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Exploration of phylogeography of Monacha cantiana s.l. continues: the populations of the Apuan Alps (NW Tuscany, Italy) (Eupulmonata, Stylommatophora, Hygromiidae)
expand article infoJoanna R. Pieńkowska, Giuseppe Manganelli§, Folco Giusti§, Debora Barbato§, Alessandro Hallgass§, Andrzej Lesicki
‡ Adam Mickiewicz University in Poznan, Poznań, Poland
§ Universitá di Siena, Siena, Italy
Open Access

Abstract

Two new lineages CAN-5 and CAN-6 were recognised in four populations of Monacha cantiana (Montagu, 1803) s.l. from the Italian Apuan Alps by joint molecular and morphological analysis. They are different from other M. cantiana lineages known from English, Italian, Austrian and French populations, i.e. CAN-1, CAN-2, CAN-3 and CAN-4, as well as from the other Italian Monacha species used for comparisons (M. parumcincta and M. cartusiana). Although a definite taxonomic and nomenclatural setting seems to be premature, we suggest that the name or names for these new lineages as one or two species should be found among 19th century names (Helix sobara Mabille, 1881, H. ardesa Mabille, 1881, H. apuanica Mabille, 1881, H. carfaniensis De Stefani, 1883 and H. spallanzanii De Stefani, 1884).

Keywords

16SrDNA, COI, H3, ITS2, molecular features, shell and genital structure, species distribution

Introduction

Study of the phylogeography of the Monacha cantiana (Montagu, 1803) s.l. by joint molecular and morphological analysis revealed a number of cryptic lineages, some of which might deserve distinct taxonomic status.

Examination of a first group of English, Italian, Austrian and French populations showed that it consisted of at least four distinct lineages (CAN-1, CAN-2, CAN-3, CAN-4) (Pieńkowska et al. 2018). One of these lineages (CAN-1) included most of the UK (5 sites) and Italian (5 sites) populations examined. Three other lineages were represented by 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). A taxonomic and nomenclatural setting is only currently available for CAN-1 and CAN-4. The lineage CAN-1 corresponds to the true M. cantiana (Montagu, 1803) because it is the only one that includes topotypical English populations. The lineage CAN-4 is attributed to M. cemenelea (Risso, 1826), for which a neotype has been designated and deposited. A definitive frame for the other two has been postponed because it requires much more research.

We have now studied some populations from the Apuan Alps at the north-western extremity of Tuscany, a well-known hotspot of diversity and endemism (Lanza 1997; Biondi et al. 2013; Garbari and Bedini 2014; Carta et al. 2017; Orsenigo et al. 2018). Molecular study revealed two more lineages (CAN-5 and CAN-6), molecularly distinct from each other and from all the others, but morphologically indistinguishable from each other and only slightly distinguishable from all the other lineages of M. cantiana.

Material and methods

Taxonomic sample

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)).

List of localities of the populations of Monacha cantiana s.l. (CAN-5 & CAN-6) used for molecular and morphological (SH shell, AN genitalia) research.

Localities Clade Revised taxonomy COI 16SrDNA H3 ITS2 PCA and RDA Figs
No. Coordinates Country and site Collector / date / no. of specimens (collection) New haplotype (no. spec.) GenBank ## New haplotype (no. spec.) GenBank ## New common sequence (no. sps) GenBank ## New common sequence (no. sps) GenBank ##
1 44°06'54.9"N 10°08'23.9"E Italy, Tuscany, Apuan Alps, Foce di Pianza (pathway from Campo Cecina to Monte Sagro), 1270 m a.s.l. A. Hallgass / 13.10.2013 / 5 / (FGC 41565) CAN-5 M. sp. COI 1 (4) MK066929 16S 1 (4) MK066947 H3 5 (3) MK066965 SH, AN 8, 9, 25–29
MK066930 MK066948 MK066966 ITS2 1 (1) MK066981
MK066931 MK066949 MK066967 ITS2 6 (1) MK066982
MK066932 MK066950 H3 6 (1) MK066968 ITS2 2 (1) MK066983
COI 3 (1) MK066933 16S 5 (1) MK066951 H3 8 (1) MK066969 ITS2 5 (1) MK066984
2 44°07'21.2"N 10°07'17.7"E Italy, Tuscany, Apuan Alps, Campo Cecina, 500 m N of Rifugio CAI Carrara, 1300 m a.s.l. A. Hallgass / 13.10.2013 / 5 / (FGC 41564) CAN-5 M. sp. COI 4 (3) MK066934 16S 6 (2) MK066952 H3 6 (1) MK066970 ITS2 10 (2) MK066985 SH, AN 10–12, 30–34
MK066953 H3 1 (1) MK066971 MK066986
MK066935 16S 7 (1) MK066954 H3 3 (1) MK066972 ITS2 5 (2) MK066987
MK066936 16S 8 (1) MK066955 H3 7 (1) MK066973 MK066988
COI 5 (1) MK066937 16S 6 (1) MK066956 H3 2 (1) MK066974 ITS2 3 (1) MK066989
3 44°05'56.8"N 10°07'08.5"E Italy, Tuscany, Apuan Alps, Piastra, 290 m a.s.l. A. Hallgass / 13.10.2013 / 5 / (FGC 41563) CAN-5 M. sp. H3 6 (2) MK066975 ITS2 11 (1) MK066990 SH, AN 6, 7, 19–24
COI 1 (3) MK066938 16S 2 (1) MK066957 MK066976 ITS2 4 (1) MK066991
MK066939 16S 3 (1) MK066958 H3 5 (1) MK066977 ITS2 12 (1) MK066992
MK066940 ITS2 2 (1) MK066993
COI 2 (1) MK066941 16S 4 (1) MK066959 ITS2 3 (1) MK066994
4 44°03'25.5"N 10°16'01.0"E Italy, Tuscany, Apuan Alps, 1 km E of Campagrina, 769 m a.s.l. A. Hallgass / 22.10.2011 / 5 (FGC 40322) CAN-6 M. sp. COI 6 (1) MK066942 16S 9 (1) MK066960 ITS2 9 (2) MK066995 SH, AN 13–15, 35–41
COI 7 (1) MK066943 16S 11 (1) MK066961 MK066996
COI 8 (3) MK066944 16S 10 (1) MK066962 H3 4 (1) MK066978 ITS2 8 (1) MK066997
MK066945 16S 12 (2) MK066963 H3 1 (1) MK066979 ITS2 13 (1) MK066998
MK066946 MK066964 H3 5 (1) MK066980 ITS2 7 (1) MK066999

