Redescription of Monacha pantanellii (De Stefani, 1879), a species endemic to the central Apennines, Italy (Gastropoda, Eupulmonata, Hygromiidae) by an integrative molecular and morphological approach

Abstract Specimens obtained from ten populations of a Monacha species from the central Apennines were compared with six molecular lineages of Monacha cantiana s. l. (CAN-1, CAN-2, CAN-3, CAN-4, CAN-5, CAN-6) and two other Monacha species (M. cartusiana and M. parumcincta), treated as outgroup, by molecular (nucleotide sequences of two mitochondrial COI and 16S rDNA as well as two nuclear ITS2 and H3 gene fragments) and morphological (shell and genital anatomy) analysis. The results strongly suggest that these populations represent a separate species for which two names are available: the older Helix pantanellii De Stefani, 1879 and the junior M. ruffoi Giusti, 1973. The nucleotide sequences created well separated clades on each phylogenetic tree. Genital anatomy included several distinctive features concerning vaginal appendix, penis, penial papilla and flagellum; instead, shell characters only enabled them to be distinguished from M. cartusiana and M. parumcincta. Remarkably, populations of M. pantanellii show high morphological variability. Shell variability mainly concerns size, some populations having very small dimensions. Genital variability shows a more intricate pattern of all anatomical parts, being higher as regards the vagina and vaginal appendix. Despite this morphological variability, the K2P distance range of COI sequences between populations is narrow (0.2–4.5%), if we consider all but three of the 53 sequences obtained. This research confirmed that the species of Monacha and their molecularly distinguished lineages can only occasionally be recognised morphologically and that they have significant inter- and intra-population variability. The possibility of using an overall approach, including shell, genital and molecular evidence, was taken in order to establish a reliable taxonomic setting.


Introduction
Land snail fauna of the central and southern Apennines of Italy includes many common, widespread and diversified helicoideans, such as the geomitrids Candidula Kobelt, 1871 and Xerogyra Monterosato, 1892, the hygromiid Monacha Fitzinger, 1833, the helicids Marmorana Hartmann, 1844 andHelix Linnaeus, 1758. Despite this, their taxonomy, systematics and phylogenetics have been challenging since the early studies exclusively based on shell features. Taxonomic revisions of the second half of the 20 th century (e.g., Forcart 1965;Giusti 1973) lumped many of the earliest described taxa on the basis of a similar gross genital morphology. However, more recent investigations using protein electrophoresis/allozymes (Marmorana: Oliverio et al. 1993) and mitochondrial and nuclear gene sequences (Marmorana: Fiorentino et al. 2010;Helix: Fiorentino et al. 2016) shed new light on these variable species and radiation may explain the relationship between the lineages or clades distinguished in the Apennines.
Continuing work on the hygromiid Monacha (Pieńkowska et al. 2015(Pieńkowska et al. , 2016(Pieńkowska et al. , 2018a(Pieńkowska et al. , 2018b(Pieńkowska et al. , 2019a(Pieńkowska et al. , 2019b, we studied species living in the mountain grasslands of the central Apennines, whence came reports of three species, the widespread M. cantiana (Montagu, 1803) and the endemic M. orsini (Villa and Villa, 1841) and M. ruffoi Giusti, 1973, and a number of taxa with uncertain taxonomic status (Alzona 1971;Manganelli et al. 1995). We conducted a joint molecular and morphological study of many populations, finding many different species or their molecular lineages. However, it was difficult to draw reliable nomenclatural and taxonomic conclusions because the identity of the earliest taxa, established in the past, were often based on non-diagnostic shell characters of specimens without any precise collecting record.
A first result of our research corroborated the specific distinctness of Monacha ruffoi Giusti, 1973, of which we discovered an overlooked senior synonym: Helix pantanellii De Stefani, 1879. The aim of the present research was: 1) to investigate phylogenetic relationships of Monacha pantanellii with other Monacha species or their molecular lineages; 2) to evaluate its morphological variability; 3) to redescribe the species.

