De Cuba (CNAP), Pelo Apoio Para Realizar Meu Doutorado

UNIVERSIDADE FEDERAL DO PARANÁ

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CURITIBA 2017

Com todo mi amor, a mis padres, a mi querido esposo a mi hija, Lorena Alvarez Castellanos y mi hijo Daniel Antonio Alvarez Castellanos, el nuevo miembro de la familia!! AGRADECIMENTOS

Aos meus pais Antonio Castellanos García e Maria Elena Iglesias e a minha querida sogra Maria Elena Perera, por toda a educação, amor, confiança e contínuo apoio à minha formação acadêmica. Sem a sua ajuda e motivação esse doutorado jamais teria sido feito.

A grande amiga e orientadora Maria Angélica Haddad, pela confiança, apoio, profissionalismo e esforço durante todos esses anos de doutorado. Seus ensinamentos me ajudaram na minha formação acadêmica e ética dentro do mundo da pesquisa, o que certamente me acompanharão no futuro profissional e pessoal.

Aos professores Laura Pioli Kremer, Leonardo Sandrini Neto, Rafael Metri, Alberto Lindner e Thaís Pires Miranda, pela disponibilidade e interesse em compor a banca avaliadora dessa tese.

Ao Programa de Pós-Graduação em Zoologia da Universidade Federal do Paraná (UFPR) e ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) pela bolsa concedida durante todo o período do meu doutorado.

Aos colegas do Instituto de Oceanologia (atualmente ICIMAR) e ao Centro Nacional de Áreas protegidas de Cuba (CNAP), pelo apoio para realizar meu doutorado. Aos coordenadores do projeto PNUD-GEF Sabana-Camaguey 2009- 2014, Mercedes Arellano e Natalia por toda a confiança durante a coordenação das tarefas do projeto e pelo apoio para os cruzeiros de campo, durante as coletas e pelo incentivo para seguir adiante com meu projeto de doutorado.

A Beatriz Martínez Daranas e Pedro M. Alcolado, pesquisadores e professores biólogos marinhos cubanos, pelo apoio constante ao meu trabalho de pesquisa.

Especial agradecimento aos meus amigos mergulhadores Angel Alberto Delgado, Sandy León de Armas e Johannes Acosta, que foram parte fundamental na minha aventura de coletar os hidroides. Aos amigos sempre presentes na minha vida profissional e pessoal, que são um incentivo muito grande para mim e apoio principalmente nos momentos difíceis, Rafael Fernández de Alaiza e Susana Aguilar Mugica.

Aos alunos de iniciação científica do curso de biologia da UFPR, Lucas Enes e Amanda Ventura Firmino, seu amor pela biologia se refletiram na sua ajuda durante a triagem das amostras de hidroides, fazendo um trabalho com qualidade.

Um agradecimento especial ao professor Dr. Dale Calder, pelo auxílio na confirmação das identificações das espécies.

A todas as colegas do Laboratório de Cnidaria da UFPR, pelo descontraído, mas sério ambiente de trabalho, que sempre me animou para seguir adiante com o difícil e constante trabalho de triagem no laboratório. Também aos alunos e funcionários do departamento e da pos-graduação de Zoologia, que me animaram sempre com um sorriso que muitas vezes diminuía meu sentimento de saudade.

Aos professores Paulo de Tarso da Cunha Chaves, Emygdio Leite de Araujo Monteiro e Marcos Barbeitos, pelo profissional ensino recebido nas aulas ministradas durante o curso.

Ao professor Paulo da Cunha Lana pela sua amizade e apoio durante os precisos momentos de minha passagem pelo CEM.

Um agradecimento especial a professora Rosana Moreira Rocha, Cesar Martins, Maikon Di Domenico e aos colegas Ana Caroline Cabral e Luiz Varzinczak, por aceitarem ser parte deste trabalho e colaborar com muita vontade para que este sonho tenha se tornado uma realidade.

Agradeço a oportunidade de ter participado no curso POLUIÇÃO DOS SISTEMAS COSTEIROS E OCEÂNICOS, no laboratório do CEM, e pelo aprendizado das técnicas e procedimentos para determinação dos marcadores químicos utilizados neste trabalho.

Agradeço a diretora do Instituo Smithsoniam de pesquisas Tropicais (Tropical Research Institute STRI), Rachel Collins, pelo seu apoio antes e durante minha participação no primeiro curso de coleta e identificação de hidrozoos do Caribe, o qual foi sem dúvida uma importante aprendizagem para meu caminho na taxonomia deste grupo. Aos professores Maria Pia Miglietta e Stefano Piraino, por brindar sua experiência e conhecimento sobre os hidroides durante esses 21 intensos dias de trabalho e pesquisa enriquecedora. A todos os hidrozoólogos colegas alunos que participamos juntos nesse curso, compartilhando os bons momentos de coletas e de identificação de hidroides e medusas até que o sono batesse em nossa porta.

A os amigos novos, muito valiosos, pessoas boas que ganhei na minha moradia no Brasil, brasileiros, venezolanos, cubanos, que me brindam com seu carinho sincero cada dia, obrigada!!

A meu amor, meu esposo, Mario Oscar Alvarez, por me acompanhar nessa aventura- projeto de vida e estar presente em cada dia, na espera eterna de minhas intensas horas de computador e de estudo, pela sua ajuda, motivação, amor, confiança e compreensão sempre. RESUMO

As crescentes alterações da biodiversidade nos ecossistemas marinhos, causada pelos impactos humanos e mudanças climáticas, impõem uma demanda de formação de zoólogos e ecólogos que pesquisem a relação das comunidades biológicas com o ambiente. Os inventários de espécies realizados por taxonomistas formam a base para os estudos ecológicos, que envolvem objetivos tanto de conservação dos recursos naturais, com vistas ao desenvolvimento sustentável, quanto da mitigação e recuperação de danos e dos ecossistemas. As alterações na composição, diversidade, abundância e riqueza das comunidades de organismos do bentos marinho mostram tanto o efeito dos impactos humanos como o funcionamento dos ecossistemas naturais na relação biota-ambiente. Assim, o estudo de grupos zoológicos menos conhecidos, como os hidroides marinhos, podem complementar a compreensão dos fundamentos de conservação da biodiversidade em áreas naturais protegidas e dos problemas ambientais em áreas impactadas. Hidroides, organismos bentônicos do filo Cnidaria, classe Hydrozoa, são considerados indicadores de impactos humanos por terem a capacidade de colonizar todo tipo de substrato natural e artificial e de modificar sua estrutura em resposta a alterações ambientais. Algumas espécies sobrevivem em ambientes extremos de contaminação. No entanto, ainda existem muitas lacunas de conhecimento sobre a diversidade e a relação da estrutura da comunidade dos hidroides com o ambiente. Nesse sentido, esta tese teve o objetivo de incrementar o conhecimento da diversidade de hidroides de Cuba e de conhecer a relação da estrutura das assembleias com fatores ambientais, em dois habitats costeiros impactados, o recife de coral de Havana e as pradarias de gramas marinhas das baias San Juan dos Remedios e Buena Vista, no centro-norte de Cuba. Como objetivo complementar, a fauna atualizada de hidroides no Grande Caribe foi analisada quanto à influência de nicho ambiental e espacial no padrão de distribuição da diversidade alfa e beta. A tese está estruturada em quatro capítulos que avaliaram 1) As novas ocorrências de hidroides Leptothecata para Cuba, mostrando fotografias das estruturas e de cada espécie, junto com as descrições e discussão necessárias em cada caso 2) o estado atual e a similaridade da biodiversidade de hidroides na região do Grande Caribe, assim como a influência do ambiente e o espaço no padrão de distribuição da diversidade alfa e beta aplicando-se o método de análise de particionamento da variação 3) o efeito da contaminação fecal sobre a abundância e a riqueza da comunidade de hidroides, através da determinação da concentração de esteroides no sedimento como variáveis preditoras 4) se os indicadores de saúde do ecossistema de gramas marinhas, que incluem as variáveis bióticas das fanerógamas (densidade de ramificações de fanerógamas, peso úmido de Thalassia testudinum, porcentagem de macroalgas calcárias e tipo de algas epífitas) junto com as variáveis abióticas do ambiente e os impactos humanos são boms preditores da riquesa, abunância e distribuição dos hidroides epífitos nesse ecosistema. Os dados desses dois últimos capítulos foram avaliados aplicando o método de GLM e de análises multivariadas para conhecer se as variaveis explicativas são boms preditores dos cambios da estrutura da assembleia dos hidroides. São apresentadas 29 novas ocorrências de hidroides tecados, incluindo 7 famílias, 14 gêneros e duas novas espécies Antennella sp. nov e Halecium sp. nov. Clytia warreni Stechow, 1919, Halecium pusillum Sars, 1856 e Halecium cf. labrosum Alder, 1859 são novas ocorrências para a região do Grande Caribe (capítulo 1). No levantamento bibliográfico para essa região (capítulo 2), foram encontradas 396 espécies, incluindo as do presente trabalho. A República Dominicana e Guatemala tiveram posição isolada devido à falta de conhecimento de sua fauna de hidroides. As famílias de hidroides de maior distribuição foram Sertulariidae, Aglaopheniidae e Plumulariidae, com registros em mais de vinte países. Aglaophenia latecarinata Allman, 1877, Sertularia marginata (Kirchenpauer, 1864) e Dynamena crisioides (Lamourox, 1824) foram registradas em mais de 50% dos países analisados. Aplicando-se o Índice de Jaccard, a similaridade da composição de hidroides entre 34 países da região resultou em oito grupos, cujo padrão de distribuição pode ser explicado pelo sistema de correntes marinhas da região. A latitude e a temperatura foram significativamente correlacionadas com a diversidade alfa. As variáveis preditoras do nicho ambiental e dispersivo não explicaram, individualmente, a variação das diversidades alfa e beta. No recife de coral de Havana (capítulo 3), hidroides foram coletados em sete locais, ao longo de três transectos por local, a 10 m de profundidade. Os locais foram classificados em contaminados, moderadamente contaminados e não contaminados, conforme a análise de cinco esteroides. Os marcadores químicos coprostanol, indicador de contaminação fecal, colestanol, colesterol, estigmasterol e brassicasterol, indicadores de matéria orgânica de origem natural, foram analisados no sedimento. A análise do GLM mostrou o efeito preditivo dos esteroides sobre a riqueza (S) e a abundância de hidroides (Hyd) nas assembleias e sobre as espécies mais abundantes do estudo. O coprostanol teve o maior valor de importância relativa (RI) para prever a variação de S e de Hyd. As espécies de maior abundância neste estudo, Plumularia floridana Nutting, 1900 e C. gracilis (M.Sars, 1850), também afetadas pelo coprostanol, não ocorreram nos locais contaminados e foram mais abundante nos locais não contaminados. C. gracilis foi mais abundante nos locais moderadamente contaminados. Obelia dichotoma (Linnaeus, 1758) e Halecium bermudense (Congdon, 1907) foram as espécies mais abundantes nos locais contaminados, onde os valores de S e Hyd foram os mais baixos. Os resultados indicaram que a contaminação fecal afetou negativamente as assembleias de hidroides, destacando a necessidade de serviços básicos de gestão das fontes da contaminação para a preservação da diversidade. Nas gramas marinhas (capítulo 4), a riqueza e a abundância de hidroides teve influência de dois fatores abióticos: a salinidade e a profundidade. O peso úmido de Thalassia testudinum, a porcentagem de algas calcárias e o tipo de algas epífitas foram os principais preditores tanto da riquesa como da abundância. O cálculo do Indice total de impactos permitiu classificar os sitios em 4 grupos Li = bajo impacto (CAI-5 e CAI-7), Lm = impacto bajo moderado (CAI-4), Hm = impacto alto moderado (CAI-2, CAI-3 e CAI-12 ) e Hi = alto impacto (CAI-1). Os impactos humanos com maior importancia sobre a comunidade de hidroides foram o Impacto da atividade de pesca de arrasto e presencia de rodovias que conectam as ilhas do arquipelago cubano. O efeito da contaminação foi importante para a riqueza dos hidriodes. A riqueza e abundância de espécies foi menor nos locais mais impactados pelos efeitos das atividades humanas e maior nos locais mais distantes das fontes de contaminação e com maior intercâmbio com as águas oceânicas.

Palavras chaves: Leptothecata-hidroides, recife de corais, gramas marinhas, Caribe, GLM, análises multivariados. ABSTRACT

The growing biodiversity damage in natural marine ecosystems caused by human impacts and climate change demand the training of zoologists and ecologists studying the relationship of biological communities with their environment. Species inventories made by taxonomists form the basis for ecological studies aiming the conservations of natural resources for sustainable development and the mitigation and recovery of damaged species and impacted ecosystems. The changes in species composition, diversity, abundance and richness of communities of marine benthic organisms show both the effect of human impacts and the functioning of natural ecosystems in the biota- environment relationship. In this sense, the study of zoological groups poorly known, such as the marine hydroids, complement information for conservation of biodiversity in protected natural areas and for the understanding of environmental problems in impacted areas. Hydroids, i.e., benthic cnidarians of the class Hydrozoa, are considered indicators of human impacts because they have the capacity to colonize all types of natural and artificial substrate and to modify its structure in response to environmental changes, where some of them surviving in extreme environments of contamination. However, there are still many gaps in knowledge about the diversity and structure relationship of the hydroids community to the environment. This thesis had as general objective to increase the knowledge of the diversity of hydroids of Cuba through the taxonomic study of the thecate hydroids , and also to know the relation of the hydroid community structure with environmental factors in two impacted coastal habitats, the Havana's coral reef and the seagrass meadows of the San Juan dos Remedios and Buena Vista bays in the north of central Cuba. As a complementary objective the current diversity status of the hydroid fauna in the Wider Caribbean was analyzed and the influence of environmental and spatial niche in the distribution pattern of alpha and beta diversity of hydroids in the region was estimated. The thesis is structured in four chapters that assessed 1) The new records of thecate hydroids for Cuba, showing photographs of the structures and of each species along with the precise descriptions and discussion in each case 2) the current state and the similarity of hydroids biodiversity in the region of the Wider Caribbean as well as the influence of the environment and the space on the distribution pattern of alpha and beta diversity, applying the analysis method of variation partitioning 3) the effect of fecal contamination on the abundance and richness of hydroids community analyzing sterols on sediments as predictor variables 4) the relationship of hydroids assemblage structure with sea grass ecosystem health indicators that include the biotic variables of phanerogams (density of phanerogams shoot, wet weight of Thalassia Testudinum, percentage of calcareous macroalgae and type of epiphytic algae and abiotic environment parameters. The data analysis in the last two chapters were performed using the GLM and multivariate methods. Is presented 29 new records of species of thecate hydroids for the Cuban marine fauna that including 7 families and 14 genera, with two new species. Clytia warreni Stechow, 1919, Halecium pusillum Sars, 1856 and Halecium cf. labrosum Alder, 1859 were new occurrences for the Caribbean region (Chapter 1). The fauna of hydroids in the region of Wider Caribbean encompassed 396 species. The similarity of the hydroids fauna among the countries of the region (34) applying the Jaccard Index resulted in eight groups. Some countries like Dominican Republic and Guatemala had an isolated position, due to the lack of knowledge of their fauna. The distribution pattern found can be explained by the system of marine currents in the Caribbean region. The most widely distributed families of hydroids were Sertulariidae, Aglaopheniidae and Plumuraliidae, with records in more than twenty countries, and the species Aglaophenia latecarinata Allman, 1877, Sertularia marginata (Kirchenpauer, 1864) and Dynamena crisioides (Lamourox, 1824) were recorded in more than 50% of the countries analyzed. Latitude and temperature were significantly correlated with alpha diversity. The environmental and dispersive niche predictors did not individually explain the variation in alpha and beta diversity. On the contrary, the shared fraction explained 23% of the alpha diversity variation and 3% of the variation of the beta diversity of the hydroids fauna in the Wider Caribbean (Chapter 2). In the coral reef of Havana the collection sites were classified as contaminated, moderately contaminated and not contaminated by the assessment of five sterols concentration. The coprostanol, indicator of fecal contamination and cholestanol, cholesterol, stigmasterol and brassicasterol as signs of organic matter of natural origin. GLM analysis showed the predictive effect of steroids on richness (S), abundance of hydroid community (Hyd) as well as on the most abundant species of the study. Coprostanol had the highest relative importance (RI) to predict the variation of S, Hyd and Plumularia floridana Nutting, 1900 and C. gracilis (M.Sars, 1850), being the first most abundant of the study and in uncontaminated sites and absent from contaminated sites. C. gracilis was also abundant in moderately contaminated sites. In contrast, Obelia dichotoma (Linnaeus, 1758) and Halecium bermudense (Congdon, 1907) were the most abundant species in contaminated sites where S and Hyd had the lowest values. The results indicated that fecal contamination negatively affects hydroid assemblages, highlighting the need for basic management services of sources of contamination for the preservation of coral reef marine diversity and the health of the local resident community of Havana. In marine grasses, species richness and abundance of hydroids was influenced by two main abiotic factors: salinity and depth. The shoot density of phanerogams, wet weight of Thalassia testudinum, % of calcareous macroalgae and type of epiphytic algae are the main predictors for abundance of hydroids. The spatial distribution of hydroid assemblage was differentiated in three groups in relation to the differences in abundance and the composition of hydroid species related to the predictor variables analyzed. Species richness and abundance were lower in sites most impacted by the effects of human activities, and higher in sites farther from sources of contamination and with greater interchange with ocean waters.

Keywords: Leptothecata hydroids, coral reef, seagrass meadows, Caribbean, GLM, multivarate analyses.

LISTA DE ARTIGOS

I. New additions of Leptothecata (Cnidaria, Hydrozoa) to the shallow- water diversity of Cuba. S. Castellanos-Iglesias, and M. A. Haddad. Manuscrito formatado para submissão segundo as normas da revista Journal of Zoology, London. II. Hydroid fauna (Leptothecata and Anthoathecata) of the Wider Caribbean region related to the spatial and environmental niche effect on its diversity and distribution. S. Castellanos-Iglesias, Varzinczak L. H, M. A. Haddad. Manuscrito formatado para submissão segundo as normas da revista Marine Biology, Berlin.

III. Organic contamination as a driver of structural changes of hydroid's assemblages of the coral reefs near to Havana Harbour, Cuba.

S. Castellanos-Iglesias , A. C. Cabral, C.C. Martins, M. Di Domenico , R. Moreira Rocha, M. A. Haddad. Manuscrito publicado na revista Marine Pollution Bulletin.

IV. Species richness, abundance, and spatial distribution of the epiphytic hydroids community related with seagrass health indicators, environmental factors and human impacts in a tropical seagrass meadow.

S. Castellanos-Iglesias, M. Di Domenico , B. Martinez- Daranas, M. A. Haddad. Manuscrito formatado para submissão segundo as normas da revista Marine Environmental Research.

SUMÁRIO

INTRODUÇÃO GERAL ...... 22

REFERÊNCIAS ...... 27

CAPÍTULO 1 ...... 34

NEW ADDITIONS OF LEPTOTHECATA (CNIDARIA, HYDROZOA) TO THE SHALLOW-WATER DIVERSITY OF CUBA...... 34

Abstract...... 35

Resumo...... 36

1. Introduction ...... 37

2. Materials and methods...... 38

3. Taxonomic report ...... 40

Order Leptothecata Cornelius, 1992 ...... 40

Family Campanulariidae Johnston, 1836 ...... 40

&O\WLDPDFURWKHFD(Perkins, 1908)40

&O\WLDSDXOHQVLV(Vanhöffen, 1910)40

&O\WLDZDUUHQLStechow, 1919 ...... 41

*DVWUREODVWDsp ...... 42

/DRPHGHDIOH[XRVDAlder, 1857 ...... 43

2UWKRS\[LVVDUJDVVLFROD(Nutting, 1915) ...... 44

Family Campanulinidae Hincks, 1868 ...... 45

&XVSLGHOODsp45

(JPXQGHOODKXPLOLVFraser, 193646

"2SHUFXODUHOODODFHUDWD(Johnston 1847)47 Family Haleciidae Hincks, 1868 ...... 47

+DOHFLXPFRQLFXPStechow, 1919 ...... 47

+DOHFLXPGLFKRWRPXPAllman,1888...... 48

+DOHFLXPcf.GLVFRLGXPGalea, 2013 ...... 48

+DOHFLXPcfODEURVXPAlder, 185949

+DOHFLXPODQNHVWHULL(Bourne, 1890)50

+DOHFLXPOLJKWERXUQLCalder, 199151

+DOHFLXPSXVLOOXP(M. Sars, 1857)51

+DOHFLXP[DQWKHOODWXPGalea, 201352

+DOHFLXPFIQDQXPAlder, 185952

+DOHFLXPODELDWXPBillard, 193353

+DOHFLXPsp. nov54

0LWURFRPLXPFLUUDWXPHaeckel, 187955

Family Hebellidae Fraser, 1912 ...... 55

6FDQGLDJLJDV(Pieper, 1884) ...... 55

Family Sertulariidae Lamouroux, 1812...... 56

7ULGHQWDWDWXPLGDAllman, 187756

6HUWXODUHOODSHFXOLDULV(Leloup, 1935)57

Superfamily 3OXPXODULRLGHD McCrady, 1859 ...... 58

Family Halopterididae Millard, 1962 ...... 58

$QWHQQHOODFXUYLWKHFDFraser, 193758

$QWHQQHOODSHFXOLDULVGalea (2013) ...... 58

$QWHQQHOODVLPLOLVGalea (2013)...... 59 $QWHQQHOODsp. nov...... 59

+DORSWHULVWHQHOOD(Verrill, 1974) ...... 61

Confirmation of species with reproductive structure recored for the first time for Cuban waters...... 61

Family Sertulariidae Lamouroux, 1812...... 61

'\QDPHQDGLVWLFKD(Bosc, 1802)61

7ULGHQWDWDGLVWDQV(Lamouroux, 1816)62

Family Plumulariidae Agassiz, 1862 ...... 63

3OXPXODULDPDUJDUHWWD(Nutting, 1900)63

4. Conclusions ...... 64

5. Acknowledgments...... 65

6. References...... 67

Figures ...... 76

Appendix ...... 98

CAPÍTULO 2 ...... 101

HYDROID FAUNA (CNIDARIA: LEPTOTHECATA AND ANTHOATHECATA) OF THE WIDER CARIBBEAN RELATED TO SPATIAL AND ENVIRONMENTAL NICHE EFFECT ON ITS DIVERSITY AND DISTRIBUTION...... 101

Abstract...... 102

1.I ntroduction ...... 104

2. Study area...... 106

3. Material and Methods...... 110

3.1. Summarizing and updating the hydroids fauna ...... 110 3.2. Data collection for the analysis of spatial and niche influence on alpha and Beta diversity of hydroids...... 110

3.3 Data analyses ...... 111

3.3.1 Jaccard index and CLUSTER analysis of hydroids distribution...... 111

3.3.2 Spatial and niche influence on alpha and beta diversity of hydroids. . 112

4. Results ...... 113

4.1 Updated distribution of the hydroid fauna (Anthoathecata and Leptothecata) in the Wider Caribbean...... 113

4.2 Number and occurrence of families and genus and species richness of hydroids in the studied region...... 113

4.3 Similarity distribution of species composition of hydroid fauna in the Wider Caribbean...... 117

4.4 Spatial and environmental niche influence on alpha and beta diversity of hydroid fauna in the Wider Caribbean...... 121

5. Discussion...... 123

6. Conclusions ...... 127

7. Acknowledgments...... 128

8. References...... 129

&$3Ë78/2

“ORGANIC CONTAMINATION AS A DRIVER OF STRUCTURAL CHANGES OF HYDROID’S ASSEMBLAGES OF THE CORAL REEFS NEAR TO HAVANA HARBOUR, CUBA.”...... 138

1. Introduction ...... 143

2. Study area...... 144

3. Material and methods...... 148 3.1. Sampling ...... 148

3.2. Biological data: sorting, counting and identification of hydroids...... 148

3.3. Sterols analyses ...... 149

3.4. Data analyses ...... 149

4. Results...... 151

4.1. Characterization of sampling sites based on sterols concentration data...... 151

4.2. Characterization of hydroids community structure ...... 153

5. Discussion...... 158

6. Conclusions ...... 162

7. Acknowledgments...... 163

8. References...... 164

9. Supplementary data...... 173

CAPÍTULO 4 ...... 193

SPECIES RICHNESS, ABUNDANCE, AND SPATIAL DISTRIBUTION OF THE EPIPHYTIC HYDROIDS COMMUNITY RELATED WITH SEAGRASS HEALTH INDICATORS, ENVIRONMENTAL FACTORS AND HUMAN IMPACTS IN A TROPICAL SEAGRASS MEADOW...... 193

Abstract...... 195

1. Introduction ...... 196

2. Study area...... 198

3. Materials and Methods...... 200

3.1 Sampling, collection data and laboratory procedures...... 200

3.2 Anthropogenic impacts assesment ...... 202 3.3 Data analyses ...... 203

4. Results ...... 204

4.1 Hydroid species richness and abundance...... 204

4.2 Hydroid species composition and level of impacts of sampling sites. ... 213

4.3 Hydroid community structure and indicators of seagrass health and abiotic variables of water quality...... 216

5. Discussion...... 218

6. Conclusions ...... 222

7. Acknowledgments...... 223

8. References...... 223

9. Supplemantary data...... 229

CONCLUSÕES GERAIS ...... 240

REFERÊNCIAS...... 245 22

INTRODUÇÃO GERAL

Os hidroides, organismos do Filo Cnidaria Verrill, 1865, clase Hydrozoa Owen, 1843, são majoritariamente marinhos. Espécies bem conhecidas, como as dos gêneros Hydra, Craspedacusta e Cordylophora vivem em água doce ou salobra (Gilli and Hughes, 1995; Schuchert, 2004; Galea, 2007; Smith et al., 2011). A palavra hidroide refere-se à fase bentônica dos Hydrozoa, que têm um ciclo de vida duplo, com as formas de pólipo sésseis e assexuadas, e as formas planctônicas, as medusas sexuadas (Bouillon et al., 2004). A ausência da medusa ou seus modos de formação têm motivado diferentes propostas de classificação taxonômica (Schuchert, 1993; Cornelius, 1995; Boero, Bouillon and Piraino, 1996; Bouillon and Boero, 2000). A classificação dos hidroides foi historicamente dividida em duas perspectivas principais, estabelecidas pelos taxonomistas especializados no estudo do pólipo ou da medusa, encontrando-se a mesma espécie com nomes diferentes para cada fase de vida (Calder, 1970). Estudos recentes de diversidade e filogenia, combinando o uso de caracteres genéticos e morfológicos, mostram a confirmação de clados já existentes e a proposição de novos, deixando novas perguntas para se compreender a evolução do grupo (Collins, 2002, 2006; Moura et al., 2010; Maronna, et al., 2016) e para a definição de um sistema único de classificação. Resultados desses estudos mostram que as análises genéticas são uma forte ferramenta na diferenciação de espécies, mas que não substituem a identificação taxonômica dos hidroides com base na morfologia das espécies (Laakmann and Holst, 2014; Cunha et al., 2015).

A maior riqueza de espécies de hidroides está nos grupos Anthoathecata e Leptothecata, esse último com o maior número de aproximadamente 2100 espécies (WoRMS, 2017), compondo mais da metade (64%) do total de espécies de Hydrozoa descritas (~ 3000), até 1997 (Schuchert, 1997). A tradicional taxonomia dos hidroides se baseia em diferentes caracteres morfológicos do pólipo em Anthoathecata e do exoesqueleto em Leptothecata. Em Leptothecata os principais caracteres taxonômicos estão relacionados a forma de hidroteca e da gonoteca (Bouillon et al., 2004).

A falta de conhecimento sobre o ciclo de vida e a grande capacidade das espécies de hidroides de alterar sua morfología em resposta a mudanças ambientais têm causado problemas de identificação em muito grupos (Allman, 1864; Calder, 1970; 1988; 1991; 1997; 23

2009; Schuchert 2005; Moura et al., 2010). Junta-se a isso os poucos pesquisadores taxonomistas dedicados ao estudo dos hidroides quando comparados com outros grupos zoologicos, principalmente de algumas regiões, como o Atlântico tropical e subtropical e as regiões Ártica e Antártica. Essas diferenças regionais trazem, por consequência, um conhecimento díspar da diversidade e da ecologia dos hidroides (Medel and López- González, 1997). Do mesmo modo, o conhecimento dos hidroides em muitos habitats tropicais rasos e costeiros não existe ou é inconspícuo quando comparado com o estudo de outros organismos marinhos (Miloslavich, 2010). As partes mesofóticas e abissal dos oceanos também têm sido exploradas (Calder and Vervoort, 1998; Medel and Vervoort, 2000; Peña-Cantero, 2004), embora o conhecimento da diversidade do grupo e o papel dos hidroides nessas comunidades seja ainda incipiente.

Investigações sobre o papel ecológico dos hidroides na cadeia alimentar dos ecossistemas (Coma et al., 1995, 1999, Orejas et al., 2001, 2013) e na sua interação com outros organismos (Brooks, 1985; Damiani, 2003; Manning and Lindquist, 2003; Bavestrello et al., 2008; Fernández-Leborans, 2013; Cerrano et al., 2015; Montano et al., 2015) ressaltam seu papel nas comunidades bentônicas e como importantes componentes do biofouling e pioneiros na sucessão das comunidades incrustantes (Gili and Hughes, 1995; Amaral, et al., 2010; Bloecher, et al., 2015), Além disso, a capacidade de gerar formas de vida planctônica, dormentes, incrustantes e resistentes, que se movimentam por grandes distâncias pelo sistema de correntes oceânicas, através do rafting (Jackson 1986; Cornelius, 1992; Calder, 1993;) ou de diversos transportes marítimos, possibilita aos hidroides se instalarem em regiões distantes da origem e se tornarem invasores (Occhipinti-Ambrogi and Savini, 2003; Choong and Calder, 2013).

O conhecimento de sua distribuição e diversidade em ambientes extremos, como no Ártico (Schuchert, 2000; Ronowicz, 2007) e na Antártica (Peña-Cantero, Boero and Piraino, 2013; Peña-Cantero and Manjón-Cabeza, 2014), corroboram a importância do estudo dos hidroides para a compreensão da sobrevivência dos organismos marinhos do bentos frente às mudanças climáticas atuais (Ronowicz, 2007; Puce et al., 2008). Alterações na estrutura das assembleias e dominância de muitas espécies em ambientes contaminados por diversos impactos humanos (Grohmann et al., 1997; Cabral, 2013; Brahim et al., 2014), junto com as características do pequeno tamanho, ciclos de vida curto e resistência a condições 24 ambientais adversas os identificam como indicadores de mudanças ambientais (Mergner, 1977; Grohmann, 2009; González-Duarte, Megina and Piraino, 2014).

A sobrevivência da fauna e flora dos ecossistemas costeiros e marinhos é ameaçada pela sinergia dos crescentes impactos humanos e os efeitos das mudanças climáticas (Johnston and Roberts, 2009; Bianchi et al., 2014). O conhecimento sobre a biodiversidade de organismos pouco estudados, como os hidroides, e a descoberta de novas ocorrências são uma forte ferramenta para a formulação de propostas de conservação, especialmente nas áreas de importantes valores naturais que estão ameaçadas pelas pressões do desenvolvimento econômico iminente.

Os hidrozoários estão distribuídos amplamente e são comuns em todos os ecossistemas marinhos rasos, tais como manguezais, bancos de sargaços, gramas marinhas e recifes de corais (Calder, 1991, 1995; Gili and Hughes, 1995; Di Camilo, et al., 2008). A maioria das espécies é carnívora (Coma et al., 1999; Orejas 2001), são grandes consumidores de larvas de peixes, crustáceos e outros organismos, de modo que têm influência na dinâmica trófica dos ecossistemas. Alguns se alimentam de bactérias, protozoários, fitoplâncton e matéria orgânica dissolvida (Orejas, 2009). Espécies associadas às algas intracelulares são produtoras de nutrientes.

Os estudos taxonômicos de hidroides datam do século XVIII, com um aumento significativo nas últimas quatro décadas do século XX (Schuchert, 1997). Ao contrário, a ecologia dos hidroides tem sido menos estudada, embora tenha ocorrido um aumento recente. O continuo aumento de artigos sobre taxonomia, ecologia, filogenia e genética de populações dos hidroides mostra o atual interesse da comunidade científica nesse grupo e a expectativa de continuar alcançando novas descobertas (Maronna et al., 2016).

O estudo da fauna de hidroides de Cuba, do Mar do Caribe e do Oceano Atlântico teve início no século XIX (Allman, 1877; Clarke, 1879; Fewkes, 1881). Inventários nas águas cubanas registram um total de 113 espécies de hidrozoários para Cuba, sendo os hidroides tecados e atecados os melhores representados, com 64 % do total conhecido. Em Cuba, a estrutura das assembleias de hidroides e sua relação com os fatores ambientais é uma lacuna de conhecimento até o presente estudo. 25

O conhecimento sobre a taxonomia e ecologia dos hidroides de Cuba pode ser usado como base para estudos de conservação, monitoramento e qualidade ambiental dos habitats marinhos e também como informação para a compreensão da biogeografia e dos padrões de distribuição da biodiversidade dos organismos marinhos nas regiões do Grande Caribe, e do Atlântico Ocidental e subtropical.

O primeiro objetivo geral da tese foi aprofundar o conhecimento da taxonomia e diversidade dos hidroides de Cuba e conhecer a relação da estrutura das assembleias das espécies relacionada com fatores ambientais. Como segundo objetivo, foi avaliado o estado da diversidade dos hidroides em Cuba e sua similaridade com os países que conformam o Grande Caribe, relacionando os diferentes fatores ambientais com o padrão de distribuição da diversidade alfa e beta do grupo na região.

A tese está estruturada em quatro capítulos redigidos em inglês, no formato de manuscrito preparado para submissão em revistas científicas:

Capítulo 1. Neste capítulo foi avaliada a taxonomia dos hidroides tecados mostrando as novas ocorrências para Cuba, com fotografias para cada espécie e descrições e discussão necessários a cada caso.

Capítulo 2. No segundo capítulo foi atualizado a fauna de hidroides no Grande Caribe e foi avaliada a similaridade da composição de espécies entre os países mediante o índice de Jaccard. Também neste capitulo foi investigado, com o método de particionamento da variação, como os diferentes fatores abióticos do nicho ambiental e dispersivo influíram sobre a diversidade alfa e beta de distribuição na região.

Capítulo 3. Neste terceiro capítulo, foi avaliada a influência da contaminação fecal sobre a riqueza e abundância da comunidade de hidroides e sobre as espécies mais abundantes utilizando-se esteroides como variáveis preditoras e aplicando-se o método de GLM. As análises multivariadas permitiram reconhecer o padrão espacial da comunidade de hidroides nesse ambiente impactado pela contaminação.

Capítulo 4. No quarto capítulo, foi determinada a relação da estrutura da comunidade de hidroides epifíticos no ecossistema das gramas marinhas. Variáveis bióticas, abióticas e impactos humanos foram avaliadas como indicadoras da saúde das gramas marinhas no 26 monitoramento do estado de conservação desse ecossistema. Assim como no terceiro capítulo, o efeito das variáveis preditoras sobre a riqueza e abundância da comunidade de hidroides foi avaliado com o método de GLM e análises multivariadas permitiram relacionar o padrão de distribuição espacial da comunidade de hidroides com o ecossistema das gramas marinhas afetado pelo impacto humano.

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CAPÍTULO 1 NEW ADDITIONS OF LEPTOTHECATA (CNIDARIA, HYDROZOA) TO THE SHALLOW-WATER DIVERSITY OF CUBA.

