A new species of Euscorpius ( Scorpiones : Euscorpiidae ) from southern Bulgaria

A new scorpion species, Euscorpius drenskii sp. nov., is described from the Western Rhodope Mts. in southern Bulgaria. It is characterized by an oligotrichous trichobothrial pattern, which shows a conspicuous loss of one trichobothrium in the external median patellar series (em = 3), also observed in E. carpathicus (Linnaeus, 1767) and the subgenus Alpiscorpius Gantenbein, Fet, Largiadèr & Scholl, 1999. Phylogenetic analysis of 16S rDNA marker sequences does not show any close relationship between these three groups, suggesting that the observed loss of a trichobothrium is an independent event.

length; Lpat\ patella length; Lmet sum of the length of all metasomal segments; Wmet: sum of the width of all metasomal segments; met.seg: metasomal seg- ment; CarA/CarP %\ average ratio of distances from center of median eyes to anterior and posterior mar- gins of the carapace; DPS\ dorsal patellar spur; DD\ distal denticle; MD\ median denticles; OD\ outer denticles; ID\ inner denticles; IAD\ inner accessory denticles; imm.\ immature specimen (in any stage of development).

History of study
The remote West Rhodope Mts.escaped   1924-  1925, they have not been studied or published.The Bulgarian populations were overlooked in the most comprehensive revision of Euscorpius (Di Caporiacco 1950).The first data on Euscorpius from the West Rhodope was published by Valle (1975) who studied specimens from Smolyan Province (which currently cannot be found in the important Valle collection at Museo Civico di Scienze Natural! "Enrico Caffi", Bergamo).Trichobothrial values given by Valle   (1975) as B2 = 6 and B3 = 8 correspond to standard values (Vachon 1974) as eb = 4/4 and = 4/4; see Fet et al. (2003: 374)  Specimens were first reported as having em = 3 by Fet (1993); it was clear already at that time that the Smolyan specimens do not belong to the standard Balkan "A.mingrelicus complex" with its et-est / estdsb trichobothrial fixed finger ratio > 1.5; this ratio was on average only about 1.02 in the Raichev speci-  (Fet, pers. obs.).However, the species was then erroneously interpreted as E. croaticus (Fet 1993,   Fet & Braunwalder 2000; see below for details).Fet & Soleglad (2002) noted that an unnamed form with em = ?>\sfound in the Rhodope Mountains in both Greece and Bulgaria belongs to an undescri- bed, basal species complex (subgenus incertae sedis).
Diagnosis.A medium-small Euscorpius species, total length 28-31 mm.Colour of adults light to medium brown/reddish, carapace darker.Reticulation or marbling varies from absent to highly marked on chelicerae, carapace, mesosoma and metasoma.The number of © Biodiversity Heritage Library, http://www.biodiversitylibrary.org/; Table 2. Genetic distances between 1 6S rDNA sequences.The number of base substitutions per site between 1 3 sequences are shown.Standard error estimates are shown in the last column.3 + F/^).The number of trichobothria on the pe- dipalp patella ventral surface usually is 6.The number of trichobothria on pedipalp patella external surface is: eA = 4, = 4, esb = 2, em = 3, est = A^et-5.The pec- tinal teeth number in males usually is 8, more rarely 9; in females usually 7, more rarely 8. Lchel/Wchel ratio is 2.60 in males and 2.70 in females.Dorsal patellar spur well-developed.Femur usually more or less as long as patella; Lfem/Lpat ratio is 0.98.Carapace more or less as long as wide; average ratio Lcar/Wcar 1.015 in males and 0.967 in females; average distance from center of median eyes to anterior margin of the carapace is 40.82 % of the carapace length.Average ratio of Lmet/ Lcar'vi 2.81 in males and 2.47 in females.
Hemispermatophore.Both right and left he- mispermatophores of five specimens were studied.
They have a well-developed lamina tapered distally; well-developed basal constriction present; truncal flexure present; median projection with primary and secondary acuminate processes, of which the secondary acuminate process is usually formed by a main tine, shaped as an elongated sickle, and from one to four secondary tines, which are more squat, and of- ten forked with two or more tines; internal projection distally with 5-7 tines in its crown.The number and the shape of tines of the crown and of the seconda- ry acuminate process varied between specimens and between the right and the left hemispermatophores.

