Abstract
Accurate delineation of biodiversity units is essential for effective conservation, particularly in species complexes with cryptic diversity and fragmented distributions. In this study, we investigated the genetic structure, phylogenetic relationships, and phylogeographic history of Iranian populations within the Vipera ursinii–renardi complex by analysing two mitochondrial genes (cyt b, ND4) and three nuclear loci (PRLR, BDNF, NT3) from 12 individuals sampled across the species’ known range. Multilocus phylogenetic analyses revealed two well-supported evolutionary lineages, including a previously unreported northwestern lineage (Lineage NW), which is sister to the Central Alborz lineage (V. ebneri sensu stricto; Lineage CA). Genetic clustering and haplotype network analyses further supported the distinctiveness of these lineages, indicating limited or no gene flow between them. Molecular-clock dating suggests that the divergence between Lineage CA and Lineage NW occurred during the Late Pleistocene, likely driven by climatic oscillations that promoted ecological isolation. Based on reciprocal mitochondrial monophyly, nuclear differentiation, and geographic separation, we recognize Lineage CA and Lineage NW as two evolutionarily significant units in Iran. Recognizing and conserving this cryptic diversity is vital for preserving the evolutionary legacy of Iranian steppe vipers and for guiding effective conservation strategies under increasing environmental and anthropogenic pressures.
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Introduction
In recent years, conservation efforts have increasingly recognized the importance of defining biodiversity units that reflect evolutionary history, making accurate taxonomy essential for maintaining species richness1,2,3,4. Taxonomic classifications often extend beyond the species level, as a single species may encompass distinct evolutionary lineages, particularly in taxa with wide or fragmented geographic ranges5,6,7,8. Therefore, identifying these lineages through phylogeographical analyses is critical for conservation, as it helps detect cryptic diversity within widespread species and reveal previously unrecognized evolutionary lineages among vertebrates and invertebrates9,10,11. This approach not only improves our understanding of species boundaries but also highlights biologically important units—such as populations with high genetic diversity or those occurring in refugial areas—that are valuable targets for conservation strategies12,13. Identifying cryptic and isolated lineages can enhance conservation management at levels below the species by focusing on distinct evolutionary units11. Ultimately, refining taxonomy and uncovering cryptic diversity enable conservationists to prioritize actions that preserve genetic diversity and adaptability within ecosystems.
The Vipera ursinii-renardi complex, encompassing both mountain and lowland steppe vipers, has an intriguing evolutionary history shaped by Pleistocene climate fluctuations, geographic isolation, and habitat specialization14,15,16. Genetic analyses reveal two major clades, comprising (i) the mountain-adapted “ursinii clade” and (ii) the lowland-dwelling “renardi clade"14,15. Vipera renardi is distributed across the Eurasian steppes and likely originated from ancestral populations in the Balkans. Driven by glacial-interglacial cycles, it spread eastward into the Caucasus and Central Asia17,18. The rapid radiation of V. renardi reflects its resilience to cold, dry steppe environments, enabling demographic expansions into vast lowland regions. In contrast, V. ursinii exhibits a deeper phylogenetic structure and higher genetic diversity17,18. During glacial maxima, its populations became isolated in mountain refugia such as the Carpathians and Balkans. These refugia preserved genetic diversity and fostered subspecies differentiation, emphasizing the clade’s strong association with high-altitude grasslands. The diversification timeline aligns with major climatic transitions of the Quaternary period, highlighting adaptive shifts to grassland and alpine habitats in response to changing environmental conditions14,16,18. The outcomes of this complex evolutionary history, such as deep genetic divergence, fragmented distributions, and ecological specialization, underscore the importance of conserving these genetically distinct populations, which are particularly vulnerable to habitat loss and climate change16,19. Therefore, this species complex serves as an excellent model organism for studying how climatic, geographic, and ecological factors have shaped the biogeography and adaptation of temperate viper species.
Iran is a remarkable center of endemism and cryptic diversity for snakes and lizards, shaped by its diverse ecozones, complex topography, and rich geological history11,20,21. Regions such as the Zagros and Alborz mountains, the Caspian Hyrcanian mixed forests, and the Central Plateau serve as biodiversity hotspots, highlighting the influence of habitat isolation, climatic fluctuations, and mountain formation over millions of years21,22. By acting as both barriers and corridors, these regions foster speciation, allowing populations to adapt to diverse environmental conditions5,21,23. One notable example is the endemic viper V. ebneri Iran, a genetically distinct lineage within the V. ursinii-renardi complex that forms part of the V. eriwanensis-V. ebneri clade14,16,24,25. This population is primarily distributed in the montane regions of Iran and is closely related to its counterpart in the Armenian uplands14,24,25. Recent phylogenetic analyses using mitochondrial DNA have demonstrated the distinctiveness of V. ebneri, emphasizing its importance in understanding the evolutionary and biogeographic dynamics of the V. renardi clade in Southwest Asia14,24,26. However, its taxonomic status remains controversial as some studies recognize it as a separate species, while others suggest it may be nested within V. eriwanensis14,24,27,28. This uncertainty is compounded by limited sampling from Iranian populations and the reliance on mitochondrial DNA, which may not fully capture the species’ genetic diversity14,24. Despite its distinct lineage, the phylogeographic patterns of V. ebneri in Iran remain underexplored, leaving gaps in our understanding of its evolutionary trajectory and the potential impacts of environmental and geographic barriers on its population structure24. This ambiguity highlights the need for comprehensive studies incorporating larger sample sizes and nuclear genetic data to resolve its taxonomic boundaries and inform conservation strategies.
In the present study, we utilized both mitochondrial markers (mtDNA: cyt b and ND4) and nuclear markers (nDNA: PRLR, BDNF, and NT3) commonly applied to the V. ursinii-renardi complex15,17 to investigate the genetic diversity and population structure of steppe Vipera populations in Iran. Our objectives were threefold: (i) to delineate the potential evolutionary lineages of Steppe Vipera across its distributional range in Iran, leveraging a relatively larger dataset from new samples collected throughout its known habitat; (ii) to elucidate the phylogenetic relationships of Iranian Steppe Vipera populations with their congeners and other members of the Vipera ursinii-renardi complex; and (iii) to examine the phylogeographic processes shaping Vipera populations, including the timing of divergence and colonization events in relation to historical climatic and geological factors. These analyses aimed to refine our understanding of species boundaries within the group and to provide new insights into the taxonomy and evolutionary history of Vipera populations in Southwest Asia. Furthermore, by integrating genetic data with ecological and biogeographic contexts, we sought to inform conservation efforts for these enigmatic populations, particularly those facing significant environmental and genetic pressures.
