Geographic patterns of microsatellite variation in Boechera stricta, a close relative of Arabidopsis

The genus Boechera is a widespread North American group with great potential for studies of ecology and evolution: Boechera is closely related to Arabidopsis and exhibits different ecological and reproductive strategies. Boechera stricta (previously Arabis drummondii) is a morphologically and genetically well-defined, perennial crucifer species. Fifteen natural populations of diploid individuals from the Rocky Mountains were analysed using 21 microsatellite loci. In accordance with our expectation for this predominately inbreeding species, a high F IS value (0.89) was observed. Furthermore, populations of B. stricta were highly differentiated, as indicated by F ST = 0.56. Three clusters were identified using structure- the majority of populations belonged to either the Northern or Southern cluster. Together, the north-south partitioning and evenness of genetic variation across the two clusters suggested multiple refugia for this perennial herb in the Rocky Mountains. Pleistocene glaciation, together with the topographically and climatologically heterogeneous cordillera, has profoundly influenced the genetic architecture of B. stricta. Genetic population structure was also influenced by relatively recent genome admixture at two levels: within species (involving individuals from the Northern and Southern clusters) and between species (with the hybridization of B. stricta and Boechera holboellii). This complexity of population structure at presumably neutral microsatellite loci located throughout the genome in B. stricta provides a baseline against which to test whether functional genetic variation is undergoing local adaptive evolution throughout the natural species range.     

Asexual reproduction in a close relative of Arabidopsis: a genetic investigation of apomixis in Boechera (Brassicaceae)

Understanding apomixis (asexual reproduction through seeds) is of great interest to both plant breeders and evolutionary biologists. The genus Boechera is an excellent system for studying apomixis because of its close relationship to Arabidopsis, the occurrence of apomixis at the diploid level, and its potentially simple inheritance by transmission of a heterochromatic (Het) chromosome. Diploid sexual Boechera stricta and diploid apomictic Boechera divaricarpa (carrying a Het chromosome) were crossed. Flow cytometry, karyotype analysis, genomic in situ hybridization, pollen staining and seed-production measurements were used to analyse the parents and resulting F1, F2 and selected F3 and test-cross (TC) generations. The F1 plant was a low-fertility triploid that produced a swarm of aneuploid and polyploid F2 progeny. Two of the F2 plants were fertile near-tetraploids, and analysis of their F3 and TC progeny revealed that they were sexual and genomically stabilized. The apomictic phenotype was not transmitted by genetic crossing as a single dominant locus on the Het chromosome, suggesting a complex genetic control of apomixis that has implications for future genetic and evolutionary analyses in this group.     

Sexual reproduction, hybridization, apomixis, and polyploidization in the genus Boechera (Brassicaceae)

Of the 340 genera in the Brassicaceae, apomictic reproduction is found only in the North American genus Boechera. We investigated phylogenetic relationships, ability to hybridize, mating system, and ploidy levels of 92 lines sampled from 85 populations and representing 19 Boechera species. Phylogenetic analyses based on chloroplast DNA sequences identified three lineages in the genus. Reciprocal crosses of each line were made to a common sexual diploid B. stricta tester. The resulting F(1) progeny were analyzed for the inheritance of polymorphic microsatellite loci, genome size, and seed production. Intraspecific B. stricta crosses confirmed that this species is mostly diploid and sexual. Interspecific crosses revealed many other species were diploid and sexual and could be successfully hybridized with the tester. We also found obligate and facultative apomictic diploid and triploid lines. De novo F(1) polyploids (either triploids or tetraploids) were derived from the union of nonreduced (from an apomictic parent) and reduced (from the tester) gametes. However, seed production of these F(1) plants was generally low, suggesting a failure in the transmission of apomixis. The creation of a wide array of segregating genetic populations will facilitate future research on the evolution and inheritance of quantitative variation in Boechera.     

