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Accurate species identification is essential for surveillance and disease risk assessment but is often hindered by morphological similarity and intraspecific variation. In Malaysia, tick studies have largely relied on morphology or single-gene barcoding, with limited taxonomic and geographic coverage.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>This study applied a multilocus mitochondrial approach using cytochrome c oxidase I (COI) and 16S ribosomal DNA (rDNA) to evaluate species boundaries among ixodid ticks from Peninsular Malaysia. Ticks were collected from wild and domestic hosts between 2022 and 2023. A total of 319 specimens representing 14 morphologically defined species were analysed using COI and 16S rDNA sequencing, with additional reference sequences retrieved from GenBank for comparative analyses. Phylogenetic reconstruction, barcode gap assessment, and four species delimitation methods (ASAP, ABGD, bPTP, and GMYC) were employed to assess genetic divergence and identification accuracy.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>Molecular analyses were largely congruent with morphological identification. Distance-based delimitation methods (ASAP and ABGD) recovered operational taxonomic units (OTUs) largely consistent with morphologically defined species, whereas tree-based approaches (bPTP and GMYC) inferred substantially higher numbers of OTUs, reflecting sensitivity to intraspecific mitochondrial structuring. These additional subdivisions are interpreted conservatively as population-level genetic differentiation rather than evidence of distinct species. Clear barcode gaps were observed for COI and the concatenated COI\u2009+\u200916S rDNA datasets, whereas partial overlap occurred with 16S rDNA alone. COI demonstrated the highest and most consistent performance for routine species identification, while concatenated datasets improved phylogenetic resolution but reduced assignment clarity under strict barcoding criteria.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>Overall, COI remains the most effective mitochondrial marker for routine species identification of ixodid ticks in Peninsular Malaysia. These findings highlight the value of integrative approaches combining morphology and molecular data to strengthen tick taxonomy and support surveillance of tick-borne pathogens in the region.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s13071-026-07546-3","type":"journal-article","created":{"date-parts":[[2026,7,3]],"date-time":"2026-07-03T13:46:10Z","timestamp":1783086370000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Mitochondrial COI and 16S rDNA barcoding improve species delimitation of ixodid ticks in Peninsular Malaysia"],"prefix":"10.1186","volume":"19","author":[{"given":"Nurul Aini","family":"Husin","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Van Lun","family":"Low","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muhammad Haiqal Syarriman","family":"AbdulRahim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muhammad Rasul","family":"Abdullah Halim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Auni Atikah","family":"AbdulHalim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muhammad Al Amin","family":"Mohd-Redzuan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Edley A.","family":"Jiliun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ahmad Khusaini","family":"Mohd Kharip Shah","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin L.","family":"Makepeace","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zubaidah","family":"Ya\u2019cob","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,7,3]]},"reference":[{"key":"7546_CR1","doi-asserted-by":"publisher","first-page":"S3","DOI":"10.1017\/S0031182004005967","volume":"129","author":"F Jongejan","year":"2004","unstructured":"Jongejan F, Uilenberg G. 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