Results
Genetic diversity
To assess the genetic diversity of steppe Vipera populations in Iran, we analyzed two mitochondrial (cyt b and ND4) and three nuclear (PRLR, BDNF, and NT3) sequences from 12 newly collected specimens (Fig. 1). Population groupings were defined based on phylogenetic relationships, which closely reflected their geographic distribution. Accordingly, two genetically distinct lineages were identified within V. ebneri sensu lato: populations from the Central Alborz region (Lineage CA), and populations from northwestern Iran (Lineage NW). Genetic diversity analyses revealed contrasting patterns between the two lineages. Lineage CA, corresponding to V. ebneri sensu stricto, comprises populations from the easternmost part of the species’ range in the Central Alborz (including Lar National Park, Zanjan, and Yush). This lineage exhibits higher genetic diversity, with seven mtDNA haplotypes, a nucleotide diversity of 0.00435, and a haplotype diversity of 0.964. In contrast, Lineage NW, which includes populations from Arasbaran, Oscoo, and Khalkhal, shows lower genetic diversity, with only three mtDNA haplotypes, a nucleotide diversity of 0.00258, and a haplotype diversity of 0.714. A similar pattern is observed in the nuclear data, where Lineage CA has a nucleotide diversity of 0.00228, compared to 0.00128 in Lineage NW (Table 1).
Geographic distribution of the Vipera ursinii–renardi complex (source: IUCN Red List, https://www.iucnredlist.org), highlighting 12 newly collected specimens from six local populations in Iran analyzed in this study. These specimens represent two genetically distinct lineages within V. ebneri sensu lato, identified through phylogenetic analyses: Lineage CA (red dots), corresponding to V. ebneri sensu stricto from the easternmost part of the species’ range in the Central Alborz (Lar National Park, Zanjan, and Yush), and Lineage NW (green dots), from northwestern Iran (Arasbaran, Oscoo, and Khalkhal). The yellow polygon indicates the overall distribution of V. ebneri in Iran based on IUCN Red List data.
Phylogenetic reconstruction
To investigate phylogenetic relationships among steppe vipers from Iran within the V. ursinii–renardi complex, we analyzed concatenated mitochondrial markers (cyt b, ND4) and nuclear markers (PRLR, BDNF, NT3), totaling 3,301 bp, using both Bayesian inference and maximum likelihood approaches. The resulting topologies were congruent across methods and showed strong support for major lineages, with high bootstrap (BS) and posterior probability (PP) values. Within Iran, two genetically distinct sister lineages of V. ebneri sensu lato were identified. Lineage CA (V. ebneri sensu stricto), comprising populations from Lar National Park, Zanjan, and Yush in the Central Alborz, formed a clade distinct from Lineage NW, which includes populations from northwestern Iran (Arasbaran, Oscoo, and Khalkhal). The closely related V. eriwanensis lineage was recovered as sister to these two V. ebneri sensu lato lineages, with strong statistical support (Fig. 2). Within the V. renardi clade, the Crimean lineage received full support as a distinct lineage (BS = 100; PP = 1.0). Similarly, the subgroup comprising V. renardi parursinii (China) and V. lotievi (Chechnya) was strongly supported (PP = 1.0; BS = 98). In the V. ursinii clade, the basal lineage represented by V. ursinii ursinii was well supported (PP = 1.0; BS = 98), while taxa including V. ursinii macrops, V. ursinii rakosiensis, and V. ursinii moldavica clustered together with a PP of 0.99 and a BS of 95 (Fig. 2).
Phylogenetic tree reconstructed using mtDNA and nDNA genes illustrates the evolutionary relationships among V. ursinii-renardi complex species. The tree shows strong branch support, with maximum likelihood bootstrap values displayed below the nodes and Bayesian posterior probabilities above the nodes. Two major Iranian lineages are highlighted: “Lineage NW” (populations from Arasbaran, Osko, and Khalkhal) and V. ebneri sensu stricto (“Lineage CA”; central Alborz populations).
Haplotype network
To further investigate population-level genetic structure among steppe vipers from Iran within the V. renardi clade, we constructed a haplotype network based on mitochondrial genes (cyt b and ND4) (Fig. 3). The network illustrates genetic relationships among Iranian populations and mirrors the phylogenetic results, revealing two distinct haplogroups. One haplogroup consists of individuals from the Central Alborz (Lar, Zanjan, and Yush), while the second includes individuals from northwestern Iran (Arasbaran, Oscoo, and Khalkhal). These haplogroups are separated by 11–14 mutational steps, indicating substantial genetic divergence. The haplogroup corresponding to V. eriwanensis is genetically distinct from both Iranian groups, separated by 16–25 mutational steps (Fig. 3).
Median-joining haplotype network of the Vipera renardi clade. Each circle represents a unique haplotype, with its size proportional to the number of individuals sharing that haplotype. Mutational steps between haplotypes are shown as black lines; when more than one step separates haplotypes, the number of mutations is indicated. Distinct colors denote species or lineages: blue, V. renardi (Crimea); cyan, V. lotievi (Chechnya); red, “Lineage NW” (populations from northwestern Iran: Arasbaran, Osko, and Khalkhal); green, V. ebneri sensu stricto (“Lineage CA”; populations from the central Alborz: Zanjan, Yush, Lar National Park); yellow, V. eriwanensis (Armenia).
Spatial genetic structure
To test the consistency of the genetic groupings identified by the phylogenetic and haplotype analyses, we conducted Bayesian clustering analyses using mitochondrial and nuclear DNA sequences. The Bayesian analysis of spatial genetic structure using mtDNA (cyt b and ND4) identifies three distinct genetic clusters. Vipera eriwanensis from Armenia formed a separate cluster, while the steppe Vipera populations in Iran were divided into two groups consisting of populations from the central Alborz, and the other comprising populations from northwestern populations of Iran (Fig. 4A). These two genetic clusters in Iran were further supported by STRUCTURE analysis based on nDNA genes (PRLR, BDNF, and NT3), which shows minimal admixture between the clusters (Fig S1 and Fig. 4B).
The genetic structure of Vipera populations based on mtDNA (Panel A) and nDNA (Panel B) markers. (A) Spatial genetic structure inferred from Voronoi tessellation, with yellow, V. eriwanensis (Armenia); red, “Lineage NW” (populations from northwestern Iran: Arasbaran, Osko, and Khalkhal); green, V. ebneri sensu stricto (“Lineage CA”; populations from the central Alborz: Zanjan, Yush, Lar National Park). (B) The result of STRUCTURE analysis, where each bar represents an individual’s genetic composition, highlighting clear clustering and minimal admixture among populations.
