The genus Alpioniscus Racovitza, 1908 in Sardinia: taxonomy and natural history (Isopoda, Oniscidea, Trichoniscidae)

Abstract The genus Alpioniscus Racovitza, 1908 (Trichoniscidae) from Sardinia is revised. Three new cave-dwelling species are described: A.onnisi Taiti & Argano, sp. n., A.stochi Taiti & Argano, sp. n., and A.sideralis Taiti & Argano, sp. n.. The genus Utopioniscus Schmalfuss, 2005 is considered to be a junior synonym of Alpioniscus, after morphological and molecular analyses. Alpioniscusfragilis (Budde-Lund, 1909) and A.kuehni from Grotta del Bue Marino are illustrated. With the new species, the genus Alpioniscus in Sardinia comprises six species: two troglobionts (A.fragilis and A.onnisi), one endogean and troglobiont (A.thanit Taiti & Argano, 2009), and three stygobionts (A.kuehni, A.stochi, and A.sideralis). All the species occur in karstic areas in the central-eastern and south-eastern part of the island. A key to all the Sardinian species of Alpioniscus is provided.


Introduction
At present, 92 species of terrestrial isopods are known from Sardinia Argano 2009, 2011), many of which strictly endemic. Several new species already identified during field investigations in the last years are waiting to be described. The aim of this study is to attempt a reconstruction of the biogeographic history of the genus Alpioniscus Racovitza, 1908 in Sardinia on the basis of new data.
The geographical range of the genus Alpioniscus is discontinuous. To date, the genus comprises 31 subterranean species in two subgenera (see Tabacaru 1966): the nominal subgenus with 14 species living in the caves of the Western Alps and the southern Balkans reaching Greece, and the subgenus Illyrionethes Verhoeff, 1927, with 17 species populating caves of Catalonia, Sardinia and the Dinaric Alps Taiti 2011, Bedek et al. 2017). In Sardinia two species are known in the subgenus Illyrionethes Argano 2009, 2011): A. fragilis (Budde-Lund, 1909), widely distributed in the karst caves of central-eastern and south-eastern areas of the island, and A. thanit Taiti & Argano, 2009, from endogean environments and some caves in the central-eastern area.
A large number of specimens have been recently collected from many new localities (mainly caves) on Sardinia, revealing a more complex taxonomic scenario. In the same part of Sardinia, Schmalfuss (2005) described a very interesting new species and genus of trichoniscids, the aquatic Utopioniscus kuehni Schmalfuss, 2005, occurring in two submarine caves. This species was considered among the most archaic forms of the family Trichoniscidae. New populations of this species have recently been examined from subterranean freshwaters in coastal and inland caves, and other stygobiotic species with intermediate characters between Alpioniscus and Utopioniscus have also been identified. In this paper three new species of Alpioniscus are described and the synonymy between Alpioniscus and Utopioniscus proposed, on the basis of both morphological and molecular analyses. Analyses performed on both morphological and molecular techniques allow an exhaustive integrative taxonomic approach, which has been effectively used in several case studies on small-sized faunal taxa (see e.g., Casu et al. 2011, Scarpa et al. 2016, 2017a.

The study area
Sardinia is the second largest island of the Mediterranean (24,090 km 2 ) with a complex geology. Karsts cover 9% of the total surface and are divided in 219 distinct areas of different ages, ranging from sea level up to 1,500 m altitude. These areas are separated from each other by non-karstic rocks, so they evolve and behave independently to one another (De Waele 2003, 2009. Alpioniscus species in Sardinia are limited to the central-eastern and south-eastern main karst groups (Gulf of Orosei, Supramonte, Tacchi, Quirra, and Sarrabus).

Collectors of materials
The specimens examined were collected by some of the authors, several biospeleologists, and the astronauts from Europe, USA, Russia, Canada, Japan, and China, participating in the ESA CAVES training courses.

