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Genetic diversity among different species of the genus Leiurus (Scorpiones: Buthidae) in Saudi Arabia and the Middle East

The molecular phylogenetic relationship among two species of genus Leiurus, from Saudi Arabia with additional comparative sequence data available from Egypt, Oman and Turkey is presented. The molecular phylogeny was performed using maximum parsimony, neighbor joining and bayesian inference. Our results indicate a clear deep splitting between the Western clade, which represented by L. quinuestriatus sequences from Egypt and those from the Eastern clade which encompassing different Leiurus species from Saudi Arabia, Oman and Turkey was shown. Also, the phylogenetic relationship represents additional support for the taxonomic status of Arabian Leiurus species.


Introduction
The genus Leiurus was originally introduced as a subgenus of Androctonus by Ehrenberg in Hemprich and Ehrenberg (1829). In 1949, Vachon elevated Leiurus to full generic rank with a single species with two subspecies represented for many years; L. quinquestriatus quinquestriatus (Ehrenberg, 1828) and L. quinquestriatus hebraeus (Birula, 1908). As of 2009, four scorpion species of genus Leiurus were added (Lourenço et al., 2002(Lourenço et al., , 2006Kovařík, 2007;Yagmur et al., 2009). Recently, Lowe et al. (2014) made a revision of the genus Leiurus, mainly with the Arabian Peninsula populations based on morphological characteristics. Subsequently, four new species have been witnessed from Africa in the past few years (Lourenço and Rossi, 2016;Lourenço et al., 2018;Lourenço, 2019;Kovařík and Lowe, 2020). Finally, two new species were described of Leiurus from Kuwait and Mauritania (Lourenço, 2020a,b). With these two new species, the total number of known species of this genus is elevated to sixteen, spread across Africa and the Middle East. Of these, L. arabicus from the central Najd plateau to the east of Saudi Arabia; L. haenggii from the coastal mountains of the Red Sea in Saudi Arabia, Yemen and Oman; and L. macroctenus from coastal plains and steppes in Oman (Lowe et al., 2014;Alqahtani et al., 2019).
Recently, several studies used the molecular phylogeny as a tool for interpreting phylogenetic relationship of many scorpions within and among populations (Ben Ali et al., 2000;Fet et al., 2003;Ben Othmen et al., 2009;Alqahtani and Badry, 2020). Moreover, the molecular analysis within scorpion species L. abdullahbayrami and L. quinquestriatus populations were constructed based on 16S rRNA sequence data (Ayhan et al., 2018;Sarhan et al., 2020). In order to the recent taxonomic changes in the genus, it is necessary to assess the taxonomic status of two Leiurus species in Saudi Arabia especially L. arabicus and L. haenggii with additional comparative sequence data available from Egypt, Oman and Turkey.

Scorpion collection
Four adult specimens of two different species belong genus Leiurus were collected from two different localities in Saudi Arabia during the period from September 2017 to July 2019 ( Fig. 1 (Williams, 1968). In the night period, the UV lights were used (Stahnke, 1972). The collected scorpions were preserved in 95% ethanol for DNA isolation as described by (Prendini et al., 2003).

DNA extraction, amplification and sequencing
Total genomic DNA was extracted from leg or pedipalp muscle tissue dissected from fresh or preserved (95% ethanol) scorpion specimens using DNeasy extraction kit (Qiagen) and following manufacturer's instructions. Fragments of the mitochondrial 16S rRNA gene were amplified by polymerase chain reaction (PCR) using the 16S rRNA scorpion specific primers as described by Gantenbein et al. (1999). Amplified products were purified and sequenced on an ABI 3500 automated sequencer (Applied Biosystems Inc., USA).

Phylogenetic analysis
Sequence were checked and analyzed using BioEdit program Ò , version 7.2.5 (Hall, 1999). Sequence data were downloaded as ingroup from GenBank for L. quinquestriatus (Ehrenberg, 1980) Table 1). Also, Androctonus crassicuda downloaded as out group (AJ277598.1). The sequence data was edited using Mega 6 (Tamura and Nei, 1993) and subsequently aligned using the default settings of ClustalW. Nucleotide composition from the ingroup sequences only was calculated. The genetic distances (p-distance)  Table 1. were performed to estimate the sequence divergence for the entire data set, using Mega 6 (Tamura and Nei, 1993). Also, The phylogenetic analyses; maximum-parsimony (MP), neighbor-joining (NJ) and Bayesian inference analyses (BI) of 16S data set (n = 13) were performed as proposed by Alqahtani and Badry (2020).

Genetic data
The 16S rRNA data set consisted of 307 aligned nucleotides were analyzed. In total 110 (35. 83%) bases were constant, 120 (39.08%) bases were variable and 77 (25.08%) were parsimonyinformative. Nucleotide composition was highly biased towards A-T. The mean values of T, C, A and G within the sequence data were 35.4, 10.8, 38.2 and 15.5%, respectively. Within the 307 bp, 190 polymorphic segregating sites were detected. The sequence divergences among Leiurus lineages ranged from 0.00 to 0.17, with an average of 0.13.

