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The evolution and conservation of biodiversity is at a crossroads: human population growth and development has already wreaked havoc on species diversity and the genetic diversity of populations, affecting evolutionary trajectories of species and whole ecosystems far into the future. At the same time, biodiversity research has seen a surge in visibility, as the public concern for the environment shifts toward an interest in preserving all branches of the tree of life; and in strength, as new genomic and bioinformatics tools for biodiversity research come into increasingly common use. At the heart of biodiversity research is the study of the evolution of species, which is providing exciting new insights into the drivers of biological diversification.

Chromosome evolution is one of the most important drivers of biodiversity. Much research into chromosome evolution has focused on physiological and ecological implications of polyploidy. Because polyploidy entails changes in DNA content and gene number, expression differences associated with polyploidization can have dramatic effects on phenotype. Another important body of research has focused on the effects of chromosome inversions on promoting species differentiation through the protection of ecologically significant gene combinations from recombination. Almost completely ignored have been the biodiversity implications of other types of chromosome rearrangements that result in chromosome number changes with little or no change in DNA content (viz., fission and fusion). HoloChromEvol project propose a unique integration of genomic, cytogenetic, and ecological experiments to investigate the effects of chromosome evolution on the biodiversity of the largest flowering plant genus of the temperate zone, the sedges of genus Carex (Cyperaceae). The genus is emerging as a model system for studying chromosomal speciation in holocentric species, species in which the centromere is not a localized structure, but distributed along the entire length of the chromosome. Our research will provide novel insights into the relationship between chromosome evolution, recombination rate, local adaptation, and life history strategies: ultimately, the ecological underpinnings of biodiversity. It will also provide the genomic and genetic resources needed to establish Carex as a model system for understanding chromosome evolution across the tree of life, in all lineages in which chromosome evolution proceeds primarily by fission, fusion, and translocations.

The overarching goal of our long-term research program is to elucidate how chromosome evolution affects biodiversity patterns across the tree of life. HoloChromEvol is an important key to this goal, and will place us in a position to make substantial, career-long contributions in the field of biodiversity science. Through an integration of cutting-edge genomic and ecological experiments in a hyper-diverse flowering plant genus with exceptional chromosomal variation, this project will tease apart the mechanisms by which holocentric chromosome evolution drives genetic diversification and identify the population dynamic elements that allow for chromosomal diversification in sedges. The result will be a model of diversification in this keystone genus that integrates the dynamics of population establishment, migration, and persistence with the chromosomal and genomic architecture of population divergence.

Background: holocentric chromosomes
In holocentric chromosomes—chromosomes with diffuse centromeres—microtubule attachment during mitosis is distributed along the length of the chromosome. In contrast, monocentric chromosomes have microtubule attachment localized to one region. Holocentric chromosome organization has been described for three of the six supergroups in the domain Eukarya (the Eukaryotes): plants (angiosperms, algae and mosses); animals (at least six numerous arthropod clades, plus velvet worms and nematodes); and Rhizaria.
It has long been recognized that chromosome fragments that would be lost in monocentric chromosomes may be propagated and become fixed in organisms with holocentric chromosomes, and inherited in Mendelian fashion. Likewise, chromosomes resulting from fusion of two holocentric chromosomes typically align and segregate correctly, whereas in organisms with monocentric chromosomes, the linkage of two chromosomes often results in the formation of dicentric chromosomes that fail to segregate properly. The result is tremendous variation in rates of chromosome evolution, including hyper-variable groups such as Agrodiaetus butterflies (2n = 10 to 250) and sedges (2n = 4 to 226).
The implications of holocentry are potentially profound, but largely unstudied. In Lepidoptera, phylogenetic comparative evidence suggests that chromosome rearrangements that accrue in allopatry play a role in reinforcing speciation. In the sedge genus Carex (Cyperaceae), chromosome rearrangements contribute to genetic diversity within species as well as lineage diversification, suggesting that holocentry is an important determinant of biodiversity patterns. But as yet, no experimental studies have confirmed experimentally that chromosome diversification drives biodiversity patterns in holocentric clades. HoloChromEvol project will fill this gap, as the first study to test the mechanism by which chromosome rearrangements affect patterns of gene flow among populations, and subsequently speciation, the engine of biodiversity.

