Polyploidy and hybridization in the Mediterranean: unravelling the evolutionary history of Centaurium (Gentianaceae).

BACKGROUND AND AIMS: Polyploidy is considered one of the main mechanisms of plant evolution and speciation. In the Mediterranean Basin, polyploidy has contributed to making this region a biodiversity hotspot, along with its geological and climatic history and other ecological and biogeographical factors. The Mediterranean genus Centaurium (Gentianaceae) comprises ~25 species, of which 60 % are polyploids, including tetraploids and hexaploids. To date, the evolutionary history of centauries has been studied using Sanger sequencing phylogenies, which have been insufficient to fully understand the phylogenetic relationships in this lineage. The goal of this study is to gain a better understanding of the evolutionary history of Centaurium by exploring the mechanisms that have driven its diversification, specifically hybridization and polyploidy. We aim to identify the parentage of hybrid species, at the species or clade level, as well as assessing whether morphological traits are associated with particular ploidy levels. METHODS: We sequenced RADseq markers from 42 samples of 28 Centaurium taxa, and performed phylogenomic analyses using maximum likelihood, summary coalescent SVDquartets and Neighbor-Net approaches. To identify hybrid taxa, we used PhyloNetworks and the fastSTRUCTURE algorithm. To infer the putative parental species of the allopolyploids, we employed genomic analyses (SNIPloid). The association between different traits and particular ploidy levels was explored with non-metric multidimensional scaling. KEY RESULTS: Our phylogenetic analyses confirmed the long-suspected occurrence of recurrent hybridization. The allopolyploid origin of the tetraploid C. serpentinicola and the hexaploids C. mairei, C. malzacianum and C. centaurioides was also confirmed, unlike that of C. discolor. We inferred additional signatures of hybridization events within the genus and identified morphological traits differentially distributed in different ploidy levels. CONCLUSIONS: This study highlights the important role that hybridization has played in the evolution of a Mediterranean genus such as Centaurium, leading to a polyploid complex, which facilitated its diversification and may exemplify that of other Mediterranean groups.

Genome constitution and evolution of Elymus atratus (Poaceae: Triticeae) inferred from cytogenetic and phylogenetic analysis.

BACKGROUND: Elymus atratus (Nevski) Hand.-Mazz. is perennial hexaploid wheatgrass. It was assigned to the genus Elymus L. sensu stricto based on morphological characters. Its genome constitution has not been disentangled yet. OBJECTIVE: To identify the genome constitution and origin of E. atratus. METHODS: In this study, genomic in situ hybridization and fluorescence in situ hybridization, and phylogenetic analysis based on the Acc1, DMC1 and matK sequences were performed. RESULTS: Genomic in situ hybridization and fluorescence in situ hybridization results reveal that E. atratus 2n = 6x = 42 is composed of 14 St genome chromosomes, 14 H genome chromosomes, and 14 Y genome chromosomes including two H-Y type translocation chromosomes, suggesting that the genome formula of E. atratus is StStYYHH. The phylogenetic analysis based on Acc1 and DMC1 sequences not only shows that the Y genome originated in a separate diploid, but also suggests that Pseudoroegneria (St), Hordeum (H), and a diploid species with Y genome were the potential donors of E. atratus. Data from chloroplast DNA showed that the maternal donor of E. atratus contains the St genome. CONCLUSION: Elymus atratus is an allohexaploid species with StYH genome, which may have originated through the hybridization between an allotetraploid Roegneria (StY) species as the maternal donor and a diploid Hordeum (H) species as the paternal donor.

Convergent evolutionary patterns of heterostyly across angiosperms support the pollination-precision hypothesis.

Since the insights by Charles Darwin, heterostyly, a floral polymorphism with morphs bearing stigmas and anthers at reciprocal heights, has become a model system for the study of natural selection. Based on his archetypal heterostylous flower, including regular symmetry, few stamens and a tube, Darwin hypothesised that heterostyly evolved to promote outcrossing through efficient pollen transfer between morphs involving different areas of a pollinator's body, thus proposing his seminal pollination-precision hypothesis. Here we update the number of heterostylous and other style-length polymorphic taxa to 247 genera belonging to 34 families, notably expanding known cases by 20%. Using phylogenetic and comparative analyses across the angiosperms, we show numerous independent origins of style-length polymorphism associated with actinomorphic, tubular flowers with a low number of sex organs, stamens fused to the corolla, and pollination by long-tongued insects. These associations provide support for the Darwinian pollination-precision hypothesis as a basis for convergent evolution of heterostyly across angiosperms.