Biogeographic distribution of polyploidy and B chromosomes in the apomictic Boechera holboellii complex

The Boechera holboellii complex comprises B. holboellii and B. drummondii, both of which can reproduce through sex or apomixis. Sexuality is associated with diploid individuals, whereas apomictic individuals are diploid or triploid and may additionally have B chromosomes. Using flow cytometry and karyotype analysis, we have shown that B chromosomes (a) occur in both diploid and triploid apomictic B. holboellii, (b) may occur in triploid B. drummondii, and (c) are dispensable for the plant. Both diploid and triploid karyotypes are found in multiple chloroplast haplotypes of both species, suggesting that triploid forms have originated multiple times during the evolution of this complex. B chromosome carriers are found in geographically and genetically distinct populations, but it is unknown whether the extra chromosomes are shared by common descent (single origin) or have originated via introgressive hybridization and repeated transitions from diploidy to triploidy. Diploid plants containing the Bs reproduce apomictically, suggesting that the supernumerary elements are associated with apomixis. Finally, our analyses of pollen size and viability suggest that irregular chromosome segregation in some triploid lineages may lead to the generation of diploid individuals which carry the B chromosomes.     

Multiple hybrid formation in natural populations: concerted evolution of the internal transcribed spacer of nuclear ribosomal DNA (ITS) in North American Arabis divaricarpa (Brassicaceae)

DNA sequence variation of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA from Arabis holboellii, A. drummondii, and its putative hybrid A. divaricarpa was analyzed to study hybrid speciation in a species system geographically covering nearly the entire North American continent. Based on molecular systematics the investigated species are better combined under the genus Boechera. Multiple intraindividual ITS copies were detected in numerous accessions of A. divaricarpa, and, to a minor extent, in the parental taxa. Comparative phylogenetic analysis demonstrates that reticulate evolution is common. Consequently, concerted evolution of ITS regions resulted in different types of ITS fragments not only in hybrid populations but also in one of the parental taxa, A. holboellii. Hybrid formation often occurred independently at different sites and at different times, which is reflected by ITS copies resampling the original parental sequence variation in different ways. Some biogeographic structuring of genetic diversity is apparent and mirrors postglacial migration routes. Hybridization, reticulation, and apomixis are assumed to be the major forces driving speciation processes in this species complex. Analysis of conserved regions and secondary structures of the ITS region provided no evidence that, in this system, hybrid ITS evolution is predominantly driven in a particular direction. However, two regions in the ITS1 and ITS2, respectively, show higher mutation rates than expected from outgroup comparisons. Strong evidence for the occurrence of apomixis in A. holboellii and A. divaricarpa has come from pollen size measurements and estimations of pollen quality, which favor the hypothesis that A. drummondii served as paternal hybridization partner more frequently than A. holboellii.     

High genetic diversity and population differentiation in Boechera fecunda, a rare relative of Arabidopsis

Conservation of endangered species becomes a critical issue with the increasing rates of extinction. In this study, we use 13 microsatellite loci and 27 single-copy nuclear loci to investigate the population genetics of Boechera fecunda, a rare relative of Arabidopsis thaliana, known from only 21 populations in Montana. We investigated levels of genetic diversity and population structure in comparison to its widespread congener, Boechera stricta, which shares similar life history and mating system. Despite its rarity, B. fecunda had levels of genetic diversity similar to B. stricta for both microsatellites and nucleotide polymorphism. Populations of B. fecunda are highly differentiated, with a majority of genetic diversity existing among populations (F(ST) = 0.57). Differences in molecular diversity and allele frequencies between western and eastern population groups suggest they experienced very different evolutionary histories.     

Comparative genetic mapping in Boechera stricta, a close relative of Arabidopsis

The angiosperm family Brassicaceae contains both the research model Arabidopsis (Arabidopsis thaliana) and the agricultural genus Brassica. Comparative genomics in the Brassicaceae has largely focused on direct comparisons between Arabidopsis and the species of interest. However, the reduced genome size and chromosome number (n = 5) of Arabidopsis complicates comparisons. Arabidopsis shows extensive genome and chromosome reshuffling compared to its close relatives Arabidopsis lyrata and Capsella rubella, both with n = 8. To facilitate comparative genomics across the Brassicaceae we recently outlined a system of 24 conserved chromosomal blocks based on their positions in an ancestral karyotype of n = 8, rather than by their position in Arabidopsis. In this report we use this system as a tool to understand genome structure and evolution in Boechera stricta (n = 7). B. stricta is a diploid, sexual, and highly self-fertilizing species occurring in mostly montane regions of western North America. We have created an F(2) genetic map of B. stricta based on 192 individuals scored at 196 microsatellite and candidate gene loci. Single-nucleotide polymorphism genotyping of 94 of the loci was done simultaneously using an Illumina bead array. The total map length is 725.8 cM, with an average marker spacing of 3.9 cM. There are no gaps greater than 19.3 cM. The chromosomal reduction from n = 8 to n = 7 and other genomic changes in B. stricta likely involved a pericentric inversion, a chromosomal fusion, and two reciprocal translocations that are easily visualized using the genomic blocks. Our genetic map will facilitate the analysis of ecologically relevant quantitative variation in Boechera. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers DU 667459 to DU 708532.     

