FRESHWATER MOLLUSC DIVERSITY AT THE ROOF OF THE WORLD: PHYLOGENETIC AND BIOGEOGRAPHICAL AFFINITIES OF TIBETAN PLATEAU VALVATA

The Tibetan Plateau (TP), the ‘Roof of the World’, is the highest (mean elevation of about 5,000 m a.s.l.) and largest (about 3.0 million km) plateau on earth (Royden et al., 2008). High-elevation areas such as the TP are characterized by extreme environmental conditions (e.g. prolonged frost periods), which might pose significant challenges for aquatic organisms such as freshwater molluscs. The freshwater molluscan fauna on the TP is composed of two pulmonate gastropod (Radix Montfort, 1810 [Lymnaeidae] and Gyraulus Charpentier, 1837 [Planorbidae]) and two bivalve genera (Pisidium C. Pfeiffer, 1821 and Musculium Link, 1807 [Sphaeriidae]) (Oheimb et al., 2011, 2013; Clewing et al., 2013). In addition, Weber (1910) discovered empty shells of a third freshwater gastropod genus, Valvata O. F. Muller, 1773 (Valvatidae), in a western TP lake (Lake Bangong Co). Valvata belongs to the superfamily Valvatoidea within the informal group ‘Lower Heterobranchia’, which is predominantly marine (e.g. Cornirostridae), although some species occur in freshwater (Valvatidae) (Dinapoli & Klussmann-Kolb, 2010). The Valvatidae include about 60 valid species (Hawe et al., 2013) and are small snails with a somewhat variable shell (Scholz & Glaubrecht, 2010), but a relatively uniform protoconch and radula type (Hawe et al., 2013). They are currently distributed in freshwaters throughout the northern hemisphere, i.e. Eastern and Central Europe, Eastern and Central Asia and North America (Hauswald et al., 2008). Herein, we report the first living non-pulmonate snails (Gastropoda: Valvatidae) from the high-elevation TP. Live valvatid specimens (Fig. 1) were collected from seven different sites at Lake Bangong Co (ca. 4,300 m a.s.l., western Tibet, China; Supplementary material Table S1) during fieldwork conducted in September 2012. Interestingly, despite four other intensive field surveys on the TP conducted between 2008 and 2011, no other records of living valvatids were obtained (Oheimb et al., 2011, 2013; Clewing et al., 2013). In order to clarify the biogeographical relationships of the specimens collected, we conducted preliminary phylogenetic analyses and an ancestral area reconstruction based on three different DNA fragments. For comparison, we used additional valvatid specimens from different locations in Siberia (including ancient Lake Baikal) as well as material from China (Lake Lugu, Yunnan) and Europe (Supplementary material Table S1). In total, this study included 21 valvatid species, including five undetermined Valvata sp. All specimens were preserved in 80% ethanol and vouchers are stored at the Systematics and Biodiversity collection of the University of Giessen (UGSB, Supplementary material Table S1). Genomic DNA was extracted using the CTAB protocol described by Wilke et al. (2006). The following genes were used for phylogenetic analyses: mitochondrial cytochrome c oxidase subunit I (COI), mitochondrial large ribosomal subunit (16S) and nuclear large ribosomal subunit (28S). For PCR amplification, the following standard primers were used: COI: LCO1490 and HCO2198 (Folmer et al., 1994); 16S: 16Sar-L and 16Sbr-H (Palumbi et al., 1991); and 28S: D23F and D6R (Park & O Foighil, 2000). Fragments were amplified using the protocol described by Hauswald et al. (2008). Sequences (forward and reverse) were visualized on an ABI 3730 XL sequencer (Life Technologies) using the Big Dye Terminator Kit (Life Technologies). Prior to the single-gene alignments, the first base pairs (bp) behind the 30 end of each primer (difficult to read) were removed, leaving a 618 bp-long COI fragment, a 429–435 bp-long 16S fragment and a 697–784 bp-long 28S fragment. The proteincoding COI sequences were aligned by eye. For alignment of the two rDNA fragments, we used the multiple sequence alignment program PRANK (Loytynoja & Goldman, 2005) with empirical base frequencies, the Tamura-Nei substitution model and default settings for gap penalties. Ambiguously aligned sites (with a posterior probability of belonging to a certain structure state of 0.8) were excluded from subsequent analyses. The resulting alignments had lengths of 357 and 633 bp for the 16S and 28S partitions, respectively. All three partitions (COI, 16S and 28S) were tested separately for substitutional saturation using the test of Xia et al. (2003) implemented in DAMBE v. 5.2. 73 (Xia & Lemey, 2009). The test showed only little saturation for all three partitions even under the assumption of an asymmetrical tree. Subsequent phylogenetic analyses were conducted with 45 specimens of Valvata spp. and one specimen of Megalovalvata piligera (Lindholm, 1909). Cornirostra pellucida (Laseron, 1954) (Gastropoda: Valvatoidea) was used as outgroup (Supplementary material Table S1) according to the heterobranch phylogeny of Dinapoli & Klussmann-Kolb (2010). Concatenated sequences were analysed in BEAST v. 1.7. 4 (Drummond & Rambaut, 2007) with the following parameters set as priors: speciation 1⁄4 Yule