Connectivity among populations of Clathrina aurea (Porifera, Calcarea) in South-Southeastern Brazil André Padua, Haydée Cunha & Michelle Klautau Universidade Federal do Rio de Janeiro, Instituto de Biologia e-mails: andreqpadua@gmail.com; haydeecunha@yahoo.com.br; mklautau@biologia.ufrj.br INTRODUCTION Sponges, as other marine sessile organisms with lecithotrophic larvae, usually present restricted distributions with strong population structure due to their low dispersal capabilities, phylopatry and inbreeding (Duran et al., 2004; Maldonado, 2006). Therefore, genetic connectivity across wide distributions is unexpected for sponges. Molecular markers have been used in population genetics studies to elucidate these issues. One of the most used molecular tools today are microsatellite loci because they are highly polymorphic, considered neutral markers, abundant in the genome and co-dominant (Ellegren, 2004). Microsatellite markers were developed for only 10 sponge species, including Clathrina aurea (Padua et al., 2013). The aim of this study was to analyze the genetic connectivity and morphological polymorphism among the South-Southeastern populations of Clathrina aurea Solé-Cava et al., 1991 (Figure 1) in the Brazilian coast. MATERIALS AND METHODS Around 30 specimens were collected in each locality and fixed in ethanol 93%. All specimens had the external morphology analysed under a stereomicroscope and spicules slides for measurements were made using a standard protocol (Klautau & Valentine, 2003). DNA was extracted and seven microsatellite loci already developed for this species (Padua et al., 2013) were genotyped. Population genetics parameters were estimated using the following softwares: FSTAT (observed and expected heterozygosities, number of alleles, FIS, Hardy-Weinberg Equilibrium and linkage disequilibrium tests), Microchecker (null alleles) and MLGSim (presence of clonal individuals). Isolation by distance, pairwise FST values and a three dimensional Factorial Correspondence Analysis (3D-FCA) were also performed using Genetix. A Bayesian approach to detect the most likely number of clusters was implemented with the software Structure (Pritchard et al., 2000). RESULTS We analysed the morphology of 115 individuals of C. aurea (Cabo Frio: 30; Cagarras: 25; Ilhabela: 30; Arvoredo: 30). All of them presented the typical morphological characteristics of the species. Spicule measurements (lenght and width) differed significantly between (length: F= 10.20; df= 3; p<0.0001; width: F= 15.86; df= 3; p<0.0001) and within (length: F= 8.82; df= 111; p<0.0001; width: F= 9.12; df= 111; p<0.0001) localities. Due to the great variability within and among localities, we propose broader values for C. aurea measurements (Table 1). For the microsatellites genotying, 112 individuals were used because two of them from Ilhabela consistently failed in the amplifications and one clonal individual was found in Cabo Frio. The number of alleles ranged from five to thirty one (mean of 20.4 alleles/locus; Table 2). Non- significant values were observed for the linkage disequilibrium test (α= 0.0023), two loci showed possible presence of null alleles (Cau_B2 and Cau_G3) and were excluded from forthcoming analyses. With the set of five loci, only Ilhabela and Arvoredo departed from Hardy-Weinberg Equilibrium and a pattern of isolation by distance was observed (r=0.905; p=0.047). While F ST indicated the presence of four populations, the 3D-FCA and the Bayesian inference indicated only three (Figure 3). REFERENCES Duran, S.; Pascual, M.; Estoup, A. & Turon, X. (2004) Strong population structure in the marine sponge Crambe crambe (Poecilosclerida) as revealed by microsatellite markers. Molecular Ecology, 13: 511-522. Ellegren, H. (2004). Microsatelliets: simple sequences with complex evolution. Nature Review – Genetics, 5: 435-445. Klautau, K. & Valentine, C. (2003). Revision of the genus Clathrina (Porifera, Calcarea). Zoological Journal of the Linnean Society, 139: 1–62. Klautau, M.; Russo, C.A.M.; Lazoski, C.; Boury-Esnault, N.; Thorpe, J.P. & Solé-Cava, A.M. (1999) Does cosmopolitanism result from conservative systematics? A case study using the marine sponge Chondrilla nucula. Evolution, 53(5): 1414-1422 . Maldonado, M. (2006). The ecology of the sponge larva. Canadian Journal of Zoology, 84: 