Boechera, a model system for ecological genomics

The selection and development of a study system for evolutionary and ecological functional genomics (EEFG) depend on a variety of factors. Here, we present the genus Boechera as an exemplary system with which to address ecological and evolutionary questions. Our focus on Boechera is based on several characteristics as follows: (i) native populations in undisturbed habitats where current environments reflect historical conditions over several thousand years; (ii) functional genomics benefitting from its close relationship to Arabidopsis thaliana; (iii) inbreeding tolerance enabling development of recombinant inbred lines, near-isogenic lines and positional cloning; (iv) interspecific crosses permitting mapping for genetic analysis of speciation; (v) apomixis (asexual reproduction by seeds) in a genetically tractable diploid; and (vi) broad geographic distribution in North America, permitting ecological genetics for a large research community. These characteristics, along with the current sequencing of three Boechera species by the Joint Genome Institute, position Boechera as a rapidly advancing system for EEFG studies.     

Extensive chloroplast haplotype variation indicates Pleistocene hybridization and radiation of North American Arabis drummondii, A. x divaricarpa, and A. holboellii (Brassicaceae)

Arabis drummondii, A. holboellii and their hybrid A. x divaricarpa are widespread perennials of open habitats in North America. A phylogenetic analysis based on noncoding chloroplast DNA sequences (trnL intron and trnL/F intergenic spacer) resolved A. drummondii as a monophyletic taxon, but found A. holboellii to bear chloroplast haplotypes from highly diverged evolutionary lineages. This raised the question of a possible polyphyletic origin of A. holboellii. Arabis x divaricarpa was found to be of recent and polytopic origin, a result consistent with its presumed hybrid origin. One hundred and three chloroplast haplotypes were detected within 719 Arabis accessions investigated. The majority of chloroplast-types were estimated to have arisen prior to the Wisconsin glaciation. Phylogeographical analysis using nested clade analysis, suggested for A. holboellii (i). past fragmentation events, partitioning genetic variation in several instances between the Sierra Nevada, the Southern Rocky Mountains and the Colorado Plateau on the one hand and the Central to Northern Rockies of the United States and adjacent Cascades on the other; and for both parental species (ii). recolonization of major areas formerly covered by the Wisconsin glaciation by three haplotypes; and (iii). restricted gene flow indicating isolation by distance in areas south of the last glacial maximum. Arabis x divaricarpa is closely codistributed with its parental species and resampled their haplotypes. The highest genetic diversity was found in the Rocky Mountains from Idaho and Montana south to Utah and Colorado. This area was further hypothesized to have played a major role in the origin of both parental species and probably represented an important glacial refugium. However, evidence for glacial refugia was also found in arctic and boreal regions of Alaska and near the Great Lakes. In comparison to nuclear ribosomal internal transcribed spacer data, chloroplast DNA divergence was very high and evidently predated the origin of A. drummondii and possibly A. holboellii. Divergence of major chloroplast lineages dates back to the middle of the Pleistocene at least. Extensive hybridization is the most likely evolutionary factor working on A. holboellii to explain the revealed discrepancy in nuclear DNA and chloroplast DNA diversification.     

