Exploring Monacha cantiana (Montagu, 1803) phylogeography: cryptic lineages and new insights into the origin of the English populations (Eupulmonata, Stylommatophora, Hygromiidae)

Abstract Molecular analysis of nucleotide sequences of mitochondrial cytochrome oxidase subunit 1 (COI) and 16S ribosomal DNA (16SrDNA) as well as nuclear histone 3 (H3) and internal transcribed spacer 2 of rDNA (ITS2) gene fragments together with morphological analysis of shell and genitalia features showed that English, French and Italian populations usually assigned to Monacha cantiana consist of four distinct lineages (CAN-1, CAN-2, CAN-3, CAN-4). One of these lineages (CAN-1) included most of the UK (five sites) and Italian (five sites) populations examined. Three other lineages represented populations from two sites in northern Italy (CAN-2), three sites in northern Italy and Austria (CAN-3), and two sites in south-eastern France (CAN-4). The taxonomic and nomenclatural setting is only currently available for lineages CAN-1 and CAN-4; a definitive frame for the other two requires much more research. The lineage CAN-1 corresponds to the true M. cantiana (Montagu, 1803) because it is the only one that includes topotypical English populations. The relationships and genetic distances support the hypothesis of the Italian origin of this lineage which was probably introduced to England by the Romans. The lineage CAN-4 is attributed to M. cemenelea (Risso, 1826), for which a neotype has been designated and deposited. Its diagnostic sequences of COI, 16SrDNA, H3 and ITS2 genes have also been deposited in GenBank. Molecular and morphological (shell and genitalia) features showed that M. parumcincta (Rossmässler, 1834) is a distinct taxon from the M. cantiana lineages.


Introduction
Monacha is a diverse genus of the trochuline hygromiids widespread in the western Palaearctic from western Europe to north Africa, Iran, and Arabia. It includes a large number of nominal species and shows its highest diversity in the eastern sector of southern Europe and in Turkey (Hausdorf 2000a, 2000b, Welter-Schultes 2012, Neiber and Hausdorf 2017.
Monacha cantiana (Montagu, 1803) is one of the westernmost species. It is a medium-sized land snail living among grass in open habitats such as grasslands, pastures, cultivated and uncultivated fields or forest edges and clearings. Its geographical distribution, probably southern European in origin, was partly shaped by anthropochorous dispersal which helped the species to reach north-western Europe. For example, in the British Isles it is considered to have been introduced and this hypothesis is supported by the absence of a Holocene fossil record in England older than the third century AD (Kerney et al. 1964, Kerney 1970, Evans 1972. The aim of the present research was: (1) to study molecular and morphological (shell and genitalia) variation of the species in order to explore its phylogeography and detect any geographical patterns; (2) to investigate relationships between molecular and morphological variability in order to characterise clades recovered by molecular study; (3) to test the hypothesis that the English populations originated from introduced propagules.

Taxonomic sample
Our analysis considered a number of populations of Monacha cantiana, mainly from Italy and England, that represent its gross morphological, geographical, and ecological variability. Some sequences deposited in GenBank were also considered for the molecular analysis. One population from the type locality of Theba cemenelea Risso, 1826 a taxon regarded as a junior synonym, subspecies or species, slightly distinct from M. cantiana, was also included. For comparison, two other Monacha species were used in the molecular analysis: Monacha cartusiana (Müller, 1774) and M. parumcincta (Rossmässler, 1834). The latter was also used in the morphological analysis. While M. cartusiana is a well-established taxon, the taxonomic and nomenclatural status of M. parumcincta is still disputed, e.g. conspecificity of Italian and Balkan populations, authorship to Rossmässler, 1834or Menke, 1828(see Forcart 1965, Manganelli et al. 1995, Welter-Schultes 2012.

