Biodiversity of extant snails (Gastropoda, Mollusca) in the Pliocene Mountain Spur Natural Reserve (Northern Apennine, Italy)

Abstract Background To date, there is a substantial lack of information about gastropods from the Tuscan-Emilian Apennine in the north of Italy, notwithstanding the availability of detailed and comprehensive literature on this molluscan class. We present a gastropod fauna from the Natural Reserve of the Pliocene Mountain Spur: to our knowledge, this is the first investigation of the extant gastropod fauna in the Tuscan-Emilian Apennine and one of the few in the Apennine's mountain chain as a whole. New information We describe a gastropod fauna comprised by 25 species, belonging to 18 genera and 10 families: the general figure which is emerging is an assemblage of European and Mediterranean-European species, with a single Asian contribution. Several shells were collected as embedded in sandy-clayey soils and showed fossilisation traces: therefore, we regard these shells as subfossil samples. Namely, subfossil shells are from the species Pomatiaselegans, Granariafrumentum, Retinellaolivetorum, Xerolentaobviaobvia, Oxychiluscf.draparnaudi, Monachacartusiana and Monachacantiana. The present checklist is the first report in the Tuscan-Emilian Apennine and Emilia-Romagna areas for six taxa: Morlinaglabraglabra, Oxychiliusalliarius, Xerosectacespitum, Fruticicolafruticum, Xerogyraspadae and Xerolentaobviaobvia.


Introduction
The order Stylommatophora is by far the best-known and largest order of the superorder Eupulmonata (Gastropoda), including approximately 25,000-30,000 terrestrial species in 104 families (Mordan and Wade 2008, Brusca et al. 2016, Doğan et al. 2020, Kosicka et al. 2022).Stylommatophorans are comprised of land snails and slugs.The shell may be completely or partly enveloped by dorsal mantle or may be absent.The crown age of stylommatophorans has been estimated around the Late Cretaceous, about 140 million years ago (Doğan et al. 2020).
Currently, 2,087 species of non-marine gastropods are known to inhabit Europe.Amongst these non-marine gastropod species, as many as 1,834 (87.88%) are eupulmonates, of which only 24 (1.31%) are non-stylommatophorans; in Italy, 345 species belonging to Eupulmonata have been detected (Welter-Schultes 2012).However, it is worth recalling that groups such as Hydrobioidea and related taxa are comprised of very small freshwater snails with really restricted ranges and diffused nomenclatural issues and only expert taxonomists are able to identify such groups that are, therefore, largely underestimated (or absent) in faunal checklists (Welter-Schultes 2012).
The area of the Pliocene Mountain Spur is a system of rocky outcrops of sandstone dating back to the Pliocene, which ranges between 2.58 and 5.33 million years ago (mya).The Pliocene Mountain Spur encompasses the valleys of rivers Setta, Savena, Zena and Idice; it spans over the Tuscan-Emilian Apennines for a length of about 15 km, covering an approximate surface area of 2,628 ha (Fig. 1).
The Natural Reserve of the Pliocene Mountain Spur was created in 2006.It covers a narrower area (around 757 ha), consisting of a small-sized, 8 km-long mountain chain between Sasso Marconi and Brento, from the beginning of the Setta Valley.It is included in the Sites of Community Importance and in the Special Protection Areas (SCI/SPA IT4050012), as well as in the Natura 2000 Network (2628 ha); moreover, it is safeguarded under the Emilia-Romagna Regional Landscape Territorial Plan and the established Faunal Oasis.A map of the Pliocene Mountain Spur Area.The Natural Reserve is highlighted in green; sampling localities are shown with numbers 1-10 and refer to those in Table 1 and shown in Fig. 2. Lower left insert shows a typical morphology of the Spur: the sandstone wall of Rocca di Badolo with its large ledge.
Detected species and relative sampling localities.Sampling sites are shown in Fig. 2. Whenever subfossils shells were found beside living specimens, this is shown in the "Subfossils" column; otherwise, only living specimens were collected.The molluscan fauna that we present herein is of relevant interest, since the Northern Apennine has seldom been an object of well-structured malacological studies.Data are lacking for the Natural Reserve presented above; comprehensive publications on molluscan fauna of the Northern Apennines (or of smaller areas within) are limited, the most detailed and in-depth study being a work on the area surrounding Pistoia (Cianfanelli 2009), to which a few other works should be added (Bodon andCianfanelli 2002, Bodon 2007).Conversely, Northern Apennine areas might turn out to be actually very interesting because of the peculiar morphological and ecological features of the region, including the mountain ridge above the tree line (approximately 2,000 m); lacustrine basins and surrounding habitats; gullies and caverns that may be found in Apennine hills.Indeed, the study of this fauna provided insightful information on gastropod ecology and biogeography.

