The First Molecular Phylogeny of Orthochirus fomichevi Kovařík, Yağmur, Fet & Hussen, 2019 (Scorpiones: Buthidae) from Duhok Province, Kurdistan Region, Iraq, as Inferred from Mitochondrial COI Gene

1. Introduction
Orthochirus Karsch, 1892 is a genus of scorpions within the family Buthidae. The genus was originally established by Karsch [1] as a replacement for the preoccupied name Orthodactylus Karsch, 1881. Since its inception, the taxonomic status of Orthochirus has been subject to considerable debate. Kraepelin [2]initially regarded it as a mere synonym of Butheolus, but it was later reinstated as a valid genus by Simon [3].
The genus Orthochirus comprises small-sized scorpions with a broad distribution across North Africa, the Middle East, Arabian Peninsula, Central Asia, and India [4]. To date, 53 species within this genus have been described [5], including three species recorded in Iraq: O. fomichevi, O. mesopotamicus, and O. iraqus [6]. According to Kachel et al. [7], each Orthochirus species in Iraq exhibits a restricted geographic range: O. fomichevi inhabits the northern region and the foothills of the Zagros Mountains; O. iraqus is distributed across the central and western plains; and O. mesopotamicus occurs in the southern humid and plain areas of the country. This spatial segregation underscores the species-specific habitat preferences and limited distributional overlap within Iraq.
O. fomichevi was first described as a new species based on morphological characteristics by Kovařík et al. [6]. Prior to this, it had been misidentified as O. zagrosensis; however, O. fomichevi can be differentiated by the presence of granules on the dorsal surface of metasomal segment V, a feature absents in O. zagrosensis [6]. The known distribution of O. fomichevi includes the Kurdistan region of Iraq and parts of Turkey. This initial misidentification underscores the limitations of relying exclusively on morphological traits for accurate species delimitation within the genus Orthochirus, highlighting the need for integrative taxonomic approaches.
DNA barcoding has emerged as a widely adopted molecular approach to overcome challenges in species identification, particularly for taxa exhibiting high morphological similarity or compromised specimen integrity. This technique primarily targets the 5′ region of the mitochondrial cytochrome c oxidase subunit I (COI) gene, which exhibits sufficient interspecific variability to serve as a standardized genetic marker for species-level discrimination, commonly referred to as the “barcode of life” [8, 9]. The effectiveness of DNA barcoding is further enhanced when integrated with detailed morphological analyses [10], providing a robust framework for accurate taxonomic resolution across diverse animal groups.
Although the mitochondrial COI gene sequence for O. fomichevi is available in the GenBank database, no published studies have yet addressed its phylogenetic placement within the genus. This gap highlights the necessity for comprehensive molecular analyses to elucidate the evolutionary relationships of O. fomichevi. Accordingly, this descriptive, cross-sectional study integrates morphological identification with mitochondrial COI gene analysis to infer the molecular phylogeny of O. fomichevi, contributing to a deeper understanding of its taxonomic and evolutionary context within Orthochirus.

2. Materials and Methods

2.1. Study area
The study area was conducted in Duhok Province in the Kurdistan region of Iraq, a predominantly mountainous region bordering Turkey and Syria (Figure 1). Geographically, it lies between latitudes 36°10’N and 37°23’N and longitudes 42°20’E and 44°19’E. Duhok Province is administratively divided into seven districts: Zakho, Sumel, Duhok, Amedi, Shekhan, Akre, and Bardarash. The overall study was conducted from June 2024 to March 2025. Ethical considerations were strictly adhered to throughout the study to ensure compliance with relevant guidelines and regulations.

2.2. Taxon sampling
O. fomichevi specimens were collected nocturnally between June and October 2024 using a high-intensity ultraviolet (UV) lamp, capitalizing on the species’ nocturnal behavior and natural fluorescence under UV light, which facilitates detection. Collected specimens were divided for preservation based on subsequent analyses: Some were fixed in 95% ethanol and stored at -20 °C for molecular studies, while others were preserved in 80% ethanol and maintained at room temperature for morphological examination. The geographical distribution of O. fomichevi was mapped using ArcGIS Pro software, version 3.0.1 (Esri, USA).

2.3. Species identification and imaging
All specimens were identified morphologically using the taxonomic key provided by Kovařík et al. [6]. Morphological identifications were verified by an experienced scorpion taxonomist. Morphological terminology follows the standards established by Sissom [11]. High-resolution images of whole specimens were captured using a Canon EOS 7D camera. Image stacking was performed using Helicon Focus software (HeliconSoft, Ukraine) to enhance depth of field. The focus stacking methodology was adapted from the Canon-Cognisys system recommended by Brecko et al. [12].

