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Two New Species of Euscorpius (Scorpiones: Euscorpiidae) from Bulgaria, Serbia, and Greece

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Two New Species of Euscorpius (Scorpiones: Euscorpiidae) from Bulgaria, Serbia, and Greece Zootaxa 3894 (1): 083–105 ISSN 1175-5326 (print edition) www.mapress.com/zootaxa/ Article ZOOTAXA Copyright © 2014 Magnolia Press ISSN 1175-5334 (online edition) http://dx.doi.org/10.11646/zootaxa.3894.1.7 http://zoobank.org/urn:lsid:zoobank.org:pub:DE6A56E8-DA80-4C1D-A5BA-634D0F53D92E Two new species of Euscorpius (Scorpiones: Euscorpiidae) from Bulgaria, Serbia, and Greece VICTOR FET1, MATTHEW R. GRAHAM2, MICHAEL M. WEBBER3 & GERGIN BLAGOEV4 1 Department of Biological Sciences, Marshall University, Huntington, West Virginia, USA 2 Department of Biology, Eastern Connecticut State University, Connecticut, USA 3 School of Life Sciences, University of Nevada, Las Vegas, Nevada, USA 4 Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada Corresponding author: Victor Fet ([email protected]) Abstract Two new species of Euscorpius Thorell, 1876 (subgenus Euscorpius s.str.) (Scorpiones: Euscorpiidae) are described based on morphology and the COI DNA barcoding marker: E. deltshevi sp. n. from northern Bulgaria and neighbouring Serbia (formerly reported as E. carpathicus) and E. solegladi sp. n. from southwestern Bulgaria and neighbouring Greece (for- merly reported as E. hadzii). Key words: Scorpions, Balkans, DNA barcoding Introduction A recent review of Bulgarian scorpions of the genus Euscorpius Thorell, 1876 (Fet & Soleglad 2007) outlined issues and problems involved with delineation and understanding of species in these enigmatic arachnids. In the first thorough molecular study of Greek Euscorpius taxa, Parmakelis et al. (2013a) demonstrated that cryptic speciation among these scorpions appears to be dramatically underestimated. Here, we apply DNA barcoding, or sequencing of the mitochondrial gene coding for cytochrome c oxidase subunit I (COI), to clarify the phylogenetic position and taxonomic identity of two new, but common, species of Euscorpius inhabiting Bulgaria, and provide descriptions of each. Material and Methods Material Studied. Thanks to the courtesy of Dr. Petar Beron, who loaned the entire Euscorpius collection of the National Museum of Natural History, Sofia (NMNHS) to V.F., we studied as many as 190 specimens of E. deltshevi sp. n. and 62 specimens of E. solegladi sp. n., which allowed us to gain a better understanding of morphological variation as well as broaden our geographic perspective. A detailed list of material with label data is provided below. Specimens used for DNA barcoding. Of 25 sequences of Euscorpius used for our phylogeny, 18 are reported here for the first time (listed below with GenBank accession numbers). Euscorpius (E.) tergestinus (C.L. Koch, 1837), SLOVENIA: under Nanos Mt., Vipava, 45°50'48"N, 13°57'47"E, 15.06.2001 (Univ. of Ljubljana), VF-0777 (AMSCO047-10, KM111249); CROATIA: Majorija, Senj, 44°59'25"N, 14°54'05"E, 550 m, 7.08.2000 (B. Sket), VF-0823 (AMSCO081-10, HM418305). Euscorpius (E.) hadzii Di Caporiacco, 1950, BOSNIA & HERZEGOVINA: Herzegovina, 15 km S Trebinje, 42°42'N, 18°20'E, 3.08.2000 (P. Trontelj), VF-0795 (AMSCO056-10, HM418290); Herzegovina, Čemerno, 43°14'14"N, 18°36'4"E, 8.09.2006 (D. Pavicevic), VF- 