Untying Gordian knots: unraveling reticulate polyploid plant evolution by genomic data using the large Ranunculus auricomus species complex.

Speciation via hybridization and polyploidization is a major evolutionary force in plant evolution but is still poorly understood for neopolyploid groups. Challenges are attributed to high heterozygosity, low genetic divergence, and missing information on progenitors, ploidy, and reproduction. We study the large Eurasian Ranunculus auricomus species complex and use a comprehensive workflow integrating reduced-representation sequencing (RRS) genomic data to unravel reticulate evolution, genome diversity and composition of polyploids. We rely on 97 312 restriction site-associated DNA sequencing (RAD-Seq) loci, 576 targeted nuclear genes (48 phased), and 71 plastid regions derived from 78 polyploid apomictic taxa and four diploid and one tetraploid putative sexual progenitor species. We applied (phylo)genomic structure, network, and single nucleotide polymorphism (SNP)-origin analyses. Results consistently showed only 3-5 supported and geographically structured polyploid genetic groups, each containing extant sexual and one unknown progenitor species. Combined analyses demonstrated predominantly allopolyploid origins, each involving 2-3 different diploid sexual progenitor species. Young allotetraploids were characterized by subgenome dominance and nonhybrid SNPs, suggesting substantial post-origin but little lineage-specific evolution. The biodiversity of neopolyploid complexes can result from multiple hybrid origins involving different progenitors and substantial post-origin evolution (e.g. homoeologous exchanges, hybrid segregation, gene flow). Reduced-representation sequencing genomic data including multi-approach information is efficient to delimit shallow reticulate relationships.

Moving beyond assumptions: Polyploidy and environmental effects explain a geographical parthenogenesis scenario in European plants.

Geographical parthenogenesis (GP) describes the phenomenon that apomicts tend to have larger distribution areas and/or occur at higher altitudes or latitudes compared to sexual relatives. However, the complex effects of genome-wide heterozygosity, ploidy, reproduction mode (sexual vs. apomictic), and environment shaping GP of plants are still not well understood. We ascertained ploidy and reproduction mode by flow cytometry of 221 populations, and added genomic RADseq data (maximum 33,165 loci) of 80 taxa of the Ranunculus auricomus polyploid plant complex in temperate Europe. We observed 7% mainly diploid sexual, 28% facultative apomictic (mean sexuality 7.1%), and 65% obligate apomictic populations. Sexuals occupied a more southern, smaller distribution area, whereas apomicts expanded their range to higher latitudes. Within the complex, we detected three main genetic clusters and highly reticulate relationships. A genetically-informed path analysis using GLMMs revealed several significant relationships. Sexuality of populations (percent of sexual seeds) was higher in diploids compared to polyploids, associated with more petals, and similar between forests and open habitats. In contrast to other apomictic plant complexes, sexuality was mainly positively correlated to solar radiation and isothermality, which fits the southern distribution. We found up to three times higher heterozygosity in polyploids compared to diploids, and generally more heterozygous individuals in forests compared with open habitats. Interestingly, we revealed a previously unknown positive association between heterozygosity and temperature seasonality, suggesting a higher resistance of polyploids to more extreme climatic conditions. We provide empirical evidence for intrinsic and extrinsic factors shaping the GP pattern in a polyploid plant complex.

The relation of meiotic behaviour to hybridity, polyploidy and apomixis in the Ranunculus auricomus complex (Ranunculaceae).

