An unexpected discovery of a new subgenus and a species of Plusiocampa ( Campodeidae , Diplura ) alongside an overview of Central European subterranean campodeids

An unexpected new subgenus and species of Campodeidae (Diplura), Plusiocampa (Pentachaetocampa) inopinata subgen. et sp. nov., a troglobitic species found in Schallsinger Höhle in an isolated karstic region in southwestern Germany is described. The new taxon shows two unique characters for the genus Plusiocampa: five dorsal femoral macrosetae and the presence of g1-glandular setae in females. Two other Plusiocampa species have been studied and taxonomic remarks made for them; both are also cave dwelling species from Germany: Plusiocampa dobati Condé in Dobat, 1975 studied from eight caves in the Swabian Alb, and one unnamed species of Plusiocampa (Plusiocampa) from four caves in the Franconian Alb. The biogeographical and taxonomic affinities among Plusiocampa species of Central Europe are discussed. The distribution of Plusiocampa species in Central Europe runs alongside the frontier of the Pleistocene glaciations, with non-troglomorphic Plusiocampa species adjacent to the glacial limits and troglomorphic Plusiocampa species below. Worthy of note is the presence only in the northeast of the Central Alps of two relict Plusiocampinae species, the already known Hystrichocampa pelletieri Condé, 1948 and the new species P. (P.) inopinata subgen. et sp. nov.


Introduction
Cave-dwelling campodeid diplurans have been known from Central Europe since the end of the nineteenth century (Joseph 1882).Nowadays, about fi fteen troglobitic species are known of.This subterranean fauna is well spread throughout the Dinaric Alps (Condé & Bareth 1996) and on both sides of the Alps, in Austria (Condé 1954) and Switzerland (Condé 1962), including the northern karstic Italian regions (Bareth & Condé 1984), and extending towards the northeast along the Inner Western Carpathian Mountains, in the north of Hungary (Stach 1929), and also within caves in the south of Slovakia (Lubomir Kováč pers. com.).In Germany only one troglobitic campodeid was found by Dobat (1975) in the Swabian Alb caves.Much later, samples from seven Swabian caves were used to properly describe (Condé 1993) the endemic species cited only by its name and a short description using a letter from Condé (Dobat 1975): Plusiocampa (s.str.) dobati Condé in Dobat, 1975.Recently some accurate biospeleological studies have been made including sampling of cave-dwelling animals in North Rhine-Westphalia, Hesse, Rhenish-Palatinate, Saarland, Baden-Württemberg (mainly Swabian Alb), and Bavaria (mainly Franconian Alb), where more than 10.000 caves and artifi cial caverns are known, resulting in the discovery in a single cave (Schallsinger Höhle, Baden-Württemberg) of a new and remarkable subgenus (Figs 1-2) of the genus Plusiocampa, already well known from subterranean habitats in the Euro Mediterranean region.Furthermore, taxonomic remarks are added on the species P. dobati, known from several northern caves in Swabian Alb, and on the uncertain Plusiocampa (Plusiocampa) sp., which has been located in caves of the Franconian Alb, Bavaria.

Sampling methods
The new material of P. dobati was collected from eight caves in the Swabian Alb, Baden-Württemberg, Germany.In the Vetterhöhle, some specimens were caught by using an exhauster and were directly transferred to 96% ethanol.In addition, two ethan-diol-1,2-traps were used to collect further specimens.The collections from the traps were also transferred to 96% ethanol.In the Bärentalhöhle, P. dobati was collected by hand and directly transferred to 96% ethanol.Plusiocampa (Pentachaetocampa) inopinata subgen.et sp.nov.was collected in the Schallsinger Höhle, southern Black Forest, Baden-Württemberg, Germany.It was collected by using a wet paint brush and transferred directly to 96% ethanol.It was only found in the "Great Hall" between 195 and 230 m away from the entrance of the cave, where the cave is highly humid.Some specimens were found on organic material, others on the surface of a cave lake, several of them already dead.

Material processing and identifi cation
The specimens were washed using distilled water and were put between slides and glass coverslips to be examined under a phase-contrast optical microscope (Leica DMLS) using Marc André II solution.The illustrations were made with a drawing tube, and the measurements were taken with an ocular micrometer.For measuring the length of the body, the specimens were mounted in toto and were measured from the base of the frontal process distal macrochaetae to the abdomen's supra-anal valve.For scanning electron microscopy (Hitachi S-4100), six specimens were coated with palladium-gold and used for scanning electronic microscopic photography and measurement of the sensilla.
The morphological descriptions and abbreviations used in this paper follow Condé (1956).We use gouge sensilla for the concavo-convexly shaped sensilla located on the antennae, described by Bareth & Condé (1981) and whose function is still unknown.For the position of macrosetae we follow the terms of Condé (1956): ma, medial-anterior; la, lateral-anterior; lp, lateral-posterior; mp, medial-posterior and post, posterior.

