Cucujus tulliae sp. n. – an endemic Mediterranean saproxylic beetle from genus Cucujus Fabricius, 1775 (Coleoptera, Cucujidae), and keys for identification of adults and larvae native to Europe

Abstract Cucujus tulliae sp. n. is described as a new member of genus Cucujus Fabricius, 1775 (Coleoptera, Cucujidae), which enumerates at present eleven species distributed in Eurasia and northern America. This saproxylic beetle is the first Cucujus species known only from Mediterranean and it is probably endemic to Calabria (Italy). The species was found especially in old–growth mountain forests of high conservation value (i.e. national parks) dominated by Calabrian pine (Pinus laricio calabrica). We hypothesize that Cucujus tulliae sp. n. probably evolved from isolated populations of Cucujus haematodes Erichson, 1845. The species is thus relictual and of high conservation value, corresponding at least to endangered (EN) category with respect to recent IUCN criterion. Cucujus tulliae sp. n. is here compared with two species native to Europe – Cucujus haematodes and Cucujus cinnaberinus (Scopoli, 1763) and with the Caucasian Cucujus haematodes caucasicus Motschulsky, 1845, which is confirmed as a valid subspecies. The male genitalia of this Caucasian form have been examined and illustrated for the first time. A comprehensive key to adults and larvae of European species is provided.

and occasionally of silver fir, Abies alba fallen trees. Adult specimens are in our study area quite uneasy to collect in nature, probably because of their early appearance in spring and short reproductive season. On the contrary, the preimaginal stages are well known for their underbark life, in the literature they are sometimes defined as "scavengers", but also predators on pupae and small larvae of other insects (Mamaev et al. 1977) or even on other subcortical beetles (Rozhkov 1970;Burakowski et al. 1986). Palm (1941) observed that C. cinnaberinus larvae are able to feed on bast and cambium, and that cannibalism on younger conspecific larvae ceases in presence of longhorned beetle (Cerambycidae) larvae, that were vigorously accepted. Straka (2008) observed C. cinnaberinus adults feeding on various insects and was able to keep larval specimens alive with mealworms -darkling beetle larvae (Tenebrionidae). Horák et al. (2010) observed in far eastern Russia that the associated guild of C. haematodes was composed by springtails (Collembola), mites (Acari), ants (Formicidae), carabids (Carabidae) and subcortical histerids (Histeridae). Mazzei et al. (2011) reared C. cinnaberinus larvae with calliphorid maggots (Diptera), but especially with fresh beef meat, and this very simple method was adopted in this study.
All the larval specimens were kept at a temperature of 20°C with fresh beef meat, their development lasted about 8 months and a maximum of six larval molts were observed before pupation.
The dissections of adult male specimens were normally performed after short KOH treatment of the terminalia and total abdomen removal. The male genitalia are in fact highly delicate and a less careful extraction may cause the loss of the typical long "flagellum", well described by Sharp and Muir (1912) and troubles in position of the sclerified structures of the internal sac. Thereafter, the male genital structures were prepared on small transparent labels and enclosed in euparal, finally the labels arranged on the same insect pin of the specimen. The photographs of the adult and larval material were made with a Zeiss Stemi SV11 Stereoscope with a Canon Power-Shot G5 five MP digital camera. The male genitalia were photographed with a Zeiss Axioskop equipped with Nomarski optic (Differential Interference Contrast, DIC), using a Nikon Coolpix 4500 four MP digital camera. The male genitalia figures were merged with help of Adobe Photoshop software.
The terminology of adult genitalia follows Wilson (1930) and Thomas (1999), the larval morphology is based on Lawrence (1991).

