A NEW MEASURE OF GENETIC IDENTITY BETWEEN POPULATIONS OF SEXUAL AND ASEXUAL SPECIES

We define a new genetic identity measure that is especially well suited for asexual polyploid species as it circumvents errors in the estimation of gene frequencies. It also can be applied to sexuals allowing the study of phylogenetic relationships in species complexes consisting of sexuals and asexuals of different ploidy levels. The measure groups genotypes into classes dependent on homozygosity vs heterozygosity and the number of ancestral allele types vs the number of presumed new mutations. Its value is related to evolutionary time since divergence. The application of the method is illustrated by using electrophoretic data on the species group Solenobia triquetrella (Lepidoptera: Psychidae). A high similarity of estimated relationships among the proposed as well as other genetic identity measures is shown in the case of diploid sexual and asexual races of this species group. The phylogenetic relationships within the group are reanalyzed and monophyletic vs polyphyletic origin of parthenogenesis in this species complex is discussed. The genetic identity values found by the proposed procedure are explained by a polyphyletic origin of parthenogenesis, though a monophyletic origin of parthenogenesis in a broader sense cannot be excluded. The explanation of the phylogenetic relationships is based on the assumption of hybridization between related species and the extinction of one ancestral species. Furthermore, the genetic diversity is compared among sexual and parthenogenetic races of the species.     

Population genetics of a polyploid: is there hybridization between lineages of Hyla versicolor?

Several studies have demonstrated that polyploid species can form recurrently from their progenitors, but few studies have evaluated gene flow between the resultant polyploid lineages. Here we examine the possibility of hybridization between lineages of the tetraploid common gray treefrog (Hyla versicolor). We utilize a polymerase chain reaction (PCR) cloning approach to estimate the genotypes of tetraploid individuals and measure genetic differentiation between (1) sympatric populations of two lineages and (2) allopatric populations of a single lineage. We find that allele frequencies in sympatric populations of two lineages do not differ, suggesting that frogs of these two lineages hybridize in areas where they co-occur.     

Association of ploidy and sexual system in Lycium californicum (Solanaceae)

In North American Lycium (Solanaceae), the evolution of gender dimorphism has been proposed as a means of restoring outcrossing after polyploidization causes the loss of self-incompatibility. Previous studies of this process in Lycium focused on comparisons between species that differ in ploidy. We examined intraspecific variation in floral morphology and DNA content in populations of L. californicum to determine correlations between sexual system and cytotype. We also used nuclear ITS and GBSSI sequence data to determine whether diploid and polyploid forms represent the same phylogenetic species, and the phylogeographic relationships among populations and ploidy levels. Within populations, no variation in ploidy was found, although among populations there was a perfect correspondence between sexual system and cytotype. Diploid populations were all hermaphroditic, whereas tetraploid populations were all gender dimorphic. There was no clear geographic pattern to the occurrence of diploid and tetraploid forms. Phylogenetic analysis confirms that L. californicum, regardless of ploidy, forms a monophyletic group within the genus Lycium. Sequences from diploid and polyploid individuals did not form reciprocally monophyletic clades, indicating either multiple gains of polyploidy, ongoing gene flow between cytotypes, or lack of lineage sorting since the evolution of polyploidy. The correspondence between ploidy and sex expression is consistent with the hypothesis that polyploidization triggers the evolution of gender dimorphism in this and other Lycium species.     

Using principle component analysis to compare genetic diversity across polyploidy levels within plant complexes: an example from British Restharrows (Ononis spinosa and Ononis repens)

The investigation of genetic diversity between related plant populations which differ in ploidy levels is problematic, with common statistical methods developed for diploids being inappropriate for polyploid species. Studies into gene flow in such complexes are critical and can shed light on the mechanisms that generate and maintain populations of different polyploidy levels. We have investigated the use of principle component (PCO) analysis as one approach to elucidate population structure within British Restharrows (Leguminsoae, Ononis spp). Restharrows were common agricultural weed species until the advent of mechanical ploughing and both diploid (2n=2x=30; O. spinosa and O. intermedia) and tetraploid (2n=4x=60; O. repens and O. maritima) taxa exist. Patterns of genetic diversity were investigated among British Restharrows using 10 microsatellite loci with 21 Restharrow populations analysed (411 individual plants) from Central and Eastern Britain. PCO analysis revealed clear genetic differentiation of the sampled plants into two groups, corresponding to O. spinosa/O. intermedia (diploid) and O. repens/O. maritima (tetraploid) plants. Evidence of genetic differentiation by distance was also revealed for O. repens/O. maritima, but not for O. spinosa/O. intermedia. The data suggest the presence of strong reproductive barriers between diploid and tetraploid Restharrows in Britain, but not within ploidy levels. This genetic isolation between ploidy levels is confirmed by a detailed analysis of a sympatric site (Harton Down Hill). These results demonstrate that PCA analysis is a suitable general tool for comparing related species of different ploidy levels.     