AS
= collection of Alberto Sendra, Alcalá de Henares, Madrid, Spain MCNB = Museum de Ciències Naturals de Barcelona, Spain ZMUC = Zoological Museum, Natural History Museum of Denmark, University of Copenhagen, Denmark

Etymology
Penta in addition to chaetae (chaeta-), both from the Greek words which means fi ve setae in reference to the noticeable fi ve dorsal macrosetae on the femur.

Phyletic affi nities of the subgenera of Plusiocampa
Recently, weak support was found for the classifi cation of Campodeidae (Sendra et al. 2017), specifi cally within Plusiocampinae Paclt, 1957, based on the sparse number of solid characters and the high intraspecifi c and interspecifi c variation of many of them.This makes the taxonomy of the Plusiocampinae a delicate subject, with only a few synapomorphic features that can be used to put some order into the problematic subfamily proposed by Paclt (1957).All Plusiocampinae have at least 1+1 ma, 1+1 la and 2+2 lp 2,3 on its pronotum, known in all the ten previously described genera (Sendra et al. 2017).Among them, Plusiocampa with 57 species distributed around the Mediterranean Basin (Condé 1956) is the most diversifi ed.The exceptions are the two Chinese species Plusiocampa (Dydimocampa) sinensis Silvestri, 1931 and Plusiocampa (Didymocampa) lipsae Condé, 1993, although both should be removed from Plusiocampa due to the macrosetal formula on urosternites II to VIII.Furthermore, a laminar telotarsal process is present in the case of P. lipsae.As a consequence, a new diagnosis of Plusiocampa is proposed: telotarsus with lateral crests and smooth-setiform lateral processes; pronotal formula with 1+1 ma and 2+2 lp macrosetae and from 2+2 to 4+4 la macrosetae; no less than 3+3 post macrosetae on urotergites VI-VII; no less than 5+5 macrosetae on urosternites II-VII and 2+2 macrosetae on urosternite VIII; and sensillum of antennomere III in a ventral position.
The genus Plusiocampa was divided into four subgenera based on two features, the dorsal macrosetae on the femur (Paclt 1957) and the ventral macrosetae on the tibia.Using these two synapomorphic features and additional ones, the following taxonomic key is proposed for the current fi ve subgenera, Pentachaetocampa subgen.nov.included.Bareth & Condé, 1984 For Plusiocampa s. str., its 47 species and 10 subspecies bear one dorsal femoral macroseta and 1-3 ventral tibial macrosetae.They are distributed from the Pontic Mountains in the northwest of the Anatolian Peninsula to the Betic Mountains on the Iberian Peninsula, colonizing the Balkan, Iberian and Italian peninsulas the Central French Massif included, and also the Aegean and most of the west-Mediterranean islands, reaching the Alps and the Carpathians Mountain ranges towards the north and in the south an isolated location in the Kabylie Mountains, northern Algeria.Most of its species inhabit subterranean ecosystems (Condé 1956) and only eight species and two subspecies can be considered soil-dwelling.

