Four new populations of Monacha cantiana s.l. were considered in our analysis of their molecular and morphological (shell and genitalia structure) variability (Table 1) and compared with the other M. cantiana lineages (Pieńkowska et al. 2018). The sequences deposited in GenBank were also considered for the molecular analysis. Two other Monacha species were used for molecular comparison (Monacha cartusiana (Müller, 1774)) and for morphological and molecular comparison (M. parumcincta (Rossmässler, 1834)).

New materials examined are listed as follows, when possible: geographic coordinates of locality, locality (country, region, site, municipality and province), collector(s), date, number of specimens with name of collection where materials are kept in parenthesis (Table 1). The materials are kept in the F. Giusti collection (FGC; Dipartimento di Scienze Fisiche, della Terra e dell’Ambiente, Università di Siena, Italy). The materials used for comparison have already been described (see Pieńkowska et al. 2018: table 1) and is now supplemented with some new nucleotide sequences (Table 2).

DNA extraction, amplification and sequencing methods are described in detail in our previous paper (Pieńkowska et al. 2018).

Two mitochondrial and two nuclear gene fragments were analysed, namely cytochrome c oxidase subunit 1 (COI), 16S ribosomal DNA (16SrDNA), histone 3 (H3) and an internal transcribed spacer of rDNA (ITS2), respectively. All new sequences were deposited in GenBank (Tables 1, 2). The COI, 16SrDNA, H3 and ITS2 sequences obtained from GenBank for comparisons are listed in Table 3.

The sequences were edited by eye using the programme BioEdit, version 7.2.6 (Hall 1999). Alignments were performed using Clustal W (Thompson et al. 1994) implemented in MEGA 7 (Kumar et al. 2016). The COI 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 trimming were 591 bp for COI, 355 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. Mitochondrial (COI and 16SrDNA) and nuclear (H3 and ITS2) sequences were combined (Table 4) before phylogenetic analysis. Finally, the sequences of COI, 16SrDNA, H3 and ITS2 were combined (Table 4) for Maximum Likelihood (ML) and Bayesian inference (BI). Before doing so, uncertain regions were removed from 16SrDNA alignment with the GBlocKs 0.91b (Castresana 2000; Talavera and Castresana 2007) using parameters for relaxed selection of blocks. This procedure shortened 16SrDNA sequences from 355 to 275 positions.

The sequences of COI obtained in this study together with other sequences from GenBank were analysed by the genetic distance Neighbour-Joining method (Saitou and Nei 1987) implemented in MEGA7 using the Kimura two-parameter model (K2P) for pairwise distance calculations (Kimura 1980). Maximum Likelihood (ML) analyses were then performed with MEGA 7. Monacha cartusiana and Monacha parumcincta were added as outgroup species in each analysis. For ML analysis of combined sequences, the following best nucleotide substitution models were specified according to the Bayesian Information Criterion (BIC): HKY+G (Hasegawa et al. 1985; Kumar et al. 2016) for COI and 16SrDNA combined sequences of 879 positions (591 COI + 288 16SrDNA), TN92+G (Tamura 1992; Kumar et al. 2016) for H3+ITS2 combined sequences of 812 positions (315 H3 + 497 ITS2), and GTR+I+G (Nei and Kumar 2000; Kumar et al. 2016) for COI+16SrDNA+H3+ITS2 combined sequences with a total length of 1677 positions (591 COI + 275 16SrDNA + 315 H3 + 496 ITS2). Bayesian analysis was conducted with the MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) using the same evolution model as for ML calculation. The GTR substitution model (Nei and Kumar 2000; Kumar et al. 2016), assuming a gamma distributed rate variation (+G) allowing for some sites to be evolutionarily invariable (+I), was identified as the best-fit substitution model using jModelTest2 (Darriba et al. 2012). Four Monte Carlo Markov chains were run for one 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).

Seventy-eight specimens of seven clades (six lineages of M. cantiana s.l.: CAN-1, CAN-2, CAN-3, CAN-4, CAN-5 and CAN-6; one lineage of M. parumcincta) were considered for shell variability (see Table 1 and Pieńkowska et al. 2018). Shell variability was analysed randomly choosing five adult specimens from each population, when possible. Twelve shell variables were measured to the nearest 0.1 mm using Adobe Photoshop 7.0.1 on digital images 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, 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 (see Pieńkowska et al. 2018: fig. 1).