Global analysis of Poales diversification - parallel evolution in space and time into open and closed habitats.

Poales are one of the most species-rich, ecologically and economically important orders of plants and often characterise open habitats, enabled by unique suites of traits. We test six hypotheses regarding the evolution and assembly of Poales in open and closed habitats throughout the world, and examine whether diversification patterns demonstrate parallel evolution. We sampled 42% of Poales species and obtained taxonomic and biogeographic data from the World Checklist of Vascular Plants database, which was combined with open/closed habitat data scored by taxonomic experts. A dated supertree of Poales was constructed. We integrated spatial phylogenetics with regionalisation analyses, historical biogeography and ancestral state estimations. Diversification in Poales and assembly of open and closed habitats result from dynamic evolutionary processes that vary across lineages, time and space, most prominently in tropical and southern latitudes. Our results reveal parallel and recurrent patterns of habitat and trait transitions in the species-rich families Poaceae and Cyperaceae. Smaller families display unique and often divergent evolutionary trajectories. The Poales have achieved global dominance via parallel evolution in open habitats, with notable, spatially and phylogenetically restricted divergences into strictly closed habitats.

Chromosomal evolution in Cryptangieae Benth. (Cyperaceae): Evidence of holocentrism and pseudomonads.

Cryptangieae has recently been revised based on morphology and molecular phylogeny, but cytogenetic data is still scarce. We conducted this study with the aim of investigating the occurrence of holocentric chromosomes and pseudomonads, as well as understanding the mode of chromosomal evolution in the tribe. We performed analyses of meiotic behavior, chromosome counts, and reconstruction of the ancestral state for the haploid number. We present novel cytogenetic data for eight potentially holocentric species: Cryptangium verticillatum, Krenakia junciforme, K. minarum, Lagenocarpus bracteosus, L. griseus, L. inversus, L. rigidus, and L. tenuifolius. Meiotic abnormalities were observed, with parallel spindles being particularly noteworthy. Intra-specific variations in chromosome number were not found, which may indicate an efficient genetic control for the elimination of abnormal nuclei. The inferred ancestral haploid number was n = 16, with dysploidy being the main evolutionary mechanism. At least five chromosomal fissions occurred in Krenakia (n = 21), followed by a further ascending dysploidy event in Lagenocarpus (n = 17). As proposed for Cyperaceae, it is possible that cladogenesis events in Cryptangieae were marked by numerical and structural chromosomal changes.

The holocentric chromosome microevolution: From phylogeographic patterns to genomic associations with environmental gradients.

Geographic isolation and chromosome evolution are two of the major drivers of diversification in eukaryotes in general, and specifically, in plants. On one hand, range shifts induced by Pleistocene glacial oscillations deeply shaped the evolutionary trajectories of species in the Northern Hemisphere. On the other hand, karyotype variability within species or species complexes may have adaptive potential as different karyotypes may represent different recombination rates and linkage groups that may be associated with locally adapted genes or supergenes. Organisms with holocentric chromosomes are ideal to study the link between local adaptation and chromosome evolution, due to their high cytogenetic variability, especially when it seems to be related to environmental variation. Here, we integrate the study of the phylogeography, chromosomal evolution and ecological requirements of a plant species complex distributed in the Western Euro-Mediterranean region (Carex gr. laevigata, Cyperaceae). We aim to clarify the relative influence of these factors on population differentiation and ultimately on speciation. We obtained a well-resolved RADseq phylogeny that sheds light on the phylogeographic patterns of molecular and chromosome number variation, which are compatible with south-to-north postglacial migration. In addition, landscape genomics analyses identified candidate loci for local adaptation, and also strong significant associations between the karyotype and the environment. We conclude that karyotype distribution in C. gr. laevigata has been constrained by both range shift dynamics and local adaptation. Our study demonstrates that chromosome evolution may be responsible, at least partially, for microevolutionary patterns of population differentiation and adaptation in Carex.

Drivers of diversification in Linum (Linaceae) by means of chromosome evolution: correlations with biogeography, breeding system and habit.

