Two new Typhloreicheia species from Sardinia and their biogeographical significance (Coleoptera, Carabidae, Scaritinae)

Abstract Typhloreicheia monacha sp. n. and Typhloreicheia ilianae sp. n. are described from two caves of Central-Eastern Sardinia (Nuoro province): the Bue Marino cave and the Nurra ‘e Pradu cave, respectively. Both caves are located in the part of the island where many highly specialised subterranean carabid beetles are localised. Typhloreicheia monacha is apparently related to two other species of the same area, i.e. Typhloreicheia onnisi Casale & Magrini, 2004 and Typhloreicheia elegans (Dodero, 1916); Typhloreicheia ilianae is closely related to Typhloreicheia henroti Jeannel, 1957, known from a cave near Dorgali. Relationships and diagnostic features among these taxa are discussed and illustrated, and a key for identification of the specialised subterranean Typhloreicheia species of Sardinia is provided. The hypothesis of adaptive radiation of Reicheiina species in Sardinia, recently proposed by the senior author of this contribution, is further elaborated in light of new data.


introduction
The subtribe Reicheiina is a lineage of endogean and hypogean carabid beetles, currently classified in the tribe Clivinini of the subfamily Scaritinae of the family Carabidae (Vigna Taglianti 2005), including so far (as genera "close to Reicheia" : Jeannel 1957) about 150 species (Balkenohl 2003, Lorenz 2005, Grebennikov et al. 2009). External structural attributes of Reicheiina are markedly homogeneous: most of the species are small-sized carabids (1-4 mm) with reduced or absent eyes, moniliform antennae, fossorial prothoracic legs (typical of Scaritinae), the posterior angles of pronotum obliterated and rounded, short pronotal peduncle and the lack of a basal annular constriction on the pronotum. A few taxa, such as the subterranean Spelaeodytes mirabilis L. Miller, 1863, living in caves of the Eastern Adriatic coast (Croatia), display markedly troglomorphic adaptive features (Casale et al. 1998 a, b).
Work with Reicheiina beetles is hampered by the small sizes of its representatives and by the scarcity of material, some taxa being known from one or a few specimens. Morphological characters currently employed in defining various members of Reicheiina are those of external body shape and male genitalia, while immature stages, for example, are still completely unknown.
Monophyly of Reicheiina has never been adequately demonstrated and a distinct possibility exists that the group is an artificial assemblage of unrelated subterranean scaritine clades (Grebennikov et al. 2009). Overall subtribe's existence and its current placement in the tribe Clivinini and generic composition are more a result of gradually evolving taxonomic treatment, initiated by Holdhaus (1924), Jeannel (1957) and Basilewsky (1980), rather than a reflection on its hypothesised monophyly.
However, the monophyly of the Euro-Mediterranean core of Reicheiina formed by the genus Typhloreicheia and its relatives is highly plausible. In Sardinia, the genus Typhloreicheia Holdhaus, 1924 is markedly diversified (Casale 2009). In this contribution, the authors describe two new morphologically specialized Typhloreicheia species from caves of Sardinia, and debate some biogeographical aspects of this discovery.

Material and methods
Newly designated Sardinian Typhloreicheia type specimens were collected during speleological or biospeleological investigations. All subsequent focused attempts to obtain further individuals by using pitfall traps or baits were unsuccessful.
Male genitalia were dissected, dehydrated in ethanol, cleared in cold KOH, examined and illustrated, using standard techniques before their definitive inclusion on microscope slides attached to the respective specimens. Line drawings were made using a camera lucida attached to stereomicroscopes Wild M-5, and a microscope Leitz Orthoplan.
Photographs of male genitalia were prepared by Paolo Magrini (Florence), with a Nikon D1 digital camera mounted on a Nikon Labophot II binocular microscope. P. Magrini also prepared distributional maps of Typhloreicheia in Sardinia.
The median lobe of aedeagus is synonym of phallus of authors. The proximal gonocoxite 1, and the more distal gonocoxite 2 (in the sense of Liebherr and Will 1998), are synonyms of stylomere 1 and stylomere 2 of authors, respectively. Acronyms:

TL
body Total length, from the anterior margin of clypeus to the apex of elytra, measured along the elytral suture. L overall body length, from apex of mandibles to apex of elytra, measured along the suture. PL/PW ratio length of Pronotum, as linear distance from the anterior to the posterior margin (peduncle included), measured along the midline to the maxi- Etymology. The Latin noun "monachus-a" (= monk) recalls the monk seal Monachus monachus (Hermann, 1779), the so-called "Bue Marino", in the tradition of the Mediterranean languages, from which the type locality cave derives its name. Monk seal is presently one of the most endangered mammalian species of the Mediterranean fauna, although it was still present in the Eastern coast of Sardinia until the '60s of the past century.