A maximum-likelihood estimation of pairwise relatedness for autopolyploids

Relatedness between individuals is central to ecological genetics. Multiple methods are available to quantify relatedness from molecular data, including method-of-moment and maximum-likelihood estimators. We describe a maximum-likelihood estimator for autopolyploids, and quantify its statistical performance under a range of biologically relevant conditions. The statistical performances of five additional polyploid estimators of relatedness were also quantified under identical conditions. When comparing truncated estimators, the maximum-likelihood estimator exhibited lower root mean square error under some conditions and was more biased for non-relatives, especially when the number of alleles per loci was low. However, even under these conditions, this bias was reduced to be statistically insignificant with more robust genetic sampling. We also considered ambiguity in polyploid heterozygote genotyping and developed a weighting methodology for candidate genotypes. The statistical performances of three polyploid estimators under both ideal and actual conditions (including inbreeding and double reduction) were compared. The software package POLYRELATEDNESS is available to perform this estimation and supports a maximum ploidy of eight.     

Likelihood analysis of ongoing gene flow and historical association

Abstract.— We develop a Monte Carlo-based likelihood method for estimating migration rates and population divergence times from data at unlinked loci at which mutation rates are sufficiently low that, in the recent past, the effects of mutation can be ignored. The method is applicable to restriction fragment length polymorphisms (RFLPs) and single nucleotide polymorphisms (SNPs) sampled from a subdivided population. The method produces joint maximum-likelihood estimates of the migration rate and the time of population divergence, both scaled by population size, and provides a framework in which to test either for no ongoing gene flow or for population divergence in the distant past. We show the method performs well and provides reasonably accurate estimates of parameters even when the assumptions under which those estimates are obtained are not completely satisfied. Furthermore, we show that, provided that the number of polymorphic loci is sufficiently large, there is some power to distinguish between ongoing gene flow and historical association as causes of genetic similarity between pairs of populations.     

Estimation of haplotype frequencies,linkage-disequilibrium measures,and combination of haplotype copies in each pool by use of pooled DNA data

Inference of haplotypes is important for many genetic approaches, including the process of assigning a phenotype to a genetic region. Usually, the population frequencies of haplotypes, as well as the diplotype configuration of each subject, are estimated from a set of genotypes of the subjects in a sample from the population. We have developed an algorithm to infer haplotype frequencies and the combination of haplotype copies in each pool by using pooled DNA data. The input data are the genotypes in pooled DNA samples, each of which contains the quantitative genotype data from one to six subjects. The algorithm infers by the maximum-likelihood method both frequencies of the haplotypes in the population and the combination of haplotype copies in each pool by an expectation-maximization algorithm. The algorithm was implemented in the computer program LDPooled. We also used the bootstrap method to calculate the standard errors of the estimated haplotype frequencies. Using this program, we analyzed the published genotype data for the SAA (n=156), MTHFR (n=80), and NAT2 (n=116) genes, as well as the smoothelin gene (n=102). Our study has shown that the frequencies of major (frequency >0.1 in a population) haplotypes can be inferred rather accurately from the pooled DNA data by the maximum-likelihood method, although with some limitations. The estimated D and D' values had large variations except when the /D/ values were >0.1. The estimated linkage-disequilibrium measure rho2 for 36 linked loci of the smoothelin gene when one- and two-subject pool protocols were used suggested that the gross pattern of the distribution of the measure can be reproduced using the two-subject pool data.     

Simple allelic-phenotype diversity and differentiation statistics for allopolyploids

The analysis of genetic diversity within and between populations is a routine task in the study of diploid organisms. However, population genetic studies of polyploid organisms have been hampered by difficulties associated with scoring and interpreting molecular data. This occurs because the presence of multiple alleles at each locus often precludes the measurement of genotype or allele frequencies. In allopolyploids, the problem is compounded because genetically distinct isoloci frequently share alleles. As a result, analysis of genetic diversity patterns in allopolyploids has tended to rely on the interpretation of phenotype frequencies, which loses information available from allele composition. Here, we propose the use of a simple allelic-phenotype diversity statistic (H') that measures diversity as the average number of alleles by which pairs of individuals differ. This statistic can be extended to a population differentiation measure (F'ST), which is analogous to FST. We illustrate the behaviour of these statistics using coalescent computer simulations that show that F'ST behaves in a qualitatively similar way to FST, thus providing a useful way to quantify population differentiation in allopolyploid species.     

