﻿Next step in Monachacantiana (Montagu, 1803) phylogeography: northern French and Dutch populations (Eupulmonata, Stylommatophora, Hygromiidae)

﻿Abstract Features of shell and genitalia as well as nucleotide sequences of selected mitochondrial and nuclear genes of specimens of Monachacantiana from ten northern French and two Dutch populations were compared with the same features of British and Italian populations. They were found to be very similar to populations previously identified as belonging to the CAN-1 lineage of M.cantiana. This confirms previous suggestions that M.cantiana was introduced to western Europe (England, France and the Netherlands) in historical times.

It has been suggested that this species was introduced to the British Isles in historical times (Kerney 1970(Kerney , 1999;;Evans 1972).Our previous research on several M. cantiana populations, using an integrative approach combining analysis of the shell structure and genital anatomy with that of nucleotide sequences of mitochondrial and nuclear gene fragments, revealed six lineages, namely CAN-1, CAN-2, CAN-3, CAN-4, CAN-5 andCAN-6 (Pieńkowska et al. 2018b, 2019a).CAN-1 (representing true M. cantiana) was found to occur in the Latium region of Italy and in Spain and Britain (Pieńkowska et al. 2018b;Čejka et al. 2020), in line with the suggestion that this lineage probably spread with the Roman conquests (Pieńkowska et al. 2018b).Populations of CAN-2 were found in regions of Italy (Emilia Romagna) north of Latium (Pieńkowska et al. 2018b) and somewhat surprisingly in Slovakia (Bratislava) (Čejka et al. 2022), while those of CAN-3 were reportedly widespread even further north in Italy (Friuli-Venezia Giulia) as far as Vienna in Austria (Pieńkowska et al. 2018b(Pieńkowska et al. , 2019b) ) and Bratislava in Slovakia (Čejka et al. 2022).The lineage CAN-4, corresponding to Monacha cemenelea (Risso, 1826), was found in south-eastern France (Pieńkowska et al. 2018b;Čejka et al. 2020).CAN-5 and CAN-6 are reported from the Apuan Alps and represent one or two different species, the naming of which requires further studies on topotypical material (Pieńkowska et al. 2019a).
Monacha cantiana has been reported from France (Kerney et al. 1983;Falkner et al. 2002;Cucherat 2005;Lecaplain 2007;Gargominy et al. 2011;Welter-Schultes 2012;Bichain et al. 2019;Brulé and Bichain 2019;INPN 2019).Brulé and Bichain (2019) carefully analysed shell and genitalia features of M. cantiana specimens collected at two sites in north-eastern France near the towns of Cutry and Longwy.However since the CAN-1, CAN-2, CAN-3, and CAN-4 lineages of M. cantiana do not differ in shell or genital features, the phylogenetic relationships of populations from north-eastern France had to be clarified by genetic analysis.Although M. cantiana is known to occur in the Netherlands (Kerney et al. 1983;Gittenberger et al. 1984;Welter-Schultes 2012), it has never been confirmed genetically.
The aim of the present research was: 1) to study morphological (shell and genitalia) and molecular variation in specimens of M. cantiana collected in northern France and the Netherlands in order to clarify their relations to the British and Italian populations; 2) to test the hypothesis that the English, French and Dutch populations originated from the same introduced propagules.