2.4. DNA extraction
Genomic DNA was extracted from approximately 20 mg of metasomal muscle tissue using the AddPrep Genomic DNA Extraction Kit (Addbio, Korea). Prior to extraction, muscle tissue was washed in physiological saline solution (PiONEER, Iraq) for 15–20 minutes to remove residual ethanol and subsequently dried on filter paper. The dried tissue was transferred to a 1.5 mL Eppendorf tube and homogenized using a propylene micropestle (Shenzhen Baimai Life Science, China). The subsequent extraction steps were conducted according to the manufacturer’s protocol. The isolated DNA was stored at -20 °C until further analysis.

2.5. Mitochondrial COI gene fragment amplification 
Polymerase chain reaction (PCR) amplification of the barcode region of the mitochondrial COI gene was performed using a TC1000-G-Pro Thermal Cycler (DLAB, China). The reaction employed the universal primers LCO1490 (5’-GGTCAACAAATCATCATAAAGATATTGG-3’) and HCO2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) as described by Folmer et al. [13]. PCR reactions were conducted in a total volume of 20 µL, containing 0.5 µL (10 pmol) of each primer, 10 µL of Addstart Taq Master Mix (2× concentration; Addbio, Korea), 2 µL of template DNA (~20 ng/µL), and 7 µL of water. The thermal cycling conditions are detailed in Table 1. Amplification success was verified by electrophoresis on a 1.5% agarose gel. PCR products were subsequently sent to Macrogen Inc. (Seoul, Korea) for Sanger sequencing.

2.6. DNA sequence processing
Sanger sequencing trace files were manually checked and trimmed by SeqTrace software, version 0.9.0 [14] to generate consensus sequences, which are then subjected to BLAST tool [15] against GenBank records to confirm DNA sequence identities and to search for closely related sequences for phylogenetic construction. The study sequences were deposited in GenBank under the accession numbers listed in Table 2.

 2.7. Phylogenetic analysis
All sequences were aligned using the ClustalW algorithm implemented in MEGA software, 11 VERSION 11 [16]. Phylogenetic relationships were inferred using the maximum likelihood (ML) method, with branch support assessed via 1,000 bootstrap replicates. The optimal substitution model was selected based on bayesian information criterion (BIC) scores [17]. The resulting ML tree generated in MEGA 11 was exported in Newick format and subsequently visualized and edited using figtree software, version 1.4.4 to produce a high-quality phylogenetic tree.
Mean genetic distances within and between O. fomichevi and closely related sequences (excluding the outgroup) were calculated using the Kimura 2-parameter model (K2P) [18] to estimate evolutionary divergence among the sequences.

3. Results

3.1. Taxonomy 
3.1.1. Family Buthidae C. L. Koch, 1837
3.1.1.1. Genus Orthochirus Karsch, 1892
3.1.1.1.1. O. fomichevi Kovařík, Yağmur, Fet & Hussen, 2019
Type locality and type depository: Iraq, Sulaymaniyah Province, Chaqzhi Khwaroo; František Kovařík, private collection, Prague, Czech Republic (FKCP).
Materials examined: Iraq, Duhok province, Zakho district, Shahida, 37°08’01.1”N 42°40’24.0”E, 506 m above sea level (ASL), 2♂2♀; Khrababk, 37°08’30.6”N 42°45’02.1”E, 526 m ASL, 1♂3♀; Salka, 37°06’50.8”N 42°39’05.7”E, 573 m ASL, 2♂3♀; Betas, 37°04’02.6”N 42°42’06.1”E, 675 m ASL, 1♂; Dubanik, 37°02’30.2”N 42°50’19.9”E, 709 m ASL., 4♂3♀; Sumel district, Sumel, 36°50’31.3”N 42°51’52.8”E, 446 m ASL, 6♂2♀; Esmahil Ava, 36°57’25.3”N 42°38’12.9”E, 504 m ASL, 13♂11♀; Duhok district, Nizarke, 36°50’20.7”N 43°04’03.5”E, 659 m ASL, 6♂7♀; Shekhan district, Atrush, 36°51’07.7”N 43°21’54.3”E, 1014 m ASL, 7♂8♀. 

