Bull 291 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Bashê, S. K. Bull. Iraq nat. Hist. Mus. (2022) 17 (2): 291-301. https://doi.org/10.26842/binhm.7.2022.17.2.0291 ORIGINAL ARTICLE DISTRIBUTION AND PHYLOGENETIC OF FRESHWATER MUSSEL UNIO TIGRIDIS BOURGUIGNAT, 1852 (BIVALVIA, UNIONIDAE) FROM GREATER ZAB RIVER, IRAQ Shwan Khursheed Bashê Biology Department, Education College, Salahaddin University-Erbil, Erbil, Iraq E-mail: shwan.raman@su.edu.krd Recived Date: 27 August 2022, Accepted Date 16 November 2022, Published Date:20 December 2022 This work is licensed under a Creative Commons Attribution 4.0 International License ABSTRACT Freshwater mussels are a guild of stationary, suspended-feeding species; they perform significant ecological functions like nitrogen cycling, bioturbation that gives oxygen and habitat that other creatures need to survive, and increasing water clearance by filtration. Knowledge of the freshwater mussel Unio tigridis Bourguignat, 1852, distribution, and molecular study in Iraq was inadequate. In the current study, this species of freshwater Mussels belonging to the family Unionidae was collected from different locations in the Greater Zab River, from April to August 2022. The average water temperature of the site was arranged between (17.8 to 36.1 C°). All previous studies in the Kurdistan Region and Iraq were based on morphological characters and the current study was the first report of Unio tigridis that was confirmed by molecular genetics and COI gene, analyzed phylogenetically using Maximum Likelihood and Maximum Parsimony Methods. Keywords: COI, Distribution, Phylogenetic, Unio tigridis, Greater Zab, Iraq INTRODUCTION Freshwater mussel Unio tigridis Bourguignat, 1852 (Mollusca: Bivalvia: Unionidae) are a guild of stationary, suspended-feeding species that play vital roles in rivers and streams (Bolotov et al., 2019; Hamli and Al Asif, 2021; Hamli et al., 2021). Its essential aquatic ecosystem constituents, which perform significant ecological functions like nitrogen cycling, bioturbation that gives oxygen and habitat that other creatures need to survive, and increasing water clearance by filtration (Zieritz et al., 2016; Klishko et al., 2017; Lopes-Lima et al., 2021). The Unionidae is the richest of the six extant freshwater mussel families, there are currently 674 species known globally, with (80%) of them being widely dispersed over temperate North America and Eurasia in addition to tropical Mesoamerica, Africa, and southeast Asia (Huber, 2010, 2015). According to the International Union for Conservation of Nature's (IUCN) assessment of the majority of freshwater mussel species (517), 6% extinct, 7% vulnerable, 9% 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.7.2022.17.2.0291 https://orcid.org/0000-0002-9043-8190 mailto:shwan.raman@su.edu.krd https://creativecommons.org/licenses/by/4.0/ https://jnhm.uobaghdad.edu.iq/index.php/BINHM/Home 292 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Distribution and phylogenetic of freshwater mussel near threatened, 10% endangered, 13% critically endangered and 37% of least concern(IUCN, 2016). Freshwater mussel descriptions from the early 19 th century up till the mid-20th century included hundreds of Unio species described from many regions of the world (Huber, 2015). U. tigridis Bourguignat, 1852, the most prevalent species, found from Syria to Iraq, had been evaluated by the IUCN Red List and reported as Least Concern (LC), containing an elongate solid shell buried obliquely in the mud (Plaziat and Younis, 2005). In Iraq, the first recorded of this species in Habbaniya Lake by Najim (1959), in Euphrates by Al-Bassam (2005), and in the Greater Zab River by Ali (2007). Previously, Unionid mussels' species depended on shell traits for identification before the development of molecular technology (Lopes-Lima et al., 2017). However, it depending alone on shell morphology found difficult, due to the morphological convergences between species and the high level of phenotypic variability within species (Klishko et al., 2017). Several newly released