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Genetic And Morphometric Rediscovery Of An Extinct Land Snail On Oceanic Islands

T. Hirano, S. Wada, H. Mori, S. Uchida, T. Saito, Satoshi Chiba
Published 2018 · Biology

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Confirming the extinction of species with only old occurrence records is often difficult, due to the scarcity of biological information. Phylogenetic position and taxonomic status of the potentially extinct species are unclear in most such cases. In the present study, we document that a species of the endemic genus Hirasea of the oceanic Ogasawara Islands, which was formerly believed to have become extinct, still survives, on the basis of combined surveys involving molecular phylogenetics and morphometrics. Hirasea is notorious for its highly diversified shell morphologies and a number of species and subspecies have already become extinct because of environmental changes and impacts of alien predators. In this study, we first reconstruct phylogenetic relationships among extant Hirasea populations. Next, on the basis of quantitative shell morphological analysis of extant populations and type specimens, we identify morphological traits that reflect phylogenetic relationships among the species. By using these morphological traits and molecular phylogenies, we demonstrate that H. nesiotica liobasis, previously considered extinct, is still extant on Chichijima. Although classifications by means of morphology and molecular phylogeny often show incongruences, judgement of the conspecific status of living and extinct populations is still possible by considering ranges of variation and phylogenetic constraints on morphological traits. INTRODUCTION Identification of the conservation status of species is a crucial issue in conservation biology. Species without occurrence records for long periods of time are considered extinct, yet it is often difficult to determine whether a focal species has actually become extinct or not (Fisher & Blomberg, 2011). In recent years molecular genetic technology has played a major role in describing biological diversity (Forest et al., 2015) and is useful for rediscovering species formerly believed to be extinct (Lee et al., 2007; Poulakakis et al., 2008; Kawakami et al., 2012). However, classification of species based on molecular phylogeny often reveals incongruences with traditional taxonomy based on morphology; therefore, classification of species of which living specimens have not been collected for a long time is difficult. In this study, we address this issue and estimate relationships between extinct and extant populations by assessing how variation in morphological traits reflects molecular phylogeny. Land snails have seriously declined on oceanic islands, because of habitat loss and the impacts of introduced non-native species (Coote & Loéve, 2003; Lydeard et al., 2004; Chiba & Cowie, 2016; Cowie et al., 2017). One example is the helicarionid land-snail genus Hirasea. Hirasea is a genus endemic to the oceanic Ogasawara Islands and exhibits remarkable diversification in shell morphology, from an extremely flat lens-like shell to a conical or even spherical shell (Azuma, 1982; Ueshima & Kurozumi, 1988; Tomiyama & Kurozumi, 1992; Chiba, 2009a). Including undescribed species, 18 species and subspecies are known in Hirasea (Table 1; Chiba, 2009b). However, ten species/subspecies are believed to have already become extinct because of habitat destruction and impacts of introduced predators (Table 1; Chiba, 2009b; Chiba, Wada & Mori, 2012). Especially on Chichijima Island, where most of the species of Hirasea were recorded in the past, all Hirasea species have been believed to have become extinct (Tomiyama & Kurozumi, 1992). On Chichijima, where numbers of surveys have been conducted since the 1940s throughout the island, H. nesiotica liobasis, H. diplomphalus profundispira, H. goniobasis, H. major and H. hypolia have not been found, and these species and subspecies have therefore been listed as EX (extinct) in the Red Data Book 2014, Threatened Wildlife of Japan (Ministry of Environment, 2014). Nevertheless, in 2007, H. acutissima and H. nesiotica nesiotica, which had been believed to be extinct, were rediscovered on Hahajima Island (Chiba, Davison & Mori, 2007; Chiba et al., 2012) and living individuals of Hirasea sp. B were found on Chichijima Island (only one population on Mt Toriyama, in the easternmost part of the island) and Higashijima Island (Chiba, 2009b; Chiba & Wada, unpublished data). A recent study on © The Author(s) 2018. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved. For Permissions, please email: journals.permissions@oup.com extant populations of Hirasea from Anijima suggests that species of Hirasea display high intraspecific variation in shell size (Chiba, 2009a). This means that morphological analysis is required for species identification in Hirasea. In addition, molecular phylogenetic analysis is useful to confirm classification of the Hirasea species. The populations of Hirasea from Chichijima and Higashijima may belong to species known to exist, but their taxonomic and phylogenetic positions are unclear because there have been no molecular phylogenetic studies on Hirasea. In this study, we investigated molecular phylogenetic relationships among the extant Hirasea species in the Ogasawara Islands. By using quantitative shell morphological analysis of extant individuals and type specimens in museums, we detect shell characteristics that reflect phylogenetic relationships among the lineages. By integrative analyses of morphology and molecular phylogeny, we show how species or subspecies described from extinct populations can be identified in extant populations, thus permitting assessment of extinction or survival of particular species or subspecies. MATERIAL AND METHODS Samples We collected 50 individuals representing eight species/subspecies of Hirasea, including unidentified species, living juvenile and empty shells (Tables 1, 2) from the Ogasawara Islands (Table 2, Fig. 1). For species identification, we referred to previous studies (Azuma, 1982; Minato, 1988; Ueshima & Kurozumi, 1988; Chiba et al., 2007, 2012; Chiba, 2009a, b; Ministry of Environment, 2014). We also obtained material of seven species in six genera of other East Asian (Japanese) helicarionid snails, Bekkochlamys perfragilis, B. micrograpta, Japanochlamys cerasina, Macrochlamys sp., Parasitala reinhardti, Trochochlamys crenulata and Urazirochlamys doenitzii, which we used as outgroups. A fragment of the foot muscle from each living individual was stored in 99.5% ethanol for DNA extraction. Genetic analyses Total DNA from 33 individuals was extracted using a NucleoSpin Tissue kit (Macherey-Nagel), following the manufacturer’s standard protocol. To estimate the phylogenetic relationships among the collected snails, we sequenced fragments of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene and 16S rRNA gene as well as nuclear ribosomal internal transcribed spacer (ITS) regions 1 and 2. Polymerase chain reaction (PCR) conditions and the primers used are shown in Table 3. PCR products were purified using Exo-SAP-IT (Amersham Biosciences, Little Chalfont, UK). Sequencing was performed using the BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA) and electrophoresed using an ABI 3130xl sequencer (Applied Biosystems). The newly generated sequences have been deposited in the DDBJ/EMBL/GenBank databases (Table 2). Phylogenetic analysis The 16S, ITS1 and 2 sequences were aligned using MUSCLE v. 3.8 (Edgar, 2004) to eliminate uncertainty in the alignments in this region (Tables 3 and 4). GBLOCKS v. 0.91b (Castresana, 2000) was used to select regions in the aligned sequences that were confidently aligned for analysis. Phylogenetic trees were generated using maximum likelihood (ML), maximum parsimony (MP) and Bayesian methods. MP analyses were performed in MEGA6 (Tamura et al., 2013) using the subtree-pruning-regrafting algorithm with search level 1, in which initial trees were obtained by random addition of sequences (10 replicates). Prior to the ML and Bayesian analyses, we used Kakusan4-4.0.2011.05.28 (Tanabe, 2007, 2011) to select the appropriate models for sequence evolution (Table 5). Based on the selected models, ML analysis was performed using TreeFinder (Jobb, 2008). Nodal support for the ML and MP analyses was assessed using bootstrap (BS) analyses with 1000 replications. The Bayesian analysis was performed using Table 1. Information on Hirasea species, including current situation, type locality and study species used. Taxa Distribution area (extinct area) Type locality Study sample Reference Hirasea chichijimana Pilsbry, 1902 Anijima (Chichijima, Hahajima) Chichijima This study Chiba (2009b) H. operculina (Gould, 1859) Chichijima, Anijima, Magoshima, Nishijima (Hahajima) Chichijima This study Chiba (2009b) H. insignis Pilsbry & Hirase, 1904 (Mukojima) Mukojima Chiba (2009b) H. diplomphalus diplomphalus Pilsbry, 1902 Anijima (Chichijima, Hahajima) Chichijima This study Chiba (2009a, b) H. d. profundispira Pilsbry, 1902 (Chichijima) Chichijima Chiba (2009b) H. acutissima Pilsbry, in Hirase, 1907 Hahajima Hahajima This study Chiba et al. (2007), Chiba (2009b) H. acuta Pilsbry, 1902 Meijima, Imotojima, Hirashima (Hahajima, Anejima) Imotojima This study Chiba (2009b) H. sinuosa Pilsbry, 1902 (Hahajima) Hahajima Chiba (2009b) H. eutheca Pilsbry, in Hirase, 1907 (Hahajima) Hahajima Chiba (2009b) H. hypolia Pilsbry, in Hirase, 1907 (Chichijima, Hahajima) Hahajima Chiba (2009b) H. planulata Pilsbry & Hirase, 1903 (Hahajima) Hahajima Chiba (2009b) H. biconcava Pilsbry, in Hirase, 1907 (Hahajima) Hahajima Chiba (2009b) H. goniobasis Pilsbry, 1902 (Chichijima) Chichijima Chiba (2009b) H. nesiotica nesiotica Pilsbry, 1902 Hahajima (Chichijima) Hahajima This study Chiba et al. (2012) H. n. liobasis Pilsbry, in Hirase, 1907 (Chichijima) Chichijima Chiba (2009b) H. major Pilsbry, 1902 (Chichijima) Chichijima Chiba (2009b) H. sp. A Nakoudojima – This study Chiba (2009b) H. sp. B Chichijima, Higashijima – T
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