Collections examined
Museo Civico di Storia Naturale, Verona, Italy (MCV); Department of Entomology the National Museum, Prague, Kunratice 1, Czech Republic (NMP); collection P. Brandmayr, conserved in the Department of Ecology of the University of Calabria (PBC). The larval material used for the taxonomic key is conserved in 70% alcohol in the "Tullia Zetto" larval collection (TZC) in the Department of Ecology of the University of Calabria.
Type material. Diagnosis. Cucujus tulliae is clearly related to the cinnaberinus-haematodes species group, its distribution seems to be restricted to the mountains of Calabrian peninsula. The closest taxon could be C. haematodes, from which it can easily be distinguished by the less prominent postgenae, the arrow head shaped prosternal apophysis, the smaller and less spiny pronotum, the typical median lobe of the aedeagus and the larval morphology, that is apparently unique because of its slender body and occipital furrows.
Description. A bright red species, resembling a small-sized haematodes in colour, but with a less serrate pronotum. Length 11.2-12.5 mm. Colour light red, legs dark brown/black, tarsi brown. Prosternum of the same colour of pronotum. Antennae black, mandibles red/orange with black apex. Head distinctly wider than pronotum, with two longer setae after the posterior border of eyes. Postgenae less swollen than in C. haematodes (Figs 1,5,9). Occipital furrow deep and long as one third of the posterior head width. Frontoclypeus with a gentle longitudinal swelling. Pronotum less spiny than in C. haematodes, distinctly prolonged at the level of the neck as a short collar (Figs 3,7,11). Prosternal apophysis elongated as an arrow head, slender than in C. haematodes (Figs 4,8,12). Elytral sides distinctly carinate at its upper external margin, almost until the apex. Elytral surface opaque, density of punctures similar to that of C. haematodes. Apex of elytra broadly rounded and with short pubescence. Metathoracic wings well developed and robust. Sutural stria slightly convex. Median lobe distinctly wider than median strut, less restricted at its apex than in haematodes  (Figs 13,16,19,21). Apical process well protruding, more than in haematodes, and triangular at the tip, like in C. cinnaberinus (Figs 14,17,20,22). Median strut 4.5 times longer than median lobe and particularly thin. Flagellum longer than the entire body, connected at its base with the male deferents, its more chitinized trait reaches the median lobe at the level of the two chitinous plates of the endophallus and bends backwards to the median strut. After bending the flagellum becomes transparent and runs across the endophallus, where, at the middle of its length, it rolls up in a sort of "ball", like a wire (Fig. 17). In figure 2 the "flagellum ball" is photographed in the median strut of C. cinnaberinus (Fig. 18).
Distribution and habitat. The larvae of this species have been collected under the bark of fallen pine trees of at least 25 cm diameter or on dead silver fir fallen trunks, but only on single locations of the National Park. The species seems to prefer cooler, northern exposed slopes and high air humidity at major elevations. In laboratory most larvae kept at 20°C died before pupation, a fact that could be explained by lower temperature preferences of the immature stages. The low number of larvae collected and the successful rearing of only two adults makes hypothesis much more difficult. Concerning geographic distribution, at the moment this taxon is known only from Calabria, in the highest part of the Sila plateau in the core of the Sila National Park, from two sites: Vallone Freddo and Serra Vurga. A single larva has been collected in the surroundings of Gambarie, in the Aspromonte National Park, at 07.06.2009, but this specimen died before pupation. There is little doubt that this new taxon may be an endemic saproxylic beetle of central and southern Calabria, with larval populations depending on the availability of high amounts of dead wood, especially of Calabrian pine, Pinus laricio var. calabrica.

Cucujus cinnaberinus (Scopoli, 1763)
http://species-id.net/wiki/Cucujus_cinnaberinus Note. We examined several specimens of this taxon, both from Italy as well as from the National Museum of Prague. This species is very abundant in the pine forest of the Sila National Park in Calabria.   VIII-1960, det. Ratti, 1971.
NMP: about 120 specimens from many countries of Europe.

Cucujus haematodes Erichson, 1845 http://species-id.net/wiki/Cucujus_haematodes
Note. This species is abundant in the Sila National Park, but less than C. cinnaberinus. We collected numerous larval stages also in the Aspromonte National Park.  Sides of pronotum, inner side of postgenae and mandibles black, mandibles before the apex somewhat lighter coloured. Maximum width of the pronotum at the front border, ventral side of the same part black, with a median yellow stripe that prolonges onto the prosternal apophysis. Head triangular, postgenae obliquely protruding backwards, occipital groove well marked and reaching postgenae, nevertheless deeper in the middle, where the punctuation is well developed. Body length 12-15.5 mm, median lobe of the aedeagus evidently restricted at the basis (Fig. 14)  Pronotum of normal size, distinctly narrower than the head, apical process of the median lobe tongue like, rounded at the tip (Fig. 17) .... haematodes Er. -Body size 15.5-16 mm, pronotum broad and robust, little smaller than the head at its posterior end. Postgenae well developed, but less protruding laterally and more backwards oriented, not surpassing the head width at the level of the eyes. Apical process of the median lobe compressed, paddle like (Fig. 22)

