Christopher M. Richards

Evolutionary Conservation of the FLOWERING LOCUS C-Mediated Vernalization Response: Evidence From the Sugar Beet (Beta vulgaris)

In many plant species, exposure to a prolonged period of cold during the winter promotes flowering in the spring, a process termed vernalization. In Arabidopsis thaliana, the vernalization requirement of winter-annual ecotypes is caused by the MADS-box gene FLOWERING LOCUS C (FLC), which is a repressor of flowering. During the vernalization process, FLC is downregulated by alteration of its chromatin structure, thereby permitting flowering to occur. In wheat, a vernalization requirement is imposed by a different repressor of flowering, suggesting that some components of the regulatory network controlling the vernalization response differ between monocots and dicots. The extent to which the molecular mechanisms underlying vernalization have been conserved during the diversification of the angiosperms is not well understood. Using phylogenetic analysis, we identified homologs of FLC in species representing the three major eudicot lineages. FLC homologs have not previously been documented outside the plant family Brassicaceae. We show that the sugar beet FLC homolog BvFL1 functions as a repressor of flowering in transgenic Arabidopsis and is downregulated in response to cold in sugar beet. Cold-induced downregulation of an FLC-like floral repressor may be a central feature of the vernalization response in at least half of eudicot species.

THE INFLUENCE OF POPULATION SIZE AND ISOLATION ON GENE FLOW BY POLLEN IN SILENE ALBA

In a series of experiments conducted over two seasons, we used arrays of experimental populations to examine the effects of flower number and distance between patches on gene flow by pollen. For this study we used the dioecious, short‐lived perennial plant Silene alba (Caryophyllaceae). This species lives in disturbed roadside and agricultural habitats and displays a weedy population dynamic with high colonization and extinction rates. The motivation for the study was to understand what factors may be influencing genetic connectedness among newly colonized populations within a regional metapopulation. By using experimental populations composed of genotypes homozygous at a diagnostic locus, it was possible to identify explicitly pollen movement into a focal patch as a function of flower number and distance to the nearest neighboring patch. Overall, the mean immigration rate (measured as the fraction of seeds sired by males outside the focal patch) at 20 m was just over 47%, whereas at 80 m immigration rates were less than 6%. In addition, by knowing the context in which each of these gene‐flow events occurred, it was possible to understand some of the factors that influenced the exchange of genes. Both the number of flowers in the focal population (target) and in the neighboring populations (source) had a significant effect on the frequency of gene flow. Our experimental data also demonstrate that factors that influence gene flow at one spatial scale may not act in the same way at another. Specifically, the influence of target size and the relative size of the target and source patches on rates of gene flow depended on whether the patches were separated by 20 m or 80 m. These data suggest that the patterns of gene flow within a metapopulation system can be complex and may vary within a growing season.

Genetic diversity and population structure in Malus sieversii, a wild progenitor species of domesticated apple

Malus sieversii (Lebed.) M. Roem. is a wild progenitor species of the domesticated apple. It is found across a mountainous region of central Asia and has been the focus of several collection expeditions by the USDA-ARS-National Plant Germplasm System. This study used microsatellite variation at seven loci to estimate diversity and differentiation within M. sieversii using several complimentary approaches. Multilocus genotypes were amplified from 949 individuals representing seedling trees from 88 half-sib families from eight M. sieversii populations collected in Kazakhstan. Apportioning of genetic variation was estimated at both the family and site level. Analyses using a hierarchical model to estimate Fst showed that differentiation among individual families is more than three times greater than differentiation among sites. In addition, average gene diversity and allelic richness varied significantly among sites. A rendering of a genetic network among all sites showed that differentiation is largely congruent with geographical location. In addition, nonhierarchical Bayesian assignment methods were used to infer genetic clusters across the collection area. We detected four genetic clusters in the data set. The quality of these assignments was evaluated over multiple Markov Chain Monte Carlo runs using both posterior likelihood and stability of the assignments. The spatial pattern of genetic assignments among the eight collection sites shows two broadly distributed and two narrowly distributed clusters. These data indicate that the southwestern collection sites are more admixed and more diverse than the northern sites.

