UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” INSTITUTO DE BIOCIÊNCIAS – CAMPUS DE BOTUCATU PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS ÁREA DE CONCENTRAÇÃO – ZOOLOGIA DISSERTAÇÃO DE MESTRADO MUDANÇAS NOS PADRÕES DE DIVERSIDADE DE CARANGUEJOS ERMITÕES COSTEIROS: ALTERAÇÕES APÓS 20 ANOS GABRIEL FELLIPE BARROS RODRIGUES ORIENTADOR: PROF. DR. ADILSON FRANSOZO BOTUCATU – SÃO PAULO 2019 ‘ MUDANÇAS NOS PADRÕES DE DIVERSIDADE DE CARANGUEJOS ERMITÕES COSTEIROS: ALTERAÇÕES APÓS 20 ANOS Gabriel Fellipe Barros Rodrigues Orientador: Prof. Dr. Adilson Fransozo Dissertação apresentada ao curso de pós- graduação em Ciências Biológicas – Zoologia, do Instituto de Biociências (IBB), Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP), campus de Botucatu, como parte dos requisitos para obtenção do título de Mestre em Ciências Biológicas – Área de Zoologia BOTUCATU - SP 2019 ‘ ‘ “O que você faz, faz a diferença, e você tem que decidir que tipo de diferença você quer fazer. ” Dra. Jane Goodall ‘ ‘ Agradecimentos Eu gostaria de começar por reconhecer a riqueza de conselhos, sugestões e principalmente críticas que recebi durante todo o período que envolveu a pós-graduação do Prof. Dr. Adilson Fransozo, propiciando todo suporte para realização e financiamento desse projeto. Espero ter atingido meu objetivo inicial de fornecer um trabalho que expanda o conhecimento atual. Agradeço à Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), pela concessão da bolsa de estudos ao nível de Mestrado Acadêmico, à FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo), pelo financiamento do projeto nos anos de 1995-1996 (proc. 95/2833-0) e referente a obtenção de um veículo para transporte, ao Núcleo de Estudos de Biologia, Ecologia e Cultivo de Crustáceos (NEBECC), pela infraestrutura dos laboratórios e materiais disponíveis, ao IBAMA (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais) e à Polícia Federal, por concederem a licença para a coleta do material nas áreas estudadas. Agradeço também ao Prof. Dr. Fernando Luis Medina Mantelatto (coordenador do projeto), Prof. Dra. Giovana Bertini Profa., Profa. Dra. Lissandra, Dr. Rogério Caetano da Costa pelos dados já levantados do projeto pertencente aos anos de 1995 e 1996 na Enseada de Ubatuba-SP. Agradeço a todos os colegas do Núcleo de Estudos de Biologia, Ecologia e Cultivo de Crustáceos (NEBECC), pelo apoio nas diferentes etapas do projeto. Agradeço aos pescadores Luciano pelas coletas em 1995-1996 e Djalma Rosa (Passarinho) comandante da embarcação “Primavera” e seus auxiliares, pela competência e auxílio durante as coletas. Agradeço à minha família, principalmente a meus pais Osni Rodrigues e Elizabete da Silva Barros Rodrigues, por todo apoio dado durante os anos da graduação e pós- graduação. Aos técnicos, funcionários e colegas do departamento que me auxiliaram e me ajudam de diversas formas. À minha namorada, Danielle Hamae Yamauchi, por estar comigo em todos os momentos e me ajudar a passar pelas situações adversas. ‘ Permaneço em débito com os professores: Dra. Maria Lúcia Negreiros-Fransozo e Dr. Fernando Luis Medina Mantelatto pelos comentários dos capítulos que compõem essa dissertação. ‘ Sumário Considerações iniciais ........................................................................................................................... 1 Ameaças à biodiversidade no ambiente marinho ........................................................................ 3 Infra-ordem Anomura MacLeay, 1838 .......................................................................................... 5 Superfamília Paguroidea Latreille, 1802 ....................................................................................... 7 Referências ....................................................................................................................................... 9 How hermit crab assemblages structure changed after 20 years in the northern coast of São Paulo state, Brazil ......................................................................................................................................... 13 ABSTRACT........................................................................................................................................ 13 1. Introduction ............................................................................................................................ 14 2. Material and methods ............................................................................................................ 17 2.1 Study Area ...................................................................................................................... 17 2.2 Biotic and environmental data collection ...................................................................... 18 2.3 Statistical analysis .......................................................................................................... 20 3. Results .................................................................................................................................... 22 3.1 Spatio-temporal trends .................................................................................................. 23 3.2 Correlation with environmental variables ..................................................................... 28 4. Discussion ............................................................................................................................... 30 Patterns of abundance and richness........................................................................................... 30 3.2 Environmental variables ................................................................................................ 34 5. Conclusion .............................................................................................................................. 35 References ...................................................................................................................................... 36 Changes in coastal hermit crab biodiversity patterns: partitioning beta diversity with an interval of 20 years ............................................................................................................................................... 43 Abstract ........................................................................................................................................... 43 3. Introduction ............................................................................................................................ 44 2. Material and Methods ................................................................................................................ 46 2.1 Study Area ............................................................................................................................. 46 2.2 Data Collection ...................................................................................................................... 47 2.3 Data Analysis ......................................................................................................................... 50 3. Results ......................................................................................................................................... 52 4. Discussion ................................................................................................................................... 59 References ...................................................................................................................................... 64 ‘ The interaction network pattern of coastal hermit crab and shells: new insights from a unique relationship ......................................................................................................................................... 69 Abstract ........................................................................................................................................... 69 Introduction .................................................................................................................................... 70 2. Material and Methods ................................................................................................................ 72 2.1 Study Area ............................................................................................................................. 72 2.2 Sampling ................................................................................................................................ 73 2.3 Network analysis ................................................................................................................... 73 3. Results ......................................................................................................................................... 76 3.1. Network-level metrics .......................................................................................................... 77 3.2. Species-level metrics and species roles ............................................................................... 77 Discussion ....................................................................................................................................... 