Discrete Phenotypes Are Not Underpinned by Genome-Wide Genetic Differentiation in the Squat Lobster Munida Gregaria
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Wang et al. BMC Evolutionary Biology (2016) 16:258 DOI 10.1186/s12862-016-0836-4 RESEARCHARTICLE Open Access Discrete phenotypes are not underpinned by genome-wide genetic differentiation in the squat lobster Munida gregaria (Crustacea: Decapoda: Munididae): a multi- marker study covering the Patagonian shelf Chen Wang1*, Shobhit Agrawal1, Jürgen Laudien1, Vreni Häussermann2,3 and Christoph Held1 Abstract Background: DNA barcoding has demonstrated that many discrete phenotypes are in fact genetically distinct (pseudo)cryptic species. Genetically identical, isogenic individuals, however, can also express similarly different phenotypes in response to a trigger condition, e.g. in the environment. This alternative explanation to cryptic speciation often remains untested because it requires considerable effort to reject the hypothesis that the observed underlying genetic homogeneity of the different phenotypes may be trivially caused by too slowly evolving molecular markers. The widespread squat lobster Munida gregaria comprises two discrete ecotypes, gregaria s. str. and subrugosa,which were long regarded as different species due to marked differences in morphological, ecological and behavioral traits. We studied the morphometry and genetics of M. gregaria s. l. and tested (1) whether the phenotypic differences remain stable after continental-scale sampling and inclusion of different life stages, (2) and whether each phenotype is underpinned by a specific genotype. Results: A total number of 219 gregaria s. str. and subrugosa individuals from 25 stations encompassing almost entire range in South America were included in morphological and genetic analyses using nine unlinked hypervariable microsatellites and new COI sequences. Results from the PCA and using discriminant functions demonstrated that the morphology of the two forms remains discrete. The mitochondrial data showed a shallow, star-like haplotype network and complete overlap of genetic distances within and among ecotypes. Coalescent-based species delimitation methods, PTP and GMYC, coherently suggested that haplotypes of both ecotypes forms a single species. Although all microsatellite markers possess sufficient genetic variation, AMOVA, PCoA and Bayesian clustering approaches revealed no genetic clusters corresponding to ecotypes or geographic units across the entire South-American distribution. No evidence of isolation-by-distance could be detected for this species in South America. Conclusions: Despite their pronounced bimodal morphologies and different lifestyles, the gregaria s. str. and subrugosa ecotypes form a single, dimorphic species M. gregaria s. l.. Based on adequate geographic coverage and multiple independent polymorphic loci, there is no indication that each phenotype may have a unique genetic basis, leaving phenotypic plasticity or localized genomic islands of speciation as possible explanations. Keywords: Phenotypic plasticity, Genetic homogeneity, Squat lobster, Microsatellites, Gene flow * Correspondence: [email protected] 1Alfred Wegener Institute, Helmholtz Center for Polar- and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany Full list of author information is available at the end of the article © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wang et al. BMC Evolutionary Biology (2016) 16:258 Page 2 of 16 Background South America, M. gregaria s. l. occurs in shallow marine Different species have different morphologies and life- waters off Patagonia, including Tierra del Fuego and the styles, which is commonly taken (but not often tested) Falkland Islands/Islas Malvinas, while in the southwestern to reflect different underlying genotypes. The advent of Pacific M. gregaria s. l. are reported from off eastern New affordable DNA sequencing and molecular barcoding Zealand and Tasmania ([22, 23] and references therein). have served to greatly intensify the crosstalk between The taxonomic status of gregaria s. str. and subrugosa molecular and taxonomic disciplines by uncovering a ecotypes has been subject to conflicting interpretations. large number of previously overlooked genotypes [1–3], Both ecotypes were often regarded as different species many of which could be shown to be associated with because of morphological differences in adult specimen equally overlooked morphotypes that in retrospect were (Fig. 1) as well as different behaviors at certain develop- identified