0796 (AMSCO057-10, HM418291); Republika Srpska, Lukavac, 44°46'13"N, 18°53'31"E, 8.09.2006 (D. Accepted by Y. Marusik: 3 Oct. 2014; published: 11 Dec. 2014 83 Pavicevic), VF-0797 (AMSCO058-10, HM418292). Euscorpius (E.) deltshevi sp. n., BULGARIA: Lovech Province, Teteven, 42°55′N, 24°16′E, 05.2007, VF-0811 (AMSCO070-10, HM418298); Montana Province, Chiprovtsi District, Gorna Laka, 43º27'N, 22º54'E, 406 m, 16.05.2005 (V. Fet & S. Fet), VF-0812 (AMSCO071- 10, HM418299); Sliven Province, Sliven, 42°41'N, 26°20'E, 2.05.2003, ca. 800 m (J.O. Rein), VF-0814 (AMSCO073-10, HM418300). Sofia Province, Beledie Han, 11 km N of Kostinbrod, 42º53'N, 23º09'E, 17.05.2005 (V. Fet & S. Fet), VF-0818 (AMSCO076-10, HM418302); Lakatnik Railway Station, 43°05'10"N, 23°23'01"E, 4.05.2005 (V. Fet & A. Popov, VF-0815 (AMSCO074-10, HM418301); Tserovo, 43.00ºN, 23.35ºE, 739 m, 4.05.2005 (V. Fet & A. Popov, VF-0817 (AMSCO075-10, KM111245); Vidin Province, Belogradchik, 43º37'38"N, 22º41'E, 538 m, 16.05.2005 (V. Fet & S. Fet), VF-0821 (AMSCO079-10, KM111243); Oreshets Railway Station, near Suhi Pech Cave, 43º29'26"N, 22º44'20"E, 16.05.2005 (V. Fet & S. Fet), VF-0822 (AMSCO080-10, KM111242); SERBIA: Nišava Province: Niš, Niška Banja, 15.04.2006 (I. Karaman), VF-0746 (AMSCO026-10, KM111244). Euscorpius solegladi sp. n., BULGARIA: Blagoevgrad Province, Rila Monastery, 42°08′N, 23°20'25"E, 1147 m, 5.06.1999 (V. Fet & V. Sakalian), VF-0799 (AMSCO060-10, HM418293); Blagoevgrad Province, Sandanska Bistritsa River, 41°34'N, 23°17'E, 26.05.2005 (V. Fet & E. Fet), VF-0801 (AMSCO062-10, KM111247); Pernik Province, Radomir District, Baikalsko, 844 m, 42°25.378'N, 22°48.862'E, 28.05.2005 (V. Fet & E. Fet), VF-0802 (AMSCO063-10, KM111246); Pernik Province, Tran District, Erma Gorge, 703 m, 42°51.679'N, 22°38.952'E, 28.05.2005 (V. Fet & E. Fet), VF-0813 (AMSCO072-10, KM111248). The remaining 7 barcodes were published recently (Graham et al. 2012a, b; Parmakelis et al. 2013a): Euscorpius (Alpiscorpius) gamma Di Caporiacco, 1950. AUSTRIA, Carinthia, Trögerner-Klamm, 46°27'28"N, 14°30'02"E, 13.06.1999 (V. Fet & B. Scherabon), VF-0732 (AMSCO014-10, HM418273). Euscorpius (E.) carpathicus (Linnaeus, 1767): ROMANIA, Caraş-Severin County, Băile Herculane, 44°52'43"N, 22°24'51"E, 4.06.2008 (F. Šťáhlavský), VF-0768 (AMSCO044-10, HM418284). Euscorpius (E.) sicanus (C.L. Koch, 1837): MALTA, Buskett Gardens, 35°51'41"N, 14°23'56"E, 17.09.2001 (P. Schembri), VF-0792 (AMSCO053-10, HM418288). ITALY, Sardinia, S. Niccolo Gerrei, near Grotta Saturru, 39.49816°N, 09.31503°E, 395 m, 04.2006 (A. v.d. Mejden), VF-0789 (AMSCO052-10, JX133089). TUNISIA, Zaghouan Governorate, Jebel Zaghouan Mts., along the trek, 36°22.423'N, 10°06'E to 36°22.924'N, 10°06.789'E, 650-780 m, mixed forest, 03.2008 (P. Stoev & N. Akkari), VF-0793 (AMSCO054-10, HM418289). Euscorpius (E.) hadzii, ALBANIA, Bjeshket e Nemuna (Prokletije) Mts., Maya e Shtegut (Theth), 21.07.2000 (T. Wraber), 189F (KC215665). Euscorpius (E.) solegladi sp. n. (as E. hadzii): BULGARIA, Blagoevgrad Province, Gorna Breznitsa, 41°45'N, 23°07'E, 27.05.2005 (V. Fet & D. Dobrev), VF-0798 (AMSCO059-10, HM880289). Depositories: FKCP, private collection of František Kovařík, Prague, Czech Republic; MNHN, Muséum national d’Histoire Naturelle, Paris, France; NHMW, Naturhistorisches Museum Wien, Vienna, Austria; NMNHS, National Museum of Natural