Taxonomic sampling
Ten populations of Monacha pantanellii (Table 1, Fig. 1) were considered in our analysis of their molecular and morphological (shell and genitalia structure) variability, and compared with the M. cantiana s. l. lineages (Pieńkowska et al. 2018a(Pieńkowska et al. , 2019b. The sequences deposited in GenBank were also considered for the molecular analysis (Table 2). Two other Monacha species were used for morphological and molecular comparison: M. cartusiana (Müller, 1774) and M. parumcincta (Rossmässler, 1834). Another 23 populations of M. pantanellii were studied on a qualitative morphological basis (they were not included in the statistical analysis) ( Table 3).

Material examined
New material examined is listed as follows, when possible: geographic coordinates (Lat & Long and UTM references) of locality, locality (country, region, site, municipality and province), collector(s), date, number of specimens (sh/s shell/shells; spcm/spcms specimen/specimens), and collection where material is kept in parenthesis (Tables 1, 3). The material is kept in the F. Giusti collection (FGC; Dipartimento di Scienze Fisiche, della Terra e dell'Ambiente, Università di Siena, Italy). The material used for comparison has already been described (see Pieńkowska et al. 2018a : table 1, 2019b: table 1).

Molecular study
Fifty-eight specimens representing ten population of Monacha pantanellii were used for molecular analysis (Table 1). DNA extraction, amplification and sequencing methods are described in detail in our previous paper (Pieńkowska et al. 2018a).
Two mitochondrial and two nuclear gene fragments were analysed, namely cytochrome c oxidase subunit 1 (COI), 16S ribosomal DNA (16S rDNA), an internal transcribed spacer of rDNA (ITS2) and histone 3 (H3), respectively. All new sequences were deposited in GenBank (Table 1). The COI, 16S rDNA, ITS2 and H3 sequences obtained from GenBank for comparison are listed in Table 2.
The sequences were edited by eye using the programme BioEdit, version 7.2.6 (Hall 1999, BioEdit 2017. Alignments were performed using CLUSTALW (Thompson et al. 1994) implemented in MEGA7 (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 592 bp for COI, 286 positions for 16S rDNA, 501 positions for ITS2 and 279 bp for H3. The sequences were collapsed to haplotypes (COI and 16S rDNA) and to common sequences (ITS2 and H3) 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 16S rDNA) and nuclear (ITS2 Table 1. List of localities of populations of Monacha pantanellii used for molecular and morphological research. A question mark before the geographical coordinates of the locality no. 3 denotes that the georeferencing was done a posteriori on the basis of the available information. No.  Table 3). Details of localities of other Monacha species and their molecular lineages were provided in previous papers (Pieńkowska et al. 2015(Pieńkowska et al. , 2018a(Pieńkowska et al. , 2018b(Pieńkowska et al. , 2019b.  and H3) sequences were concatenated (Table 4) before phylogenetic analysis. Finally, the sequences of COI, 16S rDNA, ITS2 and H3 were concatenated (Table 4) for Maximum Likelihood (ML) and Bayesian Inference (BI). Estimates of evolutionary divergence between the sequences of COI obtained in this study and other sequences from GenBank were conducted with MEGA7 using the Kimura two-parameter model (K2P) (Kimura 1980). The analysis involved 83 nucleotide sequences. All positions containing gaps and missing data were eliminated. There was a total of 615 positions in the final dataset.

Localities
Maximum Likelihood (ML) analyses were then performed with MEGA7. Monacha cartusiana and Monacha parumcincta were added as outgroup species in each analysis. For ML analysis of concatenated sequences, the following best nucleotide substitution models were specified according to the Bayesian Information Criterion (BIC): HKY+G+I (Hasegawa et al. 1985, Kumar et al. 2016 for COI and 16S rDNA concatenated sequences of 878 positions (592 COI + 286 16S rDNA), T92+G+I (Tamura 1992, Kumar et al. 2016) for ITS2+H3 concatenated sequences of 780 positions (501 ITS2 + 279 H3), and T92+G+I for COI+16S rDNA+ITS2+H3 concatenated sequences with a total length of 1658 positions (592 COI + 286 16S rDNA + 501 ITS2 + 279 H3). Bayesian analysis was conducted with MRBAYES 3.1.2 (Ronquist and Huelsenbeck 2003) using the evolution model already used for ML calculation. 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  Table 1 because they were not included in the molecular and statistical morphological analysis (apart from additional morphological analysis of M. cartusiana). A question mark before the geographical coordinates of some localities denotes that the georeferencing was done a posteriori on the basis of the available information.

