Living on the edge: morphological, karyological and genetic diversity studies of the Hungarian Plantago maxima populations and established ex situ collection.

Background: The analysis of genetic diversity of protected plant species can greatly support conservation efforts. Plantago maxima Juss. ex Jacq. is a perennial species distributed along the Eurasian steppe. The westernmost range edge of the species' distribution is located in the Pannonian basin, in Hungary where it is represented by a few, fragmented and highly endangered populations. We studied population diversity of all Hungarian range edge, natural populations, and one established ex situ population. One population from the centre of distribution (Kazakhstan) was implemented in the cpDNA haplotype study to compare the peripheral vs. central populations. We performed morphometric trait-based analysis, chromosome studies (morphometric analyses and FISH) and genetic diversity evaluations using inter simple sequence repeats (ISSR) and cpDNA trnL-trnF to evaluate differences between the in situ and ex situ populations as well as central vs. peripheral populations.

Results: Our results showed no obvious morphological differences among the in situ and ex situ populations in the period between 2018 and 2020. One ex situ subpopulation develops flowers three years in a row from 2019, which is a favourable indicator of the introduction success. Hungarian populations are exclusively diploids (2n = 2x = 12). The karyogram consists of 5 metacentric and 1 acrocentric chromosome pair. Plantago maxima has one 35S and two 5S rDNA loci, located on the acrocentric chromosome pair. Eight variable ISSR primers yielded 100 fragments, of which 74.6% were polymorphic (mean H_e = 0.220). A high level of genetic variation within population was observed (92%) while the genetic differentiation among the populations was only 8%. STRUCTURE analysis revealed that the largest Kunpeszér population separated from the rest of the Hungarian populations, indicating a high rate of admixture among the other ones. Based on the trnL-trnF sequence analysis the Hungarian populations represent a single haplotype, which can indicate a reduced diversity due to isolation and recent population decline. By contrast, Kazakh population represents a distinct haplotype compared to the Hungarian samples.

Conclusions: The present study draws the attention to the high conservation value of the Plantago maxima populations from the westernmost range edge of the species' distribution.