Genetic Diversity Within Scorpio Maurus (Scorpiones: Scorpionidae) from Morocco: Preliminary Evidence Based on CO1 Mitochondrial DNA Sequences
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Biologia 63/6: 1157—1160, 2008 Section Zoology DOI: 10.2478/s11756-008-0176-y Genetic diversity within Scorpio maurus (Scorpiones: Scorpionidae) from Morocco: Preliminary evidence based on CO1 mitochondrial DNA sequences Elsa Froufe1,PedroSousa1,2,PauloC.Alves1,2 &DavidJ.Harris1,2* 1CIBIO, Centro de Investiga¸c˘ao em Biodiversidade e Recursos Genéticos, Campus Agrário de Vair˘ao, 4485–661 Vair˘ao, Portugal; e-mail: [email protected] 2Departamento de Zoologia e Antropologia, Faculdade de Ci˛encias da Universidade do Porto, 4099–002 Porto, Portugal Abstract: The large-clawed scorpion, Scorpio maurus, is a medically important scorpion and yet nothing is known regarding genetic diversity within this species. As a preliminary analysis we determined variation within the cytochrome oxidase 1 (CO1) mitochondrial gene from specimens from Morocco. High levels of genetic diversity were found that presented some geographical coherence. Of the two identified subspecies from Morocco, S. maurus birulai and S. maurus fuliginosus,the latter included genetically distinct lineages (8.0% uncorrected sequence divergence), indicating a detailed morphological and molecular revision is needed for this species. Key words: Scorpio maurus; Mitochondrial DNA; CO1; North Africa Introduction tial, not just to accurately determine true levels of bio- diversity or to revise taxonomy to reflect phylogeny, but Scorpio maurus L., 1758 (Scorpiones: Scorpionidae) has also because the many studies assessing the biochemical a huge range from Morocco across Northern Africa, nature of scorpion venoms require precise species deter- through the Arabian Peninsula and the Middle East in- mination (e.g., for S. maurus, Kharrat et al. 1997). cluding Israel, Syria, Jordan and Turkey, to as far East Despite its widespread distribution and complex as Iraq and Iran. Despite being typically found in semi- subspecific taxonomy, levels of genetic variation within desert habitats, it can also be found in regions with S. maurus remain unknown. The aim of this study was a Mediterranean climate. In areas with cooler winter to assess genetic diversity within specimens from Mo- conditions the species hibernates in burrows. Scorpio rocco using cytochrome oxidase 1 (CO1) mtDNA se- is currently considered a monotypic genus, although 17 quence data, a gene typically included in barcoding subspecies are generally accepted (Fet et al. 2000). studies as a global bioidentification system (Hebert et In other genera of scorpions from North Africa al. 2003). This is compared to geographic and subspe- and Europe, recent analyses of mitochondrial DNA cific status of the specimens, and can be built upon by (mtDNA) and various nuclear markers have contributed future research to assess diversity across the range of extensively to delimiting cryptic species. Several new the species. species of Euscorpius have been identified from Italy and the Balkans (Gantenbein et al. 2000; Scherabon et al. 2000) and various Mediterranean islands (Ganten- Material and methods bein et al. 2001), even though some are very difficult to Specimens were collected by hand in the field and stored in define using traditional morphological characters (Ja- 96% ethanol (Table 1, Fig. 1). Subspecific status was esti- cob et al. 2004). On the other hand very low genetic mated when possible, following available keys (e.g., Vachon divergence was reported from the widespread Euscor- 1952). Sub-adult specimens generally could not be identified pius italicus (Herbst, 1800) (Fet et al. 2006). Phylogeo- beyond the species level. Genomic DNA was extracted fol- graphic analyses of Buthus occitanus (Amoreux, 1789) lowing standard high-salt protocols (Sambrook et al. 1989). A fragment of the CO1 gene was amplified by PCR using the across the Strait of Gibraltar revealed extensive varia- primers published by Palumbi (1998). Sequences were ob- tion (Gantenbein & Largiad`er 2003), and some of the tained on an automated sequencer (ABI 310). All sequences lineages recovered from the Iberian Peninsula have since were submitted to GenBank (Table 1). Alignment was per- been recognized as distinct species (Louren¸co & Vachon formed manually using Bioedit v. 5.0.9. (Hall 1999), and was 2004). Identification of such cryptic variation is essen- facile as no indels were found. Two specimens of S. maurus * Corresponding author c 2008 Institute of Zoology, Slovak Academy of Sciences 1158 E. Froufe et al. Table 1. Localities and GenBank accession numbers for specimens included in this study. Population Code Species Sex Latitude Longitude GenBank 1Sc16S. maurus birulai M 35.47107 –6.03122 FJ198057 2Sc05S. maurus ? 34.63023 –5.53805 FJ198058 3Sc46S. maurus ? 32.16880 –6.53337 FJ198059 Sc56 S. maurus fuliginosus M FJ198065 4Sc27S. maurus ? 31.66017 –6.92578 FJ198066 5Sc42S. maurus ? 