BACKGROUND AND AIMS: Chromosome evolution leads to hybrid dysfunction and recombination patterns and has thus been proposed as a major driver of diversification in all branches of the tree of life, including flowering plants. In this study we used the genus Linum (flax species) to evaluate the effects of chromosomal evolution on diversification rates and on traits that are important for sexual reproduction. Linum is a useful study group because it has considerable reproductive polymorphism (heterostyly) and chromosomal variation (n = 6-36) and a complex pattern of biogeographical distribution. METHODS: We tested several traditional hypotheses of chromosomal evolution. We analysed changes in chromosome number across the phylogenetic tree (ChromEvol model) in combination with diversification rates (ChromoSSE model), biogeographical distribution, heterostyly and habit (ChromePlus model). KEY RESULTS: Chromosome number evolved across the Linum phylogeny from an estimated ancestral chromosome number of n = 9. While there were few apparent incidences of cladogenesis through chromosome evolution, we inferred up to five chromosomal speciation events. Chromosome evolution was not related to heterostyly but did show significant relationships with habit and geographical range. Polyploidy was negatively correlated with perennial habit, as expected from the relative commonness of perennial woodiness and absence of perennial clonality in the genus. The colonization of new areas was linked to genome rearrangements (polyploidy and dysploidy), which could be associated with speciation events during the colonization process. CONCLUSIONS: Chromosome evolution is a key trait in some clades of the Linum phylogeny. Chromosome evolution directly impacts speciation and indirectly influences biogeographical processes and important plant traits.

Holocentric repeat landscapes: From micro-evolutionary patterns to macro-evolutionary associations with karyotype evolution.

Repetitive elements can cause large-scale chromosomal rearrangements, for example through ectopic recombination, potentially promoting reproductive isolation and speciation. Species with holocentric chromosomes, that lack a localized centromere, might be more likely to retain chromosomal rearrangements that lead to karyotype changes such as fusions and fissions. This is because chromosome segregation during cell division should be less affected than in organisms with a localized centromere. The relationships between repetitive elements and chromosomal rearrangements and how they may translate to patterns of speciation in holocentric organisms are though poorly understood. Here, we use a reference-free approach based on low-coverage short-read sequencing data to characterize the repeat landscape of two independently evolved holocentric groups: Erebia butterflies and Carex sedges. We consider both micro- and macro-evolutionary scales to investigate the repeat landscape differentiation between Erebia populations and the association between repeats and karyotype changes in a phylogenetic framework for both Erebia and Carex. At a micro-evolutionary scale, we found population differentiation in repeat landscape that increases with overall intraspecific genetic differentiation among four Erebia species. At a macro-evolutionary scale, we found indications for an association between repetitive elements and karyotype changes along both Erebia and Carex phylogenies. Altogether, our results suggest that repetitive elements are associated with the level of population differentiation and chromosomal rearrangements in holocentric clades and therefore likely play a role in adaptation and potentially species diversification.

Genomic hotspots of chromosome rearrangements explain conserved synteny despite high rates of chromosome evolution in a holocentric lineage.

Holocentric organisms, unlike typical monocentric organisms, have kinetochore activity distributed along almost the whole length of the chromosome. Because of this, chromosome rearrangements through fission and fusion are more likely to become fixed in holocentric species, which may account for the extraordinary rates of chromosome evolution that many holocentric lineages exhibit. Long blocks of genome synteny have been reported in animals with holocentric chromosomes despite high rates of chromosome rearrangements. Nothing is known from plants, however, despite the fact that holocentricity appears to have played a key role in the diversification of one of the largest angiosperm genera, Carex (Cyperaceae). In the current study, we compared genomes of Carex species and a distantly related Cyperaceae species to characterize conserved and rearranged genome regions. Our analyses span divergence times ranging between 2 and 50 million years. We also compared a C. scoparia chromosome-level genome assembly with a linkage map of the same species to study rearrangements at a population level and suppression of recombination patterns. We found longer genome synteny blocks than expected under a null model of random rearrangement breakpoints, even between very distantly related species. We also found repetitive DNA to be non-randomly associated with holocentromeres and rearranged regions of the genome. The evidence of conserved synteny in sedges despite high rates of chromosome fission and fusion suggests that conserved genomic hotspots of chromosome evolution related to repetitive DNA shape the evolution of recombination, gene order and crossability in sedges. This finding may help explain why sedges are able to maintain species cohesion even in the face of high interspecific chromosome rearrangements.

Founder events and subsequent genetic bottlenecks underlie karyotype evolution in the Ibero - North African endemic Carex helodes.

