Playing hard to get : two new species of subterranean Trechini beetles ( Coleoptera , Carabidae , Trechinae ) from the Dinaric Karst

Almost 200 years of continuous and systematic research in subterranean habitats of the Dinaric Karst and adjoining areas have resulted in the discovery of more than 400 specialized subterranean beetles. Among these, a special place belongs to the morphologically well distinguished and elusive, so called aphaenopsoid trechine beetles, which are characterized by a prolonged head, pronotum and appendages, and widened, ovoidshaped elytra. Two new species of aphaenopsoid trechines – Derossiella lukici sp. n. from two deep pits on Mt Biokovo, Croatia, and Adriaphaenops petrimaris sp. n. from Pištet 4 Cave, Kameno more, Montenegro – are described, illustrated, and compared with closely related congeners. Identification keys for both genera and an annotated catalogue for all Adriaphaenops species, as well as data on the distribution and the ecology of these remarkable species, are provided and discussed.


Introduction
Following the description of the first cave animal, a subterranean beetle Leptodirus hochenwartii Schmidt, 1832 (Polak 2005, Moldovan 2012), the western Balkan's Dinaric Karst became a theater of ceaseless discoveries of highly specialized, cave adapted fauna.The so called troglobionts.Here, the richness of subterranean biodiversity exceeds that of similar areas throughout the world; thus, it is considered the world's primary subterranean biodiversity hotspot (Sket et al. 2004, Culver et al. 2006, Sket 2012).Moreover, it is only here that caves (locally called jama, pećina or špilja) having more than 100 troglobiotic species are known to exist (Ozimec and Lučić 2009, Zagmajster et al. in prep).Much of this exceptional richness consists of beetles specialized for living in these (Bregović and Zagmajster 2016) resource-scarce habitats (Gibert andDeharveng 2002, Culver andPipan 2009).
To date, more than 400 species of troglobiotic beetles have been recognized in the Dinaric Karst and adjoining areas (Bregović and Zagmajster 2016, Zagmajster, personal communication).The vast majority are classified in two subfamilies: Cholevinae (family Leiodidae) and Trechinae (family Carabidae).Even though their natural histories and ecology differ, the two groups show similar distribution patterns, which might suggest that the same mechanisms triggered the emergence of the two species richness peaks in the Dinaric Karst: one in the northwest and the other in the southeast (Zagmajster et al. 2008, Bregović andZagmajster 2016).
Herein we describe two recently discovered species of the Dinaric Karst aphaenopsoid trechines.They are representatives of genera with apparently differing or even opposing distribution patterns.One species belongs to the formerly monotypic and narrowly distributed Derossiella and the other to the relatively widely distributed and species-rich genus Adriaphaenops.
