Unravelling some factors affecting sexual reproduction in rock-specialist shrub: Insight from an endemic Daphne arbuscula (Thymelaeaceae).

The role of endemic species in global biodiversity is pivotal, and understanding their biology and ecology is imperative for their fitness and long-term survival, particularly in the face of ongoing climatic oscillations. Our primary goal was to investigate the sexual reproduction level of the endangered Western Carpathian endemic Daphne arbuscula (Thymelaeaceae), which inhabits extreme rocky habitats, and to comprehend the influence of specific factors on its reproductive success. We conducted the research across four populations, varying in size and environmental conditions. Over two years, we monitored flower and fruit production, analyzed genetic variability within and among populations, and studied pollination mechanisms. Daphne arbuscula proved to be strictly self-incompatible, with significant variations in flower and fruit production among populations and seasons. The average fruit production percentage consistently remained below 50% across populations, indicating challenges in sexual reproduction. Cold and harsh weather during the reproductive phase had a substantial negative impact on sexual reproduction efficacy, leading to decreased fruit production. Nevertheless, several individuals in sheltered microhabitats displayed significantly higher fruit production, ranging from 60% to 83%, emphasizing the critical role of microhabitat heterogeneity in sustaining sexual reproduction in this species. We found no pronounced differences in genetic diversity within or among populations, suggesting that genetic factors may not critically influence the reproductive success of this endemic species. The implications of our findings might be of paramount importance for the long-term survival of D. arbuscula and offer valuable insights for the development of effective conservation strategies for this species.

Homoplastic versus xenoplastic evolution: exploring the emergence of key intrinsic and extrinsic traits in the montane genus Soldanella (Primulaceae).

Specific ecological conditions in the high mountain environment exert a selective pressure that often leads to convergent trait evolution. Reticulations induced by incomplete lineage sorting and introgression can lead to discordant trait patterns among gene and species trees (hemiplasy/xenoplasy), providing a false illusion that the traits under study are homoplastic. Using phylogenetic species networks, we explored the effect of gene exchange on trait evolution in Soldanella, a genus profoundly influenced by historical introgression. At least three features evolved independently multiple times: the single-flowered dwarf phenotype, dysploid cytotype, and ecological generalism. The present analyses also indicated that the recurring occurrence of stoloniferous growth might have been prompted by an introgression event between an ancestral lineage and a still extant species, although its emergence via convergent evolution cannot be completely ruled out. Phylogenetic regression suggested that the independent evolution of larger genomes in snowbells is most likely a result of the interplay between hybridization events of dysploid and euploid taxa and hostile environments at the range margins of the genus. The emergence of key intrinsic and extrinsic traits in snowbells has been significantly impacted not only by convergent evolution but also by historical and recent introgression events.

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.

Evolution of hygrophytic plant species in the Anatolia-Caucasus region: insights from phylogenomic analyses of Cardamine perennials.

BACKGROUND AND AIMS: Southwestern Asia is a significant centre of biodiversity and a cradle of diversification for many plant groups, especially xerophytic elements. In contrast, little is known about the evolution and diversification of its hygrophytic flora. To fill this gap, we focus on Cardamine (Brassicaceae) species that grow in wetlands over a wide altitudinal range. We aimed to elucidate their evolution, assess the extent of presumed historical gene flow between species, and draw inferences about intraspecific structure. METHODS: We applied the phylogenomic Hyb-Seq approach, ecological niche analyses and multivariate morphometrics to a total of 85 Cardamine populations from the target region of Anatolia-Caucasus, usually treated as four to six species, and supplemented them with close relatives from Europe. KEY RESULTS: Five diploids are recognized in the focus area, three of which occur in regions adjacent to the Black and/or Caspian Sea (C. penzesii, C. tenera, C. lazica), one species widely distributed from the Caucasus to Lebanon and Iran (C. uliginosa), and one western Anatolian entity (provisionally C. cf. uliginosa). Phylogenomic data suggest recent speciation during the Pleistocene, likely driven by both geographic separation (allopatry) and ecological divergence. With the exception of a single hybrid (allotetraploid) speciation event proven for C. wiedemanniana, an endemic of southern Turkey, no significant traces of past or present interspecific gene flow were observed. Genetic variation within the studied species is spatially structured, suggesting reduced gene flow due to geographic and ecological barriers, but also glacial survival in different refugia. CONCLUSIONS: This study highlights the importance of the refugial regions of the Black and Caspian Seas for both harbouring and generating hygrophytic species diversity in Southwestern Asia. It also supports the significance of evolutionary links between Anatolia and the Balkan Peninsula. Reticulation and polyploidization played a minor evolutionary role here in contrast to the European relatives.

Pervasive Introgression During Rapid Diversification of the European Mountain Genus Soldanella (L.) (Primulaceae).

