University of Isfahan Taxonomy and Biosystematics 3115-9001 17 63 2025 08 23 Genetic diversity of Namak chub: Squalius namak (Actinopterygii: Leuciscinae) in the Jajrood River using microsatellite markers تنوع ژنتیکی ماهی سفید رودخانه‌ای نمک (Squalius namak) (شعاع بالگان: سفید ماهیان) در رودخانه جاجرود با استفاده از نشانگرهای ریزماهواره 1 16 29774 10.22108/tbj.2025.144493.1299 EN Hamed Shabanloo Ph. D. Graduated, Department of Fisheries, Faculty of Natural Resources, University College of Agriculture & Natural Resources, University of Tehran, Karaj, Iran Hadi Poorbagher Professor, Department of Fisheries, Faculty of Natural Resources, University College of Agriculture & Natural Resources, University of Tehran, Karaj, Iran Soheil Eagderi Associate Professor, Department of Fisheries, Faculty of Natural Resources, University College of Agriculture & Natural Resources, University of Tehran, Karaj, Iran Journal Article 2025 03 17 The Namak chub (Squalius namak) is an endemic species of the inland waters of Iran. However, no study has been conducted on the structure and genetic diversity of its populations. To address this, 103 Namak chub (S. namak) were sampled from four stations in the Jajrood River, and five microsatellite loci (Sluc4, Sluc5, Sluc7, Sluc13, and SarN2) were used. GenAlEx and CERVUS genetic software were used to analyze the data. The number of alleles ranged from 2 to 37 (average 12.43), and expected heterozygosity ranged from 0 to 0.95 (average 0.74), both of which were higher than the averages typically reported for freshwater fish. The average FST index for genetic differentiation was estimated to be 0.19, and the mean gene flow (Nm) was 1.30. Analysis of molecular variance revealed high genetic diversity within populations and low diversity among them. All loci exhibited a pattern of deviation from Hardy–Weinberg equilibrium (HWE), likely due to the use of non-specific primers, the presence of null alleles, and possibly small sample sizes. According to the findings of the present study, the genetic diversity observed in the populations of the Namak chub at these four sites was high, and considering the relatively low level of gene flow and the high FST, there appears to be significant differentiation among the populations of this species in the studied areas. These results can support efforts in biodiversity conservation and management planning. Keywords: Population genetics, Genetic differentiation, Microsatellite, Chub, Molecular marker. Introduction The genetic diversity of a species determines its adaptive capacity and evolutionary potential. Small populations of species with restricted distributions often have low genetic diversity within populations but high genetic differentiation between populations due to genetic drift and limited gene flow (Zhai et al., 2019). Genetic diversity is now a concern in many conservation programs, as low genetic diversity is associated with increased inbreeding, accumulation of deleterious mutations, and reduced adaptive potential (McCusker & Bentzen, 2010). Microsatellite loci are standard genetic markers used for population genetic analysis (Coates et al., 2009). In freshwater ecosystems, fish biodiversity is a good indicator of ecosystem health (Zhou et al., 2024). Freshwater fish species constitute 40% of the world's total fish diversity and are one of the most endangered animal groups (Costa et al., 2021). In terms of diversity and endemism, Iran has a high richness of freshwater fish. According to the latest studies, a total of 300 fish species belonging to 38 families and 110 genera live in the inland waters of Iran (Sayyadzadeh & Esmaeili, 2024). The genus Squalius is one of the important genera in the family Leuciscidae, with five species of this genus identified in the inland waters of Iran (Sayyadzadeh & Esmaeili, 2024), one of which is the Namak chub (Squalius namak Khaefi et al., 2016). This endemic species was recently described and as a result, few studies have been conducted on it (Mouludi-Saleh & Keivany, 2018). Materials & Methods The study area was the Jajroud River, one of the important aquatic ecosystems in the northeast of Tehran (Naderi et al., 2020). Using a Samus 725 electrofishing device, 103 S. namak were collected. Fish were anesthetized using a clove oil solution. Then, caudal fin clips were obtained from the samples and transferred into the labeled tubes containing 96% ethanol. DNA