Material examined
Material examined is listed as follows, when possible: geographic coordinates of locality, locality (country, region, site, municipality and province), collector(s), date, number of specimens and collection in which material is kept in parenthesis (Table 1). Collection acronyms: FGC (F. Giusti collection, Dipartimento di Scienze Fisiche, della Terra e dell'Ambiente, Università di Siena, Italy); DCBC (Department of Cell Biology Collection, Adam Mickiewicz University, Poznań, Poland).
The amplified COI fragments consisted of 650 base pairs (bp). Polymerase chain reactions were performed in a volume of 10 μl 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_Bar-code_Analysis.pdf ). Reactions were carried out 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. The amplified 16SrDNA fragments were of about 385 positions. The amplification reactions were conducted in a volume of 10 μl according to a previously described procedure (Manganelli et al. 2005). The amplified H3 sequences consisted of 429 bp. PCR reactions (10 μl) were performed according to the procedure described by Colgan et al. (1998). The 585 position-long sequences of regions enclosing 89 positions of 3'-end of 5.8SrDNA and 496 positions of complete sequence of ITS2 were amplified according to procedure described by Almeyda-Artigas et al. (2000). The PCR products were verified by agarose gel electrophoresis (1% agarose). Prior to sequencing, samples were purified with thermosensitive Exonuclease I and FastAP Alkaline Phosphatase (Fermentas, Thermo Scientific). Finally, the amplified products were sequenced in both directions with BigDye Terminator v3.1 on an ABI Prism 3130XL Analyzer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocols.

Phylogenetic inference
All individual sequences were deposited in GenBank (Table 1) Sequences were edited by eye using the program BIOEDIT, version 7.0.6 (Hall 1999). The alignments were performed using the CLUSTAL W programme (Thompson et al. 1994) implemented in MEGA 7 (Kumar et al. 2016). The COI sequences and H3 sequences were aligned according to the translated amino acid sequences. The ends of all sequences were trimmed. The lengths of the sequences after cutting were 592 bp for COI, 287 positions for 16SrDNA, 315 bp for H3 and 496 positions for ITS2. The sequences were collapsed to haplotypes (COI and 16SrDNA) and to common sequences (H3 and ITS2) using the programme ALTER (Alignment Transformation EnviRonment) (Glez-Peña et al. 2010). Gaps and ambiguous positions were removed from alignments prior to phylogenetic analysis.
Maximum Likelihood (ML) analyses were performed with MEGA 7. For each alignment file best nucleotide substitution models were specified according to the Bayesian Information Criterion (BIC): HKY+I for COI sequences (Hasegawa et al. 1985, Kumar et al. 2016, T92+I for 16SrDNA (Tamura 1992, Kumar et al. 2016, TN93+G+I for H3 (Tamura andNei 1993, Kumar et al. 2016) and JC+G for ITS2 (Jukes andCantor 1969, Kumar et al. 2016). In parallel, the sequences of COI and 16SrDNA obtained in the present work together with other sequences obtained from GenBank were analysed by the genetic distance Neighbour-Joining method (Saitou and Nei 1987) implemented in MEGA7 (Kumar et al. 2016) using the Kimura two-parameter model (K2P) for pairwise distance calculations (Kimura 1980). Next, mitochondrial sequences of COI and 16SrDNA, and nuclear sequences of H3 and ITS2 were combined and as two data sets subjected to ML analysis. The combined sequences were of length of 879 positions for COI+16SrDNA pair and of 811 positions for H3+ITS2. The specified best nucleotide substitution models for ML analysis according to the Bayesian Information Criterion (BIC) were: HKY+I (Hasegawa et al. 1985, Kumar et al. 2016 for COI+16SrDNA combined sequences and TN93+G+I (Tamura andNei 1993, Kumar et al. 2016) for H3+ITS2. Finally, sequences of COI, 16SrDNA and H3 were combined for Bayesian inference. Before doing so, uncertain regions were removed from 16SrDNA alignment with the programme GBLOCKS 0.91b (Castresana 2000, Talavera andCastresana 2007) with parameters for relaxed selection of blocks. This procedure shortened alignment of 16SrDNA sequences from 287 to 271 positions. The combined sequences with a total length of 1178 positions (592 COI + 271 16SrDNA + 315 H3) were used to infer group phylogeny by Bayesian analysis conducted with the program MRBAYES 3.1.2 (Ronquist and Huelsenbeck 2003). Monacha cartusiana was added as an outgroup species in each analysis. Using JMODELTEST2 (Darriba et al. 2012) according to the Bayesian Information Criterion (BIC), we specified a HKY substitution model for our data set (Hasegawa et al. 1985), assuming a gamma distributed rate variation among sites. 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. In parallel, Maximum Likelihood (ML) analysis was performed with MEGA7 (Kumar et al. 2016) and calculated bootstrap values were mapped on the 50% majority rule consensus Bayesian tree.