175-194. Padua, A.; Cavalcanti, F.; Cunha, H. & Klautau, M. (2013). Isolation and characterization of polymorphic microsatellite loci fromClathrina aurea (Porifera, Calcarea). Marine Biodiversity, doi: 10.1007/s12526-013-0167-2. Pritchard, J. K.; Stephens, M. & Donnelly, P. (2000). Inference of population structure using multilocus genotype data. Genetics, 155: 945-959. Valderrama, D.; Rossi, A.L.; Solé-Cava, A.M.; Rapp, H.T. & Klautau, M. (2009) Revalidation of Leucetta floridana (Haeckel, 1872) (Porifera, Calcarea): a widespread species in the tropical western Atlantic. Zoological Journal of the Linnean Society: 157(1): 1-16. Wörheide, G.; Epp, L.S, & Macis, L. (2008) Deep genetic divergences among Indo-Pacific populations of the coral reef sponge Leucetta chagosensis (Leucettidae): Founder effects, vicariance or both? BMC Evolutionary Biology, 8: 24. DISCUSSION The spicule size in C. aurea was a very variable character within and among populations. This character is the most used for sponge taxonomy and, here, we propose broader values for C. aurea (Table 1). This wider values may be very important to recognize population, since C. aurea is widely distributed in Brazil and was recently found in Peru. This intraspecific variability of the spicule size in sponges, was already observed in Chondrilla nucula and Leucetta floridana in Brazilian waters and may be the result of plasticity related to environmental factors (Klautau et al., 1999; Valderrama et al., 2009) and not a complete separation of populations or new species. Clathrina aurea presented high average number of alleles and observed heterozygosities when compared to other sponges and even to other marine invertebrates. The results of our analysis indicated the presence of three clusters in the South-Southeastern Brazilian coast. This suggests that C. aurea can disperse its propagules between Cagarras and Ilhabela (235km apart), which may be possible by the presence of intermediate populations (Angra dos Reis, for example) in a stepping stone model, already suggested for sponge populations (Wörheide et al., 2008). The connectivity among the other clusters may be hampered by the distance and lack of substrate between sites (e.g. Ilhabela and Arvoredo) (Figure 3A), although eventual exchange of migrants may occur (Figure 3C). Figure 3: (A) Result of the Mantel test, showing a isolation by distance pattern. Results of the 3D-FCA (B) and Bayesian inference (C) analyses showing the presence of three populations in the Southeastern and South regions in Brazil. Arrows represent the individuals off one cluster but with high genotype contribution from others (>50%). STUDY AREA Four sites along the Southern and Southeastern Brazilian coast were analysed (Figure 2). One in the South Region (Arvoredo - Santa Catarina State) and three in the Southeastern Region (Ilhabela - São Paulo State; Cagarras and Cabo Frio - Rio de Janeiro State). Clathrina aurea Solé-Cava et al., 1991 The species is a calcareous sponge endemic of the Brazilian coast, although recently found in Peru. It is bright yellow and presents loosely and irregularly anastomosed tubes and only triactines. Despite the wide distribution (Figure 2), the reproduction of the species is still unknown. Figure 2: (A) Localities where C. aurea was already reported (yellow dots); (B) Detail showing the sites analysed in the present study. Figure 1: Clathrina aurea Solé-Cava et al., 1991. ACKNOWLEDGEMENTS Thanks to: Emilio Lanna, Fernanda Azevedo, Fernanda Cavalcanti, João Carraro and Paulo Paiva Table 2: Number of individuals (N), number of alleles (Na) and Number of excluvise alleles (Nex) considering the set of seven loci. Observed (HO) and Expected (HE) heterozygosities, FIS values and P values of Hardy-Weinberg Equilibrium (HWE) tests for multilocus analyses in each population considering the set of five loci. Significant values (after sequential Bonferroni correction) are in bold. Table 1: Mininum, mean and maximum values, standard deviation (S.D) and number of individuals measured (n) in each locality studied in the present work compared to the holotype of C. aurea. Values are presented as legth/width in μm. . F100 mons License. F1000 Posters: Us Creative Commons License. F1000 Posters: Use Permitted u itted under Creative Commons License. F1000 Posters: Use Permitted unde reative Commons License. F1000 Posters: Use Pe ons License. F1000 Po