SPECIES DISTINCTNESS OF LITTHABITELLA BOETERS , 1970 ( CAENOGASTROPODA : TRUNCATELLOIDEA ) FROM THE IONIAN ISLANDS

The shell, protoconch, operculum and radula of Litthabitella chilodia (Westerlund, 1886) from two localities in Croatia and two in Montenegro are presented, as well as the shell of “L. chilodia ionica (Schütt, 1980)” from Levkada island. Principal Component Analysis (PCA) of the shell showed distinct differences between the Levkada population and the other four localities, but also – although with some overlap – between the populations from Croatia and Montenegro. 18SrRNA nuclear gene sequences confirmed that Litthabitella did not belong to the Hydrobiidae. Histone H3 nuclear gene sequences confirmed distinctness of the Levkada population, and thus, combined with the shell morphometrics, it confirmed the specieslevel distinctness of Litthabitella from the Ionian islands. key words: shell morphology, morphometrics, histone H3, 18SrRNA, DNA, taxonomy, phylogeny, transadriatic and transionian distribution

The aim of the present paper was to examine the anatomy of L. chilodia, to check the taxonomic status of Litthabitella from Levkada using molecular markers, and to unravel the phylogenetic relationships of Litthabitella.

MATERIAL AND METHODS
Specimens of Litthabitella came from five localities (Fig. 1) in Croatia, Montenegro and Greece (Table 1).The snails were collected from the sediment using a metal sieve.
Individuals for molecular analyses were washed in 80% ethanol and left to stand in it for ca. 12 hours.Afterwards, the ethanol was changed twice during 24 hours and, after a few days, 80% ethanol was replaced with 96% ethanol.The samples were then stored at -20°C prior to DNA extraction.
The snails were dissected using a NIKON SMZ18 stereo-microscope with dark field and phase contrast; their shells and penes were photographed with CANON EOS 50D digital camera.The protoconchs and radulae were examined using a JEOL JSM-5410 scanning electron microscope, applying the techniques described by FAlniowski (1990).
A NIKON DS-5 digital camera measurement system was used to measure seven shell parameters (szArowskA 2006, FAlniowski et al. 2012a).The linear measurements were then logarithmically transformed.For angular measurements, the arcsine transformation was applied.Euclidean distances were calculated and the minimum spanning tree was computed (MST) using NTSYSpc (roHlF 1998).The same software was used for Principal Component Analysis (PCA), based on the correlation matrix (FAlniowski 2003).The original observations were projected into PC space, with a superimposed MST (performed for clarity, not presented in the figures) to detect local distortions in the data.Such usage of the PCA in a descriptive approach makes it possible to detect morphologically distinct groups without any a priori classification.
DNA was extracted from foot tissue of each snail.The tissue was hydrated in TE buffer (3 × 10 min.).Total genomic DNA was extracted with the SHERLOCK extracting kit (A&A Biotechnology), and the final product was dissolved in 20 μl TE buffer.
The PCR reaction was performed with the following primers: The PCR conditions were as follows: for H3 -initial denaturation step of 2 min at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 50°C, 1 min at 72°C, and after all cycles were completed, an additional elongation step of 4 min at 72°C; for 18SrRNA -initial denaturation step of 4 min at 94°C, followed by 40 cycles of 45 s at 94°C, 45 s at 51°C, 2 min at 72°C and, after all cycles were completed, an additional elongation step of 4 min at 72°C.To check the quality of the PCR products 10 μl of the product was run on a 1% agarose gel.Sequencing methods are described in szArowskA et al. ( 2014).The PCR products were purified using Clean-Up columns (A&A Biotechnology) and the purified products were amplified in both directions using BigDye Terminator v3.1 (Applied Biosystems), following the manufacturer's protocol and with the primers described above.The sequencing reaction products were purified using ExTerminator Columns   The H3 sequence of Ecrobia maritima (Milashewitsch, 1916) was used as outgroup.
