Karağaç, Rümeysa ¹ ; Eroğlu, Halil Erhan ² ; Hekimoğlu, Olcay ³ and Sevsay, Sevgi ⁴

¹Department of Biology, Institute of Science, Erzincan Binali Yıldırım University, Erzincan, Türkiye.
²Department of Biology, Faculty of Sciences and Arts, Yozgat Bozok University, Yozgat, Türkiye.
³Department of Biology, Faculty of Science, Division of Ecology, Hacettepe University, Ankara, Türkiye.
⁴✉ Department of Biology, Faculty of Sciences and Arts, Erzincan Binali Yıldırım University, Erzincan, Türkiye.

2025 - Volume: 65 Issue: 2 pages: 589-601

Original research

Keywords

Abstract

Introduction

Studies on determining the chromosome numbers of mites began many years ago. Although the first chromosome studies on mites began with Reuter's (1909) work on the oogenesis of Pediculopsis graminum, the most detailed cytological study was Schrader's (1923) research on Tetranychus bimaculatus from the Tetranychidae family (Reuter 1909; Schrader 1923). These studies continued with tarsonemid, gamasid and phytoseiid mites (Cooper 1937, 1939; Hansell et al. 1964; Oliver 1964, 1965, 1972, 1973, 1977; Patau 1934; Sokolov 1934, 1945 1954; Warren 1940; Wysoki 1973; Wysoki and Swirski 1968). Subsequent studies on Tetranychus urticae aimed to gain a deeper understanding of the biology and reproductive characteristics of these harmful mite species to develop biological control strategies (Helle and Bolland 1967). Later, chromosome studies were extended to ticks (Ixodoidea) (Oliver et al. 1973) and other mite groups (Bolland and Ueckermann 1984; Eroğlu and Per 2016; Gümüş et al. 2022; Heethoff et al. 2006; Onrat et al. 2006; Wysoki and Bolland 1983; Zalewska 1979). Specific chromosomal studies on eriophyoid mites emerged later, making them one of the first mite groups to undergo detailed cytogenetic analysis (Norton et al. 1993).

Although detailed chromosome number data are lacking for most species within the Trombidiidae family, some studies have focused on the biological characteristics of the group at the genus level. Members of this family are known to be taxonomically complex, with morphological similarities that complicate classification. Notably, species in this group exhibit a distinct life cycle: they are characterized by a complex life cycle which includes heteromorphic parasitic larvae and predatory post-larval instars (Wohltmann and Wendt 1996). However, in contrast to the available biological and life cycle data, chromosomal studies in Trombidiidae are exceedingly rare. To date, chromosomal data have been reported only for Allothrombium fuliginosum, with a diploid chromosome number of 2n=24 in both males and females (Sokolov 1954).

In general, Trombidium species exhibit high morphological similarity, which makes species-level identification challenging without the preparation of detailed microscope slides. In such morphologically cryptic groups, molecular identification—particularly DNA barcoding using the COI gene—has proven to be an effective complementary tool for distinguishing closely related species (Blaxter 2004; Davrat 2005; Knowlton 2000). Therefore, the integration of both morphological and molecular data is crucial for accurate species delimitation in Trombidiidae. The aim of this study is to provide the first cytogenetic characterization of two trombidioid mite species, Trombidium latum and Eutrombidium trigonum, and to support the identification of T. latum using mitochondrial COI barcoding data. We analyzed the chromosome numbers of T. latum from the Trombidiidae family in both the larval and adult stages, and the post-larval stage of E. trigonum from the Microtrombidiidae family. Additionally, a comprehensive list of mite species with known chromosome numbers to date is provided, serving as a valuable reference for future taxonomic and cytogenetic studies.

Materials and methods

Trombidioid mites sampling and locations

Samples were collected from Erzincan, Türkiye and preserved and prepared according to the methods described by Mąkol (2005) and Mąkol and Sevsay (2011). Specimens of E. trigonum were collected from Ahmediye village, 39°52′54″N, 39°20′31″E, 2048 m, moist soil and moss, 27 April 2021. Specimens of T. latum were collected from Demirpınar Stream Basin, Üzümlü Town. 39°57′12″N, 39°35′21″E, from soil close to the Astragalus, 22 May 2021 and from the Ahmediye village, the sampling site of E. trigonum.

Larvae of T. latum were obtained by rearing a field-collected ovigerous female under controlled laboratory conditions, solely for the purpose of obtaining specimens for cytogenetic analysis. The female was placed in a glass vial containing charcoaled Plaster-of-Paris and covered with a tight, semi-transparent lid to maintain humidity and allow visual inspection. No experimental data were recorded from this procedure beyond larval availability. The morphological identification follows Mąkol (2002) for T. latum, and Gabryś (1999) for E. trigonum.

Cytogenetic method

The cytogenetic preparations were made from the protocol presented by Imai et al. (1988) and modified by Gokhman and Quicke (1995). The samples were pretreated and crushed in hypotonic solution (1% sodium citrate containing 0.005% colchicine) for 30 min., and transferred into a fresh hypotonic solution (0.075 M KCl) for 20 min. Then the samples were fixed in freshly prepared fixative solution (acetic acid: ethanol - 3:1). After the fixation, the material was transferred to a clean slide and air-dried. Then the slides were stained with 8% Giemsa.