The haplotype network was inferred with NETWORK 5.0.0.1 to reflect all relationships between COI and 16SrDNA haplotypes. During the analysis, a median-joining calculation implemented in NETWORK 5.0.0.1 was used (Bandelt et al. 1999).

Morphological study
Approximately 70 specimens of five clades (four lineages of the M. cantiana group: CAN-1, CAN-2, CAN-3 and CAN-4; one lineage of M. parumcincta) were considered for shell variability (see Table 1). Shell variability was analysed randomly, choosing when possible five adult specimens from each population. Thirteen shell variables were measured to the nearest 0.1 mm using ADOBE PHOTOSHOP 7.0.1 on digital im-ages of apertural and umbilical standard views taken with a Canon EF 100 mm 1:2.8 L IS USM macro lens mounted on a Canon F6 camera: AH aperture height, AW aperture width, LWfW last whorl final width, LWmW last whorl medial width, LWH last whorl height, LWaH height of adapical sector of last whorl, LWmH height of medial sector of last whorl, PWH penultimate whorl height, PWfW penultimate whorl final width, PWmW penultimate whorl medial width, SD shell diameter, SH shell height, UD umbilicus diameter (Fig. 1).
Approximately 60 specimens of five clades (all lineages of the M. cantiana group plus one lineage of M. parumcincta) were analysed for anatomical variability (see Table 1). Snail bodies were dissected under the light microscope (Wild M5A or Zeiss SteREO Lumar V12). Anatomical structures were drawn using a Wild camera lucida. Acronyms: BC bursa copulatrix, BW body wall, DBC duct of bursa copulatrix, DG digitiform glands, E epiphallus (from base of flagellum to beginning of penial sheath), F flagellum, FO free oviduct, GA genital atrium, GAR genital atrium retractor, OSD ovispermiduct, P penis, V vagina, VA vaginal appendix (also known as appendicula), VAS vaginal appendix basal sac, VD vas deferens. Six anatomical variables (DBC, E, F, P, V, VA) were measured using a calliper under a light microscope (0.01 mm) (Fig. 2).
Multivariate ordination by Principal Component Analysis (PCA) was performed on shell and genitalia matrices separately in order to determine the degree of correlation between variables and their role in explaining variability. Before PCA, variables were log-transformed to obtain a linear relationship. Since variation in size is the first determinant of biometric variation (e.g., Cadima andJolliffe 1996, Klingenberg 2016), multivariate morphometrics to distinguish size and shape components by removing isometric effects are nowadays routinely applied in shell biometry studies (Madec et al. 2003, Paquette and Lapointe 2007, Fiorentino et al. 2008, Caruso and Chemello 2009. We therefore performed two PCAs for each data set (shell, genitalia), one on the original matrices and one on the Z-matrices, the latter only consider shape components according to the methods proposed by Cadima and Jolliffe (1996).
Redundancy analysis (RDA; ter Braak 1986) was then applied to the original matrices and Z-matrices in order to detect any multivariate relationships between shell/ genitalia variables and the taxonomic assignment. The factors "clade/lineage" were used as constraint factor. An ANOVA-like permutation test for constrained ordination was used to assess the significance (P-value < 0.05) of the constraint for the first two RDA axes. Vegan package (Oksanen et al. 2016) in RStudio 1.0.136 (RStudio Team 2016) was used for processing.