Materials and methods
The present work contributes to the current knowledge of extant gastropod molluscs of the Pliocene Mountain Spur, with special reference to the superorder Eupulmonata.Furthermore, we also report subfossil samples, i.e. molluscan (in the present case) samples that did not complete the long and complicated physical/chemical fossilisation process (see, for instance, Gambetta (1934), Cuscani Politi (1988), Lencioni (2006)).
Mollusc specimen collection was carried out by exploring several Reserve sites, sampling both from the surface and removing the sandy-clayey sediment derived from erosion, down to a depth of 50-80 cm.Living specimens were collected for all the species of the molluscan fauna presented here; subfossil specimens were also collected for some of them (Table 1).Specimens were collected from different sources: leaf litter, rocky outcrop crevices, lower side of rock fragments, sandy deposit along the paths and at the base of sandstone walls and soil in the proximity of water bodies.Sampling sites are detailed and shown in Table 1 and Fig. 2. Species determination was carried out on conchological characters, relying on the available malacological literature (e.g., Kerney and Cameron 1979, Cossignani and Cossignani 1995, Cianfanelli 2009, Welter-Schultes 2012, Cossignani and Cossignani 2020).
Shells were deposited and stored in the collection of one of the authors (GP).Each specimen was given a specimen ID; the abbreviation "PMS" stands for "Pliocene Mountain Spur" and each site is followed by the reference number as provided in Table 1.Notes: Shell thick, ovoidal and grossly conical; spire with 4-5 convex coils; pattern reticulated, ranging from beige to light purple; operculum calcareous or chalky.Some specimens were collected on sandy-clayey slopes that line the road connecting Monte del Frate and Monte Adone, down to a depth of 60 cm.Others were collected at similar depths in the underbrush along a creek (Fosso Raibano); finally, some specimens were collected from the underbrush floor.The species is thermophilic and euryecious; it is widespread and present on calcareous soils, in meadows and underbrush, either uncultivated or cultivated, from hills to plains (Cossignani and Cossignani 1995).Drought periods of several months do not affect survival of this species (Welter-Schultes 2012).Recalling it is a burrowing species, it requires a loose substrate (Kerney and Cameron 1979), where it can dig at least 10 cm, to estivate or hibernate (Welter-Schultes 2012).This species has been described as xerophilic and calcicolous (Boato et al. 1984); moreover, it has been found in the Quaternary dune at Capo Mele (Savona, Italy) as a fossil (Boato et al. 1984).Notes: Shell cylindrical, short, stubby, brownish.Specimens were collected on the sandy underbrush soil, at the surface.The species inhabits environments with calcareous soils, provided that herbaceous or shrubby vegetation is present (Cossignani andCossignani 1995, Nardi andNiero 2013).Granaria variabilis is a xerothermophilic species that dwells also on dry walls, from the sea level to 1,600 m a.s.l.(Kerney and Cameron 1979, Boato et al. 1984, Welter-Schultes 2012).