84 References ...................................................................................................................................... 88 Considerações finais ........................................................................................................................... 99 Anexo 1 ......................................................................................................................................... 104 Anexo 2 ......................................................................................................................................... 105 Considerações iniciais Rodrigues, G.F.B 1 Considerações iniciais O termo “biodiversidade” foi cunhado em 1985, sendo uma contração de “diversidade biológica”. Pode-se dizer que biodiversidade é toda a variedade da vida na Terra, em todas suas formas e interações. Toda essa complexidade gerada por milhões de anos de evolução é responsável por tornar o planeta habitável, provendo serviços de extremo valor para a manutenção da vida na Terra. Sabe-se que a diversidade biológica não está distribuída de forma homogênea na superfície da Terra, ou seja, existem áreas que abrigam uma enorme e fantástica riqueza de espécies, muitas das quais nunca foram sequer documentadas (Magurran, 2004). Pesquisadores buscam entender como e por que as espécies estão distribuídas do jeito que estão, para uma melhor compreensão do funcionamento da vida em nosso planeta (Schemske et al., 2009). Há muito tempo naturalistas buscam entender padrões da distribuição de espécies ao redor do globo. Por exemplo, o gradiente latitudinal de riqueza percebido por Humboldt (1808), que escreveu: “Quanto mais perto chegamos dos trópicos, maior o aumento na variedade das estruturas, na beleza das formas e na mistura das cores, assim como na juventude perpétua e no vigor da vida orgânica”, pode ser considerado o mais antigo padrão ecológico (Hawkins, 2001). Dessa forma a região tropical é vista como o coração da biodiversidade na Terra. Embora que muitas teorias elaboradas com intuito de investigar a enorme biodiversidade presente nos trópicos (teoria da heterogeneidade ambiental, teoria Considerações iniciais Rodrigues, G.F.B 2 da competição, teoria da estabilidade climática, teoria da competição e predação) (Pianka, 1966; Schemske & Mittelbach, 2017), pouco se sabe sobre a real riqueza de espécies dos trópicos. A complexidade dos ecossistemas tropicais possibilita a coexistência de um enorme número de espécies originando uma miríade de relações entre elas. Um único ecossistema florestal na mata atlântica pode ter uma configuração de vários “andares”, desde comunidades biológicas próximas ao solo até aquelas nos dosséis das árvores, cada uma com suas peculiaridades e ao mesmo tempo inter-relacionadas entre si (Yoshimura & Yamashita, 2012). Da mesma forma, recifes de corais das regiões tropicais possuem uma estrutura bastante complexa, servindo como substrato para muitas espécies sésseis e bentônicas e como refúgio para organismos de vida livre, este tipo de ecossistema possibilita nichos novos como os “limpadores”, animais que se alimentam de ectoparasitas ou tecidos injuriados da superfície corpórea de outros animais (clientes) (Guimarães et al., 2007b). A dimensão da riqueza de espécies encontradas nos trópicos é completamente diferente das demais regiões (Magurran, 2004). A ecologia de comunidades é o estudo de organismos de múltiplas espécies que interagem entre si em um determinado lugar e tempo, cujo o foco é os padrões de diversidade, abundância, composição e os processos que estão por trás desses padrões. Existem diversas abordagens para a compreensão da estrutura das comunidades. A abordagem espacial busca entender como as espécies estão distribuídas em uma determinada escala espacial. A escala delimitada para o estudo depende fundamentalmente do organismo a ser estudado, podendo variar de cm² para nematódeos presentes no solo, m² para invertebrados terrestres, e até Considerações iniciais Rodrigues, G.F.B 3 km² para a megafauna (> 40 kg). Outra abordagem é a temporal, também conhecida como “dinâmica das comunidades”, cujo o foco são as mudanças da abundância relativa das espécies de uma especificada área, incluindo extinções e adição de espécies, via competição, dispersão ou especiação (Vellend, 2010). Independentemente de quais forem as abordagens utilizadas, os padrões na composição e diversidade de espécies podem ser sumarizados em apenas quatro classes de processos: (1) seleção, (2) deriva, (3) especiação e (4) dispersão (Vellend, 2010). A seleção ocorre quando indivíduos da mesma população variam em alguma característica, e quando essas variantes se reproduzem em taxas diferentes. Assim a seleção pode favorecer a espécie A sobre a espécie B em uma dada comunidade, sendo que a espécie com maior fitness (sucesso reprodutivo diferenciado) tende a excluir as outras. O processo de deriva refere-se a mudanças aleatórias na abundância relativa das espécies dentro de uma escala de espaço ou tempo. Assim como a mutação é a principal fonte de variação genética, a especiação pode ser entendida como uma fonte de variação dentro de comunidades ecológicas (Vellend, 2010). Por fim, a dispersão, ou seja, o movimento dos organismos em uma determinada área influencia toda a comunidade biológica, assim como determina a resiliência de uma espécie frente a distúrbios ambientais. Ameaças à biodiversidade no ambiente marinho Entender as ameaças que provocam perdas na biodiversidade é o primeiro passo para reverter a situação. As principais ameaças a biodiversidade são a sobre- exploração, espécies invasoras e degradação e perda de habitat (Worm et al., 2006). No entanto, para qualquer caso particular são múltiplos tipos de ameaças Considerações iniciais Rodrigues, G.F.B 4 que contribuem para o declínio e extinção de um táxon. Atualmente a taxa de extinção de espécies é cerca de 1,000 vezes maior do que antes da dominação humana do planeta (Aldhous, 2014). A “sexta extinção em massa já começou”, e não há dúvidas que nós, seres humanos, somos os grandes responsáveis (Kolbert, 2014). O ambiente marinho cobre 2/3 da superfície terrestre e definitivamente não está isento da massiva perda da biodiversidade. Embora acreditava-se que as espécies marinhas eram mais resistentes a extinção devido à grande capacidade de dispersão, atualmente sabe-se que esses organismos são bastante impactados (Kaiser et al., 2006). Os mares e oceanos provêm uma indispensável fonte de proteínas para mais de 2.5 milhões de pessoas (Carrington, 2016) e atualmente mais da metade da área dos oceanos é pescada industrialmente (Jowit, 2018). A sobrepesca é reconhecida como uma das atividades que mais contribuem para a perda da biodiversidade. Os arrastões comerciais acabam capturando muitas espécies sem interesse econômico (bycatch) e muitas vezes de proeminente interesse para a conservação, como mamíferos marinhos, tartarugas e aves marinhas. Além disso, a pesca de arrasto tem afetado espécies bentônicas como corais e esponjas, e assim degradando o habitat de muitas outras espécies marinhas. Outro fato que ameaça a biodiversidade marinha é recorrente homogeneização taxonômica, que embora aconteça em vários habitats, é bastante perceptível nos ambientes costeiros (Thrush et al., 2016), na medida que seleciona grupos específicos capazes de suportar tal regime de distúrbios. Ao longo do último século a população humana está percorrendo enormes distâncias em um tempo cada vez mais curto, carregando consigo organismos e Considerações iniciais Rodrigues, G.F.B 5 aumentando as taxas de introduções de novas espécies em todas as partes do globo (Bax et al., 2003; Stuer-lauridsen et al., 2018). Essas espécies invasoras acabam causando o declínio de espécies nativas (principalmente as especialistas) pela perda de hábitat, competição e predação (Olden et al., 2004). Á agua de lastro presente em grandes embarcações comerciais são muitas vezes responsáveis por trazer espécies invasoras de diversas regiões do planeta (Bax et al., 2003). As biotas das ilhas são particularmente mais vulneráveis à introdução de espécies cosmopolitas, frequentemente levando a extinção de espécies endêmicas. Suspeita-se ainda que essa “homogeneização” possa estar acontecendo no nível de funcionamento do ecossistema, com menor diversidade e maior preponderância de generalistas, diminuindo o número de grupos funcionais em comunidades naturais (Olden et al., 2004; Martello et al., 2018). Percebemos, cada vez mais, que o estudo da biodiversidade é o primeiro passo para conservação e uso sustentável do meio ambiente em que estamos inseridos. Do mesmo modo, a biodiversidade é uma singular “biblioteca genética” e temos o dever moral de proteger os até então únicos seres viventes com os quais compartilhamos o universo (Ehrlich & Wilson, 1991). Infra-ordem Anomura MacLeay, 1838 A infraordem Anomura (do grego “cauda irregular”) representa um grupo altamente diversificado de crustáceos decápodes sendo representados principalmente pelos caranguejos ermitões (Paguroidea), caranguejo-topeira (Hippoidea), caranguejo-rei (Lithodidae) e porcelanídeos (Galatheoidea) (Mclaughlin et al., 2010). O registro fóssil contém representantes de quase todas Considerações iniciais Rodrigues, G.F.B 6 os grupos atuais e datam