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Universidade Federal do Rio de Janeiro 
Instituto de Biologia 
 
 
 
 
 
Evolução e biodiversidade 
de peixes recifais criptobentônicos 
do Oceano Atlântico 
 
 
Ricardo Marques Dias 
 
 
 
RIO DE JANEIRO 
2019
 I 
 
 
Ricardo Marques Dias 
 
 
Evolução e biodiversidade 
de peixes recifais criptobentônicos 
do oceano Atlântico 
 
 
 
 
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). 
 
 
 
Orientador: Dr. Paulo Cesar de Paiva 
Coorientador: Dr. Anderson Vilasboa de Vasconcellos 
 
 
 
Rio de Janeiro 
Agosto/2019 
 II 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dias, Ricardo Marques 
 Evolução e biodiversidade de peixes recifais criptobentônicos do Oceano Atlântico / 
 Ricardo Marques Dias. Rio de Janeiro: UFRJ/IB –2019 
 X + 135 fls 
 
 Tese (Doutorado) – Universidade Federal do Rio de Janeiro, Instituto de Biologia, 
 Programa de Pós-graduação em Ciências Biológicas (Biodiversidade e Biologia 
 Evolutiva), 2019. 
 
 Orientador: Paulo Cesar de Paiva 
 Coorientador: Anderson Vilasboa de Vasconcellos 
 
 Referências: f. 126–135 
 
 1. Filogeografia. 2. Blennioidei. 3. Malacoctenus. 4. Parablennius. – Tese 
 
 I. Paiva, Paulo Cesar de Paiva. II. Universidade Federal do Rio de Janeiro. Programa 
 de Pós-graduação em Ciências Biológicas (Biodiversidade e Biologia Evolutiva). III. 
 Evolução e biodiversidade de peixes recifais criptobentônicos do Oceano Atlântico 
 
 
 
 
 
 
 
 III 
 
 
Evolução e biodiversidade de peixes recifais criptobentônicos do Oceano Atlântico 
 
Ricardo Marques Dias 
Orientador: Dr. Paulo Cesar de Paiva 
Coorientador: Dr. Anderson Vilasboa de Vasconcellos 
 
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 parte dos requisitos necessários à obtenção do título de Doutor em Ciências 
Biológicas. 
 
Data: 22/08/2019 
Banca examinadora: 
 
___________________________________________________________________________ 
Prof Dr Paulo Cesar de Paiva (IB/UFRJ) – Membro titular 
 
___________________________________________________________________________ 
Prof Dr António Mateo Solé-Cava – Membro titular 
 
___________________________________________________________________________ 
Prof Dra Joana Zanol Pinheiro da Silva (MN/UFRJ) – Membro titular 
 
___________________________________________________________________________ 
Prof Dra Clarissa Coimbra Canedo (IB/UERJ) – Membro titular 
 
________________________________________________________________ 
Prof Dr William Bryan Jennings (MN/UFRJ) – Membro titular 
 
____________________________________________________________ 
Prof Dra Haydée Andrade Cunha (OB/UERJ) – Membro suplente 
 
________________________________________________________________ 
Prof Dr Cristiano Moreira Rangel (MN/UFRJ) – Membro suplente 
 
 
 
Rio de Janeiro 
Agosto de 2019 
 IV 
 
 
 
"E esse caminho 
Que eu mesmo escolhi 
É tão fácil seguir 
Por não ter onde ir 
Controlando 
A minha maluquez 
Misturada 
Com minha lucidez 
Vou ficar 
Ficar com certeza 
Maluco beleza" 
 
Raul Seixa, 1973, Maluco Beleza 
 
 
 
 
 
 
 
 
 
 
 
 V 
Dedicatória 
 
"A minha companheira Natacha, por me acompanhar nesta e em muitas as outras jornadas, 
obrigado por estar ao meu lado hoje e sempre... " 
 
 
"Ao nosso filho, por quem me esforço todos os dias para que possa ter um futuro, com mais 
peixes e menos plástico nos Rio, lagos e mares" 
 
 
"As minhas sobrinhas Giulia e Giovanna, por trazerem um pouco de alegria a esta casa" 
 
 
"A minha mãe Katia, por sempre ter acreditado e sonhado junto" 
 
 
"Ao meu pai José Fernando (in memoriam), pelos poucos e saudosos anos que 
compartilhamos e pela paixão pelo mar, que mesmo sem saber nadar conseguiu imbuir em 
minha vida" 
 
 
 
 
 
 
 
 VI 
Agradecimentos 
 
Aqui demostro meus sinceros agradecimentos a todos que, de alguma forma, colaboraram 
comigo durante o período da realização deste trabalho: 
Ao Dr. Paulo Cesar de Paiva, meu orientador, imensa gratidão pela confiança e oportunidade, 
aceitando orientar um ictiólogo, pela amizade, paciência, e por todas as oportunidades 
oferecidas. 
Ao Dr. Anderson Vilasboa de Vasconcellos, pela amizade e conhecimentos compartilhados. 
Ao Dr. Daniel Fernando de Almeida, grande amigo, pela amizade e sabedoria com as coisas 
da vida. Pelos ensinamentos pessoais e profissionais, sugestões e revisões essenciais para a 
finalização desta tese. E as agulhadas!!! 
A todos os professores do Instituto de Biologia da UFRJ, que de alguma forma contribuíram 
para minha formação. 
Agradeço especialmente os professores Sergio Lima, Marcelo Britto, Liana Mendes e 
Marcelo Gehara pelo esclarecimento de dúvidas, pela experiência compartilhada e sugestões 
essenciais para a finalização desta tese. 
A CAPES Coordenação de Aperfeiçoamento de Pessoal de Nível Superior pelo financiamento 
sob forma de bolsa, ao Conselho Nacional de Desenvolvimento Científico e Tecnológico 
(CNPq), a (CAPES) e a Fundação Carlos Chagas Filho de Amparo à pesquisa do Estado do 
Rio de Janeiro, pelo financiamento de projetos do setor de Ictiologia MN/UFRJ e Laboratório 
de Poliqueta UFRJ, fundamentais para a realização deste trabalho. 
Aos membros das bancas de Seminário, pelos comentários e sugestões apresentadas para o 
aprimoramento do projeto original. Aos curadores e/ou responsáveis pelo material das 
coleções ictiológicas: Dr. Sergio Maia Queiroz Lima (UFRN), Dr. Marcelo Ribeiro de Britto 
(MNRJ), Dr. André Levy (ISPA) e Dr. Roger Bills e Dr. Zandie Adam (SAIAB). 
 VII 
Ao Instituto Luísa Pinho Sartori, pelo apoio financeiro que me proporcionou a participação no 
XII Taller Genética de la Conservación - ReGenec. 
A todos os professores, alunos e amigos da Rede de Genética para la ConservaciónReGenec, 
por duas semanas incríveis de muito aprendizado e amizade na Bolívia. 
Aos grandes amigos Sergim e Buda pelo incentivo, apoio, sugestões, artigos, conversas 
técnicas e não técnicas, pela super ajuda com as análises e paciência, mas acima de tudo pela 
amizade. 
A todos os amigos do setor de ictiologia que por aqui estão e que por aqui passaram (Daniel, 
Décio, Emanuel, Fabio, Karina, Vanessa, Gabriel S., Gabriel A., Gustavo, Leandro, Vinícius, 
Renata, Igor, Sergio, Victor, Lucas, Raul...), e a todos os amigos do Laboratório de Poliqueta 
que por aqui estão e que por aqui passaram (Victor, Gustavo, Rodolfo, Antônio, Leonardo, 
Gisele, Monique, Natalia, Ricardo, Rominho, Carlos, Stephanie, Renata, Mirian, Rodolfo, 
Letícia...) pela amizade e companheirismo e por tornar o ambiente de trabalho agradável. 
Victinho, obrigadão pela amizade, incentivo, apoio, sugestões, artigos, revisões nos 
manuscritos, pela super ajuda com as análises e horas de discussões, mas acima de tudo pela 
amizade. 
Gustavo e Rodolfo, obrigado pela amizade e pela força dentro e fora do laboratório. 
Aos amigos de coleta, Sergio, Daniel, Liana, Tati, Fran, Gabriel, Victor, Rominho, Natacha. 
A todos os funcionários do Instituto de Biologia e da Seção de Pós-Graduação pela ajuda. 
Ao Programa de Pós-Graduação em Biodiversidade e Biologia Evolutiva (IB/UFRJ) pelos 
auxílios financeiros concedidos durante o doutorado; 
E finalmente, agradeço à minha família por ter me apoiado e incentivado desde o início de 
minha carreira científica, sem vocês não seria possível chegar até aqui! 
 