as (pseudo)cryptic species [4–6]. mental stages [24–27]. Williams (1973) on the other hand However, the popularity and large number of cryptic interpreted gregaria s. str. as a transient, pelagic ontogen- species currently being discovered have led to an etic stage that would later in life gain the physical features under-appreciation of the notion that sharply distinct of subrugosa upon adopting a permanently benthic life- morphotypes are not always the consequence of genetic style. Regardless of the taxonomic ramifications, gregaria differences but can also be invoked from the same s. str. is often found in huge pelagic swarms that subru- genotype, often called by environmental triggers. The gosa lacks [23, 28]. These differences persist even where differences between associated morphotypes and life- both ecotypes co-exist in the same habitat. Nevertheless, styles of ecotypes within the same species can be sur- on the basis of a lack of mitochondrial DNA differenti- prisingly pronounced [7–11]. ation [29], these two ecotypes are currently treated as a Proving polyphenism and rejecting cryptic speciation single polymorphic species under the name of M. gregaria as an explanation is harder than sequencing a mito- in the most recent taxonomic revision of the family [22]. chondrial gene fragment, which may in part explain the But this evidence must be considered insufficient because relatively lower number of well-studied cases of poly- the sampled region (Beagle Channel) represents a very phenism. Whilst consistent differences among different small part of the species’ distribution and the molecular morphotypes in a single mitochondrial marker suffice evidence rest exclusively on two linked mitochondrial to at least flag these clades as candidate cryptic species, markers (COI and ND1), whereas the results of the only the opposite observation (no consistent differences) is nuclear marker (ITS-1) had to be excluded from the final not a conclusive demonstration of the absence of gen- analysis of the only molecular study [29]. etic differentiation among ecotypes. In order to show In order to test whether gregaria s. str. and subrugosa that too slowly evolving markers or other artefacts (e.g. ecotypes correspond to different species of Munida or mito-nuclear discordance [12]) did not trivially cause represent a single species with variable phenotypes, we the observed lack of differentiation, considerably more employed multiple independent, fast-evolving nuclear extensive molecular evidence including multiple un- microsatellite markers [30] and an expanded set of linked nuclear loci with sufficiently high substitution mtDNA sequences. The sampled area encompasses nearly rates is required. Such extensive a posteriori knowledge is the entire distribution of gregaria s. str. and subrugosa rare (e.g. in the fully sequenced Daphnia pulex [13–15]), ecotypes in South America. In addition, we analyzed mor- but numerous experimental studies in which the genetic phological differences of both ecotypes and different onto- identity of individuals is known a priori contribute greatly genetic stages following the method of [27] in order to to our understanding of the importance of polyphenism test if the more complete geographic sampling continues and morphological plasticity, e.g. parthenogenetic aphids to support the discrete morphological clusters or if the [16, 17], marbled crayfish [18], polyembryonic armadillos boundaries between the two ecotypes vanish under more [19], inbred lines of Drosophila [20], cloned swine [21]. It complete geographic coverage. is unclear if the small number of confirmed polyphenism resulting from similar or identical genetic backgrounds is Methods a condition truly rare in nature or whether it reflects Study population mostly a discovery and/or publication bias. A total number of 219 individuals were used for both In this paper we investigate the dimorphic squat lobster, morphological and molecular analyses in this study. These Munida gregaria sensu lato (Fabricius, 1793), which is samples were collected at 25 stations from Patagonia and currently considered to comprise the ecotypes M. gregaria off the Falkland Islands/Islas Malvinas ranging from 38° S sensu stricto Miers 1881 as well as its junior synonym M. to 55° S from the shallow subtidal area to 179 m water subrugosa Dana, 1852 (see [22]). For clarity and brevity, depth by mid-water or bottom trawls (Table 1, Fig. 2). hereafter we refer to them as Munida gregaria s.l. com- Sexual maturity was identified as presence of eggs and/or prising the