Quantitative variation for apomictic reproduction in the genus Boechera (Brassicaceae)

? Premise of the study: The evolution of asexual seed production (apomixis) from sexual relatives is a great enigma of plant biology. The genus Boechera is ideal for studying apomixis because of its close relation to Arabidopsis and the occurrence of sexual and apomictic species at low ploidy levels (diploid and triploid). Apomixis is characterized by three components: unreduced embryo-sac formation (apomeiosis), fertilization-independent embryogenesis (parthenogenesis), and functional endosperm formation (pseudogamy or autonomous endosperm formation). Understanding the variation in these traits within and between species has been hindered by the laborious histological analyses required to analyze large numbers of samples. ? Methods: To quantify variability for the different components of apomictic seed development, we developed a high-throughput flow cytometric seed screen technique to measure embryo:endosperm ploidy in over 22000 single seeds derived from 71 accessions of diploid and triploid Boechera. ? Key results: Three interrelated features were identified within and among Boechera species: (1) variation for most traits associated with apomictic seed formation, (2) three levels of apomeiosis expression (low, high, obligate), and (3) correlations between apomeiosis and parthenogenesis/pseudogamy. ? Conclusions: The data presented here provide a framework for choosing specific genotypes for correlations with large "omics" data sets being collected for Boechera to study population structure, gene flow, and evolution of specific traits. We hypothesize that low levels of apomeiosis represent an ancestral condition of Boechera, whereas high apomeiosis levels may have been induced by global gene regulatory changes associated with hybridization.     

Partial shotgun sequencing of the Boechera stricta genome reveals extensive microsynteny and promoter conservation with Arabidopsis

Comparative genomics provides insight into the evolutionary dynamics that shape discrete sequences as well as whole genomes. To advance comparative genomics within the Brassicaceae, we have end sequenced 23,136 medium-sized insert clones from Boechera stricta, a wild relative of Arabidopsis (Arabidopsis thaliana). A significant proportion of these sequences, 18,797, are nonredundant and display highly significant similarity (BLASTn e-value < or = 10(-30)) to low copy number Arabidopsis genomic regions, including more than 9,000 annotated coding sequences. We have used this dataset to identify orthologous gene pairs in the two species and to perform a global comparison of DNA regions 5' to annotated coding regions. On average, the 500 nucleotides upstream to coding sequences display 71.4% identity between the two species. In a similar analysis, 61.4% identity was observed between 5' noncoding sequences of Brassica oleracea and Arabidopsis, indicating that regulatory regions are not as diverged among these lineages as previously anticipated. By mapping the B. stricta end sequences onto the Arabidopsis genome, we have identified nearly 2,000 conserved blocks of microsynteny (bracketing 26% of the Arabidopsis genome). A comparison of fully sequenced B. stricta inserts to their homologous Arabidopsis genomic regions indicates that indel polymorphisms >5 kb contribute substantially to the genome size difference observed between the two species. Further, we demonstrate that microsynteny inferred from end-sequence data can be applied to the rapid identification and cloning of genomic regions of interest from nonmodel species. These results suggest that among diploid relatives of Arabidopsis, small- to medium-scale shotgun sequencing approaches can provide rapid and cost-effective benefits to evolutionary and/or functional comparative genomic frameworks.     

Is the aneuploid chromosome in an apomictic Boechera holboellii a genuine B chromosome?

The Boechera holboellii complex comprises B. holboellii and B. drummondii, both of which can reproduce through sex or apomixis. Sexuality is associated with diploidy, whereas apomictic individuals can either be diploid, aneuploid or triploid. Aneuploid individuals are found in geographically and genetically distinct populations and contain a single extra chromosome. It is unknown whether the supernumerary chromosomes are shared by common descent (single origin) or have originated via introgressive hybridizations associated with the repeated transition from diploidy to triploidy. Diploid plants containing the extra chromosome(s) reproduce apomictically, suggesting that the supernumerary elements are associated with apomixis. In this study we compared flow cytometry data, chromosome morphology, and DNA sequences of sexual diploid and apomictic aneuploids in order to establish whether the extra chromosome fits the classical concept of a B chromosome. Karyotype analyses revealed that the supernumerary chromosome in the metaphase complement is heterochromatic and often smaller than the A chromosomes, and differs in length between apomictic plants from different populations. DNA sequence analyses furthermore demonstrated elevated levels of non-synonymous substitutions in one of the alleles, likely that on the aneuploid chromosome. Although the extra chromosome in apomictic Boechera does not go through normal reductional meiosis, in which it may get eliminated or accumulated by a B-chromosome-specific process, its variable size and heterochromatic nature does meet the remaining criteria for a genuine B chromosome in other species. Its prevalence and conserved genetic composition nonetheless implies that this chromosome, if truly a B, may be atypical with respect to its influence on its carriers.     