Manuscrito formatado para submissão segundo as normas da revista Journal of Zoology London Fator de impacto 2016: 1.819 Thomson Reuters Journal Citation Reports 2016 Qualis (Biodiversidade): A2

New additions of Leptothecata (Cnidaria, Hydrozoa) to the shallow-water diversity of Cuba. S. Castellanos-Iglesias 1, M. A. Haddad 2 1,2 Departamento de Zoologia, UFPR, CEP 05508–090 Curitiba, PR, Brazil. E-mail: [email protected] 1Corresponding author

Abstract This article presents 29 new occurrences of hydroids species of the order Leptothecata (Cornelius, 1992) for the fauna of Cuba, classified in 7 families and 14 genera. Hydroids were sampled between 2-4 m, in two habitats of the north mainland: the Havana coral reef (february/2013) and the seagrass meadow of two bays in the Archipelago Sabana Camaguey, San Juan de Los Remedios and Buena Vista (may/2013). Illustrations are given for each species. The genus Gastroblasta is confirmed for the Caribbean region, with abundant colonies (one fertile) occupying two new substrates (Bryozoa and Thalassia testudinum). One morphospecie of Othophyxis sargassicola described for Brazilian waters was recorded for the Havana coral reef. The genus Cuspidella, a new record for Cuban waters, is discussed about trophosoma similarities with the species registered in the Caribbean. Clytia warreni Stechow, 1919, Halecium pusillum Sars, 1856 and Halecium cf. labrosum Alder, 1859 were recorded for the first time for the Caribbean region. Zooxanthellae symbiosis is reported for the first time for two genus, Dynamena and Antennella. Two new species are proposed, Antennella sp. nov and Halecium sp.nov.. The distinct characters of these new species and the main differences with similar species and with those reported from the area are compared. The first occurrence of fertile colonies confirms the identification of three species for Cuba. With this study the number of Leptothecata reported for Cuba is raised to 101 species.

Key words: Hydrozoans, Thecates, Sabana-Camaguey Archipelago, Havana, zooxanthellae.

Resumo

Este artigo apresenta 29 novas ocorrências de espécies de hidroides da ordem Leptothecata (Cornelius, 1992) para a fauna de Cuba, classificadas em 7 famílias e 14 gêneros. Os hidroides foram amostrados entre 2 e 10 m, em dois habitats ao norte da ilha de Cuba: no recife de coral da Havana (fevereiro/2013) e nos prados de gramas marinhas de duas baías no Arquipélago Sabana Camaguey, Baía de San Juan de Los Remedios e de Buena Vista (maio/2013). Para cada espécie, são apresentadas fotografias e desenhos. Confima-se a ocorrência do gênero Gastroblasta para a região do Caribe, em dois novos substratos, Bryozoa e a grama Thalassia testudinum, com abundância de colônias, uma fértil. Uma das morfoespécies de Ortophyxis sargassicola descritas para Brasil foi registrada no recife de Havana. Para o gênero Cuspidella, novo registro para águas cubanas, discute-se a similaridade do trofosoma com as espécies registradas na área. Clytia warreni Stechow, 1919, Halecium pusillum Sars, 1856 e Halecium cf. labrosum Alder, 1859 foram novas ocorrências para a região do Caribe. A simbiose com zooxantelas foi registrada pela primeira vez em espécies dos gêneros Dynamena e Antennella. Duas novas espécies são propostas, Antennella sp.nov. e Halecium sp. nov., com uma análise comparativa dos caracteres das espécies semelhantes já registradas para a área. A primeira ocorrência de colônias férteis confirma a identificação de três espécies para Cuba. Com este estudo, o número de Leptothecata registrados em Cuba aumenta para 101 espécies.

1. Introduction

The first discoveries of Cuban hydroids were reported for different research cruises around the Cuban mainland and Caribbean from the late nineteenth century (Allman, 1877; Clarke, 1879; Fewkes, 1881). With a preliminary report of Bahamas expedition (Nutting 1895) Nutting (1900, 1904, 1915) started an important study of the American hydroids including some records from the deep platform of the Northwestern cuban shore. Important contributions befalled in the twentieth century, when scientists as Stechow (1912, 1914, 1919) and Fraser (1943) reported new genera and species of different hydrozoan collections from Museums. Explorations of the hydroid fauna of the Gulf of Mexico by Deveey (1950, 1954) increase the list of cuban hydroids. Despite all these important records of the deep hydroid fauna we can say that deep hydroid fauna were better characterized than that inhabiting the nearby shelf and shore. It was not until 2001 that a list of cuban cnidarian was published where the first list of hydroid was accounted (Lalana et al., 2001). After that, an inventory of the diversity and distribution of hydroids of the southern Cays of Cuba (Achipelago of Los Canarreos), in different depth and ind the shallow ecosystems as mangroves, seagrass and coral reef, added an important contribution of 21 new records for the cuban hydroid fauna (Castellanos-Iglesias, 2007, 2009, 2011). In the last twenty years, scarce and casual hydroid samples were taken in different substrates of shallow waters, but important contributions of new occurrences and also a new species (Zanclea cubensis Varela, 2012) where reported (Varela et al., 2005, 2010, Varela, 2012). Despite of all these hydroid records from cuban waters, they are not sufficiently to say that hydroids from Cuba have been well known. All reports mainly from the Hydroidolina subclass have been done in the western part of the island, being the eastern side of the country completely unknown to date. The majority of cuban hydroid species records are from Leptothecata Cornelius, 1992, as it is common for other studies of marine hydroid fauna, e.g, from the central east coast of Florida (Calder, 2013), Gulf of Mexico (Calder and Cairns, 2009), Panama (Calder and Kirkendale, 2005), Siladen Island coral reef of Indonesia (Di Camilo et al., 2008). This order is the most specious taxa of the subclass Hydroidolina (Collins, 2000), with ~2100 species described (WoRMS, 2017), probably because their morphological diversity, defense structures, reproduction mechanisms and also resistance capacity to adverse condition allow them to inhabit diverse environmental conditions (Gili and Hughes, 1995). 38

The objective of this study was to provide a report of some new records of Leptothecata hydroids from samples collected in two different shallow water zones in the north of Cuba. Both areas include two different habitats of ecological and economical importance, a coral reef and seagrass meadows (Aguilar et al., 2004, 2008; Alcolado-Prieto et al., 2012; Alcolado et al., 2007). This places have note been explored with the purpose of deepening the marine hydroid fauna. In this way, this work brings an advance towards the knowledge of the cuban hydroids that is yet poorly known.

2. Materials and methods

During the months of May 2011 and March 2013, hydroid collections were carried out in fourteen sites in two different habitats, coral reef and seagrass meadows, respectively (Table 1). In 2011, the surveys were made in two basins in the central north coast of Cuba: San Juan dos Remedios (SRB) and Buena Vista (BVB). In 2013, hydroids were collected on the fringing coral reef in the western Havana. All theses surveys were part of projects linked to the Institute of Oceanology of Cuba (IDO), as follow: 1) The study of seagrass habitat in the Sabana-Camaguey Ecosystem within the UNDP-GEF Project, stage III and 2) The monitoring of coastal marine ecosystems in the Havana shore.

Table 1. Station numbers, study area, coordinates, habitat and depths of the sampling sites.

Station Study Sampling Depths Lat (N) Long (W) Habitat: number area site (m) 1 SRB CAI-2 22 35.492 -79 23.983 S 2 2 SRB CAI-3 22 33.314 -79 22.043 S 2.5 3 SRB CAI-4 22 36.316 -79 19.305 S 2.6 4 SRB CAI-5 22 37.976 -79 19.744 S 2.8 5 SRB CAI-7 22 34.085 -79 15.043 S 2.5 6 BVB CAI-9 22 28.568 -79 16.084 S 3.1 7 BVB CAI-12 22 31.496 -79 14.46 S 2.7 8 NH HB 23.1470 -82.3586 RC 10 9 NH MA 23.1422 -82.3700 RC 10 10 NH ALM 23.1369 -82.4214 RC 10 11 NH CA16 23.1285 -82.4231 RC 10 12 NH CA70 23.1256 -82.4447 RC 10 13 NH EM 23.1064 -82.4675 RC 10 14 NH SA 23.0933 -82.5100 RC 10 39

Legend: SRB- San Juan dos Remedios Bay; BVB-Buena Vista Bay; NH-North of Havana; S=seagrass; RC=coral reef The studied areas comprise two shallow coastal areas of the Cuban marine platform, selected by the economic and environmental importance they represent as touristic zones with valuable habitats and resources (Martínez-Daranas, 2007; Montalvo et al., 2009; García- García et al., 2012). Study sites with locations are showed in Fig 1.

Figure 1. Location of the studied areas and sampling sites (see Table 1) on the fringing coral reef of Havana shore in March 2013 (region 1) and in the seagrass meadows of San Juan dos Remedios and Buena vista Bays, Villa Clara, during May 2011 (region 2).

Collections were made by snorkeling up to 3 m and SCUBA diving between 4 -10 meters deep. In the coral reef (CR), the visual method was employed for collecting all hydroids with their substrates along three 10 m transects per site. In the seagrass meadows (GR), plants were removed from within twelve 25 x 25 cm quadrats per site. Samples were anesthetized with menthol, fixed in 10% formalin and taken to the laboratory for processing and analysis.

With the help of a Leica stereomicroscope, the hydroids were separated from the substrates. Slides prepared with parts or entire colonies were magnified at a OLYMPUS- 40

BX50 biological microscope and then photographed with a CANON-CT3 camera for identification purposes.

The identification was based mainly in the taxonomic studies from Cuba cited in this work and from the Caribbean, Gulf of Mexico, Florida and tropical Western Atlantic region. Taxonomic reports from other areas were also used to solve identification problems (Schuchert, 1997, Bouillon et al., 2004).

For distribution records, the Wider Caribbean area was selected, which included Bermudas, U.S-Florida, Gulf of Mexico and the Caribbean basin.

3. Taxonomic report

Order Leptothecata Cornelius, 1992

Family Campanulariidae Johnston, 1836 Clytia macrotheca (Perkins, 1908)

(Fig 2. A-C)

Campanularia macrotheca Perkins, 1908: 146, pl. 3 figs 12–13.

Clytia macrotheca—Calder, 1991d: 64, fig. 35

Material examined. Stn 13, 10 m. DZoo-CN.400. CR., colony small and sterile on Sargassum.

Remarks. The species morphology coincides with the description of the specimens from Bermuda by Calder (1991).

Type locality. Fort Jefferson, Dry Tortugas, Florida, United States of America.

Great Caribbean records. Cuba (Havana). For other sites see Galea (2008).

Geographical distribution. Western Atlantic (Calder, 1991).

Clytia paulensis (Vanhöffen, 1910)

(Fig 2. D, E) 41

Campanularia paulensis Vanhöffen, 1910: 298, fig 19a,b. Clytia ulvae Stechow, 1919: 47, fig N. Campanularia longitheca Fraser, 1914

Material examined. Stn 14, 10 m. DZoo-CN.401. CR., sterile colony on Halimeda .

Description. Stolonal colonies with sinuous hidrorhiza. Pedicels long and thin, 9-11 basal annulations, 7 medial, 4 at the hydrothecae base. Hydrothecae slender, bell-funnel shaped; margin irregular with 10 bicupidate teeth and external visible folds; diaphragm oblique. Hydranths almost retracted into hydrothecae. Approximately 14 tentacles. Gonothecae absent.

Remarks. The characteristics of the trophosome of our material coincided with the original description of this species by Vanhöffen (1910) as Campanularia paulensis (Fig 19 a,b page 298), with Clytia ulvae by Stechow, 1919 and with C. paulensis by Cornelius (1995) and Bouillon et al. (2004).

Type locality. Kraterbecken, Saint Paul island, Southern Indian Ocean. For Pacific and Atlantic Oceans, see recently reported distribution summary in Calder (2013).

Great Caribbean records. Cuba (Havana). Gulf of Mexico (Calder & Cairns. 2009).

Geographical distribution. Circumglobal in temperate and tropical waters (Cornelius, 1995b; Ramil et al., 1998; Medel & Vervoort, 2000; Peña Cantero & García Carrascosa, 2002; Bouillon et al., 2004; Liu, 2008).

Clytia warreni Stechow, 1919

(Fig 2. F-J)

Clytia elongata Warren, 1908:339-340, fig 20- Clytia warreni Stechow, 1919, Millard, 1975, page 221 and 2, fig 73 E,F.

Material examined. Stn 8, 10m. DZoo-CN.402.CR., fertile colony on Halimeda.

Description. Colony stolonal, arising from a creeping hidrorhiza. Pedicel ringed at the base and distal ends. Hydrothecae slender, narrower at the base; margin irregular with 14 sharp cusps slightly inclined inwards; diaphragm well defined and separated from pedicel by 42 a deep campanulate basal chamber. Hydrant retracted with 14 tentacles. Gonothecae elongated with pedicel born on hydrorhiza and truncated at the distal end.

Remarks. The throphosome and gonosome of our material have characters very similar with C.elongata described by Warren (1908) and with Stechow (1919) and Millard (1975) as C.warreni.

Type locality. Algoa Bay, South Africa.

Great Caribbean records. Cuba (Havana). This species was not reported for the Caribbean before.

Geographical distribution. South Africa (Millard, 1975; WoRMS, 2017).

Observations: A comparison between the characters of Clytia species identified in the present report is showed in Appendix, Table 2.

Gastroblasta sp .

(Fig 3. A-L)

Material examined. Stn 3, Stn 4, Stn 5 and Stn 6. 5 m. DZoo-CN.403.GR., sterile colony, DZoo-CN.404.GR., fertile colony on algae, Thalassia testudinum and Bryozoa.

Description. Colonies mostly sympodial, few stolonal. Hydrocaulus monosiphonic. Pedicel with 15 rings at the base. Hydrothecae bell shaped, pedicellated, up to 5 rings at the base of a very elongated basal chamber; margin with regular and not sharp teeth, without U- shaped incisions; diaphragm well defined and thin. Hydrant robust with 14 – 16 tentacles, with a differentiated globular hypostome. Gonothecae without pedicel, arising lateraly from hydrocaulus; cone shaped with truncated end; with wrinkles from the middle part to the base; without constrictions below margin. Two medusa budding near the base of the gonothecae.

Remarks. The morphology of trophosome of our material fits with Gastroblasta sp. as described by Galea (2013) to Martinique. Despite his material has a Clytia-like polyp, the author classified the specimen in the genus Gastroblasta, taking into account that its substrate was a sponge. In this study, our specimens were found on the seagrass Thalassia testudinum, and on algae and Bryozoa from the seagrass beds. Clytia–like polyps of 43

Gastroblasta Keller, 1883 living in symbiotic associations with sponges are known only for G. raffaelei Lang, 1886 (Gravili et al. 2007). The trophosome and gonothecae morphology of our material fits also with G. raffaelei specimen described by Gravili et al., (2007), although the medusa buds are located more basaly in our material. Despite of these similarities, we agree with Galea (2013) that our species could be G. ovalis (Mayer, 1900), the congenere medusae described for Florida, until life cycle and genetic studies corroborate this hypothesis.

Great Caribbean records. Cuba (SRB Bay), Martinique, Eastern Caribbean (Galea, 2013).

Geographical distribution. The medusa stages of G. timida Keller, 1883, from the Red Sea, and G. ovalis (Mayer, 1900) is from Florida.

Laomedea flexuosa Alder, 1857

(Fig. 4 A-C)

Laomedea flexuosa Alder, 1857—Bouillon et al., 2004:198,fig. 113 G,H,J,I,K.

Material examined. Stn 11. 10 m, DZoo-CN.405.CR., sterile colony on Halimeda.

Description. Colony with two stolonal and one ramified hydrocaulus. Pedicel totally annulated in ramified hydrocaulus; up to 6-7 rings in the proximal and distal portions in the stolonal ones. Hydrothecae bell shaped with thick perisarc and smooth margin. Diaphragm straight in the basal chamber.

Remarks. Our species fit with the description of this species from Mediterranean (Bouillon et al, 2004). It is accepted inside the Family Campanularriidae Johnston, 1836 (Schuchert, WoRMS, 2017). Recently, L. flexuosa was included in the new Infraorder Obeliida and in Family Clytiidae based on the rRNA gene sequences (16S, 18S, and 28S), (Maronna et al., 2016) but the authors considered that future studies are needed to improve this new taxonomic classification.

Type locality. Northumberland and Durham (Alder, 1957).

Great Caribbean records. Cuba (Havana), Gulf of Mexico (Calder & Cairns, 2009). 44

Geographical distribution. Western and eastern Atlantic (Ramil, 1988; Altuna, 1994; Calder & Cairns, 2009) , Mediterranean (Bouillon et al., 2004). Orthopyxis sargassicola (Nutting, 1915)

(Fig. 4 D-L)

Material examined. Stn 11, 12 and 13, 10 m. DZoo-CN.406, 407.CR., fertile colonies. DZoo-CN.408.CR., sterile colony on algae. Description. Stolonal colonies arising from creeping hydrorhiza. Pedicel smooth, but with one or tow nodes of growth at different intervals. Subhydrothecal spherule present. Hydrothecae cylindrical and without thick perisarc. An annular perisarcal thickening inside the base of hydrothecae. Margin with 11-12 teeth with linguiform cusps. Gonothecae present, born on hydrorhiza, hive shaped, with about 10 well-developed corrugations; gonothecae aperture large and truncate. Remarks. This specimen was identified inside the genus Orthopyxis despite it has some characteristics frequently described for species of the Campanularia genus. The hydrothecae is depth shape, its thin perisarc wall and the smooth pedicel are characters more commonly found in Campanularia spp. Than in Orthopyxis spp. Our material is similar to the specimen identified by Calder, 2013 as Campanularia macroscypha from Florida shallow waters, which is typical of the United States and the Gulf of Mexico, although this material does not have gonothecae for a comparison. The specimen from Havana are quite different in the shape of the hydrothecae from that of C. macroscypha illustrated in Allman (1877), probably sampled in the upper 50 m or so. In addition, Fraser (1944) also illustrated this species from mostly deep waters which is much alike Allman's, and different from our material. Because of these comparisons we do not considered our specimen as Campanularia sp. In addition, our material is very similar to the lineage found in Rio de Janeiro, Paraty, identified as Orthopyxis sargassicola by Cunha et al., (2011). Using morphometric and genetic analysis, they describe great variations between populations and inside the same colonies of the Orthopyxis species. These variations were mainly in the length of the polyps, pedicel, gonothecae, degree of thickness of the perisarc and cuspids of hydrothecae. The sinuosities of pedicel have a wide variation between populations too (Cunha, et al., 2011; 2015). It is known that the variability of the Campanularidae morphology is correlated with latitude and water temperature gradient (Ralph, 1956; 1957) likewise others environmental factors. 45

Although all these variations are possible to find in Campanularidae species, we identified our material as Orthopyxis sargassicola because of the similarity of our specimen with the fertile lineage found in the coast of the State of Rio de Janeiro (Paraty), Brasil.

Type locality. Bahamas (Nutting 1915).

Great Caribbean records. Cuba (Havana). On floating Sargassum in Gulf Stream (Leloup, 1935), Bahamas (Nutting, 1915), Belize & Bermudas (Calder, 1991), Martinique (Galea, 2013), Florida (Calder, 2013).

Geographical distribution. Western Atlantic. For recent information see Cunha (2011).

Family Campanulinidae Hincks, 1868

Cuspidella sp.

(Fig. 5 A-D)

Material examined. Stn 11, 12 and 13. DZoo-CN.409, 410.CR., sterile colonies on Halimeda.

Description. Colony stolonal. Hydrothecae elongated and cylindrical shaped, sessile on hydrorhiza, occasionally regenerated with visible growth lines; distal margin sometimes flaring and demarking the distance with the operculum. Operculum conical, with 15 to 17 triangular flaps bended outward or inward.

Remarks. Trophosome of this material is alike to the specimen described by Hirohito (1995) as Cuspidella gigantea from Japan and alike to the specimens of C. procumbens Kramp, 1911 described by Calder (1970) from Canada and originally from east Greenland. This genus is represented in the Caribbean by the species Cuspidella humilis Hincks, 1866 from Colombia (Hentschel, 1923) and Gulf of Mexico (Calder & Cairns, 2009). C. humillis has also a wide distribution reported in Artic Ocean and Mediterranean sea (Van der Land et al., 2001), North Atlantic Ocean (Van der Land, 2008) and Red Sea (Vine, 1986). Cuspidella polyps may vary considerably in morphology and in life-history studies they have been linked 46 with a lot of medusoid genus around the world (Calder, 1970). In the WoRMS data base (Schuchert, 2017) (http://www.marinespecies.org/aphia.php?p=taxdetails&id=117406 17_04_2017), this species is taxon inquirendum. Hincks, 1866, page 209, plate XXXIX, do not refer nor ilustrated the regeneration of the hydrothecae and the growing lines as is observed in the specimen from Japan and in our material. The characteristics of our specimens do not fit with the description of Cuspidella humilis Hincks, 1866 reported by Vervoort (1968) and because of the geografical distances between the location of our material and that of Cuspidella gigantea and C. procumbens, we decided not name our species until genetic and/or life cycle experiments can be perfomed.

Great Caribbean records. Havana (Cuba), this study.

Egmundella humilis Fraser, 1936

(Fig. 5 E)

Egmundella humilis Fraser, 1936: 50, pl. 1 fig. 2. —Galea, 2013: 13 figs.3 H,I.

Material examined. Stn 10, Stn 11 and Stn 13. 10 m. DZoo-CN. 411, 412.CR., sterile colonies on Halimeda.

Description. Colony stolonal. Hydrothecae elongated and cylindrical shaped, rounded at the base and tubular to the top; margin little flaring; pedicel lengthy and wrinkled. Diaphragm thin. Operculum with triangular flaps.

Remarks. The trophosome of the present material fits with the specimen described by Galea (2013) from Martinique. Egmundella superba Stechow, 1921, another species recorded in the Caribbean, has a much greater pedicel (Calder, 1991) and bigger hydrothcae and nemathotecae (Stechow, 1921). Egmundella grandis Fraser, 1943, recorded in Belize (Spracklin, 1982), has a pedicel mostly smooth differing from our specimens.

Type Locality. Japan, Sagami Bay, 90 m, on Bryozoa (Fraser, 1936).

Great Caribbean records. Cuba (Havana), Martinique (Galea, 2013).

Geographical distribution. Japan (Fraser, 1936; Hirohito, 1995), Mauritania (Vervoort, 2006). 47

?Opercularella lacerata (Johnston 1847)

(Fig. 5 F- J)

Opercularella lacerata —Hincks 1868: 194, pl. 39 fig. 1; Calder 1970: 1514, pl. 3 fig. 4 —Cornelius 1995a: 173, fig. 38.

Material examined. Stn 10, 10m. DZoo-CN. 413.CR., sterile colonies on Halimeda.

Description. Colony stolonal arising from a creeping hydrorhiza. Hydrothecae tubular. Pedicel short with basal rings. Diaphragm thin. Operculum conical with 9-10 flaps.

Remarks. The present material is alike the specimen from Guadaloupe, described as Incertae Sedis by Galea (2010), found also on Halimeda. As the author comments, this species can probably be Opercularella lacerata (Johnston, 1847) reported from Bonaire Island (Leloup, 1935). The lack of gonothecae did not allow us be sure that our material is O. lacerata. For redescription of this species see Schuchert (2001).

Type Locality. St. Andrews and Berwick Bay, Scotland.

Great Caribbean records. Cuba (Havana), Bonaire (Leloup, 1935).

Geographical distribution. Artic and North Atlantic Ocean (Van der Land et al., 2001), North Sea (Muller, 2004).

Family Haleciidae Hincks, 1868

Halecium conicum Stechow, 1919

(Fig.6 A-D)

Halecium reflexum Stechow, 1919 Halecium conicum —Bouillon et al., 2004:140,fig 73 K,M Halecium minutum Motz-Kossowska, 1911 Material examined. Stn 13., 10m. DZoo-CN. 414.CR., fertile colony with female gonothecae on octocoral. 48

Remarks. The trophosome and the female gonothecae shape of our material which rise from hydrorhiza allowed us to identify this species, following the description of Bouillon et al., (2004) for Mediterranean waters.

Type Locality. Monaco.

Great Caribbean records. Cuba (Havana), St. Thomas, Vervoort (1968).

Geographical distribution. Mediterranean sea (Van der Land, J et al, 2001; Bouillon et al., 2004), North Atlantic Ocean in European coast (Van der Land et al., 2001).

Halecium dichotomum Allman,1888

(Fig. 7 A-C)

Halecium dichotomum Allman, 1888: 13, pl. 6.

Material examined. Stn 8, Stn 11, Stn 12 and Stn 13, 10 m. DZoo-CN. 415, 416.CR., fertile colonies with male and female gonothecae on Dictyota, Sargassum and Halimeda.

Remarks. Our material fits with the description of Galea (2013), reinforced by the presence and the characteristics of the female and male gonothecae. Hydrothecae of the male colony have the rim not markedly everted coinciding with the ilustration and description of Migotto (1996) for specimens from São Paulo, Brazil.

Type Locality. Simon's Bay, Cape, South Africa.

Great Caribbean records. Cuba (Havana), Martinique (Galea, 2013).

Geographical distribution. South Africa (Allman, 1888; Stechow, 1925; Millard, 1975), Brazil (Migotto, 1996).

Halecium cf. discoidum Galea, 2013

(Fig. 7 D-G)

Halecium discoidum Galea, 2013: 18 figs.5 A,B.

Material examined. Stn 8, Stn 11, Stn 12 and Stn 13, 10m. DZoo-CN. 417, 418.CR., sterile colonies on Halimeda. 49

Remarks. The shape of trophosome and hydrants of our material agree with the description of Galea (2013), in our material, the stem have sometimes up to four hydrothecae that rise as branching from the base of a central hydrothecae. Despite our material do not have gonothecae, we confirm the identification.

Type Locality. Case-Pilote, Anse Batterie, Martinique.

Geographical distribution. Cuba ( Havana), Martinique (Galea, 2013).

Halecium cf. labrosum Alder, 1859

(Fig. 7 F-J)

Halecium labrosum Alder 1859: 354, Plate 13

Material examined. Stn 13., 10 m. DZoo-CN. 419.CR., one sterile colony with one hydrocaulus on Halimeda .

Description. Colony erect, monosiphonic; stem divided in internodes of irregular sizes, node transverse or oblique as ringed bulge shape Hydrothecae bell shaped, with long pedicel (deep hydrophore) emerge from the distal part of previous internodes; internodes with three rings with distance of different size margim smooth and everted. An aberrant gonothecae arises from the stem on three short ringed pedicels.

Remarks. We note the monosiphonic stem characteristic of our material and not polysiphonic as described for this species, probalby because our colony is very tiny and young, composed by only one erect simple hydrocaulus. The trophosome agree with the material described from Mediterranean (Bouillon et al., 2004), but differ from that described by Schuchert (2005) by the absence of primary and secondary hydrothecae with corrugate walls. In the specimens from Canadian waters, Calder (1970) describes the variations from colony to colony, mainly in the degree of perisarc and internodes annulations as in the hydrothecae or branches arising from the apophyses. The common characteristics between the specimens of Calder (1970) and our material are: 1) internodes of irregular size, 2) internode annulations of irregular shape, 3) apophyses distal on internodes, 4) hydrophores with deep basal chambers, 5) margim of hydrothecae flaring and everted, 6) gonothecae oval shaped, arising from branches on short ringed pedicels, 7) presence of pseudodiaphragm. 50

Type Locality. Northumberland coast, United Kingdom.

Great Caribbean records. Cuba (Havana). Not reported for Caribbean before the present study.

Geographical distribution. North Atlantic, Artic , Mediterranean. Details in WoRMS database.

Halecium lankesterii (Bourne, 1890)

(Fig. 8 A-E)

Haloikema lankesterii Bourne, 1890: 395, pl. 26 figs 1, 2.

Material examined. Stn 1, 2 m. DZoo-CN. 420, 421.GR., fertile colonies, with male gonothecae on Thalassia testudinum.

Remarks. The colonies with both forms of growth, stolonal and erect, coincide with the trophosome description by Schuchert (2005) and Galea (2008), mainly in the characteristic athecate segments of irregular size, separated by straight nodes, and with two bulging at their ends. Our material has flaring hydrothecae with diverging walls and not everted margin. The gonothecae has the aperture in the distal part and wrinkles on the straight side, differing from the female gonothecae described by (Schuchert, 2005), which has the aperture in the median portion. We identified our material as a male gonothecae. This characteristic of the gonothecae are refered as the main difference between Halecium cf. lankesterii (Bourne, 1890) and H. nanum, because both species have zooxanthellae and for that, they can be confounded (Schuchert, 2005). The characteristics of the throphosome and of the male gonothecae let us with no doubt tha our material corresponds to the description by Galea (2008) of the specimen from Guadeloupe identified as Halecium cf. lankesterii (Bourne, 1890) and differs from H. nanum Alder, 1859 (Calder, 1991).

Type Locality. Plymouth, Great Britain.

Great Caribbean records. Cuba (SRB bay), Guadeloupe (Galea 2008). 51

Geographical distribution. Indian Ocean (Vannucci, & Navas, 1973), Mediterranean and Eastern Atlantic (Peña Cantero & García Carrascosa, 2002), and Western Atlantic, Guadeloupe (Galea, 2008).

Halecium lightbourni Calder, 1991

(Fig. 8 F-J)

Halecium lightbourni Calder, 1991: 19, figs. 10, 11, 6. —Calder & Kirkendale, 2005: 481, Galea & Ferry, 2015:8,fig. 3L-P.

Material examined. Stn 10, 12 and 13, 10m. DZoo-CN. 422, 423, 424, 425, 426.CR., fertile colonies stolonal or erect, with male and female gonothecae on Halimeda.

Description. See original description of Calder (1991).

Remarks. Our material has the trophosome alike Halecium laightbourni described by Calder (1991) and by Galea & Ferry (2015). The female gonothecae coincide with the material form Martinique (Galea & Ferry, 2015).

Type Locality. Harrington Sound, Cripplegate Cave, Bermuda.

Great Caribbean records: Cuba (Havana), Bermuda (Calder, 1991), Panamá (Calder & Kirkendale, 2005), Martinique (Galea & Ferry, 2015).

Geographical distribution. North Atlantic Region. Van der Land (2008).

Halecium pusillum (M. Sars, 1857)

(Fig. 9 A-J)

Eudendrium pusillum M. Sars, 1857: 154, pl. 1 figs 14-16.

Halecium pusillum (M. Sars, 1857) —Bouillon et al., 2004:142,fig 76 E,F. —García Corrales et al., 1978: 14, fig. 4. —Peña Cantero & García Carrascosa, 2002: 71, fig. 14D–E

Material examined. Stn 12, 10 m. DZoo-CN. 427.CR., stolonal colony, delicate and fertile, with female gonothecae on Halimeda tuna (J.Ellis & Solander) J.V.Lamouroux, 1816. 52

Remarks. See the description of this species in Bouillon et al. (2004). Our material has annulated internodes and hydrothecae with everted outward margin. The presence of vegetative propagules and the ringed and corrugated female gonothecae, without terminal aperture, are characters that distinguish this species. The trophosome is very similar with that of Halecium corrugatissimum Trebilcock, 1928 from New Zeland, but they differ in the shape of the gonothecae and in the hydrothecae margin, that is everted in H.pusillum and not everted in H. corrugatissimum (Schuchert, 2005).

Type Locality. Northumberland coast, U.K.

Great Caribbean records. Cuba (Havana). Not reported for Caribbean before this study.

Geographical distribution. Eastern and western Atlantic, Mediterranean, Indic (Bouillon et al., 2004). See a detailed distribution of this species in Peña Cantero & García Carrascosa (2002).

Halecium xanthellatum Galea, 2013

(Fig. 10 A-E)

Halecium xanthellatum Galea, 2013: 20, figs. 5 F,G, H.

Material examined. Stn 13, 10 m. DZoo-CN. 428.CR., stolonal colony, very minute and fertile with female gonothecae on Halimeda.

Remarks. The material coincide with description of Galea (2013).

Type Locality. Le Prêcheur, Pointe Lamare, Martinique.

Great Caribbean records. Cuba (Havana). Martinique (Galea 2013).

Halecium cf. nanum Alder, 1859

(Fig.11 A-G)

Halecium nanum Alder, 1859:355, pi. 14, figs. 1-4. —Galea, 2008:26, figs 5G.—Calder, 1991: 20, figs 12C. 53

Material examined. Stn 8, Stn 11, Stn 12 and Stn 13. DZoo-CN. 429.CR., fertile colony with female gonothecae on Sargassum.

Description. Tiny colonies, all stolonal, mostly with regenerated hydrothecae and hydrophore very short. Stem of the hydrothecae rise from stolon with one and up to three tiny internodes following by the smooth perisarc of primary hydrothecae. Perisarc walls of moderate thickness increasing gradually from the base to tip. Hydroteca margin not everted and with a light thickening in the base, bellow the well-defined diaphragm. Female gonothecae born on short pedicel given off in pairs from each side of the base of the hydrothecae, single from one side or from inside the secondary hydrothecae. It is sac shaped but has a distinct hump in the apical end that form a 90 grade with the hump, showing some bending marks. Lots of zooxanthellae in the coenosarc.

Remarks. The stolonal hydrocaulus of this material fits with the specimen found in Gadeloupe (Galea, 2008).The perisarc, hydrothecae and gonosome of our material are very similar with that of the specimen from Bermuda described by Calder (1991), although it is erect and dichotomously branched as refered by Calder, 1991, not always stolonal.

Type Locality. Atlantic Ocean, 34°48'N, 34°25'W, (Alder, 1859).

Great Caribbean records. Cuba (Havana). Aruba, Bonaire, Granade, Trinidad and Tobago (Leloup, 1935), Belize (Sapracklin, 1982; Calder, 1991), Bermuda (Calder, 1991), Panama (Calder & Kirkendale, 2005), Guadaloupe (Galea, 2008), Gulf of Mexico (Stechow, 1919, Calder & Cainrs, 2009), South of U.S and Florida (Deevey, 1950).

Geographical distribution. Circumglobal. Western and eastern Atlantic , Western and eastern Pacific, see Calder (1991).

Halecium labiatum Billard, 1933

(Fig. 12 A-F)

Halecium labiatum Billard, 1933: 21, fig. 8

Material examined. Stn 12. DZoo-CN. 430.CR., colony very minute and fertile with female gonothecae on Sargassum. 54

Description. See original descriptions in Billard, 1933.

Type Locality. Gulf of Suez.

Great Caribbean records. Cuba (Havana). Martinique (Galea & Ferry, 2015).

Geographical distribution. Red Sea (Vervoort, 1967) and the Gulf of Aden (Rees & Vervoort, 1987). Halecium sp. nov

(Fig. 13 A-F)

Material examined. Stn 4, 2.8 m. DZoo-CN. 431, 432, 433, 434, 435.SG., Fertile colonies with female gonothecae on Thalassia testudinum.

Description. Colony erect, with monosiphonic hydrocaulus. Hydrocaulus with three basal rings, increasing in diameter to the distal part of each internode; internodes separated by oblique nodes, but sometimes, in the medial stem, there are segments of irregular sizes separated by straight nodes. Primary hydrothecae sessile, margin not everted and with a visible ring of desmocytes. One or two secondary hydrothecae rise from the base of the adcauline wall of the primary hydrothecae, mostly in the old part of the hydrocaulus; three or more wrinkles at the base of hydrophore; shallow and abcauline wall shorter than the adcauline, that curves downward. Axilar hydrothecae present. Female pedicellated gonothecae rise from the base of the sessile hydrothecae; they are very large and elongated (may reach up to four hydrocaulus internodes size) and almost a cylinder in shape. Rounded operculum. Hydrants enlarged outside of hydrothecae, having a dome shape at the base of hypostome. Hypostome rounded; almost 22 tentacles.

