DISTRIBUTION OF MONACHA CLAUSTRALIS ( ROSSMÄSSLER , 1834 ) AND M . CARTUSIANA ( O . F . MÜLLER , 1774 ) ( EUPULMONATA : HYGROMIIDAE ) IN CENTRAL EUROPEAN AND BALKAN COUNTRIES : NEW DATA

1Department of Cell Biology, Institute of Experimental Biology, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznań, Poland (e-mail: pienkowj@amu.edu.pl, alesicki@amu.edu.pl) 2Museum of Natural History, University of Wrocław, Sienkiewicza 21, 50-335 Wrocław, Poland (e-mail: malgorzata.prockow@uwr.edu.pl) 3Faculty of Geology, University of Warsaw, Żwirki i Wigury 93, 02-089 Warszawa, Poland (e-mail: magurka@uw.edu.pl) *corresponding author

Analysis of selected gene nucleotide sequences is nowadays frequently used for species classification, delimitation and identification (faLniowski & wiLke 2001, HebeRt et al. 2003a, b, 2013, ManganeLLi et al. 2005, szaRowska 2006, fioRentino et al. 2008, 2010, 2016, saueR & HausdoRf 2009, 2012, szaRowska et al. 2014, osikowski et al. 2015, 2017, osikowski 2017, Neiber & HausdoRf 2015, 2017, Rysiewska et al. 2016) and makes it possible to verify species ranges and their distribution.Nucleotide sequences of cytochrome oxidase subunit I gene (COI) were mainly used in the above cited papers.However, COI sequences were originally used for recognition of cryptic species among lepidopterans (HebeRt et al. 2003b), and were later recognised as 'barcode' sequences (HebeRt et al. 2003a, 2013, PackeR et al. 2009, goLdstein & desaLLe 2010) to resolve several taxonomic problems among gastropods, including species of the family Hygromiidae.For example, COI was, among other genes, used for species differentiation within the genus Trochulus (DuDa et al. 2011, PRoćków et al. 2013, 2014), and also for the analysis of bio-and phylogeographic aspects of hygromiids (neibeR & HausdoRf 2015, 2017, Neiber et al. 2017).The use of COI sequences allowed to re-identify M. cantiana from the Czech Republic (HLaváč & PeLtanová 2010) as M. claustralis (Pieńkowska et al. 2015, koRábek & Juřičková pers. com.).In Poland, M. cartusiana, initially reported from several localities (cHoLewa et al. 2003, Lesicki & koRaLewska-batuRa 2007, stwoRzewicz & góRka 2012), turned out to be M. claustralis based on molecular analyses (Pieńkowska et al. 2015(Pieńkowska et al. , 2016)).As stressed before, to solve taxonomic problems, molecular features should be integrated with morphological and anatomical data (Pieńkowska et al. 2016).Nevertheless, any new data obtained as a result of verification of species identification on the basis of molecular analysis, are worth publication.These data allow to assess distribution ranges of particular species and to track their possible migration routes.Such new data, presented in this paper, reveal the occurrence of M. claustralis and M. cartusiana in some new localities in Poland, Germany, Slovakia, Austria, Hungary and a few Balkan countries.