Morphological analysis
All material collected for morphological analysis was stored in 75% ethanol. The species were illustrated with the aid of a camera lucida mounted on Wild M5 and M20 microscopes. Figures were digitally drawn following the methods described in Montesanto (2015Montesanto ( , 2016. The World Geodetic System 1984 (WGS84) was the datum used for all geographic coordinates.

Molecular analysis
Several specimens of each Sardinian species from the type localities have been tested for molecular analysis (Table 1), including one specimen of Utopioniscus kuehni studied by Schmalfuss (2005). Moreover, in order to test the assignment of the Sardinian species of Alpioniscus to the subgenus Illyrionethes we included also specimens of Alpioniscus strasseri (Verhoeff, 1927), type species of the subgenus Illyrionethes from Friuli Venezia Giulia (Italy), and of A. feneriensis (Parona, 1880), type species of the subgenus Alpioniscus Racovitza, 1908, from Piedmont (Italy). A specimen of Androniscus dentiger Verhoeff, 1908 (fam. Trichoniscidae) from Tuscany (Italy) was used as outgroup.
Molecular analyses have been performed using the COI gene (Cytochrome c Oxidase subunit I) with either the universal COI primers by Folmer et al. (1994) or new specific primers designed by the authors (H: grgatgaycaratytayaatgt, L: ctaggrtcaaaaaarcawgtgtt). DNA extraction and PCR have been performed following . Annealing temperature was set at 44° C for both of primers pairs; positive and negative controls were also used for PCR. PCR products were purified by ExoSAP-IT (USB Corporation) and sequenced using an external sequencing core service (Macrogen Inc., Europe). The sequencing runs were performed both for forward and reverse strands. Sequences were aligned using Clustal W (Thompson et al. 1994), implemented in BioEdit 7.0.5.2 software (Hall 1999). The best probabilistic model of sequence evolution was determined after evaluation by jModeltest 2.1.1 (Posada 2008), with a maximum likelihood optimized search, using the Akaike Information Criterion (AIC) and the Bayesian Information Criterion (BIC). The model TPM2uf + G has been chosen as the best fitting both AIC and BIC. Phylogenetic relationships were investigated using the Bayesian Inference (BI) and the Maximum Likelihood (ML) methods. BI was carried out using the software MrBayes 3.2.2 (Ronquist et al. 2012), setting as model parameters: N ST = 3, rates = gamma, ngam-macat = 4. Two independent runs each consisting of four Metropolis-coupled MCMC chains (one cold and three heated chains) were run simultaneously for 5,000,000 generations, sampling trees every 1,000 generations. The first 25% of sampled trees were discarded. Run was executed by means of the Cipres Phylogenetic Portal (Miller et al. 2010). Convergence of chains was checked following the procedures described by Ronquist et al. (2012) and Gelman and Rubin (1992). ML analysis were conducted using the software RAxMLGUI version 1.3 (Silvestro and Michalak 2011)   Redescription. Maximum length: ♂, 9 mm; ♀, 14 mm. Colourless body, pleon narrower than pereon (Fig. 1A). Dorsal surface distinctly granulated with ovoid scalesetae as in Fig. 1B. Many gland pores on lateral margins of pleonites 4 and 5, telson, lateral surface of uropodal protopods, and some scattered pores on dorsal surface of uropodal exopods (Fig. 1E). Cephalon (Fig. 1C, D) with suprantennal line Vshaped with rounded middle part; antennal lobes quadrangular, obliquely directed outwards with concave dorsal surface. Eyes absent. Posterior margin of pereonite 1 and 2 straight, and of pereonites 3-7 progressively more concave (Fig. 1A). Pleonites 3-5 with very short posterior points (Fig. 1A, E). Telson (Fig. 1E) approx. twice as wide as long; distal part with concave sides and very broadly rounded apex. Antennula ( Fig. 1F) with second article distinctly shorter than first and third; third article distally enlarged and bearing 12-13 apical aesthetascs. Antenna ( Fig. 1G) with fifth article as long as flagellum; flagellum of 10-13 articles with four groups of aesthetascs.  liped (Fig. 2E) basis with outer margin distally oblique and setose; palp stout, apically with tuft of setae and three tufts of setae on medial margin, basal article with two setae; endite narrow, with two apical stout setae and a large penicil. Pereopods with setose dactylar seta distally bifid (Fig. 3A), pereopod 7 with water conducting system on basis, ischium and merus. Uropod (Fig. 1E) with endopod distinctly shorter than exopod, endopod inserted proximally to exopod.