Remarks. At present, there are 23 species of Halecium spp. reported for the Wider Caribbean. Our material was compared with all of them, and with others collected in the North Atlantic, Mediterranean, South Africa and New Zeland. We concluded that our material is different of all. One similar species is H. textum Kramp, 1911, which has a strong and corrugated perisarc and a similar shaped female gonothecae, but differs from our specimen that do not have a strong corrugation and the hydrothecae margin are not an evereted as in H.textum. 55

Geographical distribution. Only known for Cuba (SRB Bay).

Family Lovenellidae Russell, 1953 Mitrocomium cirratum Haeckel, 1879

(Fig. 14 A-E)

Mitrocomium sp. —Galea, 2008:28, figs 5G, Mitrocomium cirratum—Calder, 1991: 25, figs 15–16. not Mitrocomium cirratum Haeckel, 1879: 182, pl. 11 figs 9–11.

Material examined. Stn 13 and 14. 10 m. DZoo-CN. 436, 437, 438.CR., sterile colonies on Halimeda.

Remarks.The hydrant and the trophosome of our material coincide with the description of Calder (1991) and Galea (2008).

Type Locality. Corfu (Kerkira), Greece.

Great Caribbean records. Cuba (Havana), Bermudas (Calder, 1991) and Guadeloupe (Galea, 2008).

Geographical distribution. North Atlantic Sea, Mediterranean (van der Landet et al., 2001), Western Atlantic (Calder, 1991).

Family Hebellidae Fraser, 1912 Scandia gigas (Pieper, 1884)

(Fig. 15 A-D)

Lafoea gigas Pieper, 1884: 165. Scandia gigas—Peña Cantero & García Carrascosa, 2002: 59, fig. 11E–G. — Galea, 2008:24, fig. A.

Material examined. Stn 11, 10m. DZoo-CN. 439.CR., sterile colony on algae.

Remarks. The trophosome of our material coincides with the specimen described by Galea (2008) and some of its hydrothecae have up to three short renovations of the hydrothecal rim, as in the specimen described by Peña Cantero and García Carrascosa 56

(2002). For recent redescription of this species see Galea (2008).

Type locality. Eastern coast of the Adriatic Sea.

Great Caribbean records. Cuba (Havana), Bermuda (Calder 1998, 2000) and Gulf of Mexico (Calder & Cairns, 2009) both citations as Hebellopsis michaelsarsi, Guadeloupe (Galea, 2008), U.S-Florida (Calder, 2013 as Lafoea gigas which is accepted in WoRMS as Scandia gigas (Pieper, 1884),

Geographical distribution. Mediterranean (Peña Cantero & García Carrascosa, 2002), Eastern Pacific (Calder et al, 2003), Atlantic waters off the north coast of Spain (Isasi, 1985; Altuna, 1994) and of Morocco (Patriti, 1970) and western Atlantic (Leloup, 1935; Galea, 2008).

Family Sertulariidae Lamouroux, 1812 Tridentata tumida Allman, 1877

(Fig. 14 E)

Sertularia tumida Allman, 1877: 23, pl. 16 figs 3–4. Tridentata tumida—Calder, 1991d: 109, figs 58–59. Sertularia tumida—Galea, 2008: 36, figs 7A.

Material examined. Stn 6, 1 m. DZoo-CN. 440.GR., one hydrocaulus on Thalassia testudinum.

Remarks. The present material had some characters which allow the identification of this species. They are: 1) Hydrothecae tumid with full appearance, 2) intrathecal theeth and septum lacking. For the detailed descritption of the trophosome see Calder (1991b). At present days, this species is acepted in the WoRMS data base as Sertularia tumida Allman, 1877, but we decided maintaining the validity of Tridentata genus for two reasons: 1) The comments explained by Calder (1991b) about the validity of this genus and 2) The new results from molecular studies of Leptothecata (Maronna et al., 2016) that support the non- monophyly of the genus Sertularia corroborating the possible validity of Tridentata.

Type Locality. Tortugas, Florida, United States of America. 57

Great Caribbean records. Cuba (BVB Bay). Bermuda (Calder, 1991b), Guadeloupe (Galea 2008 as Serularia tumida), Gulf of Mexico (Calder & Cairns, 2009), Tortugas Florida (Allman, 1877).

Geographical distribution. Circumglobal (Calder, 1991b).

Sertularella peculiaris (Leloup, 1935)

(Fig. 16 A-F)

Thyroscyphus intermedius f. peculiaris Leloup, 1935: 33, figs 15–17.

Sertularella parvula (Allman, 1888), in Vervoort, 1968, page 46, Fig.22.

Sertularella peculiaris Leloup, 1974: 34, footnote 1.

Sertularella conica—Calder, 1983: 11, fig. 4.—Calder, 1991d: 99, fig. 52.—Migotto, 1996: 67, fig. 12J– K.

Material examined. Stn 11, 10m DZoo-CN. 441, 442, 443, 444, 445.CR., sterile and fertile colonies, mostly common on Halimeda but also on Sargassum.

Remarks. The trophosome and gonosome of our material coincide with the description by Galea (2008) for the material from Guadeloupe. The main shared characteristics that diagnosed the species are: 1) Hydrothecae with rhomboidal margin and four triangular and pointed cups, 2) operculum composed by four flaps, 3) five large intrathecal theeth just bellow the aperture visible through the hydrothecae wall, 4) gonothecae on hydrocaulus, elongate- ovoid with a short neck.

Type Locality. Bonaire Island, Netherlands Antilles, Caribbean Sea.

Great Caribbean records. Cuba (Havana). Caribbean (Vervoort, 1968), South Carolina (Calder, 1983), Bermuda (Calder, 1991b) Guadaloupe, (Galea, 2008; 2013).

Geographical distribution. Western Atlantic (Migotto, 1996). 58

Superfamily Plumularioidea McCrady, 1859

Family Halopterididae Millard, 1962 Antennella curvitheca Fraser, 1937

(Fig. 16 D-F)

Antennella curvitheca Fraser, 1937: 4, pl. 2 fig. 7.―Fraser, 1944: 315, pl. 66 fig. 299.―Van Gemerden-Hoogeveen, 1965: 56, figs 32–33.―Schuchert, 1997: 38, fig. 13.―Calder & Kirkendale, 2005: 483. ―Galea, 2010: 25, figs. Fig. 6 I.

Material examined. Stn 8, 10m DZoo-CN. 446.CR., sterile colony with mature appearance. Epizoic on hydrocauli of the hydroid Pennaria disticha Goldfuss, 1820.

Description. See (Schuchert, 1997).

Remarks. The presence of curved adcauline side of the hydrothecae. With an internal projection, and the long distal end of the thecate internode bearing a nematothecae make this species easily distinguishable. The internal tooth observed in the inner abcauline wall by Shuchert (1997) is another distinct characteristic of this species.

Type locality. Puerto Rico

Geographical distribution. Cuba (Havana). Puerto Rican (Fraser, 1937), Saint Kitts & Nevis (Van Gemerden-Hoogeveen, 1965), Panama (Calder & Kirkendale, 2005), Guadeloupe (Galea, 2010).

Antennella peculiaris Galea (2013)

(Fig. 17 A-B)

Antennella peculiaris Galea, 2013: 26, figs. Fig. 7 A,C.

Material examined. Stn 11, 12, 13 and 14, 10 m. DZoo-CN. 447, 448, 449, 450.CR., sterile colonies on octocoral and on Sargassum, but more common on Halimeda.

Remarks. The trophosome of this species is very coincident with Antennella secundaria (Gmelin, 1791) see (Galea, 2013), but their more pigmented coenosarc and their preferencial occurrence on Halimeda they distinguish it. 59

Observations. Our material has the characteristics pointed by Galea (2013) that make this species different A. secundaria, as follows: 1) an evident pigmented pale brown coenosarc, 2) their preferential occurrence on Halimeda than in Sargassum, 3) the presence of a small rectangular intermediate internode at the base of the athecate internode, 4) a very reduced segment in the distal part of the thecate internode, just above the hydrothecae, where the axillary nematothecae is located.

Type Locality. Le Prêcheur Pointe Lamare, Martinique.

Great Caribbean records. Cuba (Havana) and Martinique (Galea, 2013).

Antennella similis Galea (2013)

(Fig. 17 C-G)

Antennella similis Galea, 2013: 30, figs. Fig. 7 O,R.

Material examined. Stn 6, 3.1 m. DZoo-CN. 451.GR., sterile colonies on Thalassia testudinum.

Remarks. The identification of our material was based in the similar characteristics of the trophosome specimen described by Galea (2013).

Observations.The characteristics that diagnosed of our specimen are as follow: 1) basal part carrying varied number of frontal nematothecae, 2) a basal constriction after the origin of stolon with several irregularly transverse nodes, 3) a second transverse incomplete node in the athecate internode, 4) two nematothecae in the athecate internode and sometimes three in the most basal part of the stem, 5) hydrothecae walls straight, 5) gonothecae with two short segment pedicel arising from the stem just below the base of the hydrothecae.

Type Locality. Le Prêcheur La Perle, Martinique.

Great Caribbean records. Cuba (BVB Bay) and Martinique (Galea, 2013).

Antennella sp. nov.

(Fig. 18 A-E) 60

Material examined. Stn 1, 4 and 6, in 2, 2.8 and 3.1 m respectively. DZoo-CN. 452, 453, 454.GR.,fertile colony on Thalassia testudinum.

Description. Colony stolonal, not ramified. Basal part of the stem without nematothecae or occasionally up to two frontal nematothecae toward the distal part of the basal stem. Very near to the basal part of the stem there is a transverse node. Additionally, three continuous transverse nodes of different sizes (big-small-big) are present in the center of the basal part of the main stem; a transverslly node toward the distal part of the stem. The last node of the stem is oblique. All hydrocaulus is heteromerous. Athecate internode with a basal transverse node and a proximal oblique node, and with two conical two chambered nematothecae, with the inner side lowered. Hydrothecae with half of adcauline wall adnate and straigth. Abcauline wall lightly convex in the basal part, almost straight distally and with thick perisarc. Margin not straight, with a lightly v-iscision in the middle part of the lateral walls, appearing two rounded large cusps. Thecate internode short with four nematothecae, one median inferior almost reaching the base of hydrothecae, two laterals that do not reach the margin of the hydrothecae and a very short axillar nematothecae. Coenosarc with zooxanthellae. Female gonothecae with two short segmented pedicel and two nematothecae at the base of the stem below the hydrothecae.

Remarks. This specimen is very similar to Antennlla peculiaris Galea (2013), to A. smilllis Galea (2013) and to A. tubitheca Galea (2013), which belong to the A. secundaria group. See Schuchert (1997) and Galea (2013). However, some distinct characteristics of their trophosoma allow us to describe our specimen as a new species. Comparing with A. smillis, the main differences are: 1) basal part of stem not carrying nematothecae, 2) hydrothecae abcauline wall not straight, but slightly convex and with a thick perisarc, 3) hydrotecae margin not rounded. The main differences from A.peculiaris are: 1) not many nematothecae at the base of the stem and when they are present, are not in parallel rows. The main difference of A. tubitheca is the hydrothecae shape not tubular, deeper and with walls not straight and of A. secundaria the main difference is the presence of a reduced proximal athecate internode, which is longer in A. secundaria. The distinct characteristics of our material from those species are the presence of zooxanthelae in the coenosarc and the hydrothecae margin with a lightly lateral v-iscision. See table 3. 61

Geographical distribution. Only known from Cuba.

Halopteris tenella (Verrill, 1974) (Fig. 19 A-H) Plumularia tenella Verrill, 1874: 731. Halopteris tenella―Schuchert, 1997: 52, fig. 16.

Material examined. Stn 1, 3 and 7, in 2, 2.6 and 2.7 m respectively. DZoo-CN. 455, 456, 457, 458.GR., fertile colonies on Thalassia testudinum.

Remarks. Our material fits with the descritption by Galea (2013). Distinct characteristics allowed us identify this material: 1) two to four nematothecae on athecate internodes of the main stem, 2) two to three nematothecae on athecate internodes of the hydrocladia, 3) three nemathotecae at the base of gonothecae. The stem and the cladia are both divided into three types of internodes: hydrothecate, long ahydrothecate and short ahydrothecate.

Type Locality. Le Prêcheur Pointe Lamare, Martinique.

Great Caribbean records. Cuba (Havana). Bonaire (Leloup, 1935), Panama (Calder, 1983), Gulf of Mexico (Calder & Cairns, 2009), Guadaloupe (Galea, 2008), Martinique, (Galea, 2013).

Geographical distribution. See Galea (2013).

Confirmation of species with reproductive structure recored for the first time for Cuban waters.

Family Sertulariidae Lamouroux, 1812

Dynamena disticha (Bosc, 1802)

(Fig. 20 A-I)

Sertularia disticha Bosc, 1802: 101, pl. 29 fig. 2. Dynamena disticha—Calder, 1991d: 93, fig. 50. 62

Material examined. Stn 1, 2, 3, 4 and 6, in 2, 2.5, 2.6, 2.8, 3.1 and 2.7 m. DZoo-CN. 459, 460, 461, 462, 463.GR., colonies on Thalassia testudinum.

Remarks. The material has some distinct characters that allowed the correct identification of this species as follow: 1) cylindrical hydrothecae with walls parallel to the stem axis and the distal half curving outward, 2) hydrothecae with triangular perisarcal projections extending proximaly 3) hydrothecae with very pronounce internal teeth and rim with two pointed lateral and a smaller adcauline ones 4) gonothecae sac-shaped arises from the base of the stem and from the hydrothecae aperture. In this species the gonothecae can rise from stolon or from the base of the stem under the first hydrothecae (Calder, 1991; Medel & Vervoort, 1998; Hirohito, 1995), and also from the hydrothecae aperture (see Galea, 2008). For the first time, female gonothecae appear in a specimen from cuban waters. Zooxanthellae were also observed in the coenosarc. For a detailed description of this species see Calder (1991).

Type Locality. See Calder (1991).

Great Caribbean records. Cuba (Havana). Confirmation of the first record of fertile colonies. Aruba, Bahamas (see Vervoort, 1968), Belize (Sprackilng, 1982 as Dynamena cornicina McCrady, 1857), Bermudas (Calder, 1991), Ilha dos paises Baixos and Curacao (Leloup, 1935), Colombia (Fraser, 1947; Vervoort, 1968; Florez, 1983), Guadeloupe (Galea, 2008), Martinique (Galea, 2013), Gulf of Mexico (Calder & Cairns, 2009), Panama (Calder & Kirkendale, 2005), Saint Kitts and Nevis, Trinidad (Van Gemerden-Hoogeveen, 1965), U.S and Florida (Vervoort, 1968; Calder, 2013).

Geographical distribution. Circumglobal distribution, Calder (1991).

Tridentata distans (Lamouroux, 1816)

(Fig. 21 A-G)

Dynamena distans Lamouroux, 1816: 180, pl. 5, figs. 1a, B. Sertularia stookeyi.—Fraser, 1943: 93. Sertularia gracilis.—Jones, 2002: 215.

Material examined. Stn 2, 3, 6, 7 in 2.5, 2.6, 3.1, 2.7 m. DZoo-CN. 464, 465, 466, 63

467.GR., stolonal and fertile colonies on Thalassia testudinum.

Remarks. Some decades ago, this species divided oppinions about their sistematic position. At the present days, it is acepted in the Worms data base as Sertularia distans (Lamouroux, 1916), but some characters allow us to identify our material as Tridentata distans. They are: 1) Hydrothecae horn shaped, concave medialy an very straight distally, 2) three teeth at the hydrothecae rim instead of two of Sertularia specimens, 3) four triangular perisarcal projections at the base of the hydrothecae, two into internode direction and two towards opposite directions, 4) strictly opposite hydrothecae in the unbranch form instead the not opposite arrangement in the species of Sertularia, 5) an abcauline diverticulum present in the contracted hydrant, which is absent in the genus Sertularia. All this characters support our oppinion of maintaining this species under the genus Tridentata Stechow, 1920. For more detail of this discussion see Calder (1991).

Type Locality. See Calder (1991).

Great Caribbean records. Cuba (Havana) confirmation record with first report of fertil colonies. Aruba, Antigua, Bahamas, Bonaire, Curacao, Saint Kitts and Nevis, Saint Barthelemy, Trinidad, Venezuela (Van Gemerden-Hoogeveen, 1965 as Sertularia distans var. gracilis), Belize (Spracklin, 1982; Calder, 1991), Bermuda (Calder, 1991), Costa Rica (Kelmo & Vargas, 2002), Martinique (Galea & Ferry, 2015), Gulf of Mexico (Calder & Cairns, 2009).

Geographical distribution. Circumglobal distribution (Calder, 1991)

Family Plumulariidae Agassiz, 1862 Plumularia margaretta (Nutting, 1900)

(Fig. 22 A-H)

Monotheca margareta, Calder, 1997: 10.Fig.2 Plumularia margareta, Galea, 2010: 29. Fig.7 H-I

Material examined. Stn 3, 4 and 6, in 2.6, 2.8 and 3.1 m. DZoo-CN. 468, 469, 470.GR., two hydrocaulus one sterile and one fertile on Thalassia testudinum. 64

Remarks. This species is very demarked by the distinct characters as follow, 1) sympodial branching with position of hydrocladias in relation with hydrocaulus. 2) the evident lateral nemathotecae that exceed the hydrothecae rim, 3) hydrothecae cup-shaped and smooth rim, except for a median adcauline notch visible in our material 4) the big size and shape of the gonothecae. The gonothecae is visualized and reported for the first time for Cuban waters. To know more details of this species see Calder (1997).

Type Locality. Little Cat Island, Bahamas.

Great Caribbean records. Cuba (Havana). Confirmation of the first record of fertile colonies. Tortugas Florida (Wallace, 1909), Aruba, Bonaire, Curacao (Leloup, 1935), Puert Rican (Freser, 1944), Tobago, St. Kitts, St. Barthelemy (Van Gemerden-Hoogeveen 1965, as P. sargass), Colombia (Florez, 1983, Bandel & Wedler, 1987), Bermuda (Calder, 1997 as Monotheca margareta), Guadeloupe, (Galea, 2010), Panama (Calder & Kirkendale, 2005 as Monotheca margareta), Gulf of Mexico (Calder & Cairns 2009 as Monotheca margareta ).

Geographical distribution. see Ansín Agís et al. (2001).

4. Conclusions

Despite the recent studies that increased the biodiversity of hydroid of Cuba (Castellanos-Iglesias, 2007, 2009, 2011; Varela, 2012; Varela et al., 2005, 2010), the results of the present study suggested that cuban hydroid fauna remains almost unknowledged until today. This survey in two impacted shallow habitats of the north of the country (a coral reef and seagrass meadows) allowed us to count more than 9000 hydrocaulus from more than 65 identified morphospecies (Castellanos-Iglesias, 2017; Castellanos-Iglesias et al., 2018), of Lepthothecata and Anthothecata. Of these, 29 new records of the order Leptothecata (Cornelius, 1992) included in 7 families and 14 genera of hydroid species are presented and discussed in this reports. The remaining Anthothecata species are being studied for another manuscript to be published.

Two hydroids of this survey represent new species, Halecium sp. and Antennella sp. The indentification of materials in this study is based in the skeletal structures, design of the gonophores when they are presented and aditional data commonly used in taxonomy. Because of all material was fixed in formalin 4%, genetics analysis are imposible to use for 65 taxonomy. In addition, the small size and the unique specimen sometimes founded prevented the study of cnidome composition. Because the identification of hydroids is very difficult mainly for the sizes of specimens in shallow tropical region (in order of μm), we recognized the importance of the collection of samples for to study species alive to incorporate the cnidome identification as an important character for taxonomy of this benthic cnidarian.

Three species are first record from Caribbean waters, Clytia warreni Stechow, 1919, Halecium pusillum Sars, 1856 and Halecium cf. labrosum Alder, 1859.The genus Gastroblasta is confirmed for the Caribbean region and is reported occuping two new substrates (Bryozoa and Thalassia testudinum) with abundant colonies, one fertile. One morphospecie of Othophyxis sargassicola described to brazilian waters is recorded for the Havana coral reef. For the genus Cuspidella is presented a discussion about the similarity of trophosoma between the species registered in the area.

Three species of Clytia are new records for cuban waters and a comparative presentation of their characters were sumarized as additional information. The observation for the first time of the reproductive structure in cuban material of Dynamena disticha (Bosc, 1802) Tridentata distans (Lamouroux, 1816) and Plumularia margaretta, (Nutting, 1900) added the confirmation of these species for cuban waters. Further, the presence of zooxanthellae in three species of the genus Dynamena and Tridentata and for Antenella sp. are reported for the first time. The present study performs not only the continued interest to study the taxonomical account of the hydroids of Cuba, but it also raises our knowledge of the hydroid fauna of the Caribbean. Considering the studies of the cuban hydroid until now and these new records at least 101 species of Lepthothecata are reported for cuban waters. An upcoming study of the Anthoathecata species surely will add more new records for the biodiversity of the cuban hydroids.

5. Acknowledgments

We want to thank all colleagues from the Oceanology Institute and friends of Cuba who helped during the sampling campaigns. Thanks to UNDP/GEF Sabana-Camaguey Project Coordinators (2009-2014), from Cuba, for the financial support to the fieldwork which made this research a reality. Special thanks to professor Dale Calder for all their revisions and comments about the identification of many species.Thanks to CNPq (Brazilian National 66

Council for Scientific and Technological Development) for the PhD scholarship of the first author and to the postgraduate Zoology program of the Paraná Federal University for all support and scientific formation. Finally, we thank the anonymous reviewers that improved the quality of the MS with their useful comments. 67

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Figures

Fig. 2. A-C: Clytia macrotheca (Perkins, 1908), hydrothecae with retracted polyp (A), detail of pedicel base (B), hydrothecae (C). D-E: Clytia paulensis (Vanhöffen, 1910), details of hydrothecae (D,E). F-J: Clytia warreni Stechow 1919, details of hydrothecae and rings on pedicel (F,G), reproductive structures (H,J). Scales 100 um. 77

Fig. 3. Gastroblasta sp. Detail of hydrothecae with polyp retracted (A), hydrothecae amplified and a new polyp (B), polyp (C), detail of hydrothecae margin and basal chamber (D,E), polyp on Thalassia testudinum (F), polyps on algae (G), polyp on Bryozoa (H), reproductive structure (J,K,L), detail of apical part of gonothecae (K), detail of basal part of gonothecae with medusa buds (L). Scales 100 um. 78

Fig. 4. A-C: Laomedea flexuosa Alder, 1857, hydrocaulus erect with hydrothecae (A), hydrothecae stolonal (B). D-L: Orthopyxis sargassicola (Nutting, 1915), variation in the hydrothecae shape (D,E), detail of the base of the hydrothecae (F), details of smooth pedicel (G,H), hydrothecae with gonothecae (J), detal of shape of two gonothecae (K). Scales 100 um. 79

Fig. 5. A-D: Cuspidella sp., detail of operculum of the hydrothecae (A), hydrothecae regenerated (B), variation of operculum shape (C), two hydrothecae. E: Egmundella humilis Fraser, 1936, three hydrothecae (E). F-J: ?Opercularella lacerata (Johnston 1847), details of hydrothecae (F,G), hydrothecae with wrinkled short pedicel (H), detail of operculum flaps (J) . Scales 100 um. 80

Fig. 6. A-D: Halecium conicum Stechow, 1919, hydrocaulus arrangement with hydrothecae (A,B), detail of gonothecae (C), stolonal hydrocaulus with gonothecae on hydrorhiza (D). Scales 100 um.

Fig. 7. A-B: Halecium dichotomum Allman,1888, detail of stolonal colony with two hydrothecae, extended polyp and female gonothecae emerged from the base of the stem of primary hydrothecae (A), male gonothecae emerged from the stem base (B). C-E: Halecium cf. discoidum Galea, 2013, variation of hydrothecae position (C,D), detail of polyp (E). F-J: Halecium cf. labrosum Alder, 1859, hydrocaulus with one hydrothecae and athecate internode (F), hydrocaulus with two hydrothecae and an apparent aberrant gonothecae (G), detail of aberrant gonothecae (H), detail of pseudodiaphragm on hydrothecae ( ,J). Scales 100 um. 82

Fig. 8. A-E: Halecium lankesterii (Bourne, 1890), female gonothecae and erect stem with two hydrothecae (A), male gonothecae (B), stolonal colony with two hydtothecae (C), detail of hydrothecae wals and margim (D), detail of colony with poliperies, gonothecae and polyps (E). F-J: Halecium lightbourni Calder, 1991, female gonothecae (F,G), hydrocaulus with nine shallow hydrothecae and three regenerated (H), detail of hydrocaulus with oblique nodes and hydrtothecae (J). Scales 100 um. 83

Fig. 9. A-J: Halecium pusillum (M.Sars, 1857), hydrocaulus arrangement with polyp (A,B), detail of polyp and vegetative propagules (C), hydrothecae with hydropohore corrugate and branching (D,E), hydrothecae with margin everted outward (F,G), minute hydrocaulus (H) and polyps female gonothecae (J). Scales 100 um. 84

Fig. 10. A-E: Halecium xanthellatum Galea, 2013, stolonal colonies with polyps on hydrorhiza (A), detail of female gonothecae emerged from a minute colony with two hydrothecae (B), hydrothecae emerged from the base of shallow hydrothecae (C,D), hydrothecae with polyps (E). Scales 100 um.

Fig. 11. A-G: Halecium cf. nanum Alder, 1859, stolonal colony with extended polyp and zooxanthellae (A) hydrothecae regenerated (B) hydrothecae with polyp (C), details das zooxanthellae in the polyp (D), detail of hypostome and tentacles of polyp (E), details of zooxanthellae on tentacles (F), gonothecae in pairs emerged from the base of hydrothecae (G). Scales 100 um. 86

Fig. 12. A-F: Halecium labiatum Billard, 1933,gonothecae and hydrothecae (A), gonothecae in pair (B,C), gonothecae with spadix (C), hydrothecae stolonal regenerated (D), hydrothecae and polyp (E), gonothecae with two hydrants (F). Scales 100 um.

Fig. 13 A-F: Halecium sp. nov, hydrocaulus with hydrothecae, gonothecae and polyps (A,B), part of hydrocaulus with the pedicelate hydrothecae and hydrothecae axillar (C), detail of margin and operculum of the gonothecae (D), hydrothecae (E), base of hydrocaulus (F). Scales 100 um.

Fig. 14. A-E: Mitrocomium cirratum Haeckel, 1879, detail of hydrothecae and rim of desmocytes (A), hydrothecae regenerated with polyp (B), hydrothecae with extended polyp and budding of a gonophore (C),hydrorhiza with two hydrothecae and polyps extended (D,E). Scales 100 um.

Fig. 15. A-D: Scandia gigas (Pieper, 1884), variation of hydrothecae shape (A-C), hydrothecae with renovations (D). E: Tridentata tumida Allman, 1877, part of hydrocaulus with one paired hydrothecae. Scales 100 um . 90

Fig. 16. A-F: Sertularella peculiaris (Leloup, 1935), hydrothecae stolonal (A), detail of distal part of hydrothecae with intrathecal teeth (B), hydrothecae with gonothecae (C), detail of operculum with four valves and intrathecal teeth (D-F). G-J: Antennella curvitheca Fraser, 1937, hydrocaulus with hydrothecaes (G), parts of hydrocaulus with athecatae internodes with nematothecaes and tecathae interndes with hydrothecaes (J), detail of hydrothecae with internal teeth. Scales 100 um (A,B, D,E,F,H) , 300 um (C,G). 91

Fig. 17. A-C: Antennella peculiaris Galea (2013), part of hydrocaulus with two hydrothecae and nematothecae on internode athecate (A), hydrocaulus with pigmented coenaosarc (B), detail of small rectangular intermediate internode and reduced distal segment in the thecate internode (C) D-J: Antennella smillis Galea (2013), stolonal unbranched hydrocaulus (D), detail of athecate internode and nematothecae (E), part of hydrocaulus with two hydrothecae and nematothecae (F), second transverse incomplete node (G), proximal part of stem with irregular transvers nodes (H), distal part of stem (J). Scales 100 um. 92

Fig. 18. A-H: Antennella sp. nov, internode athecate with nematohtecae and hydrothecae with wall not straight (A,B), base of hydrocaulus (C), Hydrothecae margin with lateral v-iscision (A,B,D), second transversal node incomplete (E), detail of zooxanthellae (F), hydrocaulus with gonothecae (G), detail of female gonothecae (H). Scales 100 um. 93

Fig. 19. A-H: Halopteris tenella (Verrill, 1974), hydrocaulus with gonothecae (A), iternode athecate of stem with three nematohtecae (B) and with four or more nematothecae (C), part of hydrocaulus with internode athecate, two axilar hyrothecae and base of female gonothecae (D), female gonothecae with three nematothecae at the base (E), enlarge of picture D and E with detail of three nematotheca at the base of gonothecae (G,H), detail of axillar nematothecae (F). Scales 100 um. 94

Fig. 20. A-J: Dynamena disticha (Bosc, 1802), part of hydrocaulus with hydrothecae and zooxanthellae on coenosarc (A,B), hydrocaulus with hydrothecaes paired (C), hydrocaulus (D), detail of hydrothecae and rim with theeth, polyp retracted (E), aberrant gonothecae given off from hydrothecae aperture (F,G), typical gonothecae on hydrocaulus base (H,J), detail of typical gonothecae with operculum (J). Scales 100 um. 95

Fig. 21. A-G: Tridentata distans (Lamouroux, 1816), hydrocaulus with hydrothecaes in opposite pairs (A), detail of pair hydrothecae (B), hydrocaulus with gonothecae on stem in the base of hydrothecae (C), details and variation of gonothecae shape (D,E), detail of hydrothecae wall with perisarcal projection (F), detail of hydrothecae with intrathecal teeth bellow margin (G). Scales 100 um (A,B, D,E,F,G) , 300 um (C). 96

Fig. 22. A-H: Plumularia margaretta (Nutting, 1900), hydrocaulus erect with hydrothecae (A), part of hydrocaulus with hydrothecae and nematothecae (B,C), detail of pair of axilar nematothecae and hydrothecae (D), detail of position of lateral nematothecae and median adcauline noch of hydrothecae (E), detail of gonothecae on base of hydrocaulus (F), hydrocaulus with gonothecae (G), detail of lateral nematothecae (H). Scales 100 um. 97

Appendix.

Table 2. Comparative characterization of the species of Clytia Lamouroux, 1812 recorded in this report.

Species of Clytia Lamouroux, 1812 Characters C. macrotheca C. paulensis (Vanhoffen, C. warreni Stechow, 1919 (Perkins, 1908) 1910)

Colony stolonal stolonal, but sometimes stolonal branching Hydrorhiza creeping sinuous creeping Pedicel smooth at center and long and thin, with rings ringed at the base and ringed at proximal at proximal, center and distal end and distal part, at the distal part perisarc moderatelly thick, without subhydrothecal spherule.

diaphragm thin, horizontal or oblique well defined with a deep slightly oblique, basal campanulate basal chamber shallow, chamber narrow, cup-shaped hydrothecae shape deep goblet-shaped, constricted basally slender, narrower at the cylindrical distally base margin eight to nine truncate ten bicupidate teeth and margin irregular with 14 cusps separated by U- external folds visible sharp cusps slightly shaped incisions inclined inwards gonothecae colony sterile in this colony sterile in this elongated with pedicel study, not seen in study, but is always born on hydrorhiza and Calder (1991) smooth (Cornelius, 1995) truncated distal end

Hydrant not seen in this study Hydrant with with 14 tentacles in this approximately 14 study tentacles NR for cuban waters x x x locality of this study Havana Havana Havana substrate and habitat on Sargassum in coral on Halimeda in coral reef on Halimeda in coral reef in this study reef Bibliography Calder, 1991 Vanhöffen, 1910; Stechow, 1919; Millard, consulted Stechow, 1919; 1975; Cornelius, 1995 ; Bouillon et al., (2004) 98

Appendix

Table 3. Comparative characters of the táxons of the group of species morphologically similar to Antennella secundaria discussed here. (-) means absence of information about the character. A. secundaria A.peculiaris A.smillis A.tubitheca A. sp.nov. (Gmelin, 1791) (Galea, 2013) (Galea, (Galea, 2013) 2013) 1. Basal stem transvers internodes: number variable variable variable ⁻ 5 one basal transversal ⁻ yes yes ⁻ yes three central short internodes ⁻ ⁻ ⁻ yes, from big to small one distal oblique ⁻ yes yes ⁻ yes nematohtecae: present yes yes yes yes not or yes number variable up to 6 up to 12 up to 4 none or 2 frontal position ⁻ not yes yes yes parallel row ⁻ yes not not not 2. Ahydrothecae internode size not short short short short not short number of nematothecae 2, rarely three 2 2, 2 2 occasionally three in basal part of the stem one basal transversal node not yes, incomplete yes, yes, yes, well marked rectangular well incomplete incomplete marked one second transversal node not not yes, not None, incomplete or incomplete not well marked 99

Table 3. (Cont.) A. secundaria A.peculiaris A.smillis A.tubitheca A. sp.nov.? (Gmelin, 1791) (Galea, 2013) (Galea, 2013) (Galea, 2013) 3. Hydrothecae internode size long not short very short, equaly of very short finished finished just athecate just after de axilar after de axilar internode nematothecae or not nematothecae distal part reach the very reduced short not reduced not reduced hydrothecae rim 4. Hydrothecae adcauline wall half adnate yes yes yes yes yes and stright abcauline wall all straight yes not yes not not abcauline distal wall straight yes not yes not yes abcauline distal wall flare not yes not yes not abcauline wall with thick not not not not yes perisarc margin circular yes yes yes yes not margin smooth yes yes yes yes not margin with lateral central v- not not not not yes insicion 5. Gonothecae Female: female shape pear cornucopia cornucopia barrel cornucopia two pedicel at the base yes yes yes yes yes 100 arising from stem at the base yes yes yes yes yes of hydrothecae two nematothecae at the yes yes yes, rarely three yes yes base perisarc thick ⁻ around aperture below rim yes, at the rim around aperture not seen yes yes yes Male: male shape rounded distal ⁻ ovoid ⁻ ovoid end arising from stem at the base yes ⁻ yes ⁻ yes of hydrothecae two nematothecae at the ⁻ ⁻ yes ⁻ yes base 6. coenosarc color transparent brown ⁻ ⁻ brown with zooxanthellae not not not not yes

CAPÍTULO 2

HYDROID FAUNA (CNIDARIA: LEPTOTHECATA AND ANTHOATHECATA) OF THE WIDER CARIBBEAN RELATED TO SPATIAL AND ENVIRONMENTAL NICHE EFFECT ON ITS DIVERSITY AND DISTRIBUTION.

Manuscrito formatado para submissão segundo as normas da revista Journal of Marine Biology (Berlin) Fator de impacto 2016: 2.37 Thomson Reuters Journal Citation Reports 2016 Qualis (Biodiversidade): A2

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Hydroid fauna (Cnidaria: Leptothecata and Anthoathecata) of the Wider Caribbean related to spatial and environmental niche effect on its diversity and distribution.

Susel Castellanos-Iglesias a *, Luiz H. Varzinczak b, Maria A. Haddad a a Programa de pós-graduação em Zoologia, Setor de Ciências Biológicas da Universidade Federal do Paraná, Centro Politécnico, 81531-990 Curitiba, PR, Brazil. b Programa de pós -graduação em Ecologia e Conservação, Universidade Federal do Paraná, Centro Politécnico, 81531-990 Curitiba, PR, Brazil. *Corresponding author. Tel.: +55 (41) 98039386. E-mail address: [email protected].