Morphological study
One hundred and thirty-four specimens representing M. pantanellii, M. cantiana s. l., M. parumcincta and M. cartusiana were considered to investigate shell variability between these four species (including six molecular lineages of M. cantiana s. l.) (see Table 1 and Pieńkowska et al. 2018aPieńkowska et al. , 2019b; the 43 specimens of nine populations of M. pantanellii (Fio1, Val, Sab, Alt, Lor, Tur2, Tur1, Car and Ani, see Table 1) were also considered to investigate shell variability between specimens of these populations. Shell variability was analysed randomly choosing five adult specimens from each population, when possible. Twelve shell variables were measured to the nearest  fig. 1). One hundred and thirty-five specimens of M. pantanellii, M. cantiana s. l. (with its six molecular lineages), M. parumcincta and M. cartusiana were analysed to examine anatomical variability between species; the 50 specimens of ten populations of M. pantanellii were also considered to investigate genital variability between populations of this species (see Table 1 and Pieńkowska et al. 2018aPieńkowska et al. , 2019b. 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 (also known as mucous 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, PP penial papilla (also known as glans), V vagina, VA vaginal appendix (also known as appendicula), VAS vaginal appendix basal sac, VS vaginal sac (only present in M. cartusiana; see Pieńkowska et al. 2015: figs 11, 12), VD vas deferens. Seven anatomical variables (DBC, E, F, P, V, VS, VA) were measured under a light microscope (0.01 mm) using callipers (see: Pieńkowska et al. 2018a: 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 shape-related Z-matrices, are described in a previous paper (Pieńkowska et al. 2018a).
Differences between species for each shell and genital character were assessed through box-plots and descriptive statistics. Overall significance of differences was obtained using the Kruskal-Wallis test; when the test proved significant, multiple comparisons between pairs of species were performed using Dunn's test. In order to control the false discovery rate (FDR), the Benjamini-Hochberg correction was used to adjust P-values for multiple comparisons. The dunn.test function with the altp = TRUE option and α = 0.01 in the dunn.test R package were used for analysis (Dinno 2017).
K2P genetic distances between COI haplotypes are summarised in Table 5

Morphological study: shell
Monacha pantanellii has a globose to sub-globose shell, variable in size, colour, and presence of paler subsutural and peripheral bands, with roundish to oval slightly descending aperture, a brownish peristome and a very small to small umbilicus (Figs 5-31). RDA with species or molecular lineage constraint on the shape and size matrix (Fig. 32) showed that RDA 1 (33%, P < 0.001) separated all the species or molecular lineages from PAR. The preliminary classic PCA revealed size as the first major source of morphological variation, since PC1 (70%) was a positive combination of all variables. On   the contrary, RDA 2 (7.3%, P < 0.001) slightly separated CAN-1, CAN-2 and CAN-3 from CAN-4, CAN-5, CAN-6 and PAN with PAR in intermediate position. In this regard, PC2 (13%) mostly accounted for contrast between LWmH vs. LWaH and PWH. RDA on the shape (Z) matrix (Fig. 33) showed a hazier separation of species or molecular lineages, confirming that size is a major source of morphological variation, although both RDA axes proved to be significant. In particular, RDA 1 separated CAR, CAN-5, CAN-6 from PAR, CAN-1 and CAN-3, with the other groups in a more or less intermediate position. Conversely, RDA 2 separated PAR and CAR from all the other species or molecular lineages. Shape-related PCA indicated that SH, LWaH and PWH vs. LWfW were the principal shape determinants on PC1 and PWmW, AH and AD vs. UD on PC2.
