New records of Plagyrona Gittenberger, 1977 (Gastropoda: Eupulmonata: Valloniidae) from Europe and problems about specific determination

the existence of two distinct species is not evident at all, at least in the Mediterranean countries.


INTRODUCTION
The genus Plagyrona Gittenberger, 1977, is one of the six European genera of small terrestrial molluscs belonging to the circumglobal family of the Valloniidae Morse, 1864, which includes about 65 species (Schileyko 1998).The genus Plagyrona includes two species only: P. placida (Shuttleworth, 1852) and P. angusta Holyoak and Holyoak, 2012.
In Cianfanelli et al. (2013) a synthesis of the knowledge on the distribution of P. placida was carried on, reporting the species collected for the first time in several Italian localities (Sardinia, Campania and Calabria); subsequently Manganelli et al. (2015Manganelli et al. ( , 2017) ) discovered populations of Plagyrona in the Tuscan Archipelago (Capraia, Elba, Montecristo, Giglio islands) and from Monte Argentario in Tuscany, always identifying them as P. placida.This research has ascertained a wider distribution for this genus, which was found in Balearic Islands in Spain (Mallorca Island), in southern France (Var department), and in some small Italian islands (Zannone and Pantelleria).The characters that distinguish the two species are not constant and therefore a morphometric study has been carried out for the specific attribution, in order to verify the diagnostic characters and update the geographical distribution of these taxa.

MATERIALS AND METHODS
Shells were collected in Mediterranean areas of Spain, France, Italy and Greece, from alluvial debris or litter; they were sieved using decreasing mesh sieves and the specimens were visually separated or through a binocular microscope.The photographs of the shells were taken with the light microscope and related software.All dimensions (H, shells height; D, shells diameter; h, aperture height; d, aperture diameter; U, umbilicus diameter) were measured using a micrometer on the light microscope.For the analysis of morphometric data (ratios between the dimensions of the shell for describing the differences in shape between the two species: H/D, h/d, U/d, U/H) the Excel software 2016 (for linear regression curves, for the purpose of verifying the isometric or allometric model of growth, verifying the best regression curve in order to apply a correction value to the rations of the parameters for the specific determination on the basis of the results in literature) and PAST 3.19 (for Principal Components Analysis, PCA, for the purpose of verifying the weight of the diagnostic parameter and how clustering the specimens/populations, and Non-metric multidimensional scaling, NMDS, for grouping together the objects (or variables), based on their similarity or correlation, so that the elements of a group are as similar as possible to each other) were used (Hammer et al. 2001).Data for PCA and NMDS were normalized.
The collection data are listed as follows: locality, collection site, altitude, municipality and province/department/region in parentheses, UTM coordinates (ED 50), denomination of the SCI (a Site of Community Importance, as defined in the European Commission Habitats Directive, 92/43/EEC), if the locality is inside a protected area, collectors and dates, number of shells and collection in parentheses.Names of the localities were taken from the following maps: for Italy, from the Istituto Geografico Militare 1:25,000 chart; for France, from Institut Géographique National 1:25,000; for Spain, Mallorca, from Kompass 1:75,000 map; for Greece, from the Greek Atlas 1:300,000, Studio F.M.B. Bologna 1991/92 edition; UTM coordinates were taken from the same maps or detected by GPS.