Abstract

Threats of human impacts on marine biodiversity and the necessity to implement conservation measures of marine resources, from local to regional levels, encourage the studies of biodiversity patterns and the use of taxonomic inventories as the base of ecology, biogeography and conservation biology. This study is the first attempt to know the distribution pattern of the hydroid fauna of Antho- and Lepthothecata orders from shallow waters (0-500 m) within the Wider Caribbean. A similarity analysis between countries with Jaccard index was applied to know the role of some factors as environmental niche (temperature, depth and chlorophyll concentration) and dispersal (distance between species records) as drivers of the distribution pattern of hydroids in the study region. An updated check list of 396 species is shown with their occurrences (presence and absence data) in 34 countries. The hydroid fauna of Wider Caribbean can be divided in eight groups of similarity: 1) Dominica and British island, 2) Granada, St. Vincent_Granadine, Barbados and Monserrat 3) Jamaica and San Martin_Marteen 4) St. Kitts and Nevis and St. Barthelemy 5) Trinidad Tobago, Curacao, Aruba and Netherlands Island 6) Haiti and Venezuela 7) Cuba, Gulf of Mexico_Yucatan and U.S_Florida and 8) Colombia, Guadeloupe, Martinique, Panama, Belize and Bermudas. Countries as Saint Lucia, Honduras, Dominican Republic and Guatemala take an isolated position, probably due to the lack of knowledge of its fauna. The hydroid fauna distribution pattern probably can be explained by the current marine system in the Caribbean province. Families better distributed were Sertulariidae, Aglahopheniidae and Plumulariidae, recorded in 103 more than twenty countries. The species Aglaophenia latecarinata Allman, 1877, Sertularia marginata (Kirchenpauer, 1864) and Dynamena crisioides (Lamouroux, 1824) were recorded in more than 50% of the analyzed countries. The variation partitioning analysis has been applied to know the influence of environmental niche and dispersal on alpha and beta diversity of the hydroid fauna. One matrix was composed of abiotic variables (environmental niche data) and the second was the distance matrix generated with PCNM analysis (spatial data). For Beta diversity a distance based (db-RDA) analysis was used to correlate the community species matrix with the two sets of matrices of our predictive variables. The R-squares method using varpar function and VEGAN package, in R software, allowed us to know the total diversity variation, explained by the two sets of predictor variables and their combinations. A Venn diagram was performed to visualize the percent variation on alpha and beta diversity. Latitude and temperature were significantly correlated with alpha diversity. As a result of variation partitioning analysis, individual fraction of each explanatory variable did not explain separately the variation of alpha and beta diversity. On the contrary, the share fraction of both variables, dispersal and environmental niche, explained 23% of the variation of alpha diversity and 3% of Beta diversity of the hydroid fauna in the Caribbean province.

Key-words: Distribution pattern, tropical hydrozoan, hydroid checklist, varpar function, PCNM, alpha-beta diversity.

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1. Introduction

Hydroids are the benthic organisms of the Class Hydrozoa, Phylum Cnidaria, mainly of the Anthoathecata and Leptothecata orders. They sum approximately 2200 species registered in the World Registered Marine Species (Schuchert 2017). Hydroids are characterized by two basic forms during their life cycle: the sessile polypoid form, usually colonial, and the planktonics stages, the larvae and the free medusa. This type of double life allows them to colonize a wide variety of habitats. They are present as epizoic on animals of a wide range of size or as epiphytic on algae and phanerogams, in coral reefs, seagrass meadows and very commonly on the mangrove roots (Calder 1991). They can colonize natural and artificial substrates, being found on rocks, piles, buoys and shipwrecks. Inside the cnidarian phylum, hydrozoans are the only group with some freshwater species among the majority of marine species (Mills et al. 1996; Grant Smith 2001; Annandale 2011) and they can live from 0 to more than 3000 m depth (Calder and Vervoort, 1998). They are present in a wide range of climate, from tropical temperatures since ~30° C to less than 0° C (e.g. in Antarctic and northern cool seas) (Gili et al. 1996; Peña Cantero 2006), that highlights their capacity to develop communities in a wider variation of environmental factors.

Differences in seawater temperatures define the distributional limit of species, although other physical factors (depth, chlorophyll seawater concentration, currents directions) are recognized in forming biogeographical barriers too (Vermeij 1991). Other factors as type of substrate, inter and intraspecific relations between species modulate the distribution of organisms (Lenihan et al. 2011). The combination of all these factors makes possible the functional role of organisms in a space conform the niche (Guisan and Zimmermann 2000).This is a function that ties the individual aptitude with their habitat (Hutchinson 1957).

The differences on distribution of hydroids in marine shallow waters, like much of marine invertebrate organisms, is influenced by latitude mainly along continental coastline. In this marine zone, the number of hydroids species are highest in low latitudes, intermediate in mid- latitudes and lowest in high latitudes in the northwestern and central Atlantic waters (Calder 1992). The influence of latitude in species distribution vary between taxa, indicating that not correlated factors with latitude as sample bias and ecological factors could be also important (Fernandez and Marques 2014). The dispersion abilities and the distance that organisms travel 105 during dispersal have strong effects on different factors that determine the species distribution, e.g., the geographic extension of a population, the spatial scale of the ecological communities and the geographic distribution of the species, between other factors (Shulman and Bermingham 1995). The life cycle with fixed gonophore, or planktonic stages as larvae and/or free medusa have been related with differences in the distribution patterns of hydroids in the Caribbean region (Calder 1992).

The Caribbean province, included in the North Western zoogeographic region of the Atlantic Ocean, has a high diversity of Antho- and Lepthothacata hydroid fauna of 255 species registered until 1998, occupying the second place of biodiversity after the well-known Mediterranean province (Medel and López González 1998). It included an ample hydroid fauna of 51% composed by species common of Western Atlantic, 18.4% Amphi-Atlantic, 15.7% widely distributed in warm waters around the world, 3.9% circumtropical and 9% cosmopolitan (Medel and López González 1998). Some distribution patterns of Antho- and Lepthothacata fauna in the Atlantic Ocean have been explained by general currents system (Medel and López González 1998). The hydroid fauna of the Caribbean, Gulf of Mexico and West Indies waters have been a topic of interest in some important taxonomical studies that recognized the ecological and genuine characteristic of this region for hydrozoans, e.g., Fewkes (1881), Versluys (1899), Leloup (1935), Fraser (1947), Van Gemerden-Hoogeveen (1965), Vervoort (1968, 1972), Cairns (1986), Calder (1990, 1992,1993) and Calder and Cairns (2009). Because countries that border the Wider Caribbean encompass a major global marine biodiversity hot spot (Miloslavich et al. 2010), intermittent studies of hydroids as part of inventories and ecological surveys in some countries of the region, increased the knowledge of this cnidarian group (Table 1). All these contributions helped to understand the importance of the niche and dispersal effects on the complex ecological structure of hydroid communities in marine ecosystems. Studies in more localities, different habitats and depths, if combined with the integration of regional marine abiotic database surely increase the understanding drivers for the establishment of the marine diversity distribution, and the marine biogeographic barriers in the region where hydroids can persist.

The increase of human impacts on marine biodiversity and the necessity to implement conservation measures to the rational use of natural marine resources motivate the study of diversity patterns using biodiversity knowledge generated in taxonomic inventories, that are the 106 basal knowledge in ecology, biogeography and conservation biology (Miloslavich et al. 2010). So, the aim of this study is to analyze the state of knowledge of hydroids species (Leptothecata and Anthoathecata) of the Wider Caribbean region (Caribbean basin, Gulf of Mexico, southern coast of United States and Florida, West Indies and adjacent Atlantic water), based on the geographic distribution of georeferenced species records and regional taxonomic lists. This is a preliminary analysis of some factors (niche and spatial component) as drivers of distribution patterns of shallow-water hydroids. An updated checklist of all species registered was built with number of occurrences by country in the region.

This paper is the first to evaluate the species richness, occurrence of species, genus and families and the similarity between communities of hydroids fauna of the Wider Caribbean region. The influence of spatial and environmental niche effects on alpha and beta diversity distribution of the hydroid fauna within Wider Caribbean region were also assessed. This work summarized and updated the diversity of hydroids fauna (Leptothecata and Anthoathecata) in the study region.

The hypothesis tested was to predict how spatial component (dispersal) and environmental niche (composed by depth, temperature, and chlorophyll concentration in seawater), can be drivers on alpha and beta diversity distribution of hydroid fauna in the Wider Caribbean region.

2. Study area

The study area includes the province of Tropical North Western Atlantic and nine ecoregions (Spalding et al, 2007) where there are all Caribbean islands, continental countries with coast to Caribbean, Gulf of Mexico, coast of U.S to the Gulf of Mexico and Florida, the West Indies and the subtropical Northwestern Atlantic waters (Table 1, Fig. 1). All this area is known as Wider Caribbean. The coastal areas are those of East Mexico, Central America, Panama, Bahamas and Antillian Archipelago, South America from Colombia to French Guiana and the Southern United States (UNEP 1984). For the purpose of this revision, 37 countries were revised, but only 34 that have hydroid records were analyzed as spacial units. 107

Fig. 1. Study area including 34 countries with coastline in the Caribbean Sea, Gulf of Mexico and adjacent Atlantic water of West Indies (Wider Caribbean Region) and where hydroid fauna have been recorded. Numbers for each country: see Table 1.

A big part of the study area is located in the Central American tropical region, which includes countries surrounding the Equator line. This region is delimited in latitude by the Tropic of Cancer in the Northern Hemisphere at 23°26′13.4″ (or 23.43706°) N and the Tropic of Capricorn in the Southern Hemisphere at 23°26′13.4″ (or 23.43706°) S. The annual mean of temperature is at least 18°C that stays constant (hot) almost all the year. There are two seasons present, a wet season and a dry season. Three countries included in the study (Mexico, U.S and Bermudas) are located in the adjoining humid north subtropical region located mainly at coastal zones. In this region, the climate is hot and humid in the summer, to mild and cold in the winter. Mean temperatures in warmest month is 22° C or higher and in the coldest month is between 0°C to 18°C.

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Table 1. Countries of Wider Caribbean and bibliography analyzed. No. Country Code Bibliography for hydroids records 1 Antigua and Ant_Bar Vervoort (1968); Cairns (1986) Barbuda 2 Barbados Bar Vervoort (1968); Cairns (1986) 3 Bermudas Ber Vervoort (1968); Cairns (1986); Calder (1988, 1991a, 1997) 4 Bahamas Bah Vervoort (1968); Cairns (1986) 5 Belize Bel Vervoort (1968); ; Sprackin (1982); Calder (1988, 1991a, 1991b); Puce et al. (2005) 6 Colombia Col Vervoort (1968); Wedler (1973, 1976); Flórez- González (1983), Cairns (1986); Bandel and Wedler (1987). 7 Costa Rica Cos Vervoort, 1968; Cairns (1986); Cortés (1996); Kelmo and Vargas (2002) 8 Cuba Cub Vervoort (1968); Cairns (1986); Castellanos- Iglesias (2007), Castellanos-Iglesias et al. ( 2009, 2011); Varela et al. (2005); Varela (2010, 2012); Varela et al. ( 2012). 9 Curação Cur Vervoort (1968); Cairns (1986) 10 Dominique Dom Vervoort (1968) 11 Dominican Rep Vervoort (1968) Republic 12 Grenade Gre Vervoort (1968) 13 Guatemala Gua not record 14 Haití (Tortuga ) Hai Vervoort (1968) 15 Honduras Hon not record 16 Jamaica Jam Vervoort (1968) 17 Martinique Mart Vervoort (1968); Cairns (1986); Galea (2013); (France) Galea and Ferry (2013, 2015). 18 Montserrat (UK) Mont Vervoort (1968) 19 Mexico, Yucatan GMx Vervoort (1968); Cairns (1986); Garza-Serrato (1984); Calder and Cairns (2009). 20 Aruba Aru Vervoort (1968); Cairns (1986) 21 Panama Pan Calder and Kinkerdale ( 2005); Cairns (1986) 22 Puerto Rico PuR Vervoort (1968); Wedler and Larson (1986), Cairns (1986) 23 Saint Kitts and StKN Vervoort (1968) Nevis 24 Santa Lucía StLu Vervoort (1968) 25 Trinidad and TriB Vervoort (1968) Tobago 26 U.S and_Florida E.U (Fla) Vervoort (1968); Cairns (1986), Calder (2013) 27 St. Vincent and StVGr Vervoort (1968) the Grenadines 109

Cont. Table 1. No. Country Code Bibliography revised for records of Hydroids 28 Venezuela Ven Vervoort (1968) (Tortuga Island, Testigos Island, Coche Island, 29 Virgin British IslVNer Vervoort (1968) Islands, Anguillas, sombrero island (UK) 30 Virgin Island (St. IslVU.S Vervoort (1968) Thomas, St, Charlotte Amalia, John), USA 31 Guadeloupe Gua Vervoort (1968); Cairns (1986); Galea (2008, (France) 2010) 32 The Netherlands IslNer Vervoort (1968); Cairns (1986) (Saba e St. Eustatius) 33 Saint- Martin StMar Vervoort (1968) (France) 34 Saint-Barthélemy StBar Vervoort (1968) (France) 35 Guyana Guy no record 36 Nicaragua Nic no record 37 Surinam Sur no record

Marine currents in Caribbean and North tropical western Atlantic is relatively well known (Shulman and Bermingham 1995). There are three main currents identified in this region: Northern Caribbean current track, extended from St Croix to San Salvador (Bahamas), southern Caribbean current track that runs along Curacao, Panama and Belize. Both currents velocity are between less than 1.25 and 1.9 km⁄h (Shulman and Bermingham 1995). In addition, there is the intense Gulf Stream current (with velocity between 2 and 2.5 km⁄h), located in the eastern coast of the U.S. A and entering by Yucatan channel to the Gulf of Mexico and continue to the Gulf at the Straits of Florida. This current is commonly named as Loop Current while it is inside the Gulf of Mexico and is named as Florida current, between Florida and Cuba. Ahead of the coast of Florida this current encounters the Antilles current, the Northern Caribbean current track. After that, the Loop current flows to eastward and continues to run parallels to 110 the coast of Florida, until reaching Cape Hateras where it leaves the coast and enters deeper Atlantic Ocean. See current map in Shulman and Bermingham (1995).

3. Material and Methods

3.1. Summarizing and updating the hydroids fauna Hydroid species lists were compiled from hydroid records of each country included in the Wider Caribbean region, taking as a base the hydroid fauna revised by Vervoort (1968). Besides this, other monographs with collections in the Caribbean and adjacent region was verified (Fewkes 1881; Fraser 1944, 1947). All available scientific papers were checked to complete the revision of each species, genus and family recorded for the region (Table 1). All records were verified in the WoRMs database between 2015 and 2017, to know the present accepted status of each táxon with information of registered locality and authors, in the software Excell, office 2010. The distribution of Millepora species complex of the Caribbean (Millepora alcicornis, Millepora complanata, Millepora striata, and Millepora squarrosa) was not strictly included in the analyses of this research, because of their wider distribution on Caribbean coral reef and the existing discussion about the status and acceptance of these species after recent results obtained from morphological and genetic evaluation of this groups in the region, where their taxonomic boundaries are still not clearly defined (Ruiz-Ramos, Weil and Schizas 2014; Schuchert, WoRMS 2017), The spatial representation of distribution and abundance of the hydroid fauna were shown with the use of software ArcMap from ArcGIS, version 10.01.

3.2. Data collection for the analysis of spatial and niche influence on alpha and Beta diversity of hydroids. Data of environmental variables (according to the niche) were obtained to build a matrix representing environmental niche dimensions from different sources. Mean annual sea surface temperature (SST) for the period 2009-2013 (composite dataset created by UNEP-WCMC) were obtained from NASA Ocean Biology (OB.DAAC, 2014), and data of chlorophyll concentration was obtained from the Moderate Resolution Imaging Spectroradiometer (MODIS) of Aqua Ocean Colour website (NASA OB.DAAC, Greenbelt, MD, USA, 2014). Bathymetry data was obtained from Space: Oceanography, Geophysics and Climate, Geoscience Professional Services, Bethesda, Maryland (Sandwell et al. 2002). In addition, 111 information of depth based on hydroid records for each geographic locations of each country included in the studied region was used to complement the information of bathymetric data.

Geographic locations (Latitude and Longitude) of hydroid records of 34 countries were utilized to build a matrix of geographical distance between sites in fossil package (Vavrek 2011) in R. They were obtained with arbitrary selection of five hydroid records from literature with information of latitude and longitude for each country. These geographic locations were plotted in the ArcMap software of ArcGis, version 10.1, to select the specific environmental data (SST, depth and chlorophyl) which corresponded for each site of hydroid recorded. All these data was used to construct the environmental matrix.

Geographic distance (spatial variables) among location of sites of each countries were used to build a spatial matrix with the Principal Coordinates Neighbour Matrix PCNM analysis (Borcard and Legendre 2002; Borcard et al. 2004). This analysis used a Euclidean distance matrix (distance in km) between each location of hydroid records to create eigenvector which were used as spatial variables. A data matrix of environmental variables (SST, depth and chlorophyll) used as the other predictive variables was prepared from online database mentioned above and standardized (mean = 0, standard deviation = 1) with package VEGAN in R, version 1.0.136.

3.3 Data analyses

3.3.1 Jaccard index and CLUSTER analysis of hydroids distribution. To analyze the similarity of hydroids communities in the study region, a data matrix recording presence or absence of each of the 396 species in 34 countries was built from species updated list in this study. We calculated in a pairwise comparison between communities their degree of similarity with the Jaccard index, which takes into account the proportion of shared species between communities in relation to the overall species present in both communities. A CLUSTER analyze by method of average group was done to determine the groups of samples (countries) formed by similarity between composition and species presence and absence.

Each location (country) was selected for having shore line toward the Caribbean and Atlantic waters, independently of their level of knowledge of the hydroid fauna. A depth range from the intertidal zone to 500 m was selected because of depth information reached by 112 hydroids recorded in the study zone. All these analyses were performed with software R and with the package VEGAN.

3.3.2 Spatial and niche influence on alpha and beta diversity of hydroids.

To test how alpha diversity (species richness of each country) was influenced by each of ourpredictive variables (latitude, longitude, SST, depth and chlorophyll concentration) we applied a series of simple Linear Regression Model.

To understand the independent contribution of environment with the space and vice versa (as predictor variables), a variation partitioning analysis was performed for two response variables, alpha diversity and beta diversity of the hydroid fauna. For alpha diversity, we analyzed the species richness of each country as our response variable and for beta diversity, we used the distance similarity community matrix of species obtained with Jaccard index as response variables. For each response variable was assessed the variation partitioning respect of two sets of explanatory variables (Peres-Neto and Legendre 2010). Those were the abiotic variables (environmental niche data) and vectors considered as dispersal variables generated from distance matrix with PCNM analysis (spatial data)

Variation partitioning analysis was done with varpart function (Partition the Variation of Community Matrix by 2, 3… Explanatory Matrices (Borcard, et al. 1992; Legendre and Legendre 1998; Peres-Neto et al. 2006).

For Beta diversity, a distance based (db-RDA) analysis was used to correlate the community species matrix with the two sets of matrices of our predictive variables. This analysis had three steps to ordination process: 1-calculate the distance matrix, 2-Run a PCoA and 3- Run a RDA on the eingenvalues obtained from the PCoA. These analyzes were done with package VEGAN in R.

Computing a PCoA with the distance community matrix generated with Jaccard index allows us use the positive eigenvalues obtained in the variation partitioning analysis as response variable for Beta diversity.

The method adjusted R squared (Peres-Neto et al. 2006) was used to assess the total diversity variation explained by the two sets of predictor variables and their combinations 113

(Peres-Neto and Legendre 2010). The fraction explained uniquely by each of predictor variables was [a] for environmental niche and [c] for space, and their joint effect as [b]. In addition, was obtained [d] as residual fraction of variation not explained by predictor variables (Borcard et al. 1992).

A Venn diagram was done for both alpha and beta diversity. The labels of intersection fractions [b] and of each individual fraction [a] and [c] with the adjusted R squared were obtained with the function varpar in the package VEGAN (Oksanen et al. 2007) with software R. This last analysis allowed to know the percentage of variation of diversity of hydroid fauna explained by dispersal as spatial variable and environmental niche.

4. Results

4.1 Updated distribution of the hydroid fauna (Anthoathecata and Leptothecata) in the Wider Caribbean. To update the hydroid fauna (Leptothecata and Anthoathecata) of the Wider Caribbean 33 articles about hydroid studies in the region were reviewed. Since the Vervoort (1968) checklist account for the Caribbean hydroids up to day, 91 changes of species names and 69 of genus names were observed. Following the database information in Worms (World Register of Marine Species, 2017), 9 species were taxom inquerenda, 2 were nomen dubium and four species were added to WoRMS list, thus updating the present status of each species. The updated checklist and the hydroid species distribution in the studied region (Wider Caribbean), with the species alphabetically ordered and their occurrence in each country indicated, are unpublished data resulted from this research, that will be show forward in the publication of this thesis.

4.2 Number and occurrence of families and genus and species richness of hydroids in the studied region.

The updated Leptothecata and Anthoathecata hydroid fauna are composed of 396 species, 125 genus and 40 families. Thecate hydroids diversity was larger with 261 species and 135 were Athecate. Numbers of species, families and genus tended to be higher in countries located at the boundary of tropical and subtropical region of the studied area. Numbers of taxa were different between countries (Fig. 2). 114

Fig. 2. Variation of taxa numbers (families, genus and species) between countries in the Wider Caribbean.

Number of families, genus and species of each country was summarized in (Table 2). Related to species richness, the Gulf of Mexico-Yucatán and U.S-Florida were better represented for hydroid fauna. The countries and localities of the region with higher number of hydroid families were the Gulf of Mexico including Yucatán, with 30 families. Ten countries had more than 20 families registered. Inside this group, Cuba was in the ninth position with twenty hydroid families (Table 2). Despite the unfrequented and scarce studies in some island of the region, e.g, Martinica and Cuba, they occupied the third and fourth position in species number. Bermuda occupied the fourth place. (Table 2).

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Table 2. Hydroids families, genus and species records in each country in the Wider Caribbean. Tabela 3. Ocorrência de famílias, gêneros e espécies em cada país da região do Grande Caribe. Countries Number of Number of Species Countries codes families genera richness Gulf of Mexico and Yucatan GMx 30 75 206 (Mexico) U.S (Florida and coast of U.S_Fla 28 71 173 Golfo de Mexico) Martinique Mart 21 49 104 Cuba Cub 20 51 102 Bermudas Ber 27 55 94 Guadeloupe Gua 21 42 74 Belize Bel 23 42 72 Panama Pan 24 42 71 Puerto Rico, Culebra Island PuR 19 47 66 Colombia Col 12 22 62 Bahamas Bah 14 29 53 Costa Rica Cost 20 38 41 Venezuela, Tortuga, Testigos Ven 10 22 35 and Coche Island Islands of the Netherlands Isl_PB 7 30 32 (Bonaire, Saba and St. Eustatius) Virgin Islands U.S (St. slVU.S 14 25 31 Thomas, St. Charlotte Amalia, John) Barbados Bar 8 20 30 Aruba Ar 9 17 27 Curação Cur 9 15 22 Haití (Tortuga ) Hai 12 18 23 Trinidad and Tobago Tri 8 13 18

Granade Gren 7 12 17 Saint Vincent and the St VGr 3 7 10 Grenadines Saint Kitts and Nevis St KN 5 6 9 Montserrat Mont 6 10 10 British Virgin Islands, slVNer 6 7 8 (Anguillas, Sombrero Island, St Eustatius) Jamaica Jam 6 7 7 Saint-Barthélemy StBr 5 6 7 Dominique Dom 4 5 6 Antigua and Barbuda Ant-Bar 5 5 5 116

Honduras Hon 1 2 3 Saint- Martin (French), Sint- StMar 3 3 3 Marteen (Dutch) Guatemala Guat 1 2 2 Saint Lucia StLu 1 2 2 Dominicana Republic Rdom 1 1 1 Guyane Guy 0 0 0 Nicaragua Nic 0 0 0 Surinam Sur 0 0 0

Families better distributed were Sertulariidae, Aglaopheniidae and Plumulariidae, as they were found in more than twenty countries. From the forty families recorded in this study, thirteen were considered rare with only five occurrences in the area (Fig. 3).

Fig. 3. Number of records of hydroids families (Anthoathecata e Lepthotecata, Cornelius 1992) in Wider Caribbean. 117

A total of 25 species had more than 30% registers in the region (Fig. 4). The most frequent species (present in more than 50% of all countries) were Aglaophenia latecarinata Allman, 1877, Dynamena crisioides (Lamouroux, 1824) and Sertularia marginata (Kirchenpauer, 1864). Otherwise, 32% (126 species) of the species were considered rare, having only one record for one country.

Fig. 4. Species of hydroids (Anthoathecata e Lepthotecata) that have more than 30 % of occurrence on Wider Caribbean countries.

4.3 Similarity distribution of species composition of hydroid fauna in the Wider Caribbean.

A total of 396 species were analyzed. Number of species tended to be highest in the northern countries, corresponding to the highest latitudes of the tropical region, intermediate in lower latitudes and lowest in the mid-latitudes. In the Cluster analysis results, using a similarity cut off point of 0.9 or less the hydroid fauna of Caribbean province was divided in eight groups (Fig. 5): 1) Dominique and British island, 2) Granada, St. Vincent_Granadine, Barbados and Monserrat 3) Jamaica and San Martin_Marteen 4) St. Kitts and Nevis and St. Barthelemy 118

5)Trinidad Tobago, Curacao, Aruba and Netherlands Island 6) Haiti and Venezuela 7) Cuba, Gulf of Mexico_Yucatan and U.S_Florida and 8) Colombia, Guadeloupe, Martinique, Panama, Belize e Bermudas. Countries as Saint Lucia, Honduras, Dominican Republic and Guatemala takes an isolated position, probably due to the lack of knowledge of its fauna. Hydroids fauna distribution patterns probably can be explained by the marine system currents in the Basin of Caribbean province,(Fig. 6)

The result of CLUSTER analysis with the application of Jaccard similarity index is show in Fig. 5.

Fig. 5. Cluster analysis of hydroid species records of the Wider Caribbean obtained by pairwise comparison of similarity degrees of the hydroid fauna communities between countries, with the Jaccard index (Sokal and Sneath, 1963). Colors and numbers indicate groups of similarity.

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Fig. 6. The pattern of the marine system currents in Basin Caribbean and general distribution of hydroid species obtained by pairwise comparison of the degrees of similarity of hydroid species communities between countries with the CLUTERS analysis using the average between groups. Colored circles indicate the groups of similarity. 121

4.4 Spatial and environmental niche influence on alpha and beta diversity of hydroid fauna in the Wider Caribbean.

Latitude and temperature were significantly correlated with species richness (number of species for each country) as linear models results can be seen in Table 3. In the studied region, richness of the hydroid fauna increased with high latitudes (F=5.74, p=0.02) (Fig. 6). In the contrary, species richness is negatively correlated with Temperature, decreasing when temperature increases (F=6.31, p=0.01) (Fig. 7).

Table 3. Results of linear model regression between Species richness (S) and Latitude (Lat) and temperature (Temp.).

Estimate R2 Error t-value Pr(>|t|) F df p-value Std adjust Intercept -20.53 27.21 -0.75 0.45 5.74 0.12 32 0.02 * S vs Lat 3.78 1.57 2.39 0.02 * Intercept 646.02 240.46 2.68 0.01 * 6.31 0.13 32 0.01 * S vs Temp. -28.98 11.53 -2.51 0.01 *

Fig. 6. Result of linear model regression of Species richness of hydroids vs Latitude. 122

Fig.7. Result of linear model regression of Species richness of hydroids vs Temperature.

Venn scheme of variation partitioning analysis shows that individual fraction of each predictor ((a) the environmental niche (SST, depth and chlorophyll) and (c) the space as dispersal) didn’t explain separately the variation of alpha and beta diversity of the hydroid fauna. The share fraction of both predictors, dispersal and environmental niche (b), explained 23% of alpha diversity variation and 3% of beta diversity variation. Fig. 8

Fig. 8. Venn scheme of variation partitioning analysis result for alpha and beta diversity of hydroid fauna of Wider Caribbean. 123

5. Discussion

As our hypotheses predicted, the highest variation in the composition of hydroid community was better explained by both environmental and spatial variables, than each separated fractions of factors. The importance of the influence of the share fraction of spatial as dispersal and environmental factors as depth, temperature and chlorophyll concentration, on alpha and beta diversity of hydroid community have been demonstrated in this study. Also the share fraction of environemental and spatial variables was the most important for the 50% of variation of community composition in 58 published data sets information of community structure (Cottenie, 2005). Also the environmental niche variables, temperature, depth and productivity were three major factors that highlight the variation of species richness marine distribution, that occur mainly in coastal zones and shelf areas with higher productivity (Chaudhary et al., 2016). Depth data is rarely considered in studies of biogeographic inference (Miranda, Genzano and Marques, 2015). For hydroids, depth is one of the causal factor of variation of distribution of species, when diversity, species composition and abundance, change with the depth (Di Camilo et al, 2008, Ronowicz, Wlodarska-Kowalczuk and Kukliński, 2013).

The distribution of species is influenced by several of physical and biological oceanographic processes (Fernandez, Navarrete and Marques 2014). Water circulation in the ocean affect larval transportation, destination and arrival moments to shores (Abelson and Denny 1997; Largier 2003; Pineda et al. 2010). Survival of larvae during dispersal depends of anatomical tolerance to the environment, food availability and predation (Morgan 1995; Shanks 1995; Vargas et al. 2006). Water temperature and geographic physical structure are drivers of dispersal barriers of species and contribute with genetic differentiation promoting speciation (Cornelius 1992b; Palumbi 1994; Schroth et al. 2002). Latitude is correlated with temperature and solar radiation which influences the primary and secondary production (Chaudhary et al. 2016) and, in consequence, on richness and abundance of species (Fernandez, Navarrete and Marques 2014).

Anthoathecata and Leptothecata species, genera and families records of Wider Caribbean was generally high in latitudes little far off the Equator line. The high number of species occurred in Gulf of Mexico, U.S Florida and Cuba which are situated at the highest latitudes of the studied area, near the division between tropical and subtropical regions. A 124 similar pattern was found in the Caribbean basin compilation of biodiversity, where the Greater Antilles has the highest number of sponges, coral and mollusks. In terms of absolute species richness, the Antilles Caribbean was more specious than the Southern and Eastern Caribbean ecoregion (Miloslavich et al. 2010). In this study, also Cuba showed a trend towards the highest concentration of species richness of the Antilles.

The general unimodal dispersion pattern of highest biodiversity in the tropics (Gaston 2000) occur from high to low latitudes and with a dip at the equator line. This pattern has been observed for some marine taxa, although it is also presented in terrestrial groups (Chaudhary, Saeedi and Costello 2016). Nowadays, the marine species richness of some taxa has an asymmetric and bimodal latitudinal pattern (Fernandez and Marques 2017, Chaudhary, Saeedi and Costello 2017) and highest values are not in the Ecuator line, but some distance away (Roy et al. 1998; Lyons and Willig 1999). The cause of species richness declining near the Ecuador line is suggested by the impact of global warming and other factors as physiological preference, food and competition (Molinos et al. 2015). The variation of species richness between taxa with latitude has been documented, suggesting the shading of general custom patterns (Platnick 1991) and the importance for species distribution of other factors not correlated with latitude (Fernandez, Navarrete and Marques 2017). For the Caribbean marine biodiversity pattern is known that some factors prevent to reflect with accuracy the true status (Miloslavich et al. 2010), mainly the incompletely inventories and the variation of sampling effort between areas.

In this study, highest species richness and high number of families occurred in countries located in the limit of tropical and subtropical region (Gulf of Mexico, U.S-Florida and Cuba). Medium-high species richness of hydroids were present in intermediate latitudes, in countries with a good knowledge of the hydroid fauna, like Martinique and Guadaloupe from Caribbean Lesser Antilles and in countries of continental platforms and important research capacity like Belize, Panama and Colombia. This result showed the importance of increase taxonomic and monitoring studies to know the biodiversity patterns at region level. Distribution of hydroid fauna in the Caribbean could be underestimated by the scarce or absent records in many location and countries, like Jamaica, Santa Lucia, Guiana, Surinam and Nicaragua, the last three without any hydroid record in the available literature. The absence of infrastructure for marine biodiversity research in some of the Caribbean countries could be one of the causes of the insufficient knowledge about their marine biodiversity (Miloslavich et al. 2010). Associated with 125 this, the lack of taxonomic experts and of sampling and accurate geographic records makes difficult a definite marine distribution and biogeographic inference (Miranda, Genzano and Marques, 2015).

Latitudinal distribution differences between species composition have been attributed to climate variation (Vergés et al. 2014), habitat differences and scarceness of substrate (Blackburn and Gaston 1996). Hydroid distribution also can be influenced by the percentages of species with fix gonophores or free medusa in their life cycle (Calder 1992). These different life cycle strategies become important in biogeographic analysis, since behavior and migratory capacity depends on oceanographic conditions (Miranda, Genzano and Marques 2015). Some hydroids have a different range of geographical distribution for polyp and for medusa stage (Cornelius 1995). A wide distribution, the existence of a polyp with asexual reproduction, an adequate recruitment and larvae formation become the main strategies for hydroids survival (Jaubet and Genzano 2011).

The province of Tropical Northwestern Atlantic, where this study has been carried out, has nine ecoregions very well identified by distinct oceanographic and topographic characteristics (Spalding et al. 2007), although not for the similarity of species composition (Miloslavich et al. 2010). In our study, from the eight groups recognized in the CLUSTER analysis of hydroid assemblages, the species composition and abundance of group 8 (Guadeloupe, Martinique, Colombia, Panamá, Belize and Bermudas) showed a relation between Eastern, South and Western Caribbean ecoregions. These zones coincide with one of the main currents identified in the area, the southern Caribbean Current track. This current runs along Curacao, Panama and Belize, passing through the Yucatan channel to the Gulf of Mexico. Then it joins to the Florida and Northern Caribbean Current tracks, reaching Cape Hateras and entering deeper Atlantic Ocean. In this area, north Atlantic Ocean circulation patterns have important subtropical gyres of warm ocean currents (Lavender et al. 2010) that could contribute to the similarity of hydroid fauna between Bermuda, Central American countries and eastern Caribbean islands (Burke and Maiden 2004, Robertson and Cramer 2014).

Dispersal over long distance is well recognized in the marine environment (Heads 2005) as happens with hydroid species of free medusa in the life cycle and those that do not have 126 and use rafting as dispersal form. They can colonize substrates as floating plastic debris, recognized as passive track of ocean circulation (Lavender et al. 2010). These could be the dispersal mechanisms for the species wider distributed, mainly of the families Sertulariidae, Aglaopheniidae and Plumulariidae, as Aglaophenia latecarinata Allman, 1877, Sertularia marginata (Kirchenpauer, 1864) and Dynamena crisioides (Lamouroux, 1824). These species reproduce sexually by fixed gonophores (Boero and Bouillon 1993).

The similarity of hydroid fauna composition of group 7 (Gulf of Mexico, U.S-Florida and Cuba) coincided with the subtropical region that is considered separate from tropical Gulf and Caribbean ecoregion (Spalding et al. 2007). This zone has marked differences of water temperatures and the flowing of the Loop Current cool waters, coming from the Gulf of Mexico and passing with intense velocity between Cuba and Florida.

Group 5 coincided with the beginning of the southern Caribbean current track and groups 1, 2, 3 and 4, concentrated in the majority of lesser Antilles, coincided with the influence of the northern Caribbean current track that extends to San Salvador, Bahamas.

For some taxa of marine species, a wide dispersal is provided by a long life and survival of the larval stages (Shulman, and Bermingham 1995). For hydroids, the wide distribution comes from sexual and asexual reproduction and diverse forms of life as polyps, medusa, planulae stages and others complex reproductive strategies (Gili and Hughes 1995). In addition, the capacity of colonizing different substrates (Calder 1991, Gili and Hughes 1995, Genzano and Rodriguez 1998) and the resistant mechanisms of dormancy to adverse environmental conditions (Cornelius 1992, Calder 1993) allow them to cross geographic barriers of different marine abiotic and biotic factors.

