Bull 105 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Hassan and Ali Bull. Iraq nat. Hist. Mus. (2024) 18 (1): 105-120. https://doi.org/10.26842/binhm.7.2024.18.1.0105 ORIGINAL ARTICLE MOLECULAR STUDY OF SOME SPECIES OF CENTIPEDES IN IRAQ WITH NEW RECORD OF LITHOBIUS CRASSIPES L. KOCH, 1862 (CHILOPODA, LITHOBIOMORPHA, LITHOBIIDAE) Shaymaa Hussein Hassan and Hayder Badri Ali Department of Biology, College of Science, University of Baghdad, Baghdad, Iraq. Corresponding author: hayder.badri@sc.uobaghdad.edu.iq Received: 20 August 2023, Revised: 7 Dec. 2023, Accepted: 28 Dec. 2023, Published:20 June 2024 This work is licensed under a Creative Commons Attribution 4.0 International License ABSTRACT The first molecular research on Iraqi centipede fauna is presented in this article. Between October 2022 and May 2023, during various climatic circumstances, centipedes were collected from several locations in four provinces of Iraq. Three families, represented by four genera, underwent molecular identification, and five species were found. From the order Scolopendromorpha family Scolopendridae, two species were recorded, Scolopendra morsitans Linnaeus, 1758, and S. cingulata Latreille, 1829, Cormocephalus sp.; while from the order Lithobiomorpha, family Lithobiidae, one species was recorded for first time in Iraq; Lithobius crassipes L. Koch, 1862 from the order Geophilomorpha family Himantariidae, one species Bothriogaster Signata Kessler, 1874. DNA was extracted from the specimens, the mtDNA fragment from the Cytochrome C Oxidase Subunit I (COI) gene was amplified by using the PCR technique with appropriate primers, and subsequently, the Basic Local Alignment Search Tool (BLAST) tool, which is accessible at the NCBI, was used. Additionally, a phylogenetic tree was built, and a distant comparison was shown. Keywords: Centipedes, Iraq, Lithobius, Molecular, New record. INTRODUCTION About 3000 species of Chilopoda have been described, and they are more frequently found in tropical and subtropical countries. Their rational evolutionary history, which has a fossil record lasting 420 million years, can explain their widespread global distribution. Recent investigations have demonstrated a hard phylogeny that supports theories about their biology that are based on evolution utilizing both morphological and molecular evidence (Edgecomb and Giribet, 2007). The inexistence centipedes are classified into five monophyletic orders. Only two species of the order Craterostigmomorpha (Pockock, 1902) are extant species, and they are both from New Zealand and Tasmania, respectively (Vélez et al., 2012). In contrast, the orders Lithobiomorpha (Pockock, 1895) and Scutigeromorpha (Pockock, 1895) contain BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Iraq Natural History Research Center & Museum, University of Baghdad https://jnhm.uobaghdad.edu.iq/index.php/BINHM/Home Copyright © Bulletin of the Iraq Natural History Museum Online ISSN: 2311-9799, Print ISSN: 1017-8678 https://doi.org/10.26842/binhm.7.2024.18.1.0105 https://orcid.org/0000-0003-4842-151X mailto:hayder.badri@sc.uobaghdad.edu.iq https://creativecommons.org/licenses/by/4.0/ https://jnhm.uobaghdad.edu.iq/index.php/BINHM/Home 106 Bull. Iraq nat. Hist. Mus. 18 (1): 105-120. Molecular study of some species of centipedes approximately 1,100 valid species each and are both found in temperate regions of the world. With over 1,300 genuine species in 14 recognized families, the order Geophilomorpha (Pockock, 1896) is the most diverse and extensive group. With more than 700 recognized species, the order Scolopendromorpha (Pocock, 1895) is diverse in lower latitudes (Edgecombe and Giribet, 2007; Edgecombe and Bonato, 2011). There are few studies on this field in Iraq. In the first papers on Chilopoda from Iraq were published by Brolemann (1921, 1922), when he studied material from Amara and Jebel Hamrin, he made a note including 14 species from the five orders. Even though Chamberlin and Hoffman (1958) studied material from different parts of Iraq and described three new species Lithobius cuklauvus (Chamberlin, 1958), M. integer (Chamberlin, 1944) and Ottobius irikensis (Chamberlin, 1958); moreover, it recorded more than 11 species from 4 orders. Advanced, Würmli (1975) recorded Thereuonema turkestana hamrinensis Verhoeff, 1905, under the synonym T. microstoma (Meinert, 1886). While Dobroruka (1976) described two new