Diagnosis of the genus Cucujus Fabricius, 1775
Larvae extremely flat bodied, length 7-8 mm in young stages, around 25-30 mm in aged ones. Colour from pale yellow to dark reddish brown. Head prognathous, always broader than pronotum, maximum width at the occiput (parietale), postgenae inflated. Epicranial suture sinuate, coronal suture short but distinct. Stemmata normally in number of six, located more or less at half of the length of head. Antennae of variable length; first antennomere robust, distal antennomere much slender and setose only at the apex. Second antennomere with a flat sensorium bordered by a chitinous ring. Fronto clypeal suture more less concave in the middle; front margin of the labrum with 4 macrochaetae; frontoclypeal suture absent. Mandibles asym-metrical, broadly triangular and irregular bidentate, the teeth of the left one not at the same level (dorsal view). Two setae on the external side of each mandible; prostheca thin and hook like, broad based. Mola tuberculate, long, anteriorly with a penicillum formed by a dense brush of short setae. The complex of mouth parts well protruding and occupying more or less one third of the head width. Maxillary and labial palps very short. Legs very short and robust, ending with one tarsungulus.
Notal sclerites distinctly bordered. Abdominal segment VIII longer than precedent, lateral margin with two processes bearing setae. Lateral portion of segment VIII with a stout protrusion below the spiraculum ("spiracular process"). Between segment VIII and IX there is a strong conical appendage with many apical setae. Tergum IX with one pair of strong urogomphi bearing a robust bifid spine at their base. Lateral ends of tergum IX with a strongly sclerotized thorn. Antennae slender, second joint distinctly longer than the first one, apical joint thin five times longer than wide at the basis. Lateral border of parietale a little swollen in correspondence of the stemmata. Epistomal front margin moderately oblique towards the mandibular basis (Figs 24-25). Urogomphi well curved and apically a little converging to the median plane (Figs 26-27). Basal tooth with minor spine directed outwards and far from the apex. Spiracular process small and little pronounced (Figs 36-38). Conical appendage very large at the basis, distinctly setose around the apical part, chitinous apex short (Fig. 37)  Head posteriorly sinuate and marked by very long antennae. Second antennomere a little longer than the first one. Apical antennomere six times longer than wide at the basis. Epistomal front margin strongly oblique towards the mandibular insertion. Frontal suture less sinuate than in previous species (Figs 32-33). Urogomphi strong and a little converging at the end, apex with three evident macrochaetae (Figs 34-35). Basal tooth of normal shape and slender. Spiracular process distinctly protrudent backwards (Figs 42-43). Lateral thorn of tergum IX strongly bent to the rear, forming an angle of ninety degrees (Fig. 44)

Conclusions
The new described taxon of saproxylic beetle, Cucujus tulliae, seems to be of limited distribution, at the moment the only two adult specimens, obtained by rearing, are known from Sila National Park, and a single larva has been captured in the Aspromonte National Park, always on dead Calabrian Pine trunks. It is not astounding that this very cryptic species escaped the capture by previous collectors, most of them were occasional visitors of the Sila plateau and this area has never been the object of a long term faunal survey. Moreover, in the older Italian reports (Ratti 1986(Ratti , 2000 both C. cinnaberinus as well as C. haematodes were collected mainly on beech (Fagus sylvatica) or silver fir and normally in single specimens or very low numbers. The only exception that is known concerns C. haematodes, collected by Prof. Sandro Ruffo in more individuals on the Mt. Pollino (Calabria and Basilicata: Italy), in the year 1953, during a kind of "nuptial flight" (personal communication to the last author). Only after 2009 these saproxylic beetles seem to have expanded their habitat also into pure pine woods (Mazzei et al. 2011), perhaps as a consequence of changes in forest management -especially hands-off approach causing higher dead wood accumulations.
The new species seems to be also habitat restricted, in fact the larvae have been found only at three sites in Sila National Park, on northern exposures or at high elevations. Thus, further research is needed to assess the ecological requirements of the species.
The systematic position of C. tulliae sp. n. indicates an affinity with C. haematodes -body colour and male genital parts, especially the connection between median lobe and median strut (phallobase of other authors) are similar in both species. The new species may have evolved during a cold climate phase (glacial period?) from isolated C. haematodes populations. The smaller body size (if confirmed by new findings) speaks for less favorable conditions, as prey density or trunk sizes.
Concerning a first hypothesis on the conservation status of this southern Italian endemic, the evaluation as endangered (EN) in the IUCN categories seems appropriate -e.g. with respect to endemic Osmoderma spp. ). C. tulliae should be "ipso facto" included in the Italian red list of saproxylic beetles.
The second outstanding result of this study is that for the first time C. haematodes caucasicus Motsch. has been recognized as a valid subspecies, or perhaps as an allopatric species of the haematodes "species aggregate", the heterogeneity of which has been emphasized especially by Horák and Chobot (2009). Also Mamaev et al. (1977), basing on larval characters, considered C. caucasicus as a separate species, and a thorough reexamination of V. de Motschoulsky's diagnosis reveals that later authors severely underestimated his statements.
The C. haematodes group reveals to be not only extremely widespread in his Palaearctic distribution, but also highly influenced by isolation and tending to local speciation. A revision of this species aggregate could be of importance for conservation biology and for the relationships between saproxylic predators and history of palearctic forests.