Subclass Heterobranchia
Superfamily Pupilloidea W. Notes: Shell small, grossly stubby and cylindrical; spire with 7-9 coils; four teeth are visible in the aperture, at different levels of development; peristome white; colour beige.Samples were collected on sandy soils beside the road to Monte Adone, at the surface, as well as in underbrush upstream to the road, near Campiuno.This subspecies inhabits calcareous soils and lives on grass and shrubs in sunny areas, as well as under rocks, in crevices and on screes (Cossignani and Cossignani 1995) Notes: Background colour from yellowish to beige to greenish; normally 1-5 spiral brown bands are present, well evident, with yellow or white grooves; peristome white, the edge being more or less folded outside; umbilicus closed in adults.The single specimen was collected in the underbrush, at the surface, below the walls of Monte Mario.This species inhabits natural meadows, shrubs, dunes, cultivated fields, as well as gardens (Cossignani andCossignani 1995, Welter-Schultes 2012) Notes: The shell is highly variable; typically, it is large, ranging from beige to brown in colour.Specimens were caught in a sandy underbrush, at the surface.This species is common in dense vegetation along water bodies, as well as in the underbrush, in mountain areas from Northern Apennine to Calabria, sometimes over the timberline (Cossignani andCossignani 1995, Welter-Schultes 2012).Notes: Shell with 5-7 coils, variable in colour and shape, with or without bands; umbilicus open; peristome brown in adults.The specimen was collected along the path in the underbrush through the small Raibano Valley, at the surface.The species inhabits dry, open places: arid meadows, ruderal habitats, hedgerows and grassland (Kerney and Cameron 1979).Cernuella virgata also lives on dunes ( Kerney and Cameron 1979): in Romagna, specimens were collected on sandy dunes in the Rimini (Italy) area (Cossignani and Cossignani 1995).This species can live at roadsides, in harvested fields and railways; it ascends vertical walls and attaches to tall plants to escape bottom heat (Welter-Schultes 2012).In Italy, this taxon is highly polymorphic, both in terms of shell structure and genital morphology, so that doubt was cast whether it is a single species or a species complex (Boato et al. 1984).

Family Geomitridae
Xerogyra spadae (Calcara, 1845) Notes: The specimen was collected in the underbrush below the walls of Monte Mario, at the surface.This species typically inhabits cool, moist underbrushes, in rocky grasslands, even at high elevation on the Apennine Mountains (Cossignani andCossignani 1995, Welter-Schultes 2012).This is the first report for the Northern Apennine and Emilia-Romagna areas.Notes: Shell thick, smooth, medium-sized and flattened; 5-6 coils in adults; background colour white or white-yellowish, with variable bands, ranging from dark brown to black.Some specimens were collected below the walls of Monte Mario at a depth of about 40 cm.The subspecies is typical of xerothermophilic habitats, such as arid meadows (Cossignani and Cossignani 1995) Notes: Shell medium-sized, very variable, ranging from grey to yellow, with variable bands and spots; 5-6 flattened coils are present; peristome whitish or reddish; umbilicus large.The species inhabits meadows, ruderal habitats, roadsides and margins of arable fields (Cossignani and Cossignani 1995, Welter-Schultes 2012, Cossignani and Cossignani 2020).It is common near coasts and in valleys, but it can inhabit sunny mountain slopes in rocky areas in Italy (Welter-Schultes 2012).This is the first report for the Tuscan-Emilian Apennine and Emilia-Romagna areas.

Family Helicodontidae Kobelt, 1904
Helicodonta obvoluta subsp.Notes: Shell medium-sized to small, hairy; umbilicus open; ranging from dark brown to brown; flattened above; peristome flattened; lips somewhat thick, white, with a callus in the aperture with three lobes.This subspecies is found in forests under fallen branches, leaf litter and between stones, usually on calcareous substrate; in the Alps, it may reach 1,900 m, but is rarely found above 1,500 m (Kerney and Cameron 1979, Giusti et al. 1985, Welter-Schultes 2012).It may weakly climb up tree trunks and hibernates in rotting wood logs (Welter-Schultes 2012).Hairs on the shell can reach 1 mm in length and are usually permanent (Welter-Schultes 2012).
The collection site of the present specimen is of particular interest, at the base of sandstone outcrops in a small, moist dell with abundant vegetation -a site which may represent a refugial micro-environment with respect to ecological features of this subspecies.