 
 
 
 VIII 
Resumo 
 
Dias, Ricardo Marques. Evolução e biodiversidade de peixes recifais criptobentônicos do 
oceano Atlântico. Rio de Janeiro, 2019. Tese(Doutorado em Ciências Biológicas - Pós 
Graduação em Biodiversidade e Biologia Evolutiva). Instituto de Biologia, Universidade 
Federal do Rio de Janeiro, Rio de Janeiro, 2019. 
 
Nesta tese foi analisada a filogeografia e sistemática molecular de três espécies de peixes 
recifais criptobentônicos amplamente distribuídas no Oceano Atlântico, mas com padrões 
biogeográficos distintos, sendo eles: Parablennius marmoreus (Poey, 1876) com distribuição 
mais restrita, endêmica do Atlântico ocidental, ocorrendo de Nova York até Santa Catarina 
(Carvalho-Filho, 1999); Malacoctenus triangulatus (Springer, 1959) com distribuição 
intermediária, ocorrendo na costa ocidental e nas ilhas oceânicas do Atlântico (Mendes, 2006) 
e Parablennius pilicornis (Cuvier, 1829) com distribuição anfi-Atlântico (Bath, 1990). Foram 
utilizados quatro marcadores moleculares com diferentes taxas de evolução - dois marcadores 
nucleares (rodopsina e o intron S7) e dois mitocondriais (citocromo oxidase I e citocromo b) - 
com o objetivo de: I) investigar a ocorrência de espécies crípticas ao longo da ampla 
distribuição das espécies; II) analisar a estrutura populacional; III) elucidar a existência de 
barreiras geográficas e seus respectivos papeis na especiação; IV) produzir instrumentos 
auxiliares para a conservação das espécies. Os resultados indicaram que as três espécies 
analisadas tratam-se de complexos de espécies: A) complexo P. marmoreus - existência de 
pelo menos duas linhagens (Caribe e costa brasileira); B) complexo M. triangulatus - pelo 
menos três linhagens altamente estruturadas (província caribenha, ilhas oceânicas do nordeste 
e outra ao longo da costa brasileira); C) complexo P. pilicornis - três linhagens alopátricas 
(Atlântico sul ocidental, Atlântico norte oriental e uma terceira restrita ao sudoeste do Oceano 
Índico). Estes resultados reforçam a importância de três conhecidas barreiras biogeográficas 
que atuam na bacia do Atlântico, sendo elas: a barreira do meso-atlântico, a pluma do 
Amazonas-Orinoco e a de Benguela, que desempenham um importante papel na origem e 
manutenção da diversificação de peixes recifais do Oceano Atlântico, não apenas restringindo 
a dispersão, mas também permitindo cruzamentos ocasionais, levando ao estabelecimento de 
novas populações e espécies em alopatria. Além disso, estes resultados implicam em uma 
distribuição mais restrita e menor tamanho populacional, consequentemente reduzindo a 
capacidade de resiliência destas linhagens. Portanto, sugerimos que essas linhagens tenham 
seu status taxonômico verificado, através de uma abordagem taxonômica integrativa, o que 
permitiria uma precisa avaliação do real status de conservação da diversidade genética destes 
complexos de espécies. 
 IX 
Abstract 
 
Dias, Ricardo Marques. Evolução e biodiversidade de peixes recifais criptobentônicos do 
oceano Atlântico. Rio de Janeiro, 2019. Tese (Doutorado em Ciências Biológicas - Pós 
Graduação em Biodiversidade e Biologia Evolutiva). Instituto de Biologia, Universidade 
Federal do Rio de Janeiro, Rio de Janeiro, 2019. 
 
This thesis analyses the phylogeography and molecular systematics of three species of 
cryptobenthic reef fishes widely distributed in the Atlantic Ocean, but with distinct 
biogeographic patterns: Parablennius marmoreus (Poey, 1876) with a more restricted 
distribution, endemic to the western Atlantic, occurring New York to Santa Catarina 
(Carvalho-Filho, 1999); Malacoctenus triangulatus (Springer, 1959) with an intermediate 
distribution, occurring on the western coast and in the Atlantic ocean islands (Mendes, 2006) 
and Parablennius pilicornis (Cuvier, 1829) with a wide amphi-Atlantic distribution (Bath, 
1990). Four molecular markers with different evolution rates were used - two nuclear markers 
(rhodopsin and the S7 intron) and two mitochondrial (cytochrome oxidase I and cytochrome 
b) - in order to: I) investigate the occurrence of cryptic species along the wide distribution of 
species; II) analyse the population structure; III) elucidate the existence of geographical 
barriers and their respective role in speciation; IV) produce auxiliary instruments for the 
conservation of species. The results indicated that the three species analysed are complexes of 
species: A) P. marmoreus complex - existence of at least two lineages (Caribbean and 
Brazilian coast); B) M. triangulatus complex - at least three highly structured strains 
(Caribbean province, northeastern ocean islands and another along the Brazilian coast); C) P. 
pilicornis complex - three allopatric strains (southwestern Atlantic, northeastern Atlantic and 
southwestern Indian Ocean). These results reinforce the importance of three known 
biogeographic barriers that act in the Atlantic basin, which are: the barrier of the mid-Atlantic, 
the plume of the Amazon-Orinoco and Benguela, which play an important role in the origin 
and maintenance of diversification of reef fish in the Atlantic Ocean, not only restricting the 
dispersion but also allowing occasional crosses, leading to the establishment of new 
populations and species in allopatry. In addition, these results imply a more restricted 
distribution and smaller population size, consequently reducing the resilience capacity of 
these lineages. Therefore, we suggest that these lineages have their taxonomic status verified, 
through an integrative taxonomic approach, which would allow an accurate assessment of the 
real conservation status of the genetic diversity of these species complexes. 
 
 
 X 
SUMÁRIO 
 
 
1. Introdução geral....................................................................................................................1 
 
 
2. Capítulos................................................................................................................................7 
 
 
 Capítulo 1.......................................................................................................................8 
Genetic structure and diversity in the seaweed blenny (Parablennius marmoreus), 
a cryptobenthic fish endemic to the western Atlantic 
 
 
Capítulo 2.....................................................................................................................47 
Different speciation processes in a cryptobenthic reef fish from the Western 
Tropical Atlantic 
 
 
Capítulo 3.....................................................................................................................73 
The evolutionary history of the ringnecks blenny (Parablennius pilicornis) an 
amphi-Atlantic cryptobenthic reef fish 
 
 
3. Discussão geral..................................................................................................................116 
 
 
4. Conclusões.........................................................................................................................123 
 
 
5. Referências bibliográficas................................................................................................126
 1 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1. Introdução geral 
 
 
 
 
 
 
 
 
 