Male. Pereopod 1-4 ( Fig. 3A) with carpus and merus bearing numerous short scales on sternal margin. Pereopod 7 (Fig. 3B) ischium with straight sternal margin covered with short setae; merus with three lobes proximally. Genital papilla (Fig. 3C) with a rounded tip. Pleopod 1 (Fig. 3C) exopod triangular with acute apex; endopod enlarged at base, distal part narrow with almost parallel sides and bearing an apical seta. Pleopod 2 (Fig. 4A) exopod triangular with convex outer margin and a small apical seta; endopod of two articles, longer than exopod, first article approx. three times longer than second, second article bearing distally rounded lobe and strong seta subapically cleft. Pleopod 3-5 exopods subtriangular with apical seta (Fig. 4B-D). Distribution. The species seems to be widely distributed in the central-eastern and south-eastern karstic areas of Sardinia. The species is mainly terrestrial even if it occasionally occurs also in water.
Remarks. This species was described by Budde-Lund (1909) as Titanethes (Alpioniscus) fragilis from a Sardinian cave on material collected by Forsyth Major. No name for a specific cave was mentioned. However, considering that Forsyth Major discovered some troglobiotic species of different invertebrates during a digging campaign in caves of the Orosei Gulf, Casale et al. (2009) suggest that the cave explored by Forsyth Major most likely was the Grotta dell'Arciprete (= Grotta di Toddeitto) or one cave nearby in the same kastic area., e.g., Grotta del Bue Marino. The species is here redescribed on specimens from this last cave.
Etymology. The new species is named after our Sardinian friend Carlo Onnis for his enthusiastic and efficient activity in collecting subterranean fauna, including part of the material treated here.
Remarks. Alpioniscus onnisi sp. n. is very similar to A. fragilis and A. thanit. It differs from the former in the smooth instead of granulated dorsal surface, the shape of the dorsal scale-setae, telson with triangular instead of trapezoidal distal part, and the male pleopod 2 endopod lacking a subapical lobe; from the latter mainly in the larger body shape and the male pleopod 1 exopod with slightly convex, instead of concave, medial margin, and shorter distal part.   Remarks. Alpioniscus kuehni was originally described as Utopioniscus kuehni gen. n., sp. n. by Schmalfuss (2005) on specimens collected in underground waters from two caves on the central-eastern coast of Sardinia, Grotta dell'Utopia and Grotta del Bel Torrente, with entrances at 30 m and 16 m below sea level, respectively. These caves are the estuaries of subterranean streams which open up in the sea (De Waele and Forti 2003). In the Grotta del Bel Torrente the specimens were collected 700 m inland, where water salinity was 1% (Schmalfuss 2005;Oertel and Patzner 2007). In the Grotta dell'Utopia they were collected 2000 m inland together with     (Schmalfuss, 2005), comb. n. from Grotta del Bue Marino, ♂: A pereopod 1 B pereopod 7 C genital papilla and pleopod 1 D pleopod 2 E pleopod 3 exopod F pleopod 4 exopod G pleopod 5 exopod. a species of the freshwater stygobiotic Stenasellus Dollfus, 1897 (Asellota, Stenasellidae). The specimens from Grotta del Bue Marino and Grotta Su Bentu here examined were collected in fresh water lakes and are morphologically identical to the specimens from the type localities. This fact confirms that A. kuehni is a freshwater stygobiotic species.
