DNA barcoding of Messor ants of Bulgaria with insights into their taxonomic diversity

Biodiversity Data Journal 13: e168586 doi: 10.3897/BDJ.13.e168586 Data Paper DNA barcoding of Messor ants of Bulgaria with insights into their taxonomic diversity Albena Lapeva-Gjonova , Monika Pramatarova , Lech Borowiec , Ilia Gjonov , Rumyana Kostova , Rostislav Bekchiev , Simeon Borissov ‡ Sofia University, Faculty of Biology, Sofia, Bulgaria § National Museum of Natural History, Bulgarian Academy of Sciences, Sofia, Bulgaria | University of Wroclaw, Wroclaw, Poland ¶ Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia, Bulgaria Corresponding author: Albena Lapeva-Gjonova ([email protected]) Academic editor: Francisco Hita Garcia Received: 12 Aug 2025 | Accepted: 20 Oct 2025 | Published: 04 Nov 2025 Citation: Lapeva-Gjonova A, Pramatarova M, Borowiec L, Gjonov I, Kostova R, Bekchiev R, Borissov S (2025) DNA barcoding of Messor ants of Bulgaria with insights into their taxonomic diversity. Biodiversity Data Journal 13: e168586. https://doi.org/10.3897/BDJ.13.e168586 Abstract Background Despite ongoing efforts to catalogue European ant species, studies focusing on the genetic diversity of Balkan ants remain limited. An integrative approach combining morphology, genetics, ecology and biogeography is preferable for accurately identifying species and resolving taxonomic uncertainties, particularly amongst challenging insect taxa, such as the ants in the genus Messor (Hymenoptera, Formicidae). New information In this study, we analyse ants of the genus Messor using DNA barcode sequences, with a particular focus on the Bulgarian fauna. A total of 85 COI sequences were examined, including 84 from Messor specimens and one from Aphaenogaster, which was used as an outgroup. Of these, 81 sequences were newly generated, while four were retrieved from GenBank. The majority of specimens were collected in Bulgaria (61), with additional ‡ ‡,§ | ‡ ‡ § ¶ © Lapeva-Gjonova A et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. samples from Greece (13), Türkiye (4), Albania (1) and North Macedonia (2), providing broader genetic and geographic representation. Althogether, 11 Messor morphospecies were identified, based on specimens used for molecular analysis. To assess the degree of congruence between morphological and molecular data, six species delimitation analyses were conducted: RESL, GMYC, ASAP, ABGD, bPTP and mPTP. In addition, haplotype network analysis of all sequences identified 35 distinct and coherently clustered haplotypes, providing insights into genetic diversity. The COI barcode region successfully distinguished Messor wasmanni Krausse, 1910, M. oertzeni Forel, 1910 and M. ibericus Santschi, 1931. In contrast, species pairs, such as M. atanassovii Atanassov, 1982 and M. creticus Salata & Borowiec, 2019, as well as M. ponticus Steiner et al., 2018 and M. hellenius Agosti & Collingwood, 1987, could not be reliably differentiated using COI data. Furthermore, Messor structor (Latreille, 1798) showed high intraspecific genetic diversity. Finally, the structor and instabilis species groups were recovered with moderate to high support in both Maximum Likelihood and Bayesian Inference analyses, confirming that M. oertzeni and M. hellenius belong to the structor group. Our results provide a reference for future research and underscore the value of integrative taxonomic approaches in ant biodiversity studies. Keywords the Balkans, COI, species delimitation, Formicidae, Myrmicinae Introduction The genus Messor, commonly known as harvester ants, consists of typically granivorous species involved in seed dispersal, nutrient cycling and microclimate modification in surface soil layers (Cammeraat et al. 2002, Plowes et al. 2013, El Boukhrissi et al. 2023). These ants typically inhabit arid and semi-arid environments, with 134 species currently recognised within the Palaearctic, Afrotropical and Oriental biogeographic regions (Branstetter et al. 2022, Salata et al. 2023, Bolton 2025). While species richness is highest in North Africa and the Middle East, the environmental conditions and biogeographic history of the Southern Balkans also favour the presence of a substantial number of Messor species (Borowiec 