 2 
Introdução Geral 
 
Os ambientes de recife abrigam aproximadamente um terço da biodiversidade global 
de peixes marinhos (Spalding & Grenfell, 1997), essa elevada biodiversidade é notável, dada 
a baixa cobertura espacial global dos ambientes recifais em comparação com o oceano aberto. 
Uma grande parte dessa diversidade (pelo menos 44%) é composta de peixes recifais 
criptobentônicos (Brandl et al., 2018). Além de imensa contribuição para a biodiversidade dos 
recifes de corais como um todo, os peixes recifais criptobentônicos podem fornecer 
importantes serviços ecossistêmicos ao recife, atuando como um elo trófico, ciclando energia 
trófica de presas microscópicas para consumidores maiores (Brandl et al., 2019). 
 As altas taxas de diversificação dos peixes recifais criptobentônicospodem ser 
associadas a aspectos de sua biologia, como: 1) pequeno tamanho corpóreo, tipicamente 
menores que 5 cm; 2) intima associação com o substrato; 3) baixa capacidade de dispersão 
(desova bentônica e curto período larval) e 4) limitada mobilidade quando adultos (Miller, 
1979; Depczynski & Bellwood, 2003), fornecendo assim mecanismos para que uma rápida 
diversificação possa ocorrer. As características 1 e 2 possibilitam a exploração de micro-
habitats podendo levar ao particionamento de nicho em múltiplas escalas, enquanto as 
características 3 e 4 tornam as espécies mais propensas a eventos de especiação alopátrica 
(Brandl et al., 2018). 
 Além da alta diversificação, tais características geralmente implicam em uma 
distribuição mais restrita (Horn et al., 1999). Em habitats marinhos, espécies cujos adultos 
possuem hábitos sedentários, pequena área de vida e reduzida capacidade de dispersão por 
longas distâncias, são bons candidatos para estudos filogeográficos (Floeter et al., 2008; 
Rocha et al., 2002). 
 A filogeografia pode ser definida como o estudo dos princípios e processos que 
determinam a distribuição geográfica de linhagens genealógicas, principalmente aqueles entre 
 3 
táxons proximamente relacionados (Avise, 2004; Martins & Domingues, 2010). Desta forma, 
podemos usar informações genealógicas e geológicas para inferir os processos históricos e 
demográficos que moldaram a evolução das linhagens (Kuchta & Meyer, 2001), o que nos 
permite formular hipóteses sobre como as populações se comportaram historicamente com as 
mudanças geográficas (Moritz & Faith, 1998; Carnaval, 2002). Estudos filogeográficos 
buscam revelar a história biogeográfica das espécies e dos habitats que elas ocupam por meio 
da associação espacial entre agrupamentos de alelos com barreiras geográficas e/ou 
ecológicas (Avise 2010). 
 A análise e interpretação da distribuição das linhagens requerem o processamento 
conjunto de informações de uma série de disciplinas, incluindo a sistemática filogenética, 
genética de populações, etologia, demografia, paleontologia, geologia e modelos 
paleogeográficos e paleoclimáticos. Dessa forma, o caráter multidisciplinar da filogeografia 
cria uma ponte entre os processos micro e macroevolutivos (Martins & Domingues, 2010). 
 Diante deste contexto, a análise da distribuição de peixes recifais sob a orientação da 
teoria filogeográfica pode elucidar o local das barreiras geográficas e seu papel na especiação 
(Randall, 1998). De fato, diversos estudos filogeográficos de peixes recifais têm contribuído 
para o maior entendimento dos padrões evolutivos e biogeográficos observados na ictiofauna 
do Atlântico (e.g. Briggs & Bowen, 2012; Bowen et al., 2013; Cowman, 2014; Neves et al., 
2016; Rodríguez-Rey et al., 2017; Gwillian et al., 2018). 
 Apesar de um aumento substancial no conhecimento sobre a biogeografia e evolução 
dos peixes recifais do Oceano Atlântico (Floeter et al., 2008; Pinheiro et al., 2018), muitas 
questões sobre os processos evolutivos e padrões biogeográficos ainda não foram resolvidos, 
principalmente envolvendo peixes recifais criptobentônicos, que devido a suas características 
crípticas tem recebido menos atenção do que espécies mais conspícuas (Ahmadia et al., 
2012). Além disso, poucos estudos tentaram delimitar linhagens inter e intraespecíficas 
usando dados moleculares, o que poderia melhorar nossa compreensão sobre as barreiras 
 4 
geográficas, a estrutura populacional e os limites geográficos das espécies de organismos 
marinhos do Atlântico (Turchetto-Zolet et al. 2013). 
 Todavia, no ambiente marinho, as barreiras ao fluxo gênico são difíceis de se 
caracterizar e raramente são absolutas (Rocha et al., 2007). Dentro da Bacia do Oceano 
Atlântico, são reconhecidas três importantes barreiras biogeográficas: meso-atlântico, pluma 
do Amazonas-Orinoco e o sistema de afloramento de Benguela. Essas barreiras desempenham 
um papel significativo na geração e manutenção da biodiversidade dos organismos marinhos 
no Oceano Atlântico (Briggs & Bowen, 2013). A mais antiga, a barreira do meso-Atlântico 
começou a se formar após a separação da África e da América do Sul, aproximadamente há 
84 milhões de anos (Pérez-Díaz & Eagles, 2017), criando uma lacuna que se expandiu até 
aproximadamente 3500 km ao longo da zona equatorial do Atlântico, representando uma 
distância extrema em relação à dispersão larval regular de organismos marinhos (Bowen et 
al., 2006; Luiz et al., 2012). A segunda barreira a se formar foi a da pluma do Amazonas-
Orinoco, que se tornou efetiva no Neógeno, aproximadamente entre 6,8 e 4,5 milhões de anos 
atrás, separando os ambientes recifais brasileiros e caribenhos, sendo reconhecida como uma 
influente barreira na formação de pares de espécies irmãs, principalmente para espécies de 
peixes recifais de águas rasas (Rocha, 2003; Floeter et al., 2008; Rodríguez-Rey et al., 2017). 
Por fim, a barreira mais recente, conhecida como o sistema de afloramento de Benguela (zona 
de ressurgência), se tornou pronunciada há aproximadamente 2 milhões de anos atrás, ao 
longo da costa atlântica do sul da África (Marlow et al., 2000). Essa barreira proporciona 
limitações ao fluxo gênico entre a população dos oceanos Atlântico e Índico meridional, 
especialmente para espécies de águas rasas (Reid et al., 2016), formando a zona de transição 
Índico/Oceano Atlântico. Essa zona de transição foi identificada como uma ruptura 
filogeográfica em algumas espécies costeiras de peixes de recife (Grant & Bowen, 1998; 
Teske et al., 2011; Henrique et al., 2012, 2014, 2015; Reid et al., 2016; Gwilliam et al., 2018). 
 Além da presença dessas barreiras, outras características tais como a íntima associação 
 5 
com o bentos, seu comportamento críptico, ovos demersais protegidos pelo macho e fase 
larval curta (Almeida et al., 1980; Depczynski & Bellwood, 2003; Beldade et al., 2007) 
podem afetar a capacidade de dispersão das espécies de peixes recifais criptobentônicos, o 
que pode levar à estruturação genética entre suas populações (Hohenlohe, 2004; Cowen & 
Sponaugle, 2009). 
 Investigações sobre filogeografia e filogenética de peixes recifais criptobentônicos do 
Atlântico ainda são raros (e.g. Muss et al., 2001; Almeida 2011; Neves et al., 2016; 
Rodríguez-Rey et al., 2017; Lastrucci et al., 2018). Estes trabalhos permitem entender os 
processos evolutivos que atuaram e influenciaram a atual distribuição das espécies (Hickerson 
et al., 2010), e tem revelado uma alta diversidade críptica, sendo as espécies muitas vezes 
concordantes com os limites biogeográficos conhecidos (Rodríguez-Rey et al., 2017; 
Lastrucci et al., 2018). 
 Com base no exposto acima, o principal objetivo deste trabalho foi investigar os 
padrões biogeográficos e os processos evolutivos que moldaram a atual distribuição dos 
peixes recifais criptobentônicos do oceano Atlântico, com base em estudos filogeográficos de 
três espécies de peixes amplamente distribuídas no Oceano Atlântico, mas com padrões 
biogeográficos distintos: Parablennius marmoreus (Poey, 1876) com distribuição mais 
restrita, endêmica do Atlântico ocidental, ocorrendo de Nova York até Santa Catarina 
(Carvalho-Filho, 1999); Malacoctenus triangulatus (Springer, 1959) com distribuição 
intermediária, ocorrendo na costa ocidental e nas ilhas oceânicas do Atlântico (Mendes, 2006) 
e Parablennius pilicornis (Cuvier, 1829) com distribuição anfiatlântica (Bath, 1990). Estes 
estudos estão organizados em três capítulos: Capítulo 1 - Genetic structure and diversity in 
the seaweed blenny (Parablennius marmoreus), a cryptobenthic fish endemic to the western 
Atlantic; Capítulo 2 - Different speciation processes in a cryptobenthic reef fish from the 
Western Tropical Atlantic (artigo já publicado no periódico Hydrobiologia); Capítulo 3 - The 
 6 
evolutionary history of the ringnecks blenny (Parablennius pilicornis)an amphi-Atlantic 
cryptobenthic reef fish. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 7 
 
 
 
 
 
 
 
 
 
 
2. Capítulos 
 
 
 
 
 
 
 
 8 
 
 
 
 
 
 
 
 
 
 
 
Capítulo 1 
 
 
Genetic structure and diversity in the seaweed blenny (Parablennius marmoreus), a 
cryptobenthic fish endemic to the western Atlantic 
Ricardo Marques Dias, Anderson Vilasboa de Vasconcellos & Paulo Cesar de Paiva 
 
 9 
Running title: Genetic structure and diversity in Parablennius marmoreus 
 
 
Genetic structure and diversity in the seaweed blenny (Parablennius marmoreus), a 
cryptobenthic fish endemic to the western Atlantic 
 
Ricardo M. Dias
1, 2 *
; Anderson Vilasboa
3
; Paulo C. Paiva
1
 
 
1
 Laboratório de Polychaeta, Universidade Federal do Rio de Janeiro, Departamento de 
Zoologia, Instituto de Biologia, Av. Brigadeiro Trompowski s/n - CCS Bloco A, Ilha do 
Fundão, RJ, Brazil 
2
 Universidade Federal do Rio de Janeiro, Museu Nacional, Setor de Ictiologia, Departamento 
de Vertebrados, Quinta da Boa vista, s/n, 20940-040, Rio de Janeiro, RJ, Brazil 
3
 Laboratório de Genética Pesqueira e da Conservação, Universidade do Estado do Rio de 
Janeiro, Departamento de Genética, Av. São Francisco Xavier, 524 - Pavilhão Haroldo 
Lisboa, sala 201, Maracanã, RJ, Brazil 
 * Corresponding author: Ricardo Marques Dias. E-mail address: ricdias77@gmail.com 
 
 
 
 
 
 
 
 
 