Materials examined

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).

New ITS2 sequences obtained from the specimens of Monacha cantiana s.l. (CAN-2 to CAN-4) and M. parumcincta (PAR) used for molecular research. Number of localities after Pieńkowska et al. (2018). Earlier data on other sequences (COI, 16SrDNA, H3 and ITS2) from these localities were published by Pieńkowska et al. (2018).

Localities Clade Revised taxonomy ITS2
No. Coordinates Country and site Collector / date / no. of specimens (collection) New common sequence No. Spec. GenBank ##
12 45°11'59.85"N 10°58'49.30"E Italy, Venetum, Sorgà (Verona) A. Hallgass / 09.2012 / 6 (FGC 42964) CAN-2 M. cantiana ITS2 14 1 MK067000
15 44°22'09.98"N 11°15'11.28"E Italy, Emilia Romagna, along Fiume Setta, upstream its confluence with Fiume Reno (Sasso Marconi, Bologna) A. Hallgass / 09.2012 / 3 (FGC 42977) CAN-3 M. sp. ITS2 15 1 MK067001
17 48°15'25.50"N 16°30'46.38"E Austria, Breitenlee, abandoned railway station M. Duda / 09.2015 / 3 (FGC 44020) CAN-3 M. sp. ITS2 16 1 MK067002
18 43°46'11.79"N 07°22'21.50"E France, Alpes-Maritimes, Vallée de Peillon, Sainte Thecle A. Hallgass / 24.10.2011/ 5 (FGC 40320) CAN-4 M. cemenelea ITS2 17 1 MK067003
ITS2 18 1 MK067004
24 40°13'25.49"N 15°52'17.07"E Italy, Basilicata, along the road from Moliterno to Fontana d’Eboli (Moliterno, Potenza) A. Hallgass / 2012 / 5 (FGC 42962) PAR M. parumcincta ITS2 19 1 MK067005

DNA extraction, amplification and sequencing

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

Phylogenetic inference

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.

GenBank sequences used for comparison in molecular analysis.

Species COI 16SrDNA H3 ITS2 References
Monacha cantiana CAN-1 KJ458539 Razkin et al. 2015
KM247375 KM247390 Pieńkowska et al. 2015
KX507234 KX495428 Neiber and Hausdorf 2015
MG208884-MG208924 MG208960-MG208995 MG209031-MG209039 MH137963-MH137978 Pieńkowska et al. 2018
MG209041-MG209048
Monacha cantiana CAN-2 MG208925-MG208932 MG208996-MG209004 MG209049-MG209052 MH137979-MH137981 Pieńkowska et al. 2018
Monacha cantiana CAN-3 HQ204502 HQ204543 Duda et al. 2011; Kruckenhauser et al. 2014
KF596907 KF596863 Cadahia et al. 2014
MG208933-MG208938 MG209005-MG209010 MG209040 MH137982-MH137983 Pieńkowska et al. 2018
MG209053-MG209057
Monacha cemenelea CAN-4 MG208939-MG208943 MG209011-MG209015 MG209058-MG209060 MH137984 Pieńkowska et al. 2018
Monacha sp. AY741419 Manganelli et al. 2005
Monacha parumcincta PAR AY741418 Manganelli et al. 2005
MG208944-MG208959 MG209016-MG209030 MG209061-MG209071 MH137985-MH137992 Pieńkowska et al. 2018
Monacha cartusiana KM247380 KM247397 Pieńkowska et al. 2015
KX507189 KX495378 Neiber and Hausdorf 2015
MG209072 MH137993 Pieńkowska et al. 2018

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.

Combined Sequences of the following gene sequences: COI+16SrDNA and H3+ITS2 for ML analysis and of COI+16SrDNA+H3+ITS2 for Bayesian analysis.