Description of the male holotype
Colouration: Whole colour light brownish with ca- rapace and pedipalps darker reddish; sternites and pectines and genital operculum very light brownish/ ivory; chelicerae very light, yellowish, palms without marbling; telson yellowish, with a longitudinal lighter line and dark reddish aculeus tip; all pedipalps ca- rinae darker, dark brown to blackish coloured; none marbling is present.
Carapace: A very fine granulation on whole sur- face is present, except in the anterior area between the anterior edge, the lateral eyes and median eyes, which is almost smooth, very finely punctated and glossy, and the lateral area behind the lateral eyes, which has a few greater granules; anterior edge granulate and more or less straight; deep and dark posterior lateral furrows; two pairs of lateral eyes (with a larger ante- rior eye), and a pair of median eyes, situated distally of the middle; distance from centre of median eyes to anterior margin is 40.91 % of carapace length.
Metasoma: Dorsal carinae on segments I-IV with spaced weakly marked granules; ventrolateral carinae absent on segment I, obsolete or smooth on segments II-IV, granulated to serrulated on segment V; ventromedian carina absent on segments I-IV, the V with spaced weakly marked granules; dorsal inter- carinal spaces with a very fine granulation, smooth on the lateral and ventral surface.
Genital operculum: The genital operculum is for- med by two longitudinally separated subtriangular sclerites; genital papillae protruding; a few microse-

Discussion
The species of the genus Euscorpius in Bulgaria have been insufficiently studied.Limited information was given mostly in relatively recent papers (Valle 1975,   Fet 2000, Teruel et al. 2004, Fet & Soleglad 2007).
Some authors assumed the possibility of new species present in Bulgaria (Teruel et al. 2004, Fet & Soleglad 2007); however, they did not focus on resolving the systematic position of these forms, but rather grouped several populations based on their morphology, and addressed them as belonging to species groups or complexes: "F.carpathicus complex", "£.
hadzii complex" and "F.mingrelicus complex".The new species described in this paper, E. dren- skiiy has not been included in the study of Parmakelis   et al. (2013).However, we used 16S rDNA to construct a phylogenetic tree, which places this species in a clade outside of the subgenus Euscorpius s.str., together with the neighbouring populations from southwestern Bulgaria and northeastern Greece (clade E4 in Parmakelis et al. 2013).This confirms that E.
drenskii, E. carpathicus (type species of the subgenus Euscorpius s.str.) and the subgenus Alpiscorpius are three distinct and strongly supported clades with a long history of independent evolution, despite of the peculiar reduced trichobothrial series em = 2.
According to our preliminary phylogeny const- ructed based on 16S rDNA data, E. drenskii, together with other populations from southwestern Bulgaria and northeastern Greece form a larger clade, with Euscorpius avcii as its closest clade.This clade is well- separated from the subgenus Polytrichobotrhius Bi- rula, 1917 (type species E. italicus) as well as from the subgenus Euscorpius Thorell, 1876 s.str. (here represented by E. carpathicus, E. tergestinus, and E. concinnus).
E. drenskii exhibits genetic distance of 3.4 % to 5.5 % from other populations of its clade (clade E4 in Parmakelis et al. 2013), which is equal or higher than among other closely related species (e.g., E. carpathicus has a genetic distance of 3.4 % and 3.1 % from E. tergestinus and E. concinnus, respectively), and 7.8 % to 14.2 % from the remaining species of our phylogenetic tree.Note the large genetic divergence shown between E. drenskii and E. carpathicus (type species of the subgenus Euscorpius), which is 7.8 %, and with E. germanus (type species of the subgenus Alpiscorpius) , which is as high as 11.9 %.It is clear that the new species does not belong to the subgenus Euscorpius s.str., and that the shared condition of em = 3 between these three groups is homoplasious.
Regarding its trichobothrial pattern, E. drenskii is one of the most oligotrichous species in the entire genus Euscorpius', in fact, only a few species of the subgenus Alpiscorpius have a lower summary number of patellar trichobothria {Pv + Pe) (e.g.E. germanus, E. alpha and E. gamma).So far, no species has been described with such low values outside of the subgenus Alpiscorpius (or related to it).With Pv = 6 and Pe = 22 (c/ = 5 and em = 3), E. drenskii.has the same trichobothrial values as E. mingrelicus s.str.and E. croaticus Di Caporiacco 1950, and an even lower value than E. mingrelicus ciliciensis Birula 1898 {Pv = 7 and Pe = 22).It should be also be noted that, among the populations phylogenetically close to E. drenskii, none have em = 3, and most have Pv = 6-9 and Pe = 23-25 {et = 5-7 and em = 4) (Tropea et al. in prep.).
Thus this character state is probably independently derived (autapomorphic).A very similar situation is presented by E. carpathicus in south-western Romania, which has em = 3, while phylogenetically close E. deltshevi from Serbia and northern Bulgaria has em - 4 (Fet et al. 2014, unpublished data of Tropea).
With its trichobothrial pattern, which should be considered the most clear diagnostic character set for E. drenskii, it can be easily distinguished from most of the other Euscorpius species.In fact, as explained above, only E. carpathicus, E. mingrelicus, and E. croaticus have exactly the same trichobothrial pattern as E. drenskii.However, E. drenskii can be quite readily differentiated from these forms as follows: From E. carpathicus, E. drenskii is distinguished mainly by: (1) the number of = 6 in E. drenskii versus normally 8 in E. carpathicus', (2) E. drenskii has Pe-et -5 versus usually 6 and 7 in E. carpathicus.In addition, E. carpathicus has a dark brown colour, and inhabits south-western Romania.
From E. mingrelicus, E. drenskii can be easily dis- tinguished by the ratio of distances between trichobothria on fixed finger, et-est / est-dsb,^\\ioh is > 1.5 in E. mingrelicus complex (Bonacina 1980), while it is just over 1 in E. drenskii.In addition, F. mingrelicus has a dark brown colour.
The last species, which has the same number of trichobothria as E. croaticus.However, while the latter groups with the subgenus Alpiscorpius, in our phylogeny E. drenskii forms a clade strongly separated from Alpiscorpius.