Molecular divergence time
To elucidate the evolutionary history of Iranian steppe viper populations within the V. ursinii–renardi complex, we reconstructed a time-calibrated phylogeny using BEAST v1.8.2. Divergence time estimates indicate that major lineages within the complex originated during the Pliocene and Pleistocene (Fig. 5). The earliest split, approximately 4.88 Ma (95% Highest Posterior Density [HPD]: 3.91–5.83 Ma), marks the separation of the V. ursinii–renardi complex from V. berus in the Pliocene. Within the V. ursinii clade, V. renardi diverged from the V. ursinii species group around 2.26 Ma (95% HPD: 1.52–3.2 Ma) in the early Pleistocene. Subsequent diversification of both mountain and lowland steppe vipers occurred during the mid-Pleistocene. Around 1.42 Ma (95% HPD: 0.9–2.02 Ma), three major lineages emerged: V. eriwanensis, Lineage NW (northwestern Iran), and Lineage CA (V. ebneri sensu stricto from the Central Alborz). These lineages separated from V. renardi. During the same period, the Crimean lineage of V. renardi originated, along with a subgroup comprising V. renardi parursinii (China) and V. lotievi (Chechnya) (Fig. 5). The two Iranian steppe viper lineages (CA and NW) subsequently diverged from each other in the late Pleistocene, approximately 0.67 Ma (95% HPD: 0.4–1.0 Ma), after previously splitting from V. eriwanensis around 0.9 Ma (Figs. 4 and 95% HPD: 0.56–1.35 Ma).
Divergence time estimation tree for the V. ursinii-renardi complex species, based on mtDNA and nDNA gene sequences. (A) The full dated tree is visualized with calibration points. (B) A pruned dated tree showing only the V. ursinii-renardi complex species. Two lineages from Iran are indicated: “Lineage NW” (northwestern Iran: Arasbaran, Osko, Khalkhal) and V. ebneri sensu stricto (“Lineage CA”; central Alborz: Zanjan, Yush, Lar National Park). Nodes are labeled with mean divergence times (in millions of years, Ma) and 95% highest posterior density (HPD) intervals (in parentheses). The timeline at the bottom highlights the geological periods (Pliocene and Pleistocene).
Evolutionary hypothesis testing
To infer the evolutionary trajectory of steppe Vipera populations in Iran and their sister lineage (V. eriwanensis), we evaluated four plausible scenarios (A–D) using Approximate Bayesian Computation (ABC). Each scenario represents an alternative sequence of divergence events involving V. eriwanensis, Lineage CA (populations from the Central Alborz), and Lineage NW (northwestern populations). In Scenario A, all three lineages—V. eriwanensis, Lineage CA, and Lineage NW—split simultaneously from a common ancestor at time t1. Scenario B proposes that Lineage NW diverged first from V. eriwanensis at time t1, followed by the emergence of Lineage CA from V. eriwanensis at the more recent time t2. In Scenario C, Lineage CA and Lineage NW branched off from each other at time t1, and V. eriwanensis later diverged from Lineage CA at time t2. Finally, Scenario D depicts an early divergence of V. eriwanensis from the common ancestor at time t1, followed by a more recent divergence between Lineage CA and Lineage NW at time t2 (Fig. 6).
Four speciation scenarios tested to explore the evolutionary relationships among Steppe Vipers using ABC analysis implemented in DIYABC. The analysis focuses on two genetic lineages of Steppe Vipers in Iran (Lineage CA and Lineage NW) and Armenian Steppe Vipers (V. eriwanensis). Scenario A: All three lineages (V. eriwanensis, Lineage NW, and Lineage CA) diverge simultaneously from a common ancestor at the same time point (t1). Scenario B: Lineage NW diverges first from the common ancestor at t2. Later, at t1, V. eriwanensis and Lineage CA lineage splits into V. eriwanensis and Lineage CA. Scenario C: V. ebneri (Lineage CA) diverges first at t2. Then, at t1, Lineage CA and V. eriwanensis split from each other. Scenario D: V. eriwanensis diverges first from the common ancestor at t2. At the more recent time t1, the remaining lineage splits into Lineage NW and Lineage CA.
The results of the ABC analysis strongly support Scenario D as the most probable evolutionary hypothesis for the divergence among V. eriwanensis, Lineage NW, and Lineage CA (Fig. 7). The direct estimation of posterior probabilities (left panel) shows that Scenario D consistently achieves the highest probability, stabilizing at approximately 0.4, while Scenarios A and B have intermediate probabilities below 0.3, and Scenario C remains the least probable at less than 0.1. Logistic regression (right panel) provides even stronger support for Scenario D, with posterior probabilities approaching 1.0 across all simulated datasets, while the other scenarios show negligible probabilities. These results indicate that Scenario D, which posits an initial divergence between Lineage NW and V. eriwanensis at time t1, followed by the divergence between Lineage NW and Lineage CA (V. ebneri sensu stricto) at time t2, best explains the observed genetic patterns among these populations.
Posterior probabilities for four evolutionary scenarios inferred through ABC. The left panel shows direct probability estimates, and the right panel displays logistic regression results. Scenario D consistently has the highest support, suggesting it as the most likely evolutionary scenario for the divergence among V. eriwanensis and the two lineages of Iran.
Species delimitation
We conducted a comprehensive set of species delimitation analyses to assess species boundaries within the V. ursinii–renardi complex, with a particular focus on evaluating whether the Steppe Vipera lineages (Lineage NW and Lineage CA) have the potential to be considered distinct species. The analyses revealed varying numbers of delimited species among populations within the V. ursinii–renardi complex based on mtDNA and nDNA data (Fig. 8). GMYC and bPTP, both relying solely on mtDNA, identified 17 species, showing the highest resolution among the methods. ASAP, a distance-based method, was applied separately to each mitochondrial marker and identified 11 putative species based on the cyt b dataset and 10 species based on the ND4 dataset. In contrast, methods incorporating concatenated mtDNA and nDNA data yielded fewer species, with SNAPP delimiting 8 species (Fig S.2) and BPP identifying 11 species.
In the steppe Vipera, however, all species delimitation methods were fairly consistent with each other. All methods detected the divergence of the Iranian Vipera populations (Lineage CA and Lineage NW) from V. eriwanensis. Moreover, with the exception of SNAPP, all methods also detected species boundaries between Lineage CA and Lineage NW.