Microsatellite Characterization of Subspecies and Their Hybrids in Culex pipiens Complex (Diptera: Culicidae) Mosquitoes Along a North-South Transect in the Central United States

ABSTRACT Mosquitoes in the Culex pipiens complex, Cx. p. pipiens L. and Cx. p. quinquefasciatus Say are morphologically similar and important vectors of West Nile and St. Louis Encephalitis viruses in the United States. The subspecies differ with respect to overwintering strategies, with Cx. p. pipiens entering diapause in response to winter conditions and Cx. p. quinquefasciatus lacking this ability, yet they hybridize when found in sympatry. Specimens (n = 646) were collected using gravid traps set along a transect from New Orleans, LA, to Chicago, IL. Microsatellite markers were used to: genetically characterize subspecies and hybrids, determine the degree and extent of hybridization to better define the hybrid zone, and examine the efficacy of hybrid detection between microsatellites and a singlegene assay based on the acetylcholinesterase.2 gene (HotAce.2). The results support the presence of two distinct genetic entities, with abroad, stable hybrid zone in between. Admixture analyses classified >40% of individuals as hybrids. Allelic richness was markedly different at the northern and southern ends of the transect, and there was a significant isolation by distance effect. The hybrid zone seems to be wider and extends further to the south than previous work indicated, and as a result, we propose new boundaries compared with those indicated by a previous study. Microsatellites detected more hybrids than the HotAce.2 assay, although the latter assay remains useful as a screening tool. We suggest that the parental subspecies and the hybrid zone are maintained by heterosis combined with selection for diapause at northern latitudes.

Species Delimitation under the General Lineage Concept: An Empirical Example Using Wild North American Hops (Cannabaceae: Humulus lupulus)

There is an emerging consensus that the intent of most species concepts is to identify evolutionarily distinct lineages. However, the criteria used to identify lineages differ among concepts depending on the perceived importance of various attributes of evolving populations. We have examined five different species criteria to ask whether the three taxonomic varieties of Humulus lupulus (hops) native to North America are distinct lineages. Three criteria (monophyly, absence of genetic intermediates, and diagnosability) focus on evolutionary patterns and two (intrinsic reproductive isolation and niche specialization) consider evolutionary processes. Phylogenetic analysis of amplified fragment length polymorphism (AFLP) data under a relaxed molecular clock, a stochastic Dollo substitution model, and parsimony identified all varieties as monophyletic, thus they satisfy the monophyly criterion for species delimitation. Principal coordinate analysis and a Bayesian assignment procedure revealed deep genetic subdivisions and little admixture between varieties, indicating an absence of genetic intermediates and compliance with the genotypic cluster species criterion. Diagnostic morphological and AFLP characters were found for all varieties, thus they meet the diagnosability criterion. Natural history information suggests that reproductive isolating barriers may have evolved in var. pubescens, potentially qualifying it as a species under a criterion of intrinsic reproductive isolation. Environmental niche modeling showed that the preferred habitat of var. neomexicanus is climatically unique, suggesting niche specialization and thus compliance with an ecological species criterion. Isolation by distance coupled with imperfect sampling can lead to erroneous lineage identification using some species criteria. Compliance with complementary pattern- and process-oriented criteria provides powerful corroboration for a species hypothesis and mitigates the necessity for comprehensive sampling of the entire species range, a practical impossibility in many systems. We hypothesize that var. pubescens maintains its genetic identity, despite substantial niche overlap with var. lupuloides, via the evolution of partial reproductive isolating mechanisms. Variety neomexicanus, conversely, will likely persist as a distinct lineage, regardless of limited gene flow with vars. lupuloides and pubescens because of ecological isolation--adaptation to the unique conditions of the Rocky Mountain cordillera. Thus, we support recognition of vars. neomexicanus and pubescens as species, but delay making a recommendation for var. lupuloides until sampling of genetic variation is complete or a stable biological process can be identified to explain its observed genetic divergence.