The species is well described by Schmalfuss (2005) and is here fully illustrated on specimens from Grotta del Bue Marino (Figs 9-11). As confirmed by molecular data (see below), the genus Utopioniscus must be considered to be a junior synonym of Alpioniscus. In fact, all the diagnostic morphological characters of the genus Alpioniscus mentioned by Vandel (1960) are present also in A. kuehni, in particular the shape of the male pleopods 1 and 2, as already pointed out by Schmalfuss (2005). Alpioniscus kuehni is mainly characterized by the enlarged shape of the pereon, the reduction of the number of aesthetascs of the antennula, the very thin antenna with the flagellum of 20 to 30 articles, the enlargement of the maxillipedal endite, and the lack of groove with scales on the pereopod 7 for the water conducting system. The last two characters are certainly adaptive for aquatic life. The enlarged maxillipedal endite is, in fact, present also in the following two new species which are both aquatic. Taiti  Description. Maximum length: ♂ and ♀, 4.5 mm. Colourless body, pleon slightly narrower than pereon (Fig. 12A). Dorsal surface smooth with some scattered scalesetae as in Fig. 12B. Some gland pores on lateral margins of pleonites 4 and 5, telson and on dorsal surface of uropodal protopod and exopod (Fig. 12F). Cephalon (Fig.  12C-E) with suprantennal line V-shaped; antennal lobes quadrangular. Eyes absent. Posterior margins of pereonite 1-4 straight, of pereonites 5-7 progressively more concave (Fig. 12A). Pleonites 3-5 with very short posterior points (Fig. 12F). Distal part of telson with concave sides and very broadly rounded apex (Fig. 12F). Antennula (Fig. 12G) with distal article narrow and bearing two apical aesthetascs. Antenna (Fig.  12H) with fifth article as long as flagellum; flagellum of five to seven articles. Mandibles with two penicils in the right (Fig. 13A) and three penicils in the left (Fig. 13B). Outer branch of maxillula with 5 + 6 teeth, apically entire, three or four of the outer group strongly bent inwards, two slender stalks, one setose; inner branch with three long penicils (Fig. 13C). Maxilla with setose apex (Fig. 13D). Maxilliped (Fig. 13E) endite quadrangular with a setose distal margin and a distinct subapical short penicil  on medial margin; palp narrow and bent in medial direction, three tufts of setae on medial margin and one apically, basal article with a single seta; basis distally enlarged with a triangular lobe on outer margin. Pereopods with setose dactylar seta (Fig. 14A). Pereopod 7 (Fig. 14B) basis with a groove covered with scales on rostral surface (water Figure 14. Alpioniscus stochi Taiti & Argano, sp. n. from Grotta Su Palu, ♂ paratype: A pereopod 1 B pereopod 7 C genital papilla and pleopod 1 D pleopod 2 E pleopod 3 exopod F pleopod 4 exopod G pleopod 5 exopod.
Etymology. The new species is named after our colleague and friend Dr. Fabio Stoch, who greatly contributed to the knowledge of Italian stygobiotic fauna and collected part of the material.
