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.

Department of Environment NT C Islamic Azad University Tehran Iran

Department of Environmental Science Faculty of Natural Resources College of Agric and Natural Resources University of Tehran Tehran Iran

Faculty of Science University of South Bohemia České Budějovice Czech Republic

Institute of Parasitology Biology Centre CAS České Budějovice Czech Republic

Zobrazit více v PubMed

Rao, M. et al. Environmental and evolutionary drivers of diversity patterns in the tea family (Theaceae s.s.) across China. PubMed DOI PMC

Fu, Q. et al. Linking evolutionary dynamics to species extinction for flowering plants in global biodiversity hotspots. DOI

Foale, S. Who cares about coral? The biological species concept and ‘cumulative intrinsic value’ in cross-cultural perspective. DOI

Qian, H., Kessler, M., Zhang, J., Jin, Y. & Jiang, M. Evolutionary causes of global patterns of species richness in regional fern floras across the world. DOI

Behrooz, R. et al. Conservation below the species level: suitable evolutionarily significant units among mountain Vipers (the PubMed DOI

Malaney, J. L. & Cook, J. A. Using biogeographical history to inform conservation: the case of preble’s meadow jumping mouse. PubMed DOI

Baird, A. B. et al. On the utility of taxonomy to reflect biodiversity: the example of Lasiurini (Chiroptera: Vespertilionidae). DOI

Nazarizadeh, M., Martinů, J., Nováková, M., Stanko, M. & Štefka, J. Phylogeography of the parasitic mite PubMed DOI PMC

Crowther, M. S., Fillios, M., Colman, N. & Letnic, M. An updated description of the Australian Dingo ( DOI

Creighton, M. J. A., Luo, A. Q., Reader, S. M. & Mooers, A. Ø. Predictors of taxonomic splitting and its role in primate conservation.

Kamali, K. et al. Integrating phylogenetic, phylogeographic, and morphometric analyses to reveal cryptic lineages within the genus PubMed DOI PMC

Apurva, J., Deepika, G. & Rakesh, V. Macro-invertebrate diversity in relation with habitat ecology with special reference to fresh water: A review. DOI

Bofill, S. I. H., Mayer, F. & Thong, V. D. (2024). Bat diversity in the Cuc Phuong National Park, Vietnam – Results from VIETBIO field training and annotated species list. PubMed PMC

Ferchaud, A. L. et al. Phylogeography of the DOI

Mizsei, E. et al. Species distribution modelling leads to the discovery of new populations of one of the least known European snakes, DOI

Lucchini, N., Kaliontzopoulou, A., Lourdais, O. & Martínez-Freiría, F. Climatic adaptation explains responses to pleistocene oscillations and diversification in European Vipers. DOI

Mizsei, E. et al. Nuclear markers support the mitochondrial phylogeny of PubMed DOI

Mizsei, E. et al. The distribution of meadow and steppe Vipers (Vipera graeca, V. renardi and V. ursinii): A revision of the new atlas of amphibians and reptiles of Europe. DOI

Luiselli, L. Meadow viper, Vipera ursinii, in italy: aspects of conservation ecology (Reptilia, Viperidae). DOI

Asadi, A. et al. Evolutionary history and postglacial colonization of an Asian pit Viper ( PubMed DOI PMC

Moradi, N., Joger, U., Bafti, S., Sharifi, S., SehhatiSabet, M. E. & A. & Biogeography of the Iranian snakes. PubMed DOI PMC

Chen, Y. et al. The niches of nuthatches affect their lineage evolution differently across latitude. PubMed DOI

Aghbolaghi, M. A., Ahmadzadeh, F., Kiabi, B. & Keyghobadi, N. The permanent inhabitant of the oak trees: phylogeography and genetic structure of the Persian squirrel ( DOI

Freitas, I. et al. Evaluating taxonomic inflation: towards evidence-based species delimitation in Eurasian Vipers (Serpentes: Viperinae). DOI

Martínez-Castro, A., Kaliontzopoulou, A., Freitas, I. & Martínez-Freiriá, F. Macroevolutionary variation and environmental correlates of scalation traits in Eurasian Vipers (Serpentes: Viperinae). DOI

Dufresnes, C. et al. Phylogenomic insights into the diversity and evolution of Palearctic Vipers. PubMed DOI

Zinenko, O. et al. Mitochondrial phylogeny shows multiple independent ecological transitions and Northern dispersion despite pleistocene glaciations in meadow and steppe Vipers ( PubMed DOI

Iskenderov, T. M. & Najafov, J. A. Present distribution and numbers of the Eastern steppe Viper ( DOI

Vörös, J. et al. Well-known species, unexpected results: high genetic diversity in declining DOI

Zinenko, O., Sovic, M., Joger, U. & Gibbs, H. L. Hybrid origin of European Vipers ( PubMed DOI PMC

Ratnikov, V. Y. Fossil remains of modern amphibian and reptile species as the material for studying of their areas history.

