A new, genetically divergent species of Pseudobaikalia Lindholm, 1909 (Caenogastropoda, Baicaliidae)

Abstract A new gastropod species, Pseudobaikalia michelae Sitnikoiva & Kovalenkova, sp. n., (family Baicaliidae) is described from Lake Baikal. This is the first new species from the Baicaliidae for forty years. The new species is distinguished from its sister taxa by means of comparative morphology as well as analyses of DNA sequences (mtDNA and an intron of alpha-subunit gene of ATP-synthase). It was found in the southern and central-eastern parts of the lake where it occurs sympatrically with three other baicaliid species. Characters of the female reproductive system (i.e., a long oviduct loop with 2–3 narrow tube-like evaginations) and the aperture (i.e., oval shape with a simple outer lip) place these snails in the genus Pseudobaikalia Lindholm, 1909. The new species is most similar in its shell morphology to the northern Baikal species Pseudobaikalia jentteriana (smooth elongated shape) but differs by a more oval aperture that is slightly angled to the columella. Combined mitochondrial and nuclear sequences in a Bayesian analysis showed that all specimens of Pseudobaikalia michelae sp. n. form a well-supported clade.


Introduction
Lake Baikal is the most ancient freshwater lake on Earth. It is inhabited by very diverse endemic species flocks (Sherbakov 1999). The family Baicaliidae Fisher, 1885 (Radoman 1983, Sitnikova et al. 2004 or subfamily Baicaliinae of the family Amnicolidae (Bouchet et al. 2005, Kantor et al. 2010 includes eight genera and nearly 40 species (Dybowski 1875, Lindholm 1909, Kozhov 1936, Sitnikova et al. 2004). The last new species description for this taxon dates back to 1975 (Beckman andStarobogatov 1975), more than 40 years ago. At present, nucleotide sequences for mitochondrial and nuclear genes are known for 23 species of baicaliids (Zubakov et al. 1997, Peretolchina et al. 2008, Kovalenkova et al. 2015. Molecular analyses of baicaliids have revealed limited phylogenetic structure based on mtDNA sequences (Zubakov et al. 1997). In this study the phylogeny inferred from sequences of cytochrome c oxidase subunit I (CO1), mitochondrial small subunit rDNA (mtSSU) and intron of alphasubunit gene of ATP-synthase confirms that specimens of Pseudobaikalia michelae sp. n. form a separate clade. Indeed, unique conchological and anatomical characters of these snails add support to their status as a new species.

