Sympatric cryptic species in the crinoid genus Cenolia (Echinodermata: Crinoidea: Comasteridae) delineated by sequence and microsatellite markers
Graphical abstract
Introduction
The problem of cryptic speciation in the marine realm has garnered a great deal of attention in the past two decades (Bucklin et al., 2011, Jones, 2011, Knowlton, 2000, Palumbi, 1994, Pola et al., 2012, Poore and Bruce, 2012, Van Soest et al., 2012). The advent of molecular techniques has dramatically increased the number of species suspected to be inadequately distinguished according to traditional taxonomy, to the extent that recognition of cryptic clades is now of significance to conservation and ecosystem monitoring efforts (Blanquer et al., 2008, Rocha et al., 2007, Sweijd et al., 2000, Waples et al., 2008). Marine invertebrates are especially prone to being inadequately distinguished in the taxonomic literature due to systematic problems such as limited sampling, poor preservation of specimens and inadequate understanding of reproductive behaviours (Knowlton, 2000). Species have been known to evolve varied reproductive and physiological traits without this being reflected in the external morphology, compounding the problem (Byrne et al., 2003).
The extant Crinoidea (feather stars) are free-living, filter-feeding echinoderms that may be either sessile or mobile in their adult phase. Most of what is known about their morphological evolution is derived from an extensive fossil record (Rouse et al., 2013). Whether or not these animals exhibit high levels of cryptic speciation is unknown, but recent extensive molecular reviews of higher level relationships in the class have drawn attention to the fact that morphological characters previously thought to be taxonomically significant are in fact highly plastic (Hemery et al., 2013, Rouse et al., 2013, Roux et al., 2013). As a consequence, traditionally understood relationships within the Crinoidea have been distorted by a high degree of homoplasy. These formerly-trusted characters are often labile, paedomorphic, and/or vary with the developmental stage of the organism, such that juveniles may readily be identified as a different species to the adult of the same taxon (Améziane and Roux, 2005, Kohtsuka and Nakano, 2005, Messing, 1984).
A molecular approach is key to resolving some of these systematic dilemmas. Relatively few studies have employed a molecular approach in regard to the possibility of cryptic species in this group (Helgen and Rouse, 2006, Hemery et al., 2012, Owen et al., 2009, Torrence et al., 2012, Wilson et al., 2007), and the majority of these are limited to mitochondrial markers only (with the notable exception of Hemery et al., 2012). Mitochondrial DNA is an excellent resource for phylogeographic studies and population genetics in animals due to its rapid evolution and lower effective population size, and is often the first line of investigation to be employed when screening for cryptic lineages. The widespread use of the mitochondrial gene cytochrome c oxidase subunit I (COI) in barcoding has made phylum-specific or even universal primers more readily available for such studies, rendering them even more attractive (Ward et al., 2008). Unfortunately, due to the acknowledged possibilities of mitochondrial introgression (Bastos-Silveira et al., 2012, Darling, 2011, Ray et al., 2008) and incomplete lineage sorting (Frade et al., 2010, Redondo et al., 2008, Smith et al., 2011), cryptic species should not be delineated by the presence of divergent mitochondrial lineages alone, particularly in the presence of uncertain morphological characters.
The problem is compounded in recently diverged species exhibiting a sympatric distribution, and further compounded when these species exist in a poorly-sampled region. The south coast of Australia is one such relatively poorly-sampled region relative to its length in spite of noteworthy efforts on the part of researchers (O’Hara and Poore, 2000, Phillips, 2001, Waters et al., 2004). One of the most striking features of the coastline in regards to diversification of marine taxa is the presence of an intermittent barrier to gene flow: Bass Strait (Fig. 1), located in the south-eastern corner of the continent between Tasmania and mainland Australia. In interglacial periods, the strait allows gene flow between the southern coast of the continent and the east coast, while during past glacial periods a landbridge formed a barrier to gene flow for marine species (Lambeck and Chappell, 2001). Intermittent barriers have been shown in multiple cases to result in the presence of sympatric lineages (Colgan and Schreiter, 2010, Golding et al., 2011, Waters et al., 2005) and a number of molecular and morphological investigations into species in this region have already revealed cryptic species complexes (Dawson, 2005, Hart et al., 2006, Kraft et al., 2010, Li et al., 2013, Naughton and O’Hara, 2009).