Combined sequence COI haplotype 16S haplotype Combined sequence H3 sequence ITS2 sequence Combined sequence COI haplotype 16S haplotype H3 sequence ITS2 sequence Locality
IT-COI16S-1 MK066929 MK066947 Italy, Tuscany, Foce di Pianza
IT-H3ITS2-6 MK066966 MK066981 IT-CS-1 MK066930 MK066948 MK066966 MK066981 Italy, Tuscany, Foce di Pianza
IT-H3ITS2-7 MK066967 MK066982 IT-CS-2 MK066931 MK066949 MK066967 MK066982 Italy, Tuscany, Foce di Pianza
IT-H3ITS2-8 MK066968 MK066983 IT-CS-3 MK066932 MK066950 MK066968 MK066983 Italy, Tuscany, Foce di Pianza
IT-COI16S-2 MK066938 MK066957 IT-H3ITS2-13 MK066976 MK066991 IT-CS-4 MK066938 MK066957 MK066976 MK066991 Italy, Piastra
IT-COI16S-3 MK066939 MK066958 IT-CS-5 MK066939 MK066958 MK066977 MK066992 Italy, Piastra
IT-COI16S-4 MK066941 MK066959 Italy, Piastra
IT-COI16S-5 MK066933 MK066951 IT-CS-6 MK066933 MK066951 MK066969 MK066984 Italy, Tuscany, Foce di Pianza
IT-COI16S-6 MK066934 MK066952 IT-H3ITS2-9 MK066970 MK066985 IT-CS-7 MK066934 MK066952 MK066970 MK066985 Italy, Tuscany, Campo Cecina
IT-COI16S-7 MK066935 MK066954 IT-H3ITS2-11 MK066972 MK066987 IT-CS-8 MK066935 MK066954 MK066972 MK066987 Italy, Tuscany, Campo Cecina
IT-COI16S-8 MK066936 MK066955 IT-H3ITS2-12 MK066973 MK066988 IT-CS-9 MK066936 MK066955 MK066973 MK066988 Italy, Tuscany, Campo Cecina
IT-COI16S-9 MK066937 MK066956 IT-H3ITS2-10 MK066974 MK066989 IT-CS-10 MK066937 MK066956 MK066974 MK066989 Italy, Tuscany, Campo Cecina
IT-COI16S-10 MK066942 MK066960 Italy, Tuscany, Campagrina
IT-COI16S-11 MK066943 MK066961 Italy, Tuscany, Campagrina
IT-COI16S-12 MK066944 MK066962 IT-H3ITS2-4 MK066978 MK066997 IT-CS-11 MK066944 MK066962 MK066978 MK066997 Italy, Tuscany, Campagrina
IT-COI16S-13 MK066945 MK066963 Italy, Tuscany, Campagrina
IT-H3ITS2-5 MK066980 MK066999 IT-CS-12 MK066946 MK066964 MK066980 MK066999 Italy, Tuscany, Campagrina
UK-COI16S-1 MG208884 MG208966 UK-H3ITS2-1 MG209031 MH137963 UK-CS-1 MG208884 MG208966 MG209031 MH137963 UK, Barrow near Barnsley
UK-COI16S-2 MG208893 MG208960 UK, Rotherham
UK-COI16S-3 MG208899 MG208976 UK-H3ITS2-2 MG209038 MH137971 UK-CS-2 MG208899 MG208976 MG209038 MH137971 UK, Sheffield
UK-COI16S-4 MG208898 MG208975 UK-H3ITS2-3 MG209037 MH137969 UK-CS-3 MG208898 MG208975 MG209037 MH137969 UK, Rotherham
UK-COI16S-5 MG208891 MG208972 UK, Cambridge
IT-COI16S-14 MG208915 MG208985 IT-H3ITS2-15 MG209045 MH137973 IT-CS-13 MG208915 MG208985 MG209045 MH137973 Italy, Latium, Valle dell’Aniene, Rome
IT-COI16S-15 MG208916 MG208987 IT-H3ITS2-16 MG209046 MH137974 IT-CS-14 MG208916 MG208987 MG209046 MH137974 Italy, Latium, Valle dell’Aniene, Rome
IT-COI16S-16 MG208917 MG208989 IT-H3ITS2-17 MG209047 MH137975 IT-CS-15 MG208917 MG208989 MG209047 MH137975 Italy, Latium, Valle dell’Aniene, Rome
IT-COI16S-17 MG208905 MG208977 IT-H3ITS2-18 MG209039 MH137972 IT-CS-16 MG208905 MG208977 MG209039 MH137972 Italy, Latium, Gole del Velino
IT-COI16S-18 MG208906 MG208979 Italy, Latium, Gole del Velino
IT-COI16S-19 MG208921 MG208990 IT-CS-17 MG208921 MG208990 MG209043 MH137976 Italy, Latium, Valle del Tronto
IT-COI16S-20 MG208923 MG208994 IT-H3ITS2-19 MG209048 MH137978 IT-CS-18 MG208923 MG208994 MG209048 MH137978 Italy, Latium, Valle del Turano
IT-COI16S-21 MG208910 MG208978 Italy, Latium, Gole del Velino
IT-COI16S-22 MG208925 MG208996 IT-H3ITS2-22 MG209050 MK067000 IT-CS-19 MG208925 MG208996 MG209050 MK067000 Italy, Venetum, Sorga
IT-COI16S-23 MG208926 MG209001 IT-H3ITS2-21 MG209049 MH137979 Italy, Venetum, Sorga
IT-COI16S-24 MG208928 MG208998 Italy, Venetum, Sorga
IT-COI16S-25 MG208932 MG209003 IT-H3ITS2-20 MG209052 MH137981 IT-CS-20 MG208932 MG209003 MG209052 MH137981 Italy, Lombardy, Rezzato
IT-COI16S-26 MG208934 MG209005 IT-H3ITS2-2 MG209040 MK067001 IT-CS-21 MG208934 MG209005 MG209040 MK067001 Italy, Emila Romagna, Fiume Setta
IT-COI16S-27 MG208933 MG209007 IT-H3ITS2-3 MG209054 MH137982 IT-CS-22 MG208933 MG209007 MG209054 MH137982 Italy, Emila Romagna, Fiume Setta
IT-COI16S-28 MG208944 MG209017 IT-H3ITS2-24 MG209061 MK067005 IT-CS-23 MG208944 MG209017 MG209061 MK067005 Italy, Basilicata, Moliterno to Fontana d’Eboli
IT-COI16S-29 MG208946 MG209019 IT-H3ITS2-23 MG209064 MH137992 Italy, Basilicata, Moliterno to Fontana d’Eboli
IT-COI16S-30 MG208949 MG209020 IT-CS-24 MG208949 MG209020 MG209067 MH137987 Italy, Tuscany, Nievole
IT-COI16S-31 MG208950 MG209028 IT-H3ITS2-25 MG209068 MH137989 Italy, Tuscany, Arezzo
IT-H3ITS2-26 MG209070 MH137990 Italy, Tuscany, Arezzo
IT-H3ITS2-27 MG209062 MH137986 Italy, Tuscany, Podere Castella
AT-COI16S-1 MG208936 MG209009 AT-H3ITS2-1 MG209055 MH137983 AT-CS-1 MG208936 MG209009 MG209055 MH137983 Austria, Breitenlee
AT-COI16S-2 MG208938 MG209008 Austria, Breitenlee
FR-COI16S-1 MG208939 MG209011 FR-H3ITS2-1 MG209058 MH137984 FR-CS-1 MG208939 MG209011 MG209058 MH137984 France, Sainte Thecle
FR-COI16S-2 MG208940 MG209012 FR-H3ITS2-2 MG209059 MK067003 FR-CS-2 MG208940 MG209012 MG209059 MK067003 France, Sainte Thecle
FR-COI16S-3 MG208941 MG209013 FR-H3ITS2-3 MG209060 MK067004 FR-CS-3 MG208941 MG209013 MG209060 MK067004 France, Sainte Thecle
HU-COI16S-1 KM247376 KM247391 HU-H3ITS2-1 MG209072 MH137993 HU-CS-1 KM247376 KM247391 MG209072 MH137993 Hungary, Kis-Balaton