An immature female of a new species was first found during speleobiological research in the cave Biokovka, Golubinjak, Mt Biokovo, Croatia in September 2007, by the Croatian speleobiologist Marko Lukić.This specimen was examined by the first author (R.L.) and provisionally placed in the genus Derossiella.Further intensive research of the deep subterranean habitats of Mt Biokovo was executed from 2015 to 2017 by members of the Croatian Biospeleological Society, DZRJ Ljubljana, and members of the SubBioLab (Bregović et al. 2015).
During one of the visits to the Pretnerova jama, Lokva, Mt Biokovo, Croatia in 2015, biology student Ester Premate found a second immature female at an approximate depth of 120 m.Finally, in June 2017, a male was collected by the second author (T.D.), again in Biokovka, at a depth of ca 300 m.Subsequent examination of all three specimens, including male genitalia, confirmed that they belong to an undescribed species of the genus Derossiella, whose description is provided below.
Whereas representatives of the genus Derossiella seem to be rare and exceptionally hard to find, almost half of the Dinaric Karst aphaenopsoid trechines, 12 out of 28 described species, are classified within the Southern Dinaric genus Adriaphaenops.The first species of the genus, Adriaphaenops antroherponomimus (Noesske, 1928), was found during the summer of 1927 in a small cave named Snježnica u Tišovom kršu (synonym = Čatol jama), Mt Bjelašnica, Gacko, Bosnia and Herzegovina by Leo Weirather, a famous Austro-Hungarian speleobiologist and an early explorer of the Dinaric Karst.Just before the Second World War, Oskar Scheibel, an entomologist from Zagreb, Croatia, described two additional species, each based on a single female specimen found in two famous caves.He described A. pretneri Scheibel, 1935 from Vjetrenica, Zavala, Popovo polje, Bosnia andHerzegovina, andA. staudacheri Scheibel, 1939 from Grbočica, Virpazar, Rijeka Crnojevića in south-eastern Montenegro.The fourth species, A. stirni Pretner, 1959, was discovered during the autumn of 1956 by a Slovenian entomologist, Jože Štirn, in Velja peć, a small cave located near Nikšić in Montenegro (Pretner 1959).By the end of the 20 th century, speleobiological investigations made on Mt Durmitor, Montenegro led to the discovery of a new species with two subspecies, A. zupcense zupcense and A. zupcense tartariensis (Pavićević 1990(Pavićević , 2001)).Intensive speleobiological investigations in Eastern Hercegovina, in the beginning of the 21 st century, resulted in the discovery of two new species, A. perreaui Quéinnec &Pavićević, 2008 from Pećina u Mravinjac, Mt Bjelašnica, Trebinje, Bos-nia andHerzegovina, andA. kevser Quéinnec, Pavićević &Ollivier, 2008 from Vilina pećina, Mt Lebršnik, Gacko, Bosnia andHerzegovina. Finally, five new species, A. albanicus, A. jasminkoi, A. mlejneki, A. njegosiensis, andA. rumijaensis, were found during speleological and speleobiological survey in the central and southern Dinarides (Bosnia and Hercegovina, Montenegro, and Albania) and were described in 2016 (Lohaj et al. 2016).
Recently, during speleobiological research performed by SubBioLab members and cavers from DZRJ Ljubljana in Pištet 4 cave (synonym = PT4), Velji Pištet, Kameno more, Risan, Montenegro in spring 2018, two specimens of the genus Adriaphaenops were found by the second author (T.D.).Subsequent examination confirmed that they belong to a new species described below.