was extracted from the fin tissue using the phenol-chloroform method (Sambrook & Russel, 2001). PCR reaction was used to amplify five pairs of primers for this work. Since no specific primer was designed for this species, the primers from related species were used. For better separation of PCR products, electrophoresis on 8% polyacrylamide gel was used. Data obtained from AlphaEaseC software were first transferred to Excel. Then genetic indices such as number of alleles per locus (Na), number of effective alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), degree of differentiation (FST) and gene flow (Nm) were calculated using GenAlEx 6.5 software (Peakall & Smouse, 2006). Also, to determine the genetic diversity within and between populations based on FST and RST, the analysis of molecular variance (AMOVA) in the GenAlEx software package was used. Finally, principal component analysis (PCA) was performed to examine the genetic relationship of the four populations and the Hardy-Weinberg equilibrium test for the four loci used with Bonferroni correction in CERVUS 3.0.7 software. Research Findings The total number of alleles at the loci level ranged from 2 to 37, with the Sluc5 locus showing the highest number of alleles (37) and the Sluc4 locus showing the lowest number (2). The average number of alleles per locus was 10.25 in the Khojir 1 population, 26.25 in the Khojir 2 population, 11.75 in the Jajrood 1 population, and 1.5 in the Jajrood 2 population. The average observed heterozygosity in the Khojir 1 population was 0.65, in the Khojir 2 population was 0.68, in the Jajrood 1 population was 0.51, and in the Jajrood 2 population was 0.75. Expected heterozygosity values (He) ranged from 0.50 to 0.96 (average across all four populations: 0.74). At the population level, the highest and lowest average values were 0.94 and 0.37, observed in the Khojir 2 and Jajrood 2 populations, respectively. At the locus level, the degree of differentiation (FST) and gene flow (Nm) were calculated, and the average gene flow among the populations was 1.30, with the highest value observed at the Sluc5 locus (1.93) and the lowest at the SarN2 locus (0.49). The average genetic differentiation was 0.19, with the highest differentiation observed at the SarN2 locus (0.33). AMOVA based on RST indicated that 93% of the observed genetic variation was related to differences among individuals within populations, and 7% was due to differences among the four studied populations. The chi-square test with Bonferroni correction, used to examine Hardy–Weinberg equilibrium, showed that all loci deviated from equilibrium. Discussion of Results & Conclusion In this study, the average heterozygosity observed in the four populations was 0.64, which is higher than the values observed in freshwater fish (0.46) (Hedayati et al., 2017). If heterozygosity values are higher than the average, the level of genetic diversity is considered high; if they are lower, genetic diversity in that population is reduced (Farasati et al., 2020). The average total number of alleles for the four populations was 12.43, which was higher than the value calculated for freshwater fish (7.5) (Hedayati et al., 2017). This value is strongly affected by the number of samples (Gorjipoor & Nazari, 2014). In the present study, the average gene flow rate was 1.30, and the reason for the high genetic diversity observed in this species can be attributed to the existence of gene flow between populations. When Nm < 1, gene flow is the dominant factor causing differentiation, and when Nm > 1, genetic drift is the main factor causing genetic differentiation (Karami Nasab et al., 2014). However, high gene flow values do not necessarily guarantee genetic diversity, because a low number of parents and populations, and consequently interbreeding among small populations, can reduce diversity (Farasati et al., 2020). However, the number obtained in this study does not represent all real populations. Another point is that the FST rate does not reach unity in most cases because the effect of polymorphism (caused by mutations) effectively reduces the FST rate (Silavi et al., 2024). Based on the available analyses, it appears that the S. namak species exhibits favorable genetic diversity in the studied areas. Given the ecological importance of this endemic species in rivers, preserving its genetic diversity is deemed necessary and important.