Family Camaenidae Pilsbry, 1895
Fruticicola Notes: Shell medium-sized, globular; umbilicus deep; ranging from white to brown in colour.The specimen was collected in the underbrush, at the surface.The species inhabits open meadows, as well as wood leaf litter, edges of woods and bushes in moist habitats (Cossignani and Cossignani 1995), while it is normally absent from open, sunny sites, since it is sensitive to drought (Kerney andCameron 1979, Welter-Schultes 2012).This species was also found in the Padan Plain (Boato et al. 1984).It feeds on dead leaves, herbs and mushrooms (Welter-Schultes 2012).This is the first report for the Tuscan-Emilian Apennine and Emilia-Romagna areas.Notes: Shell small, yellow-brown, lucent, flattened, with very few, packed coils.The specimen was collected in the underbrush.This species inhabits the leaf litter in broadleaved, deciduous forests, as well as woods, fields, rocks and gardens; rarely it also inhabits acidic places, such as conifer plantations (Kerney andCameron 1979, Cossignani andCossignani 1995).It can also be found at water margins and in cultivated areas with moist meadows (Welter-Schultes 2012).This is the first report for the Tuscan-Emilian Apennine and Emilia-Romagna areas.Notes: The shell is similar to O. alliarius, but the colour is darker.Both specimens were collected in the wood upstream to the road to Monte Adone-Brento, at a depth of 40-50 cm.This species inhabits the leaf litter in broad-leaved, deciduous forests; caves, even in depth; vegetation and rocky outcrops with a suitable level of moisture and sheltering (Boato et al. 1984, Cossignani andCossignani 1995) Some taxa are xerothermophilic, meaning that they are associated with xeric areas, which are common in the Reserve because of the microclimate of some sites, such as the base of sandstones walls, which are, in some cases, 100 m high and more (Monte Mario, Rocca di Badolo, Monte Adone).These species are Xerolenta obvia obvia, Euomphalia strigella strigella and Monachoides incarnatus incarnatus, which are definitely xerothermophilic species and should be taken as guide species for these environments, but also Granaria variabilis, Cernuella neglecta and Cernuella cf.virgata.Pomatias elegans and J. q.quadridens are also associated with, but not exclusive of, xeric environments.
It is of particular interest the discovery of Helicodonta obvoluta obvoluta, since it is associated with mountain cool, moist climates.It was collected in a small, moist dell below Monte del Frate along the road to Monte Adone, which, therefore, should be considered as a refugial zone because of its microclimatic conditions.
In broad-leaved, deciduous underbrush and along water bodies, as is the case for the small valley of Fosso Raibano (Fig. 2, site 3), there is a different microclimate, featuring cooler and moister traits.Indeed, some species are associated with that microclimate: Helix ligata, Morlina glabra glabra, Oxychilus cf.draparnaudi and Xerogyra spadae.
Several shells were collected at variables depths (20-60 cm) in sandy-clayey soils and show fossilisation traces, such as the loss of the original colour and the absorption or assimilation of that of the embedding sediment.Therefore, we consider these shells to be subfossil samples (Boato et al. 1984); they were determined as P. elegans, G. f. illyrica, Retinella olivetorum olivetorum, Xerolenta obvia obvia, Oxichilius cf.draparnaudi, Monacha cartusiana and Monacha cantiana.
It is well known (e.g.Raffi and Serpagli (1996)) that granulometry and deposition rate of the sediment significantly affect the fossilisation process that is undergoing within the sediment itself, which can be of many different types.In a nutshell, best preserved fossils are normally expected from fine-grained sediment rather than coarse ones.The organism initially poses on sediment surface and is then embedded, a process which can take place at various speeds.After burial, long chemical and physical processes drive these remains to become fossils; however, remains may be preserved even for long times, without undergoing such modifications -similar cases are known as subfossils (Brouwer 1972).
Immediately after death, organic matter decomposes; skeletal parts are disjointed, which, for Gastropods, means separating the shell from the operculum; then, maceration can take place in aquatic environments (Raffi and Serpagli 1996).These processes can occupy variable timespans and can lead to prefossilisation, resulting in subfossils.Shells become permineralised, increasing their thickness, or demineralised, reducing in thickness and robustness, because of dissolving substances.The latter phenomenon was observed, for example, in some bivalve fossil specimens from the "great ledge" of Rocca di Badolo.An example of a subfossil specimen of Pomatias elegans is shown in situ in Fig. 4.
The conservation status of subfossil specimens is compatible with the appearance of these species in the focal area in the upper Pliocene (end of the Piacenzian, which spans 3.6-2.58 million years ago) or in more recent times (Pleistocene, Holocene), pointing towards a stable presence up to extant populations.
Concluding, the present checklist is the first report in the Tuscan-Emilian Apennine and Emilia-Romagna areas for four taxa: Morlina glabra glabra, Oxychilius alliarius, Xerosecta cespitum and Fruticicola fruticum; moreover, two taxa are reported here for the first time from the entire Northern Apennine (and Emilia-Romagna as well): Xerogyra spadae and Xerolenta obvia obvia.A subfossil specimen of Pomatias elegans (blue arrowhead) in situ (site 7, Campiuno).