Geomorphological framework and locality descriptions
Plate tectonics during Eocene and Miocene triggered uplift of the so called Adriatic carbonate platform, resulting in the formation of the Dinaric Karst, a 650 km long mountain range stretching along the eastern Adriatic Sea coast (Vlahović et al. 2005, Korbar 2009) from the border with the Southern Calcareous Alps to the Albanian coast (Gams 2004).Along the Dinaric Karst, protruding mountain ridges like Mt Biokovo and Orjen (and associated Kameno more) are recognized and accompanied by numerous karstic poljes.Both ridges are predominantly formed by Jurassic and Cretaceous limestones and subordinate dolomites (Ad-amson et al. 2014, Velić et al. 2017), and are characterized by high levels of yearly precipitation (Ranković 1961).Moreover, as a part of the Orjen foothills, Kameno more is considered to be the area receiving the highest amount of precipitation in Europe (Ducić et al. 2012).In addition, both areas are highly karstified and characterized by a lack of surface waters.Another common factor in both areas is the existence of many karst springs with outlets below sea level, so called "vrulje" (Bellafiore et al. 2011, Kuhta et al. 2012), whose origins are connected to oscillations of the sea level during the Miocene and Pleistocene (Bonacci 2015).The rise and fall of glaciers during the Pleistocene largely shaped the surface morphologies of both areas (Hughes et al. 2011(Hughes et al. , Žebre et al. 2013)).Both areas include extensive karstic landscapes with numerous phenomena such as dolines, sinkholes, and a large number of caves.As a result of glacial melt water ingressing deeply into the vadose zone, deep caves were formed.On Mt Biokovo alone, more than 400 caves are known (Vedran Sudar personal communication), and 11 of them have depths exceeding 250 m.Most renowned are the caves of Mokre noge (831 m deep), discovered in 2009, and Amfora (788 m deep).No similar data on the number of caves exists for the areas of Orjen or Kameno more, but some of the deepest caves are of similar depths to those found on Mt Biokovo (e.g.Kozi Dira, 654 m deep, and PT4, 455 m deep).However, due to the thickness of the limestone beds, the large extent of yet unexplored terrain, and hydrological connections with the extensive karstic springs and vruljes in the coastal area, the potential for deep caves in both areas is likely to surpass the depth of 1 km.Due to the high endemicity of Biokovo's subterranean fauna, several speleobiological excursions were organized in recent years, culminating with the "1 st Biospeleological expedition -Biokovo 2017" (Sudar et al. 2017).The main goal of the expedition was to gather additional distributional data on the poorly known taxa and to execute speleological surveys of some of the deep caves.During four visits in 2017, from April to October, 26 caves were visited, including eight caves exceeding 250 m in depth.Some of the deep caves were visited due to the existence of pre-expedition data on ambiguous trechine belonging to a yet undescribed species of Derossiella.These caves include Biokovka, Golubinjak, Vošac, Mt Biokovo, Croatia (43.322197°N, 17.050591°E; 363 m deep) (Fig. 1a) and Pretnerova jama, Lokva, Mt Biokovo, Croatia (43.33945°N, 17.03836°E; 254 m deep) (Fig. 1b) (Bregović et al. 2015).Both caves are characterized by a series of vertical pits ingressing deeply into the vadose zone.Deeper parts of both caves, starting from the approximate depth of 75 m in Pretnerova jama and 200 m in Biokovka, are characterized by films of percolating water flowing over vertical walls; the cave hygropetric.Also, the temperature span was similar in both caves, ranging from about 4 °C in the entrance parts without direct sun influence to 5 °C in the lower parts.Subterranean fauna in both caves was sampled by using baited pitfall traps, hand collecting or by mean of an aspirator.No preserving media was used in the pitfall traps; therefore, the trapped fauna was alive and mainly released upon collecting the traps.
Similarly, the Kameno more area was visited due to the speleobiological potential, the existence of already known and peculiar troglobiotic species such as Hadesia cf.weiratheri (Perreau and Pavićević 2008), and the existence of caves with the deep vadose pits.Such is also the second deepest cave in the area, Pištet 4 cave (synonym = PT4), Velji Pištet, Kameno more, Risan (42.55183°N, 18.73864°E) (Fig. 1c), whose entrance opens beneath a boulder in the bottom of a collapsed doline.Like the described caves of Mt Biokovo, PT4 is also characterized by the existence of a series of vertical pits interconnected with a stream running through the limestone beds, reaching the phreatic waters at the maximum depth of 455 m.Deeper portions of the cave, starting already at a depth of 60 m, are characterized by the existence of the cave hygropetric.The temperature in the cave was measured only once, at an approximate depth of 200 m, and was 7.4 °C.Detailed descriptions of the cave, including morphology, hydrology, and detailed speleological maps can be found in Binding (2010Binding ( , 2011)).Due to a time constraint, the cave was sampled using hand collecting only.