https://tbj.ui.ac.ir/article_29774_d0e742452abf5dc188f9cf754b6c4978.pdf

University of Isfahan Taxonomy and Biosystematics 3115-9001 17 63 2025 08 23 Molecular identification of some Onychiuridae (Collembola: Poduromorpha) species from Iran شناسایی مولکولی برخی از گونه‌های پادمان خانواده Onychiuridae (Collembola: Poduromorpha) در ایران 17 28 29502 10.22108/tbj.2025.143520.1288 EN Maryam Salimi MSc. Student, Department of Plant Protection, Faculty of Agricultural Sciences, Sari University of Agricultural Sciences and Natural Resources (SANRU), Sari, Iran Masoumeh Shayanmehr Associate Professor, Department of Plant Protection, Faculty of Agricultural Sciences, Sari University of Agricultural Sciences and Natural Resources (SANRU), Sari, Iran Mahmoud Mohammadi Sharif Associate Professor, Department of Plant Protection, Faculty of Agricultural Sciences, Sari University of Agricultural Sciences and Natural Resources (SANRU), Sari, Iran Elham Yoosefi Lafooraki Assistant Professor, Department of Plant Protection, Faculty of Agricultural Sciences, Sari University of Agricultural Sciences and Natural Resources (SANRU), Sari, Iran Journal Article 2024 11 27 The Onychiuridae family, belonging to the order Poduromorpha within the class Collembola, consists of soil-dwelling species characterized by the absence of pigmentation, ommatidia, and furca. These traits make them challenging for morphological identification. Utilizing molecular markers as a complementary approach can facilitate species identification. To date, 34 species from 14 genera have been reported from this family, with no molecular data available for the country. The present study was conducted to perform a molecular analysis of several species collected from Gilan and Isfahan provinces, using a segment of the mitochondrial gene (COI). As a result, four genera and six species from the subfamily Onychiurinae were identified based on molecular data, and the species' gene sequences were registered in NCBI. The results demonstrated that this gene was highly effective in identifying species within the Onychiuridae family. The monophyletic status of the studied genera was confirmed through molecular data. Additionally, the morphological features used to differentiate the studied genera within this family were validated by the gene. Keywords: COI gene, DNA barcoding, Onychiurinae. Introduction The order Poduromorpha, comprising eight families, represents the most diverse and abundant group of arthropods within the class Collembola (Bellinger et al., 2025). Among these, the family Onychiuridae with 53 genera and 765 species worldwide ranks as the second-largest. The first record of Onychiuridae in Iran dates back to the work of Cox (1982). Subsequent studies, including a checklist by Shayanmehr et al. (2013), added only two species to Cox’s initial findings. However, recent years have seen increased research interest, leading to the discovery of several new species (Arbea & Kahrarian, 2017; Kaprus’ et al., 2017; Yahyapour et al., 2020; Ahmadi et al., 2023). According to the latest checklist of Poduromorpha in Iran, the family Onychiuridae now includes 34 species from 14 genera (Shayanmehr et al., 2023). Accurate species identification within Onychiuridae remains challenging due to incomplete taxonomic keys, a shortage of specialists, and logistical difficulties in sending samples abroad. To overcome these limitations, DNA barcoding can serve as a valuable complement to traditional morphological methods. Recent advances in molecular biology have greatly enhanced Collembola systematics, with mitochondrial and nuclear genomes providing key insights into evolutionary relationships (Carapelli et al., 2005; Porco et al., 2010; Pan, 2015; Zhang et al., 2017; Mateos et al., 2018; Potapov et al., 2018; Winkler et al., 2020). Given the scarcity of molecular data on Onychiuridae in Iran, this study aims to contribute molecular identifications of some species from this family. Material and Methods Springtail specimens were collected from soil and leaf litter across multiple sites in Guilan and Isfahan Provinces between 2017 and 2021. Arthropods were extracted using Berlese funnels, and Onychiuridae specimens were isolated and preserved in 70% ethanol at −20°C. For microscopic examination, specimens were cleared in 10% KOH for 2–3 minutes and mounted in Hoyer’s medium (Christiansen, 1957). Species identification was performed using established taxonomic keys (Fjellberg, 1998; Yahyapour et al., 2020). Genomic DNA was extracted using the SinaClon extraction kit, following the manufacturer’s instructions. The mitochondrial COI gene was amplified via PCR using primers described by Folmer et al., (1994), in a 25 µL reaction mixture consisting of 15.6 µL ddH₂O, 3 µL template DNA, 2.5 µL PCR buffer, 0.5 µL dNTPs (15 mM), 1 µL MgCl₂ (25 mM), 1 µL of each primer (10 µM), and 0.4 µL Taq polymerase (5 U/µL, SinaClon). The thermal cycling protocol included an initial denaturation at 95°C for 15 minutes, followed by 5 cycles at 96°C for 30 seconds, 48°C for 35 seconds, and 72°C for 40 seconds, then 30 cycles at 96°C for 30 