 
 10 
Abstract 
Parablennius marmoreus is a widely distributed cryptobenthic fish endemic to the western 
Atlantic from New York, on the northeastern coast of the USA, to Santa Catarina, on the 
southeastern coast of Brazil. However, the combination of its association with benthic 
environments, the low mobility of adult individuals, its cryptic behavior, demersal eggs, and 
short larval phase, may combine to affect the species’ dispersal ability, which may lead to 
genetic structuring among its populations. In this study, we conducted phylogeographic 
analyses of P. marmoreus based on mitochondrial (cytochrome oxidase I) and nuclear (first 
intron S7) genes, across its distribution in the western Atlantic. Our results indicated the 
existence of two consistently structured lineages within P. marmoreus: one in the 
northwestern Atlantic and the other restricted to the Brazilian provinces in the southwestern 
Atlantic, both probably isolated by the Amazon River barrier. Additionally, even though the 
Brazilian coast is a highly heterogeneous environment, no genetic structure was detected 
among P. marmoreus lineages over its large extension. However, our results suggest that the 
biodiversity of the species complex in the Tropical Northwestern Atlantic province is still 
underestimated. Together, these results provide insights on the evolutionary patterns and 
oceanographic barriers in the western tropical Atlantic. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Key words 
 
Blennioidei, Phylogeography, Evolution, Amazon river barrier, Species complex 
 11 
 
 
Introduction 
 
Phylogeographic studies have contributed to understanding the evolution and distribution 
patterns of reef fishes, via the spatial association between clusters of alleles and 
geographic/ecological barriers (Avise, 2009). In coastal marine environments, the barriers to 
gene flow are difficult to characterize and are rarely absolute (Rocha, 2007). In the Western 
Atlantic, the most important known biogeographic barrier is the freshwater discharge of the 
Orinoco and Amazon rivers that separated the Brazilian and Caribbean provinces 
approximately 7 million years ago (Mya) (Figueiredo et al., 2009; Hoorn et al., 2010). This 
barrier not only restricts dispersal, but also allows occasional crossings, leading to the 
establishment of new populations and species in allopatry or parapatry (Floeter et al., 2008). 
Similarly, several studies have recognized the Amazon-Orinoco plume in the North Brazil 
Shelf (NBS) province (sensu Spalding et al., 2007) as an influential barrier to the formation of 
pairs of sister species of shallow-reef fishes in the Western Tropical Atlantic (Rocha, 2003; 
Floeter et al., 2008; Rodríguez-Rey et al., 2017). 
Despite increased knowledge about the biogeography and evolution of southwestern 
Atlantic reef fish (Floeter et al., 2008; Pinheiro et al., 2018), many questions about 
evolutionary processes and biogeographic patterns remain to be settled. In particular, 
cryptobenthic reef fish have received far less attention than more conspicuous species, due to 
their cryptic features (Ahmadia et al., 2012), even though cryptobenthic reef fish make up a 
considerable fraction of total fish diversity on coral reefs (Depczynski et al., 2007; Eschmeyer 
et al., 2010; Allen, 2015). In addition, few studies have attempted to delimit inter- and 
intraspecific lineages using molecular data, which could improve our understanding of species 
boundaries, population structure, and geographical barriers of Atlantic marine organisms 
(Turchetto-Zolet et al., 2013). Moreover, if the marine provinces inhabited by the species 
coincide with important geographic or ecological barriers, fine-scale genetic structure may 
 12 
also occur. This is the case for different western Atlantic marine organisms: isopods (Mattos 
et al., 2018), polychaetes (Barroso et al., 2010), crabs (Gama-Maia et al., 2016; Mattos et al., 
2018), and fishes (Mai et al., 2014; Victor, 2015; Rodríguez-Rey et al., 2017; Dias et al., 
2019). Nevertheless, the patterns and processes underlying the genetic distribution of marine 
organisms in South America are largely unknown, and research on these topics can help 
understand macroecological patterns and the impacts of geological features on species 
diversification, as well as identify areas of high conservation priority (Turchetto-Zolet et al., 
2013). 
The seaweed blenny, Parablennius marmoreus (Poey, 1875), is a cryptobenthic fish 
endemic to the western Atlantic (Levy et al., 2013). According to Carvalho-Filho (1999), the 
species has a wide distribution, occurring from New York, on the northeastern coast of the 
USA, to Santa Catarina, on the southeastern coast of Brazil. Its wide distribution encompasses 
six biogeographic provinces (sensu Spalding et al., 2007), three in northwestern Atlantic 
(NWA) – Cold Temperate Northwest Atlantic, Warm Temperate Northwest Atlantic, Tropical 
Northwestern Atlantic – and three in the southwestern Atlantic (SWA) – North Brazil Shelf, 
Tropical Southwestern Atlantic, and Warm Temperate Southwestern Atlantic. These marine 
provinces are cohesive ecological units that are likely to encompass the life history of many 
taxa – from the most sedentary species to mobile and dispersive ones – via geographic 
isolation, upwelling, nutrient inputs, freshwater influx, temperature regimes, sediments, 
currents, and bathymetric or coastal complexity (Spalding et al., 2007). 
However, recent assessments of the reef fish assemblages from the North-Northeast 
subprovince on the Brazilian coast (sensu Pinheiro et al., 2018) suggest that P. marmoreus 
does not occur in this subprovince (Rocha & Rosa, 2001; Honório & Ramos, 2010; Garcia et 
al., 2015; Moura et al., 2016). Moreover, its close association with the benthos, the low 
mobility of adult individuals, its cryptic behavior, demersal eggs, and short larval phase 
(Brogan, 1994; Depczynski & Bellwood, 2003) may combine to affect the species’ dispersal 
 13 
ability, which may lead to genetic structuring among its populations (Hohenlohe, 2004; 
Cowen & Sponaugle, 2009). Currently, the seaweed blenny is listed by the International 
Union for Conservation of Nature (IUCN) as ‘least concern’ (Williams, 2014), despite it still 
being unclear whether it actually corresponds to a single species, if genetic diversity is 
partitioned evenly across the species range, or if populations are connected by geneflow. 
Our hypothesis is that P. marmoreus populations are genetically structured in two 
lineages, one occurring in the northwestern Atlantic and the other in the southwestern 
Atlantic, as a result of the influence of the Amazon-Orinoco Plume barrier. Thus, the main 
goal of this study was to investigate phylogeographic patterns in P. marmoreus in the western 
tropical Atlantic. The specific objectives were: (1) to determine the genetic structure of P. 
marmoreus across its entire geographic distribution, identifying its main genetic lineages; and 
(2) to investigate the influence of the Amazon-Orinoco Plume barrier on the genetic 
structuring of P. marmoreus. 
 
Materials and methods 
Sampling 
Parablennius marmoreus individuals (Fig. 1) were collected using hand nets or plastic bags 
during daytime SCUBA and free-dives in shallow waters. Samples were collected between 
November 2015 and October 2017 at six localities across its distribution in the western 
Atlantic: one in the NWA (USA) – Blue Heron Bridge, Florida [FL] (26°47’00”N 
80°02’26”W; N = 10); and five in the SWA (Brazil) – Frades Island, Bahia [BA] (12°46’47”S 
38°37’06”W; N = 14); Rasas Islands, Espírito Santo [ES] (20°40’40”S 40°21’55”W; N = 13); 
Arraial do Cabo, Rio de Janeiro [RJ] (22°57’54”S 42°00’30”W; N = 14); Cabras Island, São 
Paulo [SP] (23°49’47”S 45°23’31”W; N = 15); and Xavier Island, Santa Catarina [SC] 
(27°36’36”S 48°23’13”W; N = 12) (Fig. 2 and Online Resource 1). 
Specimens were anesthetized and euthanized with a eugenol-alcohol solution 
 14 
according to Fernandes et al. (2017), prior to the removal of a small sample of muscle tissue, 
which was preserved in anhydrous ethanol. All specimens were deposited in the fish 
collections of Museu Nacional/Universidade Federal do Rio de Janeiro (MNRJ) (Online 
Resource 1). Samples were obtained under permits 50598-3/2017, issued by Ministério do 
Meio Ambiente do Brasil/Instituto Chico Mendes de Conservação da Biodiversidade/Sistema 
de Autorização e Informação em Biodiversidade (SISBIO), and 02-002040, issued by the 
Florida Fish and Wildlife Conservation Commission, Division of Marine Fisheries 
Management (FWC/MFM). Additional sequences of P. marmoreus individuals from five 
localities in the NWA – Florida [FL], Blue Heron Bridge (26°47’00”N 80°02’26”W; N = 11) 
and Florida Keys (24°39’25”N 81°17’49”W; N = 1); Trinidad and Tobago [TT] (11°19’15”N 
60°32’56”W; N = 3); Mexico [MX] (21°29’09”N 86°48’14”W; N = 8); and Panama [PA] 
(9°22’30”N 79°57’32”W; N = 1) – were obtained from GenBank and BOLD (Barcode of Life 
Data System) (Online Resource 1) to extend the molecular analysis to other localities. 
 