Alpha diversity distribution pattern of the hydroid fauna in the Wider Caribbean was positively correlated with latitudes and negatively correlated with temperatures. Temperature has a symmetrical gradient with latitude suggesting a strong influence as predictor of biogeographical distribution (Chaudhary, Saeedi, and Costello 2017). Latitudinal gradient has been highly correlated with temperature for species richness of some marine taxa (Boltovskoy, and Correa 2016; Saedi et al. 2016), although contrasting patterns of distribution between taxa reveal the importance of environmental and ecological factors over the latitudinal gradient distribution in species richness. The variation of bimodal peaks of species richness in different 127 latitudes suggests that temperature is not the only abiotic factor implied in distribution patterns of marine species (Chaudhary, Saeedi and Costello 2016).

6. Conclusions

This study compiled and updated the status of knowledge of the Wider Caribbean hydroid fauna after Vervoort (1968) review. This is also the first attempt to know the similarity in the hydroid composition between the Wider Caribbean country coasts. The survey also allowed to know a preliminary distribution pattern of the hydroid fauna and explored some drivers, as environmental niche (temperature, depth, and chlorophyll sea concentration) and dispersal (spatial distance between georeferenced species records) that influence the variation of their alpha and beta diversity in the area.

The hydroid fauna (Leptothecata and Anthoathecata) of the Wider Caribbean is composed by 396 species, 40 families and 125 genera. Thecates have the higher richness with 261 species and 135 were athecates. Despite being one of the regions with the highest diversity and high percentage of endemic hydroid species of Atlantic Ocean (Medel and López- González, 1997), some Caribbean countries.have a scarce or none knowledge of these organisms mainly because of the lack of infrastructure for marine biodiversity research.

Species richness, number of families and genus tended to be high in countries located at the boundary of tropical and subtropical region of the studied area. Countries with the highest species richness were Mexico (Gulf of Mexico and Yucatan), U.S,A, (Gulf of Mexico-Florida) and Cuba (north center and western region).

The hydroid fauna composition in the Wider Caribbean can be split in eight groups of similarity. The pattern can be generally explained by the current system of the Caribbean basin, included in the Caribbean and the Tropical Northwestern Atlantic provinces. The countries that occupied an isolated position it was probably because of the lack of species registration.

Latitude and temperature were significantly correlated with alpha diversity (number of species of each country). Environmental niche and dispersal didn’t explain separately the variation of alpha and beta diversity of the hydroid fauna. On the contrary, the share fraction of both variables explained 23% and 3% respectively of the alpha and beta diversity variations. We thought that data of habitat, substrate, and reproduction type will be very important to future 128 studies of the distribution patterns of hydroid fauna in the region, because of the influence of these ecological factors in the dynamics of the community structure of this fauna.

7. Acknowledgments

We extend our special thanks to CNPq (Brazilian National Council for Scientific and Technological Development) for the PhD. Scholarship of the first author. Thanks to professor Peter Schuchert for his attentions during the revision of hydroids species status in the WoRMS data base and for the literature. Thanks to UNEP-WCMC, NASA Ocean Biology (OB.DAAC, 2014), Aqua Ocean Colour website (NASA OB.DAAC, Greenbelt, MD, USA, 2014) and Oceanography, Geophysics, and Climate, Geoscience Professional Services, Bethesda, Maryland for on line marine environmental regional data delivered.

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CAPÍTULO 3

“ORGANIC CONTAMINATION AS A DRIVER OF STRUCTURAL CHANGES OF HYDROID’S ASSEMBLAGES OF THE CORAL REEFS NEAR TO HAVANA HARBOUR, CUBA.”

Manuscrito publicado Marine Pollution Bulletin Fator de impacto 2015-2016: 3.099 Qualis (Biodiversidade): A1 139

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Organic contamination as a driver of structural changes of hydroid’s assemblages of the coral reefs near to Havana Harbour, Cuba.”

Susel Castellanos-Iglesias a, *, Ana Caroline Cabral b, Cesar.C. Martins c, Maikon Di Domenico b, c, Rosana Moreira da Rocha a, d , Maria Angélica Haddad a, d

a Post-graduate program in Zoology, Universidade Federal do Paraná, Centro Politécnico, Caixa Postal 19020, 81531-980 Curitiba, PR, Brazil.

b Post-graduate course on Estuarine and Ocean Systems (PGSISCO), Universidade Federal do Parana, Caixa Postal 61, 83255-976, Pontal do Paraná, PR, Brazil

c Centro de Estudos do Mar, Universidade Federal do Paraná, Caixa Postal 61, 83255-976 Pontal do Paraná, PR, Brazil.

d Zoology Department, Universidade Federal do Paraná, Centro Politécnico, Caixa Postal 19020, 81531-980 Curitiba, PR, Brazil.

Corresponding author

E-mail address: * [email protected] (S. Castellanos-Iglesias)

Tel.: +55 (41) 98039386

Full postal address: Zoology Department, Universidade Federal do Paraná, Centro Politécnico, Caixa Postal 19020, 81531-980 Curitiba, PR, Brazil

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Abstract

Hydroid assemblage’s responses to organic contamination were evaluated using sedimentary sterols as explanatory variables. At seven coral reef sites in the Havana west coast, hydroids were collected along three 10 m x 1 m, 10 m deep transects. Five sterols were analysed, i.e., coprostanol, an indicator of faecal contamination, and cholestanol, cholesterol, stigmasterol and brassicasterol, indicators of biogenic organic matter inputs. The sampling sites were classified by level of contamination. A total of 65 species comprised the hydroid assemblages. Hydroids community abundance and richness decreased in the contaminated sites. Coprostanol had the highest relative importance for these variables and also for Plumularia floridana and Clytia gracilis abundances. Obelia dichotoma and Halecium bermudense were relatively abundant in the contaminated sites. The results indicate that faecal contamination negatively affected the hydroid assemblages, highlighting the importance of integrated biological and chemical indicators to evaluate the environmental conditions of the Havana coral reef.

Key-words: Sterols, coprostanol, organic matter, generalized linear models, Leptothecata,

Anthoathecata.

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Highlights:

Hydroid’s richness and abundance diminish in highest contaminated sites.

Faecal sterols indicate contamination on coral reef near to Havana harbour.

GLM analysis predicted the relative importance of sterols on hydroids community.

CAP analysis showed the sensitivity of hydroids to faecal contamination.

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1. Introduction

Coastal development increases the input of pollutants to marine habitats, negatively affecting biodiversity worldwide (Johnston and Roberts, 2009) and consequently affecting human health. Monitoring environmental changes caused by organic contamination is a challenge to coastal management (Islam and Tanaka, 2004). Scientists have proposed several analyses to determine the influence of environmental and human impacts on marine benthic communities (Warwick, 1988, Anderson and Willis, 2003; Anderson et al., 2008), which are considered environmental bioindicators. They can reflect the cumulative influence of pollution by changes in the abundance, richness and diversity of their communities (Rosenberg et al., 2004, Borja and Dauer, 2008, Venturini et al., 2008).

Sterols are an important class of molecules that indicate biogenic and anthropogenic organic matter (OM), due to their relative resistance to environmental degradation, which allows them to accumulate in sediments (Takada and Eganhouse, 1998). Coprostanol (5β(H)- colestan-3β-ol) is a faecal sterol used as a chemical marker of human waste inputs by sewage effluents to coastal environments (Martins et al., 2014a; Carreira et al., 2015; Cabral et al., 2018), and it is one of the most commonly tested abiotic markers of human interference on benthic marine macrofauna (Albano et al., 2013; Barboza et al., 2015, Souza et al., 2016). Other sterols, such as cholesterol (cholest-5en-3β-ol), cholestanol (5α(Η)-cholestan-3β-ol), and brassicasterol (24-methylcholesta-5,22E-dien-3β-ol), are indicators of marine biogenic OM inputs, due to their ubiquitous presence in zooplankton and phytoplankton communities (Volkman, 2005). In addition, stigmasterol (24-ethylcholesta-5,22-E-dien-3β-ol) is a vascular plant marker and also of marine diatoms, cyanobacteria and prymnesiophycean algae (Volkman et al., 1998; Martins et al., 2011).

Hydroids are one of the less distinct organisms in coral reefs, due to their small size and uncoloured exoskeleton. Nevertheless, the morphological plasticity of some hydroids makes them a key group for detecting environmental changes in benthic marine biocoenoses (Gili and Huges, 1995). Although few studies have used hydroids as environmental bioindicators, in the tropical coral reefs of Jamaica and Siladen Island (Indonesia), stenoecious species have been suggested as indicators of light intensity and water movement (Di Camillo et al., 2008). It is known that changes in the composition and distribution of hydroid 144 communities and of resilient species are entirely connected to environmental changes (Di Camilo et al., 2008; Peña Cantero and Manjón-Cabeza, 2014).

The effects of environmental factors on hydroids were reviewed by Boero (1984). Some species resist poor water quality, and pollutants may even stimulate colony growth, while natural factors (salinity, temperature, turbidity, speed currents) can modulate the susceptibility of hydroid species to different pollutant concentrations. The sublethal responses of hydroids to pollutants include losses of hydrants and changes in growth and gonozooids production rates (Gili and Hughes, 1995). Otherwise, the effect of contamination on the biodiversity and structural changes of hydroid communities has been poorly studied until now.

Despite some environmental monitoring efforts, the port area of the Havana has been recognized as among the most contaminated aquatic ecosystems in the western coast of Havana city, Cuba (Aguilar et al., 2004, Yamiris et al., 2013) and in the wider Caribbean (Paz, 2004; Valdes, 2004; Villasol et al., 2010, UNEP-CEP, 2010, Romeu-Álvarez et al., 2012). In addition, several rivers without any treatment discharge contaminants from sewage and industrial activities directly to the marine ecosystem of Havana (Romeu, 2012), consequently affecting the health of benthic organisms and fish communities ( De la Guardia et al., 2001, Aguilar et al., 2008, Hernández-Muñoz et al., 2008; Villiers and Alcolado, 2012, Busutil and Alcolado, 2012).

Integrated assessments of environmental conditions combined with biological indicators are still lacking in the Havana coast. In this study, we determined sterol concentrations in sediments as indicators of biogenic and anthropogenic OM inputs, in order to evaluate the influence of different OM sources on the coral reefs off the west coast of Havana. We also tested whether the gradient of sewage contamination, as indicated by faecal sterols, was a driver of variations in ecological descriptors (abundance and richness) and the spatial distribution of hydroids.

2. Study area

The study area is the subtidal coral reef zone off the west coast of Havana, Cuba. Four biotopes, Echinometra, rocky plain, terrace edge and terrace base, constitute the fringing coral reef (Aguilar et al., 2004). The similarity of the ecological zones parallel to the shore and 145 the almost flat bottom of limestone conferee a relative homogeneity to this ecosystem in the study zone (González-Sansón and Aguilar, 2002; González-Díaz et al. 2003; Aguilar et al., 2004; Villiers and Alcolado, 2012). The sediments are sand and biogenic calcareous fragments (Pérez and Cánovas, 2006). Scleractinians, octocorals and sponges are common, and the macroalgae Halimeda spp. and Lobophora spp. were abundant and dominant in some of these sampling sites during survey.

The abundance and dominance of macroalgae change with the distance to the nutrient sources as contaminated rivers and submarine sewage outfalls. This was corroborated by studies of macroalgae community between clean and more impacted zones (González-Días et al., 2003) and in the determination of the environmental disturbs index between sampling sites in the coral reef of the coast of Havana Semidey (2013).

The mean sea surface temperatures (SST) in the dry (November to April) and rainy (May to October) seasons are approximately 25.7°C and 28.8 °C, respectively, with mean surface salinities of approximately 36.2 and 35.9 PSU, respectively (Cuba Oceanology Institute, data from 2009-2014). The current flows less than 1 km from the shore, and westward movements are caused by an anticyclone system (Arriaza et al., 2012), indicating that the fringing reef is affected by oceanic waters. To minimize the effects of these and other parameters as light exposition, heterogeneity, hydrodynamics, hydroids samples were collected at the same depth (10 m).

Contamination and overfishing have been the main impacts in this region for decades (Herrera and Martínez-Estalella, 1987; De la Guardia et al., 2001; González-Sansón and Aguilar, 2010). Two submarine sewage outfalls discharge domestic and industrial wastes in the area nearly 500 m offshore (Table 1). Effluents from these outfalls have also been released by ruptures at different depths and times (De la Guardia et al., 2000a,b; Hernández- Muñoz et al., 2008). Consequently, coral diseases, high percentages of macroalgae and low percentages of living coral cover prevail along with the dominant pollution-indicator species of gorgonians and sponges (González-Díaz et al. 2003, Hernández-Muñoz et al., 2008; Busutil and Alcolado, 2012).

Table 1. Characterization of sampling sites in the west coast of Havana, Cuba. 146

Distance Approximate Nearest from Latitude & Sampling sites Code distance from pollution Havana Longitude shore (m) sources Harbour (km) 23º´08’49”N Havana Bay HB 100 Havana Harbour 0.07 82º21’ 31”W 23º´08’32”N Raw sewage Maceo park MA 30-50 1.65 82º22’12”W input

Raw urban and industrial waste Almendares river 23°08’04”N ALM 50-100 from 5.9 outfall 82°24’53”W Almendares river Minor domestic 23°07’45”N 16 Street CA16 100 sewage 6.9 82°25’23”W discharge 23°06’51”N 70 Street CA70 100 - 9.2 82°26’29”W Raw wastewater Emissary 23°05’40”N from Quibú river EM 100 12.6 180 Street 82°28’05”W and sewage outfall Santana river 23°04’25”N SA 50-100 - 19.1 outfall 82°31’37”W

The seven sampling sites were distributed within the 20 km coastline and are, from the east (near the Havana Harbour entrance) towards the west, Havana Bay (HB), Maceo Park (MA), Almendares (ALM), 16 Street (CA16), 70 Street (CA70), Emissary (EM) and Santana (SA) (Fig. 1). They are located between 30 and 100 m offshore and at different distances from the pollution sources, according to previous benthos and fish community studies in the area (de la Guardia et al., 2001; Aguilar et al., 2004; González–Sansón and Aguilar, 2010) (Table 1).

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Fig. 1. Sampling sites on the coral reef in the west coast of Havana, Cuba. HB: Havana Harbour, MA: Maceo park; CA16: 16 street; CA70: 70 street; ALM: Almendares river outfall; EM: Emissary 180 street, and; SA: Santana river outfall.

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3. Material and methods.

3.1. Sampling

Hydroids were collected on March 2013 by SCUBA diving, at 10 m depth. Three 10 x 1 m transects parallel to the shore and separated from each other by at least 5 m were surveyed in each of the seven sites. Along each transect, all visualized hydroids were taken with their substrates and placed in a plastic bag. Samples were narcotized with menthol crystals and fixed in 4% formaldehyde.

Surface sediments (one sample per site) were obtained for the sterol analyses using a stainless-steel grabber. The top 2 cm of the undisturbed surface sediments were placed into pre-cleaned aluminium foil, oven dried (< 40°C, 48 h), homogenized in a mortar and stored in cleaned glass bottles until analysis.

3.2. Biological data: sorting, counting and identification of hydroids.

Hydroids were separated under a stereomicroscope. With an optical microscope, they were identified to the maximum level of taxonomic resolution and photographed with a Canon® camera (EOS-REBEL T3). The taxonomic identifications were based on studies mainly from the Caribbean and surrounding regions (Calder, 1988, 1991, 1997, Calder and Kirkendale 2005, Galea, 2008, 2010, 2013), from Europe (Schuchert, 1997, Bouillon et al., 2004, 2006) and from Brazil (Cunha et al., 2015). The colony fragments of each hydroid species were deposited in the Cnidaria collection of the Zoology Department of the Paraná Federal University. Species abundances were determined by counting all the hydrocaulus of the colonies.

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3.3. Sterols analyses

Based on the method described by Wisnieski et al. (2016), approximately 15 g of dry sediment was Soxhlet extracted over 8 h using 80 mL of a mixture of dichloromethane (DCM) and n-hexane (1:1). The 5α-androstanol was added before each blank and sample extraction and used as the surrogate standard. The concentrated extracts were purified and fractionated using 3.2 g of silica and 1.8 g of alumina column chromatography, both 5% deactivated. The samples were eluted with 5 mL of ethanol/DCM (1:9) followed by 15 mL of ethanol to obtain the polar fraction containing the sterols. The extracts were evaporated to dryness and derivatized to form trimethylsilyl ethers using BSTFA (bis(trimethylsilyl)trifluoroacetamide) with 1% TMCS (trimethylchlorosilane) for 90 min at 65 °C.

The instrumental analyses were performed using gas chromatography with an Agilent HP 7890A coupled to a flame ionization detector (FID) and a fused silica capillary column coated with 5% diphenyl/dimethylsiloxane (50 m, 0.32 mm ID and 0.17 μm film thickness). Instrumental and calibration details are described by Cabral et al. (2018). Surrogate recoveries ranged from 70 to 113% (mean = 91 ± 14). The measured concentrations of the target sterols in the IAEA-408 reference material were within 87–102% of the certified values provided by the International Atomic Energy Agency. The detection limits (DL) were <0.01 μg g-1 for all sterols analysed.

3.4. Data analyses

Species richness (S), total hydroid abundances (Hyd), and the abundance of hydroid species with more than six occurrences in the study sites were selected as response variables. Generalized linear models (GLM) were applied to predict the influence of sterols on the variations in species richness and abundance. The multicollinearity of the sterols was tested by the variance inflation factor (VIF) package (Fox and Weisberg, 2011), selecting only VIF values less than 10. Zero-inflated negative binomial distributions were used to fit the candidate models with the glmmADMB package (Skaug et al., 2011; R Development Core Team, 2015).

To account for model selection, a multi-model inference was applied to calculate the importance of all possible combinations of the explanatory variables, using the functions 150 model.sel and model.avg (MuMIn 1.12.1 package; Barton, 2015). Those models were ranked by AICc (Akaike’s Information Criterion) and delta AIC values (Zuur et al., 2009). Using the model averages, we based the significance of the results on the most robust relative importance values from the model.

The spatial correlations between samples were analysed and removed with the spatial simultaneous autoregressive (SAR) error model, and the errorsarlm function was implemented in the spdep package in R, with a neighbourhood distance of 0.005 and the “W” method (Bivand et al., 2011). The correlograms (distance vs Moran index) were created for the best fit model for each response variable. The Moran index was used to measure the similarity between pairs of sites for each distance classes (Rossi and Queneherve, 1998; Diniz-Filho et al., 2003). For the species with over-dispersion, we used the quasi-Poisson family model ranked by QAIC (Bolker, 2016).

To test the origin of sterols related to multiple biogenic (marine and/or terrestrial) sources, Pearson’s correlation coefficient (r, p < 0.05) was applied.

The concentration of coprostanol was the first criteria used to classify the sites according to faecal contamination. Sites were not contaminated (NC) when [coprostanol] < 0.10 μg g-1, moderately contaminated (MC) when [coprostanol] > 0.20 μg g-1 and contaminated (C) when [coprostanol] > 0.50 μg g-1. A ratio related to coprostanol and cholestanol concentrations [ratio I = coprostanol/(coprostanol + cholestanol)] was the second criteria used to support the assessment of faecal contamination (Martins et al., 2014a ). Values between 0.5 and 1.0 indicate sewage contamination in sediments (Leeming et al., 1998).

The species/sample abundance matrix (log+1 transformed) was the response variable used to test the effect of biogenic/anthropogenic OM (indicated by sterols) on the hydroid assemblages. The differences in the multivariate structure of the hydroid assemblages were estimated by the permutational analyses of variance (PERMANOVA+) with 9999 permutations (Anderson, 2001; McArdle and Anderson, 2001, Anderson et al., 2008). The experimental design included contamination as a fixed factor, with three levels: contaminated, moderately contaminated and not contaminated. Each of the three transects per site were sample units. A pair-wise test was applied for an a posteriori comparison. The multivariate 151 patterns of the sterols in each sampling site were assessed by using the sites as fixed factors and the sterols as vectors.

A PCO (principal coordinate analysis) (Anderson and Willis, 2003) was performed to visualize the main trends in hydroids abundance related to the contamination level. The percent contribution of each species to the dissimilarity among sites was accessed by the SIMPER analysis (Clarke, 1993). Multivariate patterns between hydroid species, sites and sterols were visualized by the canonical analysis of principal coordinates (CAP; Anderson and Willis, 2003). All these analyses were performed with the PRIMER-E and PERMANOVA software (Clarke and Gorley, 2006). To identify the individual contributions of species to multivariate patterns (Anderson and Willis, 2003), the abundance matrix of the hydroid species (log+1 transformed) was correlated with the canonical axes and used as an indirect “post hoc” test.

4. Results

4.1. Characterization of sampling sites based on sterols concentration data

Coprostanol was detected in all the sampled sites, with values ranging from 0.02 to 1.37 μg g-1. The highest coprostanol concentration occurred in HB, and the lowest coprostanol concentration occurred in SA (Table 2). There was a decreasing faecal contamination gradient from HB to CA70, east to west from the Havana coast, that was disrupted in EM and decreased again in SA.

Table 2. Sterol concentrations (μg g-1 dry weight) and diagnostic ratios in the surface sediments of the Havana coral reef, Cuba.

Sterols/Sites OM indicator HB 1 MA ALM CA16 CA70 EM SA coprostanol faecal 1.37 0.93 0.53 0.33 0.05 0.21 0.02 cholesterol marine 0.97 0.77 4.24 5.58 0.31 0.56 1.64 cholestanol marine 0.29 0.21 0.52 1.48 0.08 0.09 0.22 brassicasterol marine 0.37 0.37 2.20 2.04 0.18 0.34 0.93 stigmasterol terrestrial 0.38 0.40 1.43 1.68 0.28 0.36 0.85 Total sterols 3.38 2.68 8.92 11.11 0.90 1. 56 3.66 152

Diagnostic ratios Ratio I 2 0.83 0.82 0.50 0.18 0.38 0.70 0.08 Ratio II 3 0.30 0.27 0.12 0.27 0.26 0.16 0.13 Contamination classification 4 C C MC MC NC MC NC 1 Abbreviations are the same as those in Fig. 1 and Table 1 2 Ratio I (coprostanol/coprostanol+cholestanol) > 0.50: sewage contamination (Leeming et al., 1998) 3 Ratio II (cholestanol/cholesterol)< 0.50: fresh organic matter input; > 0.50: in situ reduction of cholesterol (Canuel and Martens, 1993; Chalaux et al., 1995) 4 (C) Contaminated; (MC) Moderately Contamination, and; (NC) Not Contaminated.

Based on the coprostanol levels (main criterion) and ratio I values (secondary criterion) (Table 2), the sites were categorized into three classes: sites CA70 and SA were NC = not contaminated (coprostanol concentration < 0.10 μg g-1 and ratio I < 0.50); sites CA16, EM and ALM were MC = moderately contaminated (coprostanol concentration > 0.20 μg g-1 and ratio I > 0.50); and sites HB and MA were C = contaminated (coprostanol concentration >0.50 μg g-1 and ratio I > 0.50). We classified ALM as MC, although its coprostanol concentration was slightly higher (0.53 μg g-1) than maximum the MC limit. CA16 was classified as MC because of its coprostanol concentrations (0.33 μg g-1), despite having a ratio I < 0.50. In addition, the cholestanol/cholesterol ratio (ratio II) was > 0.30 in all sites studied (Table 2), indicating the presence of fresh OM in the sediments.

Pearson’s analysis for sterol concentrations showed a low correlation between coprostanol and all the other natural sterols (Table S1). There was a high and positive correlation between stigmasterol (terrigenous origin) and brassicasterol (marine origin) along the Havana coast.

The highest percentages of coprostanol (cop) (over 40% of the total analysed sterols) appeared in the contaminated sites (HB and MA), and the lowest values were in SA (a not contaminated site). Sterols from marine sources (cholesterol and brassicasterol) predominated in the moderately contaminated (ALM, CA16, EM) and not contaminated sites (CA70 and SA) (Fig. S1). 153

4.2. Characterization of hydroids community structure

Sixty-five hydroid species were found along the studied area (Table S2, Supplementary Information), of which twenty-nine species composed 95% of the hydroid community abundance. A total of 9,806 hydrocaulus were counted, of which 9,755 were identified at the species level and included in the statistical analyses. Plumularia floridana Nutting, 1900 was the most abundant species (1,766 hydrocaulus), followed by Clytia gracilis (Sars, 1850) (1,539 hydrocaulus). Only 10 species had more than 200 hydrocaulus, and 26% were considered rare species (less than 10 hydrocaulus).

The contaminated sites MA and HB had the lowest mean group hydroid abundance and richness values. Sites subjected to a low input of sewage (MC-CA16 and NC-CA70) (Table 1) had the highest mean group abundance and richness values. Abundance and species richness decreased in the not contaminated site SA (Fig. 2a).

Of the sixteen models generated by GLM analysis, four (M5, M4, M9 and M6) had the best fits for richness (S) and abundance (Hyd) (Table S3). Richness was better explained by coprostanol (M5), while abundance was better explained by cholestanol + coprostanol (M4). Among all the explanatory variables (sterols), the negative effect of faecal contamination indicated by coprostanol showed the highest relative importance (RI) for predicting S (RI = 0.97) and total abundance (RI = 0.98). In addition, hydroids abundance and richness decreased along with the increment of the coprostanol concentration (Fig. 2b).

The spatial autocorrelations between the sampling sites (distance class vs Moran′s index) were positive for total abundance and for the Clytia gracilis, Antennella peculiaris and Obelia dichotoma abundances. The results in Table S3 include the spatial autocorrelation analyses.

Each of the most abundant species in this study showed different responses to the increasing sterols concentrations (Figs S2a,b).

154

Fig. 2. (A) Abundance of hydroids (number of hydrocaulus/10 x 1m transect) and species richness per transect on coral reef of the west coast of Havana, Cuba. C = contaminated, MC = moderately contaminated and NC = not contaminated sites. Sites abbreviations in Fig.1. (B) Coprostanol (cop) effect on abundance (Hyd) and species richness (S) of hydroids.

The contamination factor level was significant in the PERMANOVA test (Table 3). The pair-wise test showed (Table 3) significant species composition, abundance and richness differences between the contaminated sites and the other sites, but it did not differentiate moderately contaminated from not contaminated sites. By using the sterols as covariates and the sites as fixed factors, both coprostanol and stigmasterol significantly affected the hydroid assemblage structures (Table 3). 155

Table 3. PERMANOVA tests comparing sites by their different levels of contamination, with sterol levels as the covariates in the analysis. Level of contamination (n = 3) as the fixed factor in the design test. Factor df SS MS Pseudo-F p perms level of contamination 2 15,234 7,617 3.291 0.0003* 9901 Residuals 18 18 41,661 2,315 Total 20 20 56,894 PAIR-WISE TEST results: Groups df t p perms C, MC 13 1.824 0.0008* 4,293 C, NC 10 2.319 0.0015* 462 MC, NC 13 1.224 0.1429 4,294 Including covariates in the analysis and using sites (n = 7) as the fixed factor in the design test. Covariates df SS MS Pseudo-F p perms coprostanol 1 10,505 10,505 4.672 0.0001* 9,941 cholestanol 1 3,686 3,686 1.639 0.0925 9,927 cholesterol 1 3,749 3,749 1.667 0.0721 9,940 stigmasterol 1 7,017 7,017 3.121 0.0010* 9,912 Sites 1 1,577 1,577 0.701 0.7675 9,920 Residuals 14 31,477 2,248 Total 20 61,361

The first two PCO axes (metric MDS) explained 30.4% and 15.8% of the variability in the dissimilarity matrix (Fig. 3). There was a separation between the contaminated (HB and MA) and not contaminated sites (SA and CA70). Some transects from the moderately contaminated sites appeared scattered among the not contaminated sites, corroborating the results of the PERMANOVA pair-wise test (Table 3). 156

Fig. 3. Results of Principal Coordinate analysis (PCO) of hydroids abundance matrix (transformed with log(x+1) and Bray Curtis similarity index) showing relationship between assemblage structure and contamination level: C = contaminated sites (HB and MA), MC = moderate contamination (ALM, CA16 and EM), and NC = not contaminated (CA70 and SA). Sites abbreviation in Fig.1

The SIMPER analysis (Table S4) showed a higher dissimilarity between contaminated and the other sites. P. floridana achieved a 17.7% contribution to the C and MC dissimilarities and an 11.3% contribution to the C and NC dissimilarities because of its absence in C sites. C. gracilis contributed 10.6 and 8.82% to the dissimilarities between C and NC and between MC and NC, respectively, as it was more abundant in MC sites. Other species with important contributions were P. disticha (7.47%) and Halecium tenellum Hincks, 1861 (7.19%), which distinguished the C and MC sites, and H. tenellum (7.76%), which distinguished the C and NC sites. P. floridana was the most abundant hydroid in this study, but it was absent in the C sites. The most abundant hydroids in the C sites were O. dichotoma in HB and H. bermudense in MA. The C. gracilis abundances were similar in the MC and NC sites, but did not appear in the MC site ALM (Fig. S3) 157

CAP analysis showed a pattern of differences among the groups of sites in the plot of the first two canonical axis. The C sites were separated well and positively correlated with the coprostanol concentration. The CAP 1 axis separated the C sites from some transects of the MC sites and from all NC sites, while the CAP 2 axis separated the C sites from the other MC sites. The CAP analysis also showed the species responsible for the multivariate patterns related to the influence of different levels of contamination (Fig. 4).

Fig. 4. Canonical analysis of principal coordinates (CAP) ordination showing the axis that best discriminated hydroid species at three levels of contamination, with sterols as vector base variables. Only species present in more than six transects were considered. Site abbreviations and species in Fig.1 and Table S2.

P. floridana and H. tenellum were negatively correlated with coprostanol levels in the CAP 2 axis, while O. dichotoma and H. bermudense had a positive correlation with coprostanol and the contaminated sites. H. nanum and P. strictorcarpa were negatively correlated with cholestanol and positively correlated with the three transects of the NC sites (CAP 1 axis). O. sargassicola was positively correlated with cholestanol and the three 158 transects of the MC sites. P. disticha was positively correlated with cholestanol and brassicasterol.

5. Discussion

Our hypothesis predicted that the hydroid assemblages would respond to different levels of faecal contamination and to different types of OM sources along the Havana west coast. Changes in the assemblage structure were indeed observed, even in sites separated by the small distance of 20 km. Coprostanol indicates contamination by faecal OM and human waste inputs when it is the predominant sterol in sediments (Martins et al., 2014a; Carreira et al., 2015), and the diagnostic ratios (ratio I) can confirm indicated contamination.

The absence of a correlation between coprostanol and the other sterols (Table S3) reinforces its sewage origin. The contamination level measured by the coprostanol concentration (up to 1.37 μg g-1) was slightly higher in the study site than in other tropical/subtropical estuaries, such as the São Sebastião Channel, southeast Brazil (up to 0.62 μg g-1) (Muniz et al., 2015) and pristine environments, such as Potter Cove (0.02 – 0.08 μg g-1) (Dauner et al., 2015) and Admiralty Bay (0.03 – 0.07 μg g−1) in Antarctica (Martins et al., 2014b). Otherwise, the Havana coast contamination level was lower than that in Cienfuegos Bay, Cuba (up to 5.40 μg g-1) (Tolosa et al., 2014), and in two Brazilian estuaries, Babitonga Bay (up to 6.08 μg g-1) (Martins et al., 2014a ) and Santos Bay (up to 8.51 μg g-1) (Martins et al., 2008a,b).

Diagnostic ratios of some sterols have been reported as indicators for determining contamination sources and evaluating sewage contamination levels in sediments (Takada and Eganhouse, 1998). The coprostanol/coprostanol+cholestanol ratio (ratio I) indicates faecal contamination associated with a relatively high concentration of coprostanol (> 0.50 μg g-1) for ratios between 0.5 and 1.0 (Leeming et al., 1998). Low coprostanol concentrations and ratios below 0.3 are associated with pristine sediments, while ratios between 0.3 and 0.5 require the confirmation of other indices, due to the possible natural production of cholestanol by biogenic sources or its in situ production by the reduction of cholesterol (Grimalt et al., 1990). In fact, contaminated sites HB and MA had highest ratio I and coprostanol concentration values, while CA70 and SA had relatively low values and were classified as not contaminated sites. The cholestanol/cholesterol ratio (ratio II) was applied to indicate the OM 159 degradation in the sediments. Values < 0.5 indicate fresh OM, while values > 0.5 are related to the in situ reduction of cholesterol and degraded OM (Canuel and Martens, 1993). Low values of ratio II were found in sites HB and MA. The high cholestanol concentration in these sites and the high correlation with other marine sterols (cholesterol and brassicasterol) indicate biogenic sources. Pearson’s correlations between the sterol concentrations confirm the sources of OM in the sediments (Table S1). The high and positive correlation between stigmasterol (terrigenous origin) and brassicasterol (marine origin) can be explained by the increase in marine productivity, which is caused by the terrigenous material supply that generates the predominance of marine OM across the region.

The current patterns prevalent in the study area, with a strong influence of oceanic waters (Arriaza et al., 2012), probably causes the dilution of anthropogenic material along the west coast of Havana, explaining the high differences in faecal sterol concentrations between the C and NC sites, which were the nearest and the farthest, respectively, from the Havana Harbour (Alepúz et al., 1984; Mederos et al., 1984).

All studied sampling sites are submitted to a different levels and sources of contamination and, receive in different levels the effect of the main disturb, the high contaminated waters of the Havana Harbour. Because of this, the biodiversity of marine organisms as benthic hydroids vary as an expression of the effect of intermediary disturb hypotheses (Wilkinson, 1999). The sequence of hydroid richness and abundance not only reflect the sites distances from the Habana Harbour; but also the influence of faecal contamination from river sources.

The grouping of sites related to contaminant accumulation and contamination sources, together with dissimilarities among the benthic community assemblages, can provide powerful evidence of environmental changes (Warwick, 1988). The minimum hydroid abundance and richness were found in the contaminated sites HB and MA (Fig. 3) because high levels of faecal contamination probably eliminate individuals less adapted to these conditions, eventually reducing species recruitment.

The highest coprostanol concentration value (1.37 μg g-1) was detected in the C-HB, but unexpectedly, this site did not have the lowest hydroid abundance and richness. The lowest values were found in C-MA, which had the second highest cop value (0.97 μg g-1) (Fig. 160

3, Table 2). This result can probably be attributed to the dominant shoreline westward currents (Mosquera and Cabañas, 1985; Fernández, 1995) directing the Harbour-contaminated waters not to the nearest site, HB, but to site MA. In addition, MA is in front of the “Havana City Center”, the most populated zone in Havana, with 134,570 residents (ONE, 2015); this region discharges raw sewage and wastewater at the seashore without any treatment.

The negative effects of contamination on the hydrozoan community was previously investigated by Cifuentes et al. (2007) in marine fouling communities and by Ben-Brahim et al. (2010) in epiphytic assemblages of Posidonia oceanica, both in temperate waters. These authors allied sewage, one of the most important contaminants in polluted sites, to significant decreases in species number and percent cover. Hydroid assemblages had minor levels of species richness in harbour areas under high levels of contamination, where many hydroid species almost completely disappear (Boero, 1984).

The hydroid diversity was less affected at sites MC-CA16 and NC-CA70 and, the highest mean abundance appeared at MC-CA16. Both sites are not direct influenced by sources of high contamination levels (e.g., the Havana Harbour and Almendares and Quibú Rivers) although, it may be affected by some domestic contamination from the shore (Table 1) and (Fig. 2). In addition, they are considered as “clean” areas with high abundance of stony corals (González-Díaz et al., 2003; Perera, 2012). CA70 had the highest mean richness of hydroids favoured by a high quantities of Halimeda spp. colonized by hydroid (personal observation). Halimeda spp. are a very common substrate occupied by highly diverse epiphytic hydroid assemblages (Llobet et al., 1991).