Species delimitation results for the V. ursinii–renardi complex based on mtDNA and nDNA data using five methods: ASAP, GMYC, bPTP, SNAPP, and BPP. (A) Phylogenetic tree reconstructed using mtDNA and nDNA genes. (B) The varying number of species identified across methods highlights differences in resolution, with mtDNA-based approaches (ASAP, GMYC, bPTP) tending to over-split lineages, whereas concatenated mtDNA and nDNA methods (SNAPP, BPP) provide more conservative species boundaries.
Discussion
Genetic structure and evolutionary lineages in Iranian steppe Vipers
Our study provides new insights into the phylogeographic and evolutionary relationships within the V. renardi clade, focusing on previously underexplored populations in Iran. Using both mitochondrial and nuclear markers, we identified six evolutionary lineages consistent with earlier studies14,15,17,27 and discovered a novel lineage in northwestern Iran. This discovery underscores the role of geographic isolation in driving genetic differentiation and corroborates earlier observations of cryptic diversity in the V. ursinii-renardi complex27. Notably, the newly identified lineage (Lineage NW in northwestern Iran) shows clear genetic differentiation from its sister lineage (Lineage CA in the Central Alborz mountains), mirroring divergence patterns observed among isolated mountain populations of V. ursinii and V. renardi in the Caucasus and Tien Shan regions17,29. Furthermore, the congruence between mitochondrial and nuclear data in distinguishing these lineages align with the findings of Mizsei et al.17, highlighting the importance of multilocus approaches for resolving the complex evolutionary relationships within the V. ursinii–renardi group. In the following sections, we discuss possible explanations for these patterns in the context of the distribution and Quaternary evolution of the V. ursinii–renardi complex.
Phylogenetic relationships and phylogeographic divergence
The results of our time-calibrated phylogenetic analysis, based on concatenated mtDNA and nDNA data, indicate that the V. renardi clade diversified during the Pleistocene glacial periods (at approximately 1.42 Mya). This finding is consistent with previous studies, which have also attributed the evolutionary history of the renardi clade to Pleistocene climatic oscillations14,27,30. During the dry glaciation periods, conditions were favorable for the expansion of dry habitat species like steppe vipers, facilitating their dispersal into new regions27,31. Moreover, the V. renardi clade exhibits a shallow phylogenetic structure, indicative of relatively recent and rapid radiations compared to the V. ursinii clade. Its dispersal routes were predominantly north eastward via the Armenian uplands, resulting in diversification into areas such as the Tien-Shan mountains and the lowland steppes north of the Caucasus26,27. Additionally, the Caucasus region and adjacent mountainous zones served as refugia during colder periods, where older lineages of V. renardi clade survived and diversified14. These adaptive transitions to lowland steppe habitats were likely driven by ecological flexibility and altitude plasticity, enabling the species to thrive during climatic transitions and glacial-interglacial cycles27,32. Notably, the ability of these vipers to exploit diverse habitats underscores their remarkable adaptability in response to rapidly changing environmental conditions. Their range expansions during the Pleistocene not only highlight the dynamic nature of steppe ecosystems but also emphasize the significance of geographic barriers (such as the Caucasus Mountains) in shaping genetic structure27. Furthermore, the evolutionary trajectory of the V. renardi clade offers valuable insights into how small-bodied reptiles can undergo swift radiations and maintain wide distributions under the pressures of glacial-interglacial cycles.
Zinenko et al.27, using mitochondrial DNA genes, revealed that the divergence between V. eriwanensis and V. ebneri was estimated to have occurred approximately 1.03 Mya during the late Pleistocene. This divergence is thought to be closely tied to climatic and geological events in the region that influenced habitat connectivity and isolation. The post-Akchagyl regression, occurring in the late Pleistocene, might have played a crucial role in allowing ancestral populations to disperse across the Caspian and Black Sea basins into the Eastern Caucasus via the dry Kura Bay33,34,35. Following this dispersal, oscillating climatic conditions during the Pleistocene, such as glacial and interglacial periods, likely created ecological barriers and fragmented populations36, leading to the eventual divergence of these two lineages. Similarly, our dated tree based on concatenated mtDNA and nDNA indicates that V. eriwanensis diverged from Lineage NW (northwestern Iran) and Lineage CA (V. ebneri senso stricto in the Central Alborz) approximately 0.9 Mya, supporting the hypothesis that geographic isolation and ecological pressures during the Pleistocene were pivotal in driving the genetic differentiation of these populations. These findings underscore the interplay of climatic and tectonic events in shaping the evolutionary trajectories of species in this region.
Our molecular clock analysis shows that V. ebneri sensu stricto (Lineage CA) in Iran diverged from the northwestern Iranian lineage (Lineage NW) approximately 0.6 Mya, during the late Pleistocene. Additionally, species distribution modelling suggests that gene flow occurs from the Armenian steppe viper to the northwestern region of Iran and extends into the Alborz Mountains, where geographic isolation likely formed a barrier between Lineage CA and Lineage NW. Northwestern Iran emerges as a key corridor and biogeographical area that has facilitated the movement and diversification of various species5,20,21,37,38. This region, encompassing the Alborz and Azerbaijan mountains, served as an important refugium for cold-adapted species during glacial periods21,39. Several species with Mediterranean origins, such as Coronella and Vipera, utilized this region as a biogeographical corridor, enabling their persistence and subsequent diversification39. The climatic oscillations marked by alternating glacial and interglacial periods, likely caused substantial habitat fragmentation, promoting geographic isolation along the Alborz and Azerbaijan mountains20. Such isolation, coupled with the presence of distinct alpine steppe habitats, provided favorable conditions for genetic divergence and speciation. These highlands’ role as refugia during climatic shifts is further underscored by their notable biodiversity and endemism. Therefore, these findings align with broader evidence highlighting the Alborz mountains and adjacent regions as refugia for several reptile species5,20, underscoring the profound influence of historical climatic fluctuations on shaping the biogeography and evolution of Iranian herpetofauna.
Lineage divergence and species delimitation
The taxonomy of the V. ursinii–renardi complex has been a source of ongoing controversy due to conflicting interpretations of morphological and molecular evidence. Relying solely on morphology can be problematic, as traits such as scale patterns and body size may retain ancestral characteristics (plesiomorphies), which can obscure evolutionary relationships17,27. On the other hand, molecular studies, particularly those based on mitochondrial DNA, have added complexity by revealing unexpected phylogenetic placements. These challenges highlight the need for integrative approaches that combine nuclear and mitochondrial data with morphological and ecological evidence to resolve taxonomic ambiguities. Using this comprehensive approach, Mizsei et al.17 re-evaluated the Greek Meadow Viper (Vipera graeca), ultimately elevating it to full species status.