species from Baghdad, Lewis (1986, 2001) studied the scolopendromorph fauna of Iraq, and recorded several scolopendromorphs from different regions of Iraq. Lithobius irikensis Chamberlin, 1958, was introduced by Zapparoli (1993) as a new combination for the Ottobius irikensis described by Chamberlin and Hoffman (1958), and Zapparoli (1995) synonymized Eupolybothrus syngenes Verhoeff, 1907, under E. litoralis (L. Koch, 1867) as well. The first record of the morphology, description, and distribution preferences of the fauna of Scolopendromorpha centipedes in Kurdistan and Iraq was supplied by a number of faunistic papers (Ahmed and Hussen, 2017). Nine species from 183 taken from various locations in the central part of Iraq were recorded for the first time (Aldoori, 2020). In Basrah, south of Iraq, Al-Simary et al., (2022) gathered samples from eight stations in various regions. Out of the 105 specimens, five species (belonging to four orders and five families) were recognized. Each species' taxonomic information was also provided. The most recent and latest annotated checklist of Chilopoda from Iraq contains 27 species from 13 genera, 8 families, and 4 orders (Dyachkovi et al., 2023). In the current paper, the specimens of local centipedes were identified using genetic techniques. Utilizing the neighbor-joining method, a phylogenetic tree was created utilizing several other samples from GenBank to enhance molecular information for the centipedes in Iraq, by the present molecular study, and establish phylogenetic relationship patterns for the collected species, using molecular data from the Cytochrome c oxidase subunit (COI) analyses. MATERIALS AND METHODS Specimens' collection: The specimens collected from different geographic locations in Iraq include four provinces Baghdad, Wasit, Salahaddin, and Diyala in the middle of Iraq. All studied specimens were hand-collected in one place. The collection period was from October 107 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Hassan and Ali 2022 to May 2023, using a forceps and direct hand pickup to collect them from the soil, and under stones and remnants of mud pieces and tree roots, specimens were stored in glass tubes, then transferred to the laboratory for the purpose of killing them, and then preserved in 70% ethanol. Taxonomic nomenclature and species identification were based on the following literature: Schileyko (2007), Siriwut et al. (2015), and Ganske et al. (2018). Specimens preparing for molecular studies: The following steps were adopted for this purpose: 1- DNA extraction: Utilizing the Geneid DNA extraction kit, DNA was extracted from 11 samples using a modified version of the manufacturer's usual recommended approach. The ultimate legs of centipedes were detached from their bodies and repeatedly rinsed with distilled water to remove excess alcohol. DNA was then isolated using a DNeasy Blood and Tissue kit from the dehydrated muscle tissues of the ultimate legs. 2- Primers Preparing: Specific primers were used to obtain and amplify the partial mitochondrial Cytochrome Oxidase subunit I gene (COI), the forward LCO 1490 (5′- GGTCAACAAATCATAAAGATATTGG-3′) (Baker et al., 2019) with the reverse HCO2198, (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Oeyen, 2014; Kang et al., 2017). 3- DNA Amplification: Specific primers were used to obtain and amplify the partial mitochondrial Cytochrome Oxidase subunit I gene (COI). Polymerase Chain Reaction (PCR) typically used 5µl of DNA extract as a template for the PCR reaction, as mentioned above. After the optimization condition of the PCR cycler program (BIONEER, Crop, Daejeon, Korea), the reactions were amplified for 35 cycles. 4- Gel Electrophoresis: The amplification of 11 sample specimens’ products was confirmed by using 1.5% agarose gel electrophoresis in a 1 X TBE buffer and then stained to visualize with 4 % ethidium bromide in UV light at 320 nm, the molecular weight of the specific target bands is 710 bp. 5- Sequencing of PCR products and phylogenetic analysis: Using the partial sequence and the Sanger Sequencing Technique (Sanger et al., 1977), the forward primer LCO 1490 was applied, for sequencing, Macrogene Company, Korea, received the PCR of the mitochondrial Cytochrome Oxidase subunit I (COI) gene product to validate the identification of the 11 specimens, the phylogenetic tree was prepared using a Mega X program. RESULTS AND DISCUSSION From the family Scolopendridae, two species were recorded, Scolopendra morsitans, S. cingulate, and the genus Cormocephalus, while from the families Lithobiidae, and Himataridae, one species Lithobius crassipes, and Bothriogaster Signata, respectively, were recorded (Tab. 1). 