Rafinesque, 1815 Family Hygromiidae Tryon, 1866 Euomphalia strigella subsp. strigella (Draparnaud, 1801)
Shell solid, globular, r, Cossignani and Cossignani 1995wn; border of the aperture white; the last part of the last coil leans towards the aperture.Adult specimens of this subspecies inhabit moderately open and sunny habitats, avoiding excessive moisture, as is the case for the top of Monte Adone.It is possible, even if uncommon, to find individuals of this subspecies in deciduous woods, either in the leaf litter or on trunks or in hedgerows and scrub(Kerney and Cameron 1979, Cossignani and Cossignani 1995, Girod 2011).It lives in shrubs, between leaves and semi-dry meadows at sunny slopes, up to 1,600 m a.s.l.(Welter-Schultes 2012).
(Boato et al. 1984, Cossignani and Cossignani 1995, Cossignani and Cossignani 2020)levels, in xeric environments, less commonly on grassy and shrubby vegetation(Kerney and Cameron  1979).It tolerates extensive grazing and can be found in sheep and goat pastures, up to 2,400 m a.s.l. in the Alps (Welter-Schultes 2012).It was also reported as a Pleistocene fossil from Varazze (Savona, Italy;Boato et al. (1984)).Superfamily Helicoidea Notes:Notes: Shell globular and thin, with a maximum of six coils; globally whitish and translucent, but the last coil is reddish around the peristome; umbilicus evident, but not completely open.Some specimens were collected down to a depth of 60 cm, below Campiuno.The species inhabits open environments, typically near water bodies and at lower elevations(Boato et al. 1984, Cossignani and Cossignani 1995, Cossignani and Cossignani 2020).It is not found in woods, rather it lives in roadside verges, hedges ( Kerney and Cameron 1979), railways, dunes and well-drained calcareous soils (Welter-Schultes 2012).
(Cossignani and Cossignani 1995)ape globular; suture shallow; whorls thick and rounded; umbilicus open.Specimens were collected beside the road to Monte Adone-Brento, at the surface.This species inhabits grassy habitats, rocky areas and woodlands(Cossignani and Cossignani 1995).It can burrow in the soil and remain burrowed for long periods: in Crete, it appears for only a few days in a year at the first winter rainfall (Welter-Schultes 2012).Helix ligata O. F. Müller, 1774

