Putting the genes into community genetics.

As part of the long-term fusion of evolutionary biology and ecology (Ford, 1964), the field of community genetics has made tremendous progress in describing the impacts of plant genetic variation on community and ecosystem processes. In the "genes-to-ecosystems" framework (Whitham et al., 2003), genetically based traits of plant species have ecological consequences, but previous studies have not identified specific plant genes responsible for community phenotypes. The study by Barker et al. (2019) in this issue of Molecular Ecology uses an impressive common garden experiment of trembling aspen (Figure 1) to test for the genetic basis of tree traits that shape the insect community composition. Using a Genome-Wide Association Study (GWAS), they found that genomic regions associated with phytochemical traits best explain variation in herbivore community composition, and identified specific genes associated with different types of leaf-modifying herbivores and ants. This is one of the first studies to identify candidate genes underlying the heritable plant traits that explain patterns of insect biodiversity.

Patterns of Substitution Rate Variation at Many Nuclear Loci in Two Species Trios in the Brassicaceae Partitioned with ANOVA.

There are marked variations among loci and among lineages in rates of nucleotide substitution. The generation time hypothesis (GTH) is a neutral explanation for substitution rate heterogeneity that has genomewide application, predicting that species with shorter generation times accumulate DNA sequence substitutions faster than species with longer generation times do since faster genome replication provides more opportunities for mutations to occur and reach fixation by genetic drift. Relatively few studies have rigorously evaluated the GTH in plants, and there are numerous alternative hypotheses for plant substitution rate variation. One major challenge has been finding pairs of closely related plant species with contrasting generation times and appropriate outgroup taxa that all also have DNA sequence data for numerous loci. To test for causes of rate variation, we obtained sequence data for 256 genes for Arabidopsis thaliana, normally reproducing every year, and the biennial Arabidopsis lyrata with three closely related outgroup taxa (Brassica rapa, Capsella grandiflora, and Neslia paniculata) as well as the biennial Brassica oleracea and the annual B. rapa lineage with the outgroup N. paniculata. A sign test indicated that more loci than expected by chance have faster rates of substitution on the branch leading to the annual than to the perennial for one three-species trio but not another. Tajima's 1D and 2D tests, and a likelihood ratio test that incorporated saturation correction, rejected rate homogeneity for up to 26 genes (up to 14 genes when correcting for multiple tests), consistently showing faster rates for the annual lineage in the Arabidopsis species trio. ANOVA showed significant rate heterogeneity between the Arabidopsis and Brassica species trios (about 6 % of rate variation) and among loci (about 26-32 % of rate variation). The lineage-by-locus interaction which would be caused by locus- and lineage-specific natural selection explained about 13 % of substitution rate variation in one ANOVA model using substitution rates from genes partitioned into odd and even codons but was not a significant effect without partitioned genes. Annual/perennial lineage and species trio by annual/perennial lineage each explained about 1 % of substitution rate variation.

Properties of Weir and Cockerham's Fst estimators and associated bootstrap confidence intervals.

Weir and Cockerham introduced single locus and multiloci F(st) estimators for the parameter θ. These estimators are commonly used, but little beyond their bias and variance is known. In this work, we develop formulas that allow us to describe how the underlying value of θ and the genetic diversity of sampled loci affect the distributions of these estimators. We show that in certain settings, these estimators are close to normal, while in others they are far from normal. We use these results to analyze confidence interval construction for θ, showing that the percentile-t bootstrap works well while the BCa bootstrap works poorly. Our results are derived using a novel coalescent based method.

Parallel rate heterogeneity in chloroplast and mitochondrial genomes of Brazil nut trees (Lecythidaceae) is consistent with lineage effects.

