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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Inferring hypothesis-based transitions in clade-specific models of chromosome number evolution in sedges (Cyperaceae).

Large-scale changes in chromosome number have been associated with diversification rate shifts in many lineages of plants. For instance, several ancient rounds of polyploidization events have been inferred to promote genomic differentiation and/or isolation and, consequently, angiosperm diversification. Dysploidy, although less studied, has been suggested to also play an important role in angiosperm diversification. In this article, we aim to elucidate the role of chromosomal rearrangements on lineage diversification by analyzing a new comprehensive sedge (Cyperaceae) phylogenetic tree. Our null hypothesis is that the mode and tempo of chromosome evolution are to be homogeneous across the complete phylogeny. In order to discern patterns of diversification shifts and chromosome number changes within the family tree, we tested clade-specific chromosome evolution models for several subtrees according to previously reported increments of diversification rates. Results show that a complex, heterogeneous model composed of different clade-specific chromosome evolution transitions are significantly supported against the null hypothesis of a model with no chromosome number model transition events along the phylogeny. This could suggest a link between diversification and changes in chromosome number evolution although other possibilities are not discarded. Our methodological approach may allow identifying different patterns of chromosome evolution, as found for Cyperaceae, for other lineages at different evolutionary levels.

Do holocentric chromosomes represent an evolutionary advantage? A study of paired analyses of diversification rates of lineages with holocentric chromosomes and their monocentric closest relatives.

Despite most of the cytogenetic research is focused on monocentric chromosomes, chromosomes with kinetochoric activity localized in a single centromere, several studies have been centered on holocentric chromosomes which have diffuse kinetochoric activity along the chromosomes. The eukaryotic organisms that present this type of chromosomes have been relatively understudied despite they constitute rather diversified species lineages. On the one hand, holocentric chromosomes may present intrinsic benefits (chromosome mutations such as fissions and fusions are potentially neutral in holocentrics). On the other hand, they present restrictions to the spatial separation of the functions of recombination and segregation during meiotic divisions (functions that may interfere), separation that is found in monocentric chromosomes. In this study, we compare the diversification rates of all known holocentric lineages in animals and plants with their most related monocentric lineages in order to elucidate whether holocentric chromosomes constitute an evolutionary advantage in terms of diversification and species richness. The results showed that null hypothesis of equal mean diversification rates cannot be rejected, leading us to surmise that shifts in diversification rates between holocentric and monocentric lineages might be due to other factors, such as the idiosyncrasy of each lineage or the interplay of evolutionary selections with the benefits of having either monocentric or holocentric chromosomes.

RAD-seq linkage mapping and patterns of segregation distortion in sedges: meiosis as a driver of karyotypic evolution in organisms with holocentric chromosomes.

Meiotic drive, the class of meiotic mechanisms that drive unequal segregation of alleles among gametes, may be an important force in karyotype evolution. Its role in holocentric organisms, whose chromosomes lack localized centromeres, is poorly understood. We crossed two individuals of Carex scoparia (Cyperaceae) with different chromosome numbers (2n = 33II  = 66 × 2n = 32II  = 64) to obtain F1 individuals, which we then self-pollinated to obtain second-generation (F2) crosses. RAD-seq was performed for 191 individuals (including the parents, five F1 individuals and 184 F2 individuals). Our F2 linkage map based on stringent editing of the RAD-seq data set yielded 32 linkage groups. In the final map, 865 loci were located on a linkage map of 3966.99 cM (linkage groups ranged from 24.39 to 193.31 cM in length and contained 5-51 loci each). Three linkage groups exhibit more loci under segregation distortion than expected by chance; within linkage groups, loci exhibiting segregation distortion are clustered. This finding implicates meiotic drive in the segregation of chromosome variants, suggesting that selection of chromosome variants in meiosis may contribute to the establishment and fixation of chromosome variants in Carex, which is renowned for high chromosomal and species diversity. This is an important finding as previous studies demonstrate that chromosome divergence may play a key role in differentiation and speciation in Carex.

Allopatric speciation despite historical gene flow: Divergence and hybridization in Carex furva and C. lucennoiberica (Cyperaceae) inferred from plastid and nuclear RAD-seq data.

Gene flow among incipient species can act as a creative or destructive force in the speciation process, generating variation on which natural selection can act while, potentially, undermining population divergence. The flowering plant genus Carex exhibits a rapid and relatively recent radiation with many species limits still unclear. This is the case with the Iberian Peninsula (Spain and Portugal)-endemic C. lucennoiberica, which lay unrecognized within Carex furva until its recent description as a new species. In this study, we test how these species were impacted by interspecific gene flow during speciation. We sampled the full range of distribution of C. furva (15 individuals sampled) and C. lucennoiberica (88 individuals), sequenced two cpDNA regions (atpI-atpH, psbA-trnH) and performed genomic sequencing of 45,100 SNPs using restriction site-associated DNA sequencing (RAD-seq). We utilized a set of partitioned D-statistic tests and demographic analyses to study the degree and direction of introgression. Additionally, we modelled species distributions to reconstruct changes in range distribution during glacial and interglacial periods. Plastid, nuclear and morphological data strongly support divergence between species with subsequent gene flow. Combined with species distribution modelling, these data support a scenario of allopatry leading to species divergence, followed by secondary contact and gene flow due to long-distance dispersal and/or range expansions and contractions in response to Quaternary glacial cycles. We conclude that this is a case of allopatric speciation despite historical secondary contacts, which could have temporally influenced the speciation process, contributing to the knowledge of forces that are driving or counteracting speciation.

Niche shifts after long-distance dispersal events in bipolar sedges (Carex, Cyperaceae).