DNA extraction, amplification, and sequencing 
Genomic DNA was extracted from muscle tissue samples of each specimen using the DNeasy 
Blood & Tissue Kit (QIAGEN, Hilden, Germany). One fragment each of mitochondrial 
DNA, cytochrome c oxidase subunit I (COI), and nuclear DNA (first intron of the nuclear S7 
ribosomal protein gene, S7) were amplified using the following primers: COI – Fish F1 (5' 
TCA ACC AAC CAC AAA GAC ATT GGC AC 3') and Fish R1 (5' TAG ACT TCT GGG 
TGG CCA AAG AAT CA 3') (Ward et al., 2005), and S7 – S7RPEX1F (5' TGG CCT CTT 
CCT TGG CCG TC 3') and S7RPEX2R (5' AAC TCG TCT GGC TTT TCG CC 3') (Chow & 
Hazama, 1998). 
Amplification reactions included 1 µl DNA, 10 µl Taq Master Mix (Promega, 
Madison, WI, USA), 5 µl Miliq water, and 2 µl of each primer in a final volume of 20 µl. All 
reactions were performed with an initial denaturation step of 4 min at 94 °C, followed by 35 
 15 
cycles of 1 min of denaturation at 94 °C, 1 min of annealing at 50 °C, and 1 min of extension 
at 72 °C; and a final extension of 5 min at 72 °C. Both strands of the PCR products were 
purified and sequenced by Macrogen Inc. (Korea). The forward and reverse sequences 
obtained were edited using Seqman 5.01 (DNASTAR Inc., http://www.dnastar.com), aligned 
by the ClustalW algorithm implemented in MEGA 6.0 (Tamura et al., 2013), and checked 
manually for misalignments. The phase of diploid nuclear sequences was reconstructed using 
PHASE (Stephens & Donnelly, 2001) as implemented in DnaSP 5.10 (Librado & Rozas, 
2009), using default settings and including in the proceeding analyses only allelic states with 
probability higher than 70%, as recommended by Stephens et al. (2004). All haplotype 
sequences were deposited in GenBank (Online Resource 1). The heterozygous sequences 
were deposited in GenBank with the degenerate bases following the IUPAC ambiguity code. 
 
Phylogenetic analysis 
The phylogenetic analysis was performed by Bayesian speciation birth–death model to allow 
for inter- and intraspecific variations in the dataset following Ritchie et al. (2016), using 
BEAST 1.7 (Drummond et al., 2012). The analysis was conducted with a relaxed lognormal 
clock and uncorrelated substitution rates among branches, using default priors and run for 20 
million generations, with sampling every 2000 generations. The convergence among Markov 
Chain Monte Carlo (MCMC) was checked using Tracer 1.7 (Rambaut et al., 2018), with the 
first 15% of trees removed as burn-in, and a consensus tree assessing the posterior probability 
values of each clade was generated using TreeAnnotator 1.8.1 software (Drummond et al., 
2012). Molecular clock calibration was specific for mitochondrial fragment COI based on the 
sequence divergence between Hypsoblennius invemar Smith-Vaniz & Acero, 1980 from the 
Gulf of Mexico, Texas and Hypsoblennius brevipinnis ( nther, 1861) from the Pacific coast 
of Panama, assuming the two species diverged after the formation of the Isthmus of Panama 
(ca. 3.5 Mya). Mean net genetic divergence between this pair of species was calculated with 
 16 
MEGA 6.0 (Tamura et al., 2013) using the Kimura 2-parameter distance model (K2P) 
(Kimura, 1980). The net average distance (divergence estimate) between this pair of species 
was 0.165 substitutions per site. The substitution rate (half the divergence estimate) divided 
by 3.5 Myr gives a clock rate estimate of 0.02 substitutions per site per Myr, according to 
Levy et al. (2013). The nucleotide substitution model was selected using the Bayesian 
information criterion (BIC) as implemented in jModelTest 2.1 (Posada, 2008), which 
suggested the HKY+I and HKY+G models for the COI and S7 data sets, respectively. The 
data set included all the COI and S7 haplotypes of P. marmoreus and additional sequences of 
Lipophrys pholis (Linnaeus, 1758) (GenBank), selected as the outgroup taxon (Online 
Resource 1). Nucleotide divergences were estimated in MEGA 6.0 (Tamura et al., 2013) 
using the K2P model (Kimura, 1980). 
Two species delimitation methods were implemented on the COI data set, the 
Automatic Barcode Gap Discovery (ABGD; Puillandre et al., 2012) and the Poisson Tree 
Processes (PTP; Zhang et al., 2013). The ABGD method was calculated using K80 distance in 
the online version (http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html, last accessed 07 
December 2018), whereas the PTP method was estimated in the web interface of Bayesian 
PTP (bPTP) (http://species.h-its.org/ptp/, last accessed 07 December 2018), using the 
estimated maximum-likelihood (ML) trees as the input tree and 5×10
5
 MCMC generations. 
 
Phylogeographic analyses 
The genealogical relationships among haplotypes were assessed by constructing haplotype 
networks with PopART 1.7 (Leigh & Bryant, 2015) using the TCS method (Clement et al., 
2002). The haplotype networks were individually constructed for each marker (COI and S7). 
The genetic diversity parameters of the P. marmoreus lineages, including number of 
haplotypes (H), number of polymorphic sites (S), haplotype diversity (h), and nucleotide 
diversity (π) were estimated in ARLEQUIN 3.5 (Excoffier & Fischer, 2010). 
http://www.abi.snv.jussieu.fr/public/abgd/abgdweb.html
 17 
The population geneticstructure of P. marmoreus was first estimated by the fixation 
index (FST), with 1000 random permutations to test whether the null hypothesis of no 
population genetic structure could be rejected. Next, three different a priori hypotheses were 
tested using the analysis of molecular variance (AMOVA) on the COI data set (broader 
geographical representation). Firstly, using all samples, we tested: (1) a hypothesis based on 
the influence of the Amazon-Orinoco plume as a barrier to gene flow of shallow-reef fishes 
between the northwestern and southwestern Atlantic (NWA/SWA); and (2) a hypothesis 
based on the three provinces sampled – Tropical Northwestern Atlantic 
(FL+MX+TT)/Tropical Southwestern Atlantic (BA+ES+RJ)/Warm Temperate Southwestern 
Atlantic (SP+SC) as proposed by Spalding et al. (2007). Next, using samples from the 
southwestern Atlantic, we tested (3) a hypothesis based on the two provinces sampled in the 
Brazilian coast – Tropical Southwestern Atlantic (BA+ES+RJ)/Warm Temperate 
Southwestern Atlantic (SP+SC). The FST values and AMOVA were calculated in 
ARLEQUIN 3.5 (Excoffier & Fischer, 2010). 
 In addition, to infer multilocus genetic structure in the P. marmoreus complex, we 
performed Bayesian assignment tests using both mitochondrial (COI) and nuclear (S7) genes 
in GENELAND (Guillot et al., 2005; R Development Core Team 2006). The following 
parameters: ten independent runs, 300,000 Markov chain Monte Carlo iterations of K from 1 
to 10, sampling every 300 iterations, and the first 300 samples removed as burn-in were used 
to determine the most probable number of populations and detect possible spatial genetic 
breaks among lineages without previously assigning individuals to any cluster. 
 