Bartonova, A. et al. Range dynamics of palaearctic steppe species under glacial cycles: the phylogeography of

Trifonov, V. G. et al. Geological and biotic context of the Plio-Pleistocene evolution of the Caucasus–Caspian region (Akchagylian transgression). DOI

Richards, K. et al. Palynology and micropalaeontology of the Pliocene–Pleistocene transition in outcrop from the Western Caspian sea, azerbaijan: potential links with the Mediterranean, black sea and the Arctic ocean? DOI

Forte, A. M. et al. Late miocene to pliocene stratigraphy of the Kura Basin, a subbasin of the South Caspian basin: implications for the diachroneity of stage boundaries. DOI

Kroonenberg, S. B. et al. Pleistocene connection and Holocene separation of the Caspian and Black Seas: Data from the modern Kura Delta, Azerbaijan. In

Ashrafzadeh, M. R., Kaboli, M. & Naghavi, M. R. Mitochondrial DNA analysis of Iranian brown bears ( DOI

Yusefi, G. H., Safi, K. & Brito, J. C. Network- and distance‐based methods in bioregionalization processes at regional scale: an application to the terrestrial mammals of Iran. DOI

Noroozi, J., Talebi, A. & Doostmohammadi, M. The Alborz Mountain Range. In

Langkilde, T. & Shine, R. How much stress do researchers inflict on their study animals? A case study using a scincid lizard, PubMed DOI

Rajabizadeh, M., Nilson, G. B., Kami, H. G. & Naderi, A. R. Distribution of the subgenus

Carstens, B. C. et al. How to fail at species delimitation. PubMed DOI

Noori, S. et al. Biodiversity modelling reveals a significant gap between diversity hotspots and protected areas for Iranian reptiles. DOI

Nilson, G. & Sharifi, M.

Moritz, C. Defining ‘evolutionarily significant units’ for conservation. PubMed DOI

Crandall, K. A., Bininda-Emonds, O. R. P., Mace, G. M. & Wayne, R. K. Considering evolutionary processes in conservation biology. PubMed DOI

Fraser, D. J. & Bernatchez, L. Adaptive evolutionary conservation: towards a unified concept for defining conservation units. PubMed DOI

Joger, U. et al. Phylogeography of Western palaearctic reptiles – Spatial and Temporal speciation patterns. DOI

Townsend, T. M., Alegre, R. E., Kelley, S. T., Wiens, J. J. & Reeder, T. W. Rapid development of multiple nuclear loci for phylogenetic analysis using genomic resources: an example from squamate reptiles. PubMed DOI

Kumazawa, Y., Ota, H., Nishida, M. & Ozawa, T. Gene rearrangements in snake mitochondrial genomes: highly concerted evolution of control-region-like sequences duplicated and inserted into a tRNA gene cluster. PubMed DOI

Kumazawa, Y., Ota, H., Nishida, M. & Ozawa, T. The complete nucleotide sequence of a snake ( PubMed DOI PMC

Janke, A. & Arnason, U. The complete mitochondrial genome of PubMed DOI

Arevalo, E., Davis, S. K. & Sites, J. W. Mitochondrial DNA sequence divergence and phylogenetic relationships among eight chromosome races of the DOI

Wüster, W., Peppin, L., Pook, C. E. & Walker, D. E. A nesting of vipers: phylogeny and historical biogeography of the Viperidae (Squamata: Serpentes). PubMed DOI

Noonan, B. P. & Chippindale, P. T. Dispersal and vicariance: the complex evolutionary history of Boid snakes. PubMed DOI

Chen, Y., Ye, W., Zhang, Y. & Xu, Y. High speed BLASTN: an accelerated megablast search tool. PubMed DOI PMC

Stephens, M., Smith, N. J. & Donnelly, P. A new statistical method for haplotype reconstruction from population data. PubMed DOI PMC

Flot, J. F. & SeqPHASE A web tool for interconverting PHASE input/output files and FASTA sequence alignments. PubMed DOI