Diagnosis. Typhloreicheia monacha new species, for both its external features and
The specimens were walking on sandy, humid soil on the banks of the subterranean lakes in the inner parts of the Bue marino cave, in the same habitat as those noted for the molopine carabid beetle Speomolops sardous Patrizi, 1955 and its larva (Casale et al. 2010). The Bue Marino cave, with 48 subterranean species reported so far, is the Sardinian cave richest in hypogean fauna (Casale et al. 2008). An additional female individual was collected by Roberto Loru in the same environmental conditions during a speleological exploration in the nearby Su Spiria cave (1988 Sa/NU) 40°10'42''N -9°33'54'' E, 165 m a.s.l. (Urzulei, Codula Ilune, hypogean system of Su Palu -Grutta de Monte Longos caves), and was erroneously attributed to T. henroti (Casale & Magrini 2004).
In spite of many subsequent investigations in both these caves, including setting pitfall traps, no further individuals of this species have been obtained. T. monacha, like other species of the same genus in Sardinia (and most so-called troglobiont species: Giachino & Vailati 2010), seems to be a deep crevices dweller, which occasionally penetrate in large hypogean systems accessible to the humans, and are never collected by shifting soil.
Relationships. The following features of T. monacha sp. n. suggest its affinities with the elegans species group (Jeannel 1957;Casale and Magrini 2004): medium-large sized body, elytra with intervals 2-7 each bearing a series of erected setae and lateral margins serrate from the humeral angle to apex, shape of aedeagus and structure of endophallus. With the discovery of T. ilianae new species, however, this group appears non-monophyletic (see below, in relationships of T. ilianae). Therefore, T. elegans, T. onnisi and T. monacha sp. n. are separated in the elegans species group (in the new sense) distinct from the henroti species group (see below). Etymology. The name derives from the Oliena village at the base of the Corrasi mountain in which this new species, and many other hypogean organisms were discovered. An ancient legend says that the inhabitants (Ilienses in Latin) of Troy (Ilios in ancient Greek), after the fall of the town to the Greeks, reached Sardinia and founded a village named Iliana, which subsequently became Oliena.
Diagnosis. Typhloreicheia ilianae is the largest in size among the Sardinian congeners known so far, with its TL: 3.64 and L: 4.05 (male holotype). It is evidently related to T. henroti Jeannel, 1957 (see features of the median lobes of aedeagus in respective species: Figures 5-6), but can be distinguished from the latter mainly by its larger size (TL: 3.10 -3.65, L: 3.40 -3.86 in T. henroti); by wider and more robust head, with supra-antennal plates more prominent laterally, by more convex genae markedly constricted to the neck, by antennomeres 3-11 more thickened, and by anterior angles of both clypeus and labrum more acutely prominent; and by the different shape of the median lobe of aedeagus ( Figures 5-6).
Integument shiny, highly polished; microsculpure with fine, hardly visible microlines in form of isodiametric mesh pattern on head and elytra, almost vanished on pronotum.
Head robust, with ocular part of genae markedly convex, constricted toward the neck. Eyes absent. Anterior angles of clypeus acutely prominent. Supra-antennal plates prominent laterally, with outer margin beaded, separated from genae by deep and broad furrow; frontal furrows very deep, with shallow wrinkles in the posterior tract; vertex with an evident, convex tubercle in the middle; antennae elongate (AL: 1.58 mm in male holotype) but robust, thickened; antennomeres 6-10 slightly longer than wide.