Differences between species for each shell and genitalia characters were assessed through box-plots and descriptive statistics. The significance of differences (P < 0.01) was obtained using analysis of variance (ANOVA); where the test proved significant, an adjusted a posteriori pair-wise comparison between pairs of species was performed using Tukey's honestly significant difference (HSD) test. All variables were log transformed before analysis.

Molecular study
Thirty-nine and 18 haplotypes of COI and 16SrDNA mitochondrial gene fragments, respectively, as well as 23 and 18 common nucleotide sequences of histone H3 and ITS2 nuclear gene fragments, respectively, were established (Table 1). As a result, 77 sequences of COI as MG208883-MG208959, 71 sequences of 16SrDNA as MG208960-MG209030, 42 sequences of H3 as MG209031-MG209072 and 31 sequences of ITS2 as MH137963-MH137993 were deposited in GenBank (see also  Table 1). ML tree for combined sequences of COI and 16SrDNA (Fig. 3, Table 2) as well as Bayesian phylogenetic tree for combined sequences of COI+16SrDNA+H3 gene fragments (Fig. 4, Table 2) clustered the received combined sequences in five   Table 2). Bootstrap support above 50% from maximum likelihood analysis is marked at the nodes. Bootstrap analysis was run with 1000 replicates (Felsenstein 1985). The tree was rooted with M. cartusiana combined sequences obtained from GenBank: KM247376 and KM247391. clearly separate clades. ML tree of combined sequences of nuclear H3 and ITS2 gene fragments (Fig. 5, Table 2) clustered the combined sequences in three clades.
First clade CAN-1 includes 14 combined sequences in particular trees (Figs 3-4). The clade includes haplotypes and common sequences (Table 1) which have been found in specimens from the following UK populations: Barrow near Barnsley, East Acton, Cambridge, Rotherham and Sheffield, together with those found in specimens from Italian populations from Latium (Gole del Velino, Valle dell'Aniene, Valle del Tronto and Valle del Turano), as well as from Elba island (Tuscan Archipelago). It is noteworthy that sequences of haplotypes UK-COI 1 and UK-16S 1 are identical to sequences KM247375 and KM247390 deposited in GenBank for COI and 16SrDNA of M. cantiana, respectively (Pieńkowska et al. 2015). It is also important that UK haplotypes UK-COI 2, UK-16S 2 and UK-ITS 2 are identical to Italian IT-COI 2, Figure 4. Bayesian 50% majority-rule consensus tree obtained from analysis of the combined data set of COI, 16SrDNA, and H3 sequences (see: Table 2). Posterior probabilities (left) and bootstrap support above 50% from Maximum Likelihood analysis (right) are marked at the nodes. Bootstrap analysis was run with 1000 replicates (Felsenstein 1985). The tree was rooted with M. cartusiana combined sequences KM247376, KM247391 and MG209072.
Clade CAN-2 (Figs 3-4) includes four COI+16SrDNA combined haplotypes and four COI+16SrDNA+H3 combined sequences. All came from two north Italian populations: Sorgà in Venetum and Rezzato in Lombardy (Table 1). K2P distances between COI and 16SrDNA haplotypes of the clade CAN-2 are very small (Table 3).  Table 2). Bootstrap support above 50% from maximum likelihood analysis is marked at the nodes. Bootstrap analysis was run with 1000 replicates (Felsenstein 1985). The tree was rooted with M. cartusiana combined sequences MG209072 and MH137993.
This CAN-2 clade is not separated from CAN-1 and CAN-3 on the tree of combined nuclear gene sequences (Fig. 5).
Clade CAN-4 (Figs 3-5) includes three COI+16SrDNA, one H3+ITS2 and three COI+16SrDNA+H3 combined sequences. All were from specimens of a French population in the Maritime Alps near Nice (Sainte Thecle, Table 1). Again K2P genetic distances in this population were small (Table 3). COI sequence KF986833 deposited in GenBank by Dahirel et al. (2015) for M. cantiana from Monts d'Ardèche Natural Regional Park near Jaujac (S France) seems to belong to the same clade.