Otherwise, diversity of hydroid decreased in the site NC-SA that has lowest faecal contamination and, is the farthest of Habana Harbour. In addition, the neighbour Santana river is the least contaminated of the Havana coast (Villiers and Alcolado, 2012). Because of these characteristics, the hypothesis of intermediary disturbance (mentioned above) explains the hydroids decreasing under low impact of contamination (Svensson, 2007). Diversity of hydroid probably decreased as a result of species exclusion by species competition, with a slight increase in dominant species (Wilkinson,1999).

Beyond contamination, is known that natural factors as temperature, salinity, light intensity, water movement and food available could drive the hydroid distribution (Boero, 161

1984; Gili and Hughes, 1995; Di Camillo et al., 2008; Peña Cantero and Manjón-Cabeza, 2014). The combination of low salinity and high or moderate sewage contamination had a more adverse impact on hydroid diversity. These conditions can occur at the contaminated sites, where salinity decreases due to the influence of fresh water from the hydrographic basins of Havana Bay. Similar conditions (high contamination and low salinity) were found in the most interior sites of Babitonga Bay in the south of Brazil, where species richness and abundance decreased to the lower values (Cabral, 2013).

The type and abundance of available substrates can also modulate hydroid distribution, and explain differences in hydroid abundances and richness between sites by modifying the hydroid diversity in many ecosystems (Calder, 1991a, Genzano and Rodriguez, 1998, Ronowicz et al., 2013). Differences in abundance, cover of percentage and morfofunctional type of macroalgae, (an available substrate for colonization of hydroids), has been found between studied sites in relation with the influence of contamination (González- Días et al.,2003) corroborating MA as the more impacted site and ES as moderately impacted (Semidey, 2013).

Hydroids species also had different responses of abundance and distribution. The negative influence of faecal contamination on P. floridana, C. gracilis and H. bermudense indicates that they are sensitive to sewage and can be viewed as good bioindicators of this type of contamination (Fig. 4) P. floridana and C. gracilis, were dominant in the moderately and not contaminated sites. At the contaminated sites, P. floridana was absent and C. gracilis was rare. A detailed discussion about the response of each hydroid species to the different degrees of contamination is in the supplementary data.

Multivariate analyses determined the relationship between hydroid abundance, richness and composition with the type of OM based on sterols. The OM in sediments is important to the cyclical process of sediment resuspension in coral reef ecosystems (Burns and Brinkman, 2011). Hydroids can be omnivores, capturing all kinds of available zooplankton and phytoplankton prey (Gili et al., 2008). As sessile organisms, they can also feed on the resuspension of sediments and are therefore affected by the sediment quality. In addition, dinoflagellates, diatoms, detrital particulate organic matter and bacteria are part of hydroid diets (Coma et al., 1995, 1999; Gili et al., 1996a,b; Orejas et al., 2001). If sediments are 162 contaminated by different levels of faecal OM, as in the present study, their influence on hydroid assemblages can favour some species and disadvantage others. CAP ordination showed hydroid species typifying each level of contamination of each site group: P. floridana and H. tenellum characterized the not contaminated sites, while O. dichotoma and H. bermudense resisted the adverse conditions of the contaminated sites. C. gracilis hydroids were generalists but showed a very low specificity and abundance in contaminated sites.

The significant decrease in hydroid richness and abundance at the contaminated sites (high coprostanol levels) indicates that the hydroid community structures were affected by recent inputs of sewage contamination, as indicated by ratio II. We also observed the effect of faecal contamination in the structure of hydroid assemblages at sites near the contaminated Almendares and Quibú Rivers. A high correlation between the structure and distribution of meiofauna with contamination levels has been previously reported at the same region (Armenteros et al., 2003). The effects of urban sewage represent the primary impact on the distribution of benthic organisms, acting at spatial scales ranging from hundreds to thousands of metres (Souza et al., 2014).

6. Conclusions

Faecal contamination affected hydroid communities not only at the contaminated sites HB and MA, which are the nearest to Havana Harbour, but also at other sites that were affected mainly by the sewage discharge of the contaminated rivers along the coast (Romeu, 2012, Romeu-Álvarez et al., 2015; Villiers and Alcolado, 2012). Our results showed that hydroids are a benthic community sensitive to high contamination, which could have an increasing of abundance and biodiversity in environments with moderate faecal contamination. These results show that the marine biodiversity along the Havana coast is still affected by contamination, as it was in previous decades (Herrera and Alcolado, 1983; Alcolado and Herrera, 1987; Herrera and Martínez-Estalella, 1987; Herrera, 1990). The damage to the coral reef ecosystem is expressed by decreases in species abundance and richness and variations in the composition of marine organism assemblages, such as that of hydroids.

This is the first study about the contamination of the west coast of Havana, Cuba, assessed by analysing the sterols in sediments. These organic markers indicate different 163 levels of sewage contamination along the fringing coral reef of the Havana shore affect the structures of coral reef hydroid assemblages. The response of hydroid assemblages to contamination seems to be a useful and accurate tool to predict the influence of highly contaminated environments.

Multivariate analyses allowed the recognition of contamination patterns along the coast and indicated the contribution of individual hydroid species to the differences between the sampling sites. The assemblage compositions also varied with different levels of contamination, and the presence of some species were indicative of different environmental conditions. Plumularia floridana (1,766 hydrocaulus) and Clytia gracilis (1,539 hydrocaulus) were the most abundant hydroids in the Havana coral reef. P. floridana dominated in the not contaminated sites and was absent in the contaminated sites. C. gracilis dominated in the moderately contaminated sites. Both species were mostly influenced by high concentrations of coprostanol, indicating their sensitivity to faecal contamination. Obelia dichotoma (Linnaeus, 1758) and Halecium bermudense (Congdon, 1907) had higher abundances in the contaminated sites than in the other sites, and H. bermudense was dominant at site C-MA. The high relative importance (RI) of stigmasterol for O. dichotoma and H. bermudense indicated the influence of terrestrial OM on these hydroids distributions.

Many efforts have been made to implement management projects to reduce human impacts in the Havana Bay and coastline and to promote the sustainable use of its waters. Unfortunately, the sediment contamination in the fringing coral reef nearby, which affects the hydroid assemblage structures, shows that the impact of contamination by human activities is still high along the west coast of Havana.

7. Acknowledgments

We thank the coordinators of the The United Nations Development Programme with support from the Global Environment Facility (UNDP/GEF) Sabana-Camaguey Project who made this research a reality. We extend our special thanks to all colleagues of the Oceanology Institute of Cuba who helped with fieldwork. A.C. Cabral thanks to CAPES (Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior) and S.C. Iglesias to CNPq (Brazilian National Council for Scientific and Technological Development) for their PhD. Scholarship. CNPq research grants supported C.C. Martins (448945/2014-2) and R.M. Rocha 164

(305201/2014-0).

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9. Supplementary data.

SUPPLEMENTARY INFORMATION OF STUDY AREA

HB is the nearest site to the Havana Harbour entrance, where the water flux from the Bay reduces the influence of the contaminants (Busutil and Alcolado, 2012). MA, the second closest site to the Harbour (distance of approximately 1.6 km), receives the strongest influence by the contaminated water due to the dominant current pattern (Reyes et al., 1998), 174 and by untreated sewage discharges from Havana City. ALM is directly affected by the contaminated and nutrient rich waters of the Almendares River mouth. CA16 is in a clean zone far from the Almendares River mouth (1.5 km), although it receives some sewage and domestic discharges from the city shore (González-Diaz et al., 2003). CA70 is far from the main contamination sources (3.9 km from the Almendares River and 9.2 km from Havana Harbour) and from the most urbanized centre of Havana City. CA70 is the closest site to the shore and is used recreationally by local residents and tourists. EM is near the Quibú River mouth (810 m) and is affected by its contaminated waters (Villiers and Alcolado, 2012). SA is the farthest site from the main contaminated sources (6.5 km from the Quibú River, 13.4 km from the Almendares River and 19.1 km from Havana Harbour), and is the least affected by contamination compared with the other sampling sites of this study. The closest river to SA (1.4 km) is the Santana River, which is also considered the least contaminated river of the Havana coast, based on faecal, heterotrophic and sulfate-reducing coliform bacteria levels (Delgado, 2007).

SUPPLEMANTARY RESULTS

In the Figs. S2 a, b, the Halecium bermudense abundance decreased with increasing concentrations of cholestanol and coprostanol as related to the coprostanol in the best fit models (M5 and M7) and to cholestanol as the unique sterol for the model M16 (Table S3). Orthopyxis sargassicola (Nutting, 1915) and Antenella gracilis Allman, 1977 showed a sensitivity to faecal contamination, as indicated by the negative value of coprostanol in model M5. The Pennaria disticha Goldfuss, 1820 abundance could be explained by three significant models, including cholestanol in the best fit model (M16). Coprostanol had the highest relative importance value (RI = 0.67) in the O. dichotoma best fit model, but the species abundance decreased with high stigmasterol concentrations (Table S3 and Fig. S2b). The A. peculiaris abundance decreased with coprostanol (Fig. S2a) and cholestanol (Fig. S2b), but the cholesterol concentration had a positive effect (Fig. S2b) on the A. peculiaris abundance and the highest relative importance value (RI = 0.69) as an explanatory variable.

SUPPLEMENTARY DISCUSSION

The low RI values of sterols for A. gracilis suggest that the abundance of this species was less influenced by OM contamination, even though the best fit model selected under the 175 model.sel analysis included coprostanol.

The abundance of Antennella spp. was sensitive to faecal contamination in this study. However, they were abundant in coral reefs with apparently clean, well-lit waters with high natural nutrient enrichments, indicating that these conditions are probably a good environment for these species (Gravier-Bonnet and Bourmaud, 2006).

The H. bermudense abundance decreased in sites with high cholestanol and coprostanol concentrations, indicating the influence of faecal OM, as expressed by the cop effect. The presence of cholestanol in the best fit model (M16) suggests that H. bermudense is highly sensitive to variations in oxi/anoxic conditions, related with the OM degradation conditions expressed by the ratio II value. Additionally, the presence of cop in the M5 model shows the resistance of H. bermudense to sewage inputs in the C-MA site, with 42 of a total of only 45 hydrocaulus. According to Collins et al. (2002), Halecium species are tolerant to water quality differences without changes in abundance.

A similar effect of cholestanol was observed for the O. sargassicola and A. peculiaris abundances, but when cholestenol was combined with coprostanol, these species became sensitive to sewage contamination. Cholestanol can be derived from in situ cholesterol transformation or, minorly, from marine biogenic sources (Takada and Eganhouse, 1998). However, the predominance of “fresh” OM in all sampling sites is evidenced by the low ratio II values (< 0.50, Table 2), suggesting that the cholestanol in these coral reef sediments may be associated with high algae productivity. The high correlation between the cholestanol and sterols from marine sources (cholesterol and brassicasterol) reinforces this condition (r > 0.75; Table S1).

O. dichotoma and P. disticha had no significant RI (> 0.8) for any sterol as explanatory variables, but they did have the higest RI for coprostanol, showing that these species can be resistant to contamination by sewage inputs. P. disticha is a very widespread species in warm waters worldwide (Eldredge and Smith, 2001). It is common in human-impacted shallow waters on artificial substrates, such as docks, pilings, piers and ship hulls (Millard, 1975, Migotto, 1996). O. dichotoma and H. bermudense were the most abundant in the contaminated site assemblages (Fig. 7). These two species have a circumglobal distribution (Calder, 1991), indicating they can adapt to a wide range of environments and water 176 conditions (Boero, 1984). O. dichotoma includes up to 13 taxonomic groups in its diet but feeds mainly on faecal pellets, OM and microalgae (Orejas et al., 2013), demonstrating an adaptation to survive in contaminated coastlines (e.g., Havana) and estuaries (Paranaguá Bay, Brazil) (Cangussu et al. 2010). Additionally, in Brazil (Babitonga Bay), species of Obelia were considered indicators of sewage contamination in the winter. In the summer, warmer temperatures and greater rainfall increase their abundance and distribution levels along less contaminated sites (Cabral, 2013). This situation is similar to that of Havana during the rainy (summer) seasons, when salinity can decrease, mainly in bays and river mouths (Yamiris et al., 2013), although this phenomenon is not very drastic due the predominant current patterns, the influence of oceanic water and the narrowness of the marine platform (González Ferrer et al., 2004, Arriaza et al., 2010). Hydroids of the family Campanulariidae (Clytia spp., Obelia spp.) can colonize a great variety of substrates (Calder and Mayal, 1998; Bumbeer and Rocha, 2012) and adapt to different water quality and contamination levels (Orejas et al., 2013).

SUPPLEMENTARY REFERENCES

Arriaza L, Hernández M, Lorenzo S, Olivera J, Rodas L., Montesino D., Carrillo Y., Almeida I., Simanca J., Padrón J. N., 2012. Modelación numérica de corrientes marinas alrededor del occidente de Cuba Modeling of marine currents around Cuban western region. Serie Oceanológica. 10, 11-22.

Boero, F., 1984. The Ecology of Marine Hydroids and Effects of Environmental factors: A Review. Marine Ecology, 5 ,2, 93-118.

Bumbeer, J.A., Rocha, R.M., 2012. Detection of introduced sessile species on the near shore continental shelf in southern Brazil. Zoologia 29,2,126-134.

Busutil, L., and Alcolado, P.M. 2012. Prueba de un índice de contaminación orgánica urbana basado en comunidades de esponjas de arrecifes de Cuba. Serie Oceanolog. 10, 90- 110.

Cabral, A. C., 2013. Hidrozoários bênticos, em substrato artificial, como indicadores de condições ambientais na Baía da Babitonga, Santa Catarina. Universidade Federal do Paraná, Brasil.106p. 177

Calder, D., 1991. Shallow-water hydroids of Bermuda. The Thecata, exclusive of Plumularioidea. Rec. Ontario Mus.154, pp.140.

Calder, D.R., Mayal, E.M., 1998. Dry season distribution in a small tropical estuary, Pernambuco, Brazil. Zoologische Verhandelingen 323, 69-78.

Collins, K.J., Jensen, A.C., Mallinson, J.J., Roenelle, V. Smith, I.P., 2002. Environmental impact assessment of a scrap tyre artificial reef. Journal of Marine Science 59: 243- 249.

Cangussu, L. C, Alvater, L., Haddad M.A., Cabral, A.C., Linzmeier, H.H., M. Rocha, R. 2010. Substrate type as a selective tool against colonization by Non-native sessile invertebrates. Brazilian Journal of Oceanography, 58,3, 219-231.

Delgado, G. Y., 2007. Peligro, Vulnerabilidad y Riesgo ante la Contaminación Fecal en el Litoral Oeste de Ciudad de La Habana. Tesis presentada en opción al grado científico de Maestro en Microbiología. Universidad de La Habana, La Habana.

Eldredge L.G., Smith C.M., 2001. A guidebook of marine introduced species in Hawaii. Bishop Mus Tech Rep 21,1–80.

González-Díaz, P., de la Guardia, E., González-Sansón, G. 2003. Efectos de efluentes terrestres sobre las comunidades bentónicas de arrecifes coralinos de Ciudad de la Habana, Cuba. Rev. Inv. Mar. 24, 3, 193-204.

González-Ferrer, S., ed., 2004. Corales pétreos. Jardines Sumergidos de Cuba. España, Digital Da Vinci, 317 pp.

Gravier-Bonnet, N., Bourmaud, C. A., 2006. Hydroids (Cnidaria , Hydrozoa) of Coral Reefs : Preliminary Results on Community Structure , Species Distribution and Reproductive Biology in Juan de Nova Island ( Southwest Indian Ocean ). Wstern Indian Ocean J. Mar. Sci., 5,2, 123–132.

Migotto, A. E., 1996. Benthic shallow-water hydroids (Cnidaria, Hydrozoa) of the coast of Sao Sebastião, Brazil, including a checklist of Brazilian hydroids including a checklist of Brazilian hydroids Leiden: Nationaal Natuurhistorisch Museum. Zoologische 178

Verhandelingen, 306, 121pp.

Millard, N.A.H., 1975. Monograph on the Hydroida of southern Africa. Annals of the South African Museum, 68, 1–513.

Orejas, C., Rossi, S., Peralba, À., García, E., Gili, J. M., Lippert, H., 2013. Feeding ecology and trophic impact of the hydroid Obelia dichotoma in the Kongsfjorden (Spitsbergen, Arctic). Polar Biology, 36,1, 61–72. http://doi.org/10.1007/s00300-012-1239-7

Reyes I., Cordero J., Vega F., Rondón H., Téllez R., Martín A. Beltrán J., 1998. Caracterización oceanográfica del tramo del litoral norte habanero entre la ensenada de La Chorrera y la Calle 30 (Miramar) para la construcción de un emisario submarino (primera etapa). Informe Científico Técnico, GEOCUBA Estudios Marinos, 51 pp.

Takada, H. Eganhouse, R. P., 1998. Molecular markers of anthropogenic waste. Encyclopedia of Environmental Analysis and Remediation, 694, 2883 – 2940.

Villiers, N., and Alcolado, P. M., 2012. Size variation of Eunicea flexuosa and Plexaura kuekenthali (Cnidaria: Octocorallia) in coral reefs close to pollution sources in Havana, Cuba. SerieOceanológica. Institute of Oceanology of Cuba. 11, 91-102.

Yamiris T., Beltrán, J., Pérez, M., 2013. Control de la calidad ambiental de las aguas del litoral de Ciudad de la Habana, Cuba. Plan de vigilancia y monitoreo. Informe final de Servicio científico – técnico-CIMAB, La Habana.

179

SUPPLEMENTARY Table S1.

Pearson correlation coefficients (r) between the sterols of sediments collected in west coast of Havana, Cuba. * = indicate significant correlations at the 95% level (p < 0.05). coprostanol cholesterol cholestanol brassicasterol stigmasterol coprostanol x cholesterol -0.10 x cholesterol -0.02 0.91* x brassicasterol -0.15 0.97* 0.77* x estigmasterol -0.19 0.99* 0.85* 0.98* x

SUPPLEMENTARY Table S2.

Hydroids list of coral reef on the west coast of Havana. x- indicates species with reproduction structure Total of No. Species of hydrois Order Fertility polypery Plumularia floridana Nutting, 1 1,766 TE x 1900 2 Clytia gracilis (M.Sars, 1850) 1,539 TE x Orthopyxis sargassicola 3 734 TE x (Nutting, 1905) Antennella peculiaris Galea, 4 660 TE x 2013 Antennella gracilis Allman, 5 556 TE 1877 Obelia dichotoma (Linnaeus, 6 438 AT x 1758) Pennaria disticha Goldfuss, 7 400 TE x 1820 8 Halecium tenellum Hincks, 1861 323 TE 9 Halecium nanum Alder, 1859 313 TE x Halecium lightbourni Calder, 10 292 TE x 1991 11 Dynamena disticha (Bosc, 1802) 194 TE Halecium dichotomum Allman, 12 189 TE x 1888 Symmetroscyphus intermedius 13 177 TE (Congdon,1907) Plumularia strictocarpa 14 168 TE (Nutting, 1900) Halecium bermudense Congdon, 15 163 TE 1907 Halecium discoidum Galea, 16 149 TE 2013 180

Halopteris alternata (Nutting, 17 144 TE 1900) Sertularella peculiaris (Lelouop, 18 137 TE x 1935) Corydendrium parasiticum 19 127 AT x (Linnaeus, 1767) Campanularia hincksii Alder, 20 110 TE 1856 Cirrholovenia cf. tetranema 21 109 TE Kramp, 1959b Egmundella humilis Fraser, 22 106 TE 1936 Dynamena dalmasi (Versluys, 23 105 TE 1899) Halecium xanthellatum Galea, 24 104 TE 2013 25 Clytia linearis (Thornely, 1900) 88 TE x Eudendrium carneum Clarke, 26 79 AT x 1882 Calamphora campanulata 27 61 TE (Warren, 1908) Eudendrium calceolatum Motz- 28 54 AT x Kossowska, 1905 Nemalecium lighti (Bouillon, 29 43 TE 1986) Scandia mutabilis (Ritchie, 30 40 TE 1907) Clytia noliformis McCrady, 31 39 TE 1859 32 Scandia gigas (Pieper,1884) 32 TE Halopteris diaphana (Heller, 33 30 TE 1868) Clytia hemisphaerica (Linnaeus, 34 27 TE 1767) Turritopsis nutricula McCrady, 35 25 AT x 1859b Eudendrium bermudense Calder, 36 17 AT x 1988 37 Perarella sp. 17 AT x 38 Eudendrium sp.3 17 AT x Halecium beanii 39 14 TE (Johnston,1838) 40 Halecium sp. 1 14 TE 41 Halecium labrosum Alder, 1859 13 TE Halecium cf. plicatum Galea, 42 12 TE 2015 43 Coryne sp. 12 AT x 44 Hebella scandens (Bale, 1888) 12 TE 45 Halecium sp.2 12 TE Eudendrium capillare Alder, 46 11 AT x 1856 181

Mitrocomium cf cirratum 47 11 TE Haeckel, 1879 48 Campanularia sp.1 11 TE 49 Halecium sp.3 9 TE Sertularia distans (Lamouroux, 50 7 TE 1816) 51 Antennella sp.1 6 TE Eudendrium cf merulum 52 6 AT x Watson, 1985 53 Eudendrium sp.2 5 AT x 54 Hebella venusta (Allman, 1877) 5 TE Ventromma halecioides (Alder, 55 3 TE 1859) 56 Hydrodendron sp.1 3 TE Clytia hummelincki (Leloup, 57 2 TE 1935) Clytia macroteca (Perkins, 58 2 TE 1908) Clytia paulensis (Vanhoffen, 59 2 TE 1910) 60 Coryne pusilla Gaertner, 1774 2 AT x 61 Antennella sp.2 2 TE 62 Mitrocomium sp.1 1 TE 63 Coryne eximia Allman, 1859 1 AT x Antennella curvitheca 64 1 TE Fraser,1937a 65 Laomedea flexuosa Alder,1857 1 TE SUPPLEMENTARY Table S3. 182 Results of the analysis of model average (model.avg) from GLM and the application of zero inflated negative binomial distribution (glm.nb) ordered by AICc value and relative importance (RI) of each selected response variable. Models Intercept chol-a chol-e cop stig-e lat long df AICc QAICc delta AIC w Richness M5-S ~ cop 17.640 -11.97 - - 4 148.4 - 0.00 0.402 M4-S ~ chol-a + cop 15.84 3.81 -11.49 - - 5 150.4 - 1.98 0.149 M9-Hyd ~ cop + stig-e + chol-e 29.070 12.160 -16.24 -4142 - - 6 151.1 - 2.66 0.106 M6-Hyd ~ cop + chol-e 16.400 0.516 -11.46 - - 5 151.4 - 2.99 0.090 RI 0.32 0.28 0.97 0.29 - - Hydroid abundance 1 M4-Hyd ~ cop + chol-a 537.1 517.2 -548.1 - - 5 316.4 - 0.00 0.362 M9-Hyd ~ cop + stig-e + chol-e 1557.0 1006.00 -844.1 -3517. - - 6 318.1 - 1.63 0.160 M6-Hyd ~ cop + chol-e 517.9 105.90 -501.9 - - 5 318.9 - 2.49 0.104 M5-Hyd ~ cop 670.9 -352.5 - - 4 319.0 - 2.60 0.099 M8-Hyd ~ cop + stig-e + chol-a 733.4 899.5 -575.6 -445.4 - - 6 319.3 - 2.85 0.087 RI 0.55 0.37 0.98 0.35 - - Plumularia floridana M4-P_flo ~ cop + chol-a 55.440 188.9 -89.22 - - 5 262.3 - 0.00 0.220 M8-P_flo ~ cop + stig-e + chol-a 128.600 335.2 -129.40 -155.3 - - 6 262.6 - 0.28 0.191 M3-P_flo ~ cop + chol-a + chol-e 88.030 364.2 -48.85 -114.20 - - 6 263.4 - 1.04 0.131 M11-P_flo ~ chol-a + chol-e 53.660 402.9 -66.15 - - 5 263.6 - 1.28 0.116 M9-P_flo ~ cop + stig-e + chol-e 309.700 276.30 -171.60 -910.1 - - 6 263.7 - 1.35 0.112 M16-P_flo ~ chol-a 7.799 185.4 - - 4 264.4 - 2.05 0.079 M12-P_flo ~ chol-a + stig-e 77.770 323.5 -162.7 - - 5 265.1 - 2.82 0.054 RI 0.84 0.44 0.70 0.43 - - Clytia gracilis 1 M5-C_gra ~ cop 131.00 -114.1 - - 4 265.6 - 0.00 0.395 RI 0.68 - - Halecium bermudense M16-H_ber ~ chol-a 9.623 -3.509 - - 4 176.6 - 0.00 0.192 M5-H_ber ~ cop 9.312 -2.184 - - 4 176.6 - 0.01 0.191 183

TABLE S3. (cont.) Models Intercept chol-a chol-e cop stig-e lat long df AICc QAICc delta AIC w M15-H_ber ~ stig-e 10.060 -25.22 - - 4 176.7 - 0.06 0.186 M14-H_ber ~ chol-e 9.461 -0.6515 - - 4 176.7 - 0.09 0.184 M7-H_ber ~ cop + stig-e 13.000 -3.644 -4.042 - - 5 179.6 - 2.99 0.043 RI 0.32 0.31 0.33 0.32 - - Orthopyxis sargassicola M16-O_sar ~ chol-a 12.27 54.58 - - 4 245.2 - 0.00 0.171 M5-O_sar ~ cop 64.10 -51.83 - - 4 245.4 - 0.25 0.151 M4-O_sar ~ chol-a + cop 38.82 51.49 -44.65 - - 5 246.2 - 1.03 1.102 M12-O_sar ~ chol-a + stig-e 44.64 128.80 -86.35 - - 5 246.4 - 1.23 0.092 M11-O_sar ~ chol-a + col_e 24.52 142.50 -25.25 - - 5 246.8 - 1.64 0.075 M14-O_sar ~ chol-e 18.05 8.853 - - 4 246.9 - 1.76 0.071 M15-O_sar ~ stig-e 13.74 29.49 - - 4 247.3 - 2.06 0.061 M8-O_sar ~ cop + stig-e + chol-a 77.95 145.30 -65.36 -90.49 - - 6 247.5 - 2.34 0.053 M3-O_sar ~ cop + chol-a + chol-e 53.98 165.40 -28.160 -58.46 - - 6 247.9 - 2.73 0.044 M6-O_sar ~ cop + chol-e 46.35 7.220 -44.60 - - 5 247.9 - 2.74 0.043 M13-O_sar ~ chol-e + stig-e 95.81 102.7 -352.5 - - 5 248.0 - 2.81 0.042 M9-O_sar ~ cop + stig-e + chol-e 159.00 127.4 -84.29 -440.9 - - 6 248.0 - 2.86 0.041 RI 0.56 0.34 0.48 0.34 - - Antennella peculiaris1/ 2 M40-A_pec ~ chol-a + col_e + cop + 9.805e+00 -12.72 10.05 -6.871 -26.16 5 - 27.9 0.00 0.104 stig-e M25-A_pec ~ lat + long -1.374e+04 240.8 -99.11 3 - 28.1 0.25 0.092 M26-A_pec ~ chol-a + lat + long -1.296e+04 -0.9585 234 -91.60 4 - 29.5 1.57 0.047 M24-A_pec ~ chol-a + chol-e + cop + 3.480e+03 -7.824 1.511 -6.351 42.150 5 - 29.6 1.74 0.044 long M56-A_pec ~ chol-a + chol-e + cop + -4.558e+03 -23.59 22.55 -1.661 -59.53 -55.48 6 - 29.9 1.98 0.038 stig-e + long M62-A_pec~ chol-a + cop + stig-e + 1.150e+06 -315.30 -5.568 305.30 -15060 9726 6 - 29.9 1.98 0.038 lat + long 184

TABLE S3. (cont.) Models Intercept chol-a chol-e cop stig-e lat long df AICc QAICc delta AIC w Antennella peculiaris 1/ 2 M32-A_pec ~ chol-a +chol-e + cop 1.836e+05 -71.11 18.85 -1.046 -2454 1539 6 - 29.9 1.98 0.038 + lat + long M48-A_pec ~ chol-a +chol-e + cop 1.965e+03 -25.08 22.25 -1.949 -57.03 -84.26 6 - 29.9 1.98 0.038 + stig-e + lat M63-A_pec ~ chol-e + cop + stig-e -2.045e+05 54.08 5.005 -193.5 2477 -1786 6 - 29.9 1.98 0.038 + lat + long M60-A_pec ~ chol-a + chol-e + -4.670e+04 -16.06 26.57 -81.04 534.6 -416.8 6 - 29.9 1.98 0.038 stig-e + lat + long M57-A_pec ~ stig-e + lat + long -1.397e+04 -0.288 248 -99.89 4 - 29.9 2.05 0.037 M27-A_pec ~ chol-e + lat + long -1.388e+04 -0.0828 247.20 -99.09 4 - 30.0 2.06 0.037 M16-A_pec ~ chol-a + chol-e + cop -1.580e+03 -6.317 1.118 -4.079 68.54 5 - 30.0 2.07 0.037 + lat M29-A_pec ~ cop + lat + long -1.375e+04 9.742 240.90 -99.22 4 - 30.1 2.25 0.034 M54-A_pec ~ chol-a + cop + stig-e 4.255e+03 -6.499 -6.689 4.564 51.56 5 - 30.5 2.59 0.028 + long M46-A_pec ~ chol-a + cop + stig-e -1.871e+03 -4.924 -3.994 3.076 81.06 5 - 30.6 2.74 0.026 + lat M36-A_pec ~ chol-a + chol-e + 6.571e+00 -18.75 5.974 -12.19 4 - 30.7 2.83 0.025 stig-e M28-A_pec ~ chol-a + chol-e + lat -1.048e+04 -4.131 0.470 190 -73.80 5 - 30.8 2.94 0.024 + long RI 0.69 0.60 0.6 0.57 0.68 0.67 Antennella gracilis M5-A_gra ~ cop 48.25 -42.20 - - 4 241.4 - 0.00 0.269 M15-A_gra~ stig-e 46.44 -28.47 - - 4 243.1 - 1.69 0.116 M14-A_gra ~ chol-e 37.24 -6.216 - - 4 243.4 - 1.97 0.100 M16-A_gra ~ chol-a 26.54 -0.84 - - 4 243.9 - 2.42 0.080 M9-A_gra ~ cop + stig-e + chol-e 160.30 100.10 -85.72 -381.6 - - 5 244.2 - 2.79 0.067 M11-A_gra ~ chol-a + chol-e 50.08 105.50 -33.96 - - 6 244.3 - 2.85 0.065 M7-A_gra ~ cop + stig-e 66.69 -48.03 -21.86 - - 6 244.4 - 2.98 0.061 185

RI 0.31 0.34 0.54 0.37 - - TABLE S3. (cont.) Models Intercept chol-a chol-e cop stig-e lat long df AICc QAICc delta AIC w Obelia dichotoma 1/ 2 M34-O_dic ~ chol-a + stig-e 5.670e+00 11.67 -12.33 3 - 28.1 0.00 0.071 M35-O_dic ~ chol-e + stig-e 7.102e+00 5.3060 -20.59 3 - 28.2 0.09 0.068 M4-O_dic ~ chol-e + chol-a 3.505e+00 27.66 -7.5470 3 - 28.9 0.73 0.049 M50-O_dic ~ chol-a + stig-e + long -1.482e+03 22.36 -22.93 -18.07 4 - 29.4 1.24 0.038 M32-O_dic ~ chol-a +col_e + cop + -1.371e+05 135.80 -34.89 48.82 1625.0 -1207 6 - 29.6 1.45 0.035 lat + long 0 M56-O_dic ~ chol-a +col_e + cop + -1.890e+05 1672.0 -699.80 160.80 964.0 -2290 6 - 29.6 1.45 0.035 stig-e + long M62-O_dic ~ chol-a + cop + stig-e -1.318e+05 53.90 42.07 -49.41 1680.0 -1128 6 - 29.6 1.45 0.035 + lat + long M39-O_dic ~ chol-e + cop + stig-e 1.085e+01 9.7310 -1.292 -37.15 4 - 29.6 1.48 0.034 M38-O_dic ~ chol-a + cop + stig-e 7.543e+00 18.980 -0.896 -19.65 4 - 29.8 1.63 0.031 M51-O_dic ~ chol-e + stig-e + long -9.267e+02 8.07 -30.86 -11.35 4 - 29.8 1.67 0.031 M42-O_dic ~ chol-a + stig-e + lat 6.895e+02 16.98 -17.60 -29.52 4 - 30.0 1.82 0.029 M36-O_dic ~ chol-a + col_e + stig- 6.350e+00 5.903 2.586 -16.25 4 - 30.1 1.94 0.027 e M43-O_dic ~ chol-e + stig-e + lat 1.398e+02 5.708 -22.08 -5.72 4 - 30.2 2.08 0.025 M59-O_dic ~ chol-e + stig-e + lat + -8.049e+03 8.57 -32.70 147.6 -53.35 4 - 30.5 2.34 0.022 long M20-O_dic ~ chol-a + chol-e + long -7.903e+02 42.49 -11.49 -9.632 4 - 30.6 2.41 0.021 M12-O_dic ~ chol-a + chol-e + lat 8.496e+02 44.79 -12.11 -36.57 4 - 30.7 2.55 0.020 M8-O_dic ~ chol-a + chol-e + cop 3.509e+00 27.77 -7.574 -0.005 4 - 30.9 2.73 0.018 M24-O_dic ~ chol-a + chol-e + cop -5.911e+03 56.430 -15.13 3.986 -71.73 5 - 30.9 2.75 0.018 + long M41-O_dic ~ stig-e + lat -1.087e+03 -1.596 47.140 3 - 31.0 2.83 0.017 M58-O_dic ~ chol-a + stig-e + lat + -5.922e+03 16.720 -17.27 109.8 -41.12 4 - 31.0 2.84 0.017 long RI 0.58 0.58 0.41 0,67 0.68 0.67 Pennaria disticha 186

M16-P_dis ~ chol-a -6.4930 62.25 - - 4 235.3 - 0.00 0.375 M14-P_dis ~ chol-e -3.4040 11.70 - - 4 237.4 - 2.12 0.130 M15-P_dis ~ stig-e -9.5840 39.000 - - 4 238.0 - 2.68 0.098 M11-P_dis ~ chol-a + chol-e -1.4390 105.10 -11.44 - - 5 238.1 - 2.86 0.090 5.1890 93.98 - - - 5 238.2 - 2.91 0.087 M12-P_dis ~ chol-a + stig-e 32.580 RI 0.66 0.31 0.17 0.27 - - Only models with delta (AIC) Akaike’s Information Criterion value < 3 are shown. Species with overdispersion had a delta (QAIC) QuasiAkaike’s Information Criterion value used with family of quasipoisson distribution data. S= species richness, Hyd = hydroid abundance, cop = coprostanol sterol, chol-a = cholestanol sterol, chol-e = cholesterol sterol, lat = latitude, long = longitude RI = relative-importance of each selected variable, on a scale from 0 to 1 Parameters with relative-importance values >0.8 are highlighted in bold. (-) = not applicable variables that had significant spatial correlation, which was removed of the results here shown. 187

SUPPLEMENTARY Table S4. SIMPER result showed the species significantly contributing to the dissimilarities between groups (C-contaminated, MC-moderately contaminated and NC-not contaminated) a Species significantly contributing to the similarity within each group (average similarity/SD >1.4). Bold numbers indicated species significantly contributing to the dissimilarity between groups. Abund = Average Abundance and Cont. % = percentage of contribution.