Laboratory work and morphology
The morphological structures of the beetles were examined using Olympus SZ 60 (Olympus, Tokyo, Japan) and Leica S8 APO (Leica, Wetzlar, Germany) stereoscopic microscopes.Macrophotographs were taken using a Canon 5D Mark II camera.Male and female genitalia were dissected, cleaned, and mounted in Euparal or Dimethyl-Hydantoin formaldehyde (DMHF) on transparent slides, which were later pinned under the specimens.Fine structures of male and female genitalia were studied at magnifications up to 600× by using a Leica DM1000 light microscope (Leica, Wetzlar, Germany).Drawings were made using an attached drawing tube.

TL
total body length (measured from the anterior margin of clypeus to the apex of elytra).L overall length, from apex of mandibles to apex of elytra, measured along the suture.HL head length (measured from the anterior margin of the clypeus to the neck constriction).HW maximum width of head.

AL
antennal length (measured from the base of antennal scape to the apex of terminal antennal segment).

PL
Pronotal length (measured along the median line).

PW
Maximum width of pronotum, as greatest transverse distance.

EL
Elytral length (as linear distance measured along the suture from the elytral base to the apex).

EW
Maximum width of elytra.HL/HW Ratio head length/maximum width of head.PL/PW Ratio length of pronotum/maximum width of pronotum.EL/EW Ratio length of elytra/maximum width of elytra.
Forward slash indicates separate labels.Diagnosis.Medium-sized aphaenopsoid trechine with morphological features fully matching generic description proposed by Quéinnec (2008).Head long, parallel-sided, with complete, deep frontal furrows reaching neck constriction and two pairs of supraorbital setae, posterior setae doubled.Eyes absent, mentum with sim-ple tooth.Aphaenopsoid habitus with very narrow head and pronotum; elytra oblong-oval, 2.6 times wider than head and pronotum.Hind angles of pronotum without setae.Surface completely glabrous, striae absent, elytral chaetotaxy with macrochetae and microchetae.Cuticle depigmented, reddish-yellow, body strongly flattened dorso-ventrally, with very long and slender legs and antennae (Figs 3, 4).Closely related to the type species of the genus, Derossiella nonveilleri, from which it differs by paired posterior supraorbital setiferous punctures, differently shaped elytra with higher number of macro-and microchetae, narrower pronotum, as well as by the differently shaped aedeagus.

Genus Derossiella Quéinnec, 2008
Description.L: 5.5 mm (PT)-6.0mm (HT), TL: 4.8 mm (PT)-5.4mm (HT).Head relatively large, nearly parallel-sided, with maximum width behind middle, distinctly longer than wide (index HL/HW 1.35 (PT)-1.42(HT), slightly wider than pronotum, sparsely pubescent.Frontal furrows deep, complete, reaching neck constriction, slightly divergent posteriorly.Anterior pair of supraorbital setae situated before middle of head length, posterior supraorbital setae paired, two setae on each side of head situated close to neck constriction.Neck constriction distinct; genae gently convex.Clypeus and labrum with three pairs of setae, outer pairs longer.Antennae length 4.9 mm (HT)-4.3mm (PT), scape as long as pedicel, almost as long as terminal antennomere.Pronotum elongated, slightly longer and narrower than head, with maximum width in anterior third, index PL/PW 1.75 (PT)-1.83(HT), only slightly narrowed anteriorly, posterior part distinctly narrower than anterior.Surface glabrous, median furrow distinct, visible in whole pronotal length.Propleura visible from dorsal aspect in basal two-thirds.Anterior angles of pronotum not protruding, posterior angles obtuse.Lateral furrows well developed, deep, with one pair of anterolateral setae, situated in the apical fifth of pronotal length.