seconds, 51°C for 35 seconds, and 72°C for 40 seconds, with a final extension at 72°C for 6 minutes. Forward strand Sanger sequencing was performed by Microsynth (Switzerland). The obtained sequences were aligned in MEGA version 6 (Tamura et al., 2013), and genetic divergence was calculated. A Neighbor-Joining phylogenetic tree with 10,000 bootstrap replicates was constructed using IQ-TREE version 1.6.12, incorporating both newly obtained sequences and GenBank references (Nguyen et al., 2015). Research findings In this study, four genera and six species belonging to the subfamily Onychiurinae were collected and identified as follows: Heteraphorura iranica Kaprus’ et al., 2017, Orthonychiurus stachianus (Bagnall, 1939), Protaphorura fimata (Gisin, 1952), P. levantina (Christiansen, 1956), P. sakatoi (Yosii, 1966), and Thalassaphorura encarpata (Denis, 1931). The species T. encarpata, H. iranica, and P. levantina represent new records for Gilan Province, while P. sakatoi is reported from Isfahan Province for the first time. Eight COI gene sequences from the family Onychiuridae were obtained, corresponding to H. kaprusi, H. cf. iranica, and P. cf. sakatoi. These sequences were deposited in GenBank under accession numbers OR887479–OR887484 and OR887488–OR887489, marking the first submissions for these species. A phylogenetic tree was reconstructed using the Neighbor-Joining method with 10,000 bootstrap replicates, combining the eight new sequences with 17 reference sequences from GenBank (Figure 1). The analysis included representatives of Heteraphorura, Thalassaphorura, Protaphorura, and Deuteraphorura, all of which formed well-supported monophyletic clades. Deuteraphorura species were placed as a sister group to the other genera, distinguished by the absence of a terminal spine on abdominal segment VI. The COI gene provided strong species-level resolution, with 100% bootstrap support for all Onychiuridae lineages, including Heteraphorura sp., H. cf. iranica, Thalassaphorura biquatemata, and Protaphorura spp. Discussion of Results & Conclusion Our investigation of the Onychiuridae springtail fauna in Guilan and Isfahan provinces yielded four genera and six species. We obtained eight COI sequences representing two genera (Heteraphorura and Protaphorura), including the first submissions to GenBank for Heteraphorura kaprusi, H. cf. iranica, and Protaphorura cf. sakatoi. The COI-based phylogenetic tree strongly supported the monophyly of the four examined genera (Heteraphorura, Thalassaphorura, Protaphorura, and Deuteraphorura), and key morphological diagnostic characters were confirmed by molecular data. The COI marker proved effective for species-level identification within Onychiuridae. This study represents the first molecular investigation of this family in Iran. Given the current scarcity of molecular data on Iranian Onychiuridae, it is necessary to conduct further studies using COI gene sequences for other species of this family, as well as additional molecular markers. These efforts will help clarify interspecific relationships and enhance our understanding of the diversity of this family in Iran. Acknowledgment This study presents a part of the first author's M.Sc. thesis conducted at Sari University of Agricultural Sciences and Natural Resources. The authors gratefully acknowledge the financial and institutional support that made this research possible.

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University of Isfahan Taxonomy and Biosystematics 3115-9001 17 63 2025 08 23 Investigation of the diversity of morphological characters and their efficiency in the classification of the genus Berberis L. in Semnan Province بررسی تنوع صفات مورفولوژیکی و کارایی آن در رده‌بندی جنس زرشک (Berberis L.) در استان سمنان 29 50 29789 10.22108/tbj.2025.145544.1310 EN Najmeh Sadat Tabatabaei Qomi PhD. Student, Department of Biology, Faculty of Science, Payam Noor University, Tehran, Iran Gholamreza Bakhshi Khaniki Professor, Department of Biology, Faculty of Science, Payam Noor University, Tehran, Iran Seyed Majid Toliat Abolhasani Assistant Professor, Iranian Biological Resources Center, ACECR, Karaj, Iran Majid Ghorbani Nohooji Associate Professor, Medicinal Plants Research Center, Institute of Medicinal Plants, ACECR, Karaj, Iran Journal Article 2025 06 10 To investigate morphological diversity, 10 populations of the medicinal plant Berberis L. were collected from different regions of Semnan Province, and 34 morphological traits were analyzed. Significant differences at the 0.05 level were detected among several measured traits. The highest average raceme length (5.20 cm), number of berries per raceme (19), fruit length (1.01 cm), and thorn length (2.28 cm) were recorded in the Nekarman population. In contrast, the lowest average thorn length (1.12 cm), raceme length (1.45 cm), and number of berries per raceme (9) were recorded in the Abar village population. Principal component analysis (PCA) indicated that the first and second components together accounted for 59.06% of the total variance. Clustering and ordination analyses separated the populations