Hybridization is a key mechanism involved in lineage diversification and speciation, especially in ecosystems that experienced repeated environmental oscillations. Recently radiated plant groups, which have evolved in mountain ecosystems impacted by historical climate change provide an excellent model system for studying the impact of gene flow on speciation. We combined organellar (whole-plastome) and nuclear genomic data (RAD-seq) with a cytogenetic approach (rDNA FISH) to investigate the effects of hybridization and introgression on evolution and speciation in the genus Soldanella (snowbells, Primulaceae). Pervasive introgression has already occurred among ancestral lineages of snowbells and has persisted throughout the entire evolutionary history of the genus, regardless of the ecology, cytotype, or distribution range size of the affected species. The highest extent of introgression has been detected in the Carpathian species, which is also reflected in their extensive karyotype variation. Introgression occurred even between species with dysploid and euploid cytotypes, which were considered to be reproductively isolated. The magnitude of introgression detected in snowbells is unprecedented in other mountain genera of the European Alpine System investigated hitherto. Our study stresses the prominent evolutionary role of hybridization in facilitating speciation and diversification on the one hand, but also enriching previously isolated genetic pools. [chloroplast capture; diversification; dysploidy; European Alpine system; introgression; nuclear-cytoplasmic discordance; ribosomal DNA.].

Integrative Study of Genotypic and Phenotypic Diversity in the Eurasian Orchid Genus Neotinea.

Knowledge of population variation across species' ranges is a prerequisite for correctly assessing the overall variability of any group of organisms and provides an invaluable basis for unraveling evolutionary history, optimizing taxonomy and devising effective conservation strategies. Here, we examine the genus Neotinea, which represents a relatively recently delimited monophyletic genus of orchids, for which a detailed study of its overall variability was lacking. We applied a suite of biosystematic methods, consisting of flow cytometry, multivariate and geometric morphometrics, and analysis of genomic SNP data, to identify phylogenetic lineages within the genus, to delineate phenotypic variation relevant to these lineages, and to identify potential cryptic taxa within lineages. We found clear differentiation into four major lineages corresponding to the groups usually recognized within the genus: Neotinea maculata as a distinct and separate taxon, the Neotinea lactea group comprising two Mediterranean taxa N. lactea and Neotinea conica, the Neotinea ustulata group comprising two phenologically distinct varieties, and the rather complex Neotinea tridentata group comprising two major lineages and various minor lineages of unclear taxonomic value. N. conica constitutes both a monophyletic group within N. lactea and a distinct phenotype within the genus and merits its proposed subspecies-level recognition. By contrast, the spring and summer flowering forms of N. ustulata (var. ustulata and var. aestivalis) were confirmed to be distinct only morphologically, not phylogenetically. The most complex pattern emerged in the N. tridentata group, which splits into two main clades, one containing lineages from the Balkans and eastern Mediterranean and the other consisting of plants from Central Europe and the central Mediterranean. These individual lineages differ in genome size and show moderate degrees of morphological divergence. The tetraploid Neotinea commutata is closely related to the N. tridentata group, but our evidence points to an auto- rather than an allopolyploid origin. Our broad methodological approach proved effective in recognizing cryptic lineages among the orchids, and we propose the joint analysis of flow cytometric data on genome size and endopolyploidy as a useful and beneficial marker for delineating orchid species with partial endoreplication.

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.

Diversification dynamics and transoceanic Eurasian-Australian disjunction in the genus Picris (Compositae) induced by the interplay of shifts in intrinsic/extrinsic traits and paleoclimatic oscillations.

Understanding transcontinental biogeographic patterns has been one of the main foci of the field of biogeography. While multiple explanations for transcontinental disjunctions have been proposed, little is still known about the relative importance of intrinsic and extrinsic traits for the diversification dynamics of disjunct taxa. Here, we study the evolutionary history of the genus Picris L. (Compositae), a great model for investigating the diversification dynamics of transoceanic bipolar disjunct organisms. Ancestral state reconstructions indicate that the most recent common ancestor (MRCA) of Picris was a semelparous and heterocarpic herb that lived in unpredictable environments of North Africa and West Asia. Diversification analyses suggest a significant shift in speciation ca. 1 million years ago, likely associated with the onset of the mid-Pleistocene revolution. Longevity characters are correlated with the evolution of particular fruit types and with environmental conditions. Heterocarpic species are mostly semelparous herbs strongly linked with unpredictable habitats, while homocarpic taxa are mostly iteroparous plants occurring in predictable environments. Binary-state speciation and extinction analyses suggest that homocarpy, iteroparity, and habitats predictability accelerate diversification. Although the combination of homocarpy and iteroparity evolved in several lineages, only members of the P. hieracioides group were able to colonise Eurasia and expand to Australia by transoceanic dispersal. Those findings indicate that large-scale colonisation events depend on a complex interplay of intrinsic and extrinsic factors.

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.

On the origins of Balkan endemics: the complex evolutionary history of the Cyanus napulifer group (Asteraceae).