31.64478 –7.11481 FJ198064 6Sc74S. maurus fuliginosus F 31.14197 –8.10278 FJ198061 Sc75 S. maurus fuliginosus F FJ198062 7Sc81S. maurus fuliginosus F 30.94182 –8.11865 FJ198063 8Sc51S. maurus fuliginosus F 30.99038 –9.03982 FJ198060 Israel – S. maurus fuscus AY156584 Egypt – S. maurus palmatus AY156585 Morocco Sc1 Buthus sp. FJ198055 Morocco Sc4 Buthus sp. FJ198056 Sub-specific taxonomy and sex were determined for adult specimens only. Fig. 1. Map showing the localities of the S. maurus individuals sequenced for this study. (Prendini et al. 2003) were included from GenBank and two ing trees were combined in a 50% majority consensus tree, specimens of Buthus were used to root the trees (Table 1). in which frequency of any particular clade represents the Sequences were imported into PAUP* 4.0b10 (Swof- posterior probability (Huelsenbeck & Ronquist 2001). ford 2003). For the phylogenetic analysis we used Maxi- mum Likelihood (ML) and Maximum Parsimony (MP) anal- Results ysis with random sequence addition (100 replicate heuristic search). Support for nodes was estimated through the boot- Including the outgroups, 14 individuals were analysed strap technique (Felsenstein 1985) with 1000 replicates. The with an aligned length of 648 bp although the two sam- AIC criteria carried out in Modeltest 3.06 (Posada & Cran- ples from Genbank were 12 bp shorter. The most appro- dall 1998) was used to choose the model of evolution em- priate model of evolution for this dataset was the gen- ployed in the ML analysis. Bayesian analysis was also imple- eral time reversible model, with an estimate of invari- mented using MrBayes v.3.1 (Huelsenbeck & Ronquist 2001) able sites and a discrete approximation of the gamma with parameters estimated as part of the analysis and four incrementally heated Markov chains with the default heat- distribution. The ML analysis recovered a single tree 6 − . ing values. The analysis was run for 10 generation, saving ( ln 2270 7, Fig. 2). Two MP trees were recovered (320 one tree in each 100 generations. The log-likelihood values steps), the consensus of which differed from the ML of the sample point were plotted against the generation time tree only at nodes with <50% support in either analy- and all the trees prior to reaching stationary were discarded, sis (Fig. 2). The Bayesian analysis recovered the same ensuring that burn-in samples were not retained. Remain- tree as the ML analysis. Genetic diversity within Scorpio maurus 1159 Fig. 2. Phylogenetic relationships estimated using maximum likelihood as described in the text. ML and MP bootstrap support is indicated above nodes, Bayesian posterior probabilities below nodes. The tree was rooted using two specimens of Buthus.Codesrefer to Table 1. Discussion clade relative to the two samples of GenBank, of S. m. palmatus from Egypt and S. m. fuscus from Israel. How- As seen in North African Buthus (Gantenbein & ever, most other relationships were weakly supported, Largiad`er 2003), our results demonstrate that Scorpio as indicated by short branches and low bootstrap val- maurus as currently accepted includes highly geneti- ues. A similar situation, of genetically distinct mtDNA cally divergent mtDNA lineages within Morocco. Max- lineages but with limited evidence for relationships be- imum uncorrected pairwise divergences were up to 10% tween them, was also indicated in North African scor- (Table 2), a level far higher than is typically seen within pion genus Buthus (Buthidae) (Gantenbein & Largiad`er a species and that indicates that S. maurus may well be 2003). a species complex. Examination of the estimated phy- In Buthus, until recently Iberian Peninsula and logeny of the sampled specimens indicates there is some most Northwest African specimens were referred to B. geographic coherency. The two samples from the Rif occitanus. However, extensive molecular and morpho- mountains in North Morocco (one of which was identi- logical studies have led to the identification of many fied as S. m. birulai) are closely related, as were a group new species (e.g., Louren¸co & Geniez 2005), and sev- of samples from the High Atlas mountains (Sc 74, 75 eral previously recognized subspecies represent geneti- an 81, all S. m. fuliginosus) and another group from cally distinct lineages (Gantenbein & Largiad`er 2003), Central Morocco (Sc15, 46 and 56) that also included a Our preliminary results from S. maurus,anotherscor- specimen identified as S. m. fuliginosus.Thusatleast pion of considerable medical importance, indicate that S. m. fuliginosus includes individuals from divergent similar work is needed to fully address diversity within mtDNA lineages. All the Moroccan samples formed a this species complex. 1160 E. Froufe et al. Table 2. Uncorrected pairwise distances between Scorpio samples included in this study. S.m. fuscus S.m. palmatus Sc16 Sc5 Sc46 Sc51 Sc74 Sc75 Sc42 Sc56 S.m. fuscus – S.m. palmatus 0.0691 – Sc16 0.0941 0.0973 – Sc5 0.0924 0.0957 0.0138 – Sc46 0.0941 0.1020 0.0586 0.0586 – Sc51 0.0831 0.0895 0.0478 0.0463 0.0740 – Sc74 0.0955 0.1034 0.0694 0.0679 0.0802 0.0571 – Sc75 & Sc81 0.0893 0.1003 0.0679 0.0632 0.0771 0.0586 0.0123 – Sc42 0.0974 0.1007 0.0603 0.0556 0.0742 0.0587 0.0696 0.0711 – Sc56 0.0941 0.1021 0.0586 0.0586 0.0015 0.0740 0.0802 0.0771 0.0727 – Sc27 0.0957 0.1036 0.0602 0.0601 0.0015 0.0756 0.0817 0.0787 0.0742 0.0030 Explanations: Codes refer to Table 1.