History, Sofia, Bulgaria; NMPC, National Museum of Natural History, Prague, Czech Republic; SMF, Senckenberg-Museum, Frankfurt, Germany; VFPC, private collection of Victor Fet, Huntington, West Virginia, USA; ZMB, Zoologisches Museum Berlin, Humboldt-Universität, Berlin, Germany; ZMMSU, Zoological Museum, Moscow State University, Moscow, Russia. Morphology: Terminology and measurements (in mm) follow Stahnke (1970) and Sissom (1990); trichobothrial designations follow Fet & Soleglad (2002). Molecular Analyses Phylogenetic Analyses. We assessed the phylogenetic position of the two new species with DNA barcoding, an approach that has recently been applied to other Euscorpius taxa (Graham et al. 2012a, b; Parmakelis et al. 2013a). All DNA work was performed in the University of Guelph and new barcodes were submitted to GenBank (Accession Nos. as listed above). Barcodes are also accessible through BOLD (http://www.boldsystems.org) (Ratnasingham & Hebert 2007) under project title “Scorpions of the Ancient Mediterranean 2c (AMSCU)”. Voucher specimens are in a private collection of V. Fet (VFPC). We imported the barcodes into Geneious v. 7.0.2 (available from http://www.geneious.com/) and aligned them with the software using MUSCLE (Edgar 2004). The best-fit model of nucleotide substitution was determined for each codon position with MEGA v. 5.2.2 (Tamura et al. 2011) based on the Akaike Information Criterion (Posada 2008). Using the appropriate models (HKY for codons 1 and 2, HKY+G for codon 3), we constructed a COI phylogeny using Bayesian inference (BI) implemented in MrBayes v. 3.1.2 (Ronquist & Huelsenbeck 2003) ran 84 · Zootaxa 3894 (1) © 2014 Magnolia Press FET ET AL. through the Cyberinfrastructure for Phylogenetic Research cluster (CIPRES Gateway v 3.1) at the San Diego Supercomputer Center. We based our interpretations on runs of 20 million generations with trees sampled every 2000 generations. We set the temperature to 0.075 and discarded the first 20% of sampled trees as burn-in. Divergence Dating. We placed the COI phylogeny in a temporal context using a relaxed molecular clock implemented in BEAST v. 1.7.2. (Drummond & Rambaut 2007). We unlinked substitution models across each codon partition, but linked the tree and clock models, and ran the analysis with the same best-fit substitution models used in the MrBayes analysis. We excluded the outgroup used in the MrBayes analysis and instead rooted the chronogram by constraining a monophyletic taxon set that included all samples except the two E. tergestinus. This approach produced a midpoint root between the E. tergestinus and the clade containing all remaining samples, which was identical to the strongly supported branching pattern (1.0 pp) in the MrBayes analysis. We ran the program for 40 million generations, with trees sampled every 4000 generations. We used the uncorrelated lognormal-relaxed clock model and applied the Yule tree prior with a scorpion specific mutation rate of 0.007 substitutions/site/million years and a mean standard deviation of 0.003 (Gantenbein & Keightley 2004). Convergence was assessed using Tracer v. 1.6 (Rambaut et al. 2009). We used TreeAnnotator to construct a maximum clade credibility tree with the first 20% of trees discarded as burn-in. FIGURE 1. Geographic distribution of two new species: Euscorpius deltshevi, sp.
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