412 2003. The Journal of Arachnology 31:412–420 THE FIRST DNA PHYLOGENY OF FOUR SPECIES OF MESOBUTHUS (SCORPIONES, BUTHIDAE) FROM EURASIA Benjamin Gantenbein: Institute of Cell, Animal and Population Biology, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, United Kingdom Victor Fet: Department of Biological Sciences, Marshall University, Huntington, West Virginia 25755-2510 USA Alexander V. Gromov: Institute of Zoology, Akademgorodok, Almaty, 480060, Republic of Kazakhstan ABSTRACT. The first molecular phylogeny is presented for four species of the scorpion genus Meso- buthus, based on DNA sequences of three gene fragments (two mitochondrial and one nuclear protein coding gene, 1 kb). The inferred phylogeny based on a pooled maximum likelihood analysis revealed a clear deep splitting between the ‘‘western clade’’ consisting of M. gibbosus and M. cyprius (Greece/ Anatolia, Cyprus) and the ‘‘eastern clade’’ consisting of M. eupeus and M. caucasicus (Anatolia/Central Asia). The species M. caucasicus (recently placed in the genus Olivierus Farzanpay 1987) groups mon- ophyletically within Mesobuthus; thus, the genus Olivierus is synonymized here with Mesobuthus. Se- quences of M. eupeus and M. caucasicus sampled mainly from Kazakhstan and Uzbekistan are highly structured, indicating the possible existence of multiple species. Keywords: Scorpions, Buthidae, Mesobuthus, phylogeny, DNA, 16S, coxI, protein kinase, biogeography The genus Mesobuthus Vachon 1950 (Scor- piones, Buthidae) currently includes 12 spe- cies (Fet & Lowe 2000; Gantenbein et al. 2000b); its type species is Mesobuthus eupeus (C.L. Koch 1839). Except for M. gibbosus (Brulle ´ 1832), which is found in the Balkans and Turkey, the diversity of this genus is con- centrated in Asia. Numerous species and sub- species are distributed from Turkey to Korea, with the centers of diversity in Central Asia and Iran. Mesobuthus species are the most common and abundant scorpions in a variety of arid habitats, from sand deserts to high mountains over 3000 m (Fet 1989, 1994). They are found up to 50°N in Kazakhstan, the northern limit of the natural range of the Old World scorpions (Gromov 2001). Although Buthidae are the most diverse and medically important family of scorpions (Fet & Lowe 2000), there has been no attempt so far to produce a phylogenetic analysis of this family. Especially powerful are phylogenies based on DNA sequence data in combination with morphology (Gantenbein et al. 1999a, 2000a; Fet et al. 2001). Our first applications of this technique in Buthidae refer to the gen- era Buthus Leach 1815 (Gantenbein et al. 1999b; Gantenbein & Largiade `r 2003) and Centruroides Marx 1890 (Gantenbein et al. 2001; Towler et al. 2001); we also published a pilot phylogeny of 17 buthid genera (Fet et al. 2003). Molecular markers helped to define is- land species, where neither the biological spe- cies concept nor any other species concept can be applied (Gantenbein et al. 2000b, 2001). METHODS The currently studied available material be- longed to four species: Mesobuthus gibbosus (Greece, Turkey), M. cyprius Gantenbein & Kropf 2000 (Cyprus), M. eupeus (C.L. Koch 1839) (Turkey, Kazakhstan, Turkmenistan, Uzbekistan, China), and M. caucasicus (Nord- mann 1840) (Kazakhstan, Turkmenistan, Uz- bekistan) (see Table 1 for locality informa- tion). For DNA analyses, the total DNA was extracted from fresh or preserved (94–98% ethanol) muscle tissue using a standard phe- nol/chloroform and precipitation method (Sambrook et al. 1989). We amplified a ca. 450 base pair (bp) fragment of the 16S rRNA mitochondrial (mt) DNA by polymerase chain reaction (PCR) using the primers and condi-