do Triássico superior (Chablais et al., 2011). Esse grupo tem cativado biólogos evolucionistas devido a impressionante diversidade morfológica e adaptações ecológicas. A diversidade de arranjos corpóreos desse grupo inclui quatro configurações: 1) formas tipo-caranguejo, 2) formas de lagosta com achatamento dorso-ventral, 3) forma de caranguejo ermitão com abdome simétrico (presente em apenas uma família) e 4) formas de caranguejo ermitão com abdome assimétrico (presente em 4 famílias) (Lemaitre & McLaughlin, 2009). Os anomúros são constituídos por 17 famílias, 222 gêneros, e cerca de 2500 espécies, dos quais 54% dos gêneros e 43 % das espécies pertencem a família Paguridae (Mclaughlin et al., 2007). Esse táxon tem como clado irmão os braquiúros (Brachyura Latreille, 1802), que dominam a diversidade de crustáceos decápodes com mais 6500 espécies descritas (De Grave et al., 2009). Devido à grande complexidade morfológica e ecológica do táxon, inúmeros debates acerca da taxonomia e filogenia foram realizados nas últimas décadas. Podemos citar mudanças significativas, como por exemplo a inclusão dos caranguejos-rei (Lithodidae) na superfamília Paguroidea, devido a morfologia assimétrica de seu abdome, sendo um resquício evolutivo presente em animais com simetria bilateral (Cunningham et al., 1992) e mais recentemente foi proposto a inclusão desses organismos na família Paguridae (Tsang et al., 2011). Estudos com base genética indicam que Anomura é um grupo natural (monofilético), no entanto as superfamílias Paguroidea e Galatheoidea são polifiléticas (Tsang et al., 2011). Toda essa diversidade morfológica e debates acerca das relações de parentesco evidenciam o peculiar interesse científico presente nesse grupo. Considerações iniciais Rodrigues, G.F.B 7 Superfamília Paguroidea Latreille, 1802 Esse táxon é representado por sete famílias e 122 gêneros (McLaughlin, 2003), composto por caranguejo ermitões e caranguejo rei (Lithodidae). Os caranguejos ermitões, cujo abdome é mole e geralmente torcido, necessita estar protegido por alguma estrutura, sendo na maioria das vezes conchas de moluscos gastrópodes. A íntima relação entre ermitões e conchas de gastrópodes permite com que esses organismos realizem precisas distinções entre a qualidade das conchas encontradas no habitat, até mesmo aquelas ocupadas por outros ermitões, e a sua atual concha (Kellogg, 1976; Hazlett, 1981; Tricarico & Gherardi, 2007), visto que a sobrevivência, reprodução e crescimento dos ermitões dependem estritamente da ocupação de conchas com tamanho e forma apropriada (Hazlett, 1981). A maioria das espécies de ermitão necessitam de conchas adequadas durante toda sua ontogenia, no entanto, a espécie semi-terrestre Birgus latro (Linnaeus, 1767) (Anomura: Coenobitidae) ao atingir determinado tamanho abandona a concha, devido a adaptações morfológicas, fisiológicas e comportamentais que permitem esse evento (Greenaway, 2003). Ainda em relação às conchas alguns estudos sugerem que a assimetria do abdome (característica que possibilitou a ocupação de conchas) dos ermitões tem origem parafilética no grupo e evoluiu independentemente possivelmente até três vezes (Ahyong, 2009), no entanto descobertas mais recentes indicam que essa característica evoluiu apenas duas vezes, uma na família Parapaguridae e outra para ancestral comum de todos os outros ermitões assimétricos, indicando que a assimetria do pléon e sua descalcificação evoluiu independentemente em duas linhagens diferentes, Considerações iniciais Rodrigues, G.F.B 8 possibilitando a exploração de conchas de moluscos amonites e gastrópodes (Tsang et al., 2011). Os ermitões tornam-se organismos modelos para estudos ecológicos sobre partição de nicho, compartilhamento e avaliação do recurso (Kellogg, 1977; Laidre, 2012; Tran, 2014) devido a intrínseca relação com seu principal recurso para a sobrevivência. Em relação a importância ecológica desse grupo, embora que a maioria das espécies possuam habito detritívoro, contribuindo para a ciclagem de nutrientes nos ciclos biogeoquímicos (Duineveld et al., 1997), os ermitões podem ocupar diversas posições numa teia trófica. Desde espécies com adaptações para o habito filtrador, como o ermitão Loxopagurus loxochelis (Moreira, 1901) (Mantelatto et al., 2004; Ayres-Peres & Mantelatto, 2008), até espécie carnívoras como Petrochirus diogenes (Linnaeus, 1758) (Caine, 1975). Essas espécies compõem a fauna bentônica de ecossistemas marinhos, responsáveis por serviços ecossistêmicos indispensáveis como: estabilidade do sedimento, redução da turbidez da coluna d’água, processamento de nutrientes e carbono, e sequestro de contaminantes (Thrush & Dayton, 2002). Os ermitões estão agrupados em seis famílias: Coenobitidae Dana, 1851; Parapaguridae Smith, 1882; Pylochelidae Spence Bate, 1888; Pylojacquesidae McLaughlin e Lemaitre, 2001; Paguridae Latreille, 1802; e Diogenidae Ortmann, 1892 (Mclaughlin et al., 2007, 2010). No litoral brasileiro há registro de aproximadamente 62 espécies descritas de ermitões, pertencendo às famílias Pylochelidae (1), Diogenidae (27), Paguridae (28), e Parapaguridae (6) (Lemaitre & Tavares, 2015). Considerações iniciais Rodrigues, G.F.B 9 Considerando a crescente necessidade da preservação da fauna local de ambientes impactados pela ação antrópica e de estudos ecológicos com o foco na comunidade, o presente estudo busca uma melhor compreensão da estrutura da comunidade de ermitões na Enseada de Ubatuba, litoral norte do Estado de São Paulo. Para tanto, foi abordado no primeiro capítulo padrões de abundância, riqueza e composição de espécies capturadas na região com intervalo de 20 anos. 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Impacts of Biodiversity Loss on Ocean Ecosystem Services. Science 314: 787–791. http://www/ Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 13 How hermit crab assemblages structure changed after 20 years in the northern coast of São Paulo state, Brazil ABSTRACT Examination of spatial patterns of organisms in coastal regions is critical and necessary to understand which factors control and maintain the diversity of species. Patterns of abundance and richness of hermit crabs inhabiting Ubatuba bay were investigated in two periods separated by 20 years: 1995-1996 (P1) and 2016-2017 (P2), and the influence of environmental factors on the assemblages. We hypothesized that sites with a high degree of heterogeneity will have a higher richness when compared to homogeneous sites. In a complete randomized block design (CRBD), samples were taken monthly in five sites with trawl nets. Additionally, we collected data from environmental variables: bottom water temperature and salinity, organic matter and sediment texture. Overall, 2,165 hermit crabs were over 24 months of study (12 months for both periods), representing a total of 11 species. We obtained 419 (10 species) and 1,746 hermit crabs (7 species) in P1 and P2, respectively. We found differences among the hermit crab assemblages on sites in both periods, and also differences between the P1 and P2. The abundance and richness were inversely correlated with bottom water temperature and sediment texture. Our results show that hermit crab fauna can change considerably in a small-scale landscape. Homogenization of the substratum may have increased hermit crab abundance and reduced species Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 14 richness. In short, the hermit crab assemblage changed in 20 years, that could be caused by some environmental features and/or anthropic pressure. Keywords: Richness patterns; spatial patterns; environmental change; Paguroidea; Coastal hermit crabs 1. Introduction The knowledge of biodiversity in coastal regions is of peculiar importance considering that nowadays is where the majority of the human population lives (Small & Nicholas, 2003). Evaluating patterns of diversity over space and time, and the factors and dynamics creating and maintaining those patterns are critical for understanding the ecology of biodiversity, guiding management and conservation efforts (Zajac et al., 2013). There still many gaps in our knowledge of spatial and temporal patterns of diversity and abundance of many marine organisms, which hinder out our ability to understand ocean systems and to detect human impacts on them (Hunt et al., 2017). Seabed fauna is essential for the functioning of marine ecosystems. Bottom invertebrates (benthos) help oxygenation of the sea floor, breaking down organic material, providing habitat structure and food sources for other organisms (Tagliapietra & Sigovini, 2010). Many benthic species are sensitive to disturbance; thus, the extent and intensity of human activity in marine ecosystems can ultimately disrupt the services that benthos provide (Thrush & Dayton, 2002). Environmental drivers may act like filters that select benthic species due their Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 15 capability to tolerate the abiotic stress, some characteristics can modulate the whole benthic community and their interactions (Alsaffar et al., 2017). A clear understanding on how the composition of species and their abundance change along spatial and across environmental gradients will provide data necessary to comprehend how communities will respond