МОЛЕКУЛЯРНО-ФИЛОГЕНЕТИЧЕСКАЯ ХАРАКТЕРИСТИКА И АНАЛИЗ ГЕНЕТИЧЕСКОЙ ДИСТАНЦИИ ПОПУЛЯЦИЙ Bufotes oblongus В ЦЕНТРАЛЬНОМ УЗБЕКИСТАНЕ

УДК 575.1+591.9

Abstract

This study investigated the molecular phylogenetic characteristics of Bufotes oblongus populations collected from Central Uzbekistan based on mtDNA 16S rRNA gene sequences. The results of phylogenetic analysis showed that all B. oblongus samples, together with reference sequences retrieved from the GenBank database, formed a single monophyletic clade. In addition, genetic distance analysis revealed that the genetic distance among the Gijduvon, Romitan, Nurota, and Konimex populations ranged from 0.002 to 0.006, indicating a high level of genetic similarity among these populations. In the phylogenetic tree, the Gijduvon, Romitan, and Konimex populations formed a closely related cluster, whereas the Nurota population showed a relatively distinct position within the clade. However, the observed differences were not sufficient to consider this population as a separate taxonomic unit. The genetic distance values between B. oblongus populations and other Bufotes species were higher, confirming interspecific genetic divergence. The obtained results demonstrate that B. oblongus populations in Uzbekistan belong to a single species and maintain overall genetic cohesion across the studied regions.

Аннотация

В данном исследовании были изучены молекулярно-филогенетические характеристики популяций Bufotes oblongus, собранных на территории Центрального Узбекистана, на основе последовательностей гена 16S рРНК митохондриальной ДНК. Результаты филогенетического анализа показали, что все образцы B. oblongus вместе с референсными последовательностями, полученными из базы данных GenBank, формируют единую монофилетическую кладу. Кроме того, анализ генетических расстояний показал, что значения генетической дистанции между популяциями Гиждуван, Ромитан, Нурата и Конимех варьировали в пределах 0.002–0.006, что указывает на высокий уровень генетического сходства между этими популяциями. На филогенетическом дереве популяции Гиждуван, Ромитан и Конимех образовали тесно связанный кластер, тогда как популяция Нурата заняла относительно обособленное положение в пределах клады. Однако выявленные различия не были достаточными для выделения данной популяции в отдельную таксономическую единицу. Значения генетической дистанции между популяциями B. oblongus и другими видами рода Bufotes были выше, что подтверждает межвидовую генетическую дивергенцию. Полученные результаты демонстрируют, что популяции B. oblongus в Узбекистане относятся к одному виду и сохраняют общую генетическую целостность на исследованных территориях.

Keywords: Bufotes oblongus, 16S rRNA, mtDNA, phylogenetic analysis, genetic distance, population genetics, Central Uzbekistan.

Ключевые слова: Bufotes oblongus, 16S рРНК, мтДНК, филогенетический анализ, генетическая дистанция, популяционная генетика, Центральный Узбекистан.

Introduction

Members of the family Bufonidae represent an important component of biocenoses and play a significant role in maintaining the stability of food chains. The diverse climatic conditions of Uzbekistan provide a favorable environment for these semi-aquatic amphibians. For the optimal functioning of their life processes, several environmental factors in aquatic habitats (lakes, rivers, wetlands, and irrigation systems in rural areas), such as water temperature, salinity (mineralization level), and vegetation cover, are of crucial importance [1-7-6]. Globally, scientific studies on the molecular phylogenetic characteristics of members of the family Bufonidae have been steadily increasing in recent years. In particular, M. Vences and J.A. Holman conducted molecular genetic analyses of the amphibians of the genus Bufo, investigating the molecular taxonomy of Bufotes pewzowi and its phylogenetic relationships with closely related species [8]. S.N. Litvinchuk and colleagues performed genome-wide multilocus analyses of Bufotes phylogeography. They worked on interpreting the results of multilocus analyses (RAD-seq) in combination with mitochondrial modeling and phenotypic evidence, including bioacoustics, morphometrics, and toxin composition [5]. In Uzbekistan, the toad previously identified as Bufotes pewzowi (Bedriaga, 1898) had long been referred to as Bufo viridis (Laurenti, 1768) [8-2]. These analyses indicate that molecular phylogenetic identification studies of Bufotes pewzowi are of great importance for determining the phylogenetic placement and genetic diversity of species.

The aim of this study is to conduct a molecular phylogenetic analysis of toads belonging to the genus Bufonidae: Bufotes distributed in Central Uzbekistan and to determine their genetic relationships.

Materials and methods

The study materials were collected from various areas of the Bukhara and Navoi regions of the Republic of Uzbekistan using both route-based and stationary sampling methods. Based on biological material obtained from Bufotes oblongus specimens, molecular genetic and phylogenetic analyses were performed using the mitochondrial DNA 16S rRNA gene.

