Molecular analysis of the mitochondrial markers COI, 12S rDNA and 16S rDNA for six species of Iranian scorpions

Objectives Annually, 1.2 million humans are stung by scorpions and severely affected by their venom. Some of the scorpion species of medical importance have a similar morphology to species with low toxicity. To establish diagnostic tools for surveying scorpions, the current study was conducted to generate three mitochondrial markers, Cytochrome Oxidase I (COI gene), 12S rDNA and 16S rDNA for six species of medically important Iranian scorpions: Androctonus crassicauda, Hottentotta saulcyi, Mesobuthus caucasicus, M. eupeus, Odontobuthus doriae, and Scorpio maurus. Results Phylogenetic analyses of the obtained sequences corroborated the morphological identification. For the first time, 12S rDNA sequences are reported from Androctonus crassicauda, Hottentotta saulcyi, Mesobuthus caucasicus and M. eupeus and also the 16S rDNA sequence from Hottentotta saulcyi. We conclude that the mitochondrial markers are useful for species determination among these medically important species of scorpions.


Introduction
Scorpions comprise more than 1500 species of which fifty species are considered of medical importance [1]. Annually, 1.2 million people are stung by scorpions [2], and severely affected by their venom [3]. The geographical and climatic diversity in Iran provides suitable conditions for a substantial number of scorpion species; as many as 52 species have been reported from Iran, although this number is not definitive [4]. Of these, at least seven species are dangerous to humans: Androctonus crassicauda Olivier, 1807; Apistobuthus pterygocercus Finnegan, 1932; Hottentotta saulcyi Simon, 1880; Hottentotta schach Birula, 1905; Hemiscorpius lepturus Peters, 1861; Mesobuthus eupeus Koch, 1839 and Odonthubuthus doriae Thorell, 1876 [4]. The difficulties in identifying species based on morphological techniques [5], as well as the low number of studies [4], are two of the reasons for the uncertainty regarding the number of scorpion species existing in Iran. Using molecular markers in addition to morphological methods could be useful for a more accurate assessment of species diversity in Iran. One example where potential misidentification of scorpion species could be of importance, regards H. saulcyi and Scorpio maurus. The two species are similar in appearance, but while the venom for H. saulcyi has an LD 50 value of 0.73 mg/kg and is considered a risk for humans, S. maurus has a venom with an LD 50 value of 9.37 mg/kg that is considered harmless [6].
Among available molecular markers, the mitochondrial markers have shown to be useful for both species identification and phylogenetic evaluation of scorpion species. For example, analysis of intraspecific divergence between Androctonus scorpions using three mitochondrial markers (the COI gene, 12S rDNA and 16S rDNA), showed that these markers could explain the deep divergence sub-clades between Androctonus amoreuxi, A. australis and A. mauretanicus that also reflect differences in their venom production [7]. Also, COI and 16S rDNA markers have been used for investigating the evolution of Mesobuthus gibbosus in the Northeastern Mediterranean [8].

BMC Research Notes
The current study was carried out to determine the COI, 12S rDNA and 16S rDNA sequences of six medically important Iranian scorpions and to evaluate the utility of these markers for identification in comparison to morphology.

Taxon sampling
Six species of medical importance were collected: Androctonus crassicauda Olivier, 1807; Hottentotta saulcyi Simon, 1880; Mesobuthus caucasicus Nordmann, 1840; Mesobuthus eupeus Koch, 1839; Odontobuthus doriae Thorell, 1876 and Scorpio maurus Ehrenberg, 1828. The samples were collected from the West Azerbaijan Province (Northwestern Iran) and the Qom Province (Central Iran; Additional files 1, 2: Tables S1, S2). The scorpions were captured at night using ultraviolet light (wavelength 366.3 nm), which causes maximum fluorescence of their epicuticles. All collected specimens were stored in 96% ethanol. Specimens were identified using the keys of Farzanpay [9] and Dehghani and Valaie [10]. From the identified samples, one sample from each species at each site was selected for molecular investigations (Additional files 1, 2: Table S1, S2) and one leg was then removed from each specimen for DNA extraction.