Conclusions
In the past, the genus Euscorpius has been intensively studied; over 40 species and subspecies were descri- bed.Most of these taxa were later downgraded to subspecies status or moved to synonymy.However, since 1999, when this genus had only 4 recognized species, the number steadily increased and has gra- dually reached 17 in 2007.Thanks to further detailed studies, based both on morphological and molecular data, from 2012 to the present, the species number now increased to 43 (including E. drenskii), and se- veral other species are in press or in description.This large increase in species diversity, and in the studies that led to establishing these taxa, reflect a great degree of speciation and endemism in Euscorpius, which are often restricted to very limited areas such as a mountain range or an island, or a small group of mountains or islands.
Another interesting point that was understood during these studies, and noted for the first time by Tropea (2013), is that the existing subgeneric divisi- on of the genus Euscorpius was not consistent with the taxonomic situation.Parmakelis et al. (2013), in a much larger and detailed molecular phylogenetic study, arrived at the same result.Currently, there are a number of forms without a clear subgeneric place- ment.These include the new species described herein, E. drenskii.According to a traditional identification key, it is a part of the subgenus Euscorpius, but genetically it is completely separate, and could belong to a separate subgenus (or even genus); therefore we addressed it here as a "subgenus incertus".
Further studies, resulting in improved identification keys, are needed to bring order in this growing and complicated scorpion group.This goal could be supported by a study of hemispermatophores, which was quite decisive, e.g., in the recent revisions of scorpion genera lurus Thorell, 1876 and Protoiurus Soleglad, Fet, Kovank & Yagmur, 2012 (luridae) (Kovaffk et al. 2010, Soleglad et al. 2012).Using hemispermatophores is not an easy or universal criterion, as they are only present in males, which are usually represented in collections by fewer number than the females.In addition, to analyse these or- gans, the specimens must be dissected, and a high variability between specimens and even between the left and right hemispermatophore is present in Euscorpius (Tropea pers. obs.).Thus, to obtain a relia- ble result, a large number of adult males should be of Bulgaria.Later, Fet & Soleglad (2007) provided the first comprehensive analysis of Bulgarian scorpi- on fauna, where the new species described herein was treated under carpathicus complex".The first DNA phylogeny from Greece and adjacent regions of the Balkans published by Parmakelis et al. (2013) indica- ted that Euscorpius fauna of the Rhodope Mountains GTC); Smolyan Province, Devin District, between Mihalkovo and Devin, 550-700 m, 1-2 September 2001, leg.B. Petrov Sc V. Beshkov, 2 9 (NMNHS 198).
Fig. 21 : A phylogenetic tree of Euscorpius based on 16S rRNA mtDNA marker.See Methods and material for explanations.

