HORATIA BOURGUIGNAT, 1887: IS THIS GENUS REALLY PHYLOGENETICALLY VERY CLOSE TO RADOMANIOLA SZAROWSKA, 2006 (CAENOGASTROPODA: TRUNCATELLOIDEA)?

a : Horatia Bourguignat, 1887 was the first genus established for hydrobiid snails with valvatoid shell, and numerous valvatoid-shelled hydrobioids were classified as Horatia . The genus was the type one for some tribe/family-rank taxa. Thus it is one of the “crucial” hydrobiid genera. Horatia seems to inhabit only Croatia and Macedonia, and its type species: H. klecakiana Bourguignat, 1887, inhabits the springs in the Cetina River Valley. In the present paper the shell, operculum, soft part pigmentation, protoconch SEM microsculpture, female reproductive organs, and penis of H. klecakiana from the spring Studenci, N of Kučiće, in the valley of the Cetina River, Croatia, are described, to confirm the identity of the studied specimens with this species. Mitochondrial cytochrome oxidase subunit I (COI) and nuclear 18S ribosomal RNA gene sequences are used to infer phylogenetic relationships of Horatia , especially with Radomaniola and the sequence of Horatia from GenBank. The results suggest close relationships of the genus with Sadleriana , not with Radomaniola .

In fact, there is a number of valvatiform hydrobiid snails, often minute in size, and with hardly known anatomy, many of them assigned to Horatia.Horatia sturmi (Rosenhauer, 1856) from Spain (= Boetersiella sturmi: arconada & raMoS 2001) could be an example.Another "Horatia" described from Greece, H. hadei (gittenberger 1982) belongs to the genus Daphniola Radoman, 1973(FalniowSki & SzarowSka 2011a).Anyway, the identity and the phylogenetic position of the "real" Horatia remain enig matic.2001), introducing this sequence, and thus co--authors of this possible error, we felt obliged to check the morphology of Horatia klecakiana from the Cetina Valley, to confirm that it belongs to this species, then to check if its cytochrome oxidase subunit I (COI) sequence might represent the same species as the sequence of Horatia in GenBank, and, finally, to infer its phylogenetic relationships using molecular data.

