LANZAIOPSIS BOLE, 1989 (CAENOGASTROPODA: TRUNCATELLOIDEA): ITS PHYLOGENETIC AND ZOOGEOGRAPHIC RELATIONSHIPS

a bStract : Lanzaiopsis savinica was collected from the type locality. The shell as well as the sculpture of the proto- and teleoconch are presented. Mitochondrial cytochrome oxidase subunit I (COI) and nuclear histone 3 (H3) gene sequences were used to infer phylogeny. Lanzaiopsis clearly belongs to the Moitessieriidae but it is only distantly related to Lanzaia . Its sister clade is Moitessieria , while Bythiospeum is the sister clade of the one formed by Lanzaiopsis and Moitessieria . To our knowledge, Lanzaiopsis is the first representative of the moitessieriid non-Balkan clade whose range is so close to the Balkans.


INTRODUCTION
Lanzaiopsis Bole, 1989 is a monotypic genus introduced for a species reported from Slovenia: L. savinica Bole, 1989. Earlier, Bole & velkovrh (1984 introduced Lanzaia savinica as nomen nudum. The type locality of L. savinica is: "Kraški izvir Pečovski studenec, 1.6 km severozahodno od Luč v Savinjski dolini (Savinjske Alpe)" [karstic spring Pečovski izvir north-west of Luče in the Savinja Valley] (Bole 1989: 67). Its empty shells were collected also in nearby springs: a permanent spring about 100 m southeast of the bridge across the river Savinja (on its left bank) and deposits of temporary springs located 100-200 m north of the bridge (also on the left bank), as well as sand deposits in the cave Zavratnikova zijalka, about 1 km north of Pečovski studenec (bole 1989). The species was not found elsewhere (SlaPnik 2011). As is the case with most stygobiotic snails, the number of empty shells found far exceeds the few finds of live specimens. In two summer seasons with extremely low water levels only three males and four females were found (bole 1989) alongside hundreds of empty shells. The species is included in the Hydrobiidae Troschel, 1857, Belgrandiellinae Radoman, 1983(e.g. bole 1989, kaBaT & herShler 1993, Bank & neuBerT 2017. According to bodon et al. (1996), Lanzaiopsis may become a junior synonym of Lanzaia Brusina, 1906. The aim of our paper was to establish phylogenetic relationships of Lanzaiopsis, applying molecular markers.

MATERIAL AND METHODS
Specimens of Lanzaiopsis were collected from the type locality: spring Pečovski izvir, Luče, Ljubno ob Savinji, Slovenia; 46°21'56"N, 14°43'55"E. Two live specimens and dozens of empty shells were found. The material was fixed in 96% ethanol changed twice, and later stored in 96% ethanol.
The shells were cleaned with an ultrasonic cleaner, and examined using a HITACHI S-4700 scanning electron microscope, applying the techniques described by FalniowSki (1990).
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 extracted DNA was stored at −80 °C at the Department of Malacology, Institute of Zoology and Biomedical Research, Jagiellonian University in Kraków (Poland). Mitochondrial cytochrome oxidase subunit I (COI) and nuclear histone 3 (H3) loci were sequenced. Details of PCR conditions, primers used and sequencing were given in SzarowSka et al. (2016).
Sequences were initially aligned in the MUSCLE (edgar 2004) program in MEGA 6 (tamura et al. 2013) and then checked in BIOEDIT 7.1.3.0 (Hall 1999). The saturation test (Xia 2000, Xia et al. 2003 was performed using DAMBE (Xia 2013). In the phylogenetic analysis additional sequences from GenBank were used (Table 1). The data were analysed using approaches based on Bayesian Inference (BI) and Maximum Likelihood (ML). We applied the GTR model, whose parameters were estimated by the RaxML (STamaTakiS 2014 To apply the molecular clock, we used the data from COI. Sequences of two hydrobiids, Peringia ulvae Pennant, 1777 and Salenthydrobia ferreri Wilke, 2003 deposited in GenBank by wilke (2003) as AF478401 and AF478410, respectively, were used as outgroups (tree not shown). The divergence time between these two species was used to calibrate the molecular clock, with correction according to FalniowSki et al. (2008). The likelihoods for trees with and without the molecular clock assumption for a likelihood ratio test (LRT) (nei & kumar 2000) were calculated with PAUP (SwoFForD 2002). The relative rate test (RRT) (tajima 1993) was performed in MEGA. As Tajima's RRTs and the LRT test rejected the equal evolutionary rate throughout the tree, time estimates were calculated using a non-parametric rate smoothing (NPRS) analysis with the recommended Powell algorithm, in r8s v.1.7 for Linux (SanderSon 1997(SanderSon , 2003