Seventy-five specimens of seven clades (all lineages of M. cantiana s.l. plus one lineage of M. parumcincta) were analysed for anatomical variability (see Table 1 and Pieńkowska et al. 2018). Snail bodies were dissected under the light microscope (Wild M5A or Zeiss SteREO Lumar V12). Anatomical details 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, 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) (see Pieńkowska et al. 2018: fig. 2).

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 a previous paper (Pieńkowska et al. 2018).

Differences between species for each shell and genital character were assessed through box-plots and descriptive statistics. Significance of differences (set at p ≤ 0.01) was obtained using analysis of variance (ANOVA); when the test proved significant, an adjusted 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.

Eighteen sequences of each mitochondrial gene fragment (COI and 16SrDNA) as well as 16 and 25 sequences of nuclear gene fragments (H3 and ITS2, respectively) were deposited in GenBank as MK066929-MK066946 (COI), MK066947-MK066964 (16SrDNA), MK066965-MK066980 (H3) and MK066981-MK067005 (ITS2). Eight COI and 12 16SrDNA haplotypes were recognised among them (Table 1). Eight H3 (Table 1) and 19 ITS2 (Tables 1, 2) common nucleotide sequences were also established. ML trees for combined sequences of mitochondrial COI and 16SrDNA (Fig. 1, Table 4) and of nuclear H3 and ITS2 (Fig. 2, Table 4) gene fragments, as well as the Bayesian phylogenetic tree of combined sequences of COI+16SrDNA+H3+ITS2 gene fragments (Fig. 3, Table 4) clustered the combined sequences in two separate clades (CAN-5 and CAN-6), which were also separate from all other clades recognised previously for M. cantiana (CAN-1, CAN-2, CAN-3), M. cemenelea (CAN-4) and M. parumcincta (PAR) populations (Pieńkowska et al. 2018).

Figure 1. 

Maximum Likelihood (ML) tree of combined COI and 16SrDNA haplotypes of Monacha cantiana s.l. (see Table 4). Numbers next to the branches indicate bootstrap support above 50% calculated for 1000 replicates (Felsenstein 1985). The tree was rooted with M. cartusiana and M. parumcincta combined sequences obtained from GenBank (Table 4).

Figure 2. 

Maximum Likelihood (ML) tree of combined H3 and ITS2 common sequences of Monacha cantiana s.l. (see Table 4). Numbers next to the branches indicate bootstrap support above 50% calculated for 1000 replicates (Felsenstein 1985). The tree was rooted with M. cartusiana and M. parumcincta combined sequences obtained from GenBank (Table 4).

Figure 3. 

Bayesian 50% majority-rule consensus tree of the combined data set of COI and 16SrDNA haplotypes, and H3 and ITS2 common sequences (see Table 4). Posterior probabilities (left) and bootstrap support above 50% from ML analysis (right) are indicated next to the branches. Bootstrap analysis was run with 1000 replicates (Felsenstein 1985). The tree was rooted with M. cartusiana and M. parumcincta combined sequences obtained from GenBank (Table 4).

Networks of COI (Fig. 4) and 16SrDNA (Fig. 5) confirmed separateness of clades CAN-5 and CAN-6 and all other previously recognised clades (CAN-1 to CAN-4, PAR; Pieńkowska et al. 2018).

Figure 4. 

The median-joining haplotype network for COI haplotypes of Monacha cantiana s.l. The colours of the circles indicate Monacha species, and their size is proportional to haplotype frequencies. Small black circles are hypothetical missing intermediates. The numbers next to the branches indicate distance between taxa expressed in numbers of mutant positions. Only numbers above 10 are indicated.

Figure 5. 

Haplotype network for 16SrDNA of Monacha cantiana s.l. Other explanations as in Figure 4.

K2P genetic distances between COI haplotypes are summarised in Table 5. The smallest distances are within haplotypes of particular clades (0.2–2.2%, slightly larger 1.0–4.2% within M. parumcincta). As shown previously (Pieńkowska et al. 2018), the K2P distances between CAN-1 and CAN-2, and between CAN-3 and CAN-4, were smaller (3.3–5.1% and 5.1–6.2%, respectively) than between other clades compared in pairs (Table 5). The clades CAN-5 and CAN-6 differed considerably (12.4–14.3%). The clade CAN-5 differed to a similar degree from CAN-3 and CAN-4 clades (13.3–15.4%). Differences between these two clades (CAN-3 and CAN-4) and the clade CAN-6 were even larger (14.3–16.8%). Both CAN-5 and CAN-6 were also separated by very large genetic distances from all other clades (16.5–21.3%).