Why Carex?
Carex has several properties that make it an ideal model for testing the evolutionary implications of holocentric chromosome rearrangements:
1. Exceptionally high rate of chromosome rearrangements. Carex exhibits exceptionally high chromosome diversity, even among holocentric lineages. The two species focused on in this project (Carex scoparia and C. laevigata) exhibit high chromosome number variation. In C. scoparia, we find chromosome variants in nearly every population we study, and a phenomenal diversification among populations that diverged from each other less than 15,000 years ago. Moreover, this variation is known to be due to fissions and fusions, not polyploidy. In Carex, we are truly capturing a snapshot of chromosome speciation in process, not studying it after-the-fact.
2. High species and lineage diversity. At more than 2,000 species, Carex is among the four most speciesrich angiosperm genera worldwide. The genus’s center of diversity lies in the temperate regions of northern Hemisphere, where it dominates a wide range of wetland plant communities, and it is one of the richest angiosperm genera in North America (ca. 500 species) and the European Union (ca. 250 species). This diversity makes Carex both an important genus from a conservation standpoint and an ideal system for studying how chromosome evolution affects processes and rates of species diversification.
3. Amenable to experimentation. Our proposed study entails crosses and hybridization and F1 and F2 generation which are much more easily executed with non-woody plants than with woody plants or animals. Ability to grow plants in greenhouses for genotyping and second-generation crosses is crucial to understanding how these chromosome rearrangements affect recombination rates and diversification.

A summary description of the project objectives
For this proposal, we are addressing four fundamental questions regarding how and to what degree chromosome diversity drives biodiversity in sedges (Carex): 1) Do chromosome rearrangements protect ecologically significant genome regions from recombination? 2) To what extent do chromosome rearrangements decrease the fitness of first- and second-generation interpopulation crosses? 3) What are the relative contributions of hybrid dysfunction (decreased fitness of hybrids between individuals with differing chromosome numbers) and recombination suppression to chromosomal speciation? and 4) Do the population dynamics of sedges allow for rapid establishment of chromosome variants, even in the face of underdominance of those mutations? In combination, these questions form the core of a fully integrative view of species diversification in sedges, and position Carex as an emerging model for studying chromosomal mechanisms of diversification.

A description of the work performed since the beginning of the project
Experimental crosses, germination rates and greenhouse work
During the Spring of 2011, experimental crosses between Carex scoparia individuals with the same and different chromosome number were performed as well as selfing crosses. During the summer and fall 2011, the germination rates of the different crosses were calculated, and the F1 individuals were grown in the greenhouse. During the Spring of 2012, additional crosses were performed, and the germination rates were calculated and the individuals were grown in the greenhouse. During the Spring of 2013, the F1 individuals from four selected crosses were crossed between them to generate F2 individuals which were germinated during summer and fall of 2013 and grown in the greenhouse.
Molecular work in the DNA lab
During the Spring of 2012, the parentals and F1 individuals from three different crosses as well as closely related species of Carex scoparia were selected to performed NGS sequencing study using the technique Genotyping-By-Sequencing (GBS).
DNA was extracted from F1 individuals and parental test of F1 individuals was performed using microsatellites during the Spring of 2013.
During the Winter of 2014, DNA was extracted form ca. 200 F2 individual and there were genotyped using the NGS technique termed Restriction-Associated-DNA sequences (RADseq).
Cytogenetic study of F1 in Carex scoparia
During the summer and fall of 2013 and winter 2014 a cytogenetic study of F1 individuals has been performed.
Georeferencing individuals, genotyping and cytogenetic study
During the Spring and Summer 2014 all reproductive individuals of a population of Carex helodes (Madroñalejo, Aznalcollar, Sevilla) were georeferenced, leaf tissues and seeds were collected for DNA extraction and male flowers were collected for the cytogenetic study.

A description of the main results achieved so far
The most important results are:
- We have inferred prezygotic barriers to gene flow in Carex. The rate of germination rate is inversely proportional to the number of chromosome rearrangements between the parents or found in the cytogenetic study in the F1. This result demonstrate an important role of chromosome rearrangements in gene flow in one of the most species rich angiosperm genera.
- We have obtained enough data to study postzygotic barriers to gene flow in Carex from suppression in the recombination rates between homologous chromosomes that have suffer a rearrangement. Our new data demonstrate selection during the meiotic process. Our linkage map demonstrate that loci in rearranged regions in the genome are not inherited following Medelian ratios.
- Our last results with Carex helodes demonstrate that there is not genetic variation despite of high chromosome number variation. The 112 designed SSRs are not enough to detect intrapopulational genetic variation.

This project is providing an excellent opportunity to explore the evolution of genome structure at the population level. As holocentry is one of the least understood structural modifications of the genome, understanding its role in diversification is key to understanding genome evolution across the tree of life; elucidate the role that holocentric chromosome evolution plays in the earliest stages of genetic differentiation and speciation; and provide a model for future studies, arriving as it does at a time when next-generation genomic tools make it possible to address outstanding questions regarding genomic drivers of speciation. This project contributes to recent debate on sympatric modes of speciation, which is one of the most important topics in biodiversity research today.
The chromosomal differentiation and speciation model developed in Carex under this proposal serves as a background for studies in other organisms with holocentric chromosomes and chromosome evolution in general.