BACKGROUND AND AIMS: Despite chromosomal evolution being one of the major drivers of diversification in plants, we do not yet have a clear view of how new chromosome rearrangements become fixed within populations, which is a crucial step forward for understanding chromosomal speciation. METHODS: In this study, we test the role of genetic drift in the establishment of new chromosomal variants in the context of hybrid dysfunction models of chromosomal speciation. We genotyped a total of 178 individuals from seven populations (plus 25 seeds from one population) across the geographic range of Carex helodes (Cyperaceae). We also characterized karyotype geographic patterns of the species across the distribution range. For one of the populations, we performed a detailed study of the fine scale, local spatial distribution of its individuals and their genotypes and karyotypes. KEY RESULTS: Synergistically, phylogeographic and karyotypic evidence show two main genetic groups: southwestern Iberian Peninsula vs. northwestern African populations, and within Europe our results suggest a west-to-east expansion with signals of genetic bottlenecks. Additionally, we have inferred a pattern of descending dysploidy, plausibly as a result of a west-to-east process of post-glacial colonization in Europe. CONCLUSIONS: Our results give experimental support to the role of geographic isolation, drift, and inbreeding in the establishment of new karyotypes which is key in the speciation models of hybrid dysfunction.

Using ChromEvol to Determine the Mode of Chromosomal Evolution.

The ChromEvol software was the first to implement a likelihood-based approach, using probabilistic models that depict the pattern of chromosome number change along a specified phylogeny. The initial models have been completed and expanded during the last years. New parameters that model polyploid chromosome evolution have been implemented in ChromEvol v.2. In recent years, new and more complex models have been developed. The BiChrom model is able to implement two distinct chromosome models for the two possible trait states of a binary character of interest. ChromoSSE jointly implements chromosome evolution, speciation, and extinction. In the near future, we will be able to study chromosome evolution with increasingly complex models.

Karyotypic diversity: a neglected trait to explain angiosperm diversification?

Evolutionary changes in karyotype provide genetic support to organisms' differentiation and adaptation; however, the association between karyotype diversity and species diversification in flowering plants (angiosperms) remains to be fully elucidated. We sought evidence for this association within a phylogenetic framework using a dataset comprising > 413,000 worldwide chromosome counts of 66,000 angiosperms species. Karyotypic diversity (e.g., number of distinct chromosome numbers) explains species richness and diversification rates at both family and genus levels highlighting that chromosome evolution has probably played, at least, an important role in reinforcing speciation that was already initiated or completed by other geographical or ecological drivers. Thus, research programs investigating chromosome variation as a direct or indirect driver of diversification should be encouraged.

A non-homogeneous model of chromosome-number evolution to reveal shifts in the transition patterns across the phylogeny.

Changes in chromosome numbers, including polyploidy and dysploidy events, play a key role in eukaryote evolution as they could expediate reproductive isolation and have the potential to foster phenotypic diversification. Deciphering the pattern of chromosome-number change within a phylogeny currently relies on probabilistic evolutionary models. All currently available models assume time homogeneity, such that the transition rates are identical throughout the phylogeny. Here, we develop heterogeneous models of chromosome-number evolution that allow multiple transition regimes to operate in distinct parts of the phylogeny. The partition of the phylogeny to distinct transition regimes may be specified by the researcher or, alternatively, identified using a sequential testing approach. Once the number and locations of shifts in the transition pattern are determined, a second search phase identifies regimes with similar transition dynamics, which could indicate on convergent evolution. Using simulations, we study the performance of the developed model to detect shifts in patterns of chromosome-number evolution and demonstrate its applicability by analyzing the evolution of chromosome numbers within the Cyperaceae plant family. The developed model extends the capabilities of probabilistic models of chromosome-number evolution and should be particularly helpful for the analyses of large phylogenies that include multiple distinct subclades.

Chromosome size matters: genome evolution in the cyperid clade.

BACKGROUND AND AIMS: While variation in genome size and chromosome numbers and their consequences are often investigated in plants, the biological relevance of variation in chromosome size remains poorly known. Here, we examine genome and mean chromosome size in the cyperid clade (families Cyperaceae, Juncaceae and Thurniaceae), which is the largest vascular plant lineage with predominantly holocentric chromosomes. METHODS: We measured genome size in 436 species of cyperids using flow cytometry, and augment these data with previously published datasets. We then separately compared genome and mean chromosome sizes (2C/2n) amongst the major lineages of cyperids and analysed how these two genomic traits are associated with various environmental factors using phylogenetically informed methods. KEY RESULTS: We show that cyperids have the smallest mean chromosome sizes recorded in seed plants, with a large divergence between the smallest and largest values. We found that cyperid species with smaller chromosomes have larger geographical distributions and that there is a strong inverse association between mean chromosome size and number across this lineage. CONCLUSIONS: The distinct patterns in genome size and mean chromosome size across the cyperids might be explained by holokinetic drive. The numerous small chromosomes might function to increase genetic diversity in this lineage where crossovers are limited during meiosis.

