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

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 radiationsEspaña Ministry of Economy and Competitiveness (project CGL2016- 77401-P

Megaphylogenetic Specimen-Level Approaches to the Carex (Cyperaceae) Phylogeny Using ITS, ETS, and matK Sequences: Implications for Classification

We present the first large-scale phylogenetic hypothesis for the genus Carex based on 996 of the 1983 accepted species (50.23%). We used a supermatrix approach using three DNA regions: ETS, ITS and matK. Every concatenated sequence was derived from a single specimen. The topology of our phylogenetic reconstruction largely agreed with previous studies. We also gained new insights into the early divergence structure of the two largest clades, core Carex and Vignea clades, challenging some previous evolutionary hypotheses about inflorescence structure. Most sections were recovered as non-monophyletic. Homoplasy of characters traditionally selected as relevant for classification, historical misunderstanding of how morphology varies across Carex, and regional rather than global views of Carex diversity seem to be the main reasons for the high levels of polyphyly and paraphyly in the current infrageneric classification

Specimens at the Center: An Informatics Workflow and Toolkit for Specimen-level analysis of Public DNA database data

Major public DNA databases — NCBI GenBank, the DNA DataBank of Japan (DDBJ), and the European Molecular Biology Laboratory (EMBL) — are invaluable biodiversity libraries. Systematists and other biodiversity scientists commonly mine these databases for sequence data to use in phylogenetic studies, but such studies generally use only the taxonomic identity of the sequenced tissue, not the specimen identity. Thus studies that use DNA supermatrices to construct phylogenetic trees with species at the tips typically do not take advantage of the fact that for many individuals in the public DNA databases, several DNA regions have been sampled; and for many species, two or more individuals have been sampled. Thus these studies typically do not make full use of the multigene datasets in public DNA databases to test species coherence and select optimal sequences to represent a species. In this study, we introduce a set of tools developed in the R programming language to construct individual-based trees from NCBI GenBank data and present a set of trees for the genus Carex (Cyperaceae) constructed using these methods. For the more than 770 species for which we found sequence data, our approach recovered an average of 1.85 gene regions per specimen, up to seven for some specimens, and more than 450 species represented by two or more specimens. Depending on the subset of genes analyzed, we found up to 42% of species monophyletic. We introduce a simple tree statistic—the Taxonomic Disparity Index (TDI)—to assist in curating specimen-level datasets and provide code for selecting maximally informative (or, conversely, minimally misleading) sequences as species exemplars. While tailored to the Carex dataset, the approach and code presented in this paper can readily be generalized to constructing individual-level trees from large amounts of data for any species group

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

Comprendiendo la diversidad vegetal: un caso de estudio en Carex sección Glareosae (Cyperaceae)