2014, Janicki et al. 2016, Guénard et al. 2017, Wang et al. 2023, Juvé et al. 2025a). Comprehensive modern studies on the species composition and distribution of Messor species in Bulgaria remain insufficient. Two recent species revisions have addressed some species within the genus found in the country. One such revision, focusing on the European species of the structor group (Steiner et al. 2018), included Bulgarian material, although samples originated from only a few localities. This study recognised three 2Lapeva-Gjonova A et al species from the group in Bulgaria: Messor structor (Latreille, 1798), M. ponticus Steiner et al., 2018 (with type locality in Bulgaria) and M. ibericus Santschi, 1931. Additionally, two other species, M. mcarthuri Steiner et al., 2018 and M. hellenius Agosti & Collingwood, 1987, were recently reported from the country by Lapeva-Gjonova and Borowiec (2022). The latter was not included in the revision of this group by Steiner et al. (2018), but was recognised as such by Borowiec and Salata (2025). Furthermore, a phylogenetic analysis by Juvé et al. (2025a) revealed Messor oertzeni Forel, 1910, a well-known species from Bulgaria, as the sixth member of the structor group in the country. Messor wasmanni Krausse, 1910 and M. atanassovii Atanassov, 1982, the latter with its type locality in Bulgaria, are the only representatives of the instabilis group currently known from Bulgaria. This species group from the Eastern Mediterranean region was recently revised (Salata et al. 2023). The revision included a detailed redescription of M. atanassovii, confirming its validity as a distinct species and reporting additional localities in both Bulgaria and Greece. The latest studies on Messor in the Palaearctic Region have re-evaluated the earlier records and concluded that four species previously reported from Bulgaria (M. barbarus (Linnaeus, 1767), M. caducus (Victor, 1839), M. capitatus (Latreille, 1798) and M. concolor Santschi, 1927) do not actually occur in the Balkans. Consequently, eight Messor species are currently recognised in Bulgaria: M. atanassovii, M. wasmanni, M. oertzeni, M. structor, M. mcarthuri, M. ponticus, M. hellenius and M. ibericus ( Lapeva-Gjonova and Antonova 2022). The scarcity of historical descriptions, coupled with high morphological variability within species and occurrences of hybridisation and even xenoparity, makes the genus Messor taxonomically and biologically challenging (Schlick-Steiner et al. 2006, Steiner et al. 2011 , Romiguier et al. 2017, Steiner et al. 2018, Saar et al. 2023, Juvé et al. 2025a, Juvé et al. 2025b). This necessitates the application of complex approaches alongside the morphological one to resolve species delimitations. DNA barcoding using mitochondrial cytochrome c oxidase I (COI) gene fragments has proven to be an efficient method for species identification and biodiversity assessment, including ants of Messor genus ( Schlick-Steiner et al. 2006, Steiner et al. 2018, Strohmaier et al. 2025) and other Stenammini (Centorame et al. 2018, Gómez et al. 2018, Galkowski et al. 2019, Schifani et al. 2022, Zięcina et al. 2024). However, COI is not universally reliable for ant identification and species delimitation due to biological factors, such as incomplete lineage sorting, introgression, hybridisation, NUMTs and endosymbiont effects, as well as technical issues like gaps in reference libraries and threshold inconsistencies (Hurst and Jiggins 2005, Darras and Aron 2015, Romiguier et al. 2017). Despite these limitations, it remains a rapid, cost-effective tool with reasonable species-level resolution, supported by widely-used primers and extensive sequence repositories (deWaard et al. 2019, Martoni et al. 2024, Onyinyechi et al. 2025). Accordingly, expanding barcoding efforts in underexplored regions, such as the Balkans, is crucial for improving our understanding of species diversity and evolutionary relationships. While a large-scale barcoding project of European ants is underway DNA barcoding of Messor ants of Bulgaria with insights into their taxonomic ... 