Differential effects of polyploidy and diploidy on fitness of apomictic Boechera

The co-occurrence of apomixis (asexual reproduction) and polyploidy in plants has been the subject of debate in regard to the origin and evolution of asexuality. In recent years, polyploidy has been postulated as a maintenance and stabilization factor rather than as a source of apomixis origin. It is assumed polyploidy facilitates the compensation for mutation accumulation, and hence, the rare occurrence of diploid apomixis indirectly supports this finding. Nevertheless, diploid apomicts exist and are successful, especially in the genus Boechera. While comparing phenotypic traits, fitness-related traits and apomixis penetrance between both diploid and triploid apomicts in the genus Boechera, it was expected to find trait variance that can be attributed to ploidy. Surprisingly, little trait variation could be assigned to ploidy, but rather trait variations were mainly genotype-specific. Additionally, it is shown that paternal contribution is very important for trait success, even though all offspring are genetically identical to the mother plant. This harbors implications for the introduction of apomixis into crop plants, considering the effects of paternal contribution during asexual reproduction. Nevertheless, polyploidy is an efficient way to buffer deleterious mutations, but the flexibility of diploid apomicts of the genus Boechera for rare sexual events contributes to their success in nature.     

Intertribal hybrid plants produced from crossing Arabidopsis thaliana with apomictic Boechera

Arabidopsis thaliana and Boechera belong to different tribes of the Brassicaceae and last shared a common ancestor 13-35 million years ago. A. thaliana reproduces sexually but some Boechera accessions reproduce by apomixis (asexual reproduction by seed). The two species are reproductively isolated, preventing introgression of the trait(s) controlling apomixis from Boechera into A. thaliana and their molecular characterisation. To identify if "escapers" from such hybridisation barriers exist, we crossed diploid or tetraploid A. thaliana mothers carrying a conditional male sterile mutation with a triploid Boechera apomict. These cross-pollinations generated zygotes and embryos. Most aborted or suffered multiple developmental defects at all stages of growth, but some seed matured and germinated. Seedlings grew slowly but eventually some developed into mature plants that were novel synthetic allopolyploid hybrids. With one exception, intertribal hybrids contained three Boechera plus either one or two A. thaliana genomes (depending on maternal ploidy) and were male and female sterile. The exception was a semi-fertile, sexual partial hybrid with one Boechera plus two A. thaliana genomes. The synthesis of "escapers" that survive rigorous early developmental challenges in crosses between A. thaliana and Boechera demonstrates that the inviability form of postzygotic reproductive isolation separating these distantly related species is not impenetrable. The recovery of a single semi-fertile partial hybrid also demonstrates that hybrid sterility, another form of postzygotic reproductive isolation, can be overcome between these species.     

Boechera Summit 2011

Ecological model systems provide a conduit to understand the ecological impact of information gained from laboratory model species. Here, I review a 2011 meeting which focused on the systematic, ecological, evolutionary and developmental biology of the ecological model genus Boechera.     

Multiple origins of polyploidy and the geographic structure of Heuchera grossulariifolia

Multiple origins of polyploidy from an ancestral diploid plant species were investigated using restriction site polymorphism and sequence variation in the chloroplast DNA (cpDNA) of Heuchera grossulariifolia (Saxifragaceae). Phylogenetic analysis indicated that autopolyploidy has arisen at least twice in the evolutionary history of this species and potentially up to as many as seven times. These results suggest a greater range of independent polyploid origins as compared to a previous study of H. grossulariifolia using cpDNA restriction sites that indicated a minimum of three independent origins. Moreover, most polyploid populations did not contain cpDNA haplotypes from a single origin, but rather combined haplotypes from at least two polyploid origins. Past migration among polyploid populations of independent origin or localized polyploid formation may explain the distribution of polyploid haplotypes within and among populations. The analysis also revealed a discrepancy between relatedness and geographical location. In nearly all sympatric populations of diploids and polyploids, polyploids had the same cpDNA haplotypes as diploids from a geographically remote population. This geographical discordance has several possible explanations, including small sample sizes, extinction of parental diploid haplotypes, chloroplast introgression, and homoplasy in the cpDNA sequence data. We conclude that the recurrent formation of polyploids is an important evolutionary mechanism in the diversification of H. grossulariifolia .     