studies that used region of cytochrome oxidase subunit I gene of mitochondrial DNA (mt DNA-COI) for the biogeography and relationships of aquatic mussels in various Asiatic regions fundamentally altered our knowledge of diversity patterns (Choi, 2016; Lopes-Lima et al., 2020). Unfortunately, no recent investigation had been carried out on molecular phylogenetic data for any endemic Iraqi invertebrates especially freshwater mussels. The present investigation was conducted to study the utility of a molecular technique as a DNA barcode approach for the identification, distribution, and estimate of a phylogeny of U. tigridis, the freshwater mussels from the Greater Zab River. MATERIALS AND METHODS Specimens' collection and identification Freshwater mussels (No. 33) were collected and gathered by hand from various sites along the banks of canals, and irrigation ditches from Greater Zab River, West Erbil province, Iraq (Map 1), from April to August 2022. The water temperature was measured month wise using a temperature probe/ thermometer (HANNA instruments, HI 9811-5 for Temp., pH, EC and TDS-Romania). The collected mussel specimens brought alive and a dead shell to the laboratory. Selection has been made for the complete animals (shell not broken or eroded). The bivalves were boiled gently in 5% sodium hydroxide to be cleaned, then washed with water and dried for measurements (Abdul-Sahib and Abdul-Sahib, 2009). Morphologically, U. tigridis exhibit the traditional wedge-shaped shell used as diagnosed species (Lopes-Lima et al., 2021). DNA extraction, COI amplification, and Sequencing A biopsy was fixed at 99% ethanol and centrifuged 10,000 rpm for 3 minutes, then the ethanol was eliminated, the DNA was extracted using a commercial kit (Favorgen- Taiwan) in accordance with the manufacturer's instructions. The DNA barcoding for mussel was achieved based on amplification and sequencing of the region of (mtDNA-COI) (~700 bp) (Folmer et al., 1994), using forward primer LCO1490 and reverse HCO2198 primers. The PCR mixture (40 μl) contained: (a master mix, DNA template, 293 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Bashê, S. K. primers, and double-demonized water (ddH2O)). Under the following circumstances, PCR was carried out in a MJ Research, Applied Biosystem (AB) thermocycler: 94 C°/4 min; 35 cycles of 94 C°/30 sec; 48 C°/60 sec; 72 C°/90 sec, followed by one cycle of 72 C°/5 min. In order to see DNA under UV light, PCR products were separated on a 1.3% agarose gel and dyed with a safe dye (Bashê and Ali, 2019). DNA sequencing was performed by using an ABI 3730XLs nucleotide sequence analyzer through Macrogen Inc. (Korea). The sequences were subjected to the Basic Local Alignment Search Tool for nucleotides (Blastn) implemented in the NCBI GenBank database. In addition, 11 sequences were obtained from GenBank that available on the website: http://www.ncbi.nlm.nih.gov/genbank/. All the DNA sequences were edited and aligned with (ClustalW algorithm), available in the MUSCLE program within EMBL-EBI (https://www.ebi.ac.uk/Tools/msa/muscle/). Further estimates of COI variation and relationships among species by applying Maximum Likelihood (Kimura 2-parameter model) (Kimura, 1980), and using the method of Maximum Parsimony Branches associated with partitions that were replicated in fewer than 50% of bootstrap replicates are collapsed. In the bootstrap test (1000 replicates), the proportion of duplicate trees in which the linked taxa clustered together is displayed next to the branches (Felsenstein, 1985). The Tree-Bisection-Regrafting (TBR) technique was used to create the MP tree with search level 1 and the initial trees were created by randomly adding sequences (10 replicates) (Nei and Kumar, 2000). This analysis involved 12 nucleotide sequences; there were a total of 498 positions in the final dataset edited by Finch Tv program (version 1.4.0) and deposited in NCBI. Methods were conducted using MEGA X Kumar et al. (2018), including a sequence representative from many species currently present and available on GenBank. Map (1): Sampling sites