Description
Shell (Fig. 1) very small (0.8-1.6 mm in height (H); 1.1-1.9mm in diameter (D); 0.2-0.4mm in umbilicus diameter (U); mean ratio H/D = 0.729 and mean ratio U/D = 0.164; according to Holyoak and Holyoak 2012, fig.2), with three to four convex and slowly expanded whorls, separated by a very deep suture; spire rather elevated; last whorl not wide and slightly descending near the aperture.Protoconch not protruding, with the surface covered by many thin spiral striae and spiral groves, crossed with more spaced and less marked growth lines; teleoconch covered by dense periostracal ribs, clearly visible and equal to each other, and by thin spiral lines, sometimes scarcely visible.Aperture roundish, with oblique outer peristome, not thickened and not reflected, interrupted in the parietal portion.Umbilicus small, corresponding to 1/6 of the maximum shell diameter.Periostracum light brown in colour, with weakly whitish bands, more evident in not fresh shells.
Plagyrona placida (Shuttleworth, 1852) For literature before 2012, see Cianfanelli et al. (2013).Description Shell (Fig. 2) very small (0.7-1.6 mm in height (H); 1.0-2.3mm in diameter (D); 0.2-0.6 mm in umbilicus diameter (U); mean ratio H/D = 0.651 and mean ratio U/D = 0.229; according to Holyoak and Holyoak, 2012, fig.2), with three to three and a half convex and slowly expanded whorls, separated by a deep suture; spire not elevated; last whorl little wide and slightly descending near the aperture.Protoconch not protruding, with the surface covered by many thin spiral striae and spiral groves, crossed with more spaced and less marked growth lines; teleoconch covered by dense periostracal ribs, clearly visible and equal to each other, and by thin spiral lines, sometimes scarcely visible.Aperture roundish, with oblique outer peristome, not thickened and not reflected, interrupted in the parietal portion.Umbilicus large, corresponding to 3/10 of the maximum shell diameter.Periostracum light brown in colour, with weakly whitish bands, more evident in not fresh shells.Cianfanelli & E. Talenti leg.30/04/2013 (1 shell, 1 young shell, MZUF GC/43241, Fig. 2 A).

Habitat
The research was conducted by collecting organic substrate (litter or alluvial debris), where no alive animals were found, but only empty shells were extracted.Despite this, it was ascertained that the material was freshly deposited.The shells originated from the habitats near the collection sites, even if it was not possible to define with certainty the microhabitat where the specimens lived.The literature reports cases where the species have been found alive (Holyoak and Holyoak 2012): in rocky habitats or trees, trunks and branches covered by mosses, in the Mediterranean scrub, in holm oak forests or in mesophilic deciduous forests.Even in Italy, Plagyrona has almost always been found in intact environments, some of which are included in Sites of Community Interest (SCI), in Campania, in the Alburni Mountains and in the Cilento, in the Mediterranean scrubs with vast limestone rocky outcrops, and mostly in the mature holm oaks both on Zannone and Pantelleria islands.For example, on the highest elevation in Pantelleria, Montagna Grande (Fig. 3), there is a wet holm oak, with a microhabitat formed by mosses and lichens abundant on the trunks and branches, which recalls that of the laurel forests in Macaronesia islands.

Distribution and biogeography
The new data extend the known distribution of the genus Plagyrona already recorded in Cianfanelli et al. (2013) and confirm a Mediterranean distribution, stretching west to Macaronesia (chorotype according to Vigna Taglianti et al. 1999) (Fig. 4).From a biogeographic point of view, its presence in the islands of the Tuscan Archipelago and in Monte Argentario (Manganelli et al. 2015(Manganelli et al. , 2017) ) and, based on our data, on Mallorca, in south France, and in Zannone and Pantelleria islands is very interesting.In fact, the presence of Plagyrona in islands -especially of small dimensions -implies a good ability to colonize new areas; this genus is probably transported by birds, as already demonstrated for the genus Balea Gray, 1824 (Preece and Gittenberger 2003), with species that shares the same habitat of Plagyrona, i.e., trunks of big trees and branches covered with mosses.
Figure 5. Principal Components Analysis of the ratios between the morphometric parameters of the shells in the populations of Plagyrona spp.For populations code see Table 1.