Box plots (Fig. 34) proved that the shell characters only have discriminating value in distinguishing Monacha pantanellii from other species or molecular lineages in a few cases. In fact, according to Dunn's test with Benjamini-Hochberg adjustment (α = 0.01), no character significantly distinguished PAN from CAN-1, CAN-2 and CAN-4, only one distinguished it from CAN-5 (UD), only two from CAR (LWah, PWH), four from CAN-6 (SD, LWmW, LWfW, UD), six from CAN-3 (SH, AH, SD, AD,  LWmW, PWmW) and eight from PAR (SH, AH, SD, AD, LWmW, PWfW, LWfW, UD) (Table 6). RDA with population constraint on the shape and size matrix (Fig. 35) [Val]. On the contrary, RDA 2 (4.0%, P > 0.05) showed no significant separation of populations. The preliminary classic PCA revealed size as the first major source of morphological variation, since PC1 (67.0%) was a negative combination of all variables. RDA on the shape (Z) matrix (Fig. 36) showed no significant separation between populations, again confirming that size is a major source of morphological variation. Shape-related PCA indicated that LWaH and PWH vs. LWfW were the two principal shape determinants on PC1 and AH vs. LWmH on PC2.

Morphological study: anatomy
Monacha pantanellii has distal genitalia very similar to those of the Monacha cantiana group. The most remarkable features are the usually short vaginal appendix with mid or proximal vaginal insertion, the long flagellum and the penial papilla with thick external wall bordering a central duct without strips joining it to the external wall and with a lumen filled by many variably sized pleats (Figs 37-63).
RDA with species or molecular lineage constraint on the shape and size matrix (Fig. 64) showed that RDA 1 (27%, P < 0.001) separated M. cantiana s. l. (CAN-1, CAN-2, CAN-3, CAN-4, CAN-5 and CAN-6) from PAN, PAR and CAR. The preliminary classic PCA revealed size as the first major source of morphological variation, since PC1 (43%) accounted for VS vs. all the other variables. On the contrary, RDA 2 (22%, P < 0.001) separated CAN-5, CAN-6 and PAN from CAR and PAR. The group CAN-1, CAN-2, CAN-3 and CAN-4 was in intermediate position. In that regard, PC2 (20%) accounted for a contrast between E and VA vs. F, P, V and VS.
RDA with species or molecular lineage constraint on the shape (Z) matrix (Fig. 65) showed that RDA 1 (43%, P < 0.001) separated PAN from the group CAN-1, CAN-2, CAN-3, CAN-4 and CAN-6, with CAN-5, PAR and CAR in intermediate position, and that RDA 2 (20%, P < 0.001) separated CAR from all the others. Shape-related PCA indicated that VA and E vs. V and P were the principal shape determinants on PC1 and VS and V vs. DBC and F on PC2. In the latter case, removing the size effect altered the overall relationship patterns.  Box plots (Fig. 66) for anatomical characters showed that VA, F and P have the best discriminating value in distinguishing PAN: they distinguished 6 (VA) and 5 (F and P) species or molecular lineage pairs, respectively, according to Dunn's test with Benjamini-Hochberg adjustment (α = 0.01), followed by E and V with four and three species or molecular lineage pairs, respectively ( Table 6). The most recognisable pairs were PAN vs. CAR and PAN vs. CAN-1 (four significant characters), PAN vs. CAN-2, PAN vs. CAN-3, PAN vs. CAN-5 and PAN vs. PAR (3 significant characters). Only two characters significantly distinguished PAN vs. CAN-4 and only one PAN vs. CAN-6 ( Table 6). Anatomical characters have high discriminating value as testified by very low p values after Dunn's test: in most cases (19 of 22) P < 0.001 (Table 6). RDA with population constraint on the shape and size matrix (Fig. 67)    RDA on the shape (Z) matrix (Fig. 68) showed a less clear separation between populations. RDA 1 (43%, P < 0.001) separated the population Sab from the group of populations Tur2, Val, Alt and Lor, with Ani, Fio1, Fio2 and Car in intermediate   position. Shape-related PCA indicated that V vs. F were the two principal shape determinants on PC1 (39.5%). RDA 2 (14%, P < 0.001) separated Tur2 from Alt, Lor and Fio1, with all the other populations in a more or less intermediate position. In that regard, PC2 (24.5%) accounted for a contrast between PD and VA.