108 Bull. Iraq nat. Hist. Mus. 18 (1): 105-120. Molecular study of some species of centipedes Table (1): Include families, genera and species in the study. Family Genus Species Scolopendridae Scolopendra Linnaeus, 1758 Scolopendra morsitans Linnaeus, 1758 S. cingulata (Latreille, 1829) Cormocephalus Newport, 1844 Cormocephalus sp. Lithobiidae Lithobius Leach, 1814 Lithobius crassipes (L. Koch, 1862) Himataridae Bothriogaster (Sseliwanoff, 1879) Bothriogaster Signata (Kessler, 1874) Order, Scolopendromorpha Family, Scolopendridae Genus, Scolopendra Linnaeus, 1758 Species, Scolopendra morsitans Linnaeus, 1758 Synonyms: Scolopendra morsitans amazonica Bücherl, 1946 S. morsitans procera Haase, 1887 S. morsitans calcarata Daday, 1891 S. morsitans fasciata Attems, 1930 S. morsitans scopoliana C.L. Koch, 1841 S. brandtiana Gervais, 1837 S. bilineata Brandt, 1840 S. crassipes Brandt, 1840 S. erythrocephala Brandt, 1840 S. elegans Brandt, 1841 S. fulvipes Brandt, 1841 S. angulipes Newport, 1844 S. leachii Newport, 1844 S.colopendra longicornis Newport, 1844 S. platypoides Newport, 1844 S. tuberculidens Newport, 1844 S. algerina Newport, 1845 S. fabricii Newport, 1845 S. formosa Newport, 1845 S. richardsoni Newport, 1845 S. tigrina Newport, 1845 S. varia Newport, 1845 S. tongana Gervais, 1847 S.infesta Koch, 1847 S.planipes Koch, 1847. S. pella Wood, 1861 S.porphyratainia Wood, 1861 S. brachypoda Peters, 1862 S. mossambica Peters, 1862 S. compressipes Wood, 1862 S. carinipes Humbert & Saussure, 1870 S. afzelii Porat, 1871 S. lineata Saussure & Zehntner, 1902 S. spinosella Saussure & Zehntner, 1902 Trachycormocephalus jodhpurensis Khanna, 1977 109 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Hassan and Ali Material examined (28♂♂, 19♀♀): Baghdad Province: Al-Jadriyah, 33°16'46.6"N 44°23'06.4"E, 7♂♂, 4♀♀ 8.ii.2023; Al- Mahmudiyah 6 ♂♂ 4♀♀ 3.iii.2023, (33°3′42″N 44°21′15″E); Al- Madain, 33.0993° N, 44.5807° E, 5♂♂, 5♀♀ 22.iii.2023; Al-Tarmiah, 33.6732° N, 44.3615° E, 6♂♂, 4♀♀ 8.iv.2023. Wasit Province, Al- Azizia, 32°54'38.5"N 45°03'46.8"E, 4♂♂, 2 ♀♀ 12.ii.2023 Distribution: It's widespread in the world, including North and South America, the Caribbean Islands, Europe, Africa, the Arabian Peninsula, Pakistan, India and the Indian Ocean Islands, Southeast Asia, Australia, and New Zealand (Akkari et al., 2008; Lewis, 2010; Siriwut et al., 2015). In Iraq, Chamberlin (1944) recorded several species belonging to three orders; which represent one of them as being the first record which is S. morsitans. Plate (1): Female of Scolopendra morsitans. Species, Scolopendra cingulata Latreille, 1829 Synonyms: Rhombocephalus parvus Newport, 1845 R. viridifrons Newport, 1844 S. banatica C.L.Koch, 1847 S. cingulatoides Newport, 1844 S.doriae Pirotta, 1878 S.fulva Gervais, 1847 S. graeca C.L.Koch, 1847 S. hispanica Newport, 1845 S.italica C.L.Koch, 1837 S. nigrifrons C.L.Koch, 1847 S.obscura C.L.Koch, 1863 S. penetrans C.L.Koch, 1847 S. pulchra C.L.Koch, 1847 S.savignyii Newport, 1845 S. thracia Verhoeff, 1928 S.venefica L.Koch, 1856 S. violantis Pirotta, 1878 S. zonata C.L.Koch, 1847 S. zwickiana C.L.Koch, 1863 Material examined (2♀♀): Baghdad Province, Al-Jadriyah, 33°17′17″N 44°23′35″E, 8.ii.2023. 110 Bull. Iraq nat. Hist. Mus. 18 (1): 105-120. Molecular study of some species of centipedes Global distribution: Mediterranean area Portugal, Spain, France, Slovenia, Croatia, Bosnia- Herzegovina, Serbia ,Hungary, Romania, Bulgaria, Albania, Greece, Turkey, Sicily, Corsica, Sardinia, Crete, Cyprus, (absent in Balearics), Ukraine, S. Europe .Russia in The Caucasus, Tajikistan, Lebanon, Palestine, Jordan, Egypt, Syria, Iran. Madagascar, the Andaman, and Nicobar Islands (Wang and Mauriès, 1996). It was listed for China, referring to Haase (1887). In Iraq, Chamberlin (1944) was also the first who recorded this species. Plate (2): Female of Scolopendra cingulate. Genus, Cormocephalus Newport, 1844 Cormocephalus sp. Material examined (1♀): Baghdad Province, Al- Yusufiyah, 33.0791° N 44.2520° E, 3.i.2023. Global distribution: Southern Europe, Africa, Madagascar, Philippines, New Guinea, New Britain, the