Accurate Inference of Subtle Population Structure (and Other Genetic Discontinuities) Using Principal Coordinates

Background Accurate inference of genetic discontinuities between populations is an essential component of intraspecific biodiversity and evolution studies, as well as associative genetics. The most widely-used methods to infer population structure are model-based, Bayesian MCMC procedures that minimize Hardy-Weinberg and linkage disequilibrium within subpopulations. These methods are useful, but suffer from large computational requirements and a dependence on modeling assumptions that may not be met in real data sets. Here we describe the development of a new approach, PCO-MC, which couples principal coordinate analysis to a clustering procedure for the inference of population structure from multilocus genotype data. Methodology/Principal Findings PCO-MC uses data from all principal coordinate axes simultaneously to calculate a multidimensional “density landscape”, from which the number of subpopulations, and the membership within subpopulations, is determined using a valley-seeking algorithm. Using extensive simulations, we show that this approach outperforms a Bayesian MCMC procedure when many loci (e.g. 100) are sampled, but that the Bayesian procedure is marginally superior with few loci (e.g. 10). When presented with sufficient data, PCO-MC accurately delineated subpopulations with population Fst values as low as 0.03 (Gst>0.2), whereas the limit of resolution of the Bayesian approach was Fst = 0.05 (Gst>0.35). Conclusions/Significance We draw a distinction between population structure inference for describing biodiversity as opposed to Type I error control in associative genetics. We suggest that discrete assignments, like those produced by PCO-MC, are appropriate for circumscribing units of biodiversity whereas expression of population structure as a continuous variable is more useful for case-control correction in structured association studies.

Three types of rescue can avert extinction in a changing environment

Significance Preventing extinction of small populations in rapidly changing environments is crucial to long-term preservation of diversity, because the creation of large reserves is often not feasible. An option immediately available to managers is bringing migrants in to increase size or improve genetic composition of populations at risk. We experimentally manipulate different types and combinations of migrants to evaluate which will be most effective in rescuing populations from extinction. We find that migration of numerous individuals can reduce the probability of extinction. However, migration of just a few genetically distinct individuals both reduces probability of extinction and dramatically increases fitness and population size. We suggest managers with limited conservation resources should prioritize genetic rescue over increasing demographic size for small populations. Setting aside high-quality large areas of habitat to protect threatened populations is becoming increasingly difficult as humans fragment and degrade the environment. Biologists and managers therefore must determine the best way to shepherd small populations through the dual challenges of reductions in both the number of individuals and genetic variability. By bringing in additional individuals, threatened populations can be increased in size (demographic rescue) or provided with variation to facilitate adaptation and reduce inbreeding (genetic rescue). The relative strengths of demographic and genetic rescue for reducing extinction and increasing growth of threatened populations are untested, and which type of rescue is effective may vary with population size. Using the flour beetle (Tribolium castaneum) in a microcosm experiment, we disentangled the genetic and demographic components of rescue, and compared them with adaptation from standing genetic variation (evolutionary rescue in the strictest sense) using 244 experimental populations founded at either a smaller (50 individuals) or larger (150 individuals) size. Both types of rescue reduced extinction, and those effects were additive. Over the course of six generations, genetic rescue increased population sizes and intrinsic fitness substantially. Both large and small populations showed evidence of being able to adapt from standing genetic variation. Our results support the practice of genetic rescue in facilitating adaptation and reducing inbreeding depression, and suggest that demographic rescue alone may suffice in larger populations even if only moderately inbred individuals are available for addition.

Genetic diversity in Malus ×domestica (Rosaceae) through time in response to domestication

