épocas do ano ou que seja dependente de fatores ambientais (Decaestecker et al. 2009; Gómez & Carvalho 2000; Simon et al. 2002). Um acompanhamento anual da liberação de larvas e análises parentais em populações nativas e invasoras é recomendado para uma melhor avaliação da reprodução sexuada em Tubastraea.

A ocorrência de reprodução assexuada foi observada, através do índice de clonalidade, tanto nas populações invasoras como na população nativa avaliada, corroborando estudos preliminares (Ayre & Resing 1986). Para populações invasoras essa é uma estratégia extremamente vantajosa, uma vez que permite um crescimento rápido a partir de poucos indivíduos iniciais (Taylor & Hastings 2005). Na costa brasileira, cinco das oito localidades avaliadas para T. coccinea foram colonizadas por dois ou até um único genótipo, com a capacidade de ocupar rapidamente até 90% do substrato disponível (Mantelatto et al. 2011). A predominância da reprodução assexuada no Atlântico Sul foi, possivelmente, um dos principais fatores que levaram ao sucesso da invasão de Tubastraea. No entanto, apesar de facilitar processos de invasão, populações amplamente clonais são mais susceptíveis e podem declinar rapidamente quando expostas a um forte distúrbio (Honnay & Bossuyt 2005).

4.3 Panorama geral sobre a invasão Atlântico

O gênero Tubastraea foi introduzido no Atlântico na década de 80 e atualmente é encontrado descontinuamente ao longo de mais de 3.500 km (Creed et al. 2016; Soares et al. 2016). Embora a origem das populações do Atlântico permaneça desconhecida, acredita-se que a invasão se deu por incrustação oportunista de plataformas de petróleo, provavelmente

205 oriundas do Indo-Pacífico (Creed et al. 2016). Através da análise de amostras coletadas na costa brasileira, em vetores de introdução e uma população nativa, foi possível observar que embarcações associadas à exploração do petróleo são de fato os vetores mais prováveis de introdução das espécies de Tubastraea no Atlântico Sul. Essa hipótese é corroborada pela maior diversidade genotípica observada nos vetores em comparação com a costa, incluindo genótipos e alelos ainda não encontrados ao longo da costa. Além de introduções iniciais, o transporte de vetores contaminados pode ser responsável também por introduções secundárias, de forma que mesmo os vetores que chegaram livre de incrustações, como no caso da plataforma P14, representam um risco quando transportados de um local para outro na localidade invadida. Foram encontrados clones (colônias com genótipo multilocus idêntico) em vetores e em diferentes locais na costa, separados por até 2.000 km, uma distribuição dificilmente explicada por dispersão larval, uma vez que as larvas de Tubastraea spp. tendem a assentar próximo à colônia parental até 3 horas após a liberação (Glynn et al. 2008; de Paula et al. 2014). As hipóteses mais prováveis são (1) a ocorrência de múltiplas introduções a partir da mesma população nativa e (2) a ocorrência de introduções secundárias através do transporte de vetores.

A origem exata da maioria dos vetores analisados é desconhecida, no entanto, o padrão de diversidade genética observado sugere a ocorrência de múltiplos eventos de invasão das duas espécies, T. coccinea e T. sp. cf. T. diaphana, e a possível ocorrência de introduções secundárias ao longo da costa brasileira. Foi possível observar, por exemplo, que o Naufrágio Petroleiro do Acaraú, em Fortaleza, foi invadido por uma população da espécie T. sp. cf. T. diaphana diferente da encontrada nos demais pontos ao longo da costa brasileira. A ocorrência de múltiplas introduções tem o potencial de manter a diversidade genética de populações invasoras similar ou até maior que a observada nas populações nativas, aumentando a chance de sobrevivência das espécies no novo ambiente (Gaither et al. 2012; Kolbe et al. 2004; Lockwood et al. 2005, 2009; Roman 2006).

Os corais do gênero Tubastraea spp. são oportunistas e comumente encontrados em substratos artificiais ao longo da distribuição nativa e invasora (Castro & Pires 2001; Creed et al. 2016; Fenner & Banks 2004; Ferreira et al. 2006; Ho et al. 2017), o que potencializa seu sucesso como invasor. No Atlântico Sul, o sucesso da invasão pode ser atribuído a uma combinação de fatores que incluem as características biológicas das espécies (como crescimento rápido, maturidade reprodutiva precoce e ocorrência de reprodução assexuada) e

206 as condições favoráveis do local invadido (como escassez de predadores e competidores). No entanto, a chegada de múltiplos vetores contaminados e o transporte destes ao longo da costa, foram, sem dúvida, os principais responsáveis pela ampla distribuição das espécies de Tubastraea no Atlântico Sul. Os dados obtidos pelo presente trabalho ajudam a compreender melhor o cenário da invasão do Atlântico Sul e fornece informações cruciais para o desenvolvimento de estratégias realmente eficazes de manejo, controle e prevenção de novas invasões.

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5. Conclusões

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• Ambas as espécies avaliadas se reproduzem predominantemente por reprodução assexuada e as populações encontradas ao longo da costa são altamente clonais, demonstrando que um único indivíduo tem o potencial de colonizar grandes extensões.

• Os resultados obtidos pelo presente trabalho apontam que embarcações associadas à exploração de petróleo são os principais responsáveis pela introdução e ampla dispersão das espécies no Atlântico Sul, e que um maior controle dos vetores é imprescindível para evitar a ocorrência de novas introduções.

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