SIMPER C & MC C & NC MC & NC Groups Average 92.27 90.12 65.62 dissimilarity C MC C NC MC NC Cont. Cont. Abun Abund. Cont. Species Abund Abund. % Abund. Abund. % d. % . P. floridana 0 3.75a 17.7 0 4.36a 11.27 3.75 4.36 3.92 C. gracilis 0.55a 2.17 5.84 0.55 4.35a 10.64 2.17 4.35 8.82 O. sargassicola 0.41 1.88 5.26 0.41 2.47a 5.33 1.88 2.47 5.92 C. lineares - - - 0.0 1.98a 5.96 0.29 1.94 5.32 A. peculiaris 0 1.39 4.28 0 2.39 5.13 1.39 2.39 5.67 A. gracilis 0 1.49 3.48 0 1.92 4.34 1.49 1.92 4.99 O. dichotoma 1.2a 0.58 4.35 1.2 1.55 4.94 0.58 1.55 4.04 P. disticha 0.98a 1.68a 7.47 0.98 1.47 3.37 1.68 1.47 3.99 H. tenellum 0 2.07a 7.19 0 3.06a 7.76 2.07 3.06 4.48 H. nanum 0 0.9 3.3 0 2.54 5.17 0.9 2.54 5.2 H. lighti 0 1.95a 4.85 - - - 1.95 0.57 3.92 D. disticha 0.12 1.11 2.9 0.12 1.45 3.99 1.11 1.45 4.27 H. dichotomum 0.27 0.98 2.47 0.27 0.81 1.77 0.98 0.81 2.6 P. strictocarpa 0.48 0.39 2.31 0.48 1.38 3.42 0.39 1.38 3.31 H. bermudense 0.63 1.26 6.01 0.63 1.09 3.06 1.26 1.09 3.32 H. discodium - - - 0 1.24 3.38 0.47 1.24 3.47 H. alternata 0 0.75 2.98 - - - 0.75 0.18 2.19 S. peculiaris - - - 0 1.62 3.94 0.86 1.62 3.99 C. parasiticum 0.84a 0.5 3.49 0.84 0 1.9 - - - C. tetranema 0 0.8 2.14 - - - 0.8 0.3 2.03 188

E. humilis 0 1.25 2.47 - - - 1.25 0 2.16 H. xanthellatum 0.6 0.52 2.72 0.6 1.08 3.14 0.52 1.08 2.76 E. carneum ------0.73 0.71 2.29 C. campanulata - - - 0 1.34 2.62 0 1.34 2.71

SUPPLEMENTARY FIGURE S1.

stig-e bras-e chol-a chol-e cop

100%

80%

60%

40%

20%

Sites and level of contamination

Fig. S1. Percentages of sterols in the sediments on coral reef of the west coast of Havana, Cuba. Sterols: sitg-e = stigmasterol; bras-e = brassicasterol; chol- a = cholestenol; chol-e = cholesterol, and; cop = coprostanol. Levels of contamination: NC: not contaminated; MC: moderately contaminated, C: contaminated. Sites abbreviation in Fig.1 189

SUPPLEMENTARY FIGURE S2a.

Fig. S2a. Coprostanol (cop) effect plot based in the best fit model and RI ≥ 0.60, generated with GLM analysis for species of hydroid as response variables: 190

P_flo = Plumularia floridana, C_gra = Clytia gracilis, O_sar = Orthopyxis sargassicola, A_gra = Antennella gracilis, and H_ber = Halecium bermudense.

SUPPLEMENTARY FIGURE S2b.

191

Fig. S2b. Cholestanol (chol-a) and stigmasterol (stig-e) effect plot generated with GLM analysis for species of hydroid as response variable with RI ≥ 0.60. P_flo = Plumularia floridana, A_pec = Antennella peculiaris, H_ber = Halecium bermudense, P_dis = Pennaria disticha and O_dic = Obelia dichotoma.

SUPPLEMENTARY FIGURE S3. 192

Fig. S3. Distribution of most abundant hydroid species (number of hydrocaulus/10 x 1m transect; presence in at least six transects) in the sampling sites classified by the contamination level C = contaminated sites, MC = moderately contaminated, and NC = not contaminated. Sites abbreviations in Fig.1. 193

CAPÍTULO 4

Manuscrito formatado para submissão segundo as normas da revista Marine Environmental Research Fator de impacto 2017: 3.08 Qualis (Biodiversidade): A2 194

Species richness, abundance, and spatial distribution of the epiphytic hydroids community related with seagrass health indicators, environmental factors and human impacts in a tropical seagrass meadow.

S. Castellanos-Iglesiasa *, M. Di Domenicob , B. Martinez-Daranasc, M. A.

Haddada

a Programa de pós-graduação em Zoologia, Setor de Ciências Biológicas da

Universidade Federal do Paraná, Centro Politécnico, 81531-990 Curitiba, PR, Brazil.

b Centro de Estudos do Mar da Universidade Federal do Paraná, Caixa Postal

61, 83255-976 Pontal do Paraná, PR, Brazil.

c Centro de Investigações Marinhas, Universidade de La Habana, Cuba

*Corresponding author. Tel.: +55 (41) 98039386.

E-mail address: [email protected] (S. Castellanos-Iglesias).

195

Abstract The study assesed the relation of environmental factors with the epiphytic hydroids assemblage of the seagrass ecosystems in two impacted bays of the

Sabana Camaguey Archipelago, North of Cuba. Hydroids assemblage was composed by 14 families, 17 genera and 48 species, 30 Leptothecata and 18

Anthoathecata. Low species richness and abundance were registered in the most impacted sites and, high values were in sites far from contamination sources and with the effect of oceanic water. Salinity, depth, and epiphytic algae were the best predictors for hydroids richness and abundance using a negative binomial function.

Impacts separately (trawling and highway) had the higest RI to predict the richness and abundance of hydroid assemblages than contamination impact and the total impact index. Symmetroscyphus intermedius was the most abundant species and

Dynamena disticha the most frequent in the sampling sites. Results showed that the epiphytic hydroids communities on tropical seagrass meadows are sensitive to health condition of the seagrass (wet weight of Thalassia testudinum and epihytic algae type), water quality and human impacts.

Key-words: epiphytic Leptothecata and Anthoathecata, salinity, depth, shoot density, submerged plants, GLM.

196

1. Introduction

The seagrass ecosystems distributed in tropical and temperate regions are capable of modify the physical, chemical and biological attributes of the surrounding marine environment (Orth et al., 2006; Wright and Jones, 2006). These ecosystems can detect the environmental changes caused by the negative human influences as biological “sentinels” (Mckenzie et al., 2012). The services offered by this marine ecosystem are translated in a production of $1.9 trillion of nutrients per year (Costanza et al., 1997; Waycott et al., 2009). Examples of these services are the sediments stabilization, enrichment of the nutrients cycle and primary production that sustains a complex feeding network (Díaz et al., 2003), the fishing productivity in the coral reefs.

Seagrass meadows are used as refuge and feeding by a great diversity of birds, fishes and invertebrates and also as food by threatened species of Sirenia (sea cows) as Trichechus spp and Dugong dugon Muller, 1776 and by the green turtle Chelonia mydas (Linnaeus, 1758) (Duffy, 2006). Multiple tensors that threaten the seagrass survival include the joint effects of the coastal development, the impact of pollution on the quality of sea waters, the indiscriminate use of these ecosystems for excessive fishing and the increasing frequency of tropical storms and of the sea temperature. As a consequence, approximately 60% of the marine phanerogams have suffered a covered area decrease, since their first record in 1879. Seagrass meadows are one of the most threatened ecosystems globally (Waycott et al., 2009).

In the north central shelf of Cuba, from Matanzas to Camagüey provinces, a large system of islands and cays of the Caribbean region, composed by rich ecosystems of seagrass meadows, are connected with fringing reefs and mangroves (Alcolado et al., 2007). This ecosystem known as Sabana-Camagüey Archipelago (SCE) is composed of a chain of 2515 islands and cays, along 465 Km of the coast line of the main island of Cuba (Mancina et al., 2013). An important biodiversity has been reported to this archipelago, including species of corals, as the threatened Acropora palmata (Alcolado et al. 2007), and 65% of the whole Cuban ornitofauna 197

(Rodríguez et al., 2007 in Mancina et al., 2013). Because of the SCE diversity of associated habitats, the marine and terrestrial fauna and flora and the nearness to the high maritime traffic of Bahamas channel, this area was declared as the second Internationally Particularly Sensitive Sea Area, since 1998, by the International Maritime Organization (13 Int'l J. Marine & Coastal L. 246 (1998) ).

The studies about this region showed that marine ecosystem as the seagrass beds are affected by anthropic disturbances, as pollution and trawling fishing activity. In addition to the extreme turbulence derived from the meteorological events, these factors have been reported to cause the main impacts on the associated biological communities and on the seagrass health condition (Martínez-Daranas, 2007, Roldan, 1992).

A great diversity of marine organisms of many Phyla depends on the seagrass ecosystems (Hemminga and Duarte, 2000; Spalding et al., 2003). In the case of epiphytic organisms, some hydroid species considered as seagrass obligatory epiphyte are food source for herbivorous fish and predators, being exposed to high grazing intensity (Hughes, Johnson, and Smith, 1991). Hydroids influence on changes of fish populations was attributed to their predatory habits on small and fish larvae (Coma et al., 1994, Gili et al., 1996; Orejas et al., 2000, 2001). These ecolological interactions attribute to the epiphytic hydroids an important role in the trophic dynamics of the seagrass marine ecosystems, especially in the transfer of energy from the plankton to the benthos (Gili and Hughes, 1995).

Hydroid associated with eelgrass and seagrass beds in temperate, cold and subtropical ecosystem has been well investigated (Boero, 1981; Borowitzka et al., 1990; Coma et al., 1992; Balata et al., 2007; Ronowicz et al., 2008; Di Camillo et al., 2017), but not in tropical seagrass meadow (Hughes et al., 1991, Kaehler and Hughes, 1992). Although their ecologic and morphologic plasticity, hydroids can reflect environmental changes in dominance, specific composition, zoning patterns and seasonal succession (Genzano and Zamponi, 1997). These characteristics justify the importance of studying hydroids in seagrass habitats, mainly those 198 impacted by anthropogenic influences as traditional and commercial fishing and the increasing tourism development. This is the case of the study area of this research, San Juan dos Remedios (SRB) and Buena Vista (BVB) bays, in SCE, a part of a fishery reserve located inside the Buena Vista Biosphere Reserve declared Ramsar Convention on Wetlands of International Importance site since 2002.

In order to contribute to the knowledge of the SCE seagrass meadows diversity, focusing the conservation and sustainable use of its resources, the present study aims to identify the epiphytic hydroids and characterize their spatial distribution related to environmental factors. For this purposes, the variables used as explanatory of richness, abundance and composition of the epiphytic hydroids were considered indicators of the seagrass health and of the water quality (Mckenzie et al., 2012). Our hypothesis is that seagrass health indicators, water quality and human impacts are predictors of species richness, abundance and distribution of the epiphytic hydroids assemblages in an impacted tropical ecosystem of seagrass meadows.

2. Study area

The study area comprehends two shallow water bays in the north of Cuba: San “Juan dos Remédios” (SRB) and “Buena Vista” (BVB), both located between to 22o30’ N and 79o18’ W, in the north of the city of “Caibarien”, province of Villa Clara (Fig. 1). These bays are part of the inner connected lagoons of the Sabana- Camagüey Ecosystem (SCE), an archipelago of 2515 islands and keys located on the border of the submarine platform. These chain of keys limit the communication of these masses of water with the Atlantic Ocean (Montalvo et al., 2009), having around the development of coral reefs, seagrass medows and mangroves. This complex of habitats creates a rich biodiversity that favored the stablishment different marine protected areas (MPAs), culminanting with the Biosphere Reserve “Buenavista”, classified as a RAMSAR site since 2002. All of them are explored by touristic and fishing companies.

The predominant bottom of the bays is smooth and composed mainly by clay 199 settlement covered mainly by species Thalassia testudinum (Martínez Daranas et al., 2007).

Fig. 1. Sampling sites of hydroids in the SRB and BVB in Villa Clara Province on the north center of Cuba.

Both bays are threated by different natural and anthropogenic factors. Between natural factors are tropical torments, drought periods, increase of sea temperature and high evaporation that favors salinity increasing. There are three main anthropic impacts on the seagrasses: 1. the organic contamination and eutrophication produced by the agricultural activities and the lack of treatment of residuals from villages in the mainland; 2. the physical impacts caused by the trawling 200

"chinchorreo" activity and 3. The construction of highways that limits the exchange of waters between the bays and the ocean (Alcolado et al., 2007; Martínez-Daranas, 2007; Montalvo et al., 2004; Puga et al., 2009; Betanzos-Vega et al., 2013).

These bays are one of the most affected ecosystems of the SCE (Martínez- Daranas, 1997). Monitoring seagrass bed has been one of the goals of the PNUD/GEF Sabana Camagüey Project I, II and III (2005-2014), to implement the conservation and sustainable use of this ecosystem. Results from these monitoring showed impacts on seagrass and on associated marine fauna (Martínez-Daranas, 2007). More information about the studied area and the impacts are complemented in supplemantary information.

3. Materials and Methods

3.1 Sampling, collection data and laboratory procedures.

During May 2011, collections were made in 7 sites in the habitat of seagrass in the SRB and BVB bays, with the aid of SCUBA diving, between 3 and 5 meters deep. Those sites were selected by their differences of physical, biological and human influences (Table 1). In each place, two transects 30m length were perpendicularly placed in the shape of a cross. Along with each transect, 6 quadrats of 25 cm by side were positioned distant 5 m from each other, summing a total of 12 sampling units per site. In each square, the seagrass species composition, height of leaf, wet weight, percentage cover of macroalgae (calcareous, filamentous and crusty) in the bottom, the type of epiphyte algae (crusty and filamentous) and the type of epiphytes on seagrass leaves were estimated. All fanerogams were collected in each square and epiphyte organisms as hydroids together. The samples were placed in plastic sacs with local sea water and mentol during two hours and soon fixed in formalin 4%.

At each sampling site, hydrographic data were taken: salinity, turbidity, transparency, temperature, pH, oxygen saturation (SATO2), dissolved oxygen (DO), ammonium concentration, nitrate, phosphate, oxygen chemical and biochemical 201 demand (DQO, DBO) and type of total solids dissolved (Table S1).

A total of 84 samples were collected for sort, screening and analysis of epiphyte hydroids in the lab. With a stereo microscope, the hydroids were separated from the substrate, identified to the most specific taxonomic categories and counted. The abundance was determined by the number of hydrocaulus. Each species were photographed with a Canon EOS Rebel T3 camera, in a biological Olympus microscope. The hydroids were identified by the bibliography of taxonomic studies, especially in the region of the Caribbean, Gulf of Mexico and the tropical region of the Atlantic Ocean. (Nutting, 1900, Vervoort, 1965, Calder, 1991, 1997, Kelmo, 2001, Calder and Kinkerdale, 2005, Galea, 2008, 2009, 2010, 2013).

Table 1. Characteristics, human influences and disturbances of sampling site observed during survey. Characteristics of substrate and Sites Latitude Longitude Human influences observations during survey Poor density of Th, Trawling fishing little dense patchy of named"Chinchorreo", CAI-1 22 31.602 079 25.521 Halo. and few before to be banned macroalgae in 2003. Abundance of algae and Th with brown Protected marine leaves and detritus area for fishing, use CAI-2 22 35.492 079 23.983 (dead particulate "Chinchorreo", being organic material, as very poor and illegal well as fecal material and dead organisms) Seat clay and sand with patch of few Th, Intense dead Hal and "Chinchorreo" until CAI-3 22 33.314 079 22.043 thanatocenosis of 2009, site closer to mollusks was the highway. observed Sediment of mud and fine sand of grey Poor and illegal CAI-4 22 36.316 079 19.305 color, high density of "Chinchorreo" Th and few algae Th with high shoot density but with short Sometimes illegal CAI-5 22 37.976 079 19.744 length, clean zone, fishing with coral reef lagoon, "Chinchorreo" strong currents 202

Not arts massive of Abundance of Th. fishing from 2007, Presence of mac, lobsters nurseries, CAI-7 22 34.085 079 15.043 meg and fsh. Patches salinity and current of Halo patterns affected by the highway. Sand with Th, Intense fishing of Laurencia and CAI-12 22 31.496 079 14.460 "Chinchorreo" until Cassiopea, high 2007 salinity and turbidity Th- Thalassia testudinum, Halo- Halodule wrightii , "Chinchorreo"- trawling fishing activity of massive capture that dragged on the marine shallow bottom affecting the ecosystem of seagrass beds, mac- macroalgae, meg-megazoobenthos, fsh-fishes.

3.2 Anthropogenic impacts assesment

The contamination, construction of highways and trawling chinchorreo were analized as the main anthropic impacts in the area. As indicator of contamination was used the distance of each site to the mainland.The impact of the highways was determined by their distance to each sampling site. Both distances were measure with Mapinfo program. The impact of the trawling chinchorreo was assessed by the time that this ativity does not occur, available in scientific studies, fhising reports from local enterprises and interviews of different fisherman and stakeholders of the region. These informations was summarized and analyzed to obtain an impact value for this activity.

A human environmental index was determined to classify the sampling sites by the level of impact, (Table S2.). Three impact values were attributed per site and they were summed obtaining a value of total impact index. The lowest values correspond to the less impacted condition (1), the middle values to moderately impacted (2) and the highest values to highly impacted (3). This analysis was performed with informations of 2013, the year after the chinchorreo prohibition and the finishing of the PNUD/GEF Sabana Camagüey Projects. As a result of this assesment four groups of sites were formed (Table 2).

Table 2. Main groups of sites formed by the computation of the human total impact index (Table S2.). Level of impacted: Li = Less impacted, Lm = less 203 moderately impacted, Hm = high moderately impacted and Hi = High impacted.

Level of impact Groups Less Less High high Total impact of sites impacted moderately moderately impacted index CAI-5 5 Li CAI-7 5 Lm CAI-4 6 CAI-2 7 Hm CAI-3 7 CAI-12 7 Hi CAI-1 8

3.3 Data analyses

With the aim to assess and predict the relative importance (RI) of concurrent environmental factors (indicators of seagrass health and environmental water quality and human impact parameters) on response variables (species richness, abundance of hydroids, as number of hydroids in 30 m2, and teeming species) we used univariate methods of general linear models (GLMs). We determined the significance of each predictor using Poisson and negative binomial family of distribution. The multicollinearity of environmental variables was tested by the variance inflation factor (VIF), package car (Fox and Weisberg, 2011), selected only with VIF less than 10. To account for model selection, we adopted a multi-model inference, calculating the importance of all possible combinations of environmental factors as explanatory variables, using the functions model.sel and model.avg (package MuMIn 1.12.1; Barton, 2015). Those models were ranked by AICc (Akaike’s Information Criterion) and delta AIC values (Zuur et al., 2009). We based the significance of the results on the most robust relative importance values (RI) from each explanatory variable using the model average.

PERMANOVA analysis was applied to know the significance differences between groups of sites with level of impacts as a fixed factor. To determine the contribution of individual species to the average dissimilarity among groups of sites, 204 between different levels of impacts (Hi, Lm, Hm, Hi), we applyed the similarity percentages program SIMPER (Clarke 1993). These analysis were performed with PRIMER-E softwares (Clarke & Gorley, 2006).

The multivariate patterns of hydroid species, sites and predictor variables were visualized using canonical analysis of principal coordinates (CAP; Anderson and Willis, 2003). This method releases a constrained ordination for any distance or dissimilarity measure. All these analyses were performed with the PRIMER-E softwares (Clarke & Gorley, 2006). As an indirect “post hoc” test, the abundance matrix (sqr transformed) of hydroid species with frequency equal to three or more per site and within the 95% of the community abundance were correlated as covariate vectors in the canonical axes. This test was done to identify the species responsible for multivariate patterns (Anderson and Willis, 2003), as an indication of the individual contribution to differences among groups.

4. Results

4.1 Hydroid species richness and abundance

This study presents the first check list of hydroids of seagrass meadows in Cuba, composed of 14 families, 17 genera and 48 species and of which 30 are Leptothecata and 18 are Anthoathecata. Nine species constitute new records for the biodiversity of Cuban waters (Table S3). A total of 11.240 hydrocaulus of hydroid colonies were counted and separated from their substrates, the seagrass leaves and algae.

Species richness and abundance differed between sites (Fig. 2). The highest values were in CAI_2 and CAI_5 and the lowest values in CAI_1 and CA_7.

205

Fig. 2. Variability quadrant data of dotplot (quadrant data, mean by site and overall mean) at the sampling sites. A. Abundance of hydroids (number of hydrocaulus/25x25 cm quadrant), B. Species richness (S).

Seventeen species composed 95% of total abundance and eight species were recorded in three sites or more. The most abundant species, Symmentroscyphus intermedius (Congdon, 1907), occurred in five sites, followed by Gastroblasta sp. and Dynamena disticha (Bosc, 1802) present in 3 and 6 sites respectively. Nemalecium lighti (Hargitt, 1924), Halecium sp.7, Eudendrium 206 calceolatum Motz-Kossowska, 1905, Clytia sp.2 and Halecium sp.6 were abundant but they appeared only in one or two sites. Species with less than 41 hydrocaulus were present in less than three sites. They were: Plumularia margaretta (Nutting, 1900), Halecium sp.3, Antennella smilis Galea, 2013, Plumularia sp.1 and E. bermudense Calder, 1988. (Fig. 3).

Fig. 3. Total abundance of species which occupied 95% of community hydroid (Number of hydrocaulus/square of 25cm2) and with more than 3 records in the study sites. 207

Fig. 4. Total abundance of families and genera of hydroid assemblage (Number. of hydrocaulus/square of 25 cm2) in the study sites.

Eight families and fourteen genera were found in the seagrass meadows. The most abundant families were Thyroscyphidae, Haleciidae, Campanulariidae and Sertulariidae of Leptothecata and Eudendriidae of Anthoathecata (Fig. 4 a). Symmetroscyphus, Halecium, Gastroblasta, Dynamena and Eudendrium were the most abundant genera (Fig. 4 b). The most speciouse Leptothecate families were Haleciidae and Campanulariidae with 8 and 7 species respectively.

Salinity and depth were significant predictors for hydroids richness and 208 abundance under conditional average models and in the best fit models of GLM analysis (Table 3a.). In addition, epiphytic alga were inside the best fit models for both response varibles richness and abundance and had a RI > 0.8 for hydroid abundance. Also pH and NO2 were sgnificative for species richness. Temperature accounted for the best fit models for both response variables but was only significative for hydroids abundance. The impact index was present in the best fist models for both response variables with a higher RI (0.74) for hydroid richness.

In relation with the abundance of teeming species, total impact index was present in the best fit model of Gastroblasta sp., although the indicators of seagrass health predictors as cover of calcareus and fleshy macraolagae (Cob_cal and Cob_car respectively), fanerogam density (Den_fans_m2) and, wet weight of T. testudinum (Wet_wh_Th) were the best predictors for this species. Depth and salinity had the higest RI (1) for S. intermedius. Also, wet weight of T. testudinum (Wet_wh_Th) was significative for this species. Temperature, salinity and the epiphyte alga type were the best predictors with the higest RI (> 0.8) for D. disticha species.

Analyzing the influence of each human impact on the response variables, the trawling impact was the predictor more significative and with the higest RI (>0.8) for species richness, hydroid abundance and abundance of S. intermedius. The highway impact (Highway_Imp) was the best impact predictor for the abundance of Gastroblasta sp. and D. disticha, although do not showed a high RI for the last one. Contamination impact from glm result had a higer RI (> 0.8) for species richness and not for hydroid abundance. The total impact index was a preditor with a low RI for all variables (Table 3b.).

209

Table 3a. Best fit models obtained with model average (model.avg) analysis from GLM and the application of zero inflated negative binomial (glm.nb) and poisson distribution ordered by AICc value and relative importance (RI) of each selected predictor variable for species richness and abundance of hydroids.

Response Best fit models distribution df AIC Delta AICw Predictors p RI Variables Sal 0.005 ** 0.86 (S_Hy ~ Algae_type + nb Depth 0.0012 ** 0.86 Depth + Impact_index + 6 347.3 0.00 0.0338 Algae_type 0.0427* 0.65 Species Sal) Impact_index 0.74 richness (S_Hy ~ Impact_index pH 0.0254* 0.67 + NO2 + pH + Sal + nb 7 348.1 0.810 0.025 NO2 0.1602* 0.66 Temp) Temp 0.48 Depth 0.0003*** 1 (Abun_Hy ~ Algae_type nb 5 762.0 0.00 0.12 Sal 0.0099** 1 + Depth + Sal) Algae_type 0.022* 0.94 Hydroid (Abun_Hy ~ Algae_type abundance + Depth + Impact_index nb 6 762.4 0.394 0.028 Impact_index 0.36 + Sal) (Abun_Hy ~ Algae_type nb 6 763.3 1.304 0.177 Temp 0.038* 0.21 + Depth + Sal + Temp) (S.intermedius ~ Depth nb 6 373.5 0.00 0.0338 Depth 0.87 + pH + Sal + Temp) S. Sal 0.82 (S.intermedius ~ Depth intermedius + pH + Sal + Temp + nb 7 373.8 0.228 0.0301 Temp 0.77 Wet_wh_Th) Wet_wh_Th 0.0267* 0.71 210

Cob_cal 0.000*** 1

(Gastroblasta sp. ~ Cob_car 0.000*** 1 Cob_cal + Cob_car + Gastroblasta Dens_fan_m2 0.0133* 1 Dens_fan_m2 + poisson 8 858.2 0.00 0.04 sp. Impact_index + NO2 + Wet_wh_Th 0.000*** 1 pH + Wet_wh) NO2 0.76 Impact_index 0.44

Temp 0.96 (D.disticha ~ Algae_type D.disticha nb 5 316.5 0.00 0.16 Sal 0.96 + Sal + Temp) Algae_type 0.0129* 0.92

Models had a highest (AIC) Akaike’s Information Criterion and lowest delta and Akaike weight (AIC w). Akaike weights may be interpreted as the relative probability that a particular model would have the best fit for another set of data drawn from the same underlying processes. S_Hy=species richness of hydroids, Abun_hy=hydroid abundance, Sal-Salinity, Algae_type=type of epiphytic algae. RI= relative-importance of each selected predictor with values (>0.8), on a scale from 0 to 1, parameters are highlighted in bold. * means, p< 0.001 ‘**’ 0.01 ‘*’ 0 ‘***.

211

Table 3b. Best fit models and with delta < 3 and obtained with model average (model.avg) analysis from GLM and the application of zero inflated negative binomial (glm.nb) and poisson distribution ordered by AICc value and relative importance (RI) of each selected human impact analized as predictor variable for species richness, abundance of hydroids and teeming species. Response Best fit models distribution df AIC Delta AICw Predictors p RI Variables (S_Hy ~ 3 348.28 0.00 0.28 Trawling_Imp 0.0001*** 0.85 Trawling_Imp ) (S_Hy ~ Cont_Imp 4 349.40 1.12 0.16 Cont_Imp 0.39 + Trawling_Imp ) (S_Hy ~ nb Impact_index + 4 349.78 1.49 0.13 Impact_index 0.42 Trawling_Imp ) (S_Hy ~ Species Highway_Imp + 4 450.30 2.01 0.10 Highway_Imp 0.37 richness Trawling_Imp ) Cont_Imp 0.033 * 1 (S_Hy ~ Cont_Imp 3 382.37 0.00 0.47 + Trawling_Imp ) Trawling_Imp 0.000*** 1 poisson (S_Hy ~ Cont_Imp + Highway_Imp + 4 383.23 0.49 0.37 Highway_Imp 0.41 Trawling_Imp)

(Abun_Hy ~ Trawling_Imp 0.000*** 0.89 3 765.37 0.00 0.38 Hydroid Trawling_Imp ) nb abundance Highway_Imp 0.32 (Abun_Hy ~ 4 767.51 2.15 0.13 Impact_index 0.32 Impact_index + 212

Trawling_Imp ) (Abun_Hy ~ Cont_Imp + 4 767.52 2.15 0.13 Cont_Imp 0.30 Trawling_Imp ) (S.intermedius ~ 3 384.51 0.00 0.78 Trawling_Imp 0.015* 0.78 Trawling_Imp ) S. (S.intermedius ~ nb intermedius Highway_Imp 0.22 Impact_Index + 3 387.06 2.54 0.22 Highway_Imp ) Impact_index 0.22 Highway_Imp 0.007** 0.83 (Gastroblasta sp. ~ Gastroblasta Trawling_Imp 0.76 Highway_Imp + nb 2 252.28 0.00 0.36 sp. Trawling_Imp) Impact_index 0.52 Cont_Imp 0.47 Highway_Imp 0.0368* 0.31 (D.disticha ~ Trawling_Imp 0.38 D.disticha nb 3 345.27 0.00 0.23 Highway_Imp ) Impact_index 0.32 Cont_Imp 0.29 Models had a highest (AIC) Akaike’s Information Criterion and lowest delta and Akaike weight (AIC w). Akaike weights may be interpreted as the relative probability that a particular model would have the best fit for another set of data drawn from the same underlying processes. S_Hy=species richness of hydroids, Abun_hy=hydroid abundance, Cont_Imp=contamination impact, Imp=Impact. RI= relative-importance of each selected predictor with values (>0.8), on a scale from 0 to 1, parameters are highlighted in bold. * means, p< 0.001 ‘**’ 0.01 ‘*’ 0 ‘***’

213

4.2 Hydroid species composition and level of impacts of sampling sites.

PERMANOVA analysis showed the symilarity between the groups of sites classified by level of impacts related with the species composition of hydroids. The site CAI-1 with the highest level of impact (Hi) was significantly different from all other sites. The sites moderately impacted (Hm = CAI2, CAI3 and CAI12) were also different from the sites less impacted (Li = CAI5 and CAI7). There were not significantly differences between others sites (Hm vs Lm and Lm vs Hi), Table 5.

TTable 5. PERMANOVA tests comparing sites by their different levels of impacts, elated with the species composition of hydroids. Level of impact (n = 4) as the xed factor in the design test. Level of impacted: Li = Less impacted, Lm = less moderately impacted, Hm = high moderately impacted and Hi = High impacted. Pseudo- Factor df SS MS F p perms level of impact 3 26080 8693,5 2.9478 0.0005* 9889 2.359E Residuals 80 5 29,491 2,315 2.6201 Total 83 E5 PAIR-WISE TEST results: Groups df t p perms Hi, Hm 46 2.174 0.0009* 9920 Hi, Lm 22 1.822 0.014* 6988 Hi, Li 34 2.123 0.0017* 9921 Hm, Lm 46 1.158 0.1837 9928 Hm, Li 58 1.691 0.0067* 9931 Lm, Li 34 1.254 0.1343 9935

The SIMPER analysis (Table 6) showed a higher dissimilarity between high impacted (Hi) and the other sites. The most abundant species of this study, S. intermedius, achieved 11.89% of contribution to the Hi and Li dissimilarities and 26.5% contribution to the Hi and Lm dissimilarities. D. disticha contributed with a maximum of 23.68% to the dissimilarities between Hi and Li, and it was more 214 abundant in Li sites. E. album had the highest contribution to the dissimilarity between Hi and Li. T. distans and P. margaretta had their better contribution between moderately impacted and the others sites. Gastroblasta sp. were more abundant in moderately impacted sites and absent in the Hi and Li impacted sites. The most abundant hydroids in the Hi sites were O. dichotoma and it was exclusive of this condition. 215

Table 6. SIMPER result showed the species contributing (> 5 %) to the dissimilarities between level of impact of sampling sites: Li = Less impacted, Lm = less moderately impacted, Hm = high moderately impacted and Hi = High impacted. a = Species significantly contributing to the similarity within group (average similarity/SD >1.4). Bold numbers indicated species significantly contributing to the dissimilarity between groups. Abund = Average Abundance and Cont. % = percentage of contribution. (-)= absence of the species.

SIMPER Groups Li & Lm Li & Hm Li & Hi Hm & Lm Hi & Lm Hi & Hm Average 92.24 93.79 98.62 91.88 98.55 98.61 dissimilarity

Li Lm Li Hm Li Hi Hm Lm Hi Lm Hi Hm Cont Cont Cont. Cont. Cont. Cont. Av. Av. . % Av. Av. .% Av. Av. % Av. Av. % Av. Av. % Av. Av. % Species Ab. Ab. Ab. Ab. Ab. Ab. Ab. Ab. Ab. Ab. Ab. Ab. S. intermedius 1.72a 4,54a 24.24 1.72 3.89a 16.62 1.72 0 11.89 3.89 4.54 25.32 0 4.54 26.5 0 3.89 14.53 D. disticha 3.44a 0.79 17.90 3.44 1.20a 18.13 3.44 0.17 23.68 1.15 0.79 9.53 0.17a 0.79 8.07 0.17 1.15 13.56 E. album 0.79a 0 7.22 0.79 0.68 6.76 0.79 0 10.46 ------0 0.68 1.29 T. distans 0.29 1.01a 6.12 0.29 0.53 3.95 0.29 0.29 2.75 0.53 1.01 7.73 0.29 1.01 9.05 0.29 0.53 5.73 P. margareta 0.41a 056a 5.81 0.41 0 2.46 0.41 0.22 4.78 0 0.56 3.84 0.22 0.56 7.54 - - - Gastroblasta 0 0.46 1.68 0 3a 9.39 - - - 3.00 0.46 36.80 - - - 0 3 13.36 sp. Halecium sp1. - - - 1.52 0.24a 5.01 - - - 1.52 0.59 5.67 - - - 0 1.52 5.10 Halecium sp5. 0.45 0 4.2 0.45 0 3.21 0.45 0 5.96 ------Halecium sp3. 0.34 0.30 4.84 0.34 0.03 1.88 0.34 0 2.71 0.03 0.30 3.51 0 0.30 7.29 - - - O. bidentata ------0.36a 0 5.35 0.36 0 4.19 216

4.3 Hydroid community structure and indicators of seagrass health and abiotic variables of water quality.

The CAP analysis showed the best discriminated species of hydroid assemblages and the influence of abiotic and seagrass ecosystem predictors between dissimilarities of sites (Fig. 8a and b).

217

Fig. 8. Canonical analysis of principal coordinates (CAP) ordination showing the axes that best discriminated species of hydroid assemblages between sites. (A) With biotic (seagrass data) and abiotic (hydrochemistry data) predictor vectors, and; (B) with more abundant and rare hydroid species observation as vector variables with Pearson correlation with CAP axes.

There was a very strong relationship between variations in the hydroid assemblage’s structure and the environmental predictors (Fig. 8a). Hydroid species had an evident separation of three groups correlatedwith canonical axes accentuating differences between groups of sites. Ordination results of the Pearson correlation between the species and CAP axes (Fig. 8b) allow us to depict their environmental variables preferences (Fig. 8a). 218

Depth influenced all sites excepted the site CAI_2. This site was better correlated to density of phanerogams (Dens_fan), wet weight of Thalassia testudinum (Wet_wh_Th) and percent cover of calcareous macroalgae (Cob_cal). Also, the most abundant species, S. intermedius (Congdon, 1907) and Gastroblasta sp., were better correlated with this site. CAI_3 and CAI_5 were markedly influenced by the nutrient nitrite NO2 and the cover of fleshy macroalgae (Cob_car) and also they were correlated with D. disticha (Bosc, 1802) abundance. CAI_1, CAI_7 and CAI_12 were better correlated with depth and epiphytic algae type. Species less abundant were correlated with CAI_1, CAI_7 and CAI_4 sites. Rare species (just one occurrence) as O. bidentata, E. capilare, L. flexuosa and T. tumida were present in the high impacted (Hi) site CAI_1.