3.2. Diagnosis
The general body coloration is predominantly black, with the exception for the chelae fingers and the distal segments of the legs, which are yellow (Figure 2a, 2b, 2c and 2d). Adult body length ranges from 30.12 to 39.91 mm in males and 30.37 to 45.34 mm in females; with females generally larger than males. The trichobothrium d2 on the femur is typically absent. The movable finger of the chela bears 8–9 rows of denticles, including both internal and external denticles, as well as four subterminal denticles. The number of pectinal teeth ranges from 21 to 23 in males and 17 to 20 in females. Mesosomal sternite VII presents a granulate surface characterized by prominent granulate carinae (Figure 2e). Metasomal segments II and III lack granules on the ventral and lateral surfaces, displaying a punctate and bumpy texture (Figure 2f). Metasomal segment V is distinguished by dense granulation along the dorsal mesial surface (Figure 2g). The telson is elongated and smooth, lacks granules. The vesicle is long, with lateral and ventral punctation. The aculeus is long and curved.

3.3. Phylogenetic analysis
The barcode region of the COI gene was successfully sequenced from five O. fomichevi specimens (Table 2), collected from four distinct localities. These sequences were supplemented with 12 additional COI sequences of various Orthochirus species retrieved from the GenBank database. Additionally, a sequence of Hottentotta tamulus was included as an outgroup for phylogenetic analyses (Table 3).

A phylogenetic tree for O. fomichevi was constructed using the ML method, applying the TN93+G model [19], selected as the best-fit model by MEGA software. The ML tree with the highest log likelihood value of -1,738.62 was selected for visualization. In this tree (Figure 3), sequences of O. fomichevi formed a highly-supported clade with O. fomichevi from Iraq, as evidenced by a high bootstrap support value of 98. However, interspecific relationships among other Orthochirus species could not be well resolved due to low bootstrap values. In contrast, different O. innesi isolates did not form a clade with each other butinstead each formed a separate clade, which indicates that they belong to different taxa. Additionally, O. afghanus did not form a separate cladebut instead grouped with O. persa in a highly-supported clade (bootstrap value=98), indicating that they belong to the same taxon.

An examination of evolutionary divergence, calculated using the K2P model (Table 4), showed that the mean intraspecific divergence among members of O. fomichevi was low (0.54%), indicating a high level of genetic similarity within the species. Additionally, O. fomichevi exhibited a close genetic affinity to O. innesi from Morocco, with a mean interspecific divergence of 6.56%. In contrast, O. fomichevi was more genetically divergent from O. vachoni, with a divergence of 12.68%, compared to other Orthochirus species. Moreover, O. innesi isolates ON255632, JQ514244, and MZ669861 were highly divergent from each other, with divergence values ranging from 7.45 to 10.91%. Additionally, O. afghanus was very close to O. persa (divergence value=2.37%).