We investigated whether relative rates of divergence were correlated between the mitochondrial and chloroplast genomes as expected under lineage effects or were genome specific as expected with locus-specific effects. Five mitochondrial noncoding regions (nad1B_C, nad4exon1_2, nad7exon2_3, nad7exon3_4, and rps14-cob) for 21 samples from Lecythidaceae were sequenced. Three chloroplast regions (rpl20-5'rps12, trnS-trnG, and psbA-trnH) were sequenced to expand the taxa in an existing data set. Absolute rates of nucleotide and insertion and deletion (indel) changes were 13 times faster in the chloroplast genome than in the mitochondrial genome. Similar indel length frequency distributions for both organelles suggested that common mechanisms were responsible for generating indels. Molecular clock tests applied to phylogenetic trees estimated from mitochondrial and chloroplast sequences revealed global rate heterogeneity of nucleotide substitution. Maximum likelihood and Tajima's 1D relative rate tests show that Lecythis zabucajo exhibited a rate acceleration for both the mitochondrial and chloroplast sequences. Whereas Eschweilera romeu-cardosoi showed a significant rate slowdown for chloroplast sequences, the mitochondrial sequences for 3 Eschweilera taxa showed evidence for a rate slowdown only when compared with L. zabucajo. Significant rate heterogeneity was also observed for indel changes in the mitochondrial genome but not for the chloroplast. The lack of mitochondrial nucleotide changes for some taxa as well as chloroplast indel homoplasy may have limited the power of relative rate tests to detect rate variation. Relative ratio tests consistently indicated rate proportionality among branch lengths between the mitochondrial and chloroplast phylogenetic trees. The relative ratio tests showed that taxa possessing rate heterogeneity had parallel relative divergence rates in both mitochondrial and chloroplast sequences as expected under lineage effects. A neutral replication-dependent model of rate heterogeneity for both nucleotide and indel changes provides a simple explanation for common patterns of rate heterogeneity across the 2 organelle genomes in Lecythidaceae. The lineage effects observed here were uncoupled from annual/perennial habit because all the species from this study are perennial.

F(ST) and Q(ST) under neutrality.

A commonly used test for natural selection has been to compare population differentiation for neutral molecular loci estimated by F(ST) and for the additive genetic component of quantitative traits estimated by Q(ST). Past analytical and empirical studies have led to the conclusion that when averaged over replicate evolutionary histories, Q(ST) = F(ST) under neutrality. We used analytical and simulation techniques to study the impact of stochastic fluctuation among replicate outcomes of an evolutionary process, or the evolutionary variance, of Q(ST) and F(ST) for a neutral quantitative trait determined by n unlinked diallelic loci with additive gene action. We studied analytical models of two scenarios. In one, a pair of demes has recently been formed through subdivision of a panmictic population; in the other, a pair of demes has been evolving in allopatry for a long time. A rigorous analysis of these two models showed that in general, it is not necessarily true that mean Q(ST) = F(ST) (across evolutionary replicates) for a neutral, additive quantitative trait. In addition, we used finite-island model simulations to show there is a strong positive correlation between Q(ST) and the difference Q(ST) - F(ST) because the evolutionary variance of Q(ST) is much larger than that of F(ST). If traits with relatively large Q(ST) values are preferentially sampled for study, the difference between Q(ST) and F(ST) will also be large and positive because of this correlation. Many recent studies have used tests of the null hypothesis Q(ST) = F(ST) to identify diversifying or uniform selection among subpopulations for quantitative traits. Our findings suggest that the distributions of Q(ST) and F(ST) under the null hypothesis of neutrality will depend on species-specific biology such as the number of subpopulations and the history of subpopulation divergence. In addition, the manner in which researchers select quantitative traits for study may introduce bias into the tests. As a result, researchers must be cautious before concluding that selection is occurring when Q(ST) not equal F(ST).

Reconsidering the generation time hypothesis based on nuclear ribosomal ITS sequence comparisons in annual and perennial angiosperms.

BACKGROUND: Differences in plant annual/perennial habit are hypothesized to cause a generation time effect on divergence rates. Previous studies that compared rates of divergence for internal transcribed spacer (ITS1 and ITS2) sequences of nuclear ribosomal DNA (nrDNA) in angiosperms have reached contradictory conclusions about whether differences in generation times (or other life history features) are associated with divergence rate heterogeneity. We compared annual/perennial ITS divergence rates using published sequence data, employing sampling criteria to control for possible artifacts that might obscure any actual rate variation caused by annual/perennial differences. RESULTS: Relative rate tests employing ITS sequences from 16 phylogenetically-independent annual/perennial species pairs rejected rate homogeneity in only a few comparisons, with annuals more frequently exhibiting faster substitution rates. Treating branch length differences categorically (annual faster or perennial faster regardless of magnitude) with a sign test often indicated an excess of annuals with faster substitution rates. Annuals showed an approximately 1.6-fold rate acceleration in nucleotide substitution models for ITS. Relative rates of three nuclear loci and two chloroplast regions for the annual Arabidopsis thaliana compared with two closely related Arabidopsis perennials indicated that divergence was faster for the annual. In contrast, A. thaliana ITS divergence rates were sometimes faster and sometimes slower than the perennial. In simulations, divergence rate differences of at least 3.5-fold were required to reject rate constancy in > 80 % of replicates using a nucleotide substitution model observed for the combination of ITS1 and ITS2. Simulations also showed that categorical treatment of branch length differences detected rate heterogeneity > 80% of the time with a 1.5-fold or greater rate difference. CONCLUSION: Although rate homogeneity was not rejected in many comparisons, in cases of significant rate heterogeneity annuals frequently exhibited faster substitution rates. Our results suggest that annual taxa may exhibit a less than 2-fold rate acceleration at ITS. Since the rate difference is small and ITS lacks statistical power to reject rate homogeneity, further studies with greater power will be required to adequately test the hypothesis that annual and perennial plants have heterogeneous substitution rates. Arabidopsis sequence data suggest that relative rate tests based on multiple loci may be able to distinguish a weak acceleration in annual plants. The failure to detect rate heterogeneity with ITS in past studies may be largely a product of low statistical power.

