Bakmaz Dilara, Sönmez Serdar, Korkmaz Ertan Mahir
Department of Molecular Biology and Genetics, Faculty of Science, Sivas Cumhuriyet University, Sivas, Turkey.
Department of Biology, Faculty of Science and Letters, Adıyaman University, Adıyaman, Turkey.
PeerJ. 2025 Aug 21;13:e19870. doi: 10.7717/peerj.19870. eCollection 2025.
Accurate species delimitation is essential in morphologically conservative taxa such as harpacticoid copepods, in which cryptic diversity may go unnoticed without molecular data. The genus , common along the Turkish coastline, comprises two species: and , with overlapping ranges and subtle morphological differences. This study aimed to assess species boundaries and uncover potential hidden diversity within using the dual-marker DNA barcoding approach.
Specimens of were collected from the Mediterranean, Aegean, and Black Sea coasts of Türkiye. Nuclear DNA from a total of 46 individuals were amplified and sequenced for both mitochondrial cytochrome oxidase I () and nuclear internal transcribed spacer 2 (ITS2) markers. sequences were analysed for haplotype diversity, phylogenetic relationship, and species delimitations. ITS2 sequences were subjected to evaluation with regard to nucleotide diversity, secondary structure, and compensatory base changes (CBCs), using both sequence- and structure-based approaches. The concatenated dataset and species tree reconstruction (StarBEAST2) were employed to test gene tree-species tree congruence.
The analyses revealed a high level of haplotype diversity (21 haplotypes) and the presence of three molecular operational taxonomic units (MOTUs) within and one MOTU within , consistent with the geographic distribution patterns. ITS2 sequences exhibited relatively more conservation with nine haplotypes. These sequences revealed informative structural variation, including CBCs among candidate species. The species delimitation approaches reliably supported the identification of four to seven MOTUs, which corresponded to geographic populations. The analyses of the concatenated dataset supported four well-supported candidate species, and yielded congruent species trees, with high posterior probabilities. Morphological comparisons among MOTUs revealed subtle differences in female P5 structure and anal somite ornamentation among lineages, while MOTUs were morphologically indistinguishable.
This study provides the first integrative application of and ITS2 barcoding in and within Harpacticoida overall, combining DNA sequences and structure, and morphological data for species delimitation. The results demonstrate that is effective for detecting geographic differentiation and haplotype diversity, whereas ITS2 contributes structural resolution and potential markers of reproductive isolation through CBCs. These findings suggest the presence of a species complex within and confirm the distinct status of . Dual-marker barcoding, particularly incorporating ITS2 secondary structure, represents a valuable tool for taxonomic studies in morphologically conservative copepod groups.
在形态保守的类群（如猛水蚤桡足类）中，准确的物种界定至关重要，在这类生物中，如果没有分子数据，隐性多样性可能会被忽视。在地中海沿岸常见的[属名]属包括两个物种：[物种名1]和[物种名2]，它们的分布范围重叠且形态差异细微。本研究旨在使用双标记DNA条形码方法评估[属名]属内的物种界限，并揭示潜在的隐藏多样性。
从土耳其的地中海、爱琴海和黑海沿岸采集了[属名]属的标本。对总共46个个体的核DNA进行扩增，并对线粒体细胞色素氧化酶I（COI）和核内转录间隔区2（ITS2）标记进行测序。分析COI序列的单倍型多样性、系统发育关系和物种界定。使用基于序列和结构的方法，对ITS2序列进行核苷酸多样性、二级结构和补偿性碱基变化（CBCs）的评估。采用串联数据集和物种树重建（StarBEAST2）来检验基因树与物种树的一致性。
COI分析揭示了高水平的单倍型多样性（21个单倍型），并且在[物种名1]属内存在三个分子操作分类单元（MOTUs），在[物种名2]属内存在一个MOTU，这与地理分布模式一致。ITS2序列表现出相对较高的保守性，有九个单倍型。这些序列揭示了丰富的结构变异，包括候选物种之间的CBCs。物种界定方法可靠地支持了四到七个MOTUs的识别，这些MOTUs与地理种群相对应。串联数据集的分析支持了四个得到充分支持的候选物种，并产生了具有高后验概率的一致物种树。MOTUs之间的形态比较揭示了[物种名1]属不同谱系的雌性P5结构和肛节装饰存在细微差异，而[物种名2]属的MOTUs在形态上无法区分。
本研究首次将COI和ITS2条形码综合应用于[属名]属以及整个猛水蚤目，结合DNA序列和结构以及形态数据进行物种界定。结果表明，COI对于检测地理分化和单倍型多样性有效，而ITS2通过CBCs提供结构分辨率和生殖隔离的潜在标记。这些发现表明[属名]属内存在一个物种复合体，并确认了[物种名2]的独特地位。双标记条形码，特别是结合ITS2二级结构，是形态保守的桡足类群分类研究的有价值工具。