Material examined
The material examined originated from the populations listed in Table 1 with the following data: geographic coordinates, country and region, short description of collection site, name of collector, date, number of specimens studied and the collection where the material is stored (in brackets).The origin of the material used for comparison has been described in previous publications (Pieńkowska et al. 2015: appendix 1;Pieńkowska et al. 2018bPieńkowska et al. , 2019aPieńkowska et al. , 2019bPieńkowska et al. , 2020Pieńkowska et al. , 2022: table 1): table 1).
Detailed methods of multivariate ordination by Principal Component Analysis (PCA) and Redundancy Analysis (RDA), performed on the original shell and genitalia matrices as well as on the Z-matrices (shape-related matrices), are described in our previous papers (Pieńkowska et al. 2018b(Pieńkowska et al. , 2019a)).
We used 95% confidence interval ellipses to evaluate the uncertainty of the estimate of the population mean (centroid) of the data sample.The function ordiellipse with standard errors in the package vegan (Oksanen et al. 2022) was used.Convex hulls (function ordihull in vegan) were used to visually enclose the individuals forming each clade as a measure of data spread.All analyses were performed with RStudio (R version 4.2.1;R Core Team 2021).
Estimates of genetic distances between the COI sequences obtained in this study and other sequences from GenBank were conducted with MEGA7 using the Kimura two-parameter model (K2P) (Kimura 1980).All positions containing gaps and missing data were eliminated.There were a total of 591 positions in the final dataset.The analysis involved 53 nucleotide sequences.
For each alignment file, best nucleotide substitution models were specified according to the Bayesian Information Criterion (BIC) (see captions to figures).Phylogenetic analyses performed with IQ-Tree, RAxML and MrBayes for three sets of concatenated sequences were done dividing the data set into 2 or 4 partitions: (1) COI, (2) 16SrDNA or (1) COI, (2) 16SrDNA, (3) H3, (4) 5.8SrDNA + ITS2 + 28SrDNA.Best substitution models were inferred according to the Bayesian Information Criterion (BIC) for each of the partitions by MODELFIND-ER (Kalyaanamoorthy et al. 2017) implemented in IQ-TREE.Bayesian analysis were conducted with four Monte Carlo Markov chains running for 1 million generations, sampling every 100 generations (the first 25% of trees were discarded as 'burn-in').
The robustness of the NJ and ML trees generated by MEGA7 were assessed by bootstrap analysis with 1000 replicates (Felsenstein 1985).ML trees built by RAxML were tested by bootstrap analysis with 100 replicates.ML trees obtained with IQ-Tree were constructed under 1000 ultrafast bootstrap replicates (Minh et al. 2013).Finally, BI trees were supported by posterior probability (PP) values.Bootstrap support values from NJ and ML analysis as well as posterior probability (PP) values obtained on 50% majority rule consensus Bayesian tree were mapped onto the ML tree obtained by MEGA7.All the resulting trees were rooted with Trochulus hispidus sequences obtained from GenBank.

Morphological study: shell
Shells of French specimens of M. cantiana (Fig. 2A-D) are globose-subglobose in shape, variable in size and usually whitish or pale yellowish in colour, with slightly descending, roundish to oval aperture, similar to those of the other populations of the lineage CAN-1 (Pieńkowska et al. 2018b: figs 8-11).RDA with French specimens and "lineage" constraint on the shape and size matrix (Fig. 3B, C) showed that RDA 1 (22.2%,P < 0.01) separated CAN-6 from CAN-4, with CAN-5 and the large group CAN-1, CAN-2, CAN-3, and FRA in intermediate position, as confirmed by 95% confidence interval ellipses (Fig. 3B).The convex hull measure of data spread showed considerable overlap of some clusters.In both cases, FRA specimens fell within CAN-1 variability (Fig. 3B).The preliminary classic PCA showed that size was the first major source of morphological variation, since PC1 (69%) was a positive combination of all variables (Fig. 3A).On the contrary, RDA 2 was not significant (p > 0.05) and accounted for little morphological variation (2.6%).PC2 (15%) mostly reflected a contrast between LWaH and PWH versus LWmH and UD.

