Incidence and evolutionary relevance of autotriploid cytotypes in a relict member of the genus Daphne (Thymelaeaceae).

Odd ploidy-level cytotypes in sexually reproducing species are considered a dead end due to absent or reduced fertility. If sterility is only partial, however, their contribution to the population gene pool can be augmented by longevity and clonal growth. To test this, we investigated the cytotype origin and spatial pattern, and pollen viability in three relict shrub species of the genus Daphne (Thymelaeaceae Juss.) in central Europe. Daphne cneorum subsp. cneorum is a widespread European species that has a broad ecological amplitude, whereas D. cneorum subsp. arbusculoides and D. arbuscula are narrow endemics of the western Pannonian Plain and the Western Carpathians, respectively. Our study confirmed that all three taxa are diploid. However, of more than a thousand analysed individuals of D. cneorum subsp. cneorum, five in four different populations were triploid. Our data indicate that these triploids most likely originate from recurrent autopolyploidization events caused by the fusion of reduced and unreduced gametes. High pollen viability was observed in all three taxa and in both diploid and triploid cytotypes, ranging from 65 to 100 %. Our study highlights the significant role of odd ploidy-level cytotypes in interploidy gene flow, calling for more research into their reproduction, genetic variability, and overall fitness. Interestingly, while the endemic D. arbuscula differs from D. cneorum based on genetic and genome size data, D. cneorum subsp. arbusculoides was indistinguishable from D. cneorum subsp. cneorum. However, our study reveals that the subspecies differ in the number of flowers per inflorescence. This is the first comprehensive cytogeographic study of this intriguing genus at a regional scale, and in spite of its karyological stability, it contributes to our understanding of genomic evolution in plant species with a wide ecological amplitude.

Variability of Reproduction Pathways in the Central-European Populations of Hawthorns with Emphasis on Triploids.

The role of apomeiosis, parthenogenesis, and pseudogamy in the asexual reproduction of some plant groups has not been fully elucidated in relation to species diversification. Quantitative analyses of seed origin may help in gaining better understanding of intercytotypic interactions. Asexual reproduction associated with polyploidy and frequent hybridization plays a crucial role in the evolutionary history of the genus Crataegus in North America. In Europe, the genus represents a taxonomically complex and very difficult species group not often studied using a modern biosystematic approach. We investigated the reproduction pathways in mixed-cytotype populations of selected taxa of Crataegus in eastern Slovakia, Central Europe. The investigated accessions were characterized by seed production data and the ploidy level of mature plants as well as the embryo and endosperm tissues of their seeds determined via flow cytometry. Diploid and polyploid hawthorns reproduce successfully; they also produce high numbers of seeds. An exception is represented by an almost sterile triploid. Diploids reproduce sexually. Polyploids shift to asexual reproduction, but pseudogamy seems to be essential for regular seed development. In rare cases, fertilization of unreduced gametes occurs, which offers opportunity for the establishment of new polyploid cytotypes between diploid sexuals and polyploid asexuals. Opposite to sexual diploids, triploids are obligate, and tetraploids almost obligate apomicts. Apomixis is considered to help stabilize individual weakly differentiated polyploid microspecies. Pseudogamy is a common feature and usually leads to unbalanced maternal to paternal contribution in the endosperm of triploid accessions. Parental contribution to endosperm gene dosage is somehow relaxed in triploids. Our Crataegus plant system resembles reproduction in the diploids and polyploids of North American hawthorns. Our data provide support for the hypothesis that polyploidization, shifts in reproduction modes, and hybridization shape the genus diversity also in Central Europe.

Genome size and endoreplication in two pairs of cytogenetically contrasting species of Pulmonaria (Boraginaceae) in Central Europe.