In particular, DNA sequencing was carried out using the ABI PRISM® BigDye™ Terminator v. 3.1 reagent kit, and the reaction products were sequenced at GATC Biotech AG. The obtained nucleotide sequences were analyzed using specialized software packages, including BioEdit, ClustalX2, DNASTAR™, and PAUP*4 [3].

For constructing the phylogenetic tree, amphibian nucleotide sequences obtained from sequencing, as well as DNA sequences retrieved from the International Nucleotide Sequence Database (https://www.ncbi.nlm.nih.gov), were used. These sequences were manually edited using the Geneious Prime software. Consensus sequences were calculated using the MEGA 11 computational program.

The initial data obtained from this program, together with additional sequences retrieved from the NCBI database, were aligned using Clustal Omega 1.2.2 under standard settings and further edited in Geneious Prime. The phylogenetic tree of mtDNA 16S rRNA gene nucleotide sequences was constructed using the maximum likelihood (ML) method with 10,000 ultrafast bootstrap replicates in IQ-TREE version 1.6.12, and the analyses were performed on the CIPRES Science Gateway V3.3 platform.

For phylogenetic reconstruction of Bufotes species based on mtDNA 16S rRNA gene sequences, Peltophryne taladai (AY028495) was used as an outgroup. The resulting phylogenetic tree was visualized and analyzed using the iTOL v6.6 software (https://itol.embl.de/login.cgi) [4].

Results and their analysis

According to the results of the conducted phylogenetic analyses, the phylogenetic tree constructed based on the mitochondrial DNA 16S rRNA gene was analyzed using nucleotide sequences with a length of 609 bp (base pairs). The main nodes of the tree were supported by bootstrap values, with confidence levels ranging from 74% to 100%.

During the study, all Bufotes oblongus samples collected from the Gijduvon and Romitan districts (Bukhara region), as well as the Konimex and Nurota districts (Navoi region), were found to cluster within a single monophyletic clade together with corresponding reference sequences retrieved from the GenBank database. This result indicates the absence of significant genetic divergence between these populations and the reference sequences from global databases, as well as their close evolutionary relationships.

In particular, the phylogenetic tree showed that the samples from Gijduvon and Romitan districts were positioned very close to each other. The Konimex sample was located within a nearby clade, whereas the Nurota sample formed a relatively separate branch within the tree. However, this sample did not fall outside the main Bufotes oblongus clade (Clade I) and did not form an independent evolutionary lineage. Furthermore, bootstrap values indicated that there was no sufficiently deep genetic divergence to justify its classification as a separate species or subspecies. Therefore, the observed genetic variation in the Nurota population was interpreted as intraspecific local genetic variation.

Within Clade II, Bufo viridis samples formed a separate monophyletic group and were found to be phylogenetically closer to the Bufotes oblongus group. In addition, Bufotes sitibundus samples were observed to form an independent cluster within this clade, indicating a clear evolutionary divergence of this group.

Clades III, IV, and V of the phylogenetic tree contained Bufo balearicus, Bufotes boulengeri, and Bufo siculus, respectively. In Clade III, Bufo balearicus samples formed a distinctly separated monophyletic group. In Clade IV, Bufotes boulengeri showed low nucleotide sequence divergence and a high level of sequence similarity, indicating strong genetic stability and phylogenetic cohesion within this clade. In Clade V, Bufo siculus samples were clearly separated from other taxa, forming an independent monophyletic clade.

Figure 1. Phylogenetic tree of representatives of the genus Bufotes (Maximum Likelihood)

Overall, the results of the phylogenetic analysis based on the mitochondrial DNA 16S rRNA gene indicated that Bufotes oblongus populations in Uzbekistan belong to a single species. Although relatively minor genetic differentiation was observed in some local populations within the phylogenetic tree, particularly in samples from the Nurota district, these differences were not sufficient to justify their classification as a separate subspecies. According to the results, all analyzed Bufotes oblongus samples were assigned to a single species (Figure 1).

Genetic distance analysis. The results of genetic distance analysis revealed clear differences in the level of genetic structure and differentiation among populations belonging to the genus Bufotes. Overall, notable variation was observed between intraspecific and interspecific genetic distance values.

In particular, the genetic distance among the Gijduvon, Romitan, Nurota, and Konimex populations of Bufotes oblongus ranged from 0.002 to 0.006. This value indicates a high level of genetic similarity among these populations and suggests that they belong to nearly the same genetic cluster.

Pairwise comparison results (Gijduvon–Romitan, Gijduvon–Nurota, Romitan–Konimex, and Nurota–Konimex) consistently showed minimal genetic differentiation in all cases. Such low values indicate the absence of a pronounced genetic subdivision in the interpopulation genetic structure. This pattern may be explained by ongoing gene flow between these regions or by a relatively recent evolutionary divergence of the populations.