In the present study, we did not collect morphological measurements to minimize handling time and reduce stress to the sampled vipers, allowing for their immediate release back into their habitats40. Nevertheless, Rajabizadeh et al.41 provided the first report of steppe viper populations in northwestern Iran, examining four preserved specimens from Kiamaki Mountain, Sabalan Mountain, and Susahab village. They found no significant morphological differences between V. eriwanensis and these local populations, suggesting that V. eriwanensis extends its distribution into northwestern Iran. However, based on features such as ventral and subcaudal scales and supralabial patterning, Rajabizadeh et al.41 observed distinct differences between V. eriwanensis and the V. ebneri population in the Alborz mountains, noting that V. ebneri has fewer ventral and subcaudal scales and lacks the darkly spotted supralabials typical of V. eriwanensis. In agreement with the morphological findings, our genetic structure and phylogeny analyses confirm that V. ebneri and V. eriwanensis are distinct from each other. However, in contrast to those morphological data, our results suggest that steppe viper populations in northwestern Iran (Lineage NW) have diverged from V. eriwanensis and appear more closely related to V. ebneri as a sister lineage.
In addition, our study conducted various species delimitation analyses, combining mtDNA and nDNA, to identify potential species within the V. ursinii–renardi complex. The results from the concatenated data suggested a slightly lower number of candidate species compared to those identified using non-recombining mtDNA. The findings also revealed that, while different species delimitation methods produced largely consistent species boundaries, they were not entirely identical. Carstens et al.42 argued that such discrepancies may result either from differences in the methods’ power to detect cryptic lineages or from violations of the assumptions underlying the species delimitation approaches. They advised caution when interpreting species delimitation results to avoid defining entities that do not accurately represent evolutionary lineages. Taking a conservative approach, this study identified at least eight putative species within the V. ursinii–renardi complex. Notably, the steppe viper from the northwestern populations (Lineage NW) was recognized as a separate species by four out of five methods, while Lineage CA was consistently identified as a distinct species by all five methods.
Therefore, our findings reaffirm the validity of V. ebneri as a full species, as previously proposed by Ferchaud et al.14. Additionally, based on the morphometric analysis by Rajabizadeh et al.41, which highlighted significant differences between lineage NW (northwestern populations of Iran) and lineage CA (V. ebneri sensu stricto from the Central Alborz), our molecular data strongly support lineage CA as a candidate for elevation to a distinct endemic species for Iran. In contrast, the Steppe Vipera population from northwestern Iran may not yet warrant full species status, as its morphology closely resembles V. eriwanensis, despite being genetically recovered as a sister lineage to V. ebneri. Therefore, a comprehensive phylogenomic study—integrating the data from Dufresnes et al.26 and incorporating broader sampling of Steppe Vipera populations across Iran—is still required to reach a definitive conclusion regarding the taxonomic status of these northwestern populations.
Conservation implications and evolutionarily significant units
Current conservation measures for Iranian venomous snakes fail to account for their revised taxonomic, genetic, and distributional status, despite the fact that many of these taxa are threatened. Conservation planning in Iran has predominantly centred on large mammals and, to a lesser extent, migratory birds, while largely overlooking the country’s diverse reptile and amphibian fauna43. Vipera ebneri sensu lato is currently listed as a Vulnerable species on the IUCN Red List44, yet only a small portion of its known range, specifically populations from the central Alborz region (Lineage CA), falls within protected areas, such as Lar National Park. Furthermore, not all populations of Steppe Vipera have been identified or adequately assessed. Our study revealed a previously unrecognized lineage in northwestern Iran (Lineage NW), which had been misclassified based on morphological data as a population of V. eriwanensis41. However, our genetic analyses show that this lineage is deeply divergent and forms a sister group to V. ebneri. Given its restricted distribution and taxonomic distinctiveness, this lineage may face an increased risk of local extinction in the near future due to threats such as overexploitation for venom extraction and habitat degradation.
In the present study, we show that both major Iranian steppe-viper lineages (Lineage CA and Lineage NW) meet the criteria for designation as Evolutionarily Significant Units (ESUs). ESUs are populations that represent historically independent evolutionary lineages and therefore harbour irreplaceable components of a species’ genetic diversity45,46. They are typically diagnosed by (i) reciprocal monophyly of mitochondrial haplotypes, (ii) significant differentiation at nuclear loci, and (iii) evidence of long-term demographic or geographic isolation47. All three criteria are met by the two lineages identified in our study, underscoring their independent evolutionary trajectories and their importance for preserving the full genetic legacy of the species complex. As such, these lineages should be managed separately in any conservation or breeding programs. In particular, populations from Lineage CA and Lineage NW should not be artificially mixed—whether through captive breeding, venom extraction programs, or translocations—as doing so could erode their genetic integrity and compromise locally adapted traits. Instead, conservation actions should recognize these ESUs as distinct management units, ensuring that future interventions maintain the evolutionary and ecological uniqueness of each lineage.
Materials and methods
Permission for sampling was granted by the Department of Environment of Iran (license no. 1400.420.13938). Tissue samples from 12 V. ebneri specimens were collected in northern and northwestern Iran between 2020 and 2022 (Fig. 7). For each specimen, three scale clippings were carefully collected from the outer layer of the ventral scales to minimize harm. Following the collection process, all snakes were immediately released at their original capture locations to ensure minimal disruption to local populations. The collected tissue samples were preserved in ethanol and stored at −5 °C to maintain DNA integrity. All procedures adhered to relevant ethical guidelines and approved regulations, emphasizing the importance of minimizing the ecological impact of sampling. In addition to these 12 new samples, we included 46 additional samples representing 10 species of Vipera from GenBank to provide a broader phylogenetic and comparative context. These included two samples of V. berus berus, one sample of V. berus bosniensis, one sample of V. berus nikolskii, two samples of V. ursinii ursinii, two samples of V. ursinii ssp., three samples of V. ursinii macrops, three samples of V. ursinii rakosiensis, two samples of V. ursinii moldavica, two samples of V. renardi, and three samples of V. graeca (Table S1).
DNA extraction and amplification
DNA extractions were carried out using the DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer’s instructions. The integrity and quantity of the isolated DNA were assessed by electrophoresis on a 0.8% agarose gel and quantified using a NanoDrop spectrophotometer. We selected two mtDNA markers; cytochrome b (cyt b) and NADH dehydrogenase subunit 4 (ND4), and three nDNA; brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and prolactin receptor (PRLR). These genes have been effective in discriminating among various divergence levels in reptile species17,48,49.