Results 
We recovered 587-bp sequences for COI from 101 P. marmoreus individuals, resulting in 44 
polymorphic sites and 28 haplotypes, and 452-bp sequences for S7 from 51 individuals, with 
57 polymorphic sites and 54 phased alleles. COI nucleotide diversity was low to moderate 
 18 
(overall π = 0.01; range: 0.0002–0.0126), and haplotype diversity was low to high (overall h = 
0.622; range: 0.133–1.0) (Online Resource 2). 
Phylogenetic analysis of COI and S7 markers showed congruent topologies forming 
two major geographical clades: one comprised the samples from the northwestern Atlantic 
(NWA clade), which may be referred to as P. marmoreus sensu stricto and forms the sister 
group of a SWA clade that includes individuals from the Brazilian coast in the southwestern 
Atlantic (Fig. 3). The divergence time between clades was approximately 650 thousand years 
ago (ka) (COI) [95% Highest Posterior Density (HPD) = 0.42–0.92 ka] in the Middle 
Pleistocene. The NWA clade diverged from the SWA clade by 2.3% for COI and 3.9% for S7 
(K2P distances). 
In addition, the ABDG species delimitation method based on the COI dataset 
recovered the same two lineages (NWA and SWA) in the P. marmoreus complex. 
Considering a prior maximum divergence of intraspecific diversity (P) of 0.001–0.0046, the 
ABGD method indicated that all individuals from the NWA (MX+TT+FL) belong to the 
same lineage (NWA clade), whereas all the individuals from the Brazilian coast 
(BA+ES+RJ+SP+SC) belong to another lineage (SWA clade) (Fig. 3). Likewise, the bPTP 
method also identified that the individuals from the Brazilian coast belong to the same lineage 
(SWA clade), with a posterior probability of 0.822. However, the bPTP method indicated that 
individuals from the Caribbean province (NWA clade) belong to four different lineages (Fig. 
3). 
The COI and S7 haplotype networks showed the lack of shared haplotypes between 
samples from the NWA clade and the SWA clade, which were separated by at least 14 
mutational steps (COI) and 16 mutational steps (S7). In the SWA clade, only one haplotype 
was shared among all localities and with high frequency, whereas all others were exclusive 
haplotypes with low frequency. In contrast, in the NWA clade, three different haplogroups 
were recovered from the COI dataset one exclusive to Trinidad and Tobago, with two 
 19 
haplotypes, one exclusive to Mexico, with six haplotypes, and another, with 12 haplotypes, 
shared between Florida and Mexico (Fig. 4). 
The AMOVA performed with the COI data set showed statistically significant results 
for all hypotheses tested; however, when all samples were included, most of the genetic 
variation (K = 2, ΦCT = 80.74%) was explained by the differences between groups (NWA and 
SWA), supporting the Amazon-Orinoco barrier hypothesis. For populations within the SWA, 
the genetic variation was best explained by partitioning within populations (ΦST = 93.6%) 
(Table 1). 
The pairwise FST values revealed significant differences between the NWA clade 
(MX+TT+FL) and SWA clade (BA+ES+RJ+SP+SC) localities (FST = 0.70325–0.99205), 
between NWA localities (MX, TT, and FL) (FST = 0.38776–0.89968), and between Xavier 
Island (SC) (the southernmost coastal locality sampled here) and all other Brazilian localities 
(BA, ES, RJ, and SP) (FST = 0.10075–0.13476) (Table 2). 
In addition, the multilocus GENELAND analysis suggested two genetically different 
groups consistent with the phylogenetic analysis (K = 2, Fig. 5a). One genetic cluster 
comprises sampling localities from the NWA (FL, MX, and TT) and the other consists of 
sampling localities from the SWA (BA, ES, RJ, SP, and SC) (Fig. 5). 
 
Discussion 
Main lineages in the Parablennius marmoreus complex 
The phylogeographic analyses of the Parablennius marmoreus species complex revealed the 
presence of at least two lineages distributed throughout the western tropical Atlantic, one 
from the northwestern Atlantic and the other from the southwestern Atlantic. However, the 
biodiversity of the group in the NWA may still be underestimated, as only one of the three 
biogeographical provinces that encompass the region was sampled (Tropical Northwestern 
Atlantic). In fact, our results (bPTP species delimitation method, Fig. 3a and haplotype 
 20 
network, Fig. 4a) suggest that the biodiversity in the Tropical Northwestern Atlantic province 
is still underestimated, although a similar degree of cryptic genetic diversity was detected in 
three other cryptobenthic reef fish species studied in the same area (Victor, 2015; Dias et al., 
2019). The populations of cryptobenthic reef fishes can quickly become isolated from each 
other by the appearance of new physical barriers to gene flow. In addition, reproductive 
incompatibility may evolve rapidly due to rapid generation turnover, whereas barriers to gene 
flow may be partially permeable or temporary and can consist of strong or temporally 
transient surface currents or temporary land barriers exposed during glacioeustatic sea-level 
fluctuations (Cowman & Bellwood, 2011; Brandl et al., 2018). 
The pattern of high cryptic genetic diversity observed for the P. marmoreus lineage 
from the Tropical Northwestern Atlantic province may reflect more general biogeographic 
patterns for the Caribbean province and, according to Victor (2015), demersal-spawning 
fishes, especially the gobioid and blennioid perciforms, exhibit a pattern of cryptic speciation 
into parapatric species complexes in the Caribbean province. 
Regarding the southwestern Atlantic lineage, although the Brazilian coast is a highly 
heterogeneous environment, influenced by different marine currents and freshwater and 
sediment flows, overall no genetic structure was found over its large extension. The only 
exception was the southernmost locality (SC), where there was a weak sign of structuring 
compared to the other localities (BA, ES, RJ, and SP) (Table 2). 
 
The influence of recurring sea-level changes during Pleistocene glacial-interglacial 
cycles on the speciation of P. marmoreus 
The differentiation of P. marmoreus lineages from the NWA and SWA can be attributed to an 
allopatric speciationprocess, with the Amazon-Orinoco plume likely acting as a geographical 
barrier. This riverine complex has already been recognized by several authors as an influential 
barrier to the formation of pairs of sister species of shallow-water reef fishes in the western 
 21 
tropical Atlantic (Rocha, 2003; Rocha, 2004; Bernal & Rocha, 2011; Dias et al., 2019). 
Indeed, small-body cryptobenthic species with demersal-spawning or brooding seem to be 
most affected by the Amazon River freshwater and sediment discharge (Floeter et al., 2008). 
Using AMOVA with the mtDNA COI data set, we found support for this scenario, 
with 80.74% of all genetic variance partitioned between the NWA and SWA lineages (Table 
1). In addition, the spatial assignment of individual multilocus genotypes to the two inferred 
genetic clusters allowed identify a possible association of the Amazon barrier with the process 
of divergence between P. marmoreus (Fig. 5). However, our results indicated that the 
divergence between P. marmoreus lineages from the NWA and SWA occurred during the 
Pleistocene (approximately 650 ka, Fig. 3). This is much later than the time when the Amazon 
River began to flow to the Atlantic in the Neogene (roughly 6.8–4.5 Mya, Hoorn et al., 2010), 
becoming an influential biogeographic barrier (the Amazon-Orinoco plume) to the formation 
of pairs of sister species between tropical habitats of the Caribbean and Brazilian provinces 
(Rocha, 2003). Similarly, several pairs of sister species of shallow-water reef fish that are 
present on both sides of the Amazon River barrier diverged much later than the emergence of 
this barrier. (Rocha et al., 2002; Rocha, 2004; Robertson et al., 2006; Rodríguez-Rey et al., 
2017). The effectiveness of the Amazon barrier was strongly influenced by recurrent sea-level 
changes during Pleistocene glacial-interglacial cycles, a period of intermittent connectivity 
between the Caribbean and Brazilian provinces (Moura et al., 2016; Rodríguez-Rey et al., 
2017). Fluctuations in the effectiveness of this natural barrier may provide a mechanism for 
much of the recent diversification of reef fishes in the western Atlantic (Floeter et al., 2008). 
Although the Amazon-Orinoco plume may be an influential biogeographic barrier to 
promote divergence of P. marmoreus lineages from the Caribbean and Brazilian provinces, 
the absence of P. marmoreus populations in the North-Northeast subprovince on the Brazilian 
coast cannot be attributed to this barrier. Instead, the absence of P. marmoreus in this region 
south of the Amazon-Orinoco plume may be the result of a local extinction process, due to 
 22 
loss of habitat during the Last Glacial maximum (Ludt & Rocha, 2015). In fact, unlike the 
southeastern Brazilian continental shelf, the northeastern coastal shelf is narrow and shallow, 
and has a significantly smaller coastal area during sea-level lowstands, which might have 
resulted in a higher rate of extinction of shallow-water marine taxa in the North-Northeast 
subprovince (Rocha, 2003; Ludt & Rocha, 2015; Pinheiro et al., 2018). Therefore, the 
recurring sea-level changes during Pleistocene glacial-interglacial cycles, which have directly 
increased the effectiveness of the Amazon barrier and reduced the availability of habitable 
shallow areas, mainly in the North-Northeast subprovince, may have indirectly driven the 
divergence of P. marmoreus lineages from the Caribbean and Brazilian provinces. 
 