The two new clades of M. cantiana s.l. (CAN-5, CAN-6: Figs 6–15) have a globose-subglobose shell, variable in size and usually whitish or pale yellowish, with slightly descending, roundish to oval aperture, very similar to those of the other lineages (CAN-1, CAN-2, CAN-3, CAN-4; see Pieńkowska et al. 2018: figs 8–15), but clearly distinguished by a larger, very open umbilicus.

Figures 6–15. 

Shell variability in Monacha cantiana s.l. CAN-5 from Piastra (FGC 41563) (6, 7), Foce di Pianza (FGC 41565) (8, 9) and Campo Cecina (FGC 41564) (10–12); CAN-6 from Campagrina (FGC 40322) (13–15).

M. cantiana s.l. (lineages CAN-1 to CAN-6) is always distinguished from M. parumcincta by its umbilicus (open in M. cantiana s.l.; closed in M. parumcincta). Some populations of M. parumcincta have variably evident whitish peripheral and subsutural bands (evident if the last whorl is reddish) and/or a less glossy (more opaque) shell surface.

RDA with lineage constraint on the shape and size matrix (Fig. 16) showed that RDA 1 (44%, p < 0.001) separated the groups CAN-1, CAN-2, CAN-3, CAN-4, CAN-5 and CAN-6 from PAR. The preliminary classic PCA revealed size as the first major source of morphological variation, since PC1 (74%) was a positive combination of all variables. On the contrary, RDA 2 (7%, p < 0.01) separated CAN-1, CAN-2 and CAN-3 from CAN-4, CAN-5 and CAN-6 with PAR in intermediate position. In this regard, PC2 (11%) accounted for a contrast between LWmH vs LWaH and PWH variables.

Figures 16, 17. 

Principal component analysis (PCA) and Redundancy analysis (RDA) with lineage constraint applied to the original shell matrix (16) and Z-matrix (shape-related) (17).

RDA on the shape (Z) matrix (Fig. 17) showed no separation of lineages, confirming that size is a major source of morphological variation. Shape-related PCA indicated that LWfW and LWmW vs SH, LWaH and PWH were the two principal shape determinants on PC1 and AD vs UD on PC2.

Box plots (Fig. 18) proved the poor discriminating value of shell characters in distinguishing lineage pairs. The best discriminant character was UD that distinguished 13 clade pairs according to Tukey’s honestly significant difference test, followed by LWmH and LWmW that distinguished seven clade pairs each. The most recognizable pairs were CAN-1 vs PAR, CAN-3 vs PAR, CAN-6 vs PAR, CAN-2 vs PAR and CAN-5 vs PAR (12, 11, 10, 8 and 7 significant characters, respectively). Five significant shell characters distinguished CAN-3 vs CAN-4, four CAN-4 vs CAN-6, two CAN-1 vs CAN-4, CAN-1 vs CAN-5 or CAN-3 vs CAN-5 and only one CAN-1 vs CAN-6, CAN-2 vs CAN-6, CAN-3 vs CAN-6, CAN-4 vs CAN-5 or CAN-4 vs PAR. No significant character distinguished CAN-1 vs CAN-2, CAN-1 vs CAN-3, CAN-2 vs CAN-3, CAN-2 vs CAN-4, CAN-2 vs CAN-5 or CAN-5 vs CAN-6 (Table 6).

Figure 18. 

Box plots for shell characters of the seven Monacha clades investigated. The lower and upper limits of the rectangular boxes indicate the 25^th to 75^th percentile range, and the horizontal line within the boxes is the median (50^th percentile).

The bodies (generally pinkish or yellowish white) and mantle (with sparse brown or blackish spots near the mantle border or on the lung surface, a larger one close to the pneumostomal opening) of CAN-5 and CAN-6 are very similar to those of the other lineages of M. cantiana s.l. and M. parumcincta studied so far (Pieńkowska et al. 2018). The same is true of the distal genitalia (CAN-5: Figs 19–34; CAN-6: Figs 35–41), which as in the other lineages, have vaginal appendix (or “appendicula”) rather long, always with thin walled terminal portion and with variably evident basal sac; vaginal-atrial pilaster variably evident; epiphallus section with five to six small pleats on one side, two large pleats on the opposite side and, between them, a very small pleat; penial papilla (or glans) section with central canal wide, thin walled, internally irregularly jagged and with a sort of solid pilaster on one side; central canal connected to external wall of penial papilla by many muscular/connective strings as in the other lineages (Pienkowska et al. 2018).

Figures 19–24. 