Remarks. The specimens were collected under stones on the bottom of a subterranean stream in the Grotta Su Palu, in the eastern part of the karstic area of Supramonte. Alpioniscus stochi sp. n. is similar to A. kuehni in having a thin third article of the antennula bearing a small number of aesthetascs, and the maxilliped with a quadrangular endite and a narrow palp bent medially. It is readily distinguishable from A. kuehni by the smaller size (4.5 mm vs. 12.0 mm of the latter), narrower body shape, shorter and more thickset antenna with smaller number of flagellar articles, uropods with shorter branches, the presence of a water conducting system on pereopod 7 ischium, a more thickset merus of the male pereopod 7, a stouter male pleopod 2 endopod, and no apical seta on the exopod of the male pleopods 2-5. Taiti    Description. Maximum length: ♂, 7 mm; ♀, 7.5 mm. Body colourless, ovoidal, with pleon narrower than pereon (Fig. 15A). Dorsal surface smooth with some scattered scale-setae as in Fig. 15B. Some gland pores on lateral margins of pleonites 4 and 5 and on uropodal protopods (Fig. 15E). Cephalon (Fig. 15C, D) with suprantennal line sinuous; antennal lobes quadrangular. Eyes absent. Posterior margin of pereonite 1-3 straight, of pereonites 4-7 progressively more concave (Fig.  15A). Pleonites 3-5 with very short posterior points (Fig. 15E). Distal part of telson with slightly concave sides and truncate apex (Fig. 15E). Antennula (Fig. 15F) with distal article narrow and bearing two apical and three subapical aesthetascs. Antenna (Fig. 15G) with fifth article of peduncle shorter than flagellum; flagellum of 10-11 articles with couple of aesthetascs on second and third article. Mandibles with one penicil in the right (Fig. 16A) and three penicils in the left (Fig. 16B). Outer branch of maxillula with 5 + 6 teeth, apically entire, and two slender setose stalks; inner branch with three penicils increasing in length from distal to proximal (Fig. 16C). Maxilla with setose and bilobed apex, inner lobe very small (Fig. 16D). Maxilliped (Fig. 16E) endite quadrangular with setose distal margin and distinct subapical penicil on medial margin; palp narrow and bent in medial direction, with three tufts of setae on medial margin, tuft at apex and single seta on outer margin, basal article with two setae; basis distally enlarged with a rounded lobe on outer margin. Pereopods with bifid setose dactylar seta (Fig. 17A). Pereopod 7 (Fig. 17B) basis with water conducting system. Uropod (Fig. 15E) with protopod not grooved on outer margin; endopod distinctly shorter than exopod, exopod inserted slightly distally to endopod.
Etymology. Latin: sideralis meaning of or with respect to the distant stars. The name refers to the international team of astronauts, taking part in training courses (ESA CAVES) in Sardinia organized by the European Space Agency, who first collected this new species.
Remarks. The species occurs in subterranean freshwater ponds and streams in the karstic areas of Supramonte. It is closely related to the other two Sardinian stygobiotic species of Alpioniscus (A. kuehni and A. stochi sp. n.). It is readily distinguishable from A. kuehni by the smaller size (7.7 mm vs 12 mm), narrower body shape, shorter and more thickset antenna with smaller number of flagellar articles, presence on pereopod 7 ischium of a water conducting system, male pereopod 7 carpus shorter with more enlarged proximal part, male pleopod 1 exopod with narrower distal part, endopod shorter than exopod, male pleopod 2 exopod without setose apical seta. Alpioniscus sideralis differs from A. stochi in its larger size (7.5 mm vs. 4.5 mm of the latter), antennula with a group of subapical aesthetascs, more numerous flagellar articles of the antenna (10-11 vs. 5-7), maxillipedal basis with a rounded instead of triangular lobe on outer margin, uropods with longer exopod and endopod, male pereopod 7 with thinner merus and carpus, merus with a distinct lobe on sternal margin.

Molecular results
After alignment, a 405 bp-long sequence dataset was obtained (see Table 1 for Gen-Bank accession numbers). Since BI and ML analyses generated trees with identical topologies, only the BI tree is reported (Fig. 18 for more details). Each node is highly supported by both bootstrap values (BV) and posterior probability (PP). Among the species belonging to the genus Alpioniscus, A. (Alpioniscus) feneriensis from Piedmont is placed in the tree as the most external taxon. Its sister clade groups both A. (Illyrionethes) strasseri from Friuli Venezia Giulia and Sardinian species. All Sardinian species are clustered together in one clade. Sardinian terrestrial species (Alpioniscus fragilis, A. thanit, and A. onnisi sp. n.) set in a monophyletic group, showing a sister-taxon relationship with the clade including Sardinian aquatic species (A. stochi sp. n., A. sideralis sp. n., and A. kuehni). Within the terrestrial clade, A. onnisi sp. n. is the sister species of A. thanit. Within the aquatic clade, A. kuehni is the sister species of A. sideralis sp. n.