Ten metaphase plates with good distribution and prominent chromosomes were evaluated for each species. The plates were imaged using a DP72 digital camera mounted on an Olympus BX53 light microscope. Chromosomal measurements were made using KaryoType software and karyotype formulae were determined. The karyotype formulae consisted of the following expressions: metacentric (m), submetacentric (sm), and acrocentric (a). The ideograms were drawn from the largest chromosome to the smallest chromosome based on the chromosomal lengths. The karyotype asymmetry was calculated according to the S/A_I formula (symmetry/asymmetry index) (Eroğlu 2015).

COI sequence analysis

COI marker was used to generate sequences, as this marker has been widely used for species delimitation across various Parasitengona mites (Antonovskaia 2018; Young et al. 2012; 2019, 2021; Mąkol and Felska 2024). We generated COI sequences from four adults and a pool consisting of 20 larvae of T. latum.

DNA extraction was performed using the GeneJet Purification Kit (Thermo Fisher Scientific). A total PCR mixture of 50 μL was prepared with 17.5 μL of H₂O, 2.5 μL of each primer (10 pmol/μL), 25 μL of High Fidelity PCR Master Mix (Thermo Scientific), and 2.5 μL of DNA sample. The PCR conditions followed the protocol described by Hornok et al. (2015) using the LCO1490 and HCO2198 primers (Folmer et al. 1994). Samples were sent to Macrogen for sequencing. Sequences were aligned using the Clustal W program (Larkin et al. 2007) and compared using BLAST provided by the National Center for Biotechnology Information (NCBI) at http://blast.ncbi.nlm.nih.gov/Blast.cgi . The unique haplotype obtained in this study has been uploaded to GenBank (GenBank Accs. Number: PQ896991).

Results

In T. latum, the diploid chromosome number was 2n=12 (Figure 1). The lengths of the small metacentric and acrocentric chromosomes ranged from 1.05 to 1.92 µm. The karyotype formula was 8m + 4a. The haploid chromosome length and mean chromosome length were 8.31 and 1.39 μm, respectively. The relative length and centromeric index ranged from 12.64 to 23.10 and from 18.75 to 43.75, respectively (Table 1). The S/A_I value was 1.66. There was no satellite on the chromosomes (Figure 2).

In E. trigonum, the diploid chromosome number was 2n=14 (Figure 3). The lengths of the small metacentric, submetacentric, and acrocentric chromosomes ranged from 0.68 to 1.40 µm. The karyotype formula was 2m + 6sm + 6a. The haploid chromosome length and mean chromosome length were 6.35 and 0.91 μm, respectively. The relative length and centromeric index ranged from 10.71 to 22.05 and from 18.57 to 42.14, respectively (Table 2). The S/A_I value was 2.29. There was no satellite on the chromosomes (Figure 4).

COI sequence analysis and species identification

The comparison of COI sequences confirmed that the samples collected from Erzincan Province belong to T. latum. BLAST analysis showed 99.3% and 99.1% identity with the sequences PQ193923.1 and PQ193922.1, respectively, both derived from T. latum specimens collected in Poland (Mąkol and Felska 2024). The observed nucleotide differences (three and four base substitutions) therefore interpreted as geographic variation within the species rather than evidence of a distinct taxon.

Discussion

The selection of T. latum and E. trigonum in this study was initially based on their abundance at the sampling sites and their wide distribution in Europe (Mąkol and Wohltmann 2012), which suggests their ecological relevance. Notably, our study provides the first chromosomal data for both species, filling a significant gap in the cytogenetic knowledge of Trombidioidea. For T. latum, known issues of morphometric overlap with other congeners, especially in postlarval stages (Mąkol and Felska 2024), posed challenges for accurate identification. The integration of COI barcoding in our methodology proved essential for validating morphological diagnoses (Mąkol and Felska 2024), thereby strengthening the reliability of our karyological findings. In the case of E. trigonum, the species' distinct morphological characters facilitated identification in early developmental stages (Husband and Wohltmann 2011). By generating the first karyotypes for these taxa, our study offers a baseline for future comparative cytogenetic work and contributes to clarifying evolutionary and systematic relationships within Trombidiidae and Microtrombidiidae.

The karyotype data published to date are presented in Table 3. As illustrated in the table, there has been a gradual decline in interest in chromosomal studies over time. This decline may be attributed to the technical difficulties of cytogenetic analysis in mites and the greater focus on molecular methods in recent years. Diploid chromosome numbers in mites generally range from 4 to 30 (for detailed references, see Supplmeentary Table 1). The chromosome counts observed in T. latum and E. trigonum also fall within this range. Notably, as these represent the first chromosomal records for the families Trombidiidae and Microtrombidiidae, direct comparisons with closely related species are currently not possible. Nevertheless, the results presented here provide a valuable cytogenetic baseline and are expected to contribute significantly to the accumulation of karyological data within these understudied mite families. The S/A_I parameter is widely used in detecting karyotype asymmetry (Eroğlu 2015). The karyotype of T. latum was ''symmetric'' type by S/A_I value of 1.66 (1.0 < S/A_I ≤ 2.0). The symmetrical karyotype indicates the early stages of karyotype evolution. The karyotype of E. trigonum was ''between symmetric and asymmetric'' type by S/A_I value of 2.29 (2.0 < S/A_I ≤ 3.0).