Genitalia (proximal parts excluded) (19), internal structure of distal genitalia (20), transverse sections of medial epiphallus (21, 22) and basal and apical penial papilla (23, 24) of Monacha cantiana s.l. CAN-5 from Piastra (FGC 41563).

Figures 25–29. 

Genitalia (proximal parts excluded) (25), internal structure of distal genitalia (26), transverse sections of medial epiphallus (27) and basal and apical penial papilla (28, 29) of Monacha cantiana s.l. CAN-5 from Foce di Pianza (FGC 41565).

Figures 30–34. 

Genitalia (proximal parts excluded) (30), transverse sections of medial epiphallus (31, 32) and basal and apical penial papilla (33, 34) of Monacha cantiana s.l. CAN-5 Campo Cecina (FGC 41564).

Figures 35–41. 

Genitalia (proximal parts excluded) (35), internal structure of distal genitalia (36) and transverse sections of medial epiphallus (37, 39), basal and apical penial papilla (38, 40, 41) of Monacha cantiana s.l. CAN-6 from Campagrina (FGC 40322).

M. cantiana s.l. (lineages CAN-1 to CAN-6) is always distinguished from M. parumcincta by its vaginal appendix (rather long with thin-walled terminal portion and variably evident basal sac in M. cantiana; short, only occasionally with very short terminal portion and always without basal sac in M. parumcincta); vaginal-atrial pilaster (present and variably evident in M. cantiana s.l.; absent in M. parumcincta); penial papilla (central canal connected to external wall by many muscular/connective strings, internally jagged and with a sort of solid pilaster on one side in M. cantiana s.l.; central canal not connected to external wall, internally smooth or slightly jagged and almost completely filled by large invagination in M. parumcincta).

RDA with lineage constraint on the shape and size matrix (Fig. 42) showed that RDA 1 (36%, p < 0.001) separated the M. cantiana s.l. (CAN-1, CAN-2, CAN-3, CAN-4, CAN-5 and CAN-6) from PAR. The preliminary classic PCA revealed size as the first major source of morphological variation, since PC1 (54%) was a positive combination of all variables. On the contrary, RDA 2 (12%, p < 0.001) separated the group CAN-1, CAN-2, CAN-3, CAN-4 and PAR from the group CAN-5 and CAN-6. In that regard, PC2 (17%) accounted for a contrast between P and DBC vs F.

Figures 42, 43. 

Principal component analysis (PCA) and Redundancy analysis (RDA) with lineage applied to the original genitalia matrix (42) and Z-matrix (shape-related) (43).

RDA with species constraint on the shape (Z) matrix (Fig. 43) showed that RDA 1 (28%, p < 0.001) separated the group CAN-1, CAN-2 and CAN-3 from the group CAN-5 and CAN-6 with PAR and CAN-4 in intermediate position and that RDA 2 (11%, p < 0.001) separated PAR from CAN-4 with the large group CAN-1, CAN-2, CAN-3, CAN-5 and CAN-6 in intermediate position. Shape-related PCA indicated that P, E and DBC vs VA and F were the two principal shape determinants on PC1 and DBC and VA vs E, V and F on PC2. In the latter case, removing the size effect altered the overall relationship patterns.

Box plots (Fig. 44) for anatomical characters showed that F and VA have the best discriminating value (they distinguished 11 and 8 clade pairs, respectively, according to Tukey’s honestly significant difference test), followed by E and V (five and four pairs, respectively). The most recognizable pairs were CAN-5 vs PAR or CAN-6 vs PAR (four significant characters), CAN-1 vs PAR or CAN-4 vs PAR (3 significant characters) and CAN-2 vs CAN-5, CAN-2 vs PAR or CAN-3 vs PAR (2 significant characters). Only one significant character distinguished CAN-1 vs CAN-2, CAN-1 vs CAN-5, CAN-1 vs CAN-6, CAN-2 vs CAN-6, CAN-3 vs CAN-5, CAN-3 vs CAN-6, CAN-4 vs CAN-5 or CAN-4 vs CAN-6 and none distinguished CAN-1 vs CAN-3, CAN-1 vs CAN-4, CAN-2 vs CAN-3, CAN-2 vs CAN-4, CAN-3 vs CAN-4 or CAN-5 vs CAN-6 (Table 6).

Figure 44. 

Box plots for genitalia characters of the seven Monacha clades investigated. The lower and upper limits of the rectangular boxes indicate the 25^th to 75^th percentile range, and the horizontal line within the boxes is the median (50^th percentile).