PHYLOGENETIC POSITION OF TEFENNIA SCHÜTT ET YILDIRIM, 2003 (CAENOGASTROPODA: RISSOOIDEA)

A BSTRACT : The phylogenetic position of Tefennia tefennica Schutt et Yildirim, 2003, an endemic snail species from southwestern Turkey, was inferred with maximum likelihood analyses of DNA sequences of mitochondrial cytochrome oxidase subunit I and nuclear 18S rRNA. Tefennia belongs to the Hydrobiidae, Sadlerianinae; its sister clade comprises Grossuana Radoman, 1973, Trichonia Radoman, 1973 and Daphniola Radoman, 1973. Shell, radula and soft parts of T. tefennica are presented.


INTRODUCTION
The Hydrobiidae of Turkey are represented by the subfamilies Hydrobiinae Troschel, 1857 (2 genera); Pyrgorientalinae Radoman, 1973 (2 genera) and Sadlerianinae Radoman, 1973 (8 genera) (RADOMAN 1983, SZAROWSKA 2006, YILDIRIM et al. 2006. The monotypic genus Tefennia Schütt et Yildirim, 2003 is known from only one locality in the Burdur Province, SW. Turkey (YILDIRIM et al. 2006). In the original description it is included in the Hydrobiidae (SCHÜTT & YILDIRIM 2003), and listed among the Orientalininae by YILDIRIM et al. (2006). The type species, Tefennia tefennica Schütt et Yildirim, 2003, has minute dimensions and a peculiar anatomy of female genitalia, with a bursa copulatrix in an anterior position and only one rudimentary seminal receptacle (rs 2 ). The aim of this paper was to establish the phylogenetic position of this interesting genus using partial sequences of the mitochondrial COI and nuclear 18S rRNA genes of T. tefennica. Additionally, the shell, radula and soft-part anatomy of this species were studied. The protoconch and radula, not described earlier, were examined using SEM.

MATERIAL AND METHODS
Material: Baºpinar spring, Tefenni, Burdur, Turkey, leg. D. C. ÇAÐLAN The snails were fixed with 80% ethanol. The shells were cleaned in an ultrasonic cleaner and photographed with a CANON EOS 50D digital camera. Three adult males and three females were dissected, using a NIKON SMZ-U stereomicroscope. The female genitalia (pallial oviduct) were examined using a MOTIC light microscope. The protoconchs and radulae were examined using a JEOL JSM-5410 scan-ning electron microscope, applying the techniques described by FALNIOWSKI (1990).
DNA was extracted from foot tissue of each snail. The tissue was hydrated in TE buffer (10 mM TRIS-HCl pH 8.0, 1 mM EDTA) (3 × 10 min); then 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: LCO1490 (5'-GGTCAACAAATCATAAAGATATTGG-3') Vol. 20(4): 271-277 doi: 10.2478doi: 10. /v10125-012-0024-0 (FOLMER et al. 1994 and COR722b (5'-TAA ACTT CAGGGTGACCAAAAAATYA-3') (WILKE & DAVIS 2000) for the cytochrome oxidase subunit I (COI) mitochondrial gene; SWAM18SF1 (5'-GAATGGCTCA TTAAATCAGTCGAGGTTCCTTAGATGATCCAAAT C-3') and SWAM18SR1 (5'-ATCCTCGTTAAAGGG TTTAAAGTGTACTCATTCCAATTACGGAGC-3') for the 18S ribosomal RNA gene (PALUMBI 1996). The PCR conditions were as follows: COI -initial denaturation step of 4 min at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at 55°C, 2 min at 72°C, and a final extension of 4 min at 72°C; 18S -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 was performed. The total volume of each PCR reaction mixture was 50 µl. To check the quality of the PCR products 10 µl of the PCR product was run on 1% agarose gel. The PCR products were purified using Clean-Up columns (A&A Biotechnology) and the purified PCR 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 (A&A Biotechnology); DNA sequences then underwent electrophoresis on an ABI Prism sequencer. All the sequences were deposited in GenBank (Table 1).
The COI sequences were aligned by eye using BioEdit 5.0.0 (HALL 1999) (2000) with MACCLADE. Mutational saturation for the COI dataset was examined by plotting the numbers of transitions and transversions for all the codon positions together, and for the 3rd position separately, against the percentage sequence divergence, using DAMBE 5.2.9 (XIA 2000). We also used DAMBE 5.2.9 to perform the saturation test (XIA et al. 2003). It revealed a significant degree of saturation in the third position of the sequences. In rissooids, COI approaches saturation with about 18.6 % or 120 nucleotide differences (DAVIS et al. 1998), which seems to happen after ap-proximately 10 million years. However, to avoid a substantial loss of information in the case of closely related species, this position was not excluded from the dataset and it was used for the analysis. Initially, we performed phylogeny reconstruction for 18S and COI data separately, using the maximum likelihood (ML) technique. For each ML analysis, we used the best fit model of sequence evolution found by Modeltest v3.06 (POSA- DA & CRANDALL 1998, POSADA 2003). The best model for each dataset was chosen using the Akaike Informa-Phylogenetic position of Tefennia Schütt et Yildirim, 2003 Figs 1-11. Shells of Tefennia tefennica; bar represents 500 µm tion Criterion (AKAIKE 1974). We performed ML analyses in PAUP*4.0b10 (SWOFFORD 2002) and used a heuristic search strategy with stepwise addition of taxa, 10 random-sequence addition replicates, and tree-bisection-reconnection (TBR) branch swapping (SWOFFORD et al. 1996). Nodal support was estimated using the bootstrap (BS) approach (FELSENSTEIN 1985). Bootstrap values for ML trees were calculated using 1,000 bootstrap replicates, the "fast" heuristic search algorithm, and the same model parameters as for each ML analysis. Next, the partition homogeneity test (FARRIS et al. 1995) was performed (1,000 replicates) with PAUP*, to check whether the two genes could be analysed together. Due to its results (p>0.7253), the maximum likelihood heuristic search was then run for the combined molecular data.
In the phylogeny reconstruction, we used Gen-Bank sequences from 27 rissooid taxa (Table 1) Lateral tooth formula: 4-14. All cusps on central and lateral teeth comparatively stout and massive. Intestine course S-shaped. Stomach without caecum.
Three sequences of COI and three of 18S (Table  1)  In the COI analysis (Fig. 20) Tefennia was resolved within the Hydrobiidae Troschel, 1857, subfamily Sadlerianinae Radoman, 1977(after SZAROWSKA 2006. Its sister clade consisted of Grossuana Radoman, 1973, Trichonia Radoman, 1973, and Daphniola Radoman, 1973. The bootstrap support for this placement was 57% (Fig. 20). In an ML tree computed for all molecular data (COI and 18S) the sister group of Tefennia was the same as in Fig. 20 but its support was higher (68%: Fig. 21). On the other hand, the relationships between this clade (Tefennia, Daphniola, Grossuana and Trichonia) and the other genera of the Sadlerianinae in Fig. 21 were somewhat different from the corresponding relationships shown in Fig. 20. However, in both trees (Figs 20-21) the clade was placed within the Sadlerianinae. It has to be noted, however, that low values of supports may only suggest the pattern presented.
The molecularly-inferred phylogenetic relationships of Tefennia suggest that the loss of the distal receptacle (rs 1 ) in this genus is secondary. Within the genera included in the phylogenetic analysis in the