Key of the subgenera of Plusiocampa
Stygiocampa has six species, all of them sharing the absence of dorsal femoral macrosetae in addition to a progressive reduction of the notal and urotergal macrosetae formula.Furthermore, four of these species have an increase in the number of macrosetae on the urosternites.All of this shows remarkable troglomorphic features, its species inhabiting the subterranean ecosystems around the Dinaric Alps (Condé & Bareth 1996).
The two species of Dydimocampa share the possession of two dorsal femoral macrosetae and in both the mp meso-and metanotal macrosetae are absent.They have been found at two unique locations, a cave in the Crimean Peninsula (Silvestri 1949) and in the Movile Cave (Condé 1996) in the southeast of Romania, both near the Black Sea.
Only one species is proposed in Venetocampa: Plusiocampa (Venetocampa) paolettii Bareth & Condé, 1984, collected from a single cave in the Feltrine Alps.It is characterized by the absence of ventral tibial macrosetae and the progressive reduction of the notal and urotergal macrosetae, sharing these features with Stygiocampa, and the presence of dorsal femoral macrosetae as its differential feature (Bareth & Condé 1984).With this defi nition, another troglobitic species, Plusiocampa (Plusiocampa) dargilani (Moniez, 1984) could be included in Venetocampa.Plusiocampa dargilani was found in many caves in the Central Massif, southern France (Condé 1997), and in addition, it shares with most of the species of Stygiocampa an increase in macrosetae on the urosternites.
Finally, the new subgenus Pentachaetocampa subgen.nov.bears two synapomorphic features that separate it from other already-known Plusiocampinae: the fi ve dorsal macrosetae on the meso-and metathoracic femur and the presence of a narrow fi eld of g 1 -glandular setae on the fi rst urosternite in adult females.No other species of Plusiocampa has fi ve dorsal femoral macrosetae, although this does occur in another genus of Plusiocampinae.This is the case for Hystrichocampa Condé, 1948, a monospecifi c genus with a widely distributed species, H. pelletieri Condé, 1948, from several caves and mines from karst regions around the French and Swiss Jura (Condé 1948(Condé , 1962)).Nevertheless, many solid features differentiate Hystrichocampa from Pentachaetocampa subgen.nov.Among them the most noticeable are: the shape and pubescence of the telotarsal processes, the absence of g 1 -glandular setae in females and the unequal claws in Hystrichocampa.Furthermore, no other Plusiocampa or Plusicampinae females bear g 1 -glandular setae on the fi rst urosternite.
FEMALE UROSTERNITE I (Figs 6,[15][16].With subcylindrical appendages thinner than male appendages, each bearing up to 20 a 1 -glandular setae in a distal fi eld; and a narrow marginal fi eld with up to 25 g 1 -glandular setae in larger female adults. MALE UROSTERNITE I (FIG.5).With moderated large subcylindrical appendages, each bearing up to 70 a 1 -glandular setae in a large fi eld covering almost distal half of its ventral and apical side; and a narrow marginal fi eld with up to 35 g 1 -glandular setae in longer male adults.

Phyletic affi nities
Plusiocampa (Pentachaetocampa) inopinata subgen.et sp.nov. is a clearly recognizable species due to the presence of fi ve femoral macrosetae and the presence of g 1 -glandular setae in females.It differs strongly from members of other subgenera in the previously mentioned characters; but without these two differential features, P (P.) inopinata subgen.et sp.nov.seems to be most closely related to Plusiocampa (Plusiocampa) bonadonai Condé, 1948, based on similarities in the macrosetal formula of the thorax and abdomen and in the size and shape of the telotarsus.It is also a cave-dwelling species known from several caves, also found in endogean habitats (Condé & Poivre 1982) in the Maritime Alps, southeastern France.

Taxonomic remarks
Antennae with 28 and 29 antennomeres in adults and 28 in one juvenile.Cupuliform organ with eight complex olfatory chemoreceptor sensilla characterized by a cylindrical central structure without noticeable pores, surrounded by one concentric fold .Gouge sensilla long (25 μm), with a tip on the end (Fig. 20).Frontal process with a frontal and lateral rim (Fig. 21).The shortness of their telotarsal processes and the shape of the claws are confi rmed .The urosternites of male and female were mentioned in the original description but no drawings were produced.In the fi rst urosternite of both males and females there are no g 1 -glandular setae and the appendages support up to 54 a 1 -glandular setae in males and up to nine in females .

Phyletic affi nities
This small Plusiocampa s. str.sp.from Franconian Alb caves is characterized by its short appendages, modest claws, low number of antennomeres and cercal articles in addition to only four olfactory chemoreceptors in the cupuliform organ; all of these non-troglomorphic features indicate a not exclusively subterranean life for this species.Due to this, Plusiocampa s. str.sp.can be considered a troglophile form that can live in subterranean superfi cial habitats or humid soils.This troglophile life-style can be seen in other Plusiocampa species described from Hungarian caves (Stach 1929;Loksa 1960).This is the case for Plusiocampa (Plusiocampa) spelaeae Stach, 1929 from Baradla Cave (Gömör, northern Hungary) and Plusiocampa (Plusiocampa) breviantennata Loksa, 1960 from Lócz Cave (Balatonfüred, Hungary).Nevertheless, both cave-dwelling Hungarian species were poorly described and nowadays it is impossible to make a complete comparison with other species of Plusiocampa.Furthermore, both Hungarian species apparently lack medial posterior macrosetae on the mesonotum and metanotum.
The traits related with the small size observed in this Franconian Alb Plusiocampa s. str.sp., in addition to the presence in this species of thoracic medial posterior macrosetae, are found in a soil-dwelling species, Plusiocampa (Plusiocampa) humicola Ionescu, 1951, recently redescribed by Sendra et al. (2012).However, in the males of P. (P.) humicola g 1 -glandular setae are absent and that species was also found far from Germany in Cluj (Romania).
It is also possible to look into the affi nities of Plusiocampa s. str.sp.from Franconian caves in a species of Plusiocampa from a less distant area.The latter was informally referred to as Plusiocampa strohuali cavicola Vornatscher, 1943 and it has been reported from several Austrian caves (Condé 1954).Nevertheless, further sampling of material from the type location is the only solution to unravelling the taxonomic mess of several Plusiocampa species from Central Europe.