C. R. Boettger, 1909 Candidula unifasciata subsp. unifasciata (Poiret, 1801)
(Cossignani andBoato et al. (1984)ng from white to gr, Cossignani and Cossignani 2020)her patterns (bands or spots) on the lower region; 5-6 coils are present; peristome thick, sometimes with teeth; umbilicus narrow.Specimens were collected in the underbrush, close to the sandstone walls of Monte Mario, at the surface.Individuals of this subspecies perfer dry environments between rocks and rocky outcrops, dry meadows and walls(Cossignani and Cossignani 1995, Welter-Schultes 2012, Cossignani and Cossignani 2020)or, more generically, open habitats and areas where grasses were somewhat scraped, from the mountain plain to the vegetation limit in high altitude plain, up to 2,400 m(Kerney and Cameron 1979, Welter-Schultes 2012).This subspecies is also known as a Pleistocene fossil in the Verezzi area(Savona, Italy;Boato et al. (1984)).

Superfamily Gastrodontoidea Tryon, 1866 Family Oxychilidae Hesse, 1927 (1879) Morlina glabra subsp. glabra (Rossmässler, 1835)
Shell with five coils; d, Cossignani and Cossignani 1995ures.The single specimen of M. g. glabra was collected in a sandy underbrush below the walls of Monte Mario.Normally, this subspecies is found in underbrush, rocks and screes on open hillsides, as well as in biotopes in front of caves and in open, moist environments, but also leaf litter of deciduous and coniferous forests(Kerney and Cameron 1979, Cossignani and Cossignani 1995, Welter-Schultes 2012).It is mailny montane and can reach up to 1,850 m a.s.l. in Switzerland(Kerney and Cameron 1979).It feeds on plants and juvenile snails (Welter-Schultes 2012).This is the first report for the Northern Apennine and Emilia-Romagna areas.

Gastrodontidae Tryon, 1866 Retinella olivetorum subsp. olivetorum (Gmelin, 1791)
(Boato et al. 1984ssignani 1995 urban waste ground, ro, Cossignani and Cossignani 2020) greenhouses(Kerney and  Cameron 1979, Welter-Schultes 2012).O.draparnaudi feeds on earthworms, juvenile slugs, juvenile snails, even cat and dog food (Welter-Schultes 2012).Fossils from genus Oxychilus were found in Quaternary sites near to Pietra Ligure and Capo Mele (Savona, Italy;Boato et al. 1984).Some specimens were collected at a depth of up to 50 cm along the Raibano Valley path.Normally, R. o. olivetorum inhabits shady hill and mountain meadows in chestnut and olive woods(Cossignani and Cossignani 1995, Welter- Schultes 2012, Cossignani and Cossignani 2020).It can survive drought inside the soil and appears only during and after longer rainfalls (Welter-Schultes 2012).Still, the present data should be taken as preliminary and further investigation is needed to completely characterise pulmonate molluscs in the Pliocene Mountain Spur Natural Reserve.Taxonomic work is still needed and consensus has not been reached on some of the present names: for example, Cornu aspersum (O.F.Müller, 1774) is listed as Helix aspersa Müller, 1774 by some authors (for instance, Welter-Schultes (2012)); Morlina glabra glabra (Rossmässler, 1835) is listed as Oxychilus glaber glaber (Rossmässler, 1835) by some authors (e.g.Kerney and Cameron (1979)).However, the general figure which is emerging from this site is an assemblage of European and Mediterranean-European elements, with a single Asian species, Fruticicola fruticum(Boato et al. 1984,  Girod 2011).Furthermore, extensive field research should be carried out to further characterise the gastropod fauna of the Natural Reserve, with special reference to underrepresented groups, such as microgastropods or slugs.Many species are associated with calcareous soils, which result from Pliocene sandstone geochemistry, that commonly present calcareous components (Scarponi 2002).These species are P. elegans, Granaria frumentum illyrica, G. variabilis and Jaminia quadridens quadridens; thus, these species should be taken as good indicators of calcareous soils.