The largest and most readily observed of the unstalked crinoids on the southern coast of Australia is the comasterid crinoid Cenolia trichoptera (Müller, 1846), followed by its more cryptic congener Cenolia tasmaniae (Clark, 1918). Another member of the genus, C. benhami (Clark, 1916a), has been recorded from the south-eastern portion of the continent and it is not readily distinguished from C. trichoptera without microscopic examination (Rowe et al., 1986). It is therefore quite likely that these two species have become confused and that records of their distribution are unreliable. Furthermore, the type locality of Cenolia benhami is Preservation Inlet, New Zealand, and in the most recent revision of the genus by Rowe et al. (1986), the authors noted that they had not had the chance to examine the holotype, currently lodged in the Otago Museum, Dunedin, New Zealand. This is a matter of some concern, as features which have been shown to be taxonomically significant among comasterid genera were not well understood when this species was first described (Messing, 2001, Rouse et al., 2013). Furthermore, while Australia and New Zealand may share numerous marine species (e.g. Morgan et al., 2013, Thomas and Bell, 2013, Waters and Roy, 2003), these generally produce long-lived planktotrophic larvae. By contrast, at this stage all crinoid larvae have proven to be lecithotrophic (Améziane and Roux, 2005, Haig and Rouse, 2008, Kohtsuka and Nakano, 2005, McEdward and Miner, 2001, Nakano et al., 2003). In addition, Cenolia appears to be primarily a shallow-water genus (Rowe and Gates, 1995). Lecithotrophic larvae tend to exhibit a lower dispersal potential (at least in shallow water and intertidal environments) than planktotrophic larvae (Hemery et al., 2012, Levin, 2006), although recent work has shown that this paradigm may be overly simplistic when other environmental factors are assessed (Ayre et al., 2009, Mercier et al., 2012). This limitation on long-distance dispersal renders it less likely that the Australian species and the New Zealand species, both bearing the name C. benhami, are in fact the same species. A similar situation exists with regard to the New Zealand species C. novaezealandiae (Clark, 1931), synonymised with the Australian C. spanoschistum (Rowe et al., 1986).
In the present study we have employed a molecular and morphological examination in order to ascertain the accuracy of the current taxonomy of the genus Cenolia and whether or not appropriate morphological characters can be discerned. Due to the possibility of sympatric species along this coastline, we employed a detailed microsatellite investigation of gene flow between putative taxa, the first record of microsatellite work in the Crinoidea.
Section snippets
Taxonomy
The genus Cenolia currently contains five species in the region studied, including the Australian temperate species C. trichoptera, C. tasmaniae and C. spanoschistum (Clark, 1916b, Colgan and Schreiter, 2010, Golding et al., 2011, Waters et al., 2005), the sole subtropical representative of the genus, C. glebosa (Rowe et al., 1986), and the problematic C. benhami. One additional species, C. amezianeae (Messing, 2003), has been described from the tropical western Pacific (Messing, 2003) but no
Sequencing success
A total of 464 COI, 47 ITS-2 and 48 28S sequences were obtained for the genus Cenolia, including those derived from a single specimen of C. glebosa. The outgroups Clarkcomanthus littoralis and Comanthus wahlbergii were successfully sequenced for all three sequence markers.
Microsatellite library development
A total of 1834 candidate microsatellite marker primer sequence pairs were returned. While fifteen microsatellites were ultimately developed for Cenolia trichoptera, these did not cross-amplify for all identified lineages. A
Discussion
The targeted collection of Cenolia trichoptera resulted in the collection of three distinct lineages, while the collection of C. tasmaniae resulted in the collection of two.
Two of the five temperate Australian lineages could be assigned current species names as Cenolia trichoptera and Cenolia tasmaniae. This resulted in three currently undescribed species, one of which was formerly united with Cenolia benhami. While the New Zealand benhami holotype shows comb morphology characteristic of the
Conclusion
Cryptic, sympatric species have often been found after molecular investigation of southern Australian marine species (Dawson, 2005, Hart et al., 2006, Kraft et al., 2010, Li et al., 2013, Naughton and O’Hara, 2009, Waters et al., 2004). If the results of this study are typical, it suggests that the scale of the biodiversity problem and the challenge it presents to taxonomists is even greater than previously suspected (Costello et al., 2012) and that current estimates of species richness could
Acknowledgments
This study was funded by an ARC Linkage Grant (ARC linkage project: LP100200122 ‘Biotic connectivity within the temperate Australian marine protected area network at three levels of biodiversity — communities, populations and genes.’). We would also like to thank Sadie Mills (National Institute of Water and Atmospheric Research, New Zealand), Stephen Keable (Australian Museum, Sydney) and Emma Burns (Otago Museum, New Zealand) for the provision of specimens for morphological and, where
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Present address: Faculty of Science, Deakin University, Locked Bag 20001, Geelong, Victoria 3220, Australia.