Legs long, slender, densely pubescent.First two tarsomeres of male protarsi distinctly dilated and protracted at their internal margins.Tarsal claws very long and slender, without traces of denticulation on their internal sides.
Male genitalia.Aedeagus (Fig. 5) 0.58 mm long, relatively robust, regularly wide, lacking apical constriction, laterally flattened.Parameres relatively long and slender, longer than half of the length of aedeagus.Apex very short, tip obtuse.Each paramere at apex with three setae, two long, and one short.
Female genitalia as in Figure 5.
Etymology.Patronymic, dedicated to our dear friend Marko Lukić (Zagreb, Croatia), enthusiastic speleologist and speleobiologist, taxonomic specialist on subterranean Collembola, and collector of the first specimen of the new species.
Revision.Lohaj et al. 2016.Diagnosis.A medium-sized trechine beetle with aphaenopsoid features: head and pronotum elongate; elytra ovoid, strongly narrowed at the base, obviously wider than head and pronotum; body depigmented, strongly flattened, covered with sparse pubescence (Figs 6,7).This new species is mainly characterized by the presence of four pairs of dorsal setae (three discal and one preapical) on elytra.This character is so far present only in presumably closely related species, A. kevser and A. rumijaensis; see identification key below.
Pronotum elongate, slightly longer and wider than head, with maximum width in middle, only very slightly nar-rowed anteriorly, basal part distinctly narrower than anterior (index PL/PW 1.66 (HT)-1.57(PT)), sparsely pubescent, setae long, suberect; median furrow weakly marked, visible in the middle part of pronotum.Propleura visible from dorsal aspect only in basal half.Anterior angles of pronotum distinctly protruding, obtuse, posterior angles obtuse.Lateral furrows developed, deep, with one pair of anterolateral setae, situated in apical fourth of pronotal length.
Elytra subovate elongate, distinctly longer than wide (index EL/EW 1.74 (HT)-1.78(PT)), with maximum width in middle; elytral surface covered with very sparse, long and erect pubescence; striae absent.Site of stria 3 with four (three discal and one preapical) setae, humeral group of umbilicate pores not aggregated, first anterior pore of humeral group isolated and situated before the level of the first discal seta.Pore 5 located nearer to pore 6 than to pore 4.
Legs long, slender, densely pubescent.First two tarsomeres of male protarsi distinctly dilated and protracted at their internal margins.Tarsal claws very long and slender, without traces of denticulation on their internal sides.
Male genitalia (Fig. 8): aedeagus very long and slender, gradually narrowed towards apex in lateral aspect, sagittal aileron large, endophallus without distinct copulatory piece.Parameres long and slender, with two long setae at apex.Female genitalia as in Figure 9.
Etymology.Topotypic, referring to the toponym where the Pišet 4 cave is situated, Kameno more (in English, Sea of stone and in Latin, Mare petris).
Differential diagnosis.Genus Adriaphaenops currently comprises 13 described species, including A. petrimaris sp.n.This species, A. kevser, and A. rumijaensis form a group of species with four discal setae (three dorsal and one preapical) on elytra, with elytral pubescence in all three species sparser in comparison to the other species (Fig. 7). A. petrimaris morphologically resembles A. kevser, described from Mt Lebršnik, Bosnia & Hercegovina.These two species can be easily recognized by the shape of head, pronotum, and aedeagus (Figs