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).

Morphological study

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.

Results

Molecular study

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%).

Ranges of K2P genetic distances for COI sequences analysed (mean values in parentheses).

Comparison COI (%)
Within M. cantiana CAN-1 0.2–2.2 (0.8)
Within M. cantiana CAN-2 0.3 (0.3)
Within M. sp. CAN-3 0.2–1.9 (1.2)
Within M. cemenelea CAN-4 0.2–0.5 (0.3)
Within M. sp. CAN-5 0.2–1.7 (1.3)
Within M. sp. CAN-6 0.2–2.2 (1.6)
Within M. parumcincta 1.0–4.2 (3.0)
Within M. cartusiana 0.5
Between M. cantiana CAN-1 and M. cantiana CAN-2 3.3–5.1 (3.9)
Between M. cantiana CAN-1 and M. sp. CAN-3 17.6–19.2 (18.6)
Between M. cantiana CAN-1 and M. cemenelea CAN-4 17.2–18.7 (18.0)
Between M. cantiana CAN-1 and M. sp. CAN-5 16.5–18.2 (17.5)
Between M. cantiana CAN-1 and M. sp. CAN-6 18.0–19.2 (18.6)
Between M. cantiana CAN-1 and M. parumcincta 19.6–21.7 (20.7)
Between M. cantiana CAN-1 and M. cartusiana 18.9–20.5 (19.7)
Between M. cantiana CAN-2 and M. sp. CAN-3 17.8–18.2 (18.1)
Between M. cantiana CAN-2 and M. cemenelea CAN-4 18.2–18.7 (18.5)
Between M. cantiana CAN-2 and M. sp. CAN-5 17.6–18.2 (17.9)
Between M. cantiana CAN-2 and M. sp. CAN-6 18.3–19.0 (18.5)
Between M. cantiana CAN-2 and M. parumcincta 19.8–20.7 (20.2)
Between M. cantiana CAN-2 and M. cartusiana 21.4
Between M. sp. CAN-3 and M. cemenelea CAN-4 5.1–6.2 (5.6)
Between M. sp. CAN-3 and M. sp. CAN-5 13.3–14.4 (13.8)
Between M. sp. CAN-3 and M. sp. CAN-6 14.3–16.7 (15.7)
Between M. sp. CAN-3 and M. parumcincta 18.4–21.4 (19.6)
Between M. sp. CAN-3 and M. cartusiana 18.4–20.0 (19.1)
Between M. cemenelea CAN-4 and M. sp. CAN-5 14.8–15.4 (15.1)
Between M. cemenelea CAN-4 and M. sp. CAN-6 16.4–16.8 (16.6)
Between M. cemenelea CAN-4 and M. parumcincta 19.5–20.5 (19.9)
Between M. cemenelea CAN-4 and M. cartusiana 18.9–19.3 (19.0)
Between M. sp. CAN-5 and M. sp. CAN-6 12.4–14.3 (13.6)
Between M. sp. CAN-5 and M. parumcincta 17.3–20.2 (18.5)
Between M. sp. CAN-5 and M. cartusiana 20.6–21.3 (21.1)
Between M. sp. CAN-6 and M. parumcincta 17.6–19.1 (18.2)
Between M. sp. CAN-6 and M. cartusiana 17.3–17.8 (17.5)

Morphological study: shell

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 25th to 75th percentile range, and the horizontal line within the boxes is the median (50th percentile).