into three groups. The first group included the Tazreh, Majan, Nekarman, and Mehmandoyieh populations, which are probably attributable to the hybrid species Berberis integerrima × orthobotrys. The second group comprised the Abar village and Abar forest populations, most likely belonging to B. orthobotrys. The third group included the Aftar, Shahmirzad, Tarom, and Kamand populations, which are probably attributable to the hybrid species B. integerrima × crataegina. The observed variation in morphological traits reflects taxonomic diversity, and these traits can therefore be employed in the preparation of determination keys and in the classification of the genus Berberis. Keywords: Medicinal plants, populations, classification, morphology, hybrids. Introduction The genus Berberis L. is a medicinal and ornamental shrub in Iran and is classified within the Berberidaceae family (Mozaffarian, 2013). A comprehensive biosystematic study of this genus, based on morphological, cytological, palynological, and molecular evidence, was carried out in the provinces of North, South, and Razavi Khorasan on numerous specimens preserved in the herbaria of Ferdowsi University of Mashhad (FUMH) and the Forest and Rangeland Research Institute (TARI). In that study, three species, B. orthobotrys, B. integerrima, and B. crataegina, along with four unidentified taxa that were probably hybrids, were recognized. Two of these species were reported for the first time in the study area (Sodagar et al., 2012). Furthermore, in a separate floristic study conducted in the Hezar Jerib Protected Area (between Semnan and Mazandaran provinces), two species of this genus, namely B. orthobotrys and B. integerrima, were recorded (Azadbakht & Ghahremaninejad, 2023). Based on collections and observations made in Semnan Province and on preliminary taxonomic assessments by researchers and experts, the identification of Berberis species has been complicated by the phenomenon of introgression among different taxa, which has resulted in the emergence of numerous intermediate forms. These forms pose a serious obstacle to the delimitation of natural species boundaries (Azadi, 2009). Therefore, the present study was undertaken to evaluate the effectiveness of morphological traits through quantitative and qualitative analyses in order to identify and assess the diversity of barberry species occurring in Semnan Province. Materials & MethodsTo investigate morphological variation, samples were randomly collected from 10 habitats in Semnan Province. Different parts of the adult plants, including branches, leaves, and inflorescences, were sampled and photographed, and the latitude, longitude, and altitude of each collection site were recorded. The samples were obtained during two seasons, spring and autumn, and were subsequently transferred to the botany laboratory, where their initial identification was performed with the aid of a laboratory microscope and reference to authentic floras. Diagnostic traits of the plants were determined based on key features provided in standard identification guides, while additional traits were obtained from field observations. The populations collected from each habitat were then evaluated and measured using 34 traits, comprising 12 quantitative traits, 3 relative traits, and 19 qualitative traits. For phenetic analyses, the mean values of quantitative traits were used, whereas qualitative traits were coded as binary or multistate characters. Significant differences among populations were assessed by one-way analysis of variance (ANOVA) using SPSS version 26. Cluster analysis was conducted with the WARD method, applying either Euclidean or squared Euclidean distance as the dissimilarity coefficient (Ingrouille, 1986). Principal component analysis (PCA) was also employed to identify the most variable morphological traits among populations (Sneath & Sokal, 1973), using PAST version 3 software (Hammer et al., 2001). Research findingsThe analysis of variance indicated significant differences at the 5% level among populations for several traits, including leaf length, number of berries per raceme, number of leaves per node, brachyblast length, leaf length to thorn length ratio, and shrub height. In contrast, internode length, fruit width, and leaf length to width ratio did not differ significantly among the populations. Cluster analysis revealed three distinct groups: the first comprised the Majn, Tazreh, Nekarman, and Mehmandoyieh populations, which are most likely attributable to the hybrid species B. integerrima × orthobotrys. The second included the Jangal Abar and Village Abar populations, which corresponded more closely to B. orthobotrys. The third cluster comprised the Aftar, Shahmirzad, Tarom, and Kamand populations, which probably belong to the hybrid species B. integerrima × crataegina. Principal component analysis (PCA) showed that the first and second components had the greatest contribution to the total variance within the genus, together accounting for 59.06% of the variation. Examination of eigenvalues indicated that, in the first component, the traits of