The Polyploid Series of the Achillea millefolium Aggregate in the Iberian Peninsula Investigated Using Microsatellites

The Achillea millefolium aggregate is one of the most diverse polyploid complexes of the Northern hemisphere and has its western Eurasian boundary in the Iberian Peninsula. Four ploidy levels have been detected in A. millefolium, three of which have already been found in Iberia (diploid, hexaploid and octoploid), and a fourth (tetraploid) reported during the preparation of this paper. We collected a sample from 26 Iberian populations comprising all ploidy levels, and we used microsatellite markers analyzed as dominant in view of the high ploidy levels. Our goals were to quantify the genetic diversity of A. millefolium in the Iberian Peninsula, to elucidate its genetic structure, to investigate the differences in ploidy levels, and to analyse the dispersal of the species. The lack of spatial genetic structure recovered is linked to both high levels of gene flow between populations and to the fact that most genetic variability occurs within populations. This in turn suggests the existence of a huge panmictic yarrow population in the Iberian Peninsula. This is consistent with the assumption that recent colonization and rapid expansion occurred throughout this area. Likewise, the low levels of genetic variability recovered suggest that bottlenecks and/or founder events may have been involved in this process, and clonal reproduction may have played an important role in maintaining this genetic impoverishment. Indeed, the ecological and phenologic uniformity present in the A. millefolium agg. in Iberia compared to Eurasia and North America may be responsible for the low number of representatives of this complex of species present in the Iberian Peninsula. The low levels of genetic differentiation between ploidy levels recovered in our work suggest the absence of barriers between them.     

Differential attack on diploid, tetraploid, and hexaploid Solidago altissima L. by five insect gallmakers

Genetic variation among plants can influence host choice and larval performance in insect herbivores. Ploidy (cytotype) variation is a particularly dramatic form of plant genetic variation, and where diploid and polyploid cytotypes of a species occur in sympatry, they may provide herbivores with choices that are distinguished by profound and genome-wide genetic differences. We tested for non-random attack by five gallmaking insect herbivores on diploid, tetraploid, and hexaploid cytotypes of the goldenrod Solidago altissima L., working in seven midwestern US populations where the ploidies co-occur on spatial scales relevant to insect host choice. For four of the five herbivores, attack was non-random with respect to ploidy at one or more sites. Ploidy effects on attack were complex: the ploidy subjected to highest attack varied both across herbivores within sites and (for most herbivores) across sites within herbivores. Ploidy effects on attack will alter rates of encounter between insect herbivores—either increasing or decreasing the likelihood of two herbivores sharing a host plant ramet, compared with the case with no effects of ploidy. Plant ploidy variation appears likely to have a major impact on insect community organization, and perhaps on plant–herbivore coevolution, but that impact is likely to be spatially heterogeneous. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Frequency of local, regional, and long-distance dispersal of diploid and tetraploid Saxifraga oppositifolia (Saxifragaceae) to Arctic glacier forelands

? Premise of the Study: Climate change forces many species to migrate. Empirical small-scale data on migration and colonization in the Arctic are scarce. Retreating glaciers provide new territory for cold-adapted plant species, but the genetic consequences depend on dispersal distances and frequencies. We estimated local, regional, and long-distance dispersal frequencies, as well as their effect on levels of genetic diversity, in diploid and tetraploid individuals of Saxifraga oppositifolia. ? Methods: Samples were collected in four aged moraines in each of three glacier forelands, in surrounding areas and reference populations in the Arctic archipelago Svalbard. These samples were analyzed for neutral amplified fragment length polymorphisms (AFLPs, n = 707) and ploidy levels (n = 30). ? Key Results: Genetic clustering and ploidy analyses revealed two distinct genetic groups representing diploids and tetraploids, with few intermediate triploids. The groups were intermixed in most sampled populations. No differences in genetic diversity were found between tetraploids and diploids, or between established and glacier foreland populations. Seeds were dispersed over local, regional, and long distances, with the highest proportions of seeds originating from close sources. A minimum of 4-15 founding individuals from several source populations had initially established in each glacier foreland. ? Conclusions: Our data suggest that S. oppositifolia can rapidly colonize new deglaciated areas without losing genetic diversity. Thus, glacier forelands can be alternative habitats for cold-adapted vascular plants tracking their climatic niche. Our data show no difference in colonization success between diploid and tetraploid individuals.     

