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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