Mating system and environmental variation drive patterns of adaptation in Boechera spatifolia (Brassicaceae)

Determining the relative contribution of population genetic processes to the distribution of natural variation is a major goal of evolutionary biology. Here, we take advantage of variation in mating system to test the hypothesis that local adaptation is constrained by asexual reproduction. We explored patterns of variation in ecological traits and genome‐wide molecular markers in Boechera spatifolia (Brassicaceae), a species that contains both apomictic (asexual) and sexual individuals. Using a combination of quantitative genetics, neutral genetic (SSR) and genome‐wide single nucleotide polymorphism, we assessed the hypothesis that asexual lineages should have reduced signatures of adaptation relative to sexual conspecifics. All three measures (traits, SSRs, SNPs) demonstrated that apomicts are genetically distinct from sexuals, regardless of population location. Additionally, phylogenetic clustering revealed that the apomictic group shared a single common ancestor. Across the landscape, sexual genome‐wide SNP variation was strongly associated with latitude (r² > 0.9), indicating that sexual populations have differentiated across an environmental gradient. Furthermore, flowering time and growth rate, as assessed in a common garden, strongly covary with the elevation and latitude of the source population. Despite a wide geographic distribution that largely overlaps with sexual populations, there was little evidence for differentiation in molecular markers or quantitative characters among apomictic populations. Combined, these data indicated that, in contrast to asexual populations, sexual populations show evidence of local adaptation.     

Climate change shifts natural selection and the adaptive potential of the perennial forb Boechera stricta in the Rocky Mountains

Heritable genetic variation is necessary for populations to evolve in response to anthropogenic climate change. However, antagonistic genetic correlations among traits may constrain the rate of adaptation, even if substantial genetic variation exists. We examine potential genetic responses to selection by comparing multivariate genetic variance–covariances of traits and fitness (multivariate Robertson–Price identities) across different environments in a reciprocal transplant experiment of the forb Boechera stricta in the Rocky Mountains. By transplanting populations into four common gardens arrayed along an elevational gradient, and exposing populations to control and snow removal treatments, we simulated future and current climates and snowmelt regimes. Genetic variation in flowering and germination phenology declined in plants moved downslope to warmer, drier sites, suggesting that these traits may have a limited ability to evolve under future climates. Simulated climate change via snow removal altered the strength of selection on flowering traits, but we found little evidence that genetic correlations among traits are likely to affect the rate of adaptation to climate change. Overall, our results suggest that climate change may alter the evolutionary potential of B. stricta, but reduced expression of genetic variation may be a larger impediment to adaptation than constraints imposed by antagonistic genetic correlations.     

Polyploidy,phylogeography and Pleistocene refugia of the rockfern Asplenium ceterach: evidence from chloroplast DNA

Chloroplast DNA sequences were obtained from 331 Asplenium ceterach plants representing 143 populations from throughout the range of the complex in Europe, plus outlying sites in North Africa and the near East. We identified nine distinct haplotypes from a 900 bp fragment of trnL-trnF gene. Tetraploid populations were encountered throughout Europe and further afield, whereas diploid populations were scarcer and predominated in the Pannonian-Balkan region. Hexaploids were encountered only in southern Mediterranean populations. Four haplotypes were found among diploid populations of the Pannonian-Balkans indicating that this region formed a northern Pleistocene refugium. A separate polyploid complex centred on Greece, comprises diploid, tetraploid and hexaploid populations with two endemic haplotypes and suggests long-term persistence of populations in the southern Mediterranean. Three chloroplast DNA (cpDNA) haplotypes were common among tetraploids in Spain and Italy, with diversity reducing northwards suggesting expansion from the south after the Pleistocene. Our cpDNA and ploidy data indicate at least six independent origins of polyploids.