3 (Menchetti et al., unpublished), further research specifically targeting the genetic diversity of Balkan Messor ants will provide valuable insights into taxonomy and phylogeny. Sampling methods Sampling description:Specimens for DNA barcoding were primarily selected, based on morphology and their origin from diverse collection sites across the country. Morphological identifications followed Steiner et al. (2018) and Borowiec and Salata (2025). Molecular analyses: DNA extraction, amplification and sequencing of the standard 658 bp COI barcode region were performed by the Canadian Centre for DNA Barcoding (CCDB) using the primers LepF1 and LepR1 (Hebert et al. 2004). DNA was extracted from the hind legs of specimens preserved in ethanol. In total, 84 COI Messor sequences were analysed, of which 80 were newly generated. The following four sequences were obtained from GenBank and were included in the phylogenetic analyses: KT184551 (Messor structor), KT184569 (M. mcarthuri), KT184511 (M. ibericus) from Steiner et al. (2018) and DQ074353 (M. ponticus) from Schlick-Steiner et al. (2006). The sequence of Aphaenogaster festae Emery, 1915 generated in the current study was selected as an outgroup in the phylogenetic analyses. All 81 sequences generated in this study are deposited in the Barcode of Life Data System (BOLD) under the BGMES project, where collection information and photos of each specimen are also provided. Voucher specimens are preserved in the Zoological Collection of Sofia University (BFUS). To assess the degree of congruence between morphological identification conducted prior to the molecular data, multiple species delimitation approaches were applied to the molecular dataset. Sequence alignment and trimming were performed using MEGA v.12 (Kumar et al. 2024). In the BOLD system, the sequences were assigned to Barcode Index Numbers (BINs), an algorithm-based approach to delineate operational taxonomic units, which were automatically calculated for records by Refined Single Linkage (RESL) analysis. These BINs have a unique identifier and provide a good proxy for species (Ratnasingham and Hebert 2013). To estimate genetic distances and enable comparison, pairwise distances were calculated under the Kimura 2-parameter (K2P) model using MEGA v.12 (Kumar et al. 2024), whereafter species boundaries were tested with Assemble Species by Automatic Partitioning (ASAP) and Automatic Barcode Gap Discovery (ABGD). Subsequently, ultrametric trees were generated in BEAST v. 10.5.0 (Baele et al. 2025) with a strict clock, coalescent tree prior and 100 million generations, sampling every 1000 trees. The effective sample size (ESS) was monitored in Tracer v. 1.7.2 (Rambaut et al. 2018). Trees were summarised via TreeAnnotator (Suchard et al. 2018) removing 10% as a burn-in. Species delimitation analyses included Generalised Mixed Yule Coalescent Approach (GMYC) with a single threshold (implemented on the web server https://species.h-its.org/gmyc/, accessed on 27 July 2025), the Poisson Tree Processes (bPTP) (implemented on the web server http://species.h-its.org/ptp/, accessed on 28 July 2025) and, finally, the multi-rate Poisson Tree Processes (mPTP) (implemented on the web server http://mptp.h-its.org/#/tree, accessed on 27 July 2025) 4Lapeva-Gjonova A et al (Fujisawa and Barraclough 2013, Zhang et al. 2013, Trifinopoulos et al. 2016, Kapli et al. 2017). Phylogenetic reconstruction was performed using both Maximum Likelihood (ML) and the Bayesian Inference (BI) analyses. ML analysis was performed in IQ-TREE (Nguyen et al. 2015) on the W-IQ-TREE interface (Trifinopoulos et al. 2016). The integrated ModelFinder (Kalyaanamoorthy et al. 2017) was used to infer the best substitution model. Nodal support was obtained through a standard non-parametric bootstrap with 1000 replicates. BI analysis was run using MrBayes v.3.2.7a (Ronquist et al. 2012). Phylogenetic trees (BI and ML) were visualised using iTOL v.5 (Letunic and Bork 2021). Haplotype analysis was conducted utilising the DnaSP v.6 software (Rozas et al. 2017) and the results were visualised through the utilisation of PopArt employing TCS network analysis (Clement et al. 2002, Leigh and Bryant 2015). A map of sequence sampling sites was created in QGIS version 3.34.12-Prizren, based on the Cross Blended Hypsometric map layer (https://www.naturalearthdata.com). Geographic coverage Description:The specimens used in this study were recently collected, primarily from Bulgaria (61), with additional samples from Greece (13), Türkiye (4), Albania (1) and North Macedonia (2) to ensure broader genetic and geographic representation (Fig. 1). Coordinates:Latitude: min. 34.931 max. 43.768; Longitude: min. 19.577 max. 27.794. Figure 1. Map of sequence sampling sites.  