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.

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.

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.

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.

Polyploidy Expands the Range of Centaurium (Gentianaceae).

The Mediterranean region is one of the most important worldwide hotspots in terms of number of species and endemism, and multiple hypotheses have been proposed to explain how diversification occurred in this area. The contribution of different traits to the diversification process has been evaluated in different groups of plants. In the case of Centaurium (Gentianaceae), a genus with a center of diversity placed in the Mediterranean region, polyploidy seems to have been an important driver of diversification as more than half of species are polyploids. Moreover, ploidy levels are strongly geographically structured across the range of the genus, with tetraploids distributed towards more temperate areas in the north and hexaploids in more arid areas towards the south. We hypothesize that the diversification processes and biodiversity patterns in Centaurium are explained by the coupled formation of polyploid lineages and the colonization of different areas. A MCC tree from BEAST2 based on three nuclear DNA regions of a total of 26 taxa (full sampling, of 18 species and 8 subspecies) was used to perform ancestral area reconstruction analysis in "BioGeoBEARS." Chromosome evolution was analyzed in chromEvol and diversification in BAMM to estimate diversification rates. Our results suggest that two major clades diverged early from the common ancestor, one most likely in the western Mediterranean and the other in a widespread area including west and central Asia (but with high uncertainty in the exact composition of this widespread area). Most ancestral lineages in the western clade remained in or around the western Mediterranean, and dispersal to other areas (mainly northward and eastward), occurred at the tips. Contrarily, most ancestral lineages in the widespread clade had larger ancestral areas. Polyploidization events in the western clade occurred at the tips of the phylogeny (with one exception of a polyploidization event in a very shallow node) and were mainly tetraploid, while polyploidization events occurred in the widespread clade were at the tips and in an ancestral node of the phylogeny, and were mainly hexaploid. We show how ancestral diploid lineages remained in the area of origin, whereas recent and ancestral polyploidization could have facilitated colonization and establishment in other areas.

Tackling Rapid Radiations With Targeted Sequencing.

In phylogenetic studies across angiosperms, at various taxonomic levels, polytomies have persisted despite efforts to resolve them by increasing sampling of taxa and loci. The large amount of genomic data now available and statistical tools to analyze them provide unprecedented power for phylogenetic inference. Targeted sequencing has emerged as a strong tool for estimating species trees in the face of rapid radiations, lineage sorting, and introgression. Evolutionary relationships in Cyperaceae have been studied mostly using Sanger sequencing until recently. Despite ample taxon sampling, relationships in many genera remain poorly understood, hampered by diversification rates that outpace mutation rates in the loci used. The C4 Cyperus clade of the genus Cyperus has been particularly difficult to resolve. Previous studies based on a limited set of markers resolved relationships among Cyperus species using the C3 photosynthetic pathway, but not among C4 Cyperus clade taxa. We test the ability of two targeted sequencing kits to resolve relationships in the C4 Cyperus clade, the universal Angiosperms-353 kit and a Cyperaceae-specific kit. Sequences of the targeted loci were recovered from data generated with both kits and used to investigate overlap in data between kits and relative efficiency of the general and custom approaches. The power to resolve shallow-level relationships was tested using a summary species tree method and a concatenated maximum likelihood approach. High resolution and support are obtained using both approaches, but high levels of missing data disproportionately impact the latter. Targeted sequencing provides new insights into the evolution of morphology in the C4 Cyperus clade, demonstrating for example that the former segregate genus Alinula is polyphyletic despite its seeming morphological integrity. An unexpected result is that the Cyperus margaritaceus-Cyperus niveus complex comprises a clade separate from and sister to the core C4 Cyperus clade. Our results demonstrate that data generated with a family-specific kit do not necessarily have more power than those obtained with a universal kit, but that data generated with different targeted sequencing kits can often be merged for downstream analyses. Moreover, our study contributes to the growing consensus that targeted sequencing data are a powerful tool in resolving rapid radiations.

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.

Correction: Cryptic Species Due to Hybridization: A Combined Approach to Describe a New Species (Carex: Cyperaceae).

[This corrects the article DOI: 10.1371/journal.pone.0166949.].

Cryptic Species Due to Hybridization: A Combined Approach to Describe a New Species (Carex: Cyperaceae).