Morphological study: anatomy
French specimens of M. cantiana have distal genitalia  resembling the other populations assigned to CAN-1, which are in turn similar to those of the populations belonging to the CAN-2, CAN-3 and CAN-4 lineages (Pieńkowska et al. 2018b: figs 20-30).
RDA on the shape (Z) matrix (Figs 7E, F) showed that RDA 1 (33.7%,P < 0.001) separated the 95% confidence interval ellipses of CAN-5, CAN-6 and CAN-4 from the large group CAN-1, CAN-2, CAN-3, and FRA; RDA 2 (8%, P < 0.001) separated CAN-5 and the group CAN-1, CAN-2, CAN-3, FRA from CAN-6 and CAN-4 (Fig. 7E).Convex hulls showed some overlaps, especially in the data spread of CAN-1 (Fig. 7F).Shape-related PCA indicated that P and E vs VA and F were the two principal shape determinants on PC1 and DBC and VA vs V and F on PC2 (Fig. 7D).
The phylogenetic analysis of COI sequences obtained from the specimens and comparative sequences derived from GenBank is shown in Fig. 8.The results are consistent with previously published findings (Pieńkowska et al. 2018b(Pieńkowska et al. , 2019a(Pieńkowska et al. , 2019b(Pieńkowska et al. , 2020(Pieńkowska et al. , 2022)), distinguishing six lineages (CAN-1 -CAN-6) in M. cantiana s.l. that clustered separately from COI sequences of other species including M. parumcincta, M. pantanellii and M. cartusiana.The new COI sequences (haplotypes 1-10) from France, the Netherlands and England clustered in the CAN-1 lineage.Only the COI 11 haplotype obtained from two specimens of the Italian population from Sasso di Simone (population no.23 in Table 1) grouped with the CAN-2 lineage.K2P genetic distances (Table 2) showed small genetic differentiation between COI sequences of particular CAN-1 populations (infra-populational distances ranged from 0.2% in Dutch populations to 1.1% in French populations).The K2P distances between these populations were also small (in the range 0.5-1.2%).The K2P distances between French, Dutch, English and Italian populations of CAN-1 and CAN-2 were also small (in the range 3.5-4.1%)while the distance separating the CAN-1 populations from the CAN-3 and CAN-4 populations was much larger (in the range 18.0-18.8%).In turn, the distance separating the CAN-3 and CAN-4 populations was 5.6-6.1%.   1) were compared with COI sequences of M. cantiana s.l., M. parumcincta, M. pantanellii and M. cartusiana obtained from GenBank (Suppl.material 1).Sequences were cut to 591 bp.HKY+G+I was the best nucleotide substitution model according to the Bayesian Information Criterion (BIC).The tree was rooted with Trochulus hispidus sequences obtained from GenBank (Suppl.material 1).
Results similar to those of COI analysis were obtained for other single gene analyses (Suppl.materials 8, 9 for 16SrDNA, Suppl.material 10 for the ITS2 gene with flanking 5.8S and 28S gene fragments).Note that the newly obtained 16SrDNA sequences in Suppl.material 8 were trimmed to 292 positions in alignment length because GenBank lacks the reference long 16SrDNA sequences of the 809 positions used to construct the tree in Suppl.material 9. Analysis of newly obtained longer sequences (i.e.ITS2 flanked by 5.8SrDNA and 28SrDNA gene fragments) (ITS2 1 -ITS2 34 haplotypes) and the only comparable sequence of Neiber and Hausdorf (2017) showed that this gene did not differentiate the CAN-1, CAN-2 and CAN-3 lineages.Similar results were obtained previously using ITS2 gene sequences without flanking fragments of 5.8SrDNA and 28SrDNA (Pieńkowska et al. 2018b: fig. 64).Only in the case of sequences assigned to the CAN-4 lineage were they distinct from CAN-1, CAN-2 and CAN-3, as shown in Pieńkowska et al. (2018b: fig. 64).
The phylogenetic tree for concatenated sequences were similar in ML analyses obtained with different software.The tree for mitochondrial gene sequences (COI+16SrDNA) in Fig. 9 shows that the sequences obtained from specimens of the French, Dutch, and English populations (see also Suppl.material 5) grouped with the reference sequences for CAN-1.In a tree of concatenated nuclear genes (Fig. 10: H3+ITS2 with flanks), the sequences from the French populations grouped with CAN-1, CAN-2, and CAN-3 lineages, only sequences of the CAN-4 lineage being distinguished.However, note that the bootstrap and posterior probability values weakly supported the results of the concatenated H3+ITS2 gene sequences.The tree for the concatenated sequences of all the genes analysed in this paper (Fig. 11, see also Suppl.material 7) showed that concatenated sequences CS 1-CS 25 from northern French populations clustered together with CS 26-CS 34 and CS 35-CS 38 sequences obtained from English and Italian specimens, respectively.They all belonged to the CAN-1 lineage.The CAN-1, CAN-2, CAN-3, and CAN-4 lineages grouped separately.