Results of Tukey’s honestly significant difference (HSD) test for shell and genitalia characters (in bold Tukey’s post-hoc p ≤ 0.01).

Pairs SH AH LWmH LWaH PWH SD
CAN-1 vs CAN-2 0.99624 0.80619 0.13492 0.64537 0.99057 0.63122
CAN-1 vs CAN-3 0.52140 0.28168 0.06284 1.00000 0.99999 0.22745
CAN-1 vs CAN-4 0.08096 0.59307 0.54497 0.00097 0.00582 0.34307
CAN-1 vs CAN-5 0.81752 0.99959 0.86439 0.00006 0.44707 0.99988
CAN-1 vs CAN-6 0.77627 0.80465 0.02347 0.29268 0.99992 0.08726
CAN-1 vs PAR 0.00001 0.00000 0.00009 0.00001 0.00125 0.00032
CAN-2 vs CAN-3 0.99544 0.99999 0.99929 0.77166 0.99881 1.00000
CAN-2 vs CAN-4 0.15929 0.22915 0.01822 0.55297 0.33334 0.07297
CAN-2 vs CAN-5 0.82169 0.71176 0.57227 0.84890 0.99950 0.79654
CAN-2 vs CAN-6 0.99776 1.00000 0.99993 0.99994 0.98407 0.99242
CAN-2 vs PAR 0.00365 0.00008 0.00002 0.51420 0.51214 0.00095
CAN-3 vs CAN-4 0.00643 0.04670 0.01004 0.00675 0.03910 0.01412
CAN-3 vs CAN-5 0.10929 0.23103 0.60853 0.00526 0.85885 0.48747
CAN-3 vs CAN-6 1.00000 0.99988 0.97726 0.47647 0.99950 0.98207
CAN-3 vs PAR 0.00000 0.00000 0.00000 0.00068 0.04033 0.00001
CAN-4 vs CAN-5 0.53531 0.81117 0.17929 0.96835 0.24318 0.30059
CAN-4 vs CAN-6 0.02495 0.21289 0.00350 0.68422 0.03827 0.00540
CAN-4 vs PAR 0.94161 0.19901 0.70423 0.99998 0.99243 0.94050
CAN-5 vs CAN-6 0.30662 0.70539 0.24510 0.94331 0.73973 0.19886
CAN-5 vs PAR 0.00429 0.00004 0.00001 0.97460 0.33336 0.00072
CAN-6 vs PAR 0.00009 0.00003 0.00000 0.65314 0.05065 0.00001
Pairs AD LWmW PWmW PWfW LWfW UD
CAN-1 vs CAN-2 0.69737 0.13492 0.93036 0.87269 0.31096 0.96096
CAN-1 vs CAN-3 0.31086 0.06284 0.60648 0.41696 0.21613 0.99999
CAN-1 vs CAN-4 0.50802 0.54497 0.09498 0.68052 0.97680 0.88793
CAN-1 vs CAN-5 0.96922 0.86439 0.80483 0.97841 0.92956 0.00001
CAN-1 vs CAN-6 0.64832 0.02347 0.86310 0.28589 0.01739 0.00000
CAN-1 vs PAR 0.00015 0.00009 0.00253 0.00752 0.00003 0.00000
CAN-2 vs CAN-3 1.00000 0.99929 1.00000 1.00000 0.99951 0.95368
CAN-2 vs CAN-4 0.13909 0.01822 0.07501 0.33305 0.22490 0.65706
CAN-2 vs CAN-5 0.41336 0.57227 0.53801 0.63842 0.76317 0.27349
CAN-2 vs CAN-6 1.00000 0.99993 1.00000 0.99559 0.99073 0.00493
CAN-2 vs PAR 0.00086 0.00002 0.01749 0.02031 0.00004 0.00000
CAN-3 vs CAN-4 0.03838 0.01004 0.01014 0.09468 0.21544 0.97116
CAN-3 vs CAN-5 0.11621 0.60853 0.13645 0.18479 0.82554 0.00061
CAN-3 vs CAN-6 1.00000 0.97726 1.00000 0.99741 0.83628 0.00001
CAN-3 vs PAR 0.00001 0.00000 0.00029 0.00030 0.00000 0.00000
CAN-4 vs CAN-5 0.89567 0.17929 0.58669 0.95667 0.75219 0.00034
CAN-4 vs CAN-6 0.11242 0.00350 0.04140 0.06153 0.02534 0.00000
CAN-4 vs PAR 0.78586 0.70423 1.00000 0.96612 0.13925 0.00000
CAN-5 vs CAN-6 0.35200 0.24510 0.38979 0.13051 0.16182 0.17535
CAN-5 vs PAR 0.01180 0.00001 0.19674 0.14546 0.00001 0.00000
CAN-6 vs PAR 0.00030 0.00000 0.00588 0.00062 0.00000 0.00000
Pairs DBC V F E P VA
CAN-1 vs CAN-2 0.07018 0.99978 0.78435 0.11949 0.17040 0.00083
CAN-1 vs CAN-3 0.95915 0.99932 0.98006 0.74183 0.08763 0.23114
CAN-1 vs CAN-4 0.99996 0.63222 0.22100 0.81959 0.76747 0.89555
CAN-1 vs CAN-5 0.94079 0.99983 0.00000 0.23792 0.98466 0.98588
CAN-1 vs CAN-6 0.21936 0.02524 0.00000 0.84359 1.00000 0.13261
CAN-1 vs PAR 0.95468 0.00603 0.01845 0.00032 0.98841 0.00000
CAN-2 vs CAN-3 0.59703 0.99388 0.99743 0.91922 1.00000 0.48744
CAN-2 vs CAN-4 0.22526 0.62669 0.04688 0.04004 0.04443 0.29982
CAN-2 vs CAN-5 0.01390 0.99642 0.00000 0.98147 0.55615 0.00027
CAN-2 vs CAN-6 1.00000 0.04898 0.00000 0.97601 0.52105 0.95169
CAN-2 vs PAR 0.02181 0.16528 0.84806 0.00000 0.08682 0.00000
CAN-3 vs CAN-4 0.96675 0.90393 0.11396 0.27618 0.02653 0.99623
CAN-3 vs CAN-5 0.60068 1.00000 0.00000 0.99937 0.42618 0.08653
CAN-3 vs CAN-6 0.78328 0.14420 0.00000 1.00000 0.43860 0.99411
CAN-3 vs PAR 0.64853 0.01508 0.39875 0.00006 0.04538 0.00000
CAN-4 vs CAN-5 0.99962 0.81255 0.00036 0.08838 0.48386 0.65711
CAN-4 vs CAN-6 0.37610 0.86820 0.00508 0.37200 0.91204 0.91815
CAN-4 vs PAR 0.99956 0.00208 0.00054 0.48361 0.98179 0.00000
CAN-5 vs CAN-6 0.06177 0.06806 1.00000 0.99998 0.99871 0.05266
CAN-5 vs PAR 1.00000 0.00588 0.00000 0.00000 0.82000 0.00000
CAN-6 vs PAR 0.07869 0.00001 0.00000 0.00088 0.99850 0.00000