to environmental change, fundamental for conservation efforts (Sommer et al., 2014). Hermit crabs are decapod crustaceans which constitute an important parcel of macrobenthic fauna. Most of the hermit crabs are omnivorous and detritivorous which contribute to biogeochemical processes in ecosystems, recycling nutrients due to its ability to obtain food from several trophic levels (Nagabhushanam & Sarojini, 1968; McNatty et al., 2009; Selin et al., 2016). Some studies found that environmental variables like water temperature (Fransozo et al., 2008; Frameschi et al., 2013; Stanski et al., 2016), organic matter and sediment (Negreiros-Fransozo et al., 1997; Frameschi et al., 2013) influence the abundance of hermit crabs. Another particular feature modulates the hermit crab diversity, gastropod shells are an important and limiting factor that plays a crucial role in the interaction with the environment (Hazlett, 1981). Due their naked and no calcified abdomens, hermit crabs must find and occupy empties gastropod shells or other materials such as bivalve and scaphopod shells, polychaete tubes, sponge, corals, wood or even hollowed-out fragments of stones to protect themselves (Lancaster, 1986; Williams & McDermott, 2004). Such use makes them a hard mobile substrate provider, several animals inhabit the surface and interior of those shells, contributing to the diversity of many others invertebrate taxon. There are almost Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 16 550 invertebrate species associated with over 180 species of hermit crab species worldwide (McDermott et al., 2010). However, patterns and processes that control the richness and abundance of hermit crabs within coastal areas still are not clear. The niche diversification hypotheses proposed by Connell (1978), pointed that niche sub-divisions are a consequence of some degree of habitat heterogeneity. Different species need different aspects of habitat like food, space habitat and time of activity (called “niche space”). High biotic diversities occur in sites with a broad range of biotic and abiotic characteristics, maintaining specialist species and preventing generalist species dominance (Miserendino et al., 2018). Heterogenic substrates could permit that species occupy narrower niches, because of the effective partitioning of resources allowing the coexistence of several species. Long-term studies would significantly increase our understanding of the relative importance of abundance and richness spatial patterns, since different processes may be acting in particular sites, altering local assemblages (Vellend, 2010; Anderson et al., 2011). These singularities point towards different management and conservation options, especially if these changes are studied in relation to environmental change in coastal ecosystems (Gray, 2001). This study focuses on how hermit crab abundance and richness change over [1] a 20 years’ time-break and [2] across distinct environmental sites in a bay system, understanding the effects of environmental variables in the assemblage structure. Our hypothesis is that sites with a high degree of heterogeneity will have a higher richness when compared to homogeneous sites, according to “niche Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 17 diversification hypothesis”, which explains diversity in terms of specialization to sub-division of a habitat. 2. Material and methods 2.1 Study Area Ubatuba Bay (23°26’ S and 45°02’ W) is located along the northern coast of São Paulo State, Brazil, facing to the east and presents a constriction in its coastline, which induces wave diffraction (Mahiques et al., 1998). The proximity of the coastal range (Serra do Mar) results in small bays and headland-embayed beaches with variable orientation. This causes incident waves to approach the coastline at different angles, resulting in locally specific sediment transport patterns (Mahiques et al., 2016). In most of the area, the sediments contain mainly silt and very fine sand and few samples show coarse sand or clay modal distribution. This region presents a mixed fauna, that includes tropical, temperate and sub-Antarctic species, which are explained by the thermal regime of the coastal water (Coelho & Ramos, 1972). In 2008, a Marine Protection Area (Cunhambebe sector) was created with the intent to protect biodiversity in the north coast of São Paulo State (Proclamation No. 53525, October 8, 2008) forbidding pair trawl (a technique in which a bottom trawl is towed simultaneously by two boats). However, local fisheries are permitted and well-established, generating damages on soft-bottom communities. Also, in 2008, a closed season shrimp-fishing (IBAMA, 2008) was established, comprehending the period from march 1st to may 31th, forbidden any type of trawl fishing procedures. Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 18 Fig. 1 Location of sampling sites on Ubatuba bay. Ubatuba, São Paulo, Brazil. Black lines indicate the area surveyed. 2.2 Biotic and environmental data collection The hermit crabs were collected (trawled) utilizing a fishing boat equipped with double-rig nets (4.5 m wide at the mouth, 25 mm of body mesh size, and 15 mm of cod end mesh size). Each trawl lasted 30 minutes, covering an estimated swept area of 18.000 m². The samples were performed in same locations for both periods (1995-1996 and 2016-2017). Samples were taken, monthly, in three consecutive days, during both periods. The Ubatuba Bay was classified into five Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 19 sampling sites differing in terms of their location in relation to the bay mouth, the presence of a rock wall or a beach along the boundaries, the inflow of fresh water, the proximity of an offshore area, depth and sediment composition (Fransozo et al., 1998). A global positioning system (GPS) was used to record the location at the sampling sites, ensuring the sampling in the same sites for all months surveyed. Thus, we selected five available sites to this sampling period as follows: three sites plotted at depths of 5, 10, and 15 meters; and two sites defined perpendicular to the beach line ˗ one localized in a sheltered area, and other, in an exposed area to wave action (Mahiques et al., 1998) (Figure. 1). The individuals were kept in plastic bags, properly marked, in thermal boxes containing crushed ice. In the lab, the hermit crabs (Paguroidea) were removed from their shells, and identified, according to Melo (1999). Additionally, and based on previous evidences (Mantelatto & Franzoso, 1999; Fransozo et al., 2011) we selected four environmental variables to evaluate their probable action on the hermit crab fauna as follows: water temperature, water salinity, organic matter and sediment texture (phi). Details on methodology are available at Mantelatto & Fransozo (1999). Water samples were obtained with a Nansen bottle, from which we measured temperature (ºC) and salinity. Sediment samples were collected with a 0.06 m² van Veen grab to measure the organic matter content and sediment texture. All environmental data were collected simultaneously with biotic data in central point of each site sampled (Mantelatto and Franzoso 1999). Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 20 For sediment analysis procedures, two 50 g subsamples were taken, to which we added 250 ml of a NaOH (0.2 N) solution, aiming to lift up the silt + clay (Tucker, 1988). Afterward, we washed the subsamples using a sieve (mesh= 0.063 mm), washing away the silt + clay. The remaining sediment was dried and then submitted to a differential sieving, classifying the sediment grains according to the Wentworth (1922) scale. Phi values were calculated based on the equation phi = -log2d, where d= grain diameter (mm). Based on the obtained values, we calculated the central trend measurements, determining the most frequent grain fractions in the sediment. We calculated these values based on data graphically taken from cumulative sediment samples frequency distribution curves. We used values corresponding to the 16th, 50th and 84th percentages to determine the mean diameter (MD), using the equation MD= (φ16 + φ50 + φ84/3) (Suguio, 1973). Organic matter content was determined by loss on ignition (LOI). We put the subsamples (10g each) in porcelain containers, previously labelled and weighed. After that, we put them into an oven (500ºC for 3 hours) and weighed them again. The LOI is then calculated using the following equation: LOI = (Wi –Wf)/Wi * 100, where: LOI = organic matter content (%), Wi = initial weight of the sediment subsample; Wf = final weight after ignition (Hieri et al., 2001). 2.3 Statistical analysis We utilized a randomized complete block design (RCBD) to reduce temporal sources of variation (Halpern and McKenzie 2001), with ten sites (five for each period) randomly assigned to 12 months (blocks) at each studied period. Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 21 Species richness and abundance of all hermit crabs were compared among each site using a repeated measures nonparametric Friedman ANOVA by ranks, where the abundance and richness of each site were a repeated measure. Post hoc pairwise comparisons were then made with Conover matched-pairs tests. These procedures were adequate because no temporal autocorrelation, assessed through the Durbin– Watson statistic (p-value < 0.05), was detected. Exploratory multivariate analyses were applied to abundance data transformed and using Bray-Curtis dissimilarity coefficient to construct resemblance matrices to compare sites. These dissimilarities were represented in two dimensions using non-metric multidimensional scaling (nMDS) representing the samples as points in a two- dimensional space. A cluster analysis was utilized to describe sites based on the mean value of the abundance of each taxon per sampling site. A Permutational multivariate analysis of variance (PERMANOVA) (Anderson, 2001) was used to compare the hermit crab assemblages between years and sites. To verify the species-environment relationship in the separation of sample sites, we performed a Redundancy Analysis (RDA) (Gotelli & Ellison, 2016). Collinearity among environmental variables were assessed through variance inflation factors (VIF), since no collinearity was detected, all variable remains in the RDA. The statistical significance of eigenvalues of the RDA axis was evaluated by randomization (Monte Carlo) tests, using 9,999 randomized runs for each analysis. All analyses were carried out in R statistical program (R Core Team, 2019), using package “stats” for Friedman ANOVA test (R Core Team, 2019), “PMCMR” for Post hoc comparisons (Pohlert, 2014), “lmtest” to detect temporal autocorrelation (Zeileis Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 22 & Hothorn, 2002), “vegan” for multivariate analysis (Oksanen et al., 2015) and “PerformanceAnalytics” to detect collinearity (Peterson & Carl, 2018). 3. Results Across all sites, 2,165 hermit crabs were collected from 360 samples over 24 months of study (12 months in first period and 12 months in second period), representing a total of 11 species. During the first period (P1) 419 organisms were captured (Table 2) while in the second period (P2) we captured 1.746 paguroideans (Table 3). In general, hermit crab richness was 2.2 species per site, ranging from zero to five species. The hermit crab Dardanus insignis (de Saussure, 1858) (To pictures see supplementary material) was the most widely distributed, being found in almost all sites sampled. Table 2. Abundance of hermit crab species (family) sampled in Ubatuba Bay, from September 1995 to August 1996. Species/site 5 m 10 m 15 m Exposed Sheltered Total Family Diogenidae Ortmann, 1892 Dardanus insignis (de Saussure, 1858) 11 0 22 5 83 121 Isocheles sawayai Forest & de Saint Laurent, 1968 2 0 1 0 1 4 Loxopagurus loxochelis (Moreira, 1901) 4 5 158 5 0 172 Pseudopaguristes calliopsis (Forest and Saint Laurent,1968) 1 0 0 0 1 2 Petrochirus diogenes (Linnaeus, 1798) 35 1 4 0 57 97 Paguristes erythrops Holthuis, 1959 2 0 0 0 1 3 Paguristes tortugae Schmitt, 1933 1 0 0 0 3 4 Family Paguridae Latreille 1802 Pagurus criniticornis (Dana, 1852) 0 0 0 0 3 3 Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 23 Pagurus exilis (Benedict, 1892) 0 0 4 0 3 7 Pagurus leptonyx Forest and Saint Laurent, 1967 2 2 1 0 1 6 Total 58 8 190 10 153 419 Table 3. Abundance of hermit crab species (family) sampled in Ubatuba Bay, from September 2016 to August 2017. Species/Sites 5 m 10 m 15 m Exposed Sheltered Total Family Diogenidae Ortmann 1892 Dardanus insignis (de Saussure, 1858) 140 38 38 2 746 964 Isocheles sawayai Forest & de Saint Laurent 1968 0 2 0 0 8 10 Loxopagurus loxochelis (Moreira, 1901) 136 20 162 28 70 416 Petrochirus diogenes (Linnaeus, 1798) 12 4 2 0 26 44 Paguristes sp. 0 0 0 2 0 2 Family Paguridae Latreille 1802 Pagurus exilis (Benedict, 1892) 52 12 26 2 208 300 Pagurus criniticornis (Dana, 1852) 10 0 0 0 0 10 Total 350 76 228 34 1058 1746 3.1 Spatio-temporal trends When the same sites sampled in both P1 and P2 were compared (Pairwise comparisons), sites “5m”, “10m” and “Sheltered” had differences in total abundance of hermit crabs (Friedman chi-squared (9, 120) = 60.34, p < 0.001), having the second period greater abundance of these organisms (Figure 2). The same Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 24 procedure was applied to richness (Figure 3), and only site “10m” had difference when comparing the richness of same sites sampled in both P1 and P2 (Friedman chi-squared (9, 120) = 49.43, p < 0.001). Fig. 2 Boxplots summarizing abundance of hermit crabs in all sites selected, comprehending both periods. Asterisks (*) indicate difference between same sites through periods, assessed by Conover’s Post-hoc analysis. Black dots indicate outliers. Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 25 Fig. 3 Boxplots summarizing richness of hermit crabs in all sites selected, comprehending both periods. Asterisks (*) indicate difference between same sites through periods, assessed by Conover’s Post-hoc analysis. Black dots indicate outliers None single species was found in all sites, the most abundant species found in the first period was Loxopagurus loxochelis (41%) (To pictures see supplementary material), while in the second period was D. insignis (55%), also being the most abundant species overall. Multivariate analysis (PERMANOVA) revealed significant differences among the hermit crab assemblages on sites in P1 (PERMANOVA, Pseudo-F(4,120) = 6.631, p < 0.005) and P2 (PERMANOVA, Pseudo-F(4,120) = 7.961, p < 0.005). These differences are shown graphically in a nMDS plot (Figure 4). Also, PERMANOVA analysis revealed significant differences between P1 and P2 (PERMANOVA, Pseudo-F(1,120) = 8.297, p < 0.005) (Figure 5). Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 26 Fig. 4 Multivariate analysis nMDS plot of hermit crab assemblage structure from Ubatuba bay. Based on species abundance data and Bray-Curtis similarity measure; individual sites are displayed, ellipses = 0.75% of similarity. Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 27 Fig. 5 Multivariate analysis nMDS plot of hermit crab assemblage structure from Ubatuba bay. Based on species abundance data and Bray-Curtis similarity measure; comparison of individual sites from both periods, ellipses = 0.75% of similarity. We used the clusters analysis to compare the abundance of all sites; the Exposed and Sheltered sites showed lower similarity in both periods. In first period, sites 15 m, Exposed and Sheltered showed higher similarity while in the second period 5m and 10 m showed presented greater similarity (Figure 6). Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 28 Fig. 6 Results of hierarchical clusters analysis applied to the abundance of hermit crabs in 10 sites of Ubatuba bay, representing both periods (1995-1996 & 2016- 2017). 3.2 Correlation with environmental variables The relationship between hermit crab (abundance and richness) and environmental variables shown by RDA is represented by two axes (Figure 7). The first axis explained 96.38 % of the variance. The Monte Carlo test indicated that only first axis (p < 0.05) was significant. This test also indicated that bottom temperature and phi were significantly correlated. Based on this analysis, we may attest that the bottom water temperature and sediment grain size (phi) is inversely proportional to the hermit crab abundance and richness. It characterizes a preference for low water temperature and for heterogeneous sediment, mainly Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 29 composed by granulometric classes such as gravel, very coarse sand, coarse sand and medium sand (Table 4). Fig. 7 RDA plot. Visualizes the position of the biotic variables (Abundance and Richness) fitted to the environmental variables (bottom water temperature, bottom water salinity, organic matter and phi) affecting the entire hermit crab community (see Table 4 for statistical results). Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 30 Table 4. Redundancy Analysis (RDA). Summary results of hermit crabs and environmental variables. Asterisks (*) indicate statistical significance. Significance was inferred using α (p < 0.05): p-value based on 9,999 permutations. Axis Monte Carlo test RDA1 RDA2 F p Proportion Explained 0.963 0.036 3.598 0.011 Bottom Temperature -0.747 -0.008 7.746 0.007* Bottom Salinity 0.201 0.693 0.942 0.350 Organic Matter 0.368 -0.796 2.428 0.109 Phi -0.398 0.040 3.275 0.05* 4. Discussion Patterns of abundance and richness Several studies achieved in São Paulo State focusing on hermit crab fauna registered a richness, that ranged from six to eleven species, and evidenced Dardanus insignis as the species most found in almost all studies (Hebling et al., 1994; Fransozo et al., 1998, 2011; Mantelatto & Garcia, 2002). Other studies also investigated hermit crab communities near islands in Ubatuba region (Frameschi et al., 2013) and in south Brazilian Coast (Stanski et al., 2016) found eight and six species, respectively. The hermit crabs D. insignis and Isocheles sawayai Forest & de Saint Laurent, 1968 were the most abundant species in each area, respectively. Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 31 The results presented here pointed that hermit crab fauna can change considerably in a small-scale landscape and that these changes could be caused by some specific environmental features. The abundance of hermit crabs in the sampled sites showed a clear spatial configuration, with greater abundance in the Sheltered one (P2), probably because environmental characteristics – like soft substrate grain composition – in this site were more heterogenic (Mantelatto & Franzoso, 1999). Different habitats allow the establishment of various species, allowing an increase in local abundance. The complexity of benthic landscapes displays spatial heterogeneity with potential implications related to trophic interactions and larval recruitment (Johnson, 2015), complexes benthic structure provides a greater diversity of food resources (preys, decomposing matter, suspensive particles) and several refuges and settlement sites for multiple species (Thrush et al., 2016). Also, small environmental differences, both temporal and spatial, that would be insignificant at high population densities, may provide sufficient complexity to avoid competition at low densities (Huston, 1979). One of the main questions to be answered is “why the abundance of hermit crabs was higher in the second period of time?”. Several studies show that trawling disturbance caused by fishery activities could change the community structures of marine organisms (Mazor et al., 2017; Ramalho et al., 2017). Since 1970, the fisheries industries established in Ubatuba city, boosted by tax breaks policies and tourism, became one of the main economic activity (Vianna & Valentini, 2004). Non-selective fishery devices (trawls) are designed to penetrate the surface of the Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 32 sediment, disturbing and damaging benthic and infaunal animals (Watling & Norse, 1998). When comparing the number of fishing boats in Ubatuba between these two periods, we noted an increase of 35% (162 to 219 fishing boats) (Vianna & Valentini, 2004; Ávila-da-Silva et al., 2017). Fishery activities provide an amount of organic matter (by-catch) released near inshore sites which predators and scavenger animals may consume. Some studies found that hermit crabs are attracted to areas disturbed by trawling, rapidly migrating onto a trawled way line to increase their food intake (Kaiser & Spencer, 1996; Ramsay et al., 1996). These fisheries discards may improve the food intake by hermit crabs, increasing the abundance of these crustaceans. The hermit crabs Pagurus bernhardus (Linnaeus, 1758) populations at the northeast coast of Anglesey, Irish Sea, respond strongly to trawling disturbance, rapidly migrating onto a trawled sites (Ramsay et al., 1997). Also, the shells that hermit crabs dwells help to reduce mechanic damage occurring during trawls, leading to a low mortality rate (Kaiser & Spencer, 1995). However, fisheries disturbances could affect sensitive hermit crab species, which explain the lower richness found in the second period of time. A repeated intense disturbance will select for species with appropriate facultative responses, and communities are likely to become dominated by juvenile stages, mobile species, and rapid colonists (Thrush & Dayton, 2002). Homogenization of the substratum (indicated by higher values of phi, see Figure 8) is one of the most conspicuous long-term physical effects of bottom fishing. Loss of small-scale heterogeneity is caused by the removal of sessile organisms Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 33 capable of forming biogenic substrata (Veale et al., 2000) leading to a simpler abiotic structure, without biological structures like burrow walls, tubes, and galleries of the infauna (Schwinghamer et al., 1996). The removal of small-scale heterogeneity associated with the homogenization of habitats is, by definition, loss of biodiversity (Thrush & Dayton, 2002). Fig 8. Boxplots summarizing sediment texture (phi values) in all sampled sites, comprehending both periods. Black dots indicate outliers. In addition, multivariate analysis indicated a change in community structure, with significant differences between the first and the second period. The most abundant species changed when comparing the two periods of time (L. loxochelis in the first period to D. insignis in the second one). Generalist species tend to be less affected by fragmentation and disturbance than most of the species, some generalist species can even be benefited in these conditions (Devictor et al., 2008). The hermit crab D. insignis present some generalist characteristics like brief embryonic development, a high number of eggs produced per ovigerous female and reduced Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 34 size of the eggs (Miranda et al., 2006), which can explains the higher abundance in disturbed sites. The hermit crab L. loxochelis also increased abundance in second period, this species obtain food from suspended particles (suspension- feeder organism) (Biagi et al., 2006; Ayres-Peres & Mantelatto, 2008). Fisheries activities cause resuspension of organic matter in soft-substrates, facilitating suspensive species and resistant to trawl disturbance (L. loxochelis characteristics) (Van Denderen et al., 2015). Hermit crab fauna was dissimilar in Exposed and Sheltered sites (in both periods). This pattern can be consequence to the distinct hydrodynamics features, related to different energy levels acting distinctly over each site. The site Sheltered has more influence of marine biogenic constituents (shell, ophiuroid, echinoderm and other marine animals fragments) than others sites in the studied bay (Mahiques et al., 1998). Shell’s fragments indicate the presence of living snails which can provide shells availability to hermit crabs. Environmental variables Results in our study indicate that bottom temperature has been correlated inversely with hermit crab abundance. In Ubatuba the intrusion of the South Atlantic Central Water (SACW) toward the coast in the bottom layer of the continental shelf results in the movement of the Coastal Water (CW), rich in suspended matter from the continent (Castro Filho et al., 1987; Mahiques et al., 1998), which increases the amount of food for larvae because of the increase in primary productivity (De Léo & Pires-Vanin, 2006). SACW is a water mass characterized by low temperatures Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 35 (Castro Filho et al., 1987) which can explain the inverse correlation between bottom temperature and hermit crab abundance. Local sediments attributes may be responsible for differences in both the fauna and the overall densities in macrobenthic communities (Johnson, 2015). Sediment properties (sand, mud, gravel) is one of the most important predictors among benthos models (Mazor et al., 2017). Greater values of phi indicate the presence of finest sediment, decreasing soft-bottom sediment texture heterogeneity. Our results showed an inverse correlation between phi values and hermit crab richness. Spatial heterogeneity has long been considered to promote overall species diversity in ecosystems (Huston, 1979; Rodrigues et al., 2017). A gradient of decreasing species diversity with decreasing soft-bottom heterogeneity is well-established in some benthos communities (Mcclain & Barry, 2010; Cardone et al., 2014; Alsaffar et al., 2017). Based on our results, sediment texture must be a key factor that maintains hermit crab richness in those sites. 5. Conclusion Our investigation highlights the study of biodiversity to understand long-term changes in a well-established hermit crab assembly. It is clear the existence of small-scale variance in species composition, this shifts may be caused by some environmental features. 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R News 2: 7–10, https://cran.r-project.org/doc/Rnews/. https://cran/ Hermit crab assemblages: changes after 20 years Rodrigues, G.F.B 42 Changes in coastal hermit crab biodiversity patterns Rodrigues, G.F.B. 2018 43 Changes in coastal hermit crab biodiversity patterns: partitioning beta diversity with an interval of 20 years Abstract Changes in diversity, density, and community structure of soft-bottom hermit crab assemblages were investigated in different areas of a bay system in the southeastern Brazilian coast in two periods with an interval of 20 years. Density of several hermit crab species increased in second period. Most of the alpha diversity index did not change, when comparing the sampling periods. Partitioned beta diversity indicated that dissimilarities between the two periods (1995-1996 and 2016-2017) were higher in sites close to the coastline, and the relative importance of nestedness and turnover component were distinct among the sites. Also, the hermit crab assemblage composition was distinct between the periods. The first period assemblage was inversely correlated with sediment texture (Phi), while the second one the opposite was observed. Despite the similarities observed for alpha-diversity across the five sites, beta-diversity measures indicated consistently changes. The sedimentation change (probably due higher erosive processes) may be one of the factors that have altered the marine environment, causing notable changes in the hermit crab community structure after 20 years. Even being difficult with this data set to precise the anthropic influence on hermit crab assemblages, these results are useful to guide conservation efforts in this bay system and also to predict the premises on biodiversity changes along the time. Changes in coastal hermit crab biodiversity patterns Rodrigues, G.F.B. 2018 44 Key-words: Anomura; Temporal changes; Coastal environment; Paguroidea; Nestedness component; Turnover component. 