A holocentric twist to chromosomal speciation?

Chromosomal rearrangements trigger speciation by acting as barriers to gene flow. However, the underlying theory was developed with monocentric chromosomes in mind. Holocentric chromosomes, lacking a centromeric region, have repeatedly evolved and account for a significant fraction of extant biodiversity. Because chromosomal rearrangements may be more likely retained in holocentric species, holocentricity could provide a twist to chromosomal speciation. Here, we discuss how the abundance of chromosome-scale genomes, combined with novel analytical tools, offer the opportunity to assess the impacts of chromosomal rearrangements on rates of speciation by outlining a phylogenetic framework that aligns with the two major lines of chromosomal speciation theory. We further highlight how holocentric species could help to test for causal roles of chromosomal rearrangements in speciation.

Isolation and characterization of microsatellites markers in Centaurium grandiflorum ssp. boissieri.

BACKGROUND: Estimating outcrossing/selfing rates and characterizing genetic diversity with microsatellite markers are crucial to understanding the evolution of plant mating systems. METHODS AND RESULTS: We developed, optimized and characterized eight new primer pairs for Centaurium grandiflorum ssp. boissieri and transferred them to three subspecies of Centaurium quadrifolium. Two SSR loci were transferred from Sabatia campestris to the four Centaurium taxa. Polymorphisms, He, Ho and H-W deviations were estimated in two populations of C. grandiflorum ssp. boissieri and in seven individuals each of C. quadrifolium ssp. barrelieri, C. quadrifolium ssp. parviflorum and C. quadrifolium ssp. quadrifolium. A total of 80 individuals was used in these experiments. The number of polymorphic loci varied among species from one to ten. A total of 127 alleles was scored. The average number of alleles per locus was 12.7. He was higher than Ho in all sampled populations. Hardy-Weinberg equilibrium was found for some loci in different species. CONCLUSIONS: This is the first report of microsatellites successfully amplified in the whole Centaurium genus. They will be valuable for estimating mating system parameters and genetic diversity and exploring their relationships with the wide variation in flower morphology in the genus, especially anther-stigma separation.

An integrative monograph of Carex section Schoenoxiphium (Cyperaceae).

Carex section Schoenoxiphium (Cariceae, Cyperaceae) is endemic to the Afrotropical biogeographic region and is mainly distributed in southern and eastern Africa, with its center of diversity in eastern South Africa. The taxon was formerly recognized as a distinct genus and has a long history of taxonomic controversy. It has also an important morphological and molecular background in particular dealing with the complexity of its inflorescence and the phylogenetic relationships of its species. We here present a fully updated and integrative monograph of Carex section Schoenoxiphium based on morphological, molecular and cytogenetic data. A total of 1,017 herbarium specimens were examined and the majority of the species were studied in the field. Previous molecular phylogenies based on Sanger-sequencing of four nuclear and plastid DNA regions and RAD-seq were expanded. For the first time, chromosome numbers were obtained, with cytogenetic counts on 44 populations from 15 species and one hybrid. Our taxonomic treatment recognizes 21 species, one of them herein newly described (C. gordon-grayae). Our results agree with previous molecular works that have found five main lineages in Schoenoxiphium. We provide detailed morphological descriptions, distribution maps and analytical drawings of all accepted species in section Schoenoxiphium, an identification key, and a thorough nomenclatural survey including 19 new typifications and one nomen novum.

WOODIV, a database of occurrences, functional traits, and phylogenetic data for all Euro-Mediterranean trees.

Trees play a key role in the structure and function of many ecosystems worldwide. In the Mediterranean Basin, forests cover approximately 22% of the total land area hosting a large number of endemics (46 species). Despite its particularities and vulnerability, the biodiversity of Mediterranean trees is not well known at the taxonomic, spatial, functional, and genetic levels required for conservation applications. The WOODIV database fills this gap by providing reliable occurrences, four functional traits (plant height, seed mass, wood density, and specific leaf area), and sequences from three DNA-regions (rbcL, matK, and trnH-psbA), together with modelled occurrences and a phylogeny for all 210 Euro-Mediterranean tree species. We compiled, homogenized, and verified occurrence data from sparse datasets and collated them on an INSPIRE-compliant 10 × 10 km grid. We also gathered functional trait and genetic data, filling existing gaps where possible. The WOODIV database can benefit macroecological studies in the fields of conservation, biogeography, and community ecology.