Pronotum markedly convex, relatively wide (PL/PW: 0.95), with its maximum width at the basal third; sides moderately rounded, slightly narrowed in front, markedly constricted to the basal peduncle; anterior angles acutely prominent; median furrow very shallow; lateral furrows very narrow and superficial.
Male genitalia as in Figure 5. Median lobe of aedeagus markedly curved, with long apical lamina, which is widened, hatched-like distally. Endophallus with a reduced, inconspicuous apical copulatory piece and an elongate packet of serrate scales in the middle. Parameres each with two apical setae.
Female genitalia: unknown. Distribution and habitat. Typhloreicheia ilianae sp. n. is known only from the type locality. The holotype was sampled in wet soil under big stones in a small pit (-3.7 m), which represents the entrance of a large hypogean system, reaching the depth of 101 m. The associated subterranean fauna includes some of the most specialised troglomorphic endemic Sardinian elements, such as Sardaphaenops supramontanus supramontanus Cerruti & Henrot, 1956 (Coleoptera, Carabidae, Trechini) and Patriziella sardoa Jeannel, 1956 (Coleoptera, Cholevidae, Leptodirini), both described from a nearby cave Nurra 'e Sas Palumbas. The Nurra 'e Pradu cave is also one of the few localities of Sardinia from which the large sized, trogloxenic centipede Plutonium zwierleini Cavanna, 1881 is reported (Zapparoli 2009). We sampled in this cave the following taxa, all endemic to central-eastern Sardinia and representing new faunal records: the orthopteran Acroneuroptila cf. sardoa Baccetti, 1960, the sphodrine carabid beetle Laemostenus pippiai (G. Fiori, 1961) (A. Casale det.), the cholevid beetle Ovobathysciola majori (Reitter, 1885) (A. Casale det.), and the terrestrial snail Tacheocampylaea carotii (Paulucci 1882) (S. Birindelli det.). Subsequent trapping in this cave using pitfall traps, did not produce additional individuals of T. ilianae.
Relationships. Typhloreicheia ilianae is the largest among the Sardinian species known so far, exceeding the large size of T. kraussei (Reitter, 1914), a typical endogean, not hypogean species reaching 3.69 mm in length (Leo et al. 2005). Nevertheless, any relationship of T. ilianae sp. n. with T. kraussei (Reitter, 1914) and its adelphotaxon T. manto (Holdhaus, 1924), both typically endogean, not hypogean species, are highly unlikely. Both latter species have elytral setiferous pores present in intervals 2, 3, 5 and 7 only, and the lateral margins serrate only in the basal half. Furthermore, in these taxa the features of the median lobe of aedeagus are markedly dissimilar to those of T. ilianae sp. n., and the apical copulatory piece in endophallus is lacking in T. kraussei (Leo et al. 2005).
The new taxon appears related to T. henroti Jeannel, 1957, known from the Gurennoro cave (= Pisanu cave, 215 Sa/NU, near Dorgali). Both species are similar in having large-sized body and elytral lateral margins bearing numerous (21-24) teeth extending all the way from the humeral angle to elytral apex. Also both species share the peculiar shape of the median lobe of aedeagus, which is unique among the Sardinian species (Figures 5-6). These species form a pair of adelphotaxa very isolated from the rest of species known so far on the island, here indicated as henroti species group, excluding the other specialised hypogean species of Sardinia known so far (T. elegans, T. onnisi, and T. monacha sp. n.) treated above as elegans species group in the narrow sense.
The following operative and provisional key is provided to distinguish the cave dweller (deep hypogean, or troglophilic) Typhloreicheia species known so far in Central Eastern Sardinia: Elytra elongate, sub-parallel sided; lateral margins with slightly prominent and less numerous (14-16) teeth. Median lobe of aedeagus slightly curved, with apical lamina more developed and sub-truncate distally (Figure 4)