speed-ne: Software to simulate and estimate genetic effective population size (Ne ) from linkage disequilibrium observed in single samples.

The genetic effective population size, Ne , can be estimated from the average gametic disequilibrium (r2^) between pairs of loci, but such estimates require evaluation of assumptions and currently have few methods to estimate confidence intervals. speed-ne is a suite of matlab computer code functions to estimate Ne^ from r2^ with a graphical user interface and a rich set of outputs that aid in understanding data patterns and comparing multiple estimators. speed-ne includes functions to either generate or input simulated genotype data to facilitate comparative studies of Ne^ estimators under various population genetic scenarios. speed-ne was validated with data simulated under both time-forward and time-backward coalescent models of genetic drift. Three classes of estimators were compared with simulated data to examine several general questions: what are the impacts of microsatellite null alleles on Ne^, how should missing data be treated, and does disequilibrium contributed by reduced recombination among some loci in a sample impact Ne^. Estimators differed greatly in precision in the scenarios examined, and a widely employed Ne^ estimator exhibited the largest variances among replicate data sets. speed-ne implements several jackknife approaches to estimate confidence intervals, and simulated data showed that jackknifing over loci and jackknifing over individuals provided ~95% confidence interval coverage for some estimators and should be useful for empirical studies. speed-ne provides an open-source extensible tool for estimation of Ne^ from empirical genotype data and to conduct simulations of both microsatellite and single nucleotide polymorphism (SNP) data types to develop expectations and to compare Ne^ estimators.

Identification and characterization of microsatellite loci in the tuliptree, Liriodendron tulipifera (Magnoliaceae).

PREMISE OF THE STUDY: Twenty-three polymorphic microsatellite loci (simple sequence repeats) were identified and characterized for Liriodendron tulipifera (Magnoliaceae), a species native to eastern North America, to investigate its genetic diversity, population structure, and mating system. METHODS AND RESULTS: Using Illumina HiSeq paired-end reads from genomic DNA, searches for repeat motifs identified approximately 280,000 potentially amplifiable loci. Of 77 loci tested, 51 amplified consistently. When genotyped using 30 to 52 total adult trees from three old-growth populations in Maryland, Virginia, and New Jersey, USA, 23 loci were polymorphic. These loci exhibited four to 13 alleles, and observed and expected heterozygosities ranged from 0.233 to 0.865 and 0.272 to 0.876, respectively. CONCLUSIONS: The microsatellite marker loci presented here will be valuable in population genetic studies of L. tulipifera because they do not suffer from ascertainment bias and show high polymorphism.

Comparing relative rates of pollen and seed gene flow in the island model using nuclear and organelle measures of population structure.

We describe a method for comparing nuclear and organelle population differentiation (F(ST)) in seed plants to test the hypothesis that pollen and seed gene flow rates are equal. Wright's infinite island model is used, with arbitrary levels of self-fertilization and biparental organelle inheritance. The comparison can also be applied to gene flow in animals. Since effective population sizes are smaller for organelle genomes than for nuclear genomes and organelles are often uniparentally inherited, organelle F(ST) is expected to be higher at equilibrium than nuclear F(ST) even if pollen and seed gene flow rates are equal. To reject the null hypothesis of equal seed and pollen gene flow rates, nuclear and organelle F(ST)'s must differ significantly from their expected values under this hypothesis. Finite island model simulations indicate that infinite island model expectations are not greatly biased by finite numbers of populations (>/=100 subpopulations). The power to distinguish dissimilar rates of pollen and seed gene flow depends on confidence intervals for fixation index estimates, which shrink as more subpopulations and loci are sampled. Using data from the tropical tree Corythophora alta, we rejected the null hypothesis that seed and pollen gene flow rates are equal but cannot reject the alternative hypothesis that pollen gene flow is 200 times greater than seed gene flow.