BACKGROUND AND AIMS: The Balkan Peninsula is one of the most important centres of plant diversity in Europe. Here we aim to fill the gap in the current knowledge of the evolutionary processes and factors modelling this astonishing biological richness by applying multiple approaches to the Cyanus napulifer group. METHODS: To reconstruct the mode of diversification within the C. napulifer group and to uncover its relationships with potential relatives with x = 10 from Europe and Northern Africa, we examined variation in genetic markers (amplified fragment length polymorphisms [AFLPs]; 460 individuals), relative DNA content (4',6-diamidino-2-phenylindole [DAPI] flow cytometry, 330 individuals) and morphology (multivariate morphometrics, 40 morphological characters, 710 individuals). To elucidate its evolutionary history, we analysed chloroplast DNA (cpDNA) sequences of the genus Cyanus deposited in the GenBank database. KEY RESULTS: The AFLPs revealed a suite of closely related entities with variable levels of differentiation. The C. napulifer group formed a genetically well-defined unit. Samples outside the group formed strongly diversified and mostly species-specific genetic lineages with no further geographical patterns, often characterized also by a different DNA content. AFLP analysis of the C. napulifer group revealed extensive radiation and split it into nine allopatric (sub)lineages with varying degrees of congruence among genetic, DNA-content and morphological patterns. Genetic admixture was usually detected in contact zones between genetic lineages. Plastid data indicated extensive maintenance of ancestral variation across Cyanus perennials. CONCLUSION: The C. napulifer group is an example of a rapidly and recently diversified plant group whose genetic lineages have evolved in spatio-temporal isolation on the topographically complex Balkan Peninsula. Adaptive radiation, accompanied in some cases by long-term isolation and hybridization, has contributed to the formation of this species complex and its mosaic pattern.

Disentangling identity of species of the genus Taphrina parasitizing herbaceous Rosaceae, with proposal of Taphrina gei-montani sp. nov.

Five strains (CCY 058-007-001T, CCY 058-007-002, CCY 058-007-003, CCY 058-007-004 and CCY 058-007-005) of a novel parasitic yeast belonging to the genus Taphrina were isolated from leaf tissues of Geum montanum L. (Rosaceae), collected from the Vysoké Tatry Mountains, Slovakia. Genetic analyses revealed that these isolates differ by 15 unique substitutions in the ITS region and by six substitutions in the rns gene from all other species of the genus Taphrina analysed hitherto. The novel strains are also distinguished from all other species of the genus Taphrina by their morphology, biochemical properties and ecology. These strains represent a novel species, for which the name Taphrina gei-montani sp. nov. is proposed. The type strain is CCY 058-007-001T (=CBS 14159=BU001). The MycoBank number is MB815677. The present study also demonstrates that two distinct species of the genus Taphrina parasitize the herbaceous Rosaceae: Taphrina gei-montani sp. nov. on Geum montanum and Taphrina tormentillae on Potentilla species.

Dealing with discordant genetic signal caused by hybridisation, incomplete lineage sorting and paucity of primary nucleotide homologies: a case study of closely related members of the genus Picris subsection Hieracioides (Compositae).

We investigated genetic variation and evolutionary history of closely related taxa of Picris subsect. Hieracioides with major focus on the widely distributed P. hieracioides and its closely related congeners, P. hispidissima, P. japonica, P. olympica, and P. nuristanica. Accessions from 140 sample sites of the investigated Picris taxa were analyzed on the infra- and the inter-specific level using nuclear (ITS1-5.8S-ITS2 region) and chloroplast (rpl32-trnL(UAG) region) DNA sequences. Genetic patterns of P. hieracioides, P. hispidissima, and P. olympica were shown to be incongruent and, in several cases, both plastid and nuclear alleles transcended borders of the taxa and genetic lineages. The widespread P. hieracioides was genetically highly variable and non-monophyletic across both markers, with allele groups having particular geographic distributions. Generally, all gene trees and networks displayed only a limited and statistically rather unsupported resolution among ingroup taxa causing their phylogenetic relationships to remain rather unresolved. More light on these intricate evolutionary relationships was cast by the Bayesian coalescent-based analysis, although some relationships were still left unresolved. A combination of suite of phylogenetic analyses revealed the ingroup taxa to represent a complex of genetically closely related and morphologically similar entities that have undergone a highly dynamic and recent evolution. This has been especially affected by the extensive and recurrent gene flow among and within the studied taxa and/or by the maintenance of ancestral variation. Paucity of phylogenetically informative signal further hampers the reconstruction of relationships on the infra- as well as on the inter-specific level. In the present study, we have demonstrated that a combination of various phylogenetic analyses of datasets with extremely complex and incongruent phylogenetic signal may shed more light on the interrelationships and evolutionary history of analysed species groups.

Re-evaluation of the morphological variability of Microglossum viride and M. griseoviride sp. nov.

Studies in Microglossum viride (Pers.) Gillet revealed that the name was used incorrectly for two similar but different taxa. Analyses of morphological, ecological and molecular (sequences of ITS and LSU region of rRNA gene) characters of type and voucher specimens of M. viride and related taxa resulted in delimitation and description of a new species, Microglossum griseoviride V. Kucera, Lizon & M. Tomsovský. Lectotypes of Geoglossum viride Pers., and epitype of Geoglossum viride are designated. Species Microglossum minus Velen. and Microglossum lutescens Boud. are confirmed to be conspecific to M. viride.