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.

Allele Sorting as a Novel Approach to Resolving the Origin of Allotetraploids Using Hyb-Seq Data: A Case Study of the Balkan Mountain Endemic Cardamine barbaraeoides.

Mountains of the Balkan Peninsula are significant biodiversity hotspots with great species richness and a large proportion of narrow endemics. Processes that have driven the evolution of the rich Balkan mountain flora, however, are still insufficiently explored and understood. Here we focus on a group of Cardamine (Brassicaceae) perennials growing in wet, mainly mountainous habitats. It comprises several Mediterranean endemics, including those restricted to the Balkan Peninsula. We used target enrichment with genome skimming (Hyb-Seq) to infer their phylogenetic relationships, and, along with genomic in situ hybridization (GISH), to resolve the origin of tetraploid Cardamine barbaraeoides endemic to the Southern Pindos Mts. (Greece). We also explored the challenges of phylogenomic analyses of polyploid species and developed a new approach of allele sorting into homeologs that allows identifying subgenomes inherited from different progenitors. We obtained a robust phylogenetic reconstruction for diploids based on 1,168 low-copy nuclear genes, which suggested both allopatric and ecological speciation events. In addition, cases of plastid-nuclear discordance, in agreement with divergent nuclear ribosomal DNA (nrDNA) copy variants in some species, indicated traces of interspecific gene flow. Our results also support biogeographic links between the Balkan and Anatolian-Caucasus regions and illustrate the contribution of the latter region to high Balkan biodiversity. An allopolyploid origin was inferred for C. barbaraeoides, which highlights the role of mountains in the Balkan Peninsula both as refugia and melting pots favoring species contacts and polyploid evolution in response to Pleistocene climate-induced range dynamics. Overall, our study demonstrates the importance of a thorough phylogenomic approach when studying the evolution of recently diverged species complexes affected by reticulation events at both diploid and polyploid levels. We emphasize the significance of retrieving allelic and homeologous variation from nuclear genes, as well as multiple nrDNA copy variants from genome skim data.

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.

Multivariate morphometric analyses of the predatory ciliate genus Semispathidium (Ciliophora: Litostomatea), with description of S. longiarmatum nov. spec.

We studied morphometrical variation, species boundaries, and importance of morphometric features for a reliable separation of five African Semispathidium taxa. Altogether, 20 features traditionally used in alpha-taxonomy of the predatory genus Semispathidium were measured or scored on 85 protargol-impregnated interphase specimens, and were analyzed using hierarchical clustering as well as principal component and canonical discriminant analyses. This multivariate approach confirmed that a population found in Botswanan floodplain soil represents a distinct taxon. The new species is described here as S. longiarmatum, using live observation, protargol impregnation, and scanning electron microscopy. Semispathidium longiarmatum strongly resembles S. armatum and S. breviarmatum but it is clearly distinguished from these species by the extrusome pattern. The reliability of S. longiarmatum is also strengthened, according to the canonical discriminant analysis, by several quantitative features, viz., the number of ciliary rows, the length:width ratio of the macronucleus, and the number of dikinetids in brush row 1. Moreover, the present study documents the distinctness of all African Semispathidium species which can be separated by a combination of both qualitative and quantitative (morphometric) features. Consequently, Semispathidium species do not form a continuous complex but fairly discrete clusters in the phenotypic space.