on the Greater Zab River, north of Iraq. http://www.ncbi.nlm.nih.gov/genbank/ https://www.ebi.ac.uk/Tools/msa/muscle/ 294 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Distribution and phylogenetic of freshwater mussel RESULTS Mitochondrial DNA COI from freshwater mussels was sequenced, the results (100%) confirmed the present U. tigridis (Pl. 1), the shell solid and elongate lying obliquely in the mud, with its rear end extending from the bottom in pond, marshes channel, and rivers. The average temperatures from April-October 2022 were arranged (17.8-36.1 C°). The obtained sequencing result was deposited in GenBank database under the following accession numbers (OP087400) for U. tigridis. Plate (1): Unio tigridis, collected from Greater Zab River; (A) External shell, (B) Bivalve shell, (C) Visceral parts (Scale bar=2 cm). In Phylogeny, Kimura 2 parameter (K2P) was used for intraspecific and interspecific variations. The pairwise distances between the Unio are shown in Table (1). Intraspecific genetic diversity within U. tigridis was from zero to (0.005), within U. Delphinus (Reis et al., 2013), from zero to (0.042) and C. elongatulus (Araujo et al., 2018), was zero to (0.040). The maximum genetic distance observed Margaritifera homsensis was (0.228). Table (1): Estimates of evolutionary divergence sequences between and within U. tigridis species (Overall standard deviation is 0.14 with standard error 0.01). No. Species 1 2 3 4 5 6 7 8 9 10 11 1 U. tigridis (current study) 0.000 2 U. tigridis MZ511105.1 0.001 3 U. tigridis MZ511087.1 0.005 0.001 4 U. delphinus EF571442.1 0.040 0.036 0.038 5 U. delphinus EF571415.1 0.042 0.038 0.040 0.001 6 U. elongatulus KX399981.1 0.040 0.036 0.038 0.044 0.046 295 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Bashê, S. K. DISCUSSION The current work was the first to analyze the biogeographic and phylogenetic of U. tigridis freshwater mussels from the Greater Zab River, and it might be served as a baseline study for the investigated species and for the other available mussel species as well. Our result showed that, in the both tree maximum likelihood and parsimony, that U. tigridis was monophyletic in the same clade with (MZ511087.1) which was reported from the far East Asia by Lopes-Lima et al. (2021) (Diag. 1); but this topology might be changed with the addition of more loci, also had strongly related to (MZ511105.1); Which were also related and matched to the other deposited sequences, and the outgroup Margaritifera homsensis (KX550090.1) recorded by Vikhrev et al. (2018) that was found more divergence species with U. tigridis. Diagram (1): Both the Maximum Likelihood (Kimura 2-parameter model) and Maximum Parsimony methods were applied to infer the evolutionary history, all nodes supported by high bootstrap (ML/MP). 7 U. pictorum EU548056.1 0.044 0.040 0.042 0.040 0.042 0.034 8 U. pictorum KY930353.1 0.774 0.779 0.785 0.762 0.768 0.839 0.812 9 U. foucauldianus KX399998.1 0.042 0.038 0.040 0.023 0.025 0.038 0.038 0.778 10 U. foucauldianus KX400006.1 0.042 0.038 0.040 0.023 0.025 0.038 0.038 0.778 0.00 11 Margaritifera_homsensis KX550090.1 0.228 0.222 0.219 0.211 0.214 0.228 0.222 0.014 0.212 0.212 296 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Distribution and phylogenetic of freshwater mussel According to species divergence, the COI gene was frequently employed as a barcode since it has been subject of the most research and is considered as a standard for analyzing divergence among Unionidae species (Araujo et al., 2018) (Tab. 1). Notably, in the phylogenetic tree of U. tigridis, the rebuilt species tree coincides with the general topology of the concatenated matrix studies. DNA barcoding provided a remarkably valuable approach for confirming the identity of a species (Davison et al., 2009). The sequenced data presented in the current study were derived from the COI gene; it was currently one of the most widely used for phylogeny, systematics, and species identification. At present, there were many reference DNA barcode sequences belonging to Gastropod species available in