MATERIAL AND METHODS
Forty-nine specimens of M. claustralis and 52 specimens of M. cartusiana collected in 2014-2017 were used for anatomical and molecular examination.Their identification was confirmed based on the genitalia (Pieńkowska et al. 2015).Data on population localities, GenBank accession numbers, and the classification of the specimens used in this study are listed in Table 1.All voucher specimens preserved in 75% ethanol are deposited in the Department of Cell Biology Collection (DCBC), Adam Mickiewicz University, Poznań, Poland, except a specimen from Plovdiv kept in A. iRikov's collection (AIC), University of Plovdiv, Bulgaria.
Amplification of 650 bp long 'barcode sequence' was performed according to the modified protocol prepared by the Biodiversity Institute of Ontario for the Consortium for the Barcode of Life (http:// barcoding.si.edu/PDF/Protocols_for_High_Volume_DNA_Barcode_Analysis.pdf).Reactions were carried out in a volume of 10 µl under the following thermal profile: 1 min at 94 °C followed by 42 cycles of 40 s at 94 °C, 40 s at 53 °C, 1 min at 72 °C, and finally 5 min at 72 °C.Amplification of about 370 bp long fragment of 16SrDNA was conducted in a volume of 10 µl according to a previously described procedure (ManganeLLi et al. 2005).The PCR products were verified by agarose gel electrophoresis (1% agarose).Prior to sequencing, to improve its quality, the PCR products of COI were purified with thermosensitive Exonuclease I and FastAP Alkaline Phosphatase (Fermentas, Thermo Scientific).Amplified fragments of 16SrDNA were much shorter than the fragments of COI and sufficiently pure, therefore they did not require additional purification.Properly prepared PCR products were sequenced bidirectionally with BigDye Terminator v3.1 on an ABI Prism 3130XL Analyzer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocols.All obtained sequences were deposited in GenBank (Table 1).
Sequences were aligned and edited by eye using the programme BioEdit, version 7.   (kiMuRa 1980).An NJ tree credibility was tested by bootstrap analysis with 1,000 replicates (feLsenstein 1985).
The Bayesian analysis of combined COI and 16SrDNA sequences (together 832 positions -544 of COI + 288 of 16SrDNA) was conducted with the programme MrBayes 3.1.2(Ronquist & HueLsenbeck 2003).HKY substitution model (Hasegawa et al. 1985), assuming a gamma distributed rate variation among sites was found as the best-fit substitution model (ML) for our data set according to the Bayesian Information Criterion (BIC).Best-fit substitution model was calculated using algorithm implemented in MEGA 7. Four Monte Carlo Markov chains were run for 1 million generations, sampling every 100 generations (the first 250,000 trees were discarded as 'burn-in').This gave us a 50% majority rule consensus tree.At the same time, Maximum Likelihood (ML) analysis was performed with MEGA7 and calculated bootstrap values were placed on the 50% majority rule consensus Bayesian tree.
The above identification of M. claustralis and M. cartusiana was confirmed in the Bayesian phylogenetic tree (Fig. 3) based on combined haplotypes of COI and 16SrDNA gene sequences (Table 3).
Specimens from each population were also identified on the basis of their genital structure.Examples of the genitalia of representative specimens of M. claustralis and M. cartusiana are shown in Figs 4-5 and 6-8, respectively.
The genetic distances within M. claustralis are relatively large (Table 2: COI 0.0-5.7%,16SrDNA 0.0-3.9%),however, it is noteworthy that extreme distances refer only to a few specific populations.One is the population from Plovdiv, Bulgaria -only the COI sequence KM247386 (Pieńkowska et al. 2015) is the same as COI sequences KX258308-KX258350 (Pieńkowska et al. 2016) and COI 3 (this paper) from several populations from W. and S. Poland.Three other COI sequences: COI 7 and KM247387-KM247388 (Pieńkowska et al. 2015), found in the Plovdiv population, are either the same (COI 7 and KM247387) or very similar (COI 7 and KM247388 -K2P 1.0%) but differ at the level of 3.3-5.2%from all other populations.Similar results refer to the 16SrDNA sequence KM247395 (Pieńkowska et al. 2015) and the combined sequence Mclau-BG-h2 which are different from the other studied sequences (Figs 2C & 3).Because several Monacha species inhabiting Bulgaria (iRikov 2008, geoRgiev 2016) are difficult to identify based only on conchology, molecular research is necessary to resolve their relationships.Such investigations are under way (geoRgiev pers. comm.).
Besides, the COI 4 -COI 6 sequences, obtained in this study for specimens from Montenegro, also differ from all other analysed sequences of M. claus tralis at the level of 4.6-5.7%.They are also different (4.6-5.2%)from COI sequences KM247387-KM247388 from Bulgaria.Moreover, 16SrDNA  & 3, respectively.This confirms the previously suggested need (Pieńkowska et al. 2015) of comparative studies including topotypical material of M. subobstructa (Bourguignat, 1855), a species whose occurrence was reported from nearby Albania (feHéR & eRőss 2009), although the name is currently listed among synonyms of M. claustralis (weLteR-scHuLtes 2012).It should be added that the geological history of the region may explain the reasons for species diversity of snails (e.g., ketMaieR et al. 2006, fioRentino et al. 2010, 2016).The geological events in the Balkan Peninsula history caused separation of some truncatelloid species into Bulgarian, Montenegrin and Greek populations (osikowski et al. 2015, osikowski 2017).
Unique COI (COI 15, COI 16) and 16SrDNA (16S 10, 16S 11) sequences as well as combined sequences (Mcart-XK-h1 -Mcart-XK -h3) were obtained from Košutane (Kosovo) specimens.The genetic distances between them and other M. cartusiana sequences are small (0.3-3.2% -slightly larger 2.1-3.2% in comparison with the above mentioned sequences from Kielce, Belgrade and Bočsa specimens, but only 0.3-1.6%regarding all other studied M. cartusiana sequences).However, it is noteworthy that shells of these specimens were small, without umbilicus.They were collected in a xerothermic sward at high altitude.Nevertheless, the structure of their genitalia confirms the molecular results (Figs 7-8) and we report the Košutane population as representing M. cartusiana.
Taking into account the ranges of these two species shown in weLteR-scHuLtes (2012) and this study (Fig. 9), there are still many localities which should be revised molecularly.Since the shells of these species are very similar (HausdoRf 2000a) their conchological identification is unreliable.Moreover, genital anatomy requires very careful examination due to the overall similarities (HausdoRf 2000a).Interestingly, M. claustralis and M. cartu siana co-occur in some Polish (Kielce-Wietrznia) and Czech (Prague) populations (Pieńkowska et al. 2015(Pieńkowska et al. , 2016)).Now we add new Polish (Kielce-Lidl) and Bosnian (Kaonik) populations with such a co-oc-currence (Table 1, Fig. 9).This further emphasises the need of cautious identification confirmed by molecular and anatomical features.
We think and hope that the molecularly checked localities presented on the map (Fig. 9) will soon be supplemented by new data.
Finally, we can state that the results of this paper confirm the earlier observations that two species of Monacha (M.

Table 1 .
List of localities of Monacha claustralis All collected specimens are deposited in the Department of Cell Biology Collection (DCBC), Adam Mickiewicz University, Poznań, Poland (except a specimen from Plovdiv kept in A. iRikov collection (AIC), University of Plovdiv, Bulgaria).Table1 continued

Table 3 .
Combined COI and 16SrDNA datasets for the analysed Monacha species