Material and methods
The type material of Pseudobaikalia michelae sp. n. was collected by dredging during sampling expedition to Lake Baikal on 11 October 2009. Additionally, two specimens of the new species were defined among syntypes of Pseudobaikalia pulla pulla (=Leucosia angarensis var. pulla W. Dybowski, 1875) collected by Benedict Dybowski, probably near Kultuk settlement, place of his political exile, and hosted in the freshwater gastropod collection of Zoological Institute RAS (Saint Petersburg). Some snails were found at three further sites of the Lake (Fig. 1) and stored in Limnological Institute SB RAS (Irkutsk).
The representatives of other species of baicaliids used for molecular analyses were collected at different sites of the lake during expeditions in 2009-2014 by dredging or diving (for details see Table 1). The shells of the new species were compared to type specimens of baicaliids ( Fig. 2D-K) housed in the collection of the Zoological Institute RAS.
Anatomical study and molecular analysis were performed using snails initially fixed in 80% ethanol and then stored in 70% ethanol after one day. Seven snails were photographed and then shells of six individuals were destroyed for dissection. Micrographs of protoconchs and radulae were produced using a SEM while soft tissues were dissected under a light stereomicroscope. Morphological study and descriptive terminology are based on the review of morphological characters of hydrobioid gastropods (Radoman 1983, Hershler andPonder 1998). Measurements of the shells, radular teeth and inner organs were performed using the Image-Pro-Plus program for Windows XP. DNA was extracted from muscle tissue from the molluscan foot by using the modified method of Doyle (Doyle and Doyle 1990). We used universal pairs of primers for amplification and sequencing fragments of the mitochondrial genes CO1 (Folmer et al. 1994), and mtSSU (Katana et al. 2001), as well as segment of the nuclear gene of ATP synthetase alpha-subunit containing a single intron (ATPase α) (Jarman et al. 2002). Polymerase chain reaction (PCR) amplifications (35 cycles) were carried out using a BioRad T100 Thermal Cycler under the following conditions: pre-denaturation of DNA at 94 °C for 2 min, denaturation of DNA at 94 °C for 20 s, primer annealing 1 min at 48 °C for CO1, 50 °C for mtSSU rDNA and 54 °C for intron, and nucleotide chain elongation at 72 °C for 1 min (+ 5 min in the last cycle). The amplification products were sequenced at JSC Sintol (Moscow). The nucleotide noncoding sequences were aligned taking into account their putative secondary structures using Mafft v. 6 (Katoh et al. 2009).
Localities and GenBank accession numbers for sequenced species are given in Table 1.
Sites of mtSSU rDNA and ATPase α containing indels were excluded from the analysis. Concatenated 1529 bp long fragments (618 bp of mtDNA CO1, 445 bp of mtSSU rDNA, and 466 ATPase α) were used for phylogenetic inferences despite the lack of ATPase α data for the outgroup taxa. mtSSU rDNA data was also missing for Parabaikalia elata. Benedictia baicalensis (Benedictiidae: Caenogastropoda) was used as the closest available outgroup based on a previous phylogenetic analysis (Hausdorf et al. 2003), although the substitutions at the third codon position in CO1 may potentially impede the inferences. The nucleotide sequences of CO1 and mtSSU rDNA for B. baicalensis were taken from GenBank (accession numbers AF445330 and AF445339).
JModelTest v. 2.0.2 (Guindon andGascuel 2003, Darriba et al. 2012) was used to find the appropriate substitution model by means of the Bayesian Information Criterion. The phylogenetic tree was inferred by the Bayesian total evidence method as implemented in MrBayes 3.2.1 (Ronquist et al. 2012). Four replicate analyses of four simultaneous chains (1 cold) were run for 6,000,000 generations, sampling trees every 100 generations; the first 30% of trees were discarded. Trees sampled from the stationary phase of each replicate analysis were pooled to construct a single 50% majority rule consensus tree with Bayesian posterior probabilities. Uncorrected genetic distances (pdistances) were computed in the package APE (Paradis et al. 2004) using R (R Core Team 2015). Diagnosis. Shell elongated, height up to 10 mm at 5-6 well rounded or shouldered whorls, smooth or with transverse fine ribs, oval aperture with simple evenly rounded outer lip, without umbilicus, protoconch discoidal, lateral radular teeth with square face, its width equal to length of outer wing; length of capsule gland equal to albumen gland, loop of oviduct long, reaching the proximal end of albumen gland, oviduct loop a cluster that includes from 2-7 'tube-like evaginations', sometimes beyond the albumen gland.