Discussion
According to the data of the present study, the genus Alpioniscus is represented in Sardinia by six different species inhabiting various underground environments within a fragmented karst area of limited extension in the central-eastern and southeastern part of the island (Fig. 19). All six species have morphological characters corresponding to the diagnosis of the subgenus Illyrionethes proposed by Verhoeff (1927), i.e., the distal article of the male pleopod 2 endopod shorter than or of equal length as the second article. This conclusion based on morphology is also supported by the molecular comparison with Alpioniscus (Illyrionethes) strasseri, type species of the subgenus Illyrionethes, and A. (Alpioniscus) feneriensis. However, a definite assessment of the taxonomic status within the genus Alpioniscus can be reached only after the comparison of all, or most of the species belonging to the genus from its entire distribution area. According to the molecular phylogeny, the six Sardinian species are grouped into two distinct clades, one including the three terrestrial species (A. fragilis, A. thanit, and A. onnisi) and the other the three aquatic species (A. stochi, A. sideralis, A. kuehni). It seems most probable that the aquatic mode of life evolved once and then the aquatic species split into other species either by vicariant events related to the karst areas or by dispersal along subterranean aquifers with subsequent isolation. The dispersal in subterranean waters seems to be possible as demonstrated by the presence of A. sideralis and A. kuehni in two separated, even if close, karst areas (see Fig. 19). Dispersal of aquatic species across terrestrial habitats is unlikely. The presence of both terrestrial and aquatic species in the same genus is uncommon among Oniscidea. The only other case known so far is represented by the genus Trogloniscus Taiti & Xue, 2012, which includes three terrestrial and two aquatic species from caves in southern China (Taiti and Xue 2012).
Alpioniscus fragilis is a terrestrial species distributed along an area covering that of the whole genus in Sardinia. This species is mainly troglobiotic, but it can be found also in endogean habitats (e.g., under big stones near Lecorci Falls, Ogliastra) and in an aquatic environment (e.g., under submerged stones in Bue Marino Cave). So, the species shows a remarkable adaptive plasticity which allowed it to be distributed along a large and discontinuous area in suitable ecological conditions. All the other species have more restricted distributions. In some caves both terrestrial and aquatic species occur sympatrically. Alpioniscus fragilis coexists with two stygobiotic species, i.e., A. kuehni in the Grotta del Bue Marino, and A. sideralis in the Grotta Lovettecannas and Grotta Istirzili. In the Grotta Su Bentu A. fragilis occurs together with the two stygobiotic A. kuehni and A. sideralis, while in the Grotta Su Palu with the stygobiotic A. stochi and A. sideralis. The last two species share adaptive traits linked to the aquatic environment with A. kuehni, e.g., the quadrangular endite of the maxilliped, but also aspects that recall a previous existence in terrestrial environments, i.e., the water conducting system on the ischium of the pereopod 7. It is interesting to notice that some aquatic species co-occur in the same cave (i.e., A. kuehni and A. sideralis in the Grotta Su Bentu, and A. stochi and A. sideralis in the Grotta Su Palu), while this does not happen with the terrestrial species.
This richness in Sardinian Alpioniscus species is probably due to the complex geological and palaeoecological events that affected Sardinia, such as marine ingression or strong ecological variations that isolated the small karst islands (Zattin et al. 2008;Lichter et al. 2010), interrupting gene flow among populations and promoting speciation. Migratory events of underground fauna of the archipelago among the various geographical units may have occurred during the emersion periods with suitable environmental conditions. The co-occurrence of two aquatic species in the same cave may be due to subsequent invasions or different habitat preference. This last condition might be true for A. stochi, which occurs under stones on the bottom of the subterranean stream in the Grotta Su Palu, and A. sideralis, which is found on the submerged walls of the same stream. Two or more stygobiotic oniscidean species (genus Haloniscus Chilton, 1920) in the same subterranean aquifer are known also from some calcretes in Western Australian (Taiti and Humphreys 2001;Cooper et al. 2008).