Current knowledge of subterranean Campodeidae fauna in Central Europe
The distribution of the subterranean campodeids around the European Mediterranean region has its northern limits from Belgium to the Crimea Peninsula, going along the southern limit of the Pleistocene glaciations.This lack of subterranean fauna under the glacial infl uence was pointed out early by Racovitza (1907) and remarked on in more recent distributional studies from Europe (Culver et al. 2006, for instance).The reason for this absence of subterranean fauna was also remarked on by Racovitza (1907), citing the lack of food in the form of organic material that could not reach the caves through the network of voids in the vadose zone (Sendra & Reboleira 2014;Jiménez-Valverde et al. 2017).
In the case of Plusiocampa and Hystrichocampa, both included in the subfamily Plusiocampinae (Paclt 1957), the northern limits, always below the 50th parallel north, are occupied by four nontroglomorphic species of Plusiocampa (Fig. 26; see Supplementary File 1), all species with short antennae with doliform antennomeres plus four simple cupuliform sensilla, and also a small body with short legs and cerci.Four non-troglobitic species are distributed from west to east: P. (P.) humicola found in soil habitats in Romania (Sendra et al. 2012); P. (P.) spelaea described from two caves, one from the north of Hungary (Stach 1929) and another from the south of Slovakia (Paclt 1956); Plusiocampa (Plusiocampa) corcyraea Silvestri, 1912, another soil-dwelling species found in the Czech Republic (Paclt 1961(Paclt , 1979;;Rusek 1964) and Slovakia (Paclt 1965); and Plusiocampa (Plusiocampa) sp.known from the Franconian Alb caves in this study.Among these non-troglomorphic species, P. (P.) breviantennata, found in a single cave in the center of Hungary (Loksa 1960), can also be included (Fig. 26).
Towards the south-west of these non-troglomorphic species and surrounded by the Central Alps, in lower mountains regions and consequently less affected by glaciations, there are caves inhabited by troglobitic species of Plusiocampa and Hystrichocampa.In the Austrian Alps, caves are occupied by Plusiocampa (Plusiocampa) strouhali Silvestri, 1933 and its subspecies P. (P.) strouhali cavicola; Plusiocampa (Plusiocampa) caprai Condé, 1950, which is also known from shallow subterranean habitats, is the only species whose distribution reaches the extreme east of Switzerland (Condé 1950(Condé , 1962;;Janetschek 1952;Christian et al. 1996); and the remarkable species Plusiocampa (Plusiocampa) hoelzeli (Neuherz, 1984), described from a single cave in Carinthia (Neuherz 1984).In regions north of the Alps, German caves are occupied by three species: P. (P.) dobati in the Swabian Alb, Plusiocampa (Plusiocampa) sp.from the Franconian Alb and the new P. (Pemturochaeta) inopinata subgen.et sp.nov. in a small karst area in the south of the Black Forest.Plusiocampa (P.) dobati and the new one represent the only troglobiont diplurans from Germany, not far from the northern-most troglobiont diplurans in Europe.Silvestri, 1912 andHystrichocampa Condé, 1962) in Central Europe; blue indicates karst regions.
And fi nally, in caves of the Western Central Alps the relict species Hystrichocampa pelletieri Condé, 1962 can be found (Fig. 26).
The current phylogeny and the palaeobiogeographical data known for species of Plusicampinae suggest that the colonization of Europe ocurred during the fi rst half of the Cenozoic, probably from Asia (Sendra et al. 2004).The Plusiocampinae settled in Central Europe and suffered continuous climatic changes, mainly during the Pleistocene, that could have wiped out different Pluiocampinae species several times.This glaciation frontier is nowadays occupied by four non-troglobitic species of Plusiocampa s. str.living in subterranean and edaphic habitats.However, only slightly towards the south troglobitic Plusiocampa s. str.species appear.Surprisingly, two species from different genera appear, both considered as relict due to their northeastern distribution in Europe and their unique features: the unexpected P. (Pentachaetocampa) inopinata subgen.et sp.nov.and H. pelletieri, both troglobites inhabiting the north-east of the Central Alps.