Discussion
Molecular analysis of nucleotide sequences obtained from specimens originating from ten populations occurring in the grasslands of the central Apennines suggests that these populations represent a different species from other Italian M. cantiana s. l. lineages (CAN-1, CAN-2, CAN-3, CAN-5, CAN-6) and Monacha species (M. cartusiana and M. parumcincta), populations of which were previously subject to molecular analysis (Pieńkowska et al. 2018a(Pieńkowska et al. , 2019a(Pieńkowska et al. , 2019b. In each of the phylogenetic trees, i.e., ML of concatenated sequences for mitochondrial COI+16S rDNA (Fig. 2) and nuclear ITS2+H3 (Fig. 3) gene fragments as well as the BI tree of concatenated sequences COI+16S rDNA+ITS2+H3 (Fig. 4), sequences from these ten populations created well separated monophyletic clades. Two of these populations represent species described in the past: Monte Fionchi, Summit [Fio1]: Helix pantanellii De Stefani, 1879;Vallonina [Val]: Monacha ruffoi Giusti, 1973. Molecular analysis confirmed the validity of the species described by Giusti (1973) from the Reatini Mountains, although an older discarded name, introduced by De Stefani (1879), turned out to be available for it.
The range of K2P genetic distances between COI sequences obtained from the ten populations of M. pantanellii was 0.2-6.7% (Table 5). We previously found a similar range of K2P distances within populations of M. cantiana s. l. CAN-1/ CAN-2 (0.2-5.3%; Pieńkowska et al. 2018aPieńkowska et al. , 2019b, M. cartusiana (0.0-3.3%; Pieńkowska et al. 2015Pieńkowska et al. , 2016Pieńkowska et al. , 2018b, M. parumcincta (0.2-4.6%; Pieńkowska et al.  Pieńkowska et al. 2015Pieńkowska et al. , 2016Pieńkowska et al. , 2018b. It is worth noting that this K2P distance range was even narrower (0.2-4.5%) if we considered all but three of 53 the COI sequences obtained from M. pantanellii specimens. The three COI sequences excluded were found in single (one or two) specimens of populations from Carsoli [Car], Valle del Turano [Tur1] and Vallonina [Val], however COI sequences obtained from the other specimens of these populations were more similar to others found in M. pantanellii. This suggests higher intrapopulation variation within these three populations, which may prove speciation seen in a rapidly evolving mitochondrial genome (Thomaz et al. 1996;Remigio and Hebert 2003).
The conclusion that ten populations from the central Apennines form a different species is supported by the analysis of K2P genetic distances of COI sequences (Table 5). Although the utility of the 3% barcode threshold as a marker for species distinction, applied in the so-called "barcode method" based on COI sequences (Hebert et al. 2003a(Hebert et al. , 2003b(Hebert et al. , 2018Pentinsaari et al. 2020), is disputable (Davison et al. 2009;Hausdorf 2010, 2012;Köhler and Johnson 2012;Batomalaque et al. 2019;Koch et al. 2020), COI sequences have been used to analyse taxonomic problems in different gastropod families (e.g., Remigio and Hebert 2003;Elejalde et al. 2008;Duda et al. 2011;Breugelmans et al. 2013;Proćków et al. 2013Proćków et al. , 2019Čandek and Kuntner 2015;Walther et al. 2016;Kruckenhauser et al. 2017;Galan et al. 2018;Gladstone et al. 2019;Harl et al. 2019;Kneubühler et al. 2019;Bamberger et al. 2020). They were also useful in our previous studies on Monacha species (Pieńkowska et al. 2015(Pieńkowska et al. , 2018a(Pieńkowska et al. , 2019a(Pieńkowska et al. , 2019b. Indeed, we have always emphasised that phylogenetic analysis cannot be based on a single gene locus but should combine several mitochondrial and nuclear genes (Pieńkowska et al. 2015(Pieńkowska et al. , 2018a(Pieńkowska et al. , 2019a(Pieńkowska et al. , 2019b. Note that the conclusion that ten populations are distinct from other Monacha species at species level is not only supported by the analysis of COI sequences, but also of 16S rDNA, ITS2, and H3. Moreover, we have always stressed (Pieńkowska et al. 2015(Pieńkowska et al. , 2018a(Pieńkowska et al. , 2019a(Pieńkowska et al. , 2019b) that molecular features alone are insufficient to define species but need to be supported by morphological (shell and anatomy) features. Inconsistency between molecular and morphological features may occur among snail populations or species (Cameron 1992;Cameron et al. 1996;Sauer and Hausdorf 2012;Falniowski et al. 2020), because according to the concept of morphostatic evolution (Gittenberger 1991;Davis 1992;Koch et al. 2020) speciation may be reflected earlier in molecular than in morphological features.