DNA barcoding of Messor ants of Bulgaria with insights into their taxonomic ... 5 Taxonomic coverage Taxa included: Rank Scientific Name subfamily Myrmicinae Lepeletier de Saint-Fargeau, 1835 genus Aphaenogaster Mayr, 1853 species Aphaenogaster festae Emery, 1915 genus Messor Forel, 1890 species Messor atanassovii Atanassov, 1982 species Messor creticus Salata & Borowiec, 2019 species Messor hellenius Agosti & Collingwood, 1987 species Messor ibericus Santschi, 1931 species Messor mcarthuri Steiner, Csősz, Markó, Gamisch, Rinnhofer, Folterbauer, Hammerle, Stauffer, Arthofer & Schlick-Steiner, 2018 species Messor oertzeni Forel, 1910 species Messor ponticus Steiner, Csősz, Markó, Gamisch, Rinnhofer, Folterbauer, Hammerle, Stauffer, Arthofer & Schlick-Steiner, 2018 species Messor structor (Latreille, 1798) species Messor wasmanni Krausse, 1910 species Messor cf. structor species Messor sp. 1 species Messor sp. 2 Usage licence Usage licence:Open Data Commons Attribution License Data resources Data package title:Collection of COI sequences from Bulgarian species of the genus Messor Resource link: https://doi.org/10.5883/DS-BGMESSOR Number of data sets:1 6Lapeva-Gjonova A et al Data set name:Towards delimiting the diversity of Messor ants in Bulgaria using molecular data Data format:dwc, xml, tsv, fasta Description: The dataset constitutes a collection of sequences pertaining to Bulgarian species of the genus Messor (Hymenoptera, Formicidae). This dataset comprises all attributes and metadata in accordance with the BOLD rules and are available to the public via a Digital Object Identifier (DOI). Additional information Species delimitation and genetic diversity A total of 84 COI sequences, representing 11 morphospecies and 10 to 15 molecular lineages (depending on the species delimitation method used), were analysed, including 80 newly-generated sequences. The lengths of the DNA barcodes ranged from 579 to 658 bp, with the majority (59 sequences) being 658 bp long (Fig. 2). Haplotype network analysis of all sequences revealed 35 distinct haplotypes, which clustered coherently (Fig. 3). Figure 2. Results of species delimitation methods, based on DNA barcoding. Each vertical colour bar represents different delimitation schemes obtained with ASAP, RESL, ABGD, bPTP and mPTP methods, with the corresponding number of specimens. The tree is based on ASAP analysis, with nodes colour coded depending on their p-value (black: p < 0.001, red: p < 0.05, yellow: p > 0.1, grey: not applicable).  DNA barcoding of Messor ants of Bulgaria with insights into their taxonomic ... 7 Messor instabilis species group The morphological similarity between Messor atanassovii and M. creticus is supported by low genetic distance observed in the delimitation analyses (K2P 1.92%) (Suppl. material 1). These findings may indicate a relatively recent divergence between the two species, followed by geographic isolation and ecological differentiation. However, despite their overall closeness, M. atanassovii and M. creticus consistently differ in stable morphological traits. Specifically, in M. atanassovii, the occipital area and vertex of the head bear 12–20 large setae, whereas in M. creticus, the number is always lower, never exceeding nine. In addition, unlike M. creticus, which is restricted to the mountain regions of Crete, M. atanassovii is a thermophilous lowland species found in southern Bulgaria, Central Macedonia and some of the Ionian Islands (Borowiec and Salata 2025). An ongoing research into the evolutionary history of this divergence will clarify the timing and mechanisms underlying this particular event. Specimens from four nest samples — one from Central Macedonia in Greece and three from south-western Bulgaria — designated in this study as Messor sp. 1, exhibited morphological traits characteristic of both Messor atanassovii and