PCR amplification of the mitochondrial genes cyt b and ND4 followed established protocols optimized for the V. ursinii–renardi species complex14. The cyt b gene was amplified using the primers L14724Vb (5′-GATCTGAAAAACCACCGTTG-3′) and H15914Vb (5′-CCAGCTTTGGTTTACAAGAAC-3′), originally designed based on multiple published reptilian mitochondrial sequences50,51,52. The ND4 region was amplified using the forward primer ND4 (5′-CACCTATGACTACCAAAAGCTCATGTAGAAGC-3′) and the reverse primer H12763V (5′-TTCTATCACTTGGATTTGCACCA-3′), adapted from Arévalo et al.53. These primers were originally developed for Sceloporus lizards but widely applied and validated for Vipera species by Wüster et al.54 and Ferchaud et al.14. The PRLR gene was amplified using primers PRLR_f1 and PRLR_r349, with sequences 5′-GACARYGARGACCAGCAACTRATGCC-3′ and 5′-GACYTTGTGRACTTCYACRTAATCCAT-3′, respectively. The BDNF gene was amplified using primers BDNFf and BDNFr49, with sequences 5′-GACCATCCTTTTCCTKACTATGGTTATTTCATACTT-3′ and 5′-CTATCTTCCCCTTTTAATGGTCAGTGTACAAAC-3′, respectively. These PRLR and BDNF primers were originally developed from the Anolis carolinensis (green anole) genome and have been shown to amplify across diverse squamate lineages (e.g., iguanian, scincomorph, and anguimorph lizards)49.PCR conditions for PRLR and BDNF included an initial denaturation at 94 °C for 3 min, followed by 40 cycles of denaturation at 94 °C for 40 s, annealing at 50 °C for 30 s, and extension at 72 °C for 60 s, with a final extension at 72 °C for 7 min. The NT3 gene was amplified using primers NT3-F3 and NT3-R455,with sequences 5′-ATATTTCTGGCTTTTCTCTGTGGC-3′ and 5′-GCGTTTCATAAAAATATTGTTTGACC-3′, respectively. These NT3 primers were originally described for squamate reptiles by Noonan and Chippindale55,based on conserved exon regions from Anolis carolinensis, Python molurus bivittatus, and Boa constrictor, and have since been widely used in snake systematics. PCR conditions for NT3 were similar, except the annealing temperature was set at 48 °C. All PCR products were purified and sequenced bidirectionally using the same primers as for amplification. Sequencing was performed using direct double-strand cycle sequencing on a 3730xl DNA Analyzer (Applied Biosystems) at the DNA Sequencing Core Facility in South Korea.
Analysis of mitochondrial and nuclear DNA sequences
The quality of all Sanger sequencing reads was assessed and trimmed based on their chromatograms using Geneious Prime software v. 2024.0.7. For each gene, reference sequences were identified through BLASTn56 searches against the NCBI nucleotide database using default parameters. Heterozygous positions in the nuclear genes were manually identified by examining chromatograms for double peaks, indicating the presence of two different nucleotides at a single position. These heterozygous loci were coded using standard IUPAC ambiguity codes to represent nucleotide variations. For the construction of allele networks, sequences with more than one heterozygous position detected as two peaks of approximately equal height at single nucleotide sites in the BDNF, NT3, and PRLR genes were resolved into individual alleles using PHASE v. 2.1.157. Input data for PHASE were prepared using SeqPHASE58, which formats the data appropriately for haplotype reconstruction. PHASE was run with default settings except for the probability threshold, which was set to 0.7 to ensure a higher confidence level in the phased alleles.
All newly assembled gene sequences were aligned with other V. ursinii-renardi complex sequences from the NCBI database using the ClustalW algorithm59 in MEGA software v. 560. Furthermore, we evaluated a range of genetic statistics to characterize the genetic diversity within and between populations. These statistics included the number of haplotypes (h), haplotype diversity (Hd), number of polymorphic sites (S), nucleotide diversity (π), and the average number of nucleotide differences (K). All these genetic analyses were performed using DnaSP v. 5.161.
Phylogenetic reconstruction and genetic clustering
To facilitate comprehensive downstream phylogenetic analyses, sequences of the two mtDNA gene fragments (cyt b: 787 bp; ND4: 801 bp) and the three nuclear genes (PRLR: 552 bp; BDNF: 662 bp; NT3: 496 bp) were concatenated to yield a matrix 3,301 bp in length. The best-fitting substitution models and partition schemes for the mtDNA and nDNA genes were determined using the greedy algorithm and the corrected Akaike Information Criterion (AICc) in PartitionFinder v. 2.1.162.
Phylogenetic relationships among the Vipera populations from Iran and other vipers of the V. ursinii-renardi complex were reconstructed using Bayesian inference (BI) in MrBayes v. 3.1.263 and maximum likelihood (ML) methods in IQ-TREE64. For the BI analysis, codon-based partitioning was applied with substitution models as detailed in Table S2. Markov Chain Monte Carlo (MCMC) simulations were run for 40 million generations, sampling every 1,000 generations, and the first 25% of samples were discarded as burn-in to ensure the analyses were based on the stationary phase of the chain. Convergence of the MCMC runs was assessed by examining the average standard deviation of split frequencies and ensuring it fell below 0.01.
Additionally, effective sample sizes (ESS) were evaluated using Tracer v1.765, with ESS values > 200 considered adequate. In the ML analysis conducted with IQ-TREE, the nucleotide substitution models, and partitioning schemes specified in Table S2 were employed. Branch support was evaluated using 1,000 ultrafast bootstrap replicates66 to provide reliable estimates of node stability. Phylogenetic trees were visualized and edited using FigTree v. 1.3.167.
Genetic clustering analyses
To elucidate the population genetic relationships between V. ebneri in Iran and other closely related taxa within the V. renardi clade, we reconstructed haplotype networks using the median-joining algorithm implemented in PopArt v. 1.768, based on mtDNA sequences cyt b and ND4 genes. Moreover, we estimated the spatial clustering of individuals using a Bayesian approach in BAPS v. 6.0 (69; Bayesian Analysis of Population Structure), followed by population mixture analysis to identify genetic clusters. To determine the optimal number of clusters (K), we explored values ranging from 1 to 20 and selected the best-fit K based on log marginal likelihood scores.