It is not possible to distinguish M. pantanellii from the lineages of the M. cantiana group on the basis of shell characters, perhaps with the exception of CAN-6 (see Figs 32-34; Table 6). However, this may be biased by the fact that only one population of this lineage was available for study (Pieńkowska et al. 2019b). With regard to the other two species examined by comparison, M. cartusiana and M. parumcincta, the analysis found that distinguishing M. pantanellii from the former is difficult (only two characters have discriminating value), but from the latter is easy (eight characters have discriminating value). Anyway, these species are readily distinguished by colour pattern. M. cartusiana has a smoother more glossy shell, usually whitish, often with sharp milky-white subsutural and peripheral bands, intensely reddishbrown peristome, externally bordered by a ring of bright milky white. M. parumcincta has a shell similar to that of M. pantanellii, but less glossy and more opaque, sometimes with paler peripheral and subsutural bands and brownish peristome, externally bordered by a pale whitish ring.
The  (Table 6). Other anatomical features that distinguish M. pantanellii from the M. cantiana group, M. cartusiana and M. parumcincta were not included in the analysis, since it is impossible to quantify them. They are the insertion of the vaginal appendix, the shape of the vaginal appendix, and the section of the penial papilla (Table 7).
Intraspecific variability in M. pantanellii is high and concerns both shell and genitalia. Inter-population shell variability mainly affects the size features: some populations are distinguished by reduced size, notably the one from Carsoli [Car] (Figs 30, 31) and the slightly larger populations from Turania [Tur1] (Figs 11-14) and Vallonina [Val] (Figs 21, 22). This pattern was confirmed by RDA on the original shell matrix (Fig. 32) and by its disappearance when the size effect was removed (Fig. 33). Anatomically, these populations agree very well with the characters typical of the species (e.g., VA, PD, F) suggesting that shell size has no phylogenetic signal and cannot be used to support taxonomic distinctions. We can hypothesize that it depends on local conditions of drought, food availability and lack of refuges.
Intra-population shell variability is smaller, but the variation of UD from Via Salaria, Ornaro Alto [Alt] is notable (0.9-2.4 mm) including almost the extremes of the range (Figs 18-20).
According to RDA on the shape (Z) matrix, some of the most divergent populations are those from Montenero Sabino [Sab] and Lago del Turano [Tur2], which fall at the extremes of the ordination figure (Fig. 68).
This revision is the first result of research on the Monacha species living in the mountain grasslands of the central Apennines. It confirms the validity of the species described by Giusti (1973) from the Reatini Mountains, though an older discarded name, introduced by De Stefani (1879), turned out to be available for it.
It is evident from the above discussion that the species of Monacha and the lineages of M. cantiana s. l. can only occasionally be recognised morphologically and are also subject to significant inter-and intra-population variability. In this situation, revision based on type material consisting of shells may be not decisive. We therefore took an overall approach that considers shell, genital and molecular evidence to establish a reliable taxonomic setting. Only a multidisciplinary investigation of populations from the type locality, matching type specimens, can clarify the identity of old established Monacha taxa. This what we tried to do, although it was made difficult by the fact that the type locality was not always reported in a detailed way. Luckily this was not the case of the species described by De Stefani (1879). Thus, the investigation of specimens from the type locality, the summit of Monte Fionchi near Spoleto in Umbria, enabled us to ascertain that they have the same anatomical features as M. ruffoi. Conspecificity of the topotypical populations of M. pantanellii and M. ruffoi is also strongly supported by molecular analysis. Consequently, the latter has to be regarded as a junior synonym of De Stefani's species.