Networks of COI (Fig. 6) and 16SrDNA (Fig. 7) confirm separateness of five clades. Clades CAN-1 and CAN-2 are much closer than the others; French haplotypes of clade CAN-4 are separate from the Austrian-Italian CAN-3; clade PAR of M. parumcincta haplotypes is differentiated into two subgroups.

Morphological study: shell
The M. cantiana group (clades CAN-1, CAN-2, CAN-3, ) and that of M. parumcincta (clade PAR; Fig. 16) have a globose-subglobose shell, variable in colour and size, with roundish aperture and very small or closed umbilicus. The main   RDA with "clade/lineage" constraint on the shape and size matrix (Fig. 17) showed that RDA 1 (47%, P < 0.001) separated the groups CAN-1, CAN-2 and CAN-3 from PAR with CAN-4 in intermediate position. The preliminary classic PCA revealed size as the first major source of morphological variation, since PC1 (78%) was a positive combination of all variables. On the contrary, RDA 2 (3%, P < 0.05) showed a statistically significant separation between CAN-4 and the others; no difference was found between the CAN-1, CAN-2 and CAN-3 groups. In this regard, PC2 (9%) accounted for a contrast between LWmH and LWaH / PWH variables. RDA on the shape (Z) matrix (Fig. 18) confirmed a statistically significant separation between PAR and CAN-4 with the large group CAN-1-CAN-2-CAN-3 in intermediate position.
Shape-related PCA indicated that LWfW / LWmW / LWmH / SD / AD vs LWaH / PWH were the two principal shape determinants on PC1 and PWmW vs UD on PC2.
Box plots (Fig. 19) prove the poor discriminating value of shell characters in distinguishing species pairs (no character distinguishes more than four clade pairs according to Tukey's honestly significant difference test). The most recognisable pairs are CAN-1 vs. PAR, CAN-2 vs. PAR, and CAN-3 vs. PAR (11, 9, and 10 significant   (Table 4).

Morphological study: anatomy
The bodies (generally pinkish or yellowish white) and mantle (with sparse, variably numerous brown or blackish spots near mantle border or on the lung surface, one larger close to the pneumostomal opening) are very similar in the two species group, whereas the distal genitalia show some diagnostic features (Figs 20-50 vs. Figs 51-59): vagi- nal appendix or "appendicula" rather long, always with thin walled terminal portion and with variably evident basal sac (i.e., the "sac-like diverticulum of the appendicula vaginalis" first described by Giusti and Manganelli 1987: 135, Fig. 3A, C -   RDA with "clade/lineage" constraint on the shape and size matrix (Fig. 60) showed that RDA 1 (45%, P < 0.001) tended to separate the group CAN-1, CAN-2, CAN-3 and CAN-4 from PAR. The preliminary classic PCA revealed size as the first major source of morphological variation, since PC1 (53%) was a positive combination of all variables. On the contrary, RDA 2 (6%, P < 0.002) showed statistically significant separation of CAN-1, CAN-2, CAN-3 and PAR from CAN-4. In that regard, PC2 (20%) accounted for a contrast between F and P variables. RDA with species constraint on the shape (Z) matrix (Fig. 61) showed that RDA 1 (20%, P < 0.001) confirmed a statistically significant separation between PAR and CAN-4, while the large group CAN-1-CAN-2-CAN-3 remained completely unexplained. Shape-related PCA indicated that VA and F vs E and P were the two principal shape determinants on PC1 and V vs BCD on PC2.
Box plots (Fig. 62) for anatomical characters showed that VA has the best discriminating value (it distinguishes five clade pairs according to Tukey's honestly significant difference test), followed by E and V (three pairs). The most recognisable pairs are CAN-1 vs. PAR (four significant characters), CAN-2 vs. PAR, CAN-3 vs.   (Table 4).