BACKGROUND: Hybridization and polyploidization are powerful evolutionary factors that are associated with manifold developmental changes in plants such as irregular progression of meiosis and sporogenesis. The emergence of apomixis, which is asexual reproduction via seeds, is supposed to be connected to these factors and was often regarded as an escape from hybrid sterility. However, the functional trigger of apomixis is still unclear. Recently formed di- and polyploid Ranunculus hybrids, as well as their parental species were analysed for their modes of mega- and microsporogenesis by microscopy. Chromosomal configurations during male meiosis were screened for abnormalities. Meiotic and developmental abnormalities were documented qualitatively and collected quantitatively for statistical evaluations. RESULTS: Allopolyploids showed significantly higher frequencies of erroneous microsporogenesis than homoploid hybrid plants. Among diploids, F2 hybrids had significantly more disturbed meiosis than F1 hybrids and parental plants. Chromosomal aberrations included laggard chromosomes, chromatin bridges and disoriented spindle activities. Failure of megasporogenesis appeared to be much more frequent in than of microsporogenesis is correlated to apomixis onset. CONCLUSIONS: Results suggest diverging selective pressures on female and male sporogenesis, with only minor effects of hybridity on microsporogenesis, but fatal effects on the course of megasporogenesis. Hence, pollen development continues without major alterations, while selection will favour apomixis as alternative to the female meiotic pathway. Relation of investigated errors of megasporogenesis with the observed occurrence of apospory in Ranunculus hybrids identifies disturbed female meiosis as potential elicitor of apomixis in order to rescue these plants from hybrid sterility. Male meiotic disturbance appears to be stronger in neopolyploids than in homoploid hybrids, while disturbances of megasporogenesis were not ploidy-dependent.

Phylogenomics unravels Quaternary vicariance and allopatric speciation patterns in temperate-montane plant species: A case study on the Ranunculus auricomus species complex.

The time frame and geographical patterns of diversification processes in European temperate-montane herbs are still not well understood. We used the sexual species of the Ranunculus auricomus complex as a model system to understand how vicariance versus dispersal processes in the context of Pleistocene climatic fluctuations have triggered speciation in temperate-montane plant species. We used target enrichment sequence data from about 600 nuclear genes and coalescent-based species tree inference methods to resolve phylogenetic relationships among the sexual taxa of the complex. We estimated absolute divergence times and, using ancestral range reconstruction, we tested if speciation was enhanced by vicariance or by dispersal processes. Phylogenetic relationships among taxa were fully resolved with some incongruence in the position of the tetraploid R. marsicus. Speciation events took place in a very short time at the end of the Mid-Pleistocene Transition (830-580 thousand years ago [ka]). A second wave of intraspecific geographical differentiation occurred at the end of the Riss glaciation or during the Eemian interglacial between 200 and 100 ka. Ancestral range reconstruction suggests a widespread European ancestor of the R. auricomus complex. Vicariance has triggered allopatric speciation in temperate-montane plant species during the climatic deterioration that occurred during the last phase of the Mid-Pleistocene Transition. Vegetation restructuring from forest into tundra could have confined these forest species into isolated glacial macro- and microrefugia. During subsequent warming periods, range expansions of these species could have been hampered by apomictic derivatives and by other congeneric competitors in the same habitat.

Species delimitation 4.0: integrative taxonomy meets artificial intelligence.

Although species are central units for biological research, recent findings in genomics are raising awareness that what we call species can be ill-founded entities due to solely morphology-based, regional species descriptions. This particularly applies to groups characterized by intricate evolutionary processes such as hybridization, polyploidy, or asexuality. Here, challenges of current integrative taxonomy (genetics/genomics + morphology + ecology, etc.) become apparent: different favored species concepts, lack of universal characters/markers, missing appropriate analytical tools for intricate evolutionary processes, and highly subjective ranking and fusion of datasets. Now, integrative taxonomy combined with artificial intelligence under a unified species concept can enable automated feature learning and data integration, and thus reduce subjectivity in species delimitation. This approach will likely accelerate revising and unraveling eukaryotic biodiversity.

Population size affected by environmental variability impacts genetics, traits, and plant performance in Trifolium montanum L.