Since M. pantanellii is a Monacha species with distinctive anatomical features, we checked all the material accessible to us. This enabled us to find other populations of the species, some from the Reatini Mountains, where they were collected by one of us in the 1960s during field work, some from other more northern mountain ranges (Table 3).
Regarding relationships of M. pantanellii with other taxa described or reported from the central Apennines, research is underway. So far we can only reveal that they belong to lineages different from this species and the M. cantiana group.

Diagnosis.
A species of Monacha (s. str.) (according to the subgeneric division proposed by Neiber and Hausdorf 2017) with vaginal appendix usually short and slender (having shape and size of a digitiform gland) inserted at mid vagina; proximal vaginal sac absent; penial flagellum long to very long; penial papilla with narrow space between external walls and central duct; central duct circular in transverse section, usually rather small in diameter, not joined by strips to external walls and with its lumen almost totally filled by large pleats.
Male distal genitalia (Figs 37-63, Table 7) include vas deferens, flagellum, epiphallus and penis. Vas deferens very long and very slender. Flagellum long to very long and slender. Epiphallus long to very long and wide. Penis short and wide, enveloped by thin sheath, consisting of proximal portion (from start of penial sheath to base of penial papilla) and distal portion (from base of penial papilla to genital atrium). Penial papilla variable in shape (perhaps due to pre-mortem stress or spirit fixation), with apical opening, thick external walls and narrow space between external walls and central duct; central duct circular in section, usually rather small in diameter, not joined by strips to external walls of penial papilla and with its lumen almost totally filled with large pleats.
Genital atrium large, receiving vagina and penis, internally smooth or with variably developed longitudinal pleats. Type locality. "Sulla cima del Monte Fionghi al sud di Spoleto a circa mille metri sul livello del mare ", i.e., on the summit of Monte Fionchi, south of Spoleto, at an altitude of ca. 1000 m (municipality of Spoleto, province of Perugia), UTM references 32T UH 1726, Lat and Long: 42°40.455'N, 12°46.340'E.
Etymology. Named after Dante Pantanelli , Italian palaeontologist and geologist at the University of Modena. He published many papers on Miocene and Pliocene molluscs, some of which were co-authored by his friend Carlo De Stefani (1851-1924. He was also the secretary of the Italian Malacological Society and the editor of the Bullettino della Società Malacologica Italiana for many years (Manganelli et al. 2017, with references).
Giusti's species was named after Sandro Ruffo (1915-2010), a major Italian twentieth-century zoologist and director of the Museo Civico di Storia Naturale di Verona for many years (Latella 2011).
Distribution. Endemic to Umbria-Marche Apennines and Latium Sub-Apennines. It occurs from the Apennines of Gualdo Tadino in the north to the Aniene and Turano valleys in the south.
Ecology. Mesophile species living among grass in open habitats such as grasslands, pastures, forest edges and clearings in hill and mountain areas.
Conservation. Apparently common and widespread species within its range, but in some sites (e.g., Vallonina) it was no longer found during a field survey in the summer of 2019. Like other mesophilic species it could be sensitive to global warming.
Remarks. This species was distinguished from Monacha cantiana on the basis of a few shell characters ("more depressed, more fragile and paler shell, with fine growth lines, less rounded opening and deeper umbilicus") and was disregarded by its author as an "extreme variety" of the former. Subsequently it was only reported in two catalogues by Westerlund (1889: 95) and Pilsbry (1895: 266) so that when Alzona prepared the catalogue of Italian non-marine malacofauna, they included it as a doubtful species (Alzona 1971: 183).
On the contrary, our analysis showed that it matches a valid species, currently known as Monacha ruffoi, described from the Reatini mountains by Giusti (1973) as a Monacha species with a shell resembling that of cantiana, but with a much smaller vaginal appendix. This is an unexpected result: indeed, De Stefani's species is one of thousands of mollusc species established since the second half of the nineteenth century on the basis of very few shell features of no diagnostic value due to dramatic intra-and interpopulation variability. In describing thousands of species and varieties, past authors hit on some that remained valid.