5. Discussion

Our hypothesis predicted that seagrass health indicators (biotic factors), abiotic factors and human impacts are good predictors of species richness, abundance and distribution of the epiphytic hydroids assemblage in an impacted tropical ecosystem of seagrass meadows.

Seagrass meadows are one of the most threatened coastal ecosystems in the world, with a 7% of losses each year (Waycott et al., 2009). To propose more effective conservation and management actions to this ecossystem, there must be an increment in the studies of seagrass condition by monitoring the responses of biological health indicators because of their sensitivety to detect human disturbances and climate impacts (Gobert et al., 2009; Roca et al., 2016).

In this study, species richness and abundance of hydroids were sensitive to different health indicators of the T. testudinum meadows conditions. Epiphytic alga type and wet weight of T. testudinum were the best predictors for hydroid richness and abundance, having the highest RI values obtained by GLM analysis. Those indicators reflect degradation or recovery of the seagrass ecosystems and associated fauna (Marba et al., 2013; McCloskey and Unsworth, 2015) and being usually employed to monitoring this environment (Wood and Lavery, 2001). 219

The relation of hydroids with phanerogams has been well studied in cold and temperate waters (Hughes et al., 1991; Balata et al., 2007; Ronowicz et al., 2008; Di Camillo et al., 2017). Beyond the substrate, the plants provide a variety of benefits for epiphytic organisms as protection from predation, wave action and other disturbances (Ronowicz et al., 2008) and they are refuge for interspecific competition in a constant renewable stable substrate (Kaehler and Hughes, 1992). In addition, seasonality changes of the plants, their growth and species composition affect the epiphytic organisms as hydroids (Hughes et al., 1991).

Percent cover of macroalgae was good predictor for abundance of Gastroblasta sp.. Macroalgaes are biological indicators in monitoring programs. They detect changes of the environment quality and impacts of human activities (Dokulil, 2003; Piazzi et al., 2001; Diéz et al., 2009). The algae forest ecosystems forms a structural base for diversity in hydroid assemblages by shaping microhabitats of space, refuge or food for the species (Moura, 2015; Di Camilo et al, 2017). Calcareous algae as Halimeda spp. are some of the most common and diverse substrate colonized by epiphytic hydroids as up to 35 species on its thalli (Llobet, Gili and Hughes, 1991a; Llobet et al., 1991b). In this way, the distribution and abundance of epiphytic hydroids are affected by seasonal fluctuation, abundance and growth of the algae (Di Camilo et al., 2008).

Species richness and abundance of hydroids were influenced by two principal abiotic factors, salinity and depth, which had high relative importance (RI) for these variables (Table 3). It is well known that hydroids are very sensitive to changes of these parameters (Boero, 1984, Gili and Hughes, 1995, Peña Cantero and García Carrascosa, 2002; Gravier-Bonnet and Bourmaud, 2006, González-Duarte et al., 2014). Their influence on hydroids has been detected in different habitats and substrates, as rocky shore, coral reef, mangrove, estuaries, seagrass and artificial substrates. Under these factors influence, hydrois can also modify their morphology (Boero, 1981, 1987, Garcia Rubies, 1987, Calder, 1976, 1991b, 1993, Calder and Mayal, 1997, Di Camilo et al., 2008). Reduction of species richness is observed with the decrease of salinity mainly in estuaries, where highest values are in the mouth 220 and lowest near to fresh waters (Calder, 1976; Calder and Mayal, 1997). The distribution of hydroids varies with depth, since some species are specially sensitivity to narrow changes of this parameter, decreasing or increasing in abundance (González Duarte et al., 2014). Otherwise, abundance of resistant species to the abiotic changes can be found at each depth range (Calder, 1991a; Di Camilo, et al., 2008, Terlizzi and Schiel, 2009; Peña-Cantero and Manjón-Cabeza, 2014).

The differences in abundance, richness and composition of hydroids in each sampling sites, related to the predictors analyzed, are detailed in the supplemantary data.The assemblages of epiphytic hydroids in tropical seagrass meadows of T. testudinum have not been sufficiently studied, however, in the bays here studied, hydroids fauna was similar to that from Belize cays (Kaehler and Hughes, 1992). The family Thyroscyphidae was represented by only one species, but the most abundant S. intermedius, considered an obligate epiphyte on T. testudinum (Kaehler and Hughes, 1992). Its high abundance could probably be attributed to the symbiosis with algae (zooxanthellae) (Calder, 1986), an advantage for growth and facing competition in an intensely illuminated habitat (Di Camilo, et al., 2008). This species has a downward growth on the Thalassia basal growth leaves extending its survival time and also avoiding competition of epiphytic algae and sessile protozoa (Kaehler and Hughes, 1992).

Haleciidae and Campanulariidae, the most specious families, have been reported to environments with poor water quality (Orejas et al., 2013, Cabral, 2013) and in contaminated sites of the coral reef near Havana Harbour (Castellanos_Iglesias et al., 2018). Many Halecium spp. and Campanulariidae species have a circumglobal distribution (Calder, 1991a,b), indicating that they can adapt to a wide range of environments including polluted and different conditions of waters (Boero, 1984). L. flexuosa and O. bidentata occurred only in the site with high impact and contamination by human inputs (CAI1) (Table 1 and 2).

Multivariate analysis exhibited dissimilarity patterns of richness, abundance and species composition of hydroids between sampling sites. Spatial trends showed 221 that hydroid communties can be biological indicators linked to sensitivity to environmental factors as salinity, depth and seagrass health indicators. The plasticity and ecological specialization of hydroids, their abundance in different habitats as consolidated sessile benthic communities and modular ecosystem of seagrass meadows, establish solid arguments for considering them biological indicators of diverse environmental conditions (Boero, 1984; Mergner, 1987; Gili and Hughes, 1995, Puce et al., 2009; González-Duarte et al., 2014).

Environmenatal disturbances as contamination and human activities (trawling and highway impacts) to which the studied bays has been exposed in the last decades (Alcolado, et al., 2007; Montalvo et al., 2008, 2009) are the main factors limiting the species richness and abundance of hydroids in the seagrass meadow of T. testudinum. 222

6. Conclusions

The structure of epiphytic hydroid assemblage on Thalassia testudinum seagrass meadow was studied for the first time in the ecosystem of Sabana Camaguey Arquipelago of Cuba (SCE). The hydroid community encompassed 48 species belonging to 17 genera and 14 families, 30 are Leptothecate and 18 are Anthoathecate. The more abundant species were Synmetroscyohus intermedius, Gastroblasta sp. and Dynamena disticha in order of abundance. S. intermdius and Gastrblasta sp. were absent in the site highly impacted (Hi= CAI1) and D. disticha was present in all sites, but more abundant in the lower impacted sites (CAI5 and CAI7).

Results of the GLM univariate analysis corroborated the sensitivity of hydroid assemblages to water quality, health condition of T. testudinum and to the effect of the human impacts on this seagrass meadows. Species richness and abundance of hydroids were mainly affected by salinity and depth. Epiphytic algae type were the main predictors for the richness and abundance of hydroids and wet weight of T. testudinum was important for abundance of S. intermedius and Gastroblasta sp.. Species richness was the response variable more sensitive to contamination impact. Also, the trawling fishing activity and higway impact were the best explanatory variables for the hydroids community. Hydroid communities were more sensitive to the effects of human impacts separately, so, we sugest to evaluate their effects individualy for monitoring and conservation purposes of the seagrass ecosystems.

Multivariate analysis highlighted the distribution pattern of the hydroid community related with abundance and species composition. Canonical ordination (CAP) showed constrain distribution pattern affined with differences between the groups of sampling sites. In addition, were visualized the species that more contributed to this pattern under the health condition, water quality and human impacts effect of this studied seagrass ecosystems.

SIMPER analysis showed that S.intermedius and Gastroblasta sp. were the hydroids that better contributed to the differences between sampling sites classified by the level of human impact influences. Eudendrium album contributed 223 to the most similarity within-group. Tridentata distans, Plumularia margareta and Plumularia floridana typified the site CAI 4 , the only one moderately impacted.

High species richness and abundance of hydroids were found in sites further from contamination sources of the main island of the country, and with greater effects of oceanic water influence. Lower values were found in sites more impacted by human activities. Epiphytic alga type, was the best predictors for abundance and species richness of hydroids. Also, wet weight of Thalassia testudinum and percent cover of calcareous macroalgae were important for abundat of teeming species. Besides, salinity and depth were the main environmental predictors. Results corroborates that spatial distribution and structure of epiphytic hydroids communities on T.testudinum seagrass meadows are affected by water quality, contamination and human coastal activities impacts.

7. Acknowledgments

We extend our special thanks to colleagues from the Oceanology Institute of Cuba who that assisted in the samplings and to members of UNDP/GEF Sabana-Camaguey Project (2009-2014), from Cuba, for the funding and supporting to the fieldwork. Thanks to CNPq (Brazilian National Council for Scientific and Technological Development) for the first author PhD scholarship and to the UFPR Postgraduate Zoology Program for all support and scientific formation. Finally, we thank the anonymous reviewers that improved the quality of the MS with their useful comments.

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Ronowicz, M., Wlodarska-Kowalczuk, M., Kuklinski, P. 2008. Factors influencing hydroids (Cnidaria: Hydrozoa) biodiversity and distribution in Arctic kelp forest. Journal of the Marine Biological Association of the United Kingdom,88, 8, 1567–1575. doi:10.1017/S0025315408001495.

Spalding, M.,Taylor, M., Ravilious, C., Short, F., Green, E. 2003. Global overview: The distribution and status of seagrasses. Pages 5–26 in Green EP, Short FT, eds.World Atlas of Seagrasses: Present Status and Future Conserva- tion. Berkeley:University of California Press.

Piraino, S., Morri, C. 1990. Zonation and Ecology of Epiphytic Hydroids in a Mediterranean Coastal Lagoon : The Stagnone of Marsala ( North-West Sicily ). Marine Ecology, 11,1, 43–60.

Terlizzi, A., Schiel, D. .2009. Patterns along environmental gradients. In: Wahl M (ed) Marine hard bottom communities. Springer, New York, pp 101–112

13 Int'l J. Marine and Coastal L. 246 .1998. Cuba's Sabana Camaguey Archipelago: The second internationally recognized Particularly Sensitive Sea Area. The International Journal of Marine and Coastal Law. 246,3,2. 229

Wood, N. and Lavery, P. 2001. Monitoring Seagrass Ecosystem Health—The Role of Perception in Defining Health and Indicators. Ecosystem Helath. 6, 2, 134-148.

Wright, J.P., Jones, .G. 2006.The concept of organisms as ecosystem engineers ten years on:Progress, limitations,and challenges.BioScience 56: 203– 209.

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9. Supplemantary data.

SUPPLEMENTARY INFORMATION OF STUDY AREA

The two bays in the north of Cuba: San “Juan dos Remédios” (SRB) and “Buena Vista” (BVB) are considered hypersaline and with highest values near to the coast of mainland and in the center of the bays. The lesser values are registered to the north where the influence of oceanic water is high (Table 1). There is a dominance of ammonification over the nitrification processes, due to the high expulsion of ammonium by the sediments and the low survival of the nitrifying bacteria in these masses of water (Cotner et al., 2004). In the BVB bay there is a dominance of biosynthesis over the mineralization processes indicated by the high concentration of organic nitrogen (Montalvo et al., 2009).

There are three main anthropic impacts that cause main damages on biodiversity and seagrass: 1. the organic contamination and eutrophication produced by the agricultural activities and the lack of treatment of residuals from villages in the mainland; 2. the physical impacts caused by the trawling "chinchorreo" activity and 3. The construction of highways that limits the exchange of waters between the bays and the ocean (Alcolado et al., 2007; 230

Martínez-Daranas, 2007).

The contamination is present in the bays water in a variety of toxic organic and chemical substances such as heavy metals and phenols (Fernández-Vila and Chirino-Núñez, 1993) discharged directly into the water of rivers, without any treatment (García-García et al., 2012). The BVB bay has the highest concentrations of total inorganic and organic nitrogen and phosphorus. These values of nutrients and of organic matter expressed in oxygen biochemistry demand (DBO5) show characteristic conditions of the contaminated marine water masses (Montalvo et al., 2008, 2009). These bays have been classified as one of the most contaminated masses of water of the SCE (Alcolado et al., 1999; Pérez et al., 2003; Montalvo et al., 2004; Puga et al., 2009; Betanzos-Vega et al., 2013).

The "chinchorreo" has been a traditional fishing practice in the region with more than 30 year of implementation (Quirós et al., 2006; Obregón et al., 2007), that have strongly reduced marine biodiversity and fish communities in the SCE (Alcolado et al., 1999, Claro et al., 2003, Martínez-Daranas, 2007). This trawling was banned to each side of highways in 2003 (Resolution 233/03) and a fishing restriction area named "Zone under special regimen of use and proteção" was created inside the bays in 2009, on which all of fishing activity was forbbiden (Resolution 478/09) Fig 1. Finally, the forbiddance of "chinchorreo" was extended to the country in 2012. (Resolution 503/2).

SUPPLEMANTARY INFORMATION OF DISCUSSION

Lowest values of species richness and abundance of hydroids were found in sites with high impacts (Table 2). CAI1 had extremes values of salinity and high human impact related to the distance of this site to the coast of the Cuban mainland, where the effect of contamination and sedimentation was observed during survey (Table S1). This site had poor density of T. testudinum and the presence of patches of the phanerogam Halodule wrightii considered an eurihaline species typical of harsh ambience (Martinez-Darana et al., 2015) (Table 1). 231

In the same way CAI7 had salinity and current patterns affected by their distance to the highway that connect the cays and the Cuban mainland. This highway is a barrier that caused ecological damages in mangroves and seagrass habitats in the studied bays. The major disturb ocurred in the natural hydrological and sediment dispersion patterns as well as the migration, dispersal, recruitment and reproduction of fish and other marine biota as commercial species as lobster (Menéndez and Guzmán 2004, 2005a, Alcolado, et al., 2007). Extreme values of salinity, increased of sediment as a result of erosion of terrestrial habitat and industrial pollution are one of the main threats to seagrass health (Orth et al., 2006). These factors cause damages of phanerogam leaves and decreasing seagrass density diminish available substrate for larval recruitment (Piraino and Morri; 1990, Irving and Connel, 2002; Airoldi, 2003), and as a consequence, growth and reproduction of hydroids can be affected (Gravier-Bonnet and Bourmaud, 2006).

Grater values of species richness and abundance of hydroids were found in CAI 4 and CAI 5. These sites located at north and farthest of the coast with more contact with ocean water showing better environmental conditions and having low salinity and turbidity when comparing with extreme values found in the sites located more internally of the bays. These sites had the highest values of oxygen concentration, water transparency (Table S1) and high density of Thalassia (Table 1), characters that favored the hydroid richness and abundance. Oxygen and water transparency are important to maintain the basic function of photosynthesis of seagrass meadows (Neely, 2000, Borum et al., 2005, 2006) and also the development of some obligate epiphyte zooxanthellated hydroid as S. intermedius, the species more abundant in this study.

Low values of species of hydroids and abundance was observed in CAI3. This site had high values of DQO as an indicator of organic matter contamination and had historical high values of nitrites concentration (Martinez-Daranas et al., 2015), conditions of environment stress probably caused by their near location of the coast of Cuban mainland and urban runoff effect. These conditions corroborated the classification of this site as high moderately impact (Hm) Disturbances of water quality affected hydroids as habitat engineers of benthic 232 communities, and their important role in the trophic link between benthic and planktonic organisms (Di Camilo et al., 2017). Nitrite (NO2-) as a measure of nutrients was one of the predictors with high RI for species richness of hydroids (Table. 3a). Those abiotic factors have peculiar characteristics in marine waters of tropical ecosystems as seagrass meadows mainly in coastal regions of islands (Whittaker, 1998). Contamination can reduce rather ~40% of species richness of benthic organisms in marine habitat. In the case of seagrass, there is a high vulnerability to habitat collapse because of the sensitive responses and the interactions between engineer species, despite not many studies have shown loss of diversity in this ecosystem because of contamination (Johnston and Roberts, 2009).

The highest species richness and abundance of hydroid found in CAI2, was probably due to the availability of substrates observed likely to be colonized by hydroids, as abundant T. testudinum and macroalgae, as well as a lots of detritus (Table 1). Detritus is one of the most common component of diet of some species of hydroids when is abundant in the water column (Orejas et al., 2000).

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Fernández-Vila, L. J., Chirino-Núñez, A. L. 1993. Atlas Oceanográfico de las aguas del Archipiélago de Sabana-Camagüey. Instituto Cubano de Hidrografía, La Habana, Cuba. 235 p. 233

García-García, N., Pedraza-García, J., Montalvo, F.J., Martínez, M., Leyva, C. J. 2012. Evaluación preliminar de riesgos para la salud humana por metales pesados en las bahías de Buena Vista y San Juan de los Remedios, Villa Clara, Cuba. Revista Cubana de Química, 126-135.

Alcolado, P.M., García, E.E., Espinosa, N. (Eds.). 1999. Protección de la biodiversidad y desarrollo sostenible en el ecosistema Sabana- Camagüey. Madrid: CESYTA S.L.

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Montalvo J. F., García I., Loza S., Perigó E., Esponda S. C., Sánchez M. and Barrier A. 2009. Compuestos de nitrógeno y fósforo en agua de algunas bahías del archipiélago sabana-camagüey, Cuba. __.1-7pp

Montalvo, J.F. García I, Loza S., Esponda S. C., César María E., González de Zaya R. y Hernández, L. 2008. Oxígeno disuelto y materia orgánica en cuerpos de aguas interiores del Archipiélago Sabana-Camagüey, Cuba. Serie Oceanológica. (4), pp 71-84.

Betanzos-Vega, A., Capetillo Piñar, N., Lopeztegui Castillo, A., Martínez Daranas, B. 2013. Variación espacio-temporal de la turbidez y calidad en cuerpos de agua marina de uso pesquero, región norcentral de Cuba, 234

2008-2010. Ser. Oceanol., 12, 24-35.

Quirós Espinosa, A., Perdomo López, M.E., Rodríguez Moya, E., Arias Barreto, R., Hernández Albernas, J.I., Nieto Dopico, A., et al. 2006. Bases gnoseológicas del uso sostenible de los recursos marino-costeros de Villa Clara: CESAM-CITMA Villa Clara. http://www.forumcyt.cu/UserFiles/forum/Textos/0500487.pdf

Obregón, H.M., Pérez, I., Valle y Alcolado, P.M. 2007. Sectores económicos prioritarios en la protección y uso sostenible de la biodiversidad en el ecosistema Sabana-Camagüey. El sector de la pesca. En P. M. Alcolado, E. E. García y M. Arellano-Acosta, Ecosistema Sabana-Camagüey: Estado actual, avances y uso sostenible de la biodiversidad (pp. 124-130). La Habana: Editorial Academia.

Martínez-Daranas, B. R, Castellanos-Iglesias, S. et al., 2015. Estado del hábitat de un sector de la plataforma marina al norte de Villa Clara y Sancti Spiritus, Cuba. Informe final de proyecto PNUD/GEF Sabana – Camagüey III 2009-2014, Cuba. 62 pp.

Menéndez, L., Guzmán, J. M. 2004. «Inventario de Consolea millspaughii (Brito) Berger (Opuntia millspaughii) de la familia Cactaceae», en Informe de Resultado del Proyecto: Acciones Priorizadas para la consolidación de la protección de la biodiversidad en el ecosistema Sabana-Camagüey, ESGEF/PNUD, 179 pp.

Menéndez, L., Guzmán, J. M. 2005 «El humedal de Sabana-Camagüey: principales características de la vegetación», en Humedales de Iberoamérica: Experiencias de estudios y gestión (J. J. Neiff, ed.), Red Iberoamericana de Humedales (RIHU), 146-153.

Orth, R. J., Carruthers, T. J. B., Dennison, W. C., Duarte, C. M., Fourqurean, J. W., Heck, K. L., Olyarnik, S. 2006. A global crisis for seagrass ecosystems. Bioscience, 56,12, 987–996.

Piraino, S., Morri, C. 1990. Zonation and Ecology of Epiphytic Hydroids in a 235

Mediterranean Coastal Lagoon : The Stagnone of Marsala ( North-West Sicily ). Marine Ecology, 11,1, 43–60.

Irving, A.D., and Connel, S.D. 2002. Sedimentation and light penetration interact to maintain heterogeneity of subtidal habitats: algal versus invertebrate dominated assemblages. Marine Ecology Progress Series. 245, 83–91.

Airoldi L. 2003. The effect of sedimentation on rocky coast assemblages. Oceanography and Marine Biology. Annual Review 41, 161–236.

Gravier-bonnet, N., Bourmaud, C. A. 2006. Hydroids (Cnidaria, Hydrozoa) of Coral Reefs : Preliminary Results on Community Structure , Species Distribution and Reproductive Biology in Juan de Nova Island (Southwest Indian Ocean). Wstern Indian Ocean J. Mar. Sci., 5,2, 123–132.

Neely, M.B. 2000. Somatic, respiratory, and photosynthetic responses of the seagrass Halodule wrightii to light reduction in Tampa Bay, Florida including a whole plant carbon budget. Pp. 34–48 in Bortone, S.A (ed.), Seagrasses: Monitoring, Ecology, Physiology, and Management. CRC Press, Boca Raton, Florida. 318 pp.

Borum, J., Pedersen, O., Greve, T. M., Frankovich, T. A., Zieman, J. C., Fourqurean, J. W., Madden, C. J. 2005. The potential role of plant oxygen and sulphide dynamics in die-off events of the tropical seagrass, Thalassia testudinum. Journal of Ecology, 93, 148–158.

Borum, J., Sand-Jensen, K., Binzer, T., Pedersen, O. 2006. Oxygen Movement in Seagrasses. In: A. W. D. Larkum et al. (eds.), Seagrasses: Biology, Ecology and Conservation, pp. 255–270. (Martinez-Daranas et al., 2015)

Whittaker, R.J.1998. Island biogeography: ecology, evolution and conservation. Oxford University Press, New York, pp 285 236

Johnston, E. L., Roberts, D. A. 2009. Contaminants reduce the richness and evenness of marine communities: A review and meta-analysis. Environmental Pollution, 157, 1745–1752.

Orejas, C., Gili, J.M., Alva, V., Arntz, W., 2000. Predatory impact of an epiphytic hydrozoan in an upwelling area in the Bay of Coliumo (Dichato, Chile). Journal of Sea Research 44, 209-220.

237

SUPPLEMENTARY Table S1.

Environmental data of hydrochemistry in the sampling sites where hydroids were collected during survey of May 2013 under PNUD/GEF monitoring project Sabana-Camaguey Ecosystem III (SCE).

Depth Temp Sal DO SatO2 pH Turb. Tran. NO2 PO4 DQO TS TSS FSS Bays Sites m ºC (ups) (mg/l) (%) pH_f FTU % RB CAI_1 3.2 27.52 45.2 3.49 58.4 8.14 6.42 45 0.003 0.061 0.72 101.75 60.36 53.99 SRB CAI_2 2 27.79 43.33 4.98 80 8.19 1.35 100 0.002 0.089 1.26 62.66 68.12 56.21 SRB CAI_3 2.5 28.17 43.67 4.89 86.4 7.96 7.1 35 0.047 0.067 3.79 69.91 68.26 69.22 SRB CAI_4 2.6 27.72 38.34 5 80.8 8.3 1.45 96.2 0.003 0.045 1.44 68.34 65.79 56.21 SRB CAI_5 2.8 27.57 37.34 6.3 98 8.01 0.86 100 0.018 0.04 1.80 66.79 68.27 56.2 SRB CAI_7 2.5 26.98 43.64 5.9 90.6 8.28 2.81 100 0.019 0.033 0.72 51.34 60.42 50.63 BVB CAI_12 2.7 27.38 41.94 5.69 88 8.03 4.08 68 0.004 0.051 1.26 56.65 50.64 53.85

238

SUPPLEMENTARY Table S2.

Total impact index and values of damage from each type of impact (contamination, highway and trawling) in each samplig site. Contamination (distance from sampling site to main land shore) 1: (16-25 Km), 2: (8-16 Km) e 3: (2,5-8 Km); highways (distance to it) 1: (10-15 Km), 2: (5-10 Km) e 3: (1-5 Km) and trawling (time estimated without this activity), 1: without damage, 2: moderated damage (finished in 2003) and 3: high damaged (finished in 2012).

Total Sampling Contamination Highway Trawling impact site index CAI-1 3 3 2 8 CAI-2 2 2 3 7 CAI-3 2 3 2 7 CAI-4 1 2 3 6 CAI-5 1 1 3 5 CAI-7 1 2 2 5 CAI-12 2 3 2 7

SUPPLEMENTARY Table S3.

Hydroids list of seagrass meadows of two shallow bays (BSR and BVB) in the SCE (Sabana Camaguey Ecosystem) of Cuba. x- Indicates species with reproduction structure

Total of

Species of hydroids hydrocaulu Order Fertility . s Synmentroscyphus 1 TE intermedius 3312 2 Gastroblasta sp. 998 TE 3 Dynamena disticha 985 TE x 4 Halecium sp.1 616 TE x 5 Halecium sp.4 401 TE 6 Eudendrium album 319 AT 7 Trientata distans 204 TE x 8 E. racemosum 185 AT 9 Halopteris tenella 156 TE x 10 Nemalecium lighti 153 TE 11 Halecium sp.7 133 TE x 12 E. calceolatum 123 AT 239

13 Halecium sp.2 121 TE 14 Clytia sp.2 113 TE 15 Antennela peculiaris 87 TE 16 Halecium sp.6 74 TE x 17 Plumularia floridana 61 TE 18 Halecium sp.5 48 TE 19 Clytia hemiphaerica 44 TE 20 P. margareta 41 TE x 21 E. capilare 39 AT 22 E. molouyensis 33 AT 23 Orthopyxis sp. 30 TE 24 Halecium sp.3 29 TE 25 Eudendrium sp.1 24 AT 26 Halopteris sp.1 19 TE 27 A. smilis 17 TE x 28 Plumularia sp.1 16 TE 29 E. bermudense 15 AT 30 O. bidentata 12 TE 31 Athecata sp.2 8 AT 32 C. gracilis 5 TE 33 Antennela sp.2 4 TE 34 Antennela sp.1 3 TE 35 Athecata sp.4 3 AT 36 Eudendrium_sp.11a 3 AT 37 Scandia gigas 3 TE 38 Athecata sp.5 2 AT 39 Eudendrium sp.3 2 AT 40 Athecata sp.6 1 AT 41 Athecata sp.1 1 AT 42 Athecata sp.3 1 AT 43 Eudendrium sp.11b 1 AT 44 Eudendrium_sp.12 1 AT 45 Eudendrium sp.2 1 AT 46 Halecium sp.8 1 TE x 47 Laomedea flexuosa 1 TE 48 Tridentata tumida 1 TE

240

CONCLUSÕES GERAIS

Esta tese teve como objetivo geral incrementar o conhecimento da diversidade de hidroides de Cuba e das relações entre a estrutura das assembleias desses organismos com fatores ambientais, em dois habitats costeiros impactados. Também como objetivo complementar, foi atualizada a fauna de hidroides (Anthothecata e Leptothecata) no Grande Caribe e foi analisada a influência de nicho ambiental e espacial no padrão de distribuição da alfa e beta diversidade nessa região.

Os resultados obtidos sobre o estudo da taxonomia dos hidroides cubanos da ordem Leptothecate (Cornelius, 1992) reportaram 29 novas ocorrências de espécies, incluídas em 7 famílias e 14 gêneros, das quais duas são novas espécies para a ciência, Antennella sp.nov e Hallecium sp. nov.. A descoberta de uma morfoespécie igual a Ortophyxis sargassicola registrada para Brasil, (Parati) é apresentada, o que evidencia a variabilidade intraespecífica na estrutura dos hidroides entre diferentes zonas geográficas, amplamente reportada na literatura (Cunha, 2011; Cunha et al., 2015; Calder, 1970; Schuhert, 2005). Clytia warreni Stechow, 1919, Halecium pusillum Sars, 1856 e Halecium cf. labrosum Alder, 1859 foram novas ocorrências para a região do Caribe e a simbiosis com zooxantela e registrada pela primeira vez em espécies de Dynamena e Antennella. O total de hidroides tecados no país elevou-se para 101. Este valor posiciona Cuba entre os países de maior diversidade de hidroides no Grande Caribe, mesmo sendo a maioria dos estudos e coletas feitos ainda somente na região ocidental do pais. Faltam estudos nas regiões do Sur central e Oriental, ainda pouco exploradas para este grupo de organismos marinhos. Resultados similares sobre a alta diversidade de outros organismos marinhos, como moluscos, corais e esponjas de Cuba classificam o país como um dos mais importantes hot-spots de biodiversidade na região do Caribe (Miloslavich et al., 2010).

A revisão da fauna de hidroides na região do Grande Caribe permitiu atualizar o status da diversidade do grupo para 396 espécies, 40 famílias e 125 gêneros e analisar o padrão de distribuição a partir dos registros de ocorrências de espécies por países. Nossos resultados mostraram uma diferença clara na 241 distribuição dos hidroides em relação a composição e ocorrências das espécies, como o encontrado para as diferentes regiões do Atlântico (Calder 1992; Medel and López-González, 1997) mas também mostrou os países que ficaram separados pelo conhecimento restrito da sua fauna. Resultados semelhante têm sido registrado para outros grupos marinhos (Miloslavich et al., 2010). Com nossos resultados se corrobora a necessidade da formação de zoólogos para estudos de Taxonomia, sem os quais não se pode estabelecar os padrões de biodiversidade das espécies e compreender suas relações com os fatores e as mudanças ambientais a nível regional. Este problema já foi alertado para outras áreas (ex. Medel and López González 1998; Miloslavich et al, 2010)

Os resultados obtidos sobre a influência do nicho ambiental e espacial no padrão de distribuição preliminar dos hidroides no Grande Caribe mostrou a importância da relação da latitude e a temperatura sobre a variação da alfa diversidade (Platnick 1991; Chaudhary et al. 2016, 2017; Fernandez, Navarrete and Marques 2014, 2017), como o encontrado no Atlântico norte Ocidental para os hidrodes (Calder, 1992). A aplicação da análise de particionamento da variação permitiu reconhecer a importância de integração desses dois fatores sobre o padrão de distribuição da diversidade dos hidroides na região de estudo, com 23% de explicação para alfa e de 3% para beta diversidade. Todavia, consideramos que a porcentagem de explicação para a beta diversidade pode ser ampliada com dados de salinidade, habitat, substrato e forma de reprodução dos hidroides, uma proposta para outro trabalho, tendo em consideração a importância desses fatores sobre a distribuição dos hidroides, já amplamente demonstrada na literatura (Calder, 1976, 1991, 1992; Gili and huges, 1995; Ronowicz et al., 2013, Gibbons et al., 2010).

No estudo da relação das assembleias de hidroides com os níveis de contaminação e de outros fatores ambientais, a aplicação de diferentes abordagens de análises estatísticas permitiu avaliar a sensibilidade das assembleias, e das espécies mais abundantes dos hidroides, frente ao efeito desses fatores, impactantes ou não. Os resultados gerados mostraram uma visão mais integrada dos hidroies com os fatores ambientais, tanto no habitat do recife de coral como das gramas marinhas, expondo uma especificidade de 242 resposta a nível de espécies, segundo o estres ambiental das áreas estudadas (capítulos 3 e 4). A aplicação, pela primeira vez, das análises de marcadores esteroides no sedimento, do recife de coral da Havana, permitiu corroborar tanto o gradiente de contaminação ambiental dessa área, quanto seu impacto sobre as comunidades biológicas marinhas desse habitat já citado na literatura (Alcolado and Herrera, 1987; Aguilar et al., 2004, 2008; Busutil and Alcolado, 2012; Villiers and Alcolado, 2012; Alcolado-prieto et al, 2012). É também este estudo o primeiro a relacionar a estrutura das comunidades dos hidroides com esses marcadores químicos, indicadores de contaminação e matéria orgânica de diferentes origens.

Os resultados obtidos sobre a sensibilidade da comunidade de hidroides a contaminação corroboram os previamente reportados na literatura. A diminuição da riqueza e da abundância dos hidroides representam os efeitos dos impactos da contaminação sobre a diversidade (Boero, 1984; Mergner, 1987, Gili and Hughes, 1995). A dominância e abundância de espécies da família Campanulariidae, como O. dichotoma e Clytia spp., em ambientes contaminados já haviam sido registradas em ambientes estuarinos e relacionados com áreas portos e baías (Cabral, 2013; Grohmann, 2009), como foi no recife de coral da Havana. Plumuaria floridana, o hidroide mais abundante no recife de Havana, nos locais não contaminados e moderados, pode ser visualizado como indicação deste tipo de ambiente. Por outro lado, Halecium bermudense foi muito abundante nos locais mais contaminados, próximos à o porto da Havana, indicando a resistência das espécies desse gênero a diferentes contaminantes reportado na literatura (Collins et al., 2002). As respostas das assembleias dos hidroides do recife de coral, em locais próximos ao porto de Havana, parece ser uma ferramenta útil e precisa para monitorar e prever a influência da contaminação.

O estudo dos hidroides epifíticos nas gramas marinhas tem abordado a relação de distribuição das espécies com o substrato, principalmente nas regiões temperadas, nos ecossistemas das fanerógamas Zoostera marinha, Posidonia oceânica e Amphibolis griffithii (Borum, 1985; Boero, 1987; Borowitzka, 1990). Na região do Atlântico tropical, no entanto, onde a espécie dominante é 243

Thalassia testudinum, há muito poucos estudos sobre essa relação hidroide- substrato, e menos ainda com os fatores ambientais. Nossos resultados sobre as especies de hidroides epifitcas nas gramas marinhas coincidiram com os registros de Symmentrocyphus intermedius (a mais abundante neste estudo) e Tridentata tumida (considereada rara) classificadas como epífitas obrigatórias da fanerógama Thalasssia testudinum em Belize (Hughes, Johnson and Smith, 1991; Kaehler and Hughes, 1992).

A aplicação das análises de GLM permitiu estabelecer predições do efeito da contaminação sobre a riqueza e a abundancia dos hidroides no recife de coral e da influência da qualidade ambiental do ecossistema de gramas marinhas. Foi possível relacionar as espécies mais abundantes com o efeito da contaminação e a materia orgánica, obtendo resultados preditivos da resposta das especies em ambientes com o aumento das concentrações dos esterorides. Também os nossos resultados nas gramas marinhas corroboraram a sensibilidade dos hidroides a salinidade e a profundidade como um dos principais fatores abióticos que influenciam a riqueza e a abundancia desses organismos o que já foi observado por diversos autores (Calder, 1976, 1991; Di Camilo, 2008; Ronowicz, 2013).

As análises multivariadas permitiram uma visão mais compreensiva e integrada do impacto da contaminação ao longo da costa da Havana e do efeito dos fatores ambientais e da saúde das gramas marinhas sobre a distribuição da assembleia dos hidroides nas baias estudadas San Juan dos Remedios e Buena Vista. Também indicaram a contribuição de espécies individuais de hidroides para as diferenças entre os grupos de locais de amostragem em cada habitat estudado.

O nosso estudo sobre a estrutura da comunidade de hidroides no recife do Norte Havana, mostrou o impacto da contaminação fecal no sedimento, efeito das atividades humanas ao longo da costa Oeste da Havana, a pesar das medidas de higienização e controle estabelecidas sobre as principais fontes de contaminação na área. Igualmente os resultados obtidos no habitat das gramas marinhas mostra a afetação da comunidade de hidroides epifíticos pela contaminação e o impacto ambiental existente sobre esse ecossistema nessa 244

área, indicando a necessidade de continuar com as medidas de conservação e monitoramento por ser consideradas áreas de interesse turístico e económico para o país.

245

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