Talenti et al., 2020
Biogeographia 35: 1-15 9 Remarks about species identity While for the historical notes we refer to Holyoak and Holyoak (2012) and to Cianfanelli et al. (2013), a thorough examination of the data given by Holyoak and Holyoak (2012) and a comparison of the specimens from the various populations for a definition of the diagnostic characters, which may allow distinguishing the two species, is necessary.
Holyoak and Holyoak (2012) affirm that the shells of the two species are always identifiable if exceeding 1.5 mm in breadth and the two species can live sympatrically without showing any intermediate specimens.
According to Holyoak and Holyoak (2012), the discriminating characteristics between the shells of the two species are: Due to the lack of an evident growth stop in adult shells, the D max character is useful to recognize adult shells of P. placida, since this species reaches larger dimensions; for P. angusta this parameter is less useful and can be used only if it is possible to examine several shells, probably including adult specimens.The depth of the sutures is difficult to quantify, while the relationships between H, D and U are characters that are useful for the morphometric analysis.
By processing the relations with the Principal Components Analysis, the percentage of the variance is 88.1 on the first axis, 6.4 on the second and 5.4 on the third.The parameters that most influence the PCA are the ratios U/D (loading = 0.6501) and U/H (loading = 0.75279) on the first axis (perhaps may be weekly correlated with the dimensions of the shells, see below, regression curve), h/d (loading = 0.82443) on the second axis and H/D (loading = 0.68041) on the third axis.Therefore, the ratios relating to the diameter of the umbilicus would be the parameters of greater importance than the ratios relating to the height/diameter of the shell or of the opening.Figure 5 shows a prevalent distribution of the points on positive values of the first axis (broad umbilicus with respect to the diameter or height of the shell), such as those from the Perdasdefogu population in Sardinia of and Lao River in Calabria, while on negative values of PC1 and positive of PC2 (narrow umbilicus and greater height of the mouth or of the shell) are located the populations from Kerkyra, Greece and from Var, France.However, for the populations represented by a certain number of specimens, a strong dispersion is noted and therefore variability in relationships.Moreover, there is no clear concentration of the points that can confirm the existence of two or more distinct species.Even through Non-metric Multidimensional Scaling, the result is similar.
Based on the data of Holyoak and Holyoak (2012), the H/D and H/U ratios do not exactly follow a linear regression curve, but the equation that best correlates the trend is represented by an exponential curve (R² always higher than a straight line, in both cases, for both species) (Fig. 6).Therefore, the H/D and H/U ratios are not constant, but vary with the size of the shell, according to the exponential formula: H = 0.3088e 0.8224D and H = 0.0531e 0.9879U for P. angusta, H = 0.3526e 0.6578D and H = 0.0756e 0.9286U for P. placida.For each specimen, calculating on the basis of these exponential formulas, from the parameters "diameter D" and "umbilicus U", the theoretical dimension H for P. angusta and for P. placida, and comparing this value with the real dimension H, it is possible to evaluate the deviation respecting the theoretical regression curves, and therefore have an indicative value, useful for the attribution of the specimen to one or the other species.In Table 1 the deviation refers to the middle point between the two curves and it is expressed in percentage (%), with respect to the distance between the curve that represents the average between P. angusta and P. placida, with the curves of the respective species, with positive values if closer to P. placida, negative if closer to P. angusta.So a value of 0% means that the parameter H (or the parameter U) falls exactly in the middle between the curves of the two species, + 50% if it falls closer to the curve of P. placida ¼ than compared to ¾ from P. angusta, + 100% if it falls exactly in the curve of P. placida, + 200% if it falls beyond the curve of P. placida at a distance equal to twice the distance between the two curves (and therefore much more distant from the curve of P. angusta).Similarly, but on negative % values, if the H and U parameters are closer to the P. angusta curve.
Examining the results (Table 1), since the H/D and H/U correlations do not follow a linear trend but vary with size, some relationships give a very different result based on the exponential correlation.For example, the average H/D between the populations of Mallorca and Sardinia (Perdasdefogu) has the same value, while the result, based on the exponential correlation, attributes the two populations to two different species.However, there is a strong variability within populations, with values often close to the value limit between the two species or also discordant between the two series of H/D and H/U ratios.Whereas the specimens are quite numerous, and there are no strong variations in the parameters, it is possible to attribute them: to P. angusta for populations from Var, Kerkyra, Mallorca (although these are a little less high) and for some specimens from Campania (San Michele Arcangelo and Bianco River); to P. placida for Perdasdefogu (Sardinia) and Lao River (Calabria) specimens.Where the values of the two parameters denote a concordance and therefore the attribution to one of the two species, on the other side, in many instances, the variability or the intermediate situation do not allow the certain identification.In these cases, it would be necessary to examine additional specimens in order to better study the variability or to check whether the two distinct taxa are present in sympatry.
However, it is important to note that both the PCA analysis and the analysis using the regression curves have provided comparable results.It would also be important to verify other populations, such as those of the Tuscan Archipelago, where, at least for Capraia island, based on the published illustrations (Manganelli et al. 2015), the shape of the shell would seem to belong to P. angusta rather than P. placida.Table 1.Code and site of the populations of Plagyrona listed in this paper (*: listed in Cianfanelli et al. 2013), with the mean and ± standard deviation of H, D, h, d, U, H/D, U/D and the % of mean shifting H and U (and ± standard deviation shifting), the species attribution and the number of measured shells.The % mean shifting is referred to % distance of H or U from the mean of regression exponential line between P. placida and P. angusta (0%) and the regression exponential line of P. placida (100%) or P. angusta (-100%), calculated by data of D on the bases of regression exponential line by data of Holyoak and Holyoak (2012).