Genome size is species-specific feature and commonly constant in an organism. In various plants, DNA content in cell nucleus is commonly increased in process of endoreplication, cellular-specific multiplication of DNA content without mitosis. This leads to the endopolyploidy, the presence of multiplied chromosome sets in a subset of cells. The relationship of endopolyploidy to species-specific genome size is rarely analysed and is not fully understood. While negative correlation between genome size and endopolyploidy level is supposed, this is species- and lineage-specific. In the present study, we shed light on this topic, exploring both genome size and endoreplication-induced DNA content variation in two pairs of morphologically similar species of Pulmonaria, P. obscura-P. officinalis and P. mollis-P. murinii. We aim (i) to characterize genome size and chromosome numbers in these species using cytogenetic, root-tip squashing and flow cytometry (FCM) techniques; (ii) to investigate the degree of endopolyploidy in various plant organs, including the root, stem, leaf, calyx and corolla using FCM; and (iii) to comprehensively characterize and compare the level of endopolyploidy and DNA content in various organs of all four species in relation to species systematic relationships and genome size variation. We have confirmed the diploid-dysploid nature of chromosome complements, and divergent genome sizes for Pulmonaria species: P. murinii with 2n = 2x = 14, 2.31 pg/2C, P. obscura 2n = 2x = 14, 2.69 pg/2C, P. officinalis 2n = 2x = 16, 2.96 pg/2C and P. mollis 2n = 2x = 18, 3.18 pg/2C. Endopolyploidy varies between species and organs, and we have documented 4C-8C in all four organs and up to 32C (64C) endopolyploid nuclei in stems at least in some species. Two species with lower genome sizes tend to have higher endopolyploidy levels than their closest relatives. Endoreplication-generated tissue-specific mean DNA content is increased and more balanced among species in all four organs compared to genome size. Our results argue for the narrow relationship between genome size and endopolyploidy in the present plant group within the genus Pulmonaria, and endopolyploidization seems to play a compensatory developmental role in organs of related morphologically similar species.

Multiple Drivers of High Species Diversity and Endemism Among Alyssum Annuals in the Mediterranean: The Evolutionary Significance of the Aegean Hotspot.

The Mediterranean Basin is a significant hotspot of species diversity and endemism, with various distribution patterns and speciation mechanisms observed in its flora. High species diversity in the Mediterranean is also manifested in the monophyletic lineage of Alyssum annuals (Brassicaceae), but little is known about its origin. These species include both diploids and polyploids that grow mainly in open and disturbed sites across a wide elevational span and show contrasting distribution patterns, ranging from broadly distributed Eurasian species to narrow island endemics. Here, we investigated the evolution of European representatives of this lineage, and aimed to reconstruct their phylogeny, polyploid and genome size evolution using flow cytometric analyses, chloroplast and nuclear high- and low-copy DNA markers. The origin and early diversification of the studied Alyssum lineage could be dated back to the Late Miocene/Pliocene and were likely promoted by the onset of the Mediterranean climate, whereas most of the extant species originated during the Pleistocene. The Aegean region represents a significant diversity center, as it hosts 12 out of 16 recognized European species and comprises several (sub)endemics placed in distinct phylogenetic clades. Because several species, including the closest relatives, occur here sympatrically without apparent niche differences, we can reject simple allopatric speciation via vicariance as well as ecological speciation for most cases. Instead, we suggest scenarios of more complex speciation processes that involved repeated range shifts in response to sea-level changes and recurrent land connections and disconnections since the Pliocene. In addition, multiple polyploidization events significantly contributed to species diversity across the entire distribution range. All seven polyploids, representing both widespread species and endemics to the western or eastern Mediterranean, were inferred to be allopolyploids. Finally, the current distribution patterns have likely been affected also by the human factor (farming and grazing). This study illustrates the complexity of evolutionary and speciation processes in the Mediterranean flora.

Eunuchs or Females? Causes and Consequences of Gynodioecy on Morphology, Ploidy, and Ecology of Stellaria graminea L. (Caryophyllaceae).

Plant speciation results from intricate processes such as polyploidization, reproductive strategy shifts and adaptation. These evolutionary processes often co-occur, blurring their respective contributions and interactions in the speciation continuum. Here, relying on a large-scale study, we tested whether gynodioecy triggers the divergent evolution of flower morphology and genome between sexes, and contributes to the establishment of polyploids and colonization of ecological niches in Stellaria graminea. We found that gynodioecy in S. graminea leads to flower morphology divergence between females and hermaphrodites, likely due to sexual selection. Contrary to our expectations, gynodioecy occurs evenly in diploids and tetraploids, suggesting that this reproductive strategy was not involved in the establishment of polyploids. Both diploid and tetraploid females have a larger genome size than hermaphrodites, suggesting the presence of sex chromosomes. Finally, ecology differs between cytotypes and to a lesser extent between sexes, suggesting that the link between environment and presence of females is indirect and likely explained by other aspects of the species' life history. Our study shows that gynodioecy leads to the consistent evolution of sexual traits across a wide range of populations, cytotypes and environments within a given species, and this likely contributes to the phenotypic and genetic distinctiveness of the species from its sister clades.