Discussion
At a first glance, the shells and genitalia of the French specimens do not differ from those of the other populations assigned to CAN-1, which in turn are similar to those of the populations of the CAN-2, CAN-3 and CAN-4 lineages (see Pieńkowska et al. 2018b).This was fully confirmed by RDA and PCA: the French specimens fell entirely in CAN-1 on the basis of shell characters (Fig. 3C, F), and almost entirely, based on anatomical characters (Fig. 7C, F).  (Saitou and Nei 1987), ML-MEGA7 (Kumar et al. 2016), IQ-Tree (Trifinopoulos et al. 2016), RAxML (Stamatakis 2014), and posterior probabilities by BI (Ronquist et al. 2012).The tree was rooted with Trochulus hispidus concatenated sequences obtained from GenBank (Suppl.material 5).
The results of molecular analysis were consistent with those of morphological analysis (shell and genital structure).Both showed that the populations from northern France should be assigned to the CAN-1 lineage.In this sense, the molecular results complement the conclusions of Brulé and Bichain (2019).Consequently, their results corroborate the results of four previous papers on M. cantiana lineages and their phylogeography (Pieńkowska et al. 2018b(Pieńkowska et al. , 2019a(Pieńkowska et al. , 2019b(Pieńkowska et al. , 2020)).  1) were compared with concatenated H3 and ITS2 sequences of M. cantiana s.l.obtained from GenBank (Suppl.materials 3, 4).Length of sequences was 1054 positions (279 of H3 + 775 of ITS2).The Bayesian Information Criterion (BIC) specified T92+G+I the best nucleotide substitution model in MEGA7, or K2P+I for H3 and K3P+I for ITS2 partition in IQ-Tree, RAxML, and MrBayes.Numbers next to main branches indicate (left to right): bootstrap supports above 50% calculated by NJ-MEGA7 (Saitou and Nei 1987), ML-ME-GA7 (Kumar et al. 2016), IQ-Tree (Trifinopoulos et al. 2016), RAxML (Stamatakis 2014) and posterior probabilities by BI (Ronquist et al. 2012).The tree was rooted with Trochulus hispidus concatenated sequences obtained from GenBank (Suppl.material 6).
Prior suggestions that M. cantiana was introduced into England in historical times (Kerney 1970(Kerney , 1999;;Evans 1972;Pieńkowska et al. 2018b) appear to be correct.This allows us to speculate that the Roman conquests also spread M. cantiana in northern France (as well as in the area of modern-day Holland).The slightly greater genetic diversity of French populations compared to the English ones (expressed as slightly larger K2P distances) indicates that M. cantiana Figs

Figure 3 .
Figure 3. Analysis of French specimens with "lineage" constraint on the original matrix (A-C) and Z-matrix (shape-related) (D-F) of selected shell sections.Principal component analysis (PCA) (A, D) and redundancy analysis (RDA) with groups shown as ellipses representing 95% confidence intervals with standard errors (B, E) and as convex hull polygons (C, F).

Figure 7 .
Figure 7. Analysis of French specimens with "lineage" constraint on the original matrix (A-C) and Z-matrix (shape-related) (D-F) of selected genital sections.Principal component analysis (PCA) (A, D) and redundancy analysis (RDA) with groups shown as ellipses representing 95% confidence intervals with standard errors (B, E) and as convex hull polygons (C, F).

Figure 8 .
Figure8.Maximum Likelihood (ML) tree of COI haplotypes of Monacha cantiana.New COI sequences of M. cantiana (Table1) were compared with COI sequences of M. cantiana s.l., M. parumcincta, M. pantanellii and M. cartusiana obtained from GenBank (Suppl.material 1).Sequences were cut to 591 bp.HKY+G+I was the best nucleotide substitution model according to the Bayesian Information Criterion (BIC).The tree was rooted with Trochulus hispidus sequences obtained from GenBank (Suppl.material 1).

Table 1 .
List of localities of Monacha cantiana s.l.populations used for molecular and morphological (SH shell, AN genitalia) research.

Localities Current taxonomy Clade Designation of DNA voucher sps COI Long 16SrDNA H3 5.8SrDNA + ITS2 + 28SrDNA PCA and RDA Figs No. coordinates country and site
DCBC -the collection of the Department of Cell Biology, Adam Mickiewicz University, Poland; FGC -the Folco Giusti collection at Dipartimento di Scienze Fisiche, della Terra e dell'Ambiente, Università di Siena, Italy; MNHW -the Małgorzata Proćków collection at the Museum of Natural History, University of Wrocław, Poland.
Pieńkowska et al. 2018briables were measured to the nearest 0.1 mm using ADOBE PHOTOSHOP 7.0.1 on digital images of standard apertural and umbilical views taken with a Canon EF 100 mm 1:2.8 L IS USM macro lens mounted on a Canon F6 camera (see alsoPieńkowska et al. 2018b: fig.1): : fig.2):