leaf width, thorn length, brachyblast length, fruit width, petiole length, internode length, and shrub height had the highest loadings, while in the second component, fruit length and the ratio of leaf length to thorn length were the most influential traits in the grouping of the studied populations. Discussion of Results & ConclusionThe findings of this study demonstrated that populations collected from different parts of Semnan Province exhibited interspecific variation in several morphological traits. These observations are consistent with those of Bottini et al. (2000), who reported significant morphological variation among 13 wild Berberis L. species in southern Argentina and Chile, and with the results of Talebi et al. (2020), who documented considerable morphological diversity among Berberis populations from northeastern Iran. Similarly, in an analysis of 96 samples of B. integerrima from the Jasb region of Central Iran, based on 30 morphological traits, a high degree of variability was observed, and the groupings obtained in the present study are largely in agreement with the general patterns reported by Maleki-Meighani et al. (2025). Furthermore, several investigations of quantitative and qualitative traits of barberry populations in different regions of Iran, including Semnan Province, have revealed extensive diversity, and the present results are in strong agreement with those studies (Rezaei et al., 2018; Parvane et al., 2020). From a taxonomic perspective, the results of this research are also consistent with studies conducted in neighboring regions and support the recognition of the aforementioned species (Azadbakht & Ghahremaninejad, 2023; Sodagar et al., 2012). AcknowledgmentThe authors would like to express their sincere gratitude to the Institute of Medicinal Plants, ACECR, for providing valuable assistance and support in advancing the objectives of this research.

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University of Isfahan Taxonomy and Biosystematics 3115-9001 17 63 2025 08 23 Morphological adaptation patterns of the Euphrates stone loach, Oxynoemacheilus euphraticus, in the Persian Gulf basin الگوهای سازگاری ریختی جویبارماهی فرات، Oxynoemacheilus euphraticus در حوضه خلیج فارس 51 60 29504 10.22108/tbj.2025.144686.1302 EN Zahra Moradzadeh Kor MSc. Student, Department of Fisheries, Faculty of Natural Resources, University College of Agriculture and Natural Resources, University of Tehran, University of Tehran, Tehran, Iran Soheil Eagderi Associate Professor Department of Fisheries, Faculty of Natural Resources, University College of Agriculture and Natural Resources, University of Tehran, University of Tehran, Tehran, Iran Hadi Poorbagher Associate Professor Department of Fisheries, Faculty of Natural Resources, University College of Agriculture and Natural Resources, University of Tehran, University of Tehran, Tehran, Iran Atta Mouludi-Saleh Ph. D. Graduated. Department of Fisheries, Faculty of Natural Resources, University of Tehran, Karaj, Iran Journal Article 2025 03 15 This study investigated the morphological diversity of the Euphrates stone loach, Oxynoemacheilus euphraticus, in the Persian Gulf basin. For this purpose, samples were collected from the Khorram River (Karkhe River Sub-basin, Iran), Sirvan (the Iranian section of the Tigris Sub-basin), and the Euphrates River (tributaries of the Sultanyu River in Malatya (the type locality) and the Karasu River, Turkey), transferred to the laboratory, and 25 morphometric characteristics were measured. To investigate the morphological diversity between the studied populations, the measured traits were analyzed after standardization using one-way analysis of variance and Duncan's grouping, principal component analysis, and canonical variate analysis, with p-values obtained from the NPMANOVA test. The results showed significant differences between the studied populations in most of the measured traits, including predorsal length, preventral length, distance between the anus and the origin of the anal fin, height of the caudal peduncle, depth of the dorsal fin, length of the dorsal fin base, body depth at the head and at the origin of the dorsal fin, head width, eye diameter, and length of the barbels. PCA and CVA analyses distinguished the studied populations from each other. The population of the Sultansu River in Turkey showed greater differentiation than the other populations. According to the results, the Euphrates stone loach can adapt to various environmental characteristics by altering the aforementioned morphological traits to thrive in different habitats, indicating its high phenotypic plasticity across diverse habitats. Also, since different populations of this species cannot be distinguished from one another based on morphological characteristics, the aforementioned traits, which relate to the morphological phenotypic plasticity of this species, should be avoided when describing a new species. Keywords: Freshwater fishes, Tigris, Morphology, Multivariate analysis, Adaptation. Introduction The genus Oxynoemacheilus is widely distributed in Iranian inland