Solomon Island, Micronesia, India, Bangladesh, Sri Lanka, New Caledonia, Australia (including Tasmania), New Zealand, the Galapagos Islands, Central America, the West Indies, and South America (Koch, 1983; Edgecombe et al., 2019). In Iraq, Al-Doori et al. (2020) recorded Cormocephalus nigrificatus (Verhoeff, 1937) from Al-Qadisiyah Province which represents the only species that belongs to this genus (Dyachkov et al., 2023). Since the only specimen obtained was damaged in its last segment, we could not diagnose the model at the species level. Plate (3): Female of Cormocephalus sp. 111 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Hassan and Ali Order, Lithobiomorpha Family, Lithobiidae Genus, Lithobius Leach, 1814 Species, Lithobius crassipes L.Koch, 1862 Synonyms: Archeobius ulterior Chamberlin, 1952 Archilithobius longipes Attems, 1907 Lithobius atrifrons Silvestri, 1896 L. ephessoensis Matic, 1980 L. (Monotarsobius) ephessosensis Matic, 1980 L. (Monotarsobius) ercijasus Verhoeff, 1943 L. (Monotarsobius) olympicus Verhoeff, 1941 L. podokes Attems, 1903 L. riggioi Matic, 1968 L. stictonotus Manfredi, 1957 L (Monotarsobius) ulterior Chamberlin, 1952 Material examined (1♂): Baghdad Province, Al Husseiniya, 33°32'48.0"N 44°25'00.0"E, 23.ii.2023. Global distribution: Wang (1963) verified only one specimen of Monotarsobius crassipes from Shao Tso Kiang in Taiwan (Chao, 2010), also established the presence of this species in Taiwan. as well, it distributes in Albania, Algeria, Austria, Belgium, Bosnia-Herzegovina, Bulgaria, the Canary Islands Central European Russia, Finland, Croatia, Denmark, Germany, Great Britain, France, Greece, Hungary, Ireland, Italy, Luxembourg, Macedonia, Madeira, the netherlands, Norway, Poland, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Tunisia, Turkey, and Ukraine.( Zapparoli, 2006). In Iraq, there are three species recorded from this genus, including: Lithobius microps Meinert, 1868; L. aeruginosus (L. Koch, 1862), and L. fossipes Brölemann, 1922 (Aldoori, 2020; Dyachkovi et al., 2023), in addition to L. ferganensis (Trotzina, 1894), while this species was recorded for the first time for the centipedes in Iraq. Plate (4): Male of Lithobius crassipes. 112 Bull. Iraq nat. Hist. Mus. 18 (1): 105-120. Molecular study of some species of centipedes Order, Geophilomorpha Family, Himantariidae Genus, Bothriogaster Sseliwanoff, 1879 Bothriogaster signata (Kessler, 1874) Synonyms: Bothriogaster affinis Sseliwanoff, 1879 B. africanus Sseliwanoff, 1881 B. arasana Verhoeff, 1943 B. cyrenaica Verhoeff K.W., 1908 B. egyptiacus Attems, 1896 B. meinerti Sseliwanoff, 1879 B. porigera Verhoeff, 1901 B. thesei Attems, 1902 B. tunetanum Verhoeff, 1899 B. wohlberedti Verhoeff, 1934 Himantarium taeniatum Meinert,1886 Notiphilus sanguineus C.L. Koch, 1847 N. taeniatus C.L. Koch, 1847 Material examined (2♂♂, 1♀): Saladin Province, Al-Hamra Village, 34°45′15″N43°36′22″E, 2♂♂, 22.iii.2023, Muhazam Village, 34°43′40″N43°38′36″E, 1♀, 22.ii.2023. Global distribution: Eastern Mediterranean Region (Zapparoli, 1991; Stoev, 2000), through Western Asia, Central Asia, Turkmenistan, Uzbekistan, Tajikistan, Kazakhstan, (Kessler, 1874), Balkan Peninsula, Cyprus, North Africa, and Russia (Volgograd Region and South Kazakhstan regions). A new checklist of centipedes from Iran in the Alborz Mountains (Zarei and Rahimian, 2020). In Iraq, Attems (1929) was recorded this species. Plate (5): female of Bothriogaster signata. 113 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Hassan and Ali Molecular identification Conventional PCR results: COI (710-bp) fragment was amplified by the primer pair (LCO1490 F/ HCO2198 R). Consistently across the widest variety of invertebrates, including centipedes (Folmer, et al., 1994). Results indicated a success rate of 11/11 effective DNA amplification. The mitochondrial DNA-COI gene's highly conserved regions were amplified using DNA amplification. When products of mitochondrial DNA fragments run in 1.5% agarose gel during gel electrophoresis, the particular band with the PCR test was seen in Plate (6). Plate (6): Amplification of polymerase coding region (710bp) COI of specimens by using conventional PCR. The replicons run on agarose gel 1.5% and visualized with trans illuminator, M: DNA ladder (100bp). Sequencing Analysis: According to our investigation, 62 specimens of Chilopoda have been collected since its initiation (October 2022) to (May 2023). DNA was extracted from 11 centipedes’ specimens; the results of the DNA sequences showed only six specimens were successfully sequenced, the nucleotide sequences of specimens were analysed for alignment, and the Basic Local Alignment Search Tool (BLAST) program is available online at the National Centre for Biotechnology Information (NCBI). The acquired sequences' phylogenetic analysis showed that they were closely related to the query sequences collected from the GenBank. The GenBank records for several of these sequences revealed that they were classified as belonging to species that are phylogenetically remote from the locations of their local documented distribution. The bit score is a statistical indicator of moral similarity, and a greater value denotes a high level of resemblance. The definition of the expectation value (E) is that it provides an assessment of the frequency with which it is anticipated to encounter such coincidences and lowers the E-value. This suggests that there was a significant degree of similarity between sequences, which increases confidence; if it is very close to or equal to zero, then the sequences are the same as those in Table (2). The specimen Lithobius crassipes and the specimen Bothriogaster signata had 100 % identity by 660 bits (357) and 0.0 expect, and 607 bits (672) and 1e-176 expect, 114 Bull. Iraq nat. Hist. Mus. 18 (1): 105-120. Molecular study of some species of centipedes respectively. These two samples had a position of 654 in GenBank query. The species lithopius crassipes (Family: Lithobiidae) is a new record for the Iraqi centipede fauna. Table (2): Sequencing ID in GenBank, show score, expect, and compatibility of mitochondrial DNA sequences for six centipedes’ specimens. The study's sequence specimens and the same species' deposited (COI) gene copies in the NCBI database were compared, and the results revealed a high identity of more than 80% with the same reference species with accession numbers (Tab.3). The specimen from Baghdad that was recognized as Scolopendra morsitans demonstrated identification (97%) when compared to Scolopendra morsitans ON 923747 Vietnam and KR 705665 Thailand. This specimen has a different accession number due to the nucleotide polymorphism, which is supported by the alignment matrix. Additionally, the Scolopendra cingulata specimen revealed an identity of 82% and a score of 446 consistent with 95% identity to the specimen with accession numbers JN688413 Greece, as shown in the phylogenetic tree in Diagram (1). In general, the Scolopendridae family is a genera-rich family that made its species identification using molecular characteristics an approach in alpha taxonomy. The molecular approach, which offered 97%, 100% compatibility to the genus Lithobius crassipes and Bothriogaster signata, however, revealed that the two families, Lithubiidea and Himataridae, have many similar traits and have a rich-genera distribution. The specimen cormocephalus sp. has an identity of 81% with a score of 525 bits (284), and expect 4e-144 belongs to the family Scolopendridae that contains the sample with an identity of 97 % with MZ305647 Australia. Phylogenetic tree: The similarity matrix of COI gene local sample isolates in Table (2) serves as an analysis of the data gathered. With the phylogenetic tree, a distance comparison is also shown (Diag. 1). In the phylogenetic tree of our study, we noticed that local Lithobius crassipes is grouped into one branch with the same species from Canada, France, and Specimen No. Genus- Species GenBank ID. Score (bits) Expect Identity Position in GenBank 1 Scolopendra morsitans KR705661.1 531 2e-153 82% 49-665 2 S. morsitans KR705664.1 564 2e-163 82% 34-650 3 S. cingulata JN688413.1 446 4e-126 82% 18-532 5 Lithobius crassipes KX458776.1 660 0.0 87% 50-649 7 Cormocephal us sp MZ503647.1 525 4e-144 81% 34-680 9 Bothriogaster signata graeca AY288749.1 607 1e-176 81% 31-645 https://www.ncbi.nlm.nih.gov/nucleotide/KR705661.1?report=genbank&log$=nucltop&blast_rank=1&RID=R1D14G7R016 https://www.ncbi.nlm.nih.gov/nucleotide/KR705664.1?report=genbank&log$=nucltop&blast_rank=1&RID=R1D9GGGP013 https://www.ncbi.nlm.nih.gov/nucleotide/KP648275.1?report=genbank&log$=nuclalign&blast_rank=1&RID=9ZCY2WVJ015 https://www.ncbi.nlm.nih.gov/nucleotide/KP648275.1?report=genbank&log$=nuclalign&blast_rank=1&RID=9ZCY2WVJ015 