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A comprehensive study of a typical steppe perennial Iris pumila L. were carried out in the central zone of the European part of this species’ range, namely in Ukraine. Intraspecific differentiation, population size and isolation degree and its consequences, the threat of human impact were analyzed, as well as ecological amplitude and genetic variation in ISSR markers and selected chloroplast regions were determined. The species was found to have a low intraspecific differentiation that indicates the uniformity of the gene pool in the studied part of the range. Moreover, the results of isolation assessment, population and ecological study of I. pumila confirm the potential risk of extinction. A considerable part of the species populations exist as separated patches of rare ecosystems, isolated from the nearest neighbours due to intense plowing of the steppe zone. The generative reproduction is rare. In contrast, ISSR analysis revealed comparably high genetic diversity in all the sampled populations. Furthermore, specific plastid haplotypes were demonstrated in some of them. The inconsistency between the results of population ecological study and the data of molecular genetic analysis indicates that the loss of genetic diversity in the species caused by habitat fragmentation and isolation under increasing anthropogenic pressure is likely to be slower than it would appear judging from the assessment of population parameters, which clearly show negative trends. This result also emphasizes the necessity for integrated approach to assessment of the extinction risk for particular species and careful analysis of all determinants of extinction.

We investigated informativeness and effectiveness of different marker types (ISSR, IRAP, REMAP, RGAP and LP-PCR that employ primers based on the conservative sequences of abiotic stress response genes) to study genetic diversity of Iris pumila L. By the number of amplicons per primer, number of polymorphic amplicons per primer and resolving power index (Rp), ISSR-markers were the most efficient followed by LP-PCR-markers. In order of decreasing value of indicators of genetic diversity “the percentage of polymorphic bands”, and “the average Jaccardś genetic distance between plants”, marker systems may be arranged as follows: ISSR > RAPD > LP-PC > RGAP ≈ IRAP. For ISSR-markers, the percentage of polymorphic bands was 1.3–1.7 times higher than for the others, and the average genetic distance was 1.2–1.3 times higher. Different marker systems were ranked by the value of Neiś gene diversity and the Shannonś index as follows: ISSR > RAPD ≈ LP-PCR > RGAP ≈ IRAP, with the highest and the lowest values differing 1.4 times. Genetic population structure was investigated with program Structure 2.3. The data of all marker systems suggest that all genomes under study belonged to one population. The PCoA and cluster analyses based on genetic distances showed distinctions in clustering generated from different markers data and summarized data, as well as the lack of strong clusters. Mantel test revealed significant positive correlation between the matrices of genetic distances generated by the data of almost all marker systems. The strongest correlation was found between RGAP- and IRAP-markers (r = 0.452, p = 0.01) and between RGAP and ISSR (r = 0.430, p = 0.01). ISSR, RAPD and LP-PCR proved to be more effective for the study of I. pumila genetic diversity, nevertheless, joint use of different marker systems will provide a more comprehensive assessment of variation in different genomic regions.