Phylogenetic analyses
The topology resulting from MP is presented in Fig. 2, was identical to that of the NJ tree (Fig. 3). Also, the general topology of the maximum-parsimony tree was a little different from those obtained by BI analyses (Fig. 4). In all phylogenetic analyses (Figs. 2, 3 and 4), two major highly supported clades are represented by all Leiurus taxa. The first clade (the Eastern clade) includes all Arabian and Middle East forms. This clade is further split into two subclades, includes L. abdullahbayrami from Turkey and those of the Arabian part (Saudi arabia and Oman). the later further divided into two subclades includes; L. arabicus from Riyadh as a basal clade to L. haenggii from southwestern Saudi Arabia and L. macroctenus from Oman which grouped together as a sister group. While the second clade includes all Egyptian forms. The population of the Sinai Peninsula (Asian part of Egypt) and those of the African part of the country.

Genetic distance
The genetic distances from the entire data set was calculated and given in Table 2. The divergence between the Arabian species L. arabicus and L. haenggii had the lowest divergence (11%). Similarly, the distance between the Egyptian Leiurus was 2.0%. The highest divergence was observed between L. haenggii and other Leiurus species, ranged from 12%, 19%.

Discussion
The phylogenetic analysis based on 16S rRNA data (Figs. 1, 2, and 3) reveals high diversity of species belonging to the genus Leiurus, of which cover some species belong Africa and Asia. Our phylogenetic analyses presented here indicate a clear deep splitting between the Western clade, which represented by L. quinquestriatus sequences from Egypt and those from those of the Eastern clade which encompassing different Leiurus species from Saudi Arabia, Oman and Turkey. Furthermore, the genetic divergence supported the distinction of the L. quinquestriatus from Egypt including Sinai from the Eastern clade species (12-19%) ( Table 2). Levy and Amitai, (1980), suggested that the hypothesis of physical barrier play a role in preventing the gene flow between African and Asian populations. Similarly, Alqahtani and Badry (2020) noted that among the Egyptian and Arabian Buthacus species and suggested that it might be due to historical speciation. In addition, many authors refer to the vicariance which associated with the Red Sea formation, drove simultaneous difference between Arabian and African lineages in several faunal group (Sanmartín, 2003;Amer and Kumazawa, 2005;Derricourt, 2005;Lourenço and Rossi, 2016;Saleh et al., 2018).
Within the Eastern clade, L. abdullahbayrami was basal to the L. arabicus and L. haenggii from Saudi Arabia. While L. macroctenus from Oman nested as a sister group to L. haenggii. L. abdullahbayrami, can be distinguished from the above species as proposed by Lowe et al. (2014) by several morphological and morphometric characters. This species is distributed belong semi-arid habitats in Turkey and northern Syria (Khalil and Yagmur, 2010;Lowe et al., 2014). The divergence distance between L. abdullahbayrami and the others of genus Leiurus ranged from 12 to 14% (Table 2). Lourenço (2020a), described a new species of Leiurus from Kuwait, and noted that it might correspond to vicariant elements during the late Miocene, of which it might support the diversification among the Arabian species. Our results suggested that the same vicariant mechanisms, also acted to split off the Arabian clade from L. abdullahbayrami.
With regards to the relation between L. haenggii and L. arabicus, both have ''medial intercarinal surfaces of tergites II-III smooth or sparsely shagreened", without dense granulation. However, L. haenggii can distinguished from L. arabicus by having ''more robust leg, pedipalp and metasomal segments; and smooth or weakly granulated median carinae on sternites III-V of females" (Lowe et al., 2014). Also, the L. macroctenus, can be distinguished by large pectine teeth from the most other species of genus Leiurus (Lowe et al., 2014, Kovařík andLowe, 2020). The divergence between the Arabian species ranged from 11 to12 % (Table 2). Lowe et al. (2014) refer that L. arabicus is a more psammophilous ecomorphotype and it's consisted from the L. haenggii lineage. On contrary, our results showed that L. haenggii from southwestern Saudi Arabia and L. macroctenus from Oman grouped together in all phylogenetic analyses as a sister clade and it were predicted to have diverged from L. arabicus. It could be explained by the vicariant event caused by progressive aridification during the Late Pleistocene and the early Holocene, which served the diversification belong the Arabian species (Bray and Stokes, 2004;Lowe et al., 2014). Also, The effects of paleoclimatic conditions had a major impact on the distribution and differentiation of various species of Buthus, Mesobuthus and Tityus have been previously reported by several studies (Sousa et al., 2010(Sousa et al., , 2012Fet et al., 2018;Lourenço, 2016Lourenço, , 2020c.
In conclusion, a clear deep splitting between L. quinuestriatus sequences from Egypt and those from Arabia and Turkey. Also, the phylogenetic relationship represents additional support for the taxonomic status of Arabian Leiurus species. Furthermore, additional detailed morphological revision utilizing type specimens of ''L. jordanensis and L. brachycentrus" from the northern and southwestern parts of Saudi Arabia, is required to clarify their relationships with other species of this genus.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.