https://www.ncbi.nlm.nih.gov/nucleotide/KY587585.1?report=genbank&log$=nuclalign&blast_rank=5&RID=9ZHJRK1X015 https://www.ncbi.nlm.nih.gov/nucleotide/KY587585.1?report=genbank&log$=nuclalign&blast_rank=5&RID=9ZHJRK1X015 https://www.ncbi.nlm.nih.gov/nucleotide/JX023594.1?report=genbank&log$=nuclalign&blast_rank=1&RID=A0ZV8FNS015 https://www.ncbi.nlm.nih.gov/nucleotide/JX023594.1?report=genbank&log$=nuclalign&blast_rank=1&RID=A0ZV8FNS015 https://www.ncbi.nlm.nih.gov/nucleotide/JX023594.1?report=genbank&log$=nuclalign&blast_rank=1&RID=A0ZV8FNS015 115 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Hassan and Ali Germany, and also Bothriogaster signata is grouped into one branch with the same species from Australia. Although local species Scolopendra morsitans, S. cingulate, and unidentified species of the genus Cormocephalus sp. did not unite with its counterparts in one group, their relationships were very clear that they are close to its counterparts. We also find that this tree is very similar to the phylogenetic tree presented by Wang et al. (2022), where the relationship of order Lithobiomorpha with Geophilomorpha is closer than the relationship with Scolopendromorpha. The relationships identified in the present study are consistent with the conclusions obtained from a previous study (Hu et al., 2020; Wang et al., 2022). Diagram (1): phylogenetic tree constructed of Cytochrome Oxidase I gene sequences centipedes from different provinces in Iraq; local specimens with red marks, a measure of distance between taxa is indicated by the scale bar below the tree. 116 Bull. Iraq nat. Hist. Mus. 18 (1): 105-120. Molecular study of some species of centipedes CONCLUSIONS In the present article, 5 Chilopoda species from 3 families and 3 orders are known from Iraqi fauna. This study characterizes the first molecular study of centipedes in Iraq. The phylogenetic relationship is made by the sequence of the partial mtDNA-COI gene, and a phylogenetic tree was also constructed. The analysis resulted in a record of the species lithobius crassipes (L.koch, 1862) (Chilopoda, Lithobiomorpha, Lithobiidae) for the first time in Iraq. Our study identified a close relationship between Lithobiomorpha and Geophilomorpha. The distance comparison was also provided with the phylogenetic tree. CONFLICT OF INTEREST STATEMENT The authors have no conflicts of interest to declare. We "the authors" have followed and signed the scientific research ethics announced by the journal. LITERATURE CITED Ahmed, S. T. and Hussen, F. S. 2017. First record of two Scolopendromorph centipedes; Scolopendra subspinipes Leach (1815) and Scolopendra polymorpha Wood, (1861) with additional notes on Scolopendra cingulata Latreille, (1829) in Iraq. Zanco Journal of Pure and Applied Sciences, 28(6): 174-182. [CrossRef] Akkari, N., Stoev, P. and Lewis, J. 2008. The scolopendromorph centipedes (Chilopoda, Scolopendromorpha) of Tunisia: taxonomy, distribution and habitats. ZooKeys, 3: 77- 102. [CrossRef] Al-Doori, M. L., Azooz, H. W. and Al-Doori, N. L. 2020. A Taxonomic key for nine first record species of Chilopoda-Arthropoda in Central Iraq. Journal of Research on the Lepidoptera, 51: 596-607. [CrossRef] Al-Simary, J. A., Al-Abbad, M. Y. and Al-Qarooni, I. H. 2022. Survey and identification of centipedes (Arthropoda-Chilopoda) from Basrah Province – southern of Iraq. Annals of Forest Research, 65(1):1568-1583. [Click here] Attems, C. 1929. Die Myriopodenfauna von Albanien and Jugoslavien. Zoologische Jahrbücher, Abteilung für Systematik, Ökologie und Geographie der Tiere, 56: 269- 356. Baker, I. M., Ali, H. B. and Fadhil, H. Y. 2019. First record of the cellar spider genus Nita Huber & El-Hennawy, 2007 (Araneae, Pholcidae) from Iraq. Bulletin of the Iraq Natural History Museum, 15(4): 403-411. [CrossRef] Brolemann, H. W. 1921. Myriapods from Mesopotamia and Persia collected by Mr. PA Buxton. Journal of the Bombay Natural History Society, 28(1): 157-161. Brolemann, H. W. 1922. Myriapods collected in Mesopotamia and N.W. Persia by W. Edgar Evans, B. Sc., late Capt. R. A. M. C. Proceedings of the Royal Society of Edinburgh, 42: 54-74. http://doi.org/10.21271/ZJPAS.28.6.20 https://doi.org/10.3897/zookeys.3.51 http://dx.doi.org/10.36872/LEPI/V51I2/301119 https://www.e-afr.org/ https://doi.org/10.26842/binhm.7.2019.15.4.0403 117 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Hassan and Ali Chamberlin, R. V. 1944. Chilopods in