Hierarchical components of genetic variation at a species boundary: population structure in two sympatric varieties of Lupinus microcarpus (Leguminosae).

Lupinus microcarpus is a self-compatible annual plant that forms a species complex of morphologically variable but indeterminate varieties. In order to examine the hypothesis that varieties of L. microcarpus comprise genetically differentiated and reproductively isolated species, populations of L. microcarpus var. horizontalis and var. densiflorus were sampled from an area of sympatry in central California and genotyped using six microsatellite loci. Bayesian clustering divided the total sample into two groups corresponding to the named varieties with extremely low levels of inferred coancestry. Similarly, maximum likelihood and distance methods for genetic assignment placed individuals in two nonoverlapping groups. Evidence for isolation by distance (IBD) within each variety was found at shorter distance classes, but varieties remained differentiated in sympatry. Furthermore, coalescent estimates of divergence time indicate separation within the past 950-5050 generations, with minimal gene flow after divergence. A four-level hierarchical analysis of molecular variance (amova) found significant levels of genetic differentiation among varieties (theta(P) = 0.292), populations within varieties (theta(S) = 0.449), subpopulations within populations (theta(SS) = 0.623), and individuals within subpopulations (f = 0.421); but the greatest degree of differentiation was at the subpopulation level. Although it is sometimes assumed that the magnitude of genetic differences (e.g. F(ST)) should be greater between species than among populations or subpopulations of the same species, shared ancestral polymorphism may lead to relatively low levels of differentiation at the species level, even as the stochastic effects of genetic drift generate higher levels of differentiation at lower hierarchical levels. These results suggest that L. microcarpus var. horizontalis and var. densiflorus are recently diverged yet reproductively isolated species, with high levels of inbreeding resulting from the combined effects of limited gene flow, demographic bottlenecks, and partial selfing in finite, geographically structured populations.

Dynamics of Fst for the island model.

F(st) is a measure of genetic differentiation in a subdivided population. Sewall Wright observed that F(st)=1/1+2Nm in a haploid diallelic infinite island model, where N is the effective population size of each deme and m is the migration rate. In demonstrating this result, Wright relied on the infinite size of the population. Natural populations are not infinite and therefore they change over time due to genetic drift. In a finite population, F(st) becomes a random variable that evolves over time. In this work we ask, given an initial population state, what are the dynamics of the mean and variance of F(st) under the finite island model? In application both of these quantities are critical in the evaluation of F(st) data. We show that after a time of order N generations the mean of F(st) is slightly biased below 1/1+2Nm. Further we show that the variance of F(st) is of order 1/d where d is the number of demes in the population. We introduce several new mathematical techniques to analyze coalescent genealogies in a dynamic setting.

Steady state of homozygosity and Gst for the island model.

We examine homozygosity and G(st) for a subdivided population governed by the finite island model. Assuming an infinite allele model and strong mutation we show that the steady state distributions of G(st) and homozygosity have asymptotic expansions in the mutation rate. We use this observation to derive asymptotic expansions for various moments of homozygosity and to derive rigorous formulas for the mean and variance of G(st). We show that G(st) approximately 1/(1+2Nm), similarly to the well known formula of Wright for the infinite island model, and that the variance of G(st) goes to zero as mutation increases.

GENETIC FINGERPRINT-INFERRED POPULATION SUBDIVISION AND SPATIAL GENETIC TESTS FOR ISOLATION BY DISTANCE AND ADAPTATION IN THE COASTAL PLANT LIMONIUM CAROLINIANUM.

This paper examines two wild populations of Limonium carolinianum for population genetic subdivision and spatial patterns of genetic variation in an attempt to simultaneously test for both the action of local adaptation to tidal gradients and isolation by distance (IBD). A VNTR (variable number of tandem repeats) genetic "fingerprinting" marker was used to infer relatedness among mapped plants in two populations. Band sharing within and between populations estimated F'ST , an approximate measure of FST . Regression models were used to analyze the relationship between band sharing and spatial separation in tidal elevation and horizontal distance, as well as the relationship of fecundity differences with band sharing and spatial distance. Populations differed in band size frequency distributions and mean number of bands per profile and, therefore, likely differed in effective population size. F'ST was estimated at 0.0678 and was significantly greater than F'ST among randomly constructed subpopulations. Band sharing decreased 0.13% per meter in one population but showed no significant relation to distance in the other. In the population with significant IBD band sharing increased with increasingly different tidal elevation, contrary to an adaptive hypothesis, possibly due to directional gene flow or drift. Deme sizes were approximately 25 meters and greater than 100 meters, spanning larger areas than the entire environmental gradient. Fecundity differences were not associated with spatial parameters or band sharing. Unequal potential maternal fecundity measured as variance in number of seeds per maternal family was a significant source of genetic sampling variance. The VNTR marker employed is capable of detecting adaptation as identity by descent in ecological time and is an appropriate method for estimating the net evolutionary fate of polygenic traits. The results show that the net balance between selection along an environmental gradient and the effects of IBD and unequal maternal fecundity favor genetic differentiation by random processes in populations of Limonium.