3. Introduction Patterns of biodiversity provide insights into the organization of metacommunities and their responses to the local and regional footprints of human-induced environmental change (Zajac et al., 2013; Mantelatto et al., 2016; Hunt et al., 2017; Chatzinikolaou et al., 2018). Regional long-term studies would significantly increase our understanding of how local communities respond to environmental changes caused by both natural and anthropic processes (Angeler, 2013). Knowledge of the processes that govern the structure of communities across space and time is the key for conserving regional biodiversity (Tylianakis et al., 2005; Leprieur et al., 2009; Angeler, 2013; Chatzinikolaou et al., 2018). Several studies focused on local scales (alpha diversity), while efforts to characterize the spatial variability in species composition across sites (beta diversity) increased recently (Anderson et al., 2011). An advantageous approach to understand the changes in biodiversity across space and time is partition of beta diversity. The beta diversity is partitioned resulting in two antithetic components: nestedness that occurs when biotas of sites with smaller numbers of species are subsets of biotas at richer sites and replacement (turnover) component that implies in the replacement of some species by others in different sites (Baselga, 2010). This approach allows the implementation of different conservation guidelines. Dominating nestedness suggests the necessity of prioritizing a few high-diversity sites in conservation Changes in coastal hermit crab biodiversity patterns Rodrigues, G.F.B. 2018 45 planning, while dominating turnover requires a regional approach focusing on multiple sites (Wright & Reeves, 1992). Benthic fauna is often used as indicators for assessing the status and health of marine ecosystems (Rosenberg et al., 2004). Previous studies have found benthic communities with reduced richness and diversity in sites with human-induced disturbances like trawl fishery (Mazor et al., 2017) and contamination by pollutants (Chatzinikolaou et al., 2018). The hermit crabs are decapod crustaceans that compose an important piece of benthic fauna. These crustaceans are unique for their dependency on gastropod shells as a ‘mobile home’ to protect them from predators (Childress, 1972) which make them extremely dependent of shell supply, provided by gastropod and other mollusks communities (Hazlett, 1981). Despite its omnivorous and detritivore feeding behavior them are a key element in the trophic chain, obtaining food from several trophic levels (Selin et al., 2016). Some studies aiming to understand the biodiversity of hermit crabs and environmental variables, which modulate this patterns have been published for the Brazilian coast, being most of them in the Southern and Southeast region (Hebling et al., 1994; Negreiros-Fransozo et al., 1997; Fransozo et al., 1998, 2011; Mantelatto & Garcia, 2002; Meireles et al., 2012; Frameschi et al., 2013; Stanski et al., 2016; Fernandes & Alves, 2018). Nevertheless, an information lack still remains on how hermit crab assemblages changed over a time gap, and how the assemblage responds to environmental changes. Changes in coastal hermit crab biodiversity patterns Rodrigues, G.F.B. 2018 46 Here, we used a unique data set of coastal hermit crabs, that have been sampled in five sites in Ubatuba Bay during two periods (comprising 12 sampling months, each period) to (1) compare density, richness and alpha diversity indices between these periods, (2) investigate the influence of environmental factors in hermit crab assemblage and (3) evaluate the relative importance of turnover and nestedness components in sites, temporally. Such approach considers that temporal beta diversity can be understood as temporal variability of spatial beta diversity; that is, comparing the spatial beta diversity over time (Angeler, 2013). 2. Material and Methods 2.1 Study Area The coast of the State of São Paulo is, probably, one of the most affected by anthropogenic activities from the whole Brazilian coastline (Mahiques et al., 2016). For the northern beaches, from Ubatuba to São Sebastião municipalities, the proximity of the coastal range (Serra do Mar) results in small bays and headland-embayed beaches with variable orientation (Mahiques et al., 1998, 2016). Ubatuba Bay faces to the east and presents a constriction in its coastline, which induces wave diffraction (Mahiques et al., 1998). The artisanal fishery in the Ubatuba Bay is well-established, being an important economic source for human populations, boosted by tourism in the region. These activities result in severe impacts to the coastal environments. The Ubatuba bay is important for conservation efforts, sheltering several species from tropical, temperate and sub- Changes in coastal hermit crab biodiversity patterns Rodrigues, G.F.B. 2018 47 Antarctic faunas (Amaral & Nallin, 2017), and considered a hotspot for decapod crustaceans (Mantelatto et al., 2018). Some efforts intending to preserve local marine biodiversity includes the establishment of a Marine Protection Area (Cunhambebe sector), in 2008, and a closed season shrimp-fishing (IBAMA, 2008) that forbid any type of trawl fishing procedures from march 1st to may 31th, each year. 2.2 Data Collection The hermit crabs were collected (trawled) utilizing a fishing boat equipped with double-rig nets (4.5 m wide at the mouth, 25 mm of body mesh size, and 15 mm of cod end mesh size). Each trawl lasted 30 minutes, covering an estimated swept area of 18.000 m². Samples were taken, monthly, in three consecutive days, during two periods. The samples were performed in same locations for both periods (1995-1996 and 2016-2017). The Ubatuba Bay was classified into five sampling sites differing in terms of their location in relation to the bay mouth, the presence of a rock wall or a beach along the boundaries, the inflow of fresh water, the proximity of an offshore area, depth and sediment composition (Fransozo et al., 1998). A global positioning system (GPS) was used to record the location at the sampling sites, ensuring the sampling in the same sites for all months surveyed. Thus, we selected five available sites to this sampling period as follows: three sites plotted at depths of 5, 10, and 15 meters; and two sites defined perpendicular to the beach line ˗ one localized in a sheltered area, and other, in an exposed area to wave action (Figure 1). The individuals were kept in plastic bags, properly marked, Changes in coastal hermit crab biodiversity patterns Rodrigues, G.F.B. 2018 48 in thermal boxes containing crushed ice. In the lab, the hermit crabs (Paguroidea) were removed from their shells, and identified, according to Melo (1998). Fig. 1 Location of sampling sites on Ubatuba bay. Ubatuba, São Paulo, Brazil. Black lines indicate the area surveyed. Additionally, and based on previous evidences (Mantelatto & Franzoso, 1999; Fransozo et al., 2011) we selected four environmental variables to evaluate their probable action on the hermit crab fauna as follows: water temperature, water salinity, organic matter and sediment texture (phi). Details on methodology are Changes in coastal hermit crab biodiversity patterns Rodrigues, G.F.B. 2018 49 available at Mantelatto & Fransozo (1999). Water samples were obtained with a Nansen bottle, from which we measured temperature (ºC) and salinity. Sediment samples were collected with a 0.06 m² van Veen grab to measure the organic matter content and sediment texture. All environmental data were collected simultaneously with biotic data in central point of each site sampled (Mantelatto and Franzoso 1999). For sediment analysis procedures, two 50 g subsamples were taken, to which we added 250 ml of a NaOH (0.2 N) solution, aiming to lift up the silt + clay (Tucker, 1988). Afterward, we washed the subsamples using a sieve (mesh= 0.063 mm), washing away the silt + clay. The remaining sediment was dried and then submitted to a differential sieving, classifying the sediment grains according to the Wentworth (1922) scale. Phi values were calculated based on the equation phi = -log2d, whered= grain diameter (mm). Based on the obtained values, we calculated the central trend measurements, determining the most frequent grain fractions in the sediment. We calculated these values based on data graphically taken from cumulative sediment samples frequency distribution curves. We used values corresponding to the 16th, 50th and 84th percentages to determine the mean diameter (MD), using the equation MD= (φ16 + φ50 + φ84/3) (Suguio, 1973). Organic matter (OM) content was determined by loss on ignition (LOI). We put the subsamples (10 g each) in porcelain containers, previously labelled and weighed. After that, we put them into an oven (500ºC for 3 hours) and weighed them again. The LOI is then calculated using the following equation: LOI = (Wi – Changes in coastal hermit crab biodiversity patterns Rodrigues, G.F.B. 2018 50 Wf)/Wi * 100, where: LOI = organic matter content (%), Wi = initial weight of the sediment subsample; Wf = final weight after ignition (Hieri et al., 2001). 