Elsevier

Zoology

Volume 111, Issue 2, 15 March 2008, Pages 163-178
Zoology

Cryptic divergence and strong population structure in the colonial invertebrate Pycnoclavella communis (Ascidiacea) inferred from molecular data

https://doi.org/10.1016/j.zool.2007.06.006Get rights and content

Abstract

We studied sequence variation in the mitochondrial gene cytochrome c oxidase subunit I (COI) for 135 individuals from eight Mediterranean populations of the colonial ascidian Pycnoclavella communis across most of its presently known range of distribution in the Mediterranean. Three haplotypes from Atlantic locations were also included in the study. Phylogenetic, phylogeographic and population genetic analyses were used to unravel the genetic variability within and between populations. The study revealed 32 haplotypes for COI, 29 of them grouped within two Mediterranean lineages of P. communis (mean nucleotide divergence between lineages was 8.55%). Phylogenetic and network analyses suggest the possible existence of cryptic species corresponding to these two lineages. Population genetic analyses were restricted to the five populations belonging to the main genetic lineage, and for these localities we compared the information gleaned from COI sequence data and from eight microsatellite loci. A high genetic divergence between populations was substantiated using both kinds of markers (COI, global Fst=0.343; microsatellite loci, global Fst=0.362). There were high numbers of private haplotypes (COI) and alleles (microsatellites) in the populations studied. Restricted gene flow and inbreeding occur in the present range of distribution of the species. Microsatellite loci showed a strong incidence of failed amplifications, which we attribute to the marked intraspecies variability that hampered the application of these highly specific markers. Our results show important genetic variability at all levels studied, from within populations to between basins, possibly coupled to speciation processes. This variability is attributable to restricted gene flow among populations due to short-distance dispersal of the larvae.

Introduction

Understanding the patterns of genetic variability of benthic invertebrates is crucial to assess speciation processes, connectivity between populations, susceptibility to local disturbance, and management guidelines (Shanks et al. 2003; Palumbi, 1994, Palumbi, 2004; Kinlan and Gaines 2003). Populations of marine species may have a strong genetic structure even in groups with a supposedly high dispersal potential (Palumbi et al. 1997, Palumbi 2004; Hellberg, 1994, Hellberg, 1996), resulting from discontinuities in gene flow as a consequence of physical and biological processes. Indeed, much cryptic genetic variation in marine habitats has been uncovered by the advent of molecular techniques (Knowlton 2000; Féral 2002). Different reproductive strategies in modular organisms have the potential to influence the genetic structure of the populations (Jackson 1986; Hellberg, 1994, Hellberg, 1996; Ayre et al. 1997) and such patterns of genetic variation among populations can be used to infer gene flow and the evolutionary history of the species.

Advances in the development of molecular markers using DNA have provided a wide range of tools for estimating patterns of relatedness and connectivity among benthic populations, understanding the spatial distribution of alleles and analysing the populations’ structure and demographic history (Emerson et al. 2001; Waples and Gaggiotti 2006). Mitochondrial DNA sequence data have been used in a large number of studies for inferring the evolutionary and demographic past of both populations and species (Avise et al. 1987; Avise 2000; Palumbi et al. 1997). Mitochondrial genetic variability is most appropriate for phylogeny, phylogeography and population genetic approaches in which analyses of mtDNA are used to date events in the history of species, on the assumption that divergence within species is correlated with time (Moritz et al. 1987). However, a matrilineal phylogeny inferred from mitochondrial data gives us only a fraction of the genealogical information (Avise 1998) and, for this reason, it is advisable to combine the information from mitochondrial markers with nuclear ones in order to obtain a more comprehensive picture of phylogeographical patterns (Ballard and Whitlock 2004).

Microsatellite markers, on the other hand, have been widely employed in ecological and evolutionary studies over the past two decades. Because of their neutrality, codominance and high polymorphism, they are a powerful tool for inferring processes at the population level (Jarne and Lagoda 1996; Zane et al. 2002). For example, analyses with the sponge Crambe crambe revealed homogeneity for the cytochrome c oxidase subunit I (COI) gene between distant populations but high levels of polymorphism in microsatellite loci (Duran et al., 2002, Duran et al., 2004a). However, the marked specificity of microsatellites and the frequent occurrence of null alleles often hamper the application of these molecular tools (Dakin and Avise 2004). Some studies have shown that amplification success is, in general, inversely correlated with molecular distances (Estoup et al. 1995; Jarne and Lagoda 1996). The correlation between molecular divergence and microsatellite amplification may limit the usefulness of these markers when intraspecific genetic divergence is high.