Pseudobaikalia michelae
Type material. Holotype (dry) and 2 paratypes (dry and in alcohol) from the type locality were deposited in freshwater gastropod collection of Zoological Institute  RAS (Saint Petersburg, Russia) and are registered under Nos. 1/522-2015 (holotype) and 2/522-2015 (paratypes). Shells of six paratypes were destroyed for dissections (to study anatomy and nucleotide sequences) with images of them given here (Fig. 2B).
Body pigmented black, mantle edge light grey, ctenidium nearly 2 mm in length with 62-64 leaflets, osphradium broadly ovate, slightly narrowed in proximal end, ~ 0.5 mm length, opposite anterior part of ctenidium, a little deeper than mantle fold.
Reproductive system. Male with small bean prostate gland (Figs 5D, 3D), anterior and posterior vas deferens close together in middle part of prostate, penis light grey, muscular, elongated, with thin glandulous often non-visible fold, gradually tapering with short and small papilla. Female coiled oviduct with one spiral, loop grey pigmented with two 'tube-like evaginations' (Fig. 4A-C).
Remarks. The genus Pseudobaikalia includes seven mainly shallow water species (Fig. 2D-K); some of them were found down to 100 or 200 m depth. Two species P. jentteriana and P. cancellata were found only in the northern part of the lake, P. elegantula inhabits the northern and central parts of the lake, and three other species P. zachwatkini, P. pulla, and P. contabulata are found in a range of locations throughout of Baikal, and are sympatric to P. michelae sp. n.
The shells of P. jentteriana (Fig. 2E) and P. pulla pulla (Fig. 2F) are smooth as in the new species, while the other Pseudobaikalia have transverse ribs or lirae. The shell of P. pulla tenuicosta (from the northern part of Lake Baikal) has slightly raised ribs. Both subspecies of P. pulla have an operculum equal to aperture size (Fig. 2F-G), but the operculum of other species including P. michelae sp. n. is smaller than aperture. The new species is most similar to P. jentteriana in its size and smooth shell but differs in colour (P. jentteriana has light brown shell and body) and in the morphology of the female gonoduct (in P. jentteriana the oviduct loop is wider and includes 3-4 'tubelike evaginations'); the penis of P. jentteriana has not been investigated yet (Sitnikova 1991). The new species is similar to young Parabaikalia elata (Dybowski, 1975) in the shape of shell and aperture (Fig. 2I), but differs in adult shell size and the length of oviduct loop. P. elata is partially sympatric to a new species, they co-occur at sites 3 and 4 (Fig. 1), and thus it was included into molecular analyses (Fig. 6, Table 3). Molecular phylogeny. Six specimens of P. michelae sp. n. and thirteen specimens of sister species were analyzed using nucleotide sequences from the mitochondrial genes CO1, mtSSU rDNA and nuclear ATPase α intron segment.
JModelTest selected GTR + I + gamma for each CO1 codon positions separately HKY + I for mtSSU rDNA and HKY nucleotide substitution model for intron dataset as the best fit using the Bayesian Information Criterion.
Sequences of P. michelae sp. n. cluster in the well-supported clade (posterior probability = 1.00) (Fig. 6). The average mutation distance from the new species to the other Pseudobaikalia species was found to be appropriate for species-level distinction: 6.4 ± 1.3%, 2.1 ± 0.2% and 2.1 ± 0.6% for the mitochondrial CO1 and mtSSU rDNA and the nuclear marker, respectively. Interspecies genetic distances are known for other taxa within this family, which can be non-monophyletic.
The lowest mitochondrial genetic distance between P. contabulata and P. jentteriana are comparable to the distances between representatives of subspecies of P. pulla. They both are about 2% for CO1 and 0.03% for mtSSU rDNA. As for the CO1 data, P. mi- chelae sp. n. appears to be the sister group to P. contabulata and P. jentteriana with 4.8% of base substitutions. The minimum genetic distances in case of the intron of P. michelae sp. n. is 0.7% to the two species: P. jentteriana and P. zachwatkini (Table 3). Moreover, there is an important higher-level character difference in the new species sequence profile as all intron sequences of P. michelae sp. n. had a relatively large (57 BP) deletion.

Discussion
The new species is well-differentiated from its sister species in the multigene phylogeny produced here. This is very unusual for Baicaliidae where species with a highly distinctive morphology may be distinguished from each other by very small genetic distances between them. On the other hand, the range of intra-specific polymorphism at least in case of Baicalia carinata exceeds the inter-specific distances between several species within this group of gastropods (Zubakov et al. 1997, Peretolchina et al. 2008. Within Pseudobaikalia the interspecific mitochondrial distances are very small (about 1%) in case of P. jentteriana and P. contabulata. However, these two species are more distant from each other if estimated from the nuclear marker. Most likely it may be explained by secondary and possibly asymmetric hybridization events in the process of their speciation. Similar mechanisms might be responsible for the limited phylogenetic structure based on sequence data for Baicaliidae family as a whole. Thus the genetic separation of P. michelae sp. n. from other Pseudobaikalia species appears to be an exception.