G
. Tropea, V. Fet, A. Parmakelis, P. Kotsakiozi & I. Stathi Fet, Huntington, West Virginia, USA; ZMMSU, Zoological Museum of Moscow State University, Moscow, Russia.Material studied.A detailed list of material with label data is provided below.
early scorpion scholars, although the very first specimen de- posited in the National Museum of Natural History, Sofia, Bulgaria (NMNHS) was collected as early as 1901 by Prince Ferdinand, the founder of this important Museum in 1889.Ferdinand I (1861-1948) of Saxe-Coburg-Gotha royalty, the Knyaz (Prince Regnant) of the independent Bulgaria since 1887, and its Tsar (King) since 1908, was an amateur lepidopterist and botanist, who promoted natural science in the Balkans.Even though additional specimens from the West Rhodopes were collected by the most prominent Bulgarian arachnologist Pencho Drenski in for a detailed scheme comparing Valle's and Vachon's systems of trichobothrial notation.Valle, however, did not report em number (D4 series) for his Smolyan specimens.Independently, 16 specimens from the West Rhodope Mts.(now in ZMMSU) were donated to VF. in 1984 by Dr. Christo Deltshev.This series was collected by the late Dimitar Raichev, an amateur naturalist of Chepelare, Smolyan Province, in1981- 1983.This enigmatic population was studied by VF.
and triggered his first interest in Bulgarian scorpions.
Coxa and trochanter with tuberculated carinae.Femur: dorsal and ventral internal carinae tu- row of tarsus III with a total of 8/6 worn-out spi- nules, of increasing size from proximal to distal, ending with a decentralized spinule.Granulation well present on dorsal and ventral surface of leg femora, it is mostly marked and dark ventrally.Cheliceraei Movable finger: the dorsal distal denticle is much smaller than the ventral distal denticle;ventral edge is smooth with brush-like setae on the inner part; dorsal edge has five denticles: one large distal, two small subdistal, one large median, and a small basal.Fixed finger has four denticles: one distal, one subdistal, one median, and one basal, the last two in a fork arrangement; the internal surface has tae are present.Sternum: Pentagonal shape, type 2; more or less Pedipalps:Legs'.With two pedal spurs; no tarsal spur; ventral brush-like setae.
Parmakelis et al. (2013)ore localized E. solegladi Fet, Graham, Webber & Blagoev, 2014 (a form of "F.hadzii complex"), from south-westernBulgaria.Both of these species belong to the subgenus Eus- corpius s.str.In addition,Parmakelis et al. (2013), in a large phylogenetic study of Euscorpius from Greece and adjacent countries, included two other populations from the south-western Bulgaria, which are not closely related to two species described byFet et al.   (2014), but instead group with several populations from northeastern Greece (clade E4 in Parmakelis et al. 2013).In our current opinion, these closely related populations belong to several good species which our team is currently describing(Tropea et in al. in prep.).
Fet (1993)is E. croaticus.This form has recently been elevated to the status of species byGraham et al. (2012), and, according to their phy- logenetic tree based on COI data, it clustered with the subgenus Alpiscorpius.However, due to its ambiguous morphological features, E. croaticus has not been assigned to any subgenus (for more information seeGraham et al. 2012).Fet (1993)identified specimens of E. drenskii from Trigrad, Bulgaria, as E.
dissected.It must be pointed out, however, that in E. drenskii these organs, although variable, show a more complex secondary acuminate process than in many other Euscorpius, but are nevertheless similar to other Balkan populations related to E. drenskii(Tropea in prep.).Raichev, Vladimir Sakalian, Michael Soleglad, Pavel Stoev, and Milen Vassilev.Special thanks are to Petar Beron, who loaned the entire NMNHS collection of Euscorpius to V.F., to Alexi Popov who meticulously checked and corrected all Bulgarian toponyms, and to Michael Soleglad who kindly provided a map (Fig. 20).VF.'s initial travel to Bulgaria in 1999 was supported by a COBASE (Cooperation in Basic Science and Engineering) grant from the National Research Council, Washington, DC, USA.More exten- ded V.F.'s travel to Bulgaria in 2005 was supported by the Fulbright Scholar Award 04-11-08 from CIES (Council of International Exchange of Scholars), Washington, DC, USA, which allowed Victor and Galina Fet to travel and live in Bulgaria in January-May 2005.Help, hospitality, and friendship of numerous Bulgarian colleagues made the 1999 and 2005 visits productive and enjoyable.Especially notable was a wonderful journey of VF. across the West Rhodopes with Vlado Sakalian and Milen Vassilev in May-June 1999.