CONCLUSIONS
The discovery in Italy of new Plagyrona sites, with the first report of P. placida (Shuttleworth, 1852) for a new locality (Sicily), and the first reports of P. angusta Holyoak and Holyoak, 2012, in Spain, France, Greece and Italy (Campania and Sardinia), are the result of researches on the distribution of Palaearctic malacological fauna, not targeted but extended to many Italian and European areas.
The fact that the distribution of these two species was underestimated due to their small size, their habitat, their mimicry and to the fact of being rare and localized, had already been highlighted by Cianfanelli et al. (2013).Moreover, the species can be confused with other small terrestrial gastropods, such as those of the genera Pyramidula Fitzinger, 1833, Paralaoma Iredale, 1913, or Acanthinula Beck, 1847; also for this reason only recently the genus Plagyrona has been reported in Italy and in other Mediterranean European countries.For example, in the Alburni Mountains (Campania, Italy), a recent and detailed research on the terrestrial molluscs of this area (Maio et al. 2017), does not report its presence, when, instead, Plagyrona is present.
The doubt that P. placida may be a cryptogenic species, accidentally introduced by man in Italy, was expressed in Cianfanelli et al. (2013), but the new collected data are further and increasingly substantial elements to consider this species as autochthonous, thus excluding a passive anthropic diffusion as already occurred for other small terrestrial species.For example, Paralaoma servilis (Shuttleworth, 1852), Lucilla scintilla (Lowe, 1852), Lucilla singleyana (Pilsbry, 1890) are common species almost everywhere in the alluvial debris along the rivers (Cianfanelli et al. 2007), but if we exclude a site near a cave (Sant'Angelo a Fasanella), where Paralaoma servilis is present, no alien species were collected in all the other litter samples.Many of these sites are intact environments, far from inhabited sites, where the chance of non-native species introductions is very unlikely.Therefore, on the basis of the collected elements, Plagyrona species are considered indigenous elements in all the collection sites.
The specific identification of the various populations has been carried out on a morphometric basis, but, for some populations (see Table 1), the variability of the discriminating characters and the limited number of available specimens, are facing a difficult certain assignment.On the other hand, from the analysis carried out, confirmation of the existence of two distinct species is not at all evident, at least in the Mediterranean countries.The genital tract of Plagyrona is still unknown; in the Valloniidae the aphallia is frequent (Giusti et al. 1995, Schileyko 1998).In this case, the reproduction by parthenogenesis could express distinct morphological clones; a genetic analysis could therefore clarify the present status of the European populations.Unfortunately, the failure to find living specimens has so far prevented to carry out anatomical and genetic research on these species. 3

Figure 6 .
Figure 6.Linear and exponential regression curves based on H/D (7 A) and H/U (7 B) ratios based on the data from Holyoak and Holyoak (2012), fig.2, with the relative value of R2 and the equations of the exponential curves.