Taxonomic implications 
Molecular taxonomy gained traction in reef fish systematics in the early 2000s and, especially 
when combined with morphological approaches (Baldwin et al., 2011), led to a substantial 
increase in the number of cryptobenthic reef fish species known to science (Victor, 2015). 
Thus, several species complexes have been found in such unrelated marine organisms as fish 
(Santos et al., 2006; Baldwin et al., 2011; Rodríguez-Rey et al., 2017), polychaetes (Barroso 
et al., 2010), and crustaceans (Mattos et al., 2018). 
The two main P. marmoreus lineages (NWA and SWA) show consistent 
differentiation for both mitochondrial and nuclear markers, suggesting that these lineages 
should have their taxonomic status verified. However, to date, the single study that has 
examined the taxonomy of P. marmoreus in the Brazilian province using a morphological 
approach analyzed only specimens from the state of Rio de Janeiro (Rangel & Guimarães, 
2010), without checking for possible morphological variations across its distribution. In most 
cases, reexamining coloration, pigmentation, and morphology across genetic groups reveals 
corresponding diagnostic characters, thus reinforcing the presence of new species (Brandl et 
al., 2018). 
 23 
 
Implications for conservation 
The IUCN Red List is a critical indicator of the health of the world’s biodiversity. Far more 
than a list of species and their status, it is a powerful tool to inform and catalyze action for 
biodiversity conservation and policy change, critical to protecting natural resources (IUCN 
2019). Currently, Parablennius marmoreus is considered by the IUCN a species widely 
distributed in the western Atlantic, from New York to Brazil, over shallow rocky reef 
habitats, with a presumed large overall population with no known major threats. Although it 
has been recorded in the commercial aquarium trade, current levels of exploitation are not 
considered to represent any threat to the species as a whole. Therefore, it is listed as ‘Least 
Concern’ (Williams, 2014). 
Our results showed that P. marmoreus is actually a species complex, with at least two 
distinct lineages: the NWA clade in the Caribbean province and the SWA clade restricted to 
the Brazilian provinces. This result implies a more restricted distribution and smaller 
population size, consequently reducing the resilience capacity of both lineages. Thus, future 
efforts should be directed at evaluating the actual conservation status of the P. marmoreus 
species complex. 
 
Acknowledgements 
 We are grateful to N. Y. N. Dias, G. Araújo, R. Barroso, M. R. Britto and P. C. Paiva 
for help during field collections, to V. C. Seixas for help with analysis, and to Abudefduf 
Atividades Subaquáticas and DeepTrip for logistical support during fieldwork. This paper is 
part of the PhD Thesis of Ricardo Marques Dias at the Biodiversity and Evolutionary Biology 
Graduate Program at the Federal University of Rio de Janeiro (PPGBBE/UFRJ); RMD is 
supported by Conselho Nacional de Desenvolvimento Cientéfico e Tecnológico (CNPq) 
Grant Number 132124/2013-0 and Coordenação de Aperfeiçoamento de Pessoal de Nível 
 24 
Superior—Brasil (CAPES) Grant Number 2346/2011. PCP is supported by CNPq Grant 
Number 304321/2017-6. 
 
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 32 
Table 1 Analysis of molecular variance (AMOVA), evaluating different hypotheses for 
mtDNA Cytochrome c oxidase subunit I in Parablennius marmoreus species complex. 
Location acronyms are provided in Fig. 2. Details of the hypothesis are presented in the text. 
 
Hypothesis 
(number of groups) 
Group Composition ΦCT ΦSC ΦST 
 All Samples 
Amazon barrier (2) NWA/SWA 80.74* 10.66* 8.61* 
Provinces (3) TNA/TSA/WTSA 
 (FL+MX+TT/BA+ES+RJ/SP+SC) 70.18* 18.09* 11.73* 
 Within the Brazilian provinces (TSA) 
Provinces (2) TSA/WTSA 
 (BA+ES+RJ/SP+SC) 1.15 6.80* 92.06* 
* P < 0.05. 
 
 
 
 
 
 
 
 
 
 
 
 
 33 
 
Table 2 Pairwise FST values for Cytochrome c oxidase subunit I of the Parablennius marmoreus 
species complex in the Western Atlantic. Location acronyms are provided in Fig. 2 
 
 
MX TT FL BA ES RJ SP 
TT 0.38776* - - - - - - 
FL 0.60850* 0.89968* - - - - - 
BA 0.76952* 0.98115* 0.94691* - - - - 
ES 0.75991* 0.97990* 0.94541* 0.00021 - - - 
RJ 0.77576* 0.99205* 0.95138* -0.00000 0.00592 - - 
SP 0.77837* 0.98226* 0.94834* 0.00017 0.00076 -0.00478 - 
SC 0.70325* 0.92705* 0.92390* 0.10899* 0.10075* 0.13476* 0.11696* 
*p <0.05. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 34 
Figure Legends 
Fig. 1 Specimen of Parablennius marmoreus photographed in situ, Arraial do Cabo, RJ, 
Brazil. Photo by R. M. Dias 
 
Fig. 2 Sampled sites along the distribution of the Parablennius marmoreus species complex. 
Yellow site - type locality (Cuba); triangles - sequences obtained from GenBank and circles - 
sampled sites in the present study. Sampling sites and their acronyms: Panama - PA; Blue 
Heron Bridge and Florida Key's - FL; Mexico - MX; Trinidad and Tobago -TT, from 
Northwestern Atlantic in blue, and the Frades Island, Bahia - BA; Rasas Islands, Espírito 
Santo - ES, Arraial do Cabo, Rio de Janeiro - RJ; Cabras Island, São Paulo - SP; Xavier 
Island, Santa Catarina - SC from Southwestern Atlantic in green. Gray lines delimit marine 
ecoregions (Spalding et al., 2007). Brown area indicates the Amazon–Orinoco Plume barrier 
(Rocha, 2003) 
 
Fig. 3 Bayesian phylogenies of Parablennius marmoreus complex based on: a) COI, b) S7; 
with estimates of divergence times (horizontal bars) and a posteriori probability values for the 
existence of each clade. The horizontal blue bars indicate 95% credibility intervals of node 
age estimation. Samples from Northwestern Atlantic clade in blue, samples from 
Southwestern Atlantic clade in green, and outgroups in grey 
 
Fig. 4 Haplotype networks obtained from TCS analysis, using 95% probability of parsimony, 
for mtDNA (COI) and nuDNA (S7) of the specimens of the Parablennius marmoreus 
complex species from the Western Tropical Atlantic. Each haplotype is represented by a 
circle, with the size of the circle proportional to haplotype frequency 
 
 35 
Fig. 5 Bayesian cluster analysis output from GENELAND. (a) The main histogram shows the 
frequency of inferred K-value across runs. (b - c) Maps of posterior probabilities to belong to 
one of K = 2 clusters (b) Southwestern Atlantic clade and (c) Northwestern Atlantic clade, for 
samples of the Parablennius marmoreus complex species from the Western Tropical Atlantic. 
The axes indicate latitude and longitude. The black dots correspond to sampling sites and the 
location acronyms are provided in Fig. 2 
 
Online Resource 1. List of the specimens included in this study, including species, 
biogeographic provinces, major clades recovered in the phylogeny analyses, sampling site, 
geographic coordinates and sequence accession number. Species obtained from GenBank are 
marked with an * and from BoldSystem with an ** 
 
Online Resource 2. Molecular parameters of the Parablennius marmoreus in the Western 
Atlantic for mtDNA (COI) and nuDNA (S7). Parameters include total number of individuals 
analyzed in each location (N), number of haplotypes (H), number of polymorphic sites (S), 
haplotype diversity (h), nucleotide diversity 
 
 
 
 
 
 
 
 
 36 
 
 
Fig. 1 Specimen of Parablennius marmoreus photographedin situ, Arraial do Cabo, RJ, 
Brazil. Photo by R. M. Dias 
 
 
 
 
 
 
 
 
 
 
 
 
 37 
 
Fig. 2 Sampled sites along the distribution of the Parablennius marmoreus species 
complex. Yellow site - type locality (Cuba); triangles - sequences obtained from GenBank 
and circles - sampled sites in the present study. Sampling sites and their acronyms: Panama - 
PA; Blue Heron Bridge and Florida Key's - FL; Mexico - MX; Trinidad and Tobago -TT, 
from Northwestern Atlantic in blue, and the Frades Island, Bahia - BA; Rasas Islands, 
Espírito Santo - ES, Arraial do Cabo, Rio de Janeiro - RJ; Cabras Island, São Paulo - SP; 
Xavier Island, Santa Catarina - SC from Southwestern Atlantic in green. Gray lines delimit 
marine ecoregions (Spalding et al., 2007). Brown area indicates the Amazon–Orinoco Plume 
barrier (Rocha, 2003) 
 38 
 
Fig. 3 Bayesian phylogenies of Parablennius marmoreus complex based on: a) COI, b) S7; 
with estimates of divergence times (horizontal bar) and a posteriori probability values for the 
existence of each clade. The horizontal blue bar indicate 95% credibility intervals of node age 
estimation. Samples from Northwestern Atlantic clade in blue, samples from Southwestern 
Atlantic clade in green, and outgroups in grey 
 
 
 
 
 
 39 
 
 
Fig. 4 Haplotype networks obtained from TCS analysis, using 95% probability of parsimony, 
for mtDNA (COI) and nuDNA (S7) of the specimens of the Parablennius marmoreus 
complex species from the Western Tropical Atlantic. Each haplotype is represented by a 
circle, with the size of the circle proportional to haplotype frequency 
 
 
 
 
 