Morphological study: anatomy

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 1934; 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 25th to 75th percentile range, and the horizontal line within the boxes is the median (50th percentile).

Discussion

Pieńkowska et al. (2018) found that M. cantiana, as usually conceived, actually consists of four distinct lineages (CAN-1, CAN-2, CAN-3 and CAN-4). Examination of a group of four additional populations from the Apuan Alps revealed two more lineages (CAN-5 and CAN-6). From a molecular point of view, they are quite distinct from each other and from all the others but from a morphological point of view they are indistinguishable from each other and only slightly distinguishable from the others.

Our present results confirm that lineages CAN-1, CAN-2 and CAN-3 can be distinguished by analysis of mitochondrial gene (COI and 16SrDNA) sequences (Figs 1, 4, 5) but not by nuclear gene (H3 and ITS2) sequences (Fig. 2). On the other hand, analysis of both nucleotide sequences (of mitochondrial and nuclear genes) showed that the CAN-4, CAN-5 and CAN-6 lineages are distinct from all the others (Figs 13). Moreover, these gene sequences clearly separated M. cantiana lineages from M. parumcincta.

Based on their studies of lepidopteran relationships, Hebert et al. (2003a, b) suggested that nucleotide sequences of the mitochondrial COI gene could be a universal tool for species distinction. This so called “barcode method” has since been widely used (Tautz et al. 2003; Hebert et al. 2004, 2013; Hajibabaei et al. 2007; Packer et al. 2009; Goldstein and Desalle 2011; Čandek and Kuntner 2015; Dabert et al. 2018; Yang et al. 2018, but see e.g.: Moritz and Cicero 2004; Taylor and Harris 2012). It has also been used to solve taxonomic problems in different gastropod families (Hershler et al. 2003; Remigio and Hebert 2003; Rundell et al. 2004; Elejalde et al. 2008; Duda et al. 2011; Delicado et al. 2012; Breugelmans et al. 2013; Proćków et al. 2013, 2014). However, a 3% threshold was established arbitrarily by Hebert et al. (2003a, b) as a marker of species distinction, and in several stylommatophoran families it proves to be much higher (Davison et al. 2009; Sauer and Hausdorf 2010, 2012; Scheel and Hausdorf 2012). Moreover, we have always stressed (Pieńkowska et al. 2015, 2018) that molecular features alone are insufficient to define species but need to be supported by anatomical features.