So Closely Related and Yet So Different: Strong Contrasts Between the Evolutionary Histories of Species of the Cardamine pratensis Polyploid Complex in Central Europe.

Recurrent polyploid formation and weak reproductive barriers between independent polyploid lineages generate intricate species complexes with high diversity and reticulate evolutionary history. Uncovering the evolutionary processes that formed their present-day cytotypic and genetic structure is a challenging task. We studied the species complex of Cardamine pratensis, composed of diploid endemics in the European Mediterranean and diploid-polyploid lineages more widely distributed across Europe, focusing on the poorly understood variation in Central Europe. To elucidate the evolution of Central European populations we analyzed ploidy level and genome size variation, genetic patterns inferred from microsatellite markers and target enrichment of low-copy nuclear genes (Hyb-Seq), and environmental niche differentiation. We observed almost continuous variation in chromosome numbers and genome size in C. pratensis s.str., which is caused by the co-occurrence of euploid and dysploid cytotypes, along with aneuploids, and is likely accompanied by inter-cytotype mating. We inferred that the polyploid cytotypes of C. pratensis s.str. are both of single and multiple, spatially and temporally recurrent origins. The tetraploid Cardamine majovskyi evolved at least twice in different regions by autopolyploidy from diploid Cardamine matthioli. The extensive genome size and genetic variation of Cardamine rivularis reflects differentiation induced by the geographic isolation of disjunct populations, establishment of triploids of different origins, and hybridization with sympatric C. matthioli. Geographically structured genetic lineages identified in the species under study, which are also ecologically divergent, are interpreted as descendants from different source populations in multiple glacial refugia. The postglacial range expansion was accompanied by substantial genetic admixture between the lineages of C. pratensis s.str., which is reflected by diffuse borders in their contact zones. In conclusion, we identified an interplay of diverse processes that have driven the evolution of the species studied, including allopatric and ecological divergence, hybridization, multiple polyploid origins, and genetic reshuffling caused by Pleistocene climate-induced range dynamics.

Karyological patterns in the European endemic genus Soldanella L.: Absolute genome size variation uncorrelated with cytotype chromosome numbers.

PREMISE OF THE STUDY: Detailed knowledge about the karyological diversity of organisms undoubtedly represents one of the crucial steps toward a better understanding of their evolutionary trends and history. We investigated the cytotype and absolute genome size (AGS) patterns in the European mountain-dwelling genus Soldanella (Primulaceae) in light of its geographic distribution and ecological diversification. METHODS: Our chromosome number survey was based on 34 newly determined and 125 previously published chromosome counts. AGS was estimated on the basis of propidium iodide (PI) flow cytometry (299 individuals, 110 populations). KEY RESULTS: We confirmed the existence of two cytotypes with the same ploidy level, i.e., euploid 2n = 40 and dysploid 2n = 38. The overall infrageneric AGS variation ranged between 2.97 and 3.99 pg (25.6% variation). The 2n = 40 cytotype harbors a modest amount of continuous AGS variation. With regard to its distribution area and ecology, the cytotype is ubiquitous. By contrast, the 2n = 38 cytotype was detected only in six forest-dwelling taxa with AGS variation segregated into three discrete, geographically separated groups. The AGS variation of the 2n = 38 cytotype was strongly correlated with elevation and longitude. CONCLUSIONS: Despite the apparent morphological and ecological variation, members of the genus Soldanella have not undergone any pronounced cytotype and AGS diversification during their evolutionary history. The lack of correlation between chromosome numbers and AGS indicates that the evolutionary mechanism behind the origin of the dysploid cytotype 2n = 38 was a chromosomal fusion.