Reicheiina: current taxonomy, and biogeographical remarks at world scale.
As recently recalled (Casale 2009), the current distribution of Reicheiina in the Euro-Mediterranean area and the African continent is highly interesting from the biogeographical point of view: in fact, this lineage presents a markedly disjoint distribution, being represented by several genera and many species in mainland and islands of this area, and some taxa in East Africa, West Africa, South Africa and Madagascar (Jeannel 1957;Basilewsky 1980, Casale andVigna Taglianti 1996;Balkenhol 2003Balkenhol , 2005Bulirsch et al. 2005;Bulirsch and Magrini 2007). Grebennikov et al. (2009) stressed that the discovery of supposed Reicheiina in South-Eastern and Eastern Asia (Laos, Vietnam and Japan: Balkenohl 2005) (Figure 7) revealed a large gap in their known distribution and, implicitly, challenged the monophyly of the subtribe. The same authors recalled that, besides Reicheiina, other apparently unrelated representatives of the subfamily Scaritinae show reduced or absent eyes and wings, small size and depigmentation, in correlation with their subterranean way of life, and other blind Clivinini belong to the subtribe Clivinina (as the genus Trogloclivina Deuve, 2003 from Papua New Guinea).

Biogeographical remarks at local scale.
Two hypotheses have been proposed to explain the origin and the exceptional specific diversity of the genus Typhloreicheia in Sardinia.
The first hypothesis proposes at least two different, heterochronic colonisation events. The older one is represented by the common ancestor of the subgenus Sardoreicheia Jeannel, 1957. This subgenus was treated as synonym of Typhloreicheia by Casale and Vigna Taglianti (1996) and other authors, but separated again in the recent catalogues by Balkenhol (2003) and Lorenz (2005). The colonisation event originated from the Miocene tectonic drift of the Corso-Sardinian micro-plate in the Western Mediterranean (Alvarez 1972). Conversely, the more recent colonisation event of the common Apennine ancestor of Typhloreicheia sensu stricto occurred through land connections during the Messinian Salinity Crisis in the Mediterranen basin (Hsü et al. 1977).
This scenario should be similar to the process recently proposed by Wirta et al. (2010) concerning Scarabaeidae Scarabaeinae in Madagascar: in fact, this island has an exceptionally large fauna of more than 250 species of endemic dung beetles. Based on molecular phylogenies, the species descend from eight independent overseas colonisations, of which four have given rise to big radiations. Among them, the tribe Canthonini show three parallel radiations following the respective colonisations at 64-44 Mya.
The second hypothesis proposes that all Sardinian Typhloreicheia species are derived from a common Tyrrhenian, Miocene ancestor.
In other words, the question is: how many times have Reicheiina colonized the island of Sardinia? An adequate solution of this question based exclusively on morphological features is hardly possible. Involvement of molecular data might eventually help to generate complete phylogenetic hypotheses for Typhloreicheia and related genera and thus assist with detecting the number of colonisation events. The similarities between the male genitalia of some Sardinian species and those of species of the Apennine chain, the Elba island and Sicily, supports the hypothesis of multiple colonisations of the island by different ancestors. This is further strengthened by the remarkable absence of Typhloreicheia species in Corsica and Baleares, where the genus is replaced by Reicheia Saulcy, 1863. Furthermore, the scenario in Sardinia has been complicated by the recent description of the genus Dimorphoreicheia Magrini, Fancello & Leo, 2003 (with two species known so far), characterised by the presence of setiferous pores on the disc of the pronotum, a feature previously thought to be peculiar to the genus Reicheadella Reitter, 1913, with some species distributed in the southern Balkans (Casale et al. 1998 a). Nevertheless, this highly complicated situation could be explained by any hypotheses treated below.

Adaptive radiation of Typhloreicheia in Sardinia: previous and new hypotheses compared.
Adaptive radiation might work very rapidly. Classic examples include Hawaiian silverswords and Drosophila, Darwin's finches on the Galápagos islands, Anolis lizards on Caribbean islands, and cichlids of the East African Great Lakes, among others (Givnish and Sytsma 1997;Schluter 2000;Gavrilets and Losos 2009). Gavrilets and Losos (2009) recognize two main components of adaptive radiation: the production of new species by allopatric or non-allopatric speciation, and their subsequent adaptation to a diversity of ecological niches. This may happen when an ancestral taxon finds itself in a vacant area, where resources are abundant and underutilized (e.g., islands or lakes).
The genus Typhloreicheia in Sardinia shows a spectacular diversity much exceeding that in all other Tyrrhenian areas (Iberian Peninsula, Baleares, Apennines, Sicily) where the genus is represented. This fact already induced Holdhaus (1924) and Jeannel (1957), even with much scarcer data to believe that "the genus should have originated in Sardinia". Some years later Magistretti (1965), in his catalogue of the carabid fauna of Italy, was able to list only 13 Typhloreicheia species from Sardinia, whereas they are presently 46 (see Figure 8 a, b).
A similar process -e.g. the colonization of Sardinia by a continental Reicheia-like ancestor, maybe of Iberian origin through the rotation of the Corsica-Sardinia microplate (Alvarez 1972), an adaptive radiation in the island, the extinction of the colonizer on the continent, and then a re-colonization of the close, circum Mediterranean-areas through land connections during the Messinian Salinity Crisis in the Mediterranen basin (Hsü et al. 1977) cannot be excluded. In fact Reicheiina, for their peculiar distribution in the Euro-Mediterranean area, appear as a remarkably ancient lineage of Paleo-Mediterranean, Tertiary forest dweller scaritid beetles, in which remote cladogenetic events are presently documented by distribution of their relic, sometimes monotypic extant genera.

Conclusions
Available data, and discoveries in progress (including two further species not yet described, but indicated in the map of Figure 8), suggest several reasons to hypothesize that Reicheiina in Sardinia formed a remarkably diversified clade through the process of adaptive radiation.
The speciation events that produced these taxa should have been originated by isolation of small populations in micro-geographic areas, in deep soils and caves, and an exceptional extent of adaptive colonisation and diversification into a variety of soil and underground compartments and ecological niches induced by the Plio-Pleistocene climatic changes, with some cases of subsequent overlap of distributions in wet, forested phases (Casale 2009).
The hypothesis here proposed, that Typhloreicheia (in the current sense) radiated in Sardinia, and then re-colonized the close, circum-Mediterranean islands and main-land, remains of course a mere hypothesis, that should be tested. Unfortunately, as recalled in Introduction, the study of this group is difficult owing both to the small sizes of its representatives, and particularly the scarcity of material, some taxa being known from a few or only one specimens, so that molecular data on them -like fossils, or larval stages -are so far fully absent. Therefore, future investigations will need further, strong efforts both in the field and laboratory.