Disappearance of diagnostic morphological characters due to hybridization is considered to be one of the causes of the complex taxonomy of the species-rich (ca. 2000 described species) genus Carex (Cyperaceae). Carex furva s.l. belongs to section Glareosae. It is an endemic species from the high mountains of the Iberian Peninsula (Spain and Portugal). Previous studies suggested the existence of two different, cryptic taxa within C. furva s.l. Intermediate morphologies found in the southern Iberian Peninsula precluded the description of a new taxa. We aimed to determine whether C. furva s.l. should be split into two different species based on the combination of morphological and molecular data. We sampled ten populations across its full range and performed a morphological study based on measurements on herbarium specimens and silica-dried inflorescences. Both morphological and phylogenetic data support the existence of two different species within C. furva s.l. Nevertheless, intermediate morphologies and sterile specimens were found in one of the southern populations (Sierra Nevada) of C. furva s.l., suggesting the presence of hybrid populations in areas where both supposed species coexist. Hybridization between these two putative species has blurred morphological and genetic limits among them in this hybrid zone. We have proved the utility of combining molecular and morphological data to discover a new cryptic species in a scenario of hybridization. We now recognize a new species, C. lucennoiberica, endemic to the Iberian Peninsula (Sierra Nevada, Central system and Cantabrian Mountains). On the other hand, C. furva s.s. is distributed only in Sierra Nevada, where it may be threatened by hybridization with C. lucennoiberica. The restricted distribution of both species and their specific habitat requirements are the main limiting factors for their conservation.

Phylogeny, systematics, and trait evolution of Carex section Glareosae.

UNLABELLED: • PREMISE OF THE STUDY: The circumboreal Carex section Glareosae comprises 20-25 currently accepted species. High variability in geographic distribution, ecology, cytogenetics, and morphology has led to historical problems both in species delimitation and in circumscribing the limits of the section, which is one of the major tasks facing caricologists today.• METHODS: We performed phylogenetic reconstructions based on ETS, ITS, G3PDH, and matK DNA sequences from 204 samples. Concatenation of gene regions in a supermatrix approach to phylogenetic reconstruction was compared to coalescent-based species-tree estimation. Ancestral state reconstructions were performed for eight morphological characters to evaluate correspondence between phylogeny and traits used in traditional classification within the section.• KEY RESULTS: The results confirm the existence of a core Glareosae comprising 23-25 species. Most species constitute exclusive lineages, and relationships among species are highly resolved with both the supermatrix and coalescent-based species-tree approaches. We used ancestral state reconstruction to investigate sources of homoplasy underlying traditional taxonomy and species circumscription. We found that even species apparently not constituting exclusive lineages are morphologically homogeneous, raising the question of whether paraphyly of species is a phylogenetic artifact in our study or evidence of widespread homoplasy in characters used to define species.• CONCLUSIONS: This study demonstrates the monophyly of Carex section Glareosae and establishes a phylogenetic framework for the section. Homoplasy makes many of morphological characters difficult to apply for taxon delimitation. The strong concordance between supermatrix and species-tree approaches to phylogenetic reconstructions suggests that even in the face of incongruence among molecular markers, section-level or species-level phylogenies in Carex are tractable.

Genome size stability despite high chromosome number variation in Carex gr. laevigata.

PREMISE OF THE STUDY: In organisms with holocentric chromosomes like Carex species, chromosome number evolution has been hypothesized to be a result of fission, fusion, and/or translocation events. Negative, positive, or the absence of correlations have been found between chromosome number and genome size in Carex. METHODS: Using the inferred diploid chromosome number and 80 genome size measurements from 26 individuals and 20 populations of Carex gr. laevigata, we tested the null hypothesis of chromosome number evolution by duplication and deletion of whole chromosomes. KEY RESULTS: Our results show a significant positive correlation between genome size and chromosome number, but the slope of such correlation supports the hypothesis of proliferation and removal of repetitive DNA fragments to explain genome size variation rather than duplication and deletion of whole chromosomes. CONCLUSIONS: Our results refine the theory of the holokinetic drive: this mechanism is proposed to facilitate repetitive DNA removal (or any segmental deletion) when smaller homologous chromosomes are preferentially inherited, or repetitive DNA proliferation (or any segmental duplication) when larger homologs are preferred. This study sheds light on how karyotype evolution plays an important role in the diversification of the species of the genus Carex.