The phylogenetic utility of karyotype data in Trombidioidea remains difficult to assess due to the paucity of comparable chromosomal records within the group. While the diploid chromosome numbers and karyotype symmetry values presented here offer important baseline data for T. latum and E. trigonum, they cannot yet be reliably used for phylogenetic inference at higher taxonomic levels. Even in relatively better-studied mite groups, such as Ixodidae, significant intraspecific variation has been observed (e.g., Ixodes ricinus, Kotsarenko et al. 2020), suggesting that chromosomal characteristics may be subject to microevolutionary change. As such, the current variability within and across mite taxa cautions against overinterpretation of karyotype traits in the absence of broader comparative frameworks. Nonetheless, with future studies expanding cytogenetic datasets in mites, especially through standardized protocols, karyotype characters may eventually contribute to resolving phylogenetic relationships when combined with molecular and morphological data. The most important of these karyotype characters are basic and diploid chromosome numbers, chromosome type, and karyotype asymmetry. Especially chromosome type (monocentric or holocentric) constitutes an important differentiation model in many insect groups (Heethoff et al. 2006; Melters et al. 2012). While the chromosomes of T. latum and E. trigonum are monocentric, there are reports of holocentric chromosomes in mites (Eroğlu and Per 2016; Gümüş et al. 2018, 2022). However, monocentric chromosomes, which allow the determination of chromosome types, karyotype formula and karyotype asymmetry, are more prominent in terms of understanding karyotype evolution.

COI barcoding has emerged as a promising and increasingly valuable tool for accurately identifying mite species, particularly those within taxonomically challenging families like Trombidiidae. This molecular technique offers a standardized and complementary approach to traditional morphological identification, especially in morphologically cryptic groups. However, its effectiveness remains limited by the currently sparse sequence data available for many mite taxa, including Parasitengona. Only a limited number of species within the Trombidiidae family have reliable reference sequences available in public databases, which currently constrains the accuracy of molecular species delimitation (Young et al. 2019, 2021). In this study, COI barcoding successfully identified the specimens collected from Erzincan Province as T. latum, with 99.1–99.3% sequence identity to reference sequences from Poland. These values fall within the widely accepted threshold for intraspecific variation (≤2%) for species-level identification (Hebert et al. 2003; Young et al. 2019, 2021), supporting conspecificity despite three to four nucleotide differences. These minor variations likely reflect geographic differentiation within the species. Our results not only confirm the identity of T. latum through molecular means but also contribute to closing the molecular data gap in Parasitengona, underscoring the need for further studies with broader geographic sampling and additional genetic markers to explore population structure and evolutionary relationships in this understudied group.

While our results provide preliminary cytogenetic and molecular data for two trombidioid mite species, further integrative studies will help to more fully evaluate the potential of combining these approaches for species delimitation. The current findings offer a valuable reference point; however, certain limitations should be acknowledged. Chromosomal analyses were based on a limited number of individuals, and further studies are needed to assess intraspecific karyotypic variability. Likewise, although COI barcoding was informative for species identification, reliance on a single gene and a limited reference dataset restricts broader phylogenetic inference. In addition, we acknowledge that many of the available COI reference sequences are tied to species hypotheses that were originally based on limited morphological datasets, often involving a small number of specimens from single localities. In Parasitengona, intraspecific variation in morphological traits is rarely assessed, and other informative criteria such as reproductive isolation or life cycle data are largely absent. Consequently, COI comparisons may reflect similarity to potentially unstable or insufficiently defined species concepts. While we interpret our results as strong support for the identification of T. latum, we also recognize that molecular species delimitation in mites—especially in understudied groups—remains tentative unless supported by integrative evidence. This reinforces the importance of developing broader reference frameworks and expanding comparative analyses that combine molecular, morphological, and ecological data (Blaxter 2004; Dayrat 2005; Knowlton 2000). Future research incorporating multi-locus approaches and comparative karyotype analyses across related taxa will be critical for advancing our understanding of mite evolution and taxonomy.

Acknowledgments

This study is part of the master's thesis of the first author and was presented as a short summary (only T. latum) at 3rd International Symposium on Biodiversity Research 20-22 October 2021, Erzurum, Türkiye. This study was financially supported by the Scientific Research Fund of Erzincan Binali Yıldırım University (EBYU), research project number FYL-2020-748. We are very grateful to EBYU for its financial support. We would like to thank Alper Torunlar for his assistance in collecting T. latum specimens during fieldwork, and Nisa Gümüş for her help with the laboratory work. Also, we are very grateful to the two anonymous referees for their valuable comments, which also contributed significantly to the improvement of the manuscript.

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