In light of the above, we underline that the interspecific genetic distances in COI sequences between both, CAN-5 and CAN-6, and all other lineages of M. cantiana s.l. (CAN-5 vs CAN-1/CAN-2/CAN-3/CAN-4 – 13.3–18.2%, CAN-6 vs CAN-1/CAN-2/CAN-3/CAN-4 – 14.3–19.2%; Table 5) are an order of magnitude greater than Hebert’s 3% threshold (Hebert et al. 2003a, b). It is also an order of magnitude greater than intraspecific divergence (“barcode gap”, see Hebert et al. 2004; Čandek and Kuntner 2015) within CAN-5 and CAN-6 lineages, 1.3% and 1.6%, respectively (Table 5). The analysis of mitochondrial COI and 16SrDNA sequences (Figs 1, 4, 5) are supported by the results of nuclear ITS2 and H3 sequences (Fig. 2). This suggests that CAN-5 and CAN-6 lineages taken together create a taxon separate from the other lineages of M. cantiana s.l. Despite CAN-5 differs from CAN-6 at a similarly high level (COI 12.4–14.3%) there are no morphological differences between specimens of both lineages. The speciation of CAN-5 and CAN-6 lineages therefore seems to emerge more promptly in molecular (mitochondrial gene sequences) than in morphological (shell, genitalia) features, probably because of a rapidly evolving mitochondrial genome (Thomaz et al. 1996; Remigio and Hebert 2003). As mentioned above, molecular data alone cannot be used to distinguish species. It must be supported by morphological features of shells and/or genital anatomy before any decision is made about taxonomy or nomenclature.

Statistical analysis of 12 shell and six anatomical characters showed that CAN-5 and CAN-6 cannot be distinguished from each other by morphology (no character shows statistically significant differences according to Tukey’s honestly significant difference test). They are only marginally distinct from CAN-1, CAN-2, CAN-3 and CAN-4, but clearly distinct from M. parumcincta, used for comparison: two or three characters distinguish the group CAN-5 plus CAN-6 from CAN-1, CAN-2 and CAN-3; one character distinguishes CAN-5 from CAN-4; five characters distinguish CAN-6 from CAN-4; 11–14 characters distinguish the group CAN-5 plus CAN-6 from PAR. It is possible that the small sample available for lineages CAN-4 and CAN-6 (one population for each) biased comparison of these two lineages. The best discriminant characters separating the group CAN-5 plus CAN-6 from all the other lineages are umbilicus diameter (UD) and flagellum length (F). In both cases the lineages CAN-5 and CAN-6 have the highest values (Table 7).

The best discriminant morphological characters distinguishing Monacha cantiana lineages (UD umbilicus diameter, F flagellum length).

CAN-1 CAN-2 CAN-3 CAN-4 CAN-5 CAN-6 PAR
UD mean ± S.D. 1.2 ± 0.4 1.3 ± 0.2 1.2 ± 0.4 1.0 ± 0.1 1.9 ± 0.5 2.6 ± 0.4 0.0 ± 0.0
Range 0.3–2.0 1.1–1.6 0.8–1.9 0.8–1.1 1.1–2.8 2.2–3.1 0.0–0.0
number of specimens 28 4 9 5 15 5 12
F mean ± S.D. 8.5 ± 1.5 7.6 ± 1.0 8.0 ± 1.2 10.2 ± 1.1 14.9 ± 2.5 14.9 ± 1.7 6.9 ± 1.0
range 5.3–12.2 6.2–8.9 6.2–10.0 8.9–11.4 10.8–18.7 13.6–17.8 5.4–8.2
number of specimens 23 7 9 5 15 5 11

As in the case of other lineages, the greatest bias of morphological analysis was the small sample available for lineages CAN-2, CAN-3, CAN-4 and CAN-6, which prevented a realistic account of their variability. As far as we know, this newly recognised group only occurs in the Apuan Alps and consists of two differentiated lineages (CAN-5 and CAN-6). Although examination of additional populations is desirable, intra-Apuan differentiation is also known for other organisms such as plants (Bedini et al. 2011) and animals (Zinetti et al. 2013).

Six available names have been introduced for Monacha cantiana s.l. from north-western Tuscany (see Appendix 1). The oldest, Helix anconae, was established by Issel (1872) for specimens reported from a wide area extending northward to Arenzano in Liguria and southward to island of Elba and the Maremma of Tuscany. However, all the localities quoted are in coastal and lowland Liguria and Tuscany, while the populations including the group CAN-5 plus CAN-6 are from mountain sites. This would exclude a relationship of this nominal taxon with these lineages.

All the other names were established by Mabille (1881) and De Stefani (1883–1888) for specimens collected in the Apuan Alps. Syntypes of the three nominal taxa introduced by Mabille (1881) are in Bourguignat’s collection at the Muséum d’histoire naturelle, Genève (Switzerland) (Figs 45–47). Syntypes of the two nominal taxa established by De Stefani (1883–1888) are not known and probably lost. Umbilicus diameter of the shells of the syntypes of Mabille’s species and the specimens illustrated by De Stefani is consistent for at least five of these nominal taxa with that of Monacha of the group CAN-5 plus CAN-6 (Helix sobara Mabille, 1881, Helix ardesa Mabille, 1881, Helix apuanica Mabille, 1881, Helix carfaniensis De Stefani, 1883 and Helix spallanzanii De Stefani, 1884). Mabille’s three nominal taxa have precedence over those of De Stefani, and because the former were published simultaneously in the same paper, their relative precedence can only be determined by the first revisor (ICZN 1999: Art. 24). Although all three match Monacha of the group CAN-5 plus CAN-6, the best correspondence is with Helix sobara. Nevertheless, the availability of these names for the lineages CAN-5 and CAN-6 is somewhat difficult and not immediate. These nominal taxa were only established on shell characters, but no shell character shows statistically significant differences between CAN-5 and CAN-6. Their relationships could only therefore be established by molecular study of topotypes, but unfortunately Mabille (1881) did not quote any precise collection site. In some cases, the identity and relationships of extinct taxa have been addressed and clarified through study of ancient DNA from dried tissue (e.g. Villanea et al. 2016; Vogler et al. 2016). Unfortunately, this approach is not applicable for Mabille’s syntypes because they consist only of shells devoid of any dried tissue. Thus, the case can only be solved by appeal to article 75.5 of the Code (ICZN 1999).