4. Discussion
In the present study, eighty-one specimens of O. fomichevi were collected from multiple locations across Duhok Province. These scorpions were predominantly encountered in open plain habitats characterized by sparse vegetation. Morphological analysis consistently confirmed species identification based on diagnostic traits, while molecular analysis of the COI gene revealed that O. fomichevi forms a strongly supported clade (bootstrap=98) distinct from other Orthochirus species. The intraspecific divergence was low (0.54%), indicating high genetic homogeneity, while interspecific divergence with other species ranged from 6.56% to 12.68%. Notably, sequences labeled as O. innesi, O. persa, and O. afghanus in GenBank showed evidence of misidentification. 
Previous records have documented O. fomichevi in the provinces of Duhok, Erbil, and Sulaymaniyah within the Kurdistan region of Iraq, as well as in Hakkari Province from Turkey [6, 20, 21]. Morphologically, O. fomichevi can be distinguished from other Orthochirus species by several diagnostic features, including a densely granulated mesosomal sternite VII with well-developed granulate carinae; a metasomal segment V exhibiting dense granulation along the dorsal mesial surface; and metasomal segments II and III that are smooth, punctate, and bumpy ventrally and laterally, lacking granules [6]. All examined specimens in this study exhibited these defining morphological characteristics.
To complement morphological identification, phylogenetic analysis based on the mitochondrial COI gene was performed. The resulting phylogenetic tree demonstrated that the specimens collected in this study and previously sequenced isolates of O. fomichevi form a strongly supported clade (bootstrap value=98), distinct from other Orthochirus species. Estimates of genetic divergence corroborate these findings; the mean intraspecific divergence within O. fomichevi was 0.54%, reflecting high genetic similarity among conspecific individuals. In contrast, mean interspecific divergence between O. fomichevi and other Orthochirus species ranged from 6.56% to 12.68%, with O. innesi from Morocco (ON255632) exhibiting the lowest divergence, thereby representing the closest relative to O. fomichevi.
These results are consistent with previous molecular studies on the genus Orthochirus, such as Jafari et al. [22], who analyzed COI and 16S rRNA gene sequences from O. iranus, O. farzanpayi, O. zagrosensis, and O. stockwelli populations in Iran. Their findings similarly reported low intraspecific divergence alongside relatively higher interspecific divergence, supporting the phylogenetic patterns observed in the present study. However, they did not deposit their data in GenBank; that’s why we did not include them in our analysis. Similarly, Barahoei [23] also reported low intraspecific divergence, particularly in O. persa. The Orthochirus fauna of Iraq includes two other species: O. mesopotamicus and O. iraqus [7]. In this study, we were unable to infer their phylogenetic relationships due to the unavailability of their COI data in GenBank.
Additionally, the three isolates of O. innesi (ON255632, JQ514244, and MZ669861) retrieved from GenBank did not group together; instead, each formed a separate clade. Genetic divergence analysis further supported these findings, in which the divergence between them was relatively high (7.45–10.91%). The phylogenetic relationships between the isolates JQ514244 (unknown) and MZ669861 (Egypt) were previously inferred by Mohammed-Geba et al. [24], in which the two isolates grouped under the same clade. However, their study was based on a wide range of divergent species from different genera, and no Orthochirus species other than the ones mentioned; therefore, they were grouped together. The type locality of O. innesi is Cairo, Egypt [3]; therefore, we conclude that the isolate MZ669861 represents the true O. innesi, while the isolates ON255632 and JQ514244 belong to two misidentified specimens which could not be assigned due to limited COI data in GenBank.
Furthermore, O. afghanus from Afghanistan failed to form a distinct clade but instead formed a highly supported clade (bootstrap value=98) with the O. persa isolates from Iran. This indicates that they belong to the same species. The interspecific divergence further supports these findings, in which the divergence value between them was very low (2.37%) , considered two separate species. The localities of the isolates of O. afghanus (ON255631) and O. persa (PP574852, PP574854, and PP574853) analyzed in this study are close to each other [23, 25]; therefore, it was not possible to conclude whether they belong to O. afghanus or O. persa. Generally, species misidentification is very common in the case of scorpions, even in dedicated literature [26], which has led to many incorrect placements of scorpions in the GenBank database [27].
A limitation of this study was the inability to comprehensively investigate certain districts due to the political instability, their distance from our research center, and the logistical challenges associated with sample collection in these areas. Additionally, the limited availability of COI sequence data for Orthochirus species in GenBank restricted comparative molecular analyses.

5. Conclusion
In conclusion, this study provides robust evidence for the accurate identification and phylogenetic placement of O. fomichevi within the genus Orthochirus, confirmed by consistent morphological traits and a strongly supported phylogenetic clade with low intraspecific genetic divergence. The unexpected divergence and placements of isolates labeled O. innesi, O. persa, and O. afghanus highlight the need for thorough taxonomic revision and expanded genetic sampling. To address challenges from misidentifications in public databases, we recommend integrating morphological and molecular tools in scorpion taxonomy and expanding molecular datasets to include additional Orthochirus species, which will improve database accuracy and enhance biodiversity knowledge in the region

Ethical Considerations

Compliance with ethical guidelines
The study protocol was approved by the Animal Ethics Committee of College of Science, University of Zakho, Zakho Iraq (Code: AEC-031).

Data availability
The COI sequence data are available in GenBank at under the accession numbers PV219459, PV219460, PV219461, PV219462, and PV219463.

Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors.

Authors' contributions
Study conceptualization and study design: All authors; Data acquisition, interpretation, statistical analysis and writing the original draft: Farhad Ramadhan Ali; Supervision, review and editing: Hamid Saeid Kachel.

Conflict of interest
The authors declared no conflicts of interest.

Acknowledgements
The authors would like to express their sincere gratitude to Ersen Aydın Yağmur (Manisa Celal Bayar University, Turkey) for kindly photographing the specimens used in this study and for his valuable assistance in confirming their identification. Special appreciation is also extended to the Biology Research Center at the University of Zakho for kindly granting permission to conduct this research on their premises.

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