To assess whether the genetic clustering inferred from mtDNA data was consistent with nuclear nDNA markers, we conducted analyses using the program STRUCTURE v. 2.3.470. All Vipera samples from Iran were included in this analysis. We converted the genotype data to STRUCTURE format using Fasta2Structure71. In the STRUCTURE analyses, we assumed an admixture model with correlated allele frequencies. The lambda (λ) parameter, representing the Dirichlet prior for allele frequencies, was fixed at the value estimated by initially running STRUCTURE with K = 1 and allowing λ to be inferred. We analyzed the data for K values from 1 to 5, performing five replicates for each K to ensure consistency. Each run consisted of a burn-in period of 100,000 iterations followed by 1 million Markov Chain Monte Carlo (MCMC) iterations. The optimal number of clusters was determined using the ΔK method described by Evanno et al.72, which identifies the most likely K based on the rate of change in the log probability of data between successive K values. Visualization of the STRUCTURE output was performed using DISTRUCT software v. 2.373, allowing for graphical representation of individual assignments to genetic clusters.
Molecular dating
To estimate divergence times between Steppe Vipera populations in Iran and other species from the V. ursinii–renardi complex, molecular dating analyses were conducted using BEAST v. 1.8.274. The analysis was based on five gene regions (cyt b, ND4, PRLR, BDNF, and NT3), resulting in a total alignment of 3,301 base pairs. The best-fitting substitution models and partition schemes for the dataset were identified using PartitionFinder v. 2.1.162 (see Table S2). Each gene and each codon position for protein-coding genes was assigned to a separate partition. In BEAUti, substitution models were unlinked across partitions, while a single uncorrelated log-normal relaxed clock model and a birth–death tree prior were applied across the dataset to accommodate rate heterogeneity and speciation processes.
Three calibration points were applied to estimate divergence dates within the Viperidae family, following the approach of Wüster et al.54. The first calibration point corresponded to the uplift of the Panamanian Isthmus, which separated populations of the genus Porthidium approximately 3.5 million years ago (Ma)75. This event was modeled using a normal distribution with a mean of 3.5 Ma and a standard deviation of 0.51 Ma, providing a 95% confidence interval of 2.5–4.5 Ma. The second calibration point involved the initial divergence of the Eurasian viper clade (comprising the genera Macrovipera, Montivipera, and Vipera), dated to 20 Ma based on fossil evidence76. This calibration was modeled with a lognormal prior, a zero offset of 20 Ma, a lognormal mean of 1, and a lognormal standard deviation of 1. The third calibration point was the divergence between the genera Sistrurus and Crotalus, estimated to have occurred before 9 Ma based on fossil vertebrae of Sistrurus77. This point was also modeled with a lognormal prior, a zero offset of 9 Ma, a lognormal mean of 1, and a lognormal standard deviation of 1. The BEAST analysis was run for 200 million MCMC, sampling every 5,000 generations. The first 25% of samples were discarded as burn-in to ensure the analyses were based on the stationary phase of the MCMC runs. Convergence and ESS above 250 were assessed using Tracer v. 1.7 to confirm adequate mixing and stationarity.
Speciation scenario modeling using approximate bayesian computation
To investigate the speciation and gene flow between V. ebneri sensu lato populations in Iran and V. eriwanensis populations in Armenia, we employed Approximate Bayesian Computation (ABC) using DIYABC software v. 2.1.078, which evaluates the most likely hypothesis among multiple evolutionary scenarios by comparing observed genetic parameters (priors) with simulated data. Four evolutionary scenarios were defined to estimate divergence times and potential gene flow among these populations (Fig. 8). In Scenario A, V. eriwanensis and V. ebneri senso lato (Lineage CA and Lineage NW) diverged simultaneously from a common ancestor at time t1. Scenario B hypothesizes that the northwestern population (Lineage NW) diverged first from the common ancestor at t2, followed by a split between V. eriwanensis and Lineage NW at t1. Scenario C proposes that Lineage CA diverged first from the common ancestor at t2, with V. eriwanensis and Lineage NW diverging later at t1. Finally, Scenario D suggests that V. eriwanensis diverged first from the ancestral population at t2, followed by a divergence between Lineage CA and the Lineage NW at t1. The most probable scenario was identified using posterior probability values and logistic regression to compare the observed data with simulated models.
The analysis was conducted mtDNA haplotype markers due to the unavailability of nuclear nDNA data for V. eriwanensis. Mutation models appropriate for haplotype markers were applied, with all loci modeled under the infinite sites model (ISM). Prior distributions were defined for demographic parameters, including divergence times (t1 and t2), effective population sizes, and mutation rates78. Mutation rates were set within a range of 10 − 8 to 10− 9 substitutions per site per generation, consistent with typical rates for mtDNA5,79.
Coalescent-based simulations were performed for each scenario to generate 10,000,000 datasets. Summary statistics, including haplotype frequencies, genetic diversity indices, and pairwise FST values, were computed for both the observed and simulated datasets to evaluate the fit of each scenario. Posterior probabilities were estimated by comparing the observed data to the simulated datasets, retaining the 1% of simulations that most closely matched the observed data (tolerance level of 0.01). The scenario with the highest posterior probability was identified as the most likely evolutionary model and validated through cross-validation analyses and goodness-of-fit tests. To ensure robustness, pseudo-observed datasets (PODs) were generated to estimate Type I and Type II error rates, providing additional support for the reliability of the selected scenario80.
Species delimitation
To identify species boundaries within the V. ursinii–renardicomplex, we performed various species delimitation analyses using mtDNA and nDNA sequencing data. Initially, we applied single-locus species delimitation methods for mtDNA, employing both tree-based and genetic distance approaches. Species delimitation was conducted using three independent methods: the Generalized Mixed Yule Coalescent model (GMYC81,82;), the Bayesian implementation of the Poisson Tree Processes model (bPTP83;), and Assemble Species by Automatic Partitioning (ASAP84;).