Figures 45–47. 

Syntypes and original labels of Monacha species from Apuan Alps established by Mabille (1881). Helix apuanica (45) (MHNG-MOLL-115981), Helix ardesa (46) (MHNG-MOLL-115982), Helix sobara (47) (MHNG-MOLL-116022) (by courtesy of E. Tardy, Muséum d’histoire naturelle, Genève, Switzerland).

However, before proposing a definitive nomenclatural taxonomic setting, it is necessary to examine other populations of the group. In the meantime, these lineages should continue to be defined informally, in order to avoid creating settings based on partial and insufficient data. This approach has also been used for other gastropods, such as Carychium minimum Müller, 1774 and Carychium tridentatum (Risso, 1826) (see Weigand et al. 2012), Ancylus fluviatilis (Müller, 1774) (see Pfenninger et al. 2003; Albrecht et al. 2006) and Rumina decollata (Linnaeus, 1758) (see Prévot et al. 2016).

Acknowledgements

We are grateful to Michael Duda (Naturhistorisches Museum Wien, Austria) for providing specimens. We thank Helen Ampt (Siena, Italy) for revising the English, Giovanni Cappelli (Siena, Italy) for taking photographs of shells, Emmanuel Tardy (Muséum d’histoire naturelle, Genève, Switzerland) for providing information and photos of Mabille’s syntypes in Bourguignat’s collection. Many thanks also to Robert AD Cameron (University of Sheffield, United Kingdom) and to Bernhard Hausdorf (University of Hamburg, Germany) for their valuable comments on the manuscript.

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Appendix 1

Nominal taxa of Monacha cantiana group established from north-western Tuscany

Helix anconae Issel, 1872: 63–65

Type locality: “[...] Montecatini, Val di Nievole, non lungi da Cecina nella Maremma toscana, nell’isola d’Elba, a Genova, ad Arenzano ed in altre località della Toscana e della Liguria.”

Type material: probably lost.

Status: listed as subspecies of Monacha cantiana by Alzona (1971).

Helix sobara Mabille, 1881: 126–127

Type locality: “in Alpibus Apuanis.”

Type material: one syntype (MHNG-MOLL-116022) is in Bourguignat’s collection at Muséum d’histoire naturelle, Genève (Switzerland).

Note: assigned to J. Bourguignat.

Status: listed as junior synonym of Monacha cantiana anconae by Alzona (1971).

Helix ardesa Mabille, 1881: 127

Type locality: “in Alpibus Apuanis.”

Type material: one syntype (MHNG-MOLL-115982) is in Bourguignat’s collection at Muséum d’histoire naturelle, Genève (Switzerland).

Note: assigned to J. Bourguignat.

Status: listed as junior synonym of Monacha cantiana anconae by Alzona (1971).

Helix apuanica Mabille, 1881: 127–128

Type locality: “in Alpibus Apuanis.”

Type material: one syntype (MHNG-MOLL-115981) is in Bourguignat’s collection at Muséum d’histoire naturelle, Genève (Switzerland).

Note: assigned to J. Bourguignat.

Status: listed as junior synonym of Monacha cantiana anconae by Alzona (1971).

Helix (Monacha) carfaniensis De Stefani, 1883: 53–54 (as “Helix carfaniensis”), 1884: 231, 1888: fig. 8.

Type locality: Serchio Valley, Vagli. De Stefani (1884: 231) stated that the type is from Serchio Valley and depicted a shell from Vagli.

Type material: probably lost.

Status: listed as junior synonym of Monacha cantiana anconae by Alzona (1971).

Helix (Monacha) carfaniensis subvar. minor De Stefani, 1883: 54 (as “subvar. minor”)

Type locality: “App[ennino]. San Pellegrino 1464 [m].”

Type material: probably lost.

Note: First reported by De Stefani (1875: 43–44) as Helix cantiana var. minor Albers.

Status: not available because this name denotes an infrasubspecific taxon.

Helix (Eulota) cemenelea forma isselii De Stefani, 1883: 55–59 (as “Helix cemenelea forma issellii”)

Type locality: see Helix spallanzanii below.

Type material: probably lost.

Status: not available because junior homonym of Helix isseli Morelet, 1872; renamed as Helix spallanzanii De Stefani, 1884.

Helix spallanzanii De Stefani, 1884: 208, 231, 1888: fig. 7.

Type locality: Apuan Alps, Vagli. De Stefani (1884: 231) stated that the type is from Apuan Alps and depicted a shell from Vagli.

Type material: probably lost.

Status: new name for Helix (Eulota) cemenelea forma isselii De Stefani, 1883, junior homonym of Helix isseli Morelet, 1872.

Status: listed as junior synonym of Monacha cantiana anconae by Alzona (1971).