Population size, genetic diversity, and performance have fundamental importance for ecology, evolution, and nature conservation of plant species. Despite well-studied relationships among environmental, genetic, and intraspecific trait variation (ITV), the influence of population size on these aspects is less understood. To assess the sources of population size variation, but also its impact on genetic, functional trait, and performance aspects, we conducted detailed population size estimations, assessed 23 abiotic and biotic environmental habitat factors, performed population genetic analyses using nine microsatellite markers, and recorded nine functional traits based on 260 Trifolium montanum individuals from 13 semi-dry grassland locations of Central Europe. Modern statistical analyses based on a multivariate framework (path analysis) with preselected linear regression models revealed that the variation of abiotic factors (in contrast to factors per se) almost completely, significantly explained fluctuations in population size (R 2 = .93). In general, abiotic habitat variation (heterogeneity) was not affected by habitat area. Population size significantly explained genetic diversity (N A: R 2 = .42, H o: R 2 = .67, H e: R 2 = .43, and I: R 2 = .59), inbreeding (F IS: R 2 = .35), and differentiation (G ST: R 2 = .20). We also found that iFDCV (ITV) was significantly explained by abiotic habitat heterogeneity, and to a lesser extent by genetic diversity H e (R 2 = .81). Nevertheless, habitat heterogeneity did not statistically affect genetic diversity. This may be due to the use of selectively neutral microsatellite markers, and possibly by insufficient abiotic selective pressures on habitats examined. Small T. montanum populations in nonoptimal habitats were characterized by reduced genetic and functional trait diversity, and elevated genetic inbreeding and differentiation. This indicates reduced adaptability to current and future environmental changes. The long-term survival of small populations with reduced genetic diversity and beginning inbreeding will be highly dependent on habitat protection and adequate land-use actions.

Phylogenomics of Southern European Taxa in the Ranunculus auricomus Species Complex: The Apple Doesn't Fall Far from the Tree.

The taxonomic status of many Southern European taxa of the Ranunculus auricomus complex remains uncertain despite this region's proximity to the native ranges of the sexual progenitor species of the complex. We investigated whether additional sexual progenitor species are present in the Mediterranean region. Utilizing target enrichment of 736 single-copy nuclear gene regions and flow cytometry, we analyzed phylogenomic relationships, the ploidy level, and the reproductive mode in representatives of 16 populations in Southern Europe, with additional sequence data from herbarium collections. Additionally, phased sequence assemblies from suspected nothotaxa were mapped to previously described sexual progenitor species in order to determine hybrid ancestry. We found the majority of Mediterranean taxa to be tetraploid, with hybrid populations propagating primarily via apomixis. Phylogenomic analysis revealed that except for the progenitor species, the Mediterranean taxa are often polyphyletic. Most apomictic taxa showed evidence of mixed heritage from progenitor species, with certain progenitor genotypes having mapped more to the populations from adjacent geographical regions. Geographical trends were found in phylogenetic distance, roughly following an east-to-west longitudinal demarcation of the complex, with apomicts extending to the southern margins. Additionally, we observed post-hybridization divergence between the western and eastern populations of nothotaxa in Southern Europe. Our results support a classification of apomictic populations as nothotaxa, as previously suggested for Central Europe.

Geometric Morphometric Versus Genomic Patterns in a Large Polyploid Plant Species Complex.

Plant species complexes represent a particularly interesting example of taxonomically complex groups (TCGs), linking hybridization, apomixis, and polyploidy with complex morphological patterns. In such TCGs, mosaic-like character combinations and conflicts of morphological data with molecular phylogenies present a major problem for species classification. Here, we used the large polyploid apomictic European Ranunculus auricomus complex to study relationships among five diploid sexual progenitor species and 75 polyploid apomictic derivate taxa, based on geometric morphometrics using 11,690 landmarked objects (basal and stem leaves, receptacles), genomic data (97,312 RAD-Seq loci, 48 phased target enrichment genes, 71 plastid regions) from 220 populations. We showed that (1) observed genomic clusters correspond to morphological groupings based on basal leaves and concatenated traits, and morphological groups were best resolved with RAD-Seq data; (2) described apomictic taxa usually overlap within trait morphospace except for those taxa at the space edges; (3) apomictic phenotypes are highly influenced by parental subgenome composition and to a lesser extent by climatic factors; and (4) allopolyploid apomictic taxa, compared to their sexual progenitor, resemble a mosaic of ecological and morphological intermediate to transgressive biotypes. The joint evaluation of phylogenomic, phenotypic, reproductive, and ecological data supports a revision of purely descriptive, subjective traditional morphological classifications.