Phylogenetic analysis
All amplicons of the three fragments were sequenced and acquired consensus sequences were analyzed using BLAST (http://blast .ncbi.nlm.nih.gov/Blast .cgi) in order to find and include similar sequences in the phylogenetic analysis. The phylogenetic relationship of the scorpions in the current study was inferred by using the Maximum Likelihood tree building algorithm and the Tamura-Nei model [14]. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a Fig. 1 Morphological characteristics of the scorpion species of the current study. From the family Scorpionidae, which is characterized by a pentagonal sternum (a), is the only representative Scorpio maurus which is characterized by no-granule prosoma (b1) and a short and bulky pincer (b2). Species belonging to the family Buthidae have a triangular sternum as a diagnostic character (c). The Hottentotta saulcyi has a dark colored Prosoma (d1) and regular teeth of the abdominal region of the fifth segment of the post-abdominal and caudal vesicle (d2). The morphological characteristics of Androctonus crassicauda are the dark color of this species and the special tufts in the tail (e1) and, most importantly, the existence of three granules under the tooth of the fingers of the pincer (e2). Odontobuthus doriae has circular-end long teeth in the abdominal region of the tail (f). Mesobuthus eupeus has special decorations of the Prosoma (g1), absence of protrusion of the end pellet in the tergite (g2) and irregular teeth with a specific design in the abdominal region of the tail (g3). Mesobuthus caucasicus has granules along each other in carapace (h1), the complete protrusion of the end granule in the tergite region (h2), as well as the presence of an outer sub-granule being smaller than the inner sub-granule in the dental rows of the pincer (h3) (See figure on next page.) matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. Evolutionary analyses were conducted in MEGA X [15].

Results and discussion
In the current study, six species of Buthidae and Scorpionidae scorpions from northwestern and central Iran were identified based on morphological characteristics (Fig. 1). We amplified the COI gene, 12S rDNA and 16S rDNA and a representative subset was deposited in GenBank; accession numbers are shown in Additional files 1, 2: Table S1, S2 and in Fig. 2. The acquired sequences of 12S rDNA fragments in the species Androctonus crassicauda, Hottentotta saulcyi, Mesobuthus caucasicus, M. eupeus as well as 16S rDNA in Hottentotta saulcyi are novel. We also consider our COI sequence of H. saulcyi to be the first for this species, since the existing COI sequence in GenBank (KU341989) seems to be from a misidentified specimen (Fig. 2a). Additionally, the sequences of 16S rDNA of A. crassicauda, M. caucasicus, and M. eupeus, the 12S rDNA sequence of Odontobuthus doriae, and all three mitochondrial gene sequences of Scorpio maurus are the first reported from Iran.
In this study, we successfully used 16S rDNA to differentiate between genera, and sequences clustered well on the species level (Fig. 2b). Likewise, Cytochrome Oxidase I (COI) and 16S rDNA fragments had the appropriate capability for the differentiation of four Eurasian scorpion species (Mesobuthus caucasicus, M. cyprius, M. eupeus, and M. gibbosus; [16], which is in accordance with our results showing effective differentiation of Mesobuthus at the species level using all three mitochondrial markers (Fig. 2).
There has been taxonomic controversy over the placement of Mesobuthus caucasicus, which has been proposed to belong to the genus Olivierius [4,9,[17][18][19] while other researchers prefer to maintain the original scientific name Mesobuthus caucasicus [16,[20][21][22]. Our phylogenies of all three mitochondrial markers strongly suggest that Mesobuthus caucasicus should indeed be included in the genus Mesobuthus (Fig. 2) and confirmed by placing Opisthacanthus asper as a rooted outgroup (Fig. 2).
In all, we have in this paper shown that COI, 12S rDNA and 16S rDNA are efficient markers for phylogenetic discrimination of these Iranian scorpions and set the foundations for the correct identification of these medically important species.

Limitations
Due to the fact that the samples analyzed in this analysis were collected from limited areas, the collection of samples and species from wider areas could better indicate potential differences in morphological characteristics and genetic markers. Also, the selection of one sample of any scorpion species in each location for molecular investigation, as a limiting factor in the present study, should be considered.