RESULTS AND DISCUSSION
The shell of Lanzaiopsis savinica (Figs 1-2) is turriform, with characteristically shaped aperture surrounded by a broad, continuous and folded lip. The protoconch (Figs 3-4) is smooth, all the teleoconch covered with characteristic macrosculpture (Figs  5-6), composed of prominent radial growth lines, and spiral cords. This macrosculpture resembles the one visible in Costellina turrita Kuščer, 1933, Lanzaia bosnica Bole, 1970, or Paladilhiopsis bosniaca (Clessin, 1910, but not the one characteristic of Moitessieria Bourguignat, 1863 . The microsculpture of the teleoconch (Figs 7-8) is composed of irregularly distributed small pores, resembling the ones found in Marstoniopsis van Regteren Altena, 1936, and Dianella Gude, 1913(FalniowSki & SzarowSka 1995. The anatomy of the genus Lanzaia was described by bole (1970) on the basis of L. vjetrenicae Kuščer, 1933 andL. bosnica Bole, 1970. Lanzaiopsis was distinguished from Lanzaia by several traits: in Lanzaiopsis the bursa copulatrix is proportionally bigger, the receptaculum (rs 1 , distal one) smaller, the accessory gland complex of the female reproductive organs divided in two sections, but not folded, and the penis triangular and relatively short (bole 1989). The shell of Lanzaiopsis, conical and elongated, with strongly expanded, peristome and very oblique aperture axis resembles the shell of Lanzaia, except for the less prominent radial sculpture and spiral microsculpture (bole 1989).
Our COI tree ( Fig. 9) (GenBank Accession numbers MN272428-MN272429) clearly confirmed the systematic position of Lanzaiopsis within the Moitessieriidae Bourguignat, 1863, not within the Hydrobiidae, Belgrandiellinae, as postulated by numerous authors (e.g. bole 1989, kaBaT & herShler 1993, Bank & neuBerT 2017. The sister clade of Lanzaiopsis is Moitessieria. Both genera form a well supported (bootstrap support 75%, Bayesian probability 1.0) clade, close to Bythiospeum Bourguignat, 1882. The COI-based tree does not confirm the monophyly of the Moitessieriidae, with the Cochliopidae Tryon, 1866 falling between the two main moitessieriid clades (although bootstrap supports were low), as already reported by  and FalniowSki et al. (2019). The inclusion of the nuclear histone H3 sequences (Fig. 10) (GenBank Accession numbers MN272430-MN272431), how- Fig. 9. Maximum Likelihood tree inferred from mitochondrial cytochrome oxidase subunit I (COI). Bootstrap supports and Bayesian probabilities given ever, resulted in confirmation of the moitessieriid monophyly in both the H3 tree (bootstrap support 75%, Fig. 10) and the tree computed for the two concatenated sequences (bootstrap support 75%, Fig. 11 1927, Paladilhiopsis Pavlovic, 1913 and Lanzaia. While representatives of the former clade were found only in subterranean habitats in central and southern Europe (Germany, Switzerland, Austria, Italy, France) so far, i.e. outside the Balkans, the latter inhabit the Balkans, but also Hungary and Romania. The single known locality of Lanzaiopsis, although outside the Balkans, is within the range of this second, Balkan clade, and geographically is close to the Balkans. On the other hand, its locality belongs to a young Alpine arch named Southern Calcareous Alps, not to the Dinarides.
Despite the sister-clade relationships between Lanzaiopsis and Moitessieria, the divergence between those two taxa took place rather long time ago. The application of molecular clock resulted in estima-tion of the time of divergence as 5.96±0.83 Mya (Fig. 11). This is the latest Miocene, around the time of the Messinian Salinity Crisis. The closure of the present Strait of Gibraltar resulted in desiccation of the Mediterranean Sea, thus the climate became drier and hotter (hSü 1983, krijgSman et al. 1999, garcia-caStellanoS et al. 2009). This probably resulted in extinction of the cold water fauna within a large area, possibly isolating the ancestors of the two genera in distant refuges.