Incorporation of mineral nitrogen into the soil food web as affected by plant community composition.

Although nitrogen (N) deposition is increasing globally, N availability still limits many organisms, such as microorganisms and mesofauna. However, little is known to which extent soil organisms rely on mineral-derived N and whether plant community composition modifies its incorporation into soil food webs. More diverse plant communities more effectively compete with microorganisms for mineral N likely reducing the incorporation of mineral-derived N into soil food webs. We set up a field experiment in experimental grasslands with different levels of plant species and functional group richness. We labeled soil with 15NH4 15NO3 and analyzed the incorporation of mineral-derived 15N into soil microorganisms and mesofauna over 3 months. Mineral-derived N incorporation decreased over time in all investigated organisms. Plant species richness and presence of legumes reduced the uptake of mineral-derived N into microorganisms. In parallel, the incorporation of mineral-derived 15N into mesofauna species declined with time and decreased with increasing plant species richness in the secondary decomposer springtail Ceratophysella sp. Effects of both plant species richness and functional group richness on other mesofauna species varied with time. The presence of grasses increased the 15N incorporation into Ceratophysella sp., but decreased it in the primary decomposer oribatid mite Tectocepheus velatus sarekensis. The results highlight that mineral N is quickly channeled into soil animal food webs via microorganisms irrespective of plant diversity. The amount of mineral-derived N incorporated into soil animals, and the plant community properties affecting this incorporation, differed markedly between soil animal taxa, reflecting species-specific use of food resources. Our results highlight that plant diversity and community composition alter the competition for N in soil and change the transfer of N across trophic levels in soil food webs, potentially leading to changes in soil animal population dynamics and community composition. Sustaining high plant diversity may buffer detrimental effects of elevated N deposition on soil biota.

Plant intraspecific functional trait variation is related to within-habitat heterogeneity and genetic diversity in Trifolium montanum L.

Intraspecific trait variation (ITV), based on available genetic diversity, is one of the major means plant populations can respond to environmental variability. The study of functional trait variation and diversity has become popular in ecological research, for example, as a proxy for plant performance influencing fitness. Up to now, it is unclear which aspects of intraspecific functional trait variation (iFDCV) can be attributed to the environment or genetics under natural conditions. Here, we examined 260 individuals from 13 locations of the rare (semi-)dry calcareous grassland species Trifolium montanum L. in terms of iFDCV, within-habitat heterogeneity, and genetic diversity. The iFDCV was assessed by measuring functional traits (releasing height, biomass, leaf area, specific leaf area, leaf dry matter content, Fv/Fm, performance index, stomatal pore surface, and stomatal pore area index). Abiotic within-habitat heterogeneity was derived from altitude, slope exposure, slope, leaf area index, soil depth, and further soil factors. Based on microsatellites, we calculated expected heterozygosity (He) because it best-explained, among other indices, iFDCV. We performed multiple linear regression models quantifying relationships among iFDCV, abiotic within-habitat heterogeneity and genetic diversity, and also between separate functional traits and abiotic within-habitat heterogeneity or genetic diversity. We found that abiotic within-habitat heterogeneity influenced iFDCV twice as strong compared to genetic diversity. Both aspects together explained 77% of variation in iFDCV ( R adj 2  = .77, F 2, 10 = 21.66, p < .001). The majority of functional traits (releasing height, biomass, specific leaf area, leaf dry matter content, Fv/Fm, and performance index) were related to abiotic habitat conditions indicating responses to environmental heterogeneity. In contrast, only morphology-related functional traits (releasing height, biomass, and leaf area) were related to genetics. Our results suggest that both within-habitat heterogeneity and genetic diversity affect iFDCV and are thus crucial to consider when aiming to understand or predict changes of plant species performance under changing environmental conditions.