the Collections of Field Museum of Natural History. Chicago: Field Museum of Natural History, p. 175-216. Chamberlin, R.V. and Hoffman, R. L. 1958. Checklist of the millipedes of North America. United States National Museum Bulletin, 212: 1-236. Chao, J. L., Lee, K. S. and Chang, H. W. 2010. Lithobius (Monotarsobius) meifengensis, a new species of centipede from high altitude forest in central Taiwan (Chilopoda, Lithobiomorpha, Lithobiidae). ZooKeys, 741:181-192. [CrossRef] Dobroruka, L. J. 1976 Einige Chilopoden aus Irak. Věstník Československé Společnosti Zoologické, 40(4): 259-262. Dyachkov, Y. V., Al-Yacoub, G. A. and Al-Khazali, A. M. 2023. A preliminary annotated checklist of Chilopoda from Iraq. Ecologica Montenegrina, 63: 59-78. [CrossRef] Edgecombe, G. D. and Giribet, G. 2007. Evolutionary biology of centipedes (Myriapoda: Chilopoda). Annual Review of Entomology, 52: 151-170. [CrossRef] Edgecombe, G. D. and Bonato, L. 2011. Scolopendromorpha. In: Minelli, A. (Ed.), Treatise on Zoology. Vol. 1. The Myriapoda. Brill, Leiden, p. 392-407. Edgecombe, G. D., Huey, J. A., Humphreys, W. F., Hillyer, M., Burger, M. A., Volschenk, E. S. and Waldock, J. M. 2019. Blind scolopendrid centipedes of the genus Cormocephalus from subterranean habitats in Western Australia (Myriapoda: Scolopendromorpha: Scolopendridae). Invertebrate Systematics, 33(6): 807-824. [CrossRef] Folmer, R. H. A., Nilges, M., Folkers, P. J. M., Konings, R. N. H. and Hilbers, C. W. 1994. A model of the complex between single-stranded DNA and the single-stranded DNA binding protein encoded by gene V of filamentous bacteriophage M13. Journal of Molecular Biology, 240(4): 341-357. [Click here] Ganske, A. -S, Edgecombe, G. D. and Akkari, N. 2018. The peristomatic structures as a source of systematic characters in the genus Lithobius Leach, 1814 (Myriapoda, Chilopoda). ZooKeys, 741: 49-75. [CrossRef] Haase, E. 1887. Die indisch-australischen Myriopoden. I. Chilopoden. Abhandlungen und Berichte des Königlichen Zoologischen und Anthropologisch – Ethnographischen Museums zu Dresden, 5: 1-118. Hu, C., Wang, S., Huang, B., Liu, H., Xu, L., Hu, Z., and Liu, Y. 2020. The complete mitochondrial genome sequence of Scolopendra mutilans L. Koch, 1878 https://doi.org/10.3897/zookeys.741.21036 https://doi.org/10.37828/em.2023.63.6 http://dx.doi.org/10.1146/annurev.ento.52.110405.091326 https://doi.org/10.1071/IS19015 https://www.sciencedirect.com/science/article/abs/pii/S0022283684714495 https://doi.org/10.3897/zookeys.741.21706 118 Bull. Iraq nat. Hist. Mus. 18 (1): 105-120. Molecular study of some species of centipedes (Scolopendromorpha, Scolopendridae), with a comparative analysis of other centipede genomes. ZooKeys, 925: 73-88. [Click here] Kang, S., Liu, Y. and Zeng, X. 2017. Taxonomy and identification of the genus scolopendra in china using integrated methods of external morphology and molecular phylogenetics. Scientific Reports, 7(1): 16032. Kessler, K. O. 1874. On Russian centipedes (Scolopendridae et Geophilidae). Trudy Russkago Entomologicheskago Obshchestva, 8(1): 28-45. [In Russian] Koch, L. E. 1983. Revision of the Australian centipedes of the genus Cormocephalus Newport (Chilopoda: Scolopendridae: Scolopendrinae). Australian Journal of Zoology, 31(5): 799-833. [CrossRef] Lewis, J. G. E. 1986. The genus Trachycormocephalus a junior synonym of Scolopendra, with remarks on the validity of other genera of the tribe Scolopendrini (Chilopoda: Scolopendromorpha). Journal of Natural History, 20: 1083-1088. Lewis, J. G. E. and Ëüþèñ, Ä. Ã. 2001. Scolopendromorph centipedes (Chilopoda: Scolopendromorpha) collected in northern Iraq by Dr Hywel Roberts with a review of previous records. Arthropoda Selecta, 10(2): 151-154. [CrossRef] Lewis, J. G. E. 2010. A key and annotated list of the Scolopendra species of the old world with a reappraisal of Arthrorhabdus (Chilopoda: Scolopendromorpha: Scolopendridae). International Journal of Myriapodology, 3: 83-122. [CrossRef] Oeyen, J. P., Funke, S., Bo¨hme, W., and Wesener, T. 2014. The evolutionary history of the rediscovered Austrian population of the giant centipede Scolopendra cingulata Latreille (Chilopoda, Scolopendromorpha). PLoS ONE, 9(9): e108650. [CrossRef] Sanger, F., Nicklen, S. and Coulson, A. R. 1977. DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences of the United States of America, 74(12): 5463-5467. [CrossRef] Schileyko, A. A. 2007. The