MATERIAL AND METHODS
About twenty specimens of Horatia klecakiana were collected, using a sieve (0.5 mm mesh diameter), from the spring Studenci, N of Kučiće, in the valley of the Cetina River, Croatia, 43°26'41.3"N,16°48'25.5"E,45 m a. s. l., on the 21st of June 2011.
Snails were washed twice in 80% ethanol and left to stand in it for around 12 hours.Then the ethanol was changed twice more within 24 hours and finally, after a few days, the 80% solution was replaced with a 96% one, in which the samples were stored at -20°C.The shells were photographed with a CANON EOS 50D digital camera.Five adult males and five females were dissected, using a NIKON SMZ-U stereoscope microscope.The penis and female genitalia (pallial oviduct) were examined using a MOTIC light microscope.The protoconch (after ultrasonic cleaning) was examined using a JEOL JSM-5410 scanning electron microscope, applying the techniques described by FalniowSki (1990).
DNA was extracted from foot tissue of seven specimens not presented in the photographs.The tissue was hydrated in TE buffer (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'-GGTCAACAAATCATAAAGA-TATTGG-3') (FolMer et al. 1994) and COR722b (5'-TAAACTTCAGGGTGACCAAAAAATYA-3') (wilke & daviS 2000) for the cytochrome oxidase subunit I (COI) mitochondrial gene and SWAM18SF1 (5'-GAATGGCTCATTAAATCAGTCGAGGTTCCT-TAGATGATCCAAATC-3'), and SWAM18SR1 (5'-ATCCTCGTTAAAGGGTTTAAAGTGTACT-CATTCCAATTACGG AGC-3') for the 18S rRNA 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 were then amplified in both directions (HilliS et al. 1996) 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.The sequences were deposited in GenBank (Table 1).
Five sequences of COI of Radomaniola from the GenBank (wilke et al. 2001(wilke et al. , FalniowSki et al. 2012) ) were used, together with the one of Horatia from GenBank, and of our Horatia were used to calculate pairwise p-distances with MEGA5.10 (taMura et al. 2011).MEGA was also used to infer maximum likelihood (ML) tree, with the same methodology as described below.
In the phylogeny reconstruction, we used sequences from 29 rissooid taxa from GenBank (Table 1).Seven of them, used as an outgroup, represented the main non-hydrobiid lineages within the Rissooidea The COI sequences were aligned by eye using BioEdit 5.0.0 (Hall 1999) and edited with MACCLADE 4.05 (MaddiSon & MaddiSon 2002).For 18S, an initial alignment was performed using CLUSTALX 1.82 (tHoMPSon et al. 1997) and edited 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 approximately 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.In fact, the analysis conducted on 2nd and 3rd position only resulted in similar deep phylogeny, but with several polytomies within more terminal nodes.
Initially, we performed phylogeny reconstruction for 18S and COI data separately, using the maximum likelihood (ML) technique.Next, the partition homogeneity test (FarriS et al. 1995) was performed (1,000 replicates) with PAUP*4.0b10(SwoFFord 2002), to check whether the two genes could be analysed together.Since p>0.783, the maximum likelihood heuristic search was then run for the combined molecular data.Following the recommendations of PoSada & buckley (2004) and Sober (2002), the best model for each dataset was chosen using the Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC); both chose the same model.We performed ML analyses in PAUP* and used a heuristic search strategy with stepwise addition of taxa, 10 random-sequence addition replicates, and tree-bisection-reconnection (TBR) branch swap-ping (SwoFFord et al. 1996), andwith MEGA5.10 (taMura et al. 2011).Nodal support was estimated using the bootstrap (BS) approach (FelSenStein 1985).Bootstrap values for ML trees were calculated using 10,000 bootstrap replicates, with MEGA5.10 and the same model parameters as for ML analysis.
Mantle intensively pigmented black (Figs 1, 4), or its pigmentation is less intensive (Figs 3,5,6), there is nearly no pigment on the head, eyes are present.
Seven sequences of cytochrome oxidase subunit I (COI) were identical -there was no intrapopulation variability.There was also no variability between 18S sequences.
Pairwise p-distances between COI sequences of our Horatia, five species of Radomaniola, and the sequence of Horatia from GenBank (Table 2) were characteristic of the species level between all the species of Radomaniola and between all of them and Horatia from GenBank, but more than twice higher between all six species and our Horatia.In the inferred ML tree (with the model of Tamura 3-parameter +Γ) Horatia from the GenBank clustered within the Radomaniola group (Fig. 12), and the bootstrap support for the clade including all the species but our Horatia was as high as 81%.
Both the female reproductive organs and the penis resemble the ones drawn and described by boeterS (1974( ), radoMan (1983( ), bole (1993( ) and bodon et al. (2001)).radoMan (1983) presented a wide range of variability of the penis in H. klecakiana.All the morphological data confirm the assignment of our specimens to the type species of the genus Horatia.This, coupled with as many as seven specimens sequenced, all of them identical, strengthens our results.
Molecular data confirm that H. klecakiana belongs to the Hydrobiidae, not Cochliopidae as suggested by taylor (1966), and it belongs to Sadlerianinae, as proposed by SzarowSka (2006).Both p-distances' values and ML tree for Radomaniola, our Horatia, and Horatia from GenBank clearly show that the latter belongs to Radomaniola.We can only speculate about the reasons for this mistake.In our phylogeny the sister taxon of H. klecakiana is Sadleriana, certainly not Radomaniola.In this case molecularly based systematics resembles the morphology-based one of radoMan (1973,1983).
wilke et al. (2001), applying molecular data, inferred phylogenetic position of Horatia, very close to Radomaniola, and the same was confirmed in recent study (wilke et al. 2013).However, this placement raises doubts.The single sequence in GenBank, used several times, also in our studies (e.g.SzarowSka 2006, FalniowSki & SzarowSka 2011b, SzarowSka & FalniowSki 2011), always with the same placing of Horatia very close to Radomaniola Szarowska, 2006, was intriguing.As co-authors of the study of wilke et al. ( (wilke et al. 2001); the other seven taxa represented the Hydrobiinae (including "Pyrgulinae": SzarowSka et al. 2005).The remaining taxa were chosen to represent all the main lineages within the European Sadlerianinae (SzarowSka 2006).

Table 1 .
Taxa used for phylogenetic analyses, with their GenBank Accession Numbers and references