 
 40 
 
 
Fig. 5 Bayesian cluster analysis output from GENELAND. (a) The main histogram shows the 
frequency of inferred K-value across runs. (b - c) Maps of posterior probabilities to belong to 
one of K = 2 clusters (b) Southwestern Atlantic clade and (c) Northwestern Atlantic clade, for 
samples of the Parablennius marmoreus complex species from the Western Tropical Atlantic. 
The axes indicate latitude and longitude. The black dots correspond to sampling sites and the 
location acronyms are provided in Fig. 2 
 
 
 
 
 
 
 
 
 
 
 41 
Online Resource 1 List of the specimens included in this study, including species, biogeographic provinces, major clades recovered in the 
phylogeny analyses, sampling site, geographic coordinates and sequence accession number. Species obtained from GenBank are marked with an * 
and from BoldSystem with an ** 
 
 
Species Voucher N˚ Specimen Sampling site Longitude Latitude COI S7 
Parablennius marmoreus MNRJ49574 PM_FL_01 Blue Heron Bridge, Florida, EUA -80.041 26.783 waiting for n˚ waiting for n˚ 
Parablennius marmoreus MNRJ49574 PM_FL_02 Blue Heron Bridge, Florida, EUA -80.041 26.783 waiting for n˚ waiting for n˚ 
Parablennius marmoreus MNRJ49574 PM_FL_03 Blue Heron Bridge, Florida, EUA -80.041 26.783 waiting for n˚ waiting for n˚ 
Parablennius marmoreus MNRJ49574 PM_FL_04 Blue Heron Bridge, Florida, EUA -80.041 26.783 waiting for n˚ waiting for n˚ 
Parablennius marmoreus MNRJ49574 PM_FL_05 Blue Heron Bridge, Florida, EUA -80.041 26.783 – waiting for n˚ 
Parablennius marmoreus MNRJ49574 PM_FL_06 Blue Heron Bridge, Florida, EUA -80.041 26.783 waiting for n˚ waiting for n˚ 
Parablennius marmoreus MNRJ49574 PM_FL_07 Blue Heron Bridge, Florida, EUA -80.041 26.783 waiting for n˚ waiting for n˚ 
Parablennius marmoreus MNRJ49574 PM_FL_08 Blue Heron Bridge, Florida, EUA -80.041 26.783 waiting for n˚ waiting for n˚ 
Parablennius marmoreus MNRJ49574 PM_FL_09 Blue Heron Bridge, Florida, EUA -80.041 26.783 waiting for n˚ waiting for n˚ 
Parablennius marmoreus MNRJ49574 PM_FL_10 Blue Heron Bridge, Florida, EUA -80.041 26.783 waiting for n˚ waiting for n˚ 
Parablennius marmoreus MNRJ49574 PM_FL_11 Blue Heron Bridge, Florida, EUA -80.041 26.783 waiting for n˚ waiting for n˚ 
Parablennius marmoreus MNRJ49574 PM_FL_12 Blue Heron Bridge, Florida, EUA -80.041 26.783 waiting for n˚ – 
Parablennius marmoreus MNRJ49574 PM_FL_13 Blue Heron Bridge, Florida, EUA -80.041 26.783 JQ841950.1* waiting for n˚ 
Parablennius marmoreus MNRJ49574 PM_FL_14 Blue Heron Bridge, Florida, EUA -80.041 26.783 JQ842630.1* waiting for n˚ 
Parablennius marmoreus MNRJ49574 PM_FL_15 Blue Heron Bridge, Florida, EUA -80.041 26.783 JQ842629.1* – 
Parablennius marmoreus MNRJ49574 PM_FL_16 Blue Heron Bridge, Florida, EUA -80.041 26.783 JQ842628.1* – 
Parablennius marmoreus MNRJ49574 PM_FL_17 Blue Heron Bridge, Florida, EUA -80.041 26.783 JQ842627.1* – 
Parablennius marmoreus MNRJ49574 PM_FL_18 Blue Heron Bridge, Florida, EUA -80.041 26.783 JQ842626.1* – 
Parablennius marmoreus MNRJ49574 PM_FL_19 Blue Heron Bridge, Florida, EUA -80.041 26.783 JQ842625.1* – 
Parablennius marmoreus MNRJ49574 PM_FL_20 Blue Heron Bridge, Florida, EUA -80.041 26.783 JQ842624.2* – 
Parablennius marmoreus MNRJ49574 PM_FL_21 Blue Heron Bridge, Florida, EUA -80.041 26.783 JQ842623.1* – 
 42 
Parablennius marmoreus SMSA7389 PM_FL_22 Blue Heron Bridge, Florida, EUA -80.041 26.783 JQ842621.1* – 
Parablennius marmoreus f9pm322 PM_FL_23 Keys, Florida, EUA -81.297 24.657 LIDMA179-09** – 
Parablennius marmoreus ECOCH7153 PM_MX_01 Isla Mujeres, Quintana Roo, Mexico -86.804 21.486 MXV129-11** – 
Parablennius marmoreus ECOCH7199 PM_MX_02 Isla Mujeres, Quintana Roo, Mexico -86.798 21.492 MXV197-11** – 
Parablennius marmoreus ECOCH7199 PM_MX_03 Isla Mujeres, Quintana Roo, Mexico -86.798 21.492 MXV198-11** – 
Parablennius marmoreus ECOCH7208 PM_MX_04 Isla Mujeres, Quintana Roo, Mexico -86.798 21.492 MXV208-11** – 
Parablennius marmoreus ECOCH7209 PM_MX_05 Isla Mujeres, Quintana Roo, Mexico -86.798 21.492 MXV209-11** – 
Parablennius marmoreus ECOCH7210 PM_MX_06 Isla Mujeres, Quintana Roo, Mexico -86.798 21.492 MXV210-11** – 
Parablennius marmoreus ECOCH7239 PM_MX_07 Isla Mujeres, Quintana Roo, Mexico -86.798 21.492 MXV271-11** – 
Parablennius marmoreus ECOCH7247 PM_MX_08 Isla Mujeres, Quintana Roo, Mexico -86.804 21.486 MXV280-11** – 
Parablennius marmoreus USNM:FISH:TOB9278 PM_TT_01 Pirates Bay, Tobago, Trinidad and Tobago -60.549 11.321 TOBA278-09** – 
Parablennius marmoreus USNM:FISH:TOB9279 PM_TT_02 Pirates Bay, Tobago, Trinidad and Tobago -60.549 11.321 TOBA279-09** – 
Parablennius marmoreus USNM:FISH:TOB9365 PM_TT_03 Arnos Vale Beach, Tobago, Trinidad and Tobago -60.763 11.226 TOBA365-09** – 
Parablennius marmoreus PASTPM1 PM_PA_01 Panama -79.959 9.375 – JQ697303.1* 
Parablennius marmoreus MNRJ45884 PM_BA_01 Ilha dos Frades, Salvador, Bahia, Brazil -38.618 -12.780 waiting for n˚ waiting for n˚ 
Parablennius marmoreus MNRJ45884 PM_BA_02 Ilha dos Frades, Salvador, Bahia, Brazil -38.618 -12.780 waiting for n˚ waiting for n˚ 
Parablennius marmoreus MNRJ45884 PM_BA_03 Ilha dos Frades, Salvador, Bahia, Brazil -38.618 -12.780 waiting for n˚ – 
Parablennius marmoreus MNRJ45884 PM_BA_04 Ilha dos Frades, Salvador, Bahia, Brazil -38.618 -12.780 waiting for n˚ – 
Parablennius marmoreus MNRJ45884 PM_BA_05 Ilha dos Frades, Salvador, Bahia, Brazil -38.618 -12.780 waiting for n˚ waiting for n˚ 
Parablennius marmoreus MNRJ45884 PM_BA_06 Ilha dos Frades, Salvador, Bahia, Brazil -38.618 -12.780 waiting for n˚ – 
Parablennius marmoreus MNRJ45884 PM_BA_07 Ilha dos Frades, Salvador, Bahia, Brazil -38.618 -12.780 waiting for n˚ – 
Parablennius marmoreus MNRJ45884 PM_BA_08 Ilha dos Frades, Salvador, Bahia, Brazil -38.618 -12.780 waiting for n˚ – 
Parablennius marmoreus MNRJ45884 PM_BA_09 Ilha dos Frades, Salvador, Bahia, Brazil -38.618 -12.780 waiting for n˚ – 
Parablennius marmoreus MNRJ45884 PM_BA_10 Ilha dos Frades, Salvador, Bahia, Brazil -38.618 -12.780 waiting for n˚ – 
Parablennius marmoreus MNRJ45884 PM_BA_11 Ilha dos Frades, Salvador, Bahia, Brazil -38.618 -12.780 waiting for n˚ waiting for n˚ 
Parablennius marmoreus MNRJ45884 PM_BA_12 Ilha dos Frades, Salvador, Bahia, Brazil -38.618 -12.780 waiting for n˚ waiting for n˚ 
Parablennius