Discussion
The finding that M. cantiana, as usually conceived, actually consists of four distinct lineages (CAN-1, CAN-2, CAN-3, CAN-4) is an absolute novelty. One of these lineages (CAN-1) included most of the populations examined (11 populations). It is widespread   (60) and Z-matrix (shape-related) (61). Ellipses show the 95% confidence intervals associated with each group. and reported from the United Kingdom, Spain and Italy. The other three lineages include only two (CAN-2 and CAN-4) or three (CAN-3) populations, respectively, and at present have a narrow distribution, being known only from two sites in northern Italy (CAN-2), three sites in northern Italy and Austria (CAN-3) and two sites in south-eastern France (CAN-4) (Fig. 63). If these lineages were treated as distinct species, a taxonomical and nomenclatural setting would only be possible for CAN-1 and CAN-4 at present (a definitive framework for the other two requires more research).
Statistical analysis of a series of shell and anatomical characters shows that at least three lineages (CAN-1, CAN-2, CAN-3) cannot be distinguished from each other based on morphology and that one lineage (CAN-4) is only marginally distinct. On the contrary, these four lineages are anatomically well distinct from the Monacha species used for comparison (M. parumcincta), and three of them (CAN-1, CAN-2, CAN-3) are also conchologically distinct on the basis of many significant characters (11, 9, and 10, respectively). The major bias of morphological analysis was the small sample available for lineages CAN-2, CAN-3, and CAN-4, which prevented a realistic account of their variability. Sequences characteristic of clade CAN-1 formed a well-separated group in ML and Bayesian trees (Figs 3-5). Although they were all from UK and Italian populations, they are mixed together in the trees without separate branches for UK and Italian populations. Interestingly, three pairs of haplotypes or common sequences are identical: UK-COI 2 / IT-COI 2, UK-16S 2 / IT-16S 1 and UK-ITS2 2 / IT-ITS2 1. This and small K2P genetic distances within this clade (0.9% in COI, 0.5% in 16SrDNA) suggest that the clade represents one taxon. CAN-1 corresponds to the true M. cantiana because it is the only clade that includes topotypical English populations. Close rela-  Table 1 for locality numbers).
tions between the sequences studied (clade CAN-1 in Figs 3-5) support the conclusion that the populations have a common Mediterranean origin (Neiber and Hausdorf 2017), which in view of available fossil record (Kerney et al. 1964, Kerney 1970, Evans 1972, may be postulated to date back to the Roman conquest. The same is also true for the Spanish populations from Pais Vasco (Sopelana), whose sequences (KX507234 and KJ458539 / KX495428), deposited in GenBank for COI and 16SrDNA of M. cantiana Hausdorf 2015, Razkin et al. 2015), respectively, were located between our UK and Italian (Latium sites close to Rome) populations in our ML trees (Fig. 64). Nevertheless further studies on molecular characteristics of M. cantiana populations from Scotland, N France, N Germany, Belgium, and The Netherlands are necessary in order to test this hypothesis.  (Felsenstein 1985). Numbers on branches represent bootstrap support above 50%. A the COI sequences of Monacha cartusiana KM247389, KM247376 and KX507189 were used as an outgroup, and those of M. cantiana KF986833, KX507234 and HQ204502 as reference sequences. 592-bp sequences of new COI haplotypes (Table 1) were shortened to a 556-bp fragment for alignment with the GenBank sequences used as outgroup or references B the 16SrDNA sequences of Monacha cartusiana KM247391, KM247397 and KX495378 sequences were chosen as outgroup. M. cantiana AY741419, HQ204543, KJ458539 and KX495428 as well as M. parumcincta AY741418 sequences were used as references. The final dataset contained 287 positions C the ITS2 tree was rooted with Monacha cartusiana sequence MH137993 D the H3 tree was rooted with Monacha cartusiana sequence MG209072. Monacha cantiana KF596955 was used as a reference.