UNLABELLED • PREMISE OF THE STUDY Patterns of genetic diversity in domesticated plants are affected by geographic region of origin and cultivation, intentional artificial selection, and unintentional genetic bottlenecks. While bottlenecks are mainly associated with the initial domestication process, they can also affect diversity during crop improvement. Here, we investigate the impact of the improvement process on the genetic diversity of domesticated apple in comparison with other perennial and annual fruit crops.• METHODS Apple cultivars that were developed at various times (ranging from the 13th through the 20th century) and 11 of the 15 apple cultivars that are used for 90% of the apple production in the United States were surveyed for genetic diversity based on either 9 or 19 simple sequence repeats (SSRs). Diversity was compared using standard metrics and model-based approaches based on expected heterozygosity (He) at equilibrium. Improvement bottleneck data for fruit crops were also collected from the literature.• KEY RESULTS Domesticated apples showed no significant reduction in genetic diversity through time across the last eight centuries. Diversity was generally high, with an average He > 0.7 for apples from all centuries. However, diversity of the apples currently used for the bulk of commercial production was lower.• CONCLUSIONS The improvement bottleneck in domesticated apples appears to be mild or nonexistent, in contrast to improvement bottlenecks in many annual and perennial fruit crops, as documented from the literature survey. The low diversity of the subset of cultivars used for commercial production, however, indicates that an improvement bottleneck may be in progress for this perennial crop.

Distinguishing Terminal Monophyletic Groups from Reticulate Taxa: Performance of Phenetic, Tree-Based, and Network Procedures

Hybridization is a well-documented, natural phenomenon that is common at low taxonomic levels in the higher plants and other groups. In spite of the obvious potential for gene flow via hybridization to cause reticulation in an evolutionary tree, analytical methods based on a strictly bifurcating model of evolution have frequently been applied to data sets containing taxa known to hybridize in nature. Using simulated data, we evaluated the relative performance of phenetic, tree-based, and network approaches for distinguishing between taxa with known reticulate history and taxa that were true terminal monophyletic groups. In all methods examined, type I error (the erroneous rejection of the null hypothesis that a taxon of interest is not monophyletic) was likely during the early stages of introgressive hybridization. We used the gradual erosion of type I error with continued gene flow as a metric for assessing relative performance. Bifurcating tree-based methods performed poorly, with highly supported, incorrect topologies appearing during some phases of the simulation. Based on our model, we estimate that many thousands of gene flow events may be required in natural systems before reticulate taxa will be reliably detected using tree-based methods of phylogeny reconstruction. We conclude that the use of standard bifurcating tree-based methods to identify terminal monophyletic groups for the purposes of defining or delimiting phylogenetic species, or for prioritizing populations for conservation purposes, is difficult to justify when gene flow between sampled taxa is possible. As an alternative, we explored the use of two network methods. Minimum spanning networks performed worse than most tree-based methods and did not yield topologies that were easily interpretable as phylogenies. The performance of NeighborNet was comparable to parsimony bootstrap analysis. NeighborNet and reverse successive weighting were capable of identifying an ephemeral signature of reticulate evolution during the early stages of introgression by revealing conflicting phylogenetic signal. However, when gene flow was topologically complex, the conflicting phylogenetic signal revealed by these methods resulted in a high probability of type II error (inferring that a monophyletic taxon has a reticulate history). Lastly, we present a novel application of an existing nonparametric clustering procedure that, when used against a density landscape derived from principal coordinate data, showed superior performance to the tree-based and network procedures tested.

Retention of agronomically important variation in germplasm core collections: implications for allele mining

The primary targets of allele mining efforts are loci of agronomic importance. Agronomic loci typically exhibit patterns of allelic diversity that are consistent with a history of natural or artificial selection. Natural or artificial selection causes the distribution of genetic diversity at such loci to deviate substantially from the pattern found at neutral loci. The germplasm utilized for allele mining should contain maximum allelic variation at loci of interest, in the smallest possible number of samples. We show that the popular core collection assembly procedure “M” (marker allele richness), which leverages variation at neutral loci, performs worse than random assembly for retaining variation at a locus of agronomic importance in sugar beet (Betavulgaris L. subsp. vulgaris) that is under selection. We present a corrected procedure (“M+”) that outperforms M. An extensive coalescent simulation was performed to demonstrate more generally the retention of neutral versus selected allelic variation in core subsets assembled with M+. A negative correlation in level of allelic diversity between neutral and selected loci was observed in 42% of simulated data sets. When core collection assembly is guided by neutral marker loci, as is the current common practice, enhanced allelic variation at agronomically important loci should not necessarily be expected.