To generate an ultrametric gene tree for analysis, we constructed a phylogenetic tree based on the concatenated alignment of the cyt b and ND4 genes using BEAST v1.8.2. The analysis was conducted under an uncorrelated lognormal relaxed molecular clock assumption, with a constant population size coalescent model as the tree prior. The UCLD mean prior followed an exponential distribution with a mean of 10 and an initial value of 1. We performed two independent runs, each consisting of 100 million generations, with sampling every 5000 generations. Convergence and sampling adequacy were evaluated using Tracer v1.6, ensuring ESS values exceeded 200. To refine the results, the first 20–25% of trees were discarded as burn-in, and the remaining trees were combined using LogCombiner v1.8.285. A maximum clade credibility (MCC) tree with mean heights was generated using TreeAnnotator v1.8.286. The GMYC approach distinguishes interspecific stochastic birth-death processes (pure-birth Yule process) from intraspecific neutral coalescent processes by examining the timing of branching events in single-gene trees. This method requires a well-sampled, accurately estimated ultrametric tree that represents true species genealogy, independent of population structure and size fluctuations. To assess the fit of our data to the GMYC model, we used the P2C2M.GMYC R package87. The GMYC single-threshold analysis was performed in RStudio v4.288 using the Splits R package89, with the ultrametric tree from the BEAST analysis as input. To avoid branches of length zero, we applied the function multi2di. Unlike GMYC, the bPTP method does not require an ultrametric tree and instead relies on a standard phylogenetic tree. For this analysis, we used the maximum likelihood (ML) phylogenetic tree reconstructed in IQ-TREE and ran the bPTP analysis using the online server at http://species.h-its.org/. For genetic distance-based delimitation (ASAP), the concatenated mitochondrial DNA (cyt b and ND4) was treated as a single partition to infer putative species. ASAP clusters specimens into species using pairwise genetic distances without requiring predefined hypotheses. The concatenated sequences of both genes were uploaded to the web server at https://bioinfo.mnhn.fr/abi/public/asap, and the analysis was conducted using uncorrected pairwise genetic distances. Species groups were identified based on a probability threshold of less than 0.01.
To compare species delimitation models based on the concatenated mtDNA and nDNA dataset, we employed Bayes Factor Delimitation (BFD90,) analyses using the SNAPP package implemented in BEAST291,92. This approach uses marginal likelihood scores to evaluate competing models by calculating Bayes factors within the multispecies coalescent (MSC) framework. We examined a nested series of hypotheses, ranging up to the maximum number of potential species, with each lineage treated as a distinct species.
BFD analyses were conducted in BEAST2 using SNAPP with fixed unsampled mutation rates (u, v) set to 1, alpha at 1, beta at 250, and lambda at 20. The coalescence rate was initially set at 10 but was allowed to vary during the analysis. Default settings were applied for all remaining parameters. Marginal likelihoods for each candidate model were estimated through path sampling using 48 steps, with subsequent MCMC runs executed for 200,000 generations, sampled every 1,000 generations. We ranked and compared the resulting marginal likelihoods using Bayes factors93.
To assess the influence of prior settings, we repeated analyses with default priors for mutation rates (u, v), alpha, beta, lambda, and coalescence rate. The highest-ranked model from the BFD analysis was then used for final species tree estimation with SNAPP. For this final estimation, the MCMC analysis ran for 1,000,000 generations, with sampling every 1,000 steps. Tracer was employed to verify convergence and ensure high effective sample size (ESS) values. Finally, an MCC tree was constructed using TreeAnnotator v.1.7.5, discarding the first 25% of samples as burn-in.
Additionally, we applied a multi-locus approach for species delimitation using the Bayesian Phylogenetics and Phylogeography (BPP) program, v. 3.494. This analysis incorporated mtDNA markers (cyt b and ND4) and nDNA markers (PRLR, BDNF, and NT3) within the framework of the multispecies coalescent model. This model is particularly effective in accounting for incomplete lineage sorting and gene tree heterogeneity, making it ideal for resolving complex evolutionary relationships. The BPP analysis was initiated with a random starting tree, ensuring unbiased exploration of possible tree topologies. Uniform priors were assigned to substitution rates (0, 100) and gamma shape parameters (0, 10) to account for rate variation among sites, reflecting the heterogeneity in evolutionary dynamics across loci. These priors were chosen based on recommendations from the BPP manual and empirical studies to balance computational efficiency and model realism. Scenario 2, which hypothesizes population divergence with gene flow, was implemented to test phylogeographic structure and evaluate whether gene flow has contributed to genetic differentiation among populations94. This scenario is particularly relevant for investigating recently diverged lineages where migration may obscure clear genetic boundaries. The analysis was run under the reversible-jump MCMC algorithm to explore the posterior probabilities of alternative species delimitations, allowing for direct comparison of different hypotheses about species limits. Convergence of the MCMC chains was assessed by running multiple independent replicates and checking for consistency in posterior probabilities. Additionally, posterior predictive checks were performed to validate the fit of the model to the data, ensuring robust and reliable species delimitation94.
Data availability
The datasets generated during the current study are available from the corresponding author on reasonable request. All sequence data are deposited in NCBI under accession codes PV330333 - PV330392.
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Acknowledgements
We are grateful to the conservation managers of the Department of Environment of Iran and rangers of protected areas in northern and northwestern Iran for their help and expertise during the fieldwork. We would like to thank Mohammad Reza Masoud for his contribution to fieldwork and Faezeh Fatemizadeh for providing insightful comments on the manuscript. Computational resources were provided by the e-INFRA CZ project (ID:90254), supported by the Ministry of Education, Youth and Sports of the Czech Republic.
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Study concept and design: MK, MN, and SAJ. Acquisition of data and sampling: MF and MK. Data analyses: MF, MN, and MK. Conducting lab works: MF and MN. Writing of the manuscript: MF, MN, SAJ, and MK. Obtaining research permits: MK and MZ. Finalizing the manuscript: MN, SAJ, MZ, and MK. All authors read and approved the final manuscript. MF: Mohammad Faridie (mfaridi@iau.ac.ir)SAJ: Seyed Ali Jozi (sa.jozi@iau.ac.ir)MZ: Mojgan Zaeimdar (mojganzaeimdar@iau.ac.ir) MN: Masoud Nazarizadeh (Masoud.Nazarizadeh@paru.cas.at)MK: Mohammad Kaboli (mkaboli@ut.ac.ir).
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Sample collection required approval from the Department of Environment of Iran. The research ethics committee at the zoological museum of the Faculty of Natural Resources and Environment of the University of Tehran (NEZMUT) certifies that this study was carried out according to the ethical framework and regulations of the sampling permit (No. 1400.420.13938) issued by the Department of Environment of Iran.
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Faridi, M., Jozi, S.A., Zaeimdar, M. et al. Molecular phylogeny and species delimitation unravel climate-driven diversification and cryptic radiation in Iranian steppe Vipers (Vipera ursinii-renardi complex). Sci Rep 15, 42148 (2025). https://doi.org/10.1038/s41598-025-26158-y
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DOI: https://doi.org/10.1038/s41598-025-26158-y