waters. So far, 62 species have been described in this genus worldwide (van der Laan, 2021), of which 17 species are found in Iranian inland waters (Eagderi et al., 2022). The wide distribution range of the members of this genus has resulted in the description of many new species, creating numerous taxonomic challenges. The Euphrates stone loach, Oxynoemacheilus euphraticus, has a wide distribution range in the Euphrates–Tigris river system within the Persian Gulf basin (Freyhof & Özuluĝ, 2017) and, in recent years, has been reported from the Tigris River drainage in the Lesser and Greater Zab and Sirvan sub-basins (Mouludi-Saleh et al., 2022). This species is also found in the Tigris River drainage in Iraq (Çiçek et al., 2023). Despite some studies on the distribution of this species, the morphological mechanisms associated with its adaptation to diverse habitats remain largely unknown. Therefore, this study aims to fill this gap by analyzing morphometric data from different populations. For this purpose, the present study was conducted to investigate the morphological patterns of the Euphrates stone loach in the Persian Gulf basin. Materials and methods To investigate the morphological adaptation patterns of the Euphrates stone loach, 48 specimens were collected from rivers in three sub-basins of the Persian Gulf, including Khorramrud (Karkhe sub-basin, 15 specimens), Sirvan (the Iranian section of the Tigris sub-basin, 15 specimens), and the Euphrates River (Turkey, Sultansu tributaries in Malatya, the type locality of the species) and Karasu in Erzincan, comprising 18 specimens, using an electrofishing device (Samus MP750) (Table 1). After anesthesia, the samples were fixed in a 4% buffered formalin solution and then transported to the laboratory. In the laboratory, 25 morphological characteristics were measured using a digital caliper with an accuracy of 0.1 mm, according to Armbruster (2012). After photography and biometry of the fish samples, their abdomens were opened, and the sex of the samples was determined. To eliminate the effects of allometric growth, raw morphometric data were standardized based on the allometric method (allometric vs. standard algorithm). The efficiency of the corrected data was assessed by testing the significance of the correlation between the variables and the standard length. To investigate the patterns and possible morphological differences between the studied populations, one-way analysis of variance, Duncan's range, Principal Component Analysis (PCA), and Canonical Variate Analysis (CVA) were used based on the p-value obtained from the NPMANOVA test. Standardization and all statistical analyses were performed using PAST-2.17b, SPSS 19, and Excel 2016 software. The significance level in this study was set at 0.05. Research Findings Due to the non-normality of the data, the Kruskal–Wallis test was used to compare each morphological trait separately between the studied populations. The results revealed significant differences in many morphological traits. Specifically, the findings indicated that significant differences were observed in the predorsal length, preanal length, distance between the anus and the origin of the anal fin, caudal peduncle height, dorsal fin depth, dorsal fin base length, body depth at the head and at the origin of the dorsal fin, head width, eye diameter, and barbel length (Table 2). In the PCA, to investigate potential morphological patterns between the studied populations, the first component emerged as the most influential component. The PCA plot based on the two components, PC1 and PC2, distinctly separated all populations (Fig. 1). In the CVA analysis, all populations were thoroughly distinguished from one another, with the Sultansu River population in Turkey significantly separated from the others (Fig. 2). The results of the CVA test of body shape indicated a significant difference in the morphometric characteristics of the Euphrates stone loach, based on the p-value obtained from the NPMANOVA test. Discussion of Results & Conclusion Intraspecific variation exists both within each population of a species and between different populations due to geographical variation. Therefore, the formation of a new species signifies the transformation of variation within a species into differences between two species (Ridley, 1996). However, geographical isolation does not necessarily serve as an isolating barrier to the creation of a new species, because geographical variation in species can arise from adaptation to their habitat conditions. Consequently, the morphological differences and color patterns observed between the studied populations of the Euphrates stone loach can be regarded as morphological differences stemming from geographical separation. In conclusion, it can be stated that, given that different populations of this species are indistinguishable from each other in terms of morphological characteristics, one should not limit the description of a taxon to only morphological traits that reflect the flexibility of the species.

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