Discussion
The vast landscape of the Dinaric Karst is characterized by the existence of numerous karstic fields and mountain ridges reaching well above 2000 m a.s.l.While most of the lowland areas, including most of the karstic fields, were intensively sampled already in the early days of speleobiology, high mountainous areas received far less attention, mostly due to their physical remoteness and the challenging logistical demands intrinsic to their exploration.However, in the last 25 years many new beetle species and even genera have been found and described from such areas (Monguzzi 1993, Casale et al. 2012, Lohaj et al. 2016).Their discovery was largely dependent on the improvements of caving techniques, which enabled speleological surveys and speleobiological sampling in the deep vadose caves of the Dinaric Karst that reach a maximum depth of 1471 m (Lukina jama-Trojama system, Nothern Velebit, Croatia) (Bakšić et al. 2013).
Sixteen of 30 Dinaric aphaenopsoid trechines, including those described here, were discovered in vertical pits.Moreover, if we consider only those species discovered after 1980, 16 of 23 species (70%) were discovered in caves where vertical caving equipment is needed.The rest were predominantly discovered in remote and hardly accessible karstic areas, while only a few of them were found after systematic sampling of already known caves.Along with the aphaenopsoid trechine beetles discovered throughout the Dinaric Karst, new species and genera of Cholevinae were also discovered.Some genera include morphologically and ecologically specialized hygropetricolous Cholevinae: Radziella Casale & Jalžić, 1988;Tartariella Nonveiller & Pavićević, 1999;Croatodirus Casale, Giachino & Jalžić, 2000;Nauticiella Moravec & Mlejnek, 2002;Velebitodromus Casale, Giachino &Jalžić, 2004 andKircheria Giachino &Vailati, 2006.All of these filter-feeding genera are dependent on the constant influx of percolating waters in the vadose zone, which enables functioning of the cave hygropetric (Sket 2004, Giachino andVailati 2006).
Genera morphologically similar to the Dinaric aphaenopsoid trechines are also found among subterranean trechines distributed in the Alps or Pyrenees (Faille et al. 2013).Common morphology of these genera is characterized by elongated appendages, head, and a pronotum, with ovoid and basally strongly narrowed elytra, which are wider than the head or pronotum.Such morphological characteristics are present also in other groups of Balkan, American, and Asian specialized Carabidae (Gómez et al. 2016, Luo et al. 2018, Vrbica et al. 2018) and are probably a consequence of directional selection driving the convergent evolution of elaborated traits in the subterranean fauna.However, such morphological derivations still lack a proper evolutionary or functional explanation (but see Luo et al. 2018).
Representatives of the Dinaric aphaenopsoid trechines are generally hard to find, inaccessible, and mostly known from a few individuals caught by hand or, as in several cases, "accidentally" by long-term pitfall trapping.The only exceptions to this rule are representatives of the genera Acheroniotes (Lohaj and Lakota 2012) and Dalmataphaenops (our own unpublished data).The lat-ter genera are known to locally form large populations.Although the Dinaric aphaenopsoid trechines are often considered to be specialized predators (Quéinnec 2008), little is known about their biology or their role in the subterranean communities.Moreover, as they are not prone to pit-fall trapping, most of our knowledge on their biology and behavior is based on scarce field observations.At present, we are not aware of their phylogenetic relations and biogeography or evolutionary trends, as only several samples of Dinaric taxa were included into phylogenetic analyses on the continental scale (Faille et al. 2013).Some of the genera, like Scotoplanetes (Lakota et al. 2010), and the species of Derossiella described above, have elongate legs and claws.Also, when compared to other aphaenopsoid genera in the Dinaric Karst, these genera are characterized by thin integument (Quéinnec 2008).These characters can to some extent be connected with specialization to the habitat these animals most probably live in.Most representatives of these genera, including the specimens described here as D. lukici sp.n., were collected directly from 'moonmilk' or the cave hygropetric (Lakota et al. 2010, our data).Such morphological adaptations offer possibilities to hypothesize the adaptive value of these characters to predation on specialized taxa found in the cave hygropetric (hygropetricolous beetles, springtails, leeches, and crustaceans).Unfortunately, such hypotheses remain untested as most of the specimens were preserved in low concentration ethanol or even vinegar, causing degradation of DNA material and minimizing the possibilities for later DNA isolation of the gut content.
Another interesting hypothesis to be tested is the adaptive value of divergent morphological characters, as Derossiella and Adriaphaenops are known to exist in sympatry with other specialized aphaenopsoid trechines, Dalmataphaenops and Scotoplanetes, respectively.This raises the question of niche differentiation among the specialized subterranean beetles, similarly to what was already shown in other subterranean taxa (Trontelj et al. 2012, Vergnon et al. 2013).However, with the extremely low number of specimens and the already mentioned lack of a phylogenetic context for the Dinaric aphaenopsoid trechines we are unable to place such hypotheses in their proper evolutionary context.Although data on the Dinaric aphaenopsoid trechines are lacking, only the publication of all known data will enable recognition of the distributional patterns of the group, the mechanisms underlying these patterns, general biology of the taxa, and the potential need to employ suitable conservation policies.

Figure 1 .
Figure 1.Cross sections of the caves from which the newly described species were collected: a) Biokovka, Golubinjak, Mt Biokovo, Croatia; b) Pretnerova jama, Lokva, Mt Biokovo, Croatia; c) Pištet 4 cave (PT4), Velji Pištet, Kameno more, Risan, Montenegro.Approximate finding localities are presented with red points.The original cave surveys of Biokovka and PT4 were made by Ivan Glavaš and the University of Bristol Spelaeological Society, respectively.