The three percent threshold for genetic distance between COI barcode sequences was established by Hebert et al. (2003aHebert et al. ( , 2003b) as a criterion for the description of a new taxon at species level. There are many papers concerning usefulness of barcoding in taxonomy (e.g., Ebach and Holdrege 2005, Gregory 2005, Goldstein and DeSalle 2010 and showing that 3% threshold should be higher (4% or even higher) for stylommatophoran gastropods (Davison et al. 2009, Sauer andHausdorf 2012 and references cited therein). Aware of it we think that the slightly exceeded barcode threshold in K2P distances between COI sequences of CAN-1 and CAN-2 clades together with the lack of significant differences in shell (Fig. 19) and genitalia features (Fig. 62), do not permit to introduce a distinct taxon, even at subspecies level. Rather, the K2P distances show that some Italian populations of the M. cantiana group are in a process of speciation and differentiation.
The cases of the clades CAN-3 and CAN-4 are completely different, since K2P genetic distances distinguish the haplotypes of these two clades from the others (CAN-1, CAN-2, PAR) and were well above Hebert's threshold (even enlarged according to Davison et al. 2009). However, due to the lack of differences in anatomical and conchological features between CAN-3 and clades CAN-1 and CAN-2, we treat CAN-3 as mitochondrially distinct lineage only. Any taxonomic conclusion would be premature.
The situation of clade CAN-4 is distinct because this lineage includes a French population which can be considered topotypical of Theba cemenelea. Live specimens were collected by one of us (AH) at Sainte Thecle, Vallée de Peillon, a site located 10 km NE of Risso's original locality: Colline de Cimiez at Nice, now in the urban area of Nice. It was regarded as a junior synonym or at least a subspecies of M. cantiana until the early 2000s, when Falkner et al. (2002) separated it again on the basis of the presence of well evident basal sac of the vaginal appendix considered instead absent in M. cantiana. Since type material of T. cemenelea no longer exists (Chevallier 1976, Arnaud 1977, only designation of a neotype can ensure correct univocal application of Risso's name. We therefore select a specimen collected at Sainte Thecle in Vallée de Peillon as the neotype. The neotype is deposited in the malacological collection of the Museo di Storia Naturale dell'Accademia dei Fisiocritici, Siena (MOLL/3309). Its shell is illustrated in Fig. 16 and its genital anatomy in Figs 38-41. The separation of CAN-4 (M. cemenelea) is strongly supported by nucleotide sequence analysis of both mitochondrial and nuclear genes (Figs 3-5, 64). Therefore haplotypes of COI and 16SrDNA as well as sequences of H3 and ITS2 gene fragments characteristic of specimens from this population have been deposited in GenBank (accession Numbers for FR-COI 1-4: MG208939-MG208943; for FR-16S 1-2: MG209011-MG209015; for FR-H3 1-3: MG209058-MG209060; for FR-ITS2 1: MH137984).
Designation of the neotype is in line with the current concept of this Monacha species (e.g., Falkner et al. 2002) i.e., a species distinguished by a well evident basal sac of the vaginal appendix. Contrarily to what has been stated by Falkner et al. (2002), this basal sac is present but smaller or sometimes absent in M. cantiana. Moreover this taxonomic setting based on genitalia features is supported by molecular features of mitochondrial and nuclear genes.
A singular sequence AY741419 from Podere Grania, Asciano, Siena deposited in GenBank by Manganelli et al. (2005) for 16SrDNA (Fig. 64B, Table 1) as well as our not yet published molecular results for certain Italian populations (from Alpi Apuane, Tuscany) suggest that Italian M. cantiana may include other lineages.
All our results, namely shell (Figs 17-19) and genital (Figs 60-62) structures and molecular evidence of separate clades for each tree (Figs 3-5, 64), show that M. parumcincta and M. cantiana are distinct taxa. However the definitive taxonomic and nomenclatural setting of M. parumcincta is still unclear (see Forcart 1965, Manganelli et al. 1995, Welter-Schultes 2012. This and its infraspecific variation will be the subject of further studies.