The impact of microsatellite electromorph size homoplasy on multilocus population structure estimates in a tropical tree (Corythophora alta) and an anadromous fish (Morone saxatilis).

Microsatellite allelic states are determined by electrophoretic sizing of polymerase chain reaction fragments to define electromorphs. Numerous studies have documented that identical microsatellite electromorphs are potentially heterogeneous at the DNA sequence level, a phenomenon called electromorph size homoplasy. Few studies have examined the impact of electromorph size homoplasy on estimates of population genetic parameters. We investigated the frequency of microsatellite electromorph size homoplasy for 12 loci in the tropical tree Corythophora alta and 11 loci in the anadromous fish Morone saxatilis by sequencing 14-23 homozygotes per locus sampled from multiple populations for a total of 453 sequences. Sequencing revealed no homoplasy for M. saxatilis loci. Seven C. alta loci exhibited homoplasy, including single and compound repeat motifs both with and without interruptions. Between 12.5 and 42.9% of electromorphs sampled per locus showed size homoplasy. Two methods of correction for homoplasy in C. alta generally produced little or no change in single-locus estimates of RST, except for two loci in which some additional differentiation among populations was revealed. Twelve-locus estimates of RST (including the seven loci corrected for homoplasy) were slightly greater than estimates from uncorrected data, although the 95% confidence intervals overlapped. The frequency of methodological errors such as clerical mistakes or sample mislabelling per genotype scored was estimated at 5.4 and 7.3% for C. alta and M. saxatilis, respectively. Simulations showed that the increase in RST produced by homoplasy correction was only slightly larger than variation in RST estimates expected to be caused by methodological errors.

Patterns and relative rates of nucleotide and insertion/deletion evolution at six chloroplast intergenic regions in new world species of the Lecythidaceae.

Insertions and deletions (indels) in chloroplast noncoding regions are common genetic markers to estimate population structure and gene flow, although relatively little is known about indel evolution among recently diverged lineages such as within plant families. Because indel events tend to occur nonrandomly along DNA sequences, recurrent mutations may generate homoplasy for indel haplotypes. This is a potential problem for population studies, because indel haplotypes may be shared among populations after recurrent mutation as well as gene flow. Furthermore, indel haplotypes may differ in fitness and therefore be subject to natural selection detectable as rate heterogeneity among lineages. Such selection could contribute to the spatial patterning of cpDNA haplotypes, greatly complicating the interpretation of cpDNA population structure. This study examined both nucleotide and indel cpDNA variation and divergence at six noncoding regions (psbB-psbH, atpB-rbcL, trnL-trnH, rpl20-5'rps12, trnS-trnG, and trnH-psbA) in 16 individuals from eight species in the Lecythidaceae and a Sapotaceae outgroup. We described patterns of cpDNA changes, assessed the level of indel homoplasy, and tested for rate heterogeneity among lineages and regions. Although regression analysis of branch lengths suggested some degree of indel homoplasy among the most divergent lineages, there was little evidence for indel homoplasy within the Lecythidaceae. Likelihood ratio tests applied to the entire phylogenetic tree revealed a consistent pattern rejecting a molecular clock. Tajima's 1D and 2D tests revealed two taxa with consistent rate heterogeneity, one showing relatively more and one relatively fewer changes than other taxa. In general, nucleotide changes showed more evidence of rate heterogeneity than did indel changes. The rate of evolution was highly variable among the six cpDNA regions examined, with the trnS-trnG and trnH-psbA regions showing as much as 10% and 15% divergence within the Lecythidaceae. Deviations from rate homogeneity in the two taxa were constant across cpDNA regions, consistent with lineage-specific rates of evolution rather than cpDNA region-specific natural selection. There is no evidence that indels are more likely than nucleotide changes to experience homoplasy within the Lecythidaceae. These results support a neutral interpretation of cpDNA indel and nucleotide variation in population studies within species such as Corythophora alta.