In addition, genetic distance values between B. oblongus populations and other Bufotes species (B. viridis, B. sitibundus, B. balearicus, and B. siculus) were considerably higher, ranging from 0.055 to 0.114. These results indicate clear interspecific genetic differentiation and suggest that these taxa represent evolutionarily distant lineages.

Notably, the highest genetic distance (0.355) was observed between representatives of the genus Bufotes and Peltophryne taladai. This value confirms the strong evolutionary divergence of this taxon from the other studied groups and its position as a distinctly separate phylogenetic lineage.

Figure 2. Genetic distance values among Bufotes populations ranged from 0.002 to 0.355.

Overall, the obtained results indicate that Bufotes oblongus populations form a genetically stable, closely related group with weakly differentiated internal population structure. At the same time, significant interspecific genetic divergence was confirmed, and it can be noted that active phylogenetic and evolutionary branching processes are occurring within the genus Bufotes (Figure 2).

Discussion

Molecular phylogenetic analyses conducted in this study demonstrated that the mitochondrial DNA 16S rRNA gene serves as an important marker for determining the evolutionary relationships of populations belonging to the genus Bufotes. The obtained results are consistent with previous studies reporting the complex phylogenetic structure and high level of genetic diversification within the Bufotes group [2-5].

Studies conducted in Uzbekistan also support these findings. In particular, Aliyev et al. (2025) [1], reported genetic similarity among populations and the formation of monophyletic clades in Bufotes representatives inhabiting aquatic habitats of Central and Southern Uzbekistan, indicating a relatively stable genetic structure of Bufotes species in this region.

In the present study, Bufotes oblongus populations from Gijduvon, Romitan, Konimex, and Nurota were found to cluster within a single monophyletic clade, confirming their common evolutionary origin and genetic integrity. In the phylogenetic tree, the Gijduvon and Romitan populations formed the closest cluster, while the Konimex population was positioned nearby. The Nurota population showed a relatively separate branch; however, it did not fall outside the main clade and did not form an independent evolutionary lineage.

Genetic distance analysis fully supported these results, revealing very low genetic divergence (0.002–0.006) among Bufotes oblongus populations. This indicates a high level of genetic similarity and their assignment to a single genetic cluster. It also suggests the possible continuation of gene flow among populations.

Comparative analysis with other Bufotes and Bufo species revealed clear phylogenetic separation. Bufotes viridis and Bufotes sitibundus formed distinct monophyletic clades, showing significant evolutionary divergence from B. oblongus. Similarly, Bufo balearicus, Bufotes boulengeri, and Bufo siculus were positioned in separate independent clades, indicating a high level of phylogenetic diversification.

Overall, the obtained results confirm that Bufotes oblongus populations in Central Uzbekistan represent a genetically stable and unified evolutionary unit, with very low intraspecific genetic differentiation. At the same time, significant phylogenetic divergence and ongoing evolutionary diversification within the genus Bufotes were observed.

Conclusion

Phylogenetic analyses based on the mitochondrial DNA 16S rRNA gene revealed a high level of genetic similarity among Bufotes oblongus populations in Uzbekistan. Samples collected from Gijduvon, Romitan, Konimex, and Nurota were grouped within a single monophyletic clade, confirming their assignment to a single species. The closest phylogenetic relationship was observed between the Gijduvon and Romitan populations. Although some local genetic differences were detected in the Nurota population, these variations were not sufficient, based on bootstrap values, to justify its classification as a separate subspecies or distinct evolutionary lineage.

Genetic distance analysis also supported the phylogenetic results. In particular, the genetic distance among the Gijduvon, Romitan, Nurota, and Konimex populations ranged from 0.002 to 0.006, indicating a high level of genetic similarity and low genetic divergence among these populations. This confirms that the studied populations belong to a single genetic group and maintain overall genetic cohesion. In the phylogenetic tree, other representatives of the family Bufonidae formed separate monophyletic clades, indicating their independent evolutionary origins. In particular, clear phylogenetic separation was observed in Bufotes sitibundus, Bufotes boulengeri, Bufotes balearicus, and Bufotes siculus. Compared with these taxa, B. oblongus populations showed higher genetic distance values, confirming significant interspecific genetic divergence. Thus, the obtained molecular genetic data demonstrate that Bufotes oblongus populations in Central Uzbekistan belong to a single species, maintain a high level of genetic similarity, and are clearly differentiated from other representatives of the genus Bufotes both genetically and phylogenetically.

Acknowledgements. This research was carried out within the framework of the scientific research program funded by the state budget of the Institute of Zoology of the Academy of Sciences of the Republic of Uzbekistan for 2025–2029, entitled “1.2. Creation of a digital information system of the fauna of the Bukhara and Navoi regions.” The authors express their sincere gratitude to the administration of the Institute for providing the necessary conditions for the successful completion of the planned research work.

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