Thompson, J. D., Higgins, D. G. & Gibson, T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. PubMed DOI PMC

Tamura, K. et al. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. PubMed DOI PMC

Librado, P. & Rozas, J. DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. PubMed DOI

Lanfear, R., Frandsen, P. B., Wright, A. M., Senfeld, T. & Calcott, B. PartitionFinder 2: new methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. PubMed

Ronquist, F. & Huelsenbeck, J. P. MrBayes 3: bayesian phylogenetic inference under mixed models. PubMed DOI

Minh, B. Q. et al. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. PubMed DOI PMC

Rambaut, A., Drummond, A. J., Xie, D., Baele, G. & Suchard, M. A. Posterior summarization in bayesian phylogenetics using tracer 1.7. PubMed DOI PMC

Hoang, D. T., Chernomor, O., von Haeseler, A., Minh, B. Q. & Vinh, L. S. UFBoot2: improving the ultrafast bootstrap approximation. PubMed DOI PMC

Rambaut, A. & Drummond, A. FigTree v1.3.1.

Leigh, J. W. & Bryant, D. P. O. P. A. R. T. Full-feature software for haplotype network construction. DOI

Corander, J., Marttinen, P., Sirén, J. & Tang J. BAPS: Bayesian analysis of population structure. Manual version 1–27 (2005).

Pritchard, J. K., Stephens, M. & Donnelly, P. Inference of population structure using multilocus genotype data. PubMed DOI PMC

Bessa-Silva, A. Fasta2Structure: a user-friendly tool for converting multiple aligned FASTA files to STRUCTURE format. PubMed DOI PMC

Evanno, G., Regnaut, S. & Goudet, J. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. PubMed DOI

Rosenberg, N. A. & DISTRUCT A program for the graphical display of population structure. DOI

Drummond, A. J. & Rambaut, A. BEAST: bayesian evolutionary analysis by sampling trees. PubMed DOI PMC

Wüster, W., Salomão, M. G., Quijada-Mascareñas, J. A., Thorpe, R. S. & Campbell, J. A. Origins and evolution of the South American pitviper fauna: evidence from mitochondrial DNA sequence analysis. In

Szyndlar, Z. & Rage, J. C. West Palearctic cobras of the genus DOI

Parmley, D. & Holman, J. A. Earliest fossil record of a pigmy rattlesnake (Viperidae: DOI

Cornuet, J. M. et al. DIYABC v2.0: A software to make approximate bayesian computation inferences about population history using single nucleotide polymorphism, DNA sequence and microsatellite data. PubMed DOI

Bromham, L. Molecular clocks in reptiles: life history influences rate of molecular evolution. PubMed DOI

Cornuet, J. M., Ravigné, V. & Estoup, A. Inference on population history and model checking using DNA sequence and microsatellite data with the software DIYABC (v1.0). PubMed DOI PMC

Pons, J. et al. Sequence-based species delimitation for the DNA taxonomy of undescribed insects. PubMed DOI

Fontaneto, D. et al. Independently evolving species in asexual bdelloid rotifers. PubMed DOI PMC

Zhang, J., Kapli, P., Pavlidis, P. & Stamatakis, A. A general species delimitation method with applications to phylogenetic placements. PubMed DOI PMC

Puillandre, N., Brouillet, S. & Achaz, G. ASAP: assemble species by automatic partitioning. PubMed DOI

Rambaut, A. & Drummond, A. J. LogCombiner v1.8.2.

Helfrich, P. et al. Versatile visual annotation of hierarchical text relations. in

Fonseca, E. M., Duckett, D. J. & Carstens, B. C. P2C2M.GMYC: an R package for assessing the utility of the generalized mixed Yule coalescent model. DOI

RStudio Team. RStudio: Integrated Development for R.

Ezard, T., Fujisawa, T. & Barraclough, T. SPLITS: Species’ limits by threshold statistics.

Leaché, A. & Ogilvie, H. A. Bayes factor delimitation of species (

Bouckaert, R. et al. BEAST 2: a software platform for bayesian evolutionary analysis. PubMed DOI PMC

Bryant, D., Bouckaert, R., Felsenstein, J., Rosenberg, N. A. & RoyChoudhury, A. Inferring species trees directly from biallelic genetic markers: bypassing gene trees in a full coalescent analysis. PubMed DOI PMC

Kass, R. E. & Raftery, A. E. Bayes factors. DOI

Yang, Z. The BPP program for species tree Estimation and species delimitation. DOI