PREMISE OF THE STUDY: Bipolar species represent the greatest biogeographical disjunction on Earth, raising many questions about the colonization and adaptive processes behind such striking distribution. We investigated climatic niche differences of five Carex bipolar species in North and South America to assess niche shifts between these two regions. Moreover, we assessed potential distribution changes with future climate change. METHODS: We used 1202 presence data points from herbarium specimens and 19 bioclimatic variables to assess climatic niche differences and potential distributions among the five species using ordination methods and Maxent. KEY RESULTS: The niche overlap analyses showed low levels of niche filling and high climatic niche expansion between North and South America. Carex macloviana and C. maritima showed the greatest niche expansion (60% and 96%, respectively), followed by C. magellanica (45%) and C. microglochin (39%). Only C. canescens did not colonize new environments (niche expansion = 0.2%). In contrast, all species but C. magellanica had niche filling that was <40%; hence, they are absent in the south from many environments they inhabit in North America. Climate change will push all species toward higher latitudes and elevation, reducing the availability of suitable environments. CONCLUSIONS: The colonization of South America seems to have involved frequent climatic niche shifts. Most species have colonized new environments from those occupied in the North. Observed niche shifts appear congruent with time since colonization and with current genetic structure within species. In these cold-dwelling species, climate change will most likely decrease their suitable environments in the future.

Long-distance dispersal explains the bipolar disjunction in Carex macloviana.

PREMISE OF THE STUDY: The sedge Carex macloviana d'Urv presents a bipolar distribution. To clarify the origin of its distribution, we consider the four main hypotheses: long-distance dispersal (either by mountain hopping or by direct dispersal), vicariance, parallel evolution, and human introduction. METHODS: Phylogenetic, phylogeographic, and divergence time estimation analyses were carried out based on two nuclear ribosomal (ETS and ITS) regions, one nuclear single copy gene (CATP), and three plastid DNA regions (rps16 and 5'trnK introns, and psbA-trnH spacer), using Bayesian inference, maximum likelihood, and statistical parsimony. Bioclimatic data were used to characterize the climatic niche of C. macloviana. KEY RESULTS: Carex macloviana constitutes a paraphyletic species, dating back to the Pleistocene (0.62 Mya, 95% highest posterior density: 0.29-1.00 Mya). This species displays strong genetic structure between hemispheres, with two different lineages in the Southern Hemisphere and limited genetic differentiation in Northern Hemisphere populations. Also, populations from the Southern Hemisphere show a narrower climatic niche with regards to the Northern Hemisphere populations. CONCLUSIONS: Carex macloviana reached its bipolar distribution by long-distance dispersal, although it was not possible to determine whether it was caused by mountain hopping or by direct dispersal. While there is some support that Carex macloviana might have colonized the Northern Hemisphere by south-to-north transhemisphere dispersal during the Pleistocene, unlike the southwards dispersal pattern inferred for other bipolar Carex L. species, we cannot entirely rule out north-to-south dispersion.

Bipolar distributions in vascular plants: A review.

Bipolar disjunct distributions are a fascinating biogeographic pattern exhibited by about 30 vascular plants, whose populations reach very high latitudes in the northern and southern hemispheres. In this review, we first propose a new framework for the definition of bipolar disjunctions and then reformulate a list of guiding principles to consider how to study bipolar species. Vicariance and convergent evolution hypotheses have been argued to explain the origin of this fragmented distribution pattern, but we show here that they can be rejected for all bipolar species, except for Carex microglochin. Instead, human introduction and dispersal (either direct or by mountain-hopping)-facilitated by standard and nonstandard vectors-are the most likely explanations for the origin of bipolar plant disjunctions. Successful establishment after dispersal is key for colonization of the disjunct areas and appear to be related to both intrinsic (e.g., self-compatibility) and extrinsic (mutualistic and antagonistic interactions) characteristics. Most studies on plant bipolar disjunctions have been conducted in Carex (Cyperaceae), the genus of vascular plants with the largest number of bipolar species. We found a predominant north-to-south direction of dispersal, with an estimated time of diversification in agreement with major cooling events during the Pliocene and Pleistocene. Bipolar Carex species do not seem to depend on specialized traits for long-distance dispersal and could have dispersed through one or multiple stochastic events, with birds as the most likely dispersal vector.

Chromosomal rearrangements in holocentric organisms lead to reproductive isolation by hybrid dysfunction: The correlation between karyotype rearrangements and germination rates in sedges.

PREMISE OF THE STUDY: Understanding the drivers of speciation is a central task of evolutionary biology. Chromosomal rearrangements are known to play an important role in species diversification, but the role of rearrangements of holocentric chromosomes-chromosomes without localized centromeres-is poorly understood. METHODS: We made numerous artificial crosses between Carex scoparia individuals of different diploid chromosome numbers and, for comparison, between individuals of the same chromosome number. We studied chromosome pairing and chromosomal rearrangements in the F1 individuals using light microscopy. We then estimated germination rates as a function of geographic distance, genetic distance, chromosome number differences in parents, and pairing irregularities in F1 individuals, using generalized least squares to fit alternative regression models. KEY RESULTS: The most informative predictors of germination rates in the F1 generation are chromosome number differences and minimum number of chromosome pairing irregularities in the F1 individuals. Genetic and geographic distances between parents are not significant predictors. CONCLUSIONS: Holocentric chromosomal rearrangements play an important role in postzygotic reproductive isolation in Carex through F1 hybrid inviability and sterility. Hybrid dysfunction seems to be a suitable model for chromosomal speciation when there are several chromosomal rearrangements between parents. However, we have not tested the hypothesis that genome rearrangements may also play an important role in suppressing recombination between cytogenetically divergent populations.