Marlene Hahn

Genotyping-by-sequencing as a tool to infer phylogeny and ancestral hybridization: A case study in Carex (Cyperaceae)

Determining phylogenetic relationships among very closely related species has remained a challenge for evolutionary biologists due to interlocus phylogenetic discordance and the difficulty of obtaining variable markers. Here, we used a Genotyping-by-Sequencing (GBS) approach to sample a reduced representation genomic data set and infer the phylogeny of seven closely related species in the genus Carex (Cyperaceae). Past attempts to reconstruct phylogenetic relationships among these species produced conflicting and poorly-supported results. We inferred a robust phylogeny based on >3000 GBS loci and >1300 SNPs (with a minimum sequence depth within individuals of 10) using maximum likelihood and Bayesian inference. We also tested for historical introgression using the D-statistic test. We compared these analyses with partitioned RAD analysis, which is designed to identify suboptimal trees reflecting secondary phylogenetic signal that may be obscured by the dominant signal in the data. Phylogenetic analyses yielded fully resolved trees with high support. We found two main clades, one grouping Carex scoparia populations and C. waponahkikensis, and a second clade grouping C. longii, C. vexans, C. suberecta and C. albolutescens. We detected marginally significant signals of introgression between C. scoparia and C. suberecta or C. albolutescens, and we rejected a hybrid origin hypothesis for C. waponahkikensis. Our results demonstrate the power of NGS data sets for resolving some of the most difficult phylogenetic challenges where traditional phylogenetic markers have failed.

Sympatric parallel diversification of major oak clades in the Americas and the origins of Mexican species diversity

Oaks (Quercus, Fagaceae) are the dominant tree genus of North America in species number and biomass, and Mexico is a global center of oak diversity. Understanding the origins of oak diversity is key to understanding biodiversity of northern temperate forests. A phylogenetic study of biogeography, niche evolution and diversification patterns in Quercus was performed using 300 samples, 146 species. Next-generation sequencing data were generated using the restriction-site associated DNA (RAD-seq) method. A time-calibrated maximum likelihood phylogeny was inferred and analyzed with bioclimatic, soils, and leaf habit data to reconstruct the biogeographic and evolutionary history of the American oaks. Our highly resolved phylogeny demonstrates sympatric parallel diversification in climatic niche, leaf habit, and diversification rates. The two major American oak clades arose in what is now the boreal zone and radiated, in parallel, from eastern North America into Mexico and Central America. Oaks adapted rapidly to niche transitions. The Mexican oaks are particularly numerous, not because Mexico is a center of origin, but because of high rates of lineage diversification associated with high rates of evolution along moisture gradients and between the evergreen and deciduous leaf habits. Sympatric parallel diversification in the oaks has shaped the diversity of North American forests.

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

Abstract 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.

White-Tailed Deer are a Biotic Filter During Community Assembly, Reducing Species and Phylogenetic Diversity

Promotional Statement: White-tailed deer browsing has been implicated in the loss of species diversity from forests throughout eastern North America. We build on this previous research by examining how browsing also affects phylogenetic community structure. With this approach, we can better understand the role of deer browsing in the community assembly process. In browsed plots, we found that reductions in phylogenetic diversity were much greater than reductions in species richness or diversity. Species persisting in browsed communities were also closely-related. Our findings indicate deer browsing acts as a biotic filter during the community assembly process.

Phylogeny and biogeography of East Asian evergreen oaks (Quercus section Cyclobalanopsis; Fagaceae): Insights into the Cenozoic history of evergreen broad-leaved forests in subtropical Asia

The evolutionary history of Quercus section Cyclobalanopsis, a dominant lineage in East Asian evergreen broadleaved forests (EBLFs), has not been comprehensively studied using molecular tools. In this study, we reconstruct the first comprehensive phylogeny of this lineage using a genomic approach (restriction-site associated DNA sequencing, RAD-seq), sampling 35 of the ca. 90 species currently recognized, representing all main morphological groups of section Cyclobalanopsis. In addition, 10 other species of Quercus and two outgroups were also sampled. Divergence times were estimated using a relaxed clock model and two fossil calibrations. Ancestral areas and dispersal routes were inferred using statistical dispersal-vicariance analysis and the dispersal-extinction-cladogenesis (DEC) model. The phylogeny of Quercus section Cyclobalanopsis demonstrates the section to be monophyletic, comprising two main lineages and six subclades that are well supported by anatomical traits. Biogeographical reconstructions indicate that the wide northern hemisphere distribution of Quercus was disrupted in the Late Eocene, leading to the main extant groups at about 33 Ma. The earliest divergences in section Cyclobalanopsis correspond to the phased uplift of the Himalayas and lateral extrusion of Indochina at the transition of the Oligocene and Miocene, where the highest rate of diversification occurred in the late Miocene. Dispersal from Sino-Himalaya and the Palaeotropics to Sino-Japan in the Miocene was facilitated by the increased intensity of East Asian summer monsoons and by the Middle Miocene Climatic Optimum. Our results highlight the importance of climatic changes and Indo-Eurasian collision-induced tectonic activities from the Neogene onward to the spatial-temporal diversification patterns of Asian EBLF lineages.

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.

A nuclear DNA barcode for eastern North American oaks and application to a study of hybridization in an Arboretum setting

Abstract DNA barcoding has proved difficult in a number of woody plant genera, including the ecologically important oak genus Quercus. In this study, we utilized restrictionsite‐associated DNA sequencing (RAD‐seq) to develop an economical single nucleotide polymorphism (SNP) DNA barcoding system that suffices to distinguish eight common, sympatric eastern North American white oak species. Two de novo clustering pipelines, PyRAD and Stacks, were used in combination with postclustering bioinformatic tools to generate a list of 291 potential SNPs, 80 of which were included in a barcoding toolkit that is easily implemented using MassARRAY mass spectrometry technology. As a proof‐of‐concept, we used the genotyping toolkit to infer potential hybridization between North American white oaks transplanted outside of their native range (Q. michauxii, Q. montana, Q muehlenbergii/Q. prinoides, and Q. stellata) into a horticultural collection surrounded by natural forests of locally native trees (Q. alba and Q. macrocarpa) in the living collection at The Morton Arboretum (Lisle, IL, USA). Phylogenetic and clustering analyses suggested low rates of hybridization between cultivated and native species, with the exception of one Q. michauxii mother tree, the acorns of which exhibited high admixture from either Q. alba or Q. stellata and Q. macrocarpa, and a hybrid between Q. stellata that appears to have backcrossed almost exclusively to Q. alba. Together, RAD‐seq and MassARRAY technologies allow for efficient development and implementation of a multispecies barcode for one of the more challenging forest tree genera.

Specimens at the Center: An Informatics Workflow and Toolkit for Specimen-Level Analysis of Public DNA Database Data

Abstract 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.

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.

Proceedings Introduction: Phylogeny and Ecological Diversification in Carex

Andrew L. Hipp, Pedro Jiménez-Mejías, Marcia J. Waterway, Marlene Hahn, and Eric H. Roalson The Morton Arboretum, Lisle, Illinois 60532, U.S.A. The Field Museum, Chicago, Illinois 60605, U.S.A. Washington State University, Pullman, Washington 99164, U.S.A. Current address: The New York Botanical Garden, Bronx, New York 10458, U.S.A. McGill University, Macdonald Campus, Ste-Anne-de-Bellevue, QC, H9X 3V9, Canada Author for correspondence (ahipp@mortonarb.org)