Breeding system of diploid sexuals within the Ranunculus auricomus complex and its role in a geographical parthenogenesis scenario.

The larger distribution area of asexuals compared with their sexual relatives in geographical parthenogenesis (GP) scenarios has been widely attributed to the advantages of uniparental reproduction and polyploidy. However, potential disadvantages of sexuals due to their breeding system have received little attention so far. Here, we study the breeding system of five narrowly distributed sexual lineages of Ranunculus notabilis s.l. (R. auricomus complex) and its effects on outcrossing, inbreeding, female fitness, and heterozygosity. We performed selfing and intra- and interlineage crossings by bagging 481 flowers (59 garden individuals) followed by germination experiments. We compared seed set and germination rates, and related them to genetic distance and genome-wide heterozygosity (thousands of RADseq loci). Selfings (2.5%) unveiled a significantly lower seed set compared with intra- (69.0%) and interlineage crossings (69.5%). Seed set of intra- (65%) compared to interpopulation crossings (78%) was significantly lower. In contrast, all treatments showed comparable germination rates (32%-43%). Generalized linear regressions between seed set and genetic distance revealed positive relationships in general and between lineages, and a negative one within lineages. Seed set was the main decisive factor for female fitness. Germination rates were not related to genetic distance at any level, but were positively associated with heterozygosity in interlineage crossings. Experiments confirmed full crossability and predominant outcrossing among sexual R. notabilis s.l. lineages. However, up to 5% (outliers 15%-31%) of seeds were formed by selfing, probably due to semi-self-compatibility in a multi-locus gametophytic SI system. Less seed set in intrapopulation crossings, and higher seed set and germination rates from crossings of genetically more distant and heterozygous lineages (interlineage) indicate negative inbreeding and positive outbreeding effects. In GP scenarios, sexual species with small and/or isolated populations can suffer from decreased female fitness due to their breeding system. This factor, among others, probably limits range expansion of sexuals.

Desert-like badlands and surrounding (semi-)dry grasslands of Central Germany promote small-scale phenotypic and genetic differentiation in Thymus praecox.

Environmental heterogeneity among sites can generate phenotypic and genetic variation facilitating differentiation and microevolution of plant populations. Badlands are desert-like, predominantly vegetation-poor habitats often embedded in (semi-)dry grasslands. The desert-like conditions of badlands demand extreme adaptation of plants, that is, phenotypic modifications in short-term and/or natural adaptation in long-term. However, detailed knowledge is missing about both plant phenotypic and genetic differentiation in this unique and widely occurring habitat type. The present study focused on the largest known badlands systems in Central Europe located in the "Drei Gleichen" region, a designated nature conservation area in Central Germany. Locations were suitable for this study in terms of having co-occurring badlands and (semi-)dry grassland habitats (sites) occupied by the pioneer plant Thymus praecox. Here, we studied the environmental preferences, morphological and functional trait variation, and genetic variation using microsatellite markers of T. praecox. Results revealed significant, mainly site-dependent environmental, phenotypic, and genetic differentiation. In general, individuals in badlands are shorter in height and have lower patch sizes (length × width), relative growth rates, and smaller stomata. The PCA additionally unveiled slightly increased leaf robustness, trichome density, decreased stomatal conductance, fewer females, and earlier phenology in badlands. We interpret differentiation patterns as adaptive responses to light, temperature, drought, and nutrient stress conditions supported by reviewed literature. Genetic differentiation was strongest between local badlands and grassland sites, and clearly weaker among locations and between sites (in total) as indicated by G ST, AMOVA, PCoA, and population structure. Our study supports the importance of small-scale microhabitat conditions as a driver of microevolutionary processes, and the population's need for sufficient phenotypic variation and genetic resources to deal with environmental changes. We demonstrated that badlands are an appropriate model system for testing plant response to extreme habitats and that more research is needed on these fascinating landscapes.