Maximum likelihood (ML) approach was conducted in RAxML v8.0.24 (stAmAtAkis 2014).One thousand searches were initiated with starting trees obtained through randomised stepwise addition maximum parsimony method.The tree with the highest likelihood score was considered to be the best representation of the phylogeny.Bootstrap support was calculated with 1,000 replicates and summarised on the best ML tree.RAxML analyses were performed using free computational resources of the CIPRES Science Gateway (miller et al. 2010).The Bayesian analyses were run with MrBayes ver.3.2.3(ronquist et al. 2012) with default priors.Two simultaneous analyses were performed, each lasting 40,000,000 generations with one cold chain and three heated chains, starting from random trees and sampling trees every 1,000 generations.The first 25% of trees were discarded as burnin.The analyses were summarised on a 50% majority-rule tree.
The protoconch of L. chilodia from Sotonići (Figs 15-17) is formed by 1¾ whorls, growing slowly and regularly, with a broad apex and no macrosculpture .The protoconch microsculpture (Fig. 19) is in the form of delicate irregularities of the surface.The border between the proto-and teleoconch is well marked.The operculum (Fig. 20) is elongate-ellipsoidal, spiral, paucispiral, with submarginal nucleus, its growth lines  are flat but well marked.
The radula  has the central tooth formula: 4 -1 -4 2 -2 The central cusp is no more than twice longer than the adjacent cusps, all the cusps are rather blunt.There are two pairs of basal cusps .Their arrangement is noteworthy: instead of lying one by one along a line more or less parallel to the plate of the tooth, like in nearly all the hydrobioids, or to the lateral margins like in the Bithyniidae, in Litthabitella they are arranged one behind the other (Figs 24-25).The lateral tooth (Figs 23-24 and 26) fulfills the formula: 4-1-4, with the cusps similar as on the rhachis.There are about 24 long and sharp cusps on the inner marginal tooth (Figs 24 and 26), and about 8 on the outer marginal tooth.
The penis (Figs 27-28) has a broad and massive base, and terminates with a vast lobe and a narrow, stylet-like terminal part including the vas deferens; ventrally there are two big folds on the lobe (Fig. 28).The female reproductive organs (not shown) are identical to those presented by bole (1971), boeters (1974) and szArowskA (2006).A few specimens of L. chilodia from Levkada ("ionica") were used for molecular analysis, thus it was impossible to check their soft parts anatomy and morphology.
The shell measurements (Table 2) and, especially, the PCA of the shell (Fig. 14) clearly separate the Levkada specimens from the other four populations (L.chilodia chilodia).They also show -although less well marked -differences between the populations of L. chilodia chilodia from Montenegro vs. Croatia; those are visible mostly in the proportions, but also in the shell size.
In total, we obtained 13 sequences of H3 (283 bp; GenBank Accession numbers KY215955-KY215967) and six sequences of 18SrRNA (401 bp; GenBank Accession numbers KY215947-KY215952).The saturation tests of XiA et al. ( 2003) for H3 revealed no saturation.In all analyses, the topologies of the resulting phylograms were identical in both the maximum likelihood and Bayesian inference.
According to the H3 analyses, Litthabitella was divided into four main clades (Fig. 29).The most distinct was Litthabitella from Levkada, with p-distances to other Litthabitella clades ranging from 0.016 to 0.021 (Table 3).Such high divergence, characteristic of the species level, confirmed that Litthabitella from Levkada was a distinct species.At the same time, p-distances between three L. chilodia chilodia clades were 0.006.The sequences from Trsteno in Croatia and Tomići from Montenegro (about 100 km apart) were identical.Small differences were obtained for the sequences from Sotonići which was closest to Tomići (separated by only 3 km).The most dis-   The geographical distance between L. chilodia ionica and the nearest localities of L. chilodia chilodia is long (about 420 km).
The PCA confirmed the distinctness of the shells of Litthabitella chilodia and Litthabitella from Levkada.Similarly, histone H3 confirmed the distinctness of the two taxa.Unfortunately, there were no COI sequences, since we failed to amplify the product for this locus, perhaps due to the rather long time from collection of the material.Anyway, the molecular data confirmed the species-level distinctness of Litthabitella from Levkada.As stated above, the name ionica must not be used for this taxon which should be described as new for the science.

Fig. 1 .
Fig. 1.Localities of the studied populations of Litthabitella, numbers as in Table 1

Table 2 .
Shell morphometrics of Litthabitella: localities as in Table1, measurements as in Fig.14