Recent invasion and low level of divergence between diploid and triploid forms of <Emphasis Type="Italic">Carassius auratus</Emphasis> complex in Croatia

Carassius auratus is an invasive species in European waters, comprising a complex of diploid and polyploid forms with different modes of reproduction. However, the evolutionary history and relationships between the diploids and polyploids are still unresolved. In this study, 51.5% diploids and 48.5% triploids, including four triploid males, were discovered among the 363 individuals sampled in Croatia. We used eight microsatellite loci and mitochondrial displacement loop sequences to analyze the structure and origin of populations; and to attempt to infer the evolutionary history of the two different forms in Croatia. Microsatellite analyses revealed high allelic and clonal diversity, corroborating that high propagule vectors can compensate for the negative effects of genetic bottlenecks in successful invasive species. The absence of significant population structuring confirmed recent origin and rapid spreading of populations. No evidence was found for the existence of native European populations. Distances between individuals using both nuclear and mtDNA markers revealed the absence of substantial clustering on the ploidy level, while the split between the different ploidies on population level was only partial, suggesting that the reproductive isolation between the two forms is either of a very recent origin, or that there exists uni-, or bidirectional gene flow between the diploid and triploid forms.     

Determination of Ploidy Level and Isolation of Genes Encoding Acetyl-CoA Carboxylase in Japanese Foxtail (Alopecurus japonicus)

Ploidy level is important in biodiversity studies and in developing strategies for isolating important plant genes. Many herbicide-resistant weed species are polyploids, but our understanding of these polyploid weeds is limited. Japanese foxtail, a noxious agricultural grass weed, has evolved herbicide resistance. However, most studies on this weed have ignored the fact that there are multiple copies of target genes. This may complicate the study of resistance mechanisms. Japanese foxtail was found to be a tetraploid by flow cytometer and chromosome counting, two commonly used methods in the determination of ploidy levels. We found that there are two copies of the gene encoding plastidic acetyl-CoA carboxylase (ACCase) in Japanese foxtail and all the homologous genes are expressed. Additionally, no difference in ploidy levels or ACCase gene copy numbers was observed between an ACCase-inhibiting herbicide-resistant and a herbicide-sensitive population in this study.     

Genetic similarity between natural populations of Drosophila ananassae from Kerala and Andaman and Nicobar Islands

In order to study the degree of genetic differentiation in natural populations of Drosophila ananassae, the mean genetic identity has been computed on the basis of differences in the gene arrangement frequencies. The estimates of genetic identity suggest that the populations from Keral, South India are genetically similar to the populations from the Andaman and Nicobar Islands though they are isolated by nearly 2 000 km of water. However, the South Indian populations are genetically more differentiated than the North Indian populations.     

Gene Flow among Populations of Two Rare Co-Occurring Fern Species Differing in Ploidy Level

Differences in ploidy levels among different fern species have a vast influence on their mating system, their colonization ability and on the gene flow among populations. Differences in the colonization abilities of species with different ploidy levels are well known: tetraploids, in contrast to diploids, are able to undergo intra-gametophytic selfing. Because fertilization is a post-dispersal process in ferns, selfing results in better colonization abilities in tetraploids because of single spore colonization. Considerably less is known about the gene flow among populations of different ploidy levels. The present study examines two rare fern species that differ in ploidy. While it has already been confirmed that tetraploid species are better at colonizing, the present study focuses on the gene flow among existing populations. We analyzed the genetic structure of a set of populations in a 10×10 km study region using isoenzymes. Genetic variation in tetraploid species is distributed mainly among populations; the genetic distance between populations is correlated with the geographical distance, and larger populations host more genetic diversity than smaller populations. In the diploid species, most variability is partitioned within populations; the genetic distance is not related to geographic distance, and the genetic diversity of populations is not related to the population size. This suggests that in tetraploid species, which undergo selfing, gene flow is limited. In contrast, in the diploid species, which experience outcrossing, gene flow is extensive and the whole system behaves as one large population. Our results suggest that in ferns, the ability to colonize new habitats and the gene flow among existing populations are affected by the mating system.     

The effects of pollen and seed migration on nuclear-dicytoplasmic systems. II. A new method for estimating plant gene flow from joint nuclear-cytoplasmic data

A new maximum-likelihood method is developed for estimating unidirectional pollen and seed flow in mixed-mating plant populations from counts of joint nuclear-cytoplasmic genotypes. Data may include multiple unlinked nuclear markers with a single maternally or paternally inherited cytoplasmic marker, or with two cytoplasmic markers inherited through opposite parents, as in many conifer species. Migration rate estimates are based on fitting the equilibrium genotype frequencies under continent-island models of plant gene flow to the data. Detailed analysis of their equilibrium structures indicates when each of the three nuclear-cytoplasmic systems allows gene flow estimation and shows that, in general, it is