2.3 Data Analysis Tests for homoscedasticity (Levene tests) and normality (Shapiro–Wilk tests) were first performed as pre-requisites for the statistical test (α = 0.05). Temporal changes in in total density (ind/km²), richness (S) and alpha diversity indexes (described below) were assessed using Student’s t-test for paired samples (Zar, 1999). Total density comparisons of each hermit crab was assessed using Wilcoxon test (nonparametric data) for pairwise comparisons between both periods (Hollander & Wolfe, 1999). 2.3.1 Ecological indexes Alpha diversity was estimated for hermit crabs at all sites at Ubatuba bay. The alpha diversity indexes included: Shannon index (H’), Simpson index (D’), taxonomic diversity (∆) (Clarke & Warwick, 1998) and taxonomic distinctness (Δ+) (Clarke & Warwick, 2001). The lasts two indexes incorporate phylogenetic information. A composite phylogeny of hermit crabs in the Southeastern Brazilian Coast classification scheme was compiled from Lemaitre & Tavares (2015). Four levels of classification (Superfamily, Family, Genus and Species) were used for the measurement of taxonomic distance between species in the study region. The taxonomic diversity ( Δ ) measured as: ∆ = ∑ ∑ 𝜔𝑖𝑗𝑥𝑖𝑥𝑗/ ∑ ∑ 𝑥𝑖𝑥𝑗 + ∑ 𝑛(𝑛−1)𝑖𝑖>𝑗 2𝑖>𝑗 where xi denotes the abundance of the ith species, n is the total number of Changes in coastal hermit crab biodiversity patterns Rodrigues, G.F.B. 2018 51 individuals in the sample and wij is the “distinctness weight” given to the path length linking species I and j in the hierarchical classification. The Taxonomic distinctness (Δ+) based on presence/absence of species in study sites is given by the equation Δ+ = ∑ ∑ 𝜔𝑖𝑗𝑖>𝑗 S(S−1)/2 , where s denotes the number of species observed and ωij is the weight given to the path length linking species I and j in the taxonomy. 2.3.2. Partitioning beta diversity Overall beta diversity can be partitioned into two additive components: (1) turnover (βsim sensu Baselga, 2010), which is conceptually associated with species replacement; and (2) nestedness (βnes sensu Baselga, 2010), akin to species addition. These two components of overall beta diversity are opposing factors which represent either compositional distinctness (βsim), or the degree to which a site species pool is determined by, or nested within, a neighbouring species pools (βnes). Both spatial turnover and nestedness contribute to overall compositional difference between assemblages (βsor = βsim + βnes). To assess overall partitioned beta diversity, we used package “betapart” (Baselga & Orme, 2012). Using species presence–absence data, we calculated total species beta diversity (βsor) and its two additive components: turnover (βsim) and nestedness (βnes). Partitioned beta diversity coefficients were calculated separately for each sites comparing P1(n = 12) vs. P2 (n = 12). 2.3.3. Correlation between environmental variables Distance-based redundancy analysis (dbRDA) was applied to visualize the position of the periods fitted to the significant environmental predictor variables affecting the biological assemblage data by analyzing marginal and sequential Changes in coastal hermit crab biodiversity patterns Rodrigues, G.F.B. 2018 52 tests. The analysis included bottom water temperature, bottom water salinity, sediment texture (phi), and OM (%). The direction and magnitude of the relationships between environmental variables and biological assemblages were visually presented in a dbRDA biplot. The statistical significance of eigenvalues of the dbRDA axis was evaluated by randomization (Monte Carlo) tests, using 9,999 randomized runs for each analysis. To understand the effect of natural or human induced sedimentation we compared the phi values of each site between the first and second period. Sediment grain size was evaluated following the Suguio (1973) approach, were higher values of phi indicate finer sediments. All analyses were carried out in R statistical program (R Core Team, 2019), using package “stats” for Wilcoxon test (R Core Team, 2019), “vegan” to calculate alpha diversity indexes and for multivariate analysis (Oksanen et al., 2015) and “betapart” to assess beta diversity components. 3. Results Descriptive statistics of the biological data and environmental factors measured at each sampling site are shown in Table I. Hermit crab assemblage sampled across the Ubatuba Bay yielded 11 species belonging to 2 families, six of which were common for both periods. Table II lists the hermit crab fauna present at each location and indicates the dominant species on each sampled site for both periods. We found a density (ind x km-2) of 387.96 in the first period and 1616.67 in second one (Table III). The hermit crabs Dardanus insignis (de Saussure, 1858), Loxopagurus loxochelis (Moreira, 1901) and Pagurus exilis (Benedict, 1892) were the species most frequent during the overall periods surveyed. Clear changes in Changes in coastal hermit crab biodiversity patterns Rodrigues, G.F.B. 2018 53 species compositions were detect comparing the periods. The hermit crabs Pseudopaguristes calliopsis (Forest & de Saint Laurent, 1968), Paguristes erythrops Holthuis, 1959, Paguristes tortugae Schmitt, 1933 and Pagurus leptonyx Forest & de Saint Laurent, 1968 were found exclusively in the first period. Six species were shared between first and second period communities, whereas the three most abundant species contributed more to the hermit crab assemblage composition, representing a 91.45% of total density. Table I. Biological and environmental characterization of the identified hermit crab assemblages in each sampled site at the coast of Ubatuba, State of São Paulo, Brazil, and in each period (1995-1996 and 2016-2017). 1995-1996 5 m 10 m 15 m Exposed Sheltered Depth (m) 5 10 15 7.5 10 Latitudinal (decimal º) -23.437 -23.435 -23.435 -23.424 -23.452 Longitudinal (decimal º) -45.044 -45.031 -45.015 -45.016 -45.026 Water column Bottom temperature (ºC) 23.96 ± 2.6 23.4 ± 2.7 22.55 ± 2.8 23.40 ± 2.8 23.74 ± 2.7 Salinity 33.15 ± 1.8 33.4 ± 1.7 33.75 ± 1.5 33.33 ± 1.7 33.25 ± 1.8 Sediment properties Sediment texture (Phi) 3.34 ± 0.8 3.24 ± 0.7 3.32 ± 0.2 3.29 ± 0.2 1.81 ± 0.2 Organic matter (%) 14.55 ± 2.2 13.22 ± 1.5 5.52 ± 5.1 5.35 ± 3.0 18.54 ± 9.6 Biological variables Richness (S) 2.08 ± 1.3 1.5 ± 0.5 2.08 ± 1.0 1.00 ± 0.0 2.66 ± 1.07 Total no. Ind. 58.00 8.00 190.00 10.00 153.00 Density (ind/km²) 26.38 ± 18.3 8.33 ± 2.7 87.96 ± 131.8 9.25 ± 4.5 69.44 ± 59.7 H’ 0.48 ± 0.5 0.33 ± 0.3 0.33 ± 0.3 0.00 ± 0 0.69 ± 0.25 Simpson 0.37 ± 0.39 0.34 ± 0.39 0.32 ± 0.31 0 ± 0.00 0.62 ± 0.20 2016 – 2017 5 m 10 m 15 m Exposed Sheltered Depth (m) 5 10 15 7.5 10 Latitudinal (decimal º) -23.437 -23.435 -23.435 -23.424 -23.452 Longitudinal (decimal º) -45.044 -45.031 -45.015 -45.016 -45.026 Water column Bottom temperature (ºC) 24.08 ± 2.4 23.87 ± 1.8 23.37 ± 1.7 23.91 ± 2.2 23.46 ± 2.0 Salinity 35.25 ± 1.2 34.91 ± 1.9 34.83 ± 2.12 34.83 ± 1.94 35.33 ± 1.7 Changes in coastal hermit crab biodiversity patterns Rodrigues, G.F.B. 2018 54 Sediment properties Sediment texture (Phi) 5.83 ± 0.6 4.9 ± 0.7 4.50 ± 0.9 4.90 ± 0.4 3.00 ± 1.6 Organic matter (%) 9.5 ± 2.85 6.57 ± 2.96 4.75 3.53 6.09 ± 2.21 6.22 ± 3.12 Biological variables Richness (S) 2.63 ± 1.1 2.11 ± 0.7 2.00 ± 0.9 1.40 ± 0.8 2.83 ± 1.4 Total no. Ind. 350.00 76.00 228.00 34.00 1058.00 Density (ind/km²) 215.74 ± 17.05 53.7 ± 3.79 194.44 ± 17.80 31.48 ± 2.80 896.29 ± 63.72 H’ 0.56 ± 0.5 0.67 ± 0.3 0.47 ± 0.5 0.15 ± 0.3 0.33 ± 0.31 Simpson 0.59 ± 0.30 0.59 ± 0.24 0.43 ± 0.32 0.14 ± 0.33 0.46 ± 0.30 Table II. Hermit crab species present at each site Ubatuba Bay surveyed in both periods. ‘X’ indicates presence; ‘D’ indicates a dominant species on that particular site; dash (–) indicates absence. 1995-1996 2016-2017 Species 5 m 10 m 15 m Exposed Sheltered 5 m 10 m 15 m Exposed Sheltered Family Diogenidae Ortmann 1892 Dardanus insignis (de Saussure, 1858) X - X D D D D D X D Isocheles sawayai Forest & de Saint Laurent 1968 X - X - X - X - - X Loxopagurus loxochelis (Moreira, 1901) X D D D - X X D D X Pseudopaguristes calliopsis (Forest and Saint Laurent, 1968) X - - - X - - - - - Petrochirus diogenes (Linnaeus, 1798) D X X - X X X X - X Paguristes erythrops Holthuis, 1959 X - - - X - - - - - Paguristes tortugae Schmitt, 1933 X - - - X - - - - - Paguristes sp. - - - - - - - - - X Family Paguridae Latreille 1802 Pagurus criniticornis (Dana, 1852) - - - - X X - - - - Changes in coastal hermit crab biodiversity patterns Rodrigues, G.F.B. 2018 55 The density of the three dominant species was higher in second period (Wilcoxon test, p-value < 0.05). Only hermit crab Petrochirus diogenes (Linnaeus, 1758) had lower abundance in second period (Mann-Whitney, p-value < 0.05). Overall, some ecological indexes (H’, Simpson, ∆) did not differ between the two surveyed periods. Only taxonomic distinctness (∆+) values were different (Mann-Whitney; p-value < 0.05), reaching higher values in the second period (Figure 2). However, we found lower number of species in the second period (10 species in the first and 7 in second periods). Table III. Hermit crabs’ density (ind/km²) sampled by trawl at the coast of Ubatuba, State of