GenBank which could be used for identification purposes. To date, there has been no study in Iraq regarding the molecular and phylogenetic of this investigated species. Nevertheless, the conclusions of this single-locus or multilocus DNA species delimitation algorithms could not be directly converted into new taxonomies. The Unionidae have frequently been recovered as paraphyletic by phylogenetic analysis throughout the last decade (Graf and Cummings, 2006). However, their findings were unsubstantiated and based on insufficient taxon and character sampling (Whelan et al., 2011); only two molecular character sets had been used to assess Unionoida family-group level associations through COI gene (Whelan et al., 2011), Other Unionidae phylogenetic analysis had relied on specific gene region. While, Lopes-Lima et al. (2017) hypothesized the subfamily's evolutionary connection based on COI. The worldwide distribution and diversity of mussels species is to several reasons considering; habitat destruction, degradation, and alteration caused by increasing populations of human, industrialization, and changes in land use in addition to spread of non-native exotic species (Zieritz et al., 2016 - Hamli et al., 2021), enrichment water with nutrients resulting in phytoplankton and macrophyte blooms (Sharip and Zakaria, 2007), and heavy metal concentrations often reach deadly thresholds for freshwater mussels (Nobles and Zhang, 2015). According to the present findings, water temperatures fluctuated ranges between (17.8 to 36.1 C°), this result was similar to Payton et al. (2016) in USA, which recorded highest temperatures in summer average (35 to 40 C°) during particularly hot weather, which was near or exceeds the high temperature tolerant ranges of several aquatic organisms, and substantial changes in temperature might had an impact on the balance of ecosystem. That results in species-specific variations in sensitivity to factors including physiological tolerance, limited adaptability to abiotic stresses, and life history aspects. In another way, observed an increase of only ~3°C above normal water temperatures was enough to cause 33% mortality. CONCLUSIONS In the current study, the first report based on molecular technique COI for confirmation diagnosis of Unio tigridis, distribution along Greater Zab River, and phylogenetic methods (Maximum Likelihood and Maximum Parsimony) with other species. 297 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Bashê, S. K. AKNOWLEDGMENTS I wish to thanks Dr. Rebwar Hamasalih, Ph. D. in Molecular Biology, for his valuable help and to Biology department for facilitating way for conducting this research. CONFLICT OF INTEREST STATMENT "The author has no conflict of interest to declare." LITERATURE CITED Abdul-Sahib, I. and Abdul-Sahib, E. 2009. 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(2022) 17 (2): 291-301. يه العبةةاملبلح ل العالقة النشوئية التوزيع و UNIO TIGRIDIS BOURGUIGNAT, 1852 (BIVALVIA, UNIONIDAE) بير، العراقكمن نهر الزاب ال شوان خورشيد ةهش ى .، أربيل، العراقأربيل-قسم علوم الحياة، كلية التربية، جامعة صالح الدين 20/12/2022، تأريخ النشر: 16/11/2022القبول: ، تأريخ 27/8/2022تأريخ االستالم: الخالصة في املياه العذبة، بلح البحر عبارة عن طائفة من األنواع الثابتة واملعلقة التي تتغذى بيئية مهمة مثل دورة النيتروجين ، واالضطراب الحيوي الذي يمنح التي تؤدي وظائف خرى للبقاء على قيد الحياة ، وزيادة األكسجين واملوائل التي تحتاجها الكائنات األ Unio tigridisتصفية املياه عن طريق الترشيح. كانت املعرفة لنوع بلح املياه العذبة Bourguignat, 1852 .التوزيع، والدراسة الجزيئية في العراق غير كافية لتحديد النوع ، ذي ينتمي إلى عائلة الو في الدراسة الحالية، جمعت عينات هذا النوع من بلح البحر Unionidae شهر املياه العذبة لعدد من املواقع املختلفة في نهر الزاب األكبر، من في إلى 17.8درجة حرارة املاء في املوقع مابين . كانت متوسط 2022 بآإلى 2022 نيسان م36.1 o . ملظهرية، استندت جميع الدراسات السابقة في إقليم كوردستان والعراق إلى الصفات ا استنادا الى U. tigridisفي حين ان الدراسة الحالية كانت أول تقرير عن تشخيص النوع ، حيث تم تحليل النشوء والتطور باستخدام طرق الحد COIجين الوراثة الجزيئية و .Parsimonyاألقص ى من االحتمالية والحد األقص ى من