M. wasmanni, specifically the setosity of the former and the larger size of the latter. However, all species delimitation analyses strongly supported their separation from both species and indicated Figure 3. TCS haplotype network, based on COI sequences of Messor species. Each circle represents a unique haplotype; size corresponds to the number of individuals. Lines indicate single mutational steps. Species-specific colour and letter coding follow the phylogenetic tree.  8Lapeva-Gjonova A et al a closer genetic affinity to M. wasmanni, with K2P distances of 6.28% and 5%, respectively (Suppl. material 1). Currently, M. atanassovii and M. wasmanni are the only known representatives of the instabilis group in this region. Whether the specimens designated as Messor sp. 1 represent cases of hybridogenesis or belong to a distinct species will be investigated in a future study. The most widespread species of the instabilis group, Messor wasmanni, is represented in this study by a larger number of specimens (13) from the widest geographical range — spanning Bulgaria, Türkiye and Greece (including Crete). It exhibits an intraspecific genetic distance up to 0.81% (mean: 0.18%) (Suppl. material 1). Messor structor species group Species delimitation analyses were consistent in supporting the distinctiveness of Messor oertzeni, M. mcarthuri and unidentified species close to M. oertzeni (named Messor sp. 2), as well as one molecular lineage within Messor structor represented by four sequences — three from western Bulgaria and one from North Macedonia. Further evaluation is also needed for a single sequence (BGANT032-23) obtained from a nest sample in the western Balkan Mountains (Vrachanski Balkan). Although this specimen is morphologically similar to M. structor and clearly separated from all recognised taxa in the analyses, the small sample size and absence of reproductive specimens make its taxonomic affinity still unclear. Messor ibericus was consistently recognised in distance-based methods (ASAP, RESL, ABGD) and the coalescent-based method GMYC, exhibiting an intraspecific genetic distance up to 0.46% (mean 0.2%), but not in the tree-based methods bPTP and mPTP. This discrepancy can be attributed to differences in methodological assumptions and sensitivity to genetic variation (Hubert et al. 2024). It should be noted that our study analysed only the worker caste of M. ibericus, which, as recently shown by Juvé et al. (2025b), are hybrids with M. structor. Nevertheless, since they inherit the COI marker from the maternal lineage, the genetic patterns observed in our study remain consistent with their maternal identity. Previous studies investigating Messor structor across its broad geographic distribution — spanning Austria, Bulgaria, Czechia, France, Hungary, Romania and Slovenia — revealed the existence of multiple mitochondrial lineages within the species (SchlickSteiner et al. 2006, Steiner et al. 2018, Strohmaier et al. 2025). These earlier findings align closely with the results of the present study, which detected high intraspecific genetic diversity (with K2P distance from 0 to 5.07%, mean 2.58%) and identified ten haplotypes (Fig. 3, Suppl. material 1). While several species delimitation methods (ASAP, ABGD, bPTP, mPTP) recognised two molecular lineages, the RESL algorithm distinguished five BINs within M. structor, further supporting the presence of deep genetic structuring, with multiple lineages and haplotypes suggesting potential cryptic diversity across its range or long-term population isolation within the species. DNA barcoding of Messor ants of Bulgaria with insights into their taxonomic ... 9 • Zięcina D, Menchetti M, Borowiec L, Bračko G, Lapeva-Gjonova A, Villa R, Salata S (2024) Taxonomic revision of the Aphaenogaster subterranea species group (Hymenoptera: Formicidae). Annales Zoologici 74 (2): 237‑282. https://doi.org/ 10.3161/00034541ANZ2024.74.2.002 Supplementary material Suppl. material 1: Distance analyses, GMYC summary and Bayesian Inference tree Authors: Albena Lapeva-Gjonova, Monika Pramatarova, Lech Borowiec, Ilia Gjonov, Rumyana Kostova, Rostislav Bekchiev, Simeon Borissov Data type: genomic, phylogenetic Download file (3.42 MB) 16 Lapeva-Gjonova A et al