scolopendromorph centipedes (Chilopoda) of Vietnam, with contributions to the faunas of Cambodia and Laos. Part 3. Arthropoda Selecta, 16(2): 71-95. [ResearchGate] Siriwut, W., Edgecombe, G. D., Sutcharit, C. and Panha, S. 2015. The centipede genus Scolopendra in mainland Southeast Asia: molecular phylogenetics, geometric morphometrics and external morphology as tools for species delimitation. PLoS One, 10(8): e0135355. https://zookeys.pensoft.net/article/47820/ https://doi.org/10.1071/ZO9830799 http://dx.doi.org/10.11646/zootaxa.3734.2.5 https://doi.org/10.1163/187525410X12578602960380 https://doi.org/10.1371/journal.pone.0108650 https://doi.org/10.1073/pnas.74.12.5463 https://www.researchgate.net/publication/292103494_The_scolopendromorph_centipedes_Chilopoda_of_Vietnam_with_contributions_to_the_faunas_of_Cambodia_and_Laos_Part_3 119 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Hassan and Ali Stoev, P. 2000. On the centipedes (Chilopoda) of Albania, 2. Arthropoda Selecta, 9: 199-206. [Click here] Wang, D. Q., and Mauriès, J. P. 1996. Review and perspective of study on myriapodology of China. Acta Myriapodologica, 169:81-99. [Click here] Vélez, S., Mesibov, R. and Giribet, G. 2012. Biogeography in a continental island: population structure of the relict endemic centipede Craterostigmus tasmanianus (Chilopoda, Craterostigmomorpha) in Tasmania using 16S rRNA and COI. Journal of Heredity, 103(1): 80-91. Wang, Y. H. M. 1963. Serica 1q: Millipedes and centipedes of Quemoy, Fukien Province and Taiwan Island, Botel Tobago (Lan Yu), Taiwan Province and of Singapore. Quarterly Journal of the Taiwan Museum, (1-2): 89-96. Wang, Y., Yin, C., Zhang, H., Kamau, P. M., Dong, W., Luo, A. and Lai, R. 2022. Venom resistance mechanisms in centipede show tissue specificity. Current Biology, 32(16): 3556-3563. [CrossRef] Würmli, M. 1975. Revision der Hundertfüsser-Gattung Thereuonema (Chilopoda: Scutigeridae). Entomologica Germanica, 19: 189-196. [CrossRef] Zapparoli, M. 1991. Note su alcune specie di Chilopodi della regione palestinese. Fragmenta Entomologica, Roma, 23(1): 15-33. Zapparoli, M. 1993. The present knowledge of the centipede fauna of Anatolia (Chilopoda). Biogeographia–The Journal of Integrative Biogeography, 20(1): 105-177. [CrossRef] Zapparoli, M. 1995. Geophilomorpha from Israel. In: Nitzu, E., Nitzu, E., Por, F.D., Dimentman, Ch., Decu, V. (eds.). Soil fauna of Israel. I. Bucharest, p.41-46. Zapparoli, M. 2006. A catalogue of the centipedes (Chilopoda) of Central Apennines (Italy). Bollettino del Museo Civico di Storia Naturale di Verona, Botanica Zoologia, 30: 165- 273. Zarei, R., Rahimian, H., Mirmonsef, H. and Bonato, L. 2020. Geophilomorpha from Alborz Mountains and a checklist of Chilopoda from Iran. Zootaxa, 4780(1): 132-146. [CrossRef] https://www.gbif.org/publisher/7ce8aef0-9e92-11dc-8738-b8a03c50a862 https://www.semanticscholar.org/paper/Review-and-perspective-of-study-on-myriapodology-of-Wang%20Mauri%C3%A8s/742be72b23e27bd9c892f90f48d61125372060d9 https://doi.org/10.1016/j.cub.2022.06.074 https://doi.org/10.1127/entom.germ/2/1975/189 https://doi.org/10.21426/B6110130 https://doi.org/10.11646/zootaxa.4780.1.6 120 Bull. Iraq nat. Hist. Mus. 18 (1): 105-120. Molecular study of some species of centipedes Bull. Iraq nat. Hist. Mus. (2024) 18 (1): 105-120. ( في العراق مع تسجيل Centipedesدراسة جزيئية لبعض انواع مئوية االقدام ) Lithobius Crassipes L. Koch, 1862جديد للنوع (Chilopoda, Lithobiomorpha, Lithobiidae) شيماء حسين حسن و حيدر بدري علي لعلوم, جامعة بغداد, بغداد,العراق.اقسم علوم الحياة, كلية 20/6/2024، النشر: 28/12/2023القبول: ، 7/12/2023املراجعة: ، 20/8/2023الم: االست الخالصة جمعت قدمت هذه الدراسة أول عمل جزيئي حول حيوانات مئويات األقدام في العراق. 2022محافظات عراقية للفترة ما بين تشرين األول 4مئويات األقدام من تجمعات مختلفة في عائالت ممثلة بـاربع 3في ظروف مناخية مختلفة. تم إجراء التعريف الجزيئي لـ 2023ومايو Scolopendra morsitans كل من:, سجل Scolopendromorphaمن رتبة أنواع. 5 وأجناس Linnaeus, 1758 وS. cingulata Latreille, 1829 و Cormocephalus sp. , سجل ألول كما , Geophilomorpha. ومن رتبة ,1862Lithobius crassipes (L. Koch) مرة في العراق النوع استخلص الحمض النووي من .Bothriogaster Signata Kessler 1874سجل نوع واحد وهو Cytochrome C Oxidase Subunit I (COI ), من جين mtDNAالعينات , وضخم جزء باستخدام البادئ املحدد, وبعد ذلك تم تطبيق برنامج أداة املحاذاة املحلية األساسية (BLAST التابع للموقع )NCBIرسمت شجرة النشوء والتطور باستخدام مقارنة املسافة ..