Programa de Doctorado en Estudios MedioambientalesDesde su origen en el Jurásico (ca. 140 Ma) las plantas con flores han experimentado una remarcable radiación y, hoy en día, se estima que hay más de 250.000 especies de angiospermas. Comprender los mecanismos que explican tan increíble biodiversidad ha sido el objetivo de múltiples estudios por parte de naturalistas. Entre las angiospermas, el género Carex es uno de los géneros más diversificados en las regiones templadas. Hemos elegido la sección Glareosae, un linaje dentro del género formado por 26 especies, como modelo para el estudio de la diversificación en las plantas con flores. Este grupo presenta una remarcable variabilidad morfológica, citogenética, geográfica y ecológica. La combinación de estudios morfológicos y herramientas moleculares nos han permitido comprender en profundidad cómo la evolución está actuando para originar tal diversidad en un linaje tan reciente. Primero, hemos aclarado los límites de la sección y sus especies, así como las relaciones filogenéticas entre ellas, a través de la secuenciación y análisis de cuatro regiones de ADN. En segundo lugar, se llevaron a cabo análisis de tiempos de divergencia, biogeográficos y de diversificación, usando una reconstrucción filogenética analizando 14 regiones de ADN, con objeto de entender la distribución actual de las especies y sus patrones de diversificación. Posteriormente, una nueva especie de la sección Glareosae, Carex lucennoiberica, endémica de la Península Ibérica, fue descrita basándonos en la combinación de evidencias morfológicas y moleculares. Esta especie fue críptica hasta ahora a causa del flujo génico, y la consecuente presencia de morfotipos intermedios, con la especie más cercana, C. furva. Finalmente, el estudio filogeográfico del complejo C. lucennoiberica ¿ C. furva usando secuenciación de Sanger de haplotipos plastidiales y secuenciación de alto rendimiento (RADseq), nos han permitido inferir contactos secundarios durante el Pleistoceno tras la especiación alopátrica ocurrida en dicho complejo. La secuenciación mediante RADseq ha sido crucial para desenmarañar esta complicada historia filogeográfica. Destacamos los procesos geográficos y ecológicos, así como la hibridación, como generadores de la actual biodiversidad de la sección Glareosae del género Carex en particular, y de las angiospermas en general. Todos estos motores evolutivos están lejos de actuar independientemente. Por el contrario, operan juntos, a veces en sinergia, a veces en discordancia.Universidad Pablo de Olavide. Departamento de Biología Molecular e Ingeniería BioquímicaPostprin

Origin and diversification of flax and their relationship with heterostyly across the range

Aim: Understanding plant diversity and how different traits have shaped the current biodiversity setting across the world is one of the major challenges for evolutionary biology. In this sense, genus Linum have been broadly studied as a model of species showing heterostyly, but no studies have attempted to investigate possible correlations between heterostyly and diversification. Thus, in this study we aim to explore the origin and diversification of the genus Linum and to determine its centre of diversity and potential source for the colonisation of other areas. Also, we aim to study how heterostyly may have shaped diversification rates and colonisation patterns in the genus. Location: Worldwide (focused on the Western Palearctic). Taxon: Genus Linum L. (Linaceae). Methods: We analysed nuclear ITS and plastid ndhF, matK and trnL-F DNA regions in a total of 103 samples of 93 different species of the genus Linum, as well as seven samples from other Linaceae. We performed divergence time analysis in BEAST2 under a birth-death tree model, then used the resulting tree for an ancestral area reconstruction using the R package “BioGeoBEARS.” Finally, we estimated diversification rates using BAMM and examined the correlation between diversification rates and geographic ranges and heterostyly. Results: Linum s.l. diverged during the late Eocene to mid Oligocene (27.2–38.29 Ma at 95% highest posterior density interval) in the Western Palearctic, where most species diversified. Within-area speciation is the main mechanism of diversification in the genus. Most dispersal events occurred from the Western Palearctic to other regions, probably through long-distance dispersal (LDD). No changes were found regarding diversification rates in specific clades or in relation with reproductive system (heterostyly) or geographic areas. Colonisation of new areas was achieved mostly by monomorphic lineages, whereas heterostylous species mostly remained in the ancestral Western Paleartic area. Main conclusions: The Western Palearctic acted as a source of dispersal in seven out of nine total dispersal events but never as a sink. All species or lineages that colonised new areas after LDD were monomorphic for style length, as predicted by the theory on reproductive traits of colonising species. Thus, heterostyly is shown to affect establishment success in a new area. Neither biogeographic changes nor analysed trait changes can explain speciation nor extinction rates in Linum. Our results confirm that the evolution of heterostyly is related to the paleogeographic history and are not consistent with the idea that a key innovation or “dispersification” has shaped the diversification patterns in Linum.Ministerio de Ciencia e Innovación CGL2013-45037- P, PGC2018-099608- B- 10

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.Gobierno de España AP2012-2189, CGL2012-38744, CGL2016- 77401-PEuropean Community Research Infrastructures Program GB-TAF-2523Junta de Andalucía RNM2763National Science Foundation 125590