Although modular marine invertebrates with complex life cycles and asexual reproduction are good candidates for the study of genetic variation at several scales using microsatellites (Calderon et al. 2007), most studies to date have been accomplished using allozymes (e.g. McFadden 1997; Yund and O’Neil 2000; Lazoski et al. 2001; Whalan et al. 2005). In particular, there are only three species of colonial ascidians in which microsatellite markers have been isolated (Pancer et al. 1994; Stoner et al. 1997; Maclean et al. 2004; Pérez-Portela et al. 2006).

Colonial ascidians are modular invertebrates widely distributed in marine systems, whose complex life cycles include sexual and asexual reproduction events, colonial fusion and chimerism (Sommerfeldt et al. 2003; Rinkevich 2005). Most colonial species are brooders and the larvae have a restricted dispersal capability since planktonic lifespan varies from a few minutes to hours (Millar 1971; Svane and Young 1989; Davis and Butler 1989), restricting connectivity between geographically close populations (Ayre et al. 1997). Frequent asexual reproduction and short dispersal distances led to the prediction that populations maintained by highly localised dispersal and a high degree of relatedness exist at local scales (Jackson 1986; Grosberg 1987; Ayre et al. 1997; Yund and O’Neil 2000). For instance, populations of the colonial species Botryllus schlosseri are characterised by deficiencies of heterozygotes, a feature characteristic of species with a considerable level of inbreeding (Stoner et al., 1997, Stoner et al., 2002; Ben-Shlomo et al.). However, post-larval dispersal may increase substantially the dispersal capabilities of a species. For instance, rafting can have an important role in long-range dispersal of colonial ascidian species (Worcester 1994; Muniz et al. 2006). Human-mediated transport is also a common mechanism enhancing dispersal of marine invertebrates (Carlton 2003). These mechanisms, however, are especially likely to affect species settling on drift material such as sea grass blades or seaweeds, or on artificial substrata (ship hulls, platforms).

Our study is focused on the recently described colonial ascidian Pycnoclavella communis (Pérez-Portela et al. 2007), which is common but patchily distributed on open rocky littoral habitats along the eastern Atlantic (Canary Islands and Madeira) and the western Mediterranean (Pérez-Portela et al. 2007). This is one of the few ascidian species for which microsatellite markers have been identified (Pérez-Portela et al. 2006). Although several colour morphs were described in the Mediterranean, a phylogenetic study of the genus (Pérez-Portela et al. 2007) showed that this species featured a high degree of genetic variation which was not correlated with differences in colour. The goal of the present study is to analyse the genetic structure of this variable species in the western Mediterranean, using phylogenetic inference, network estimation and population genetics, in order to explore geographical patterns and evolutionary events that can explain the distribution of genetic diversity of this species. To this end, we applied two different sets of molecular markers, mtDNA and microsatellites, to explore the degree of resolution of these different markers at the intraspecific level. As the species lives attached to rocky substrates and is not present in marinas or on other artificial structures, we do not expect post-larval processes to have a noticeable influence on its dispersal range. We hypothesise, therefore, that genetic variation will be found at different scales, and that genetic differentiation can build up even between geographically close populations.

Section snippets

Sampling and DNA extraction

Individuals morphologically attributable to the yellow morph of P. communis were collected from eight western Mediterranean localities within two areas of the Spanish littoral covering most of the presently known geographical range of the species in the Mediterranean: Palamós 41°5′N, 2°08′E (P); Blanes 41°40′N, 2°48.2′E (BL) and Tossa 41°43.2′N, 2°56.4′E (T) (northern Spanish littoral); Punta de la Mona 36°43.4′N, 3°08′W (PM); Cerro Gordo 36°43.15′N, 2°08′W (CG); Cabo de Palos 37°37.9′N,

Phylogenetic analyses of COI

The final length of the COI sequences after alignment and trimming was 546 bp. A total of 32 different haplotypes were obtained from 138 specimens, collected along the western Mediterranean and eastern Atlantic coasts (see Supplement I Appendix A), indicating a high degree of polymorphism. Overall, there were 92 polymorphic sites (16.8%), and nine substitutions resulted in non-synonymous changes. Nucleotide variation was scattered across the entire sequenced region and was mainly restricted to

Discussion

The phylogenetic tree and the haplotype network revealed significant genetic differences between the studied populations of P. communis, with the two main lineages found being separated by 8.55% sequence divergence in the COI gene. Molecular data have shown a prevalence of cryptic and sibling species in marine environments that had gone undetected in previous morphological studies (Palumbi 1994; Knowlton 2000; Féral 2002), but the criteria used to distinguish species based on molecular data are

Acknowledgments

We thank Dr. Cruz Palacín for her help with underwater work and Dr. Marta Pascual for her help with the microsatellite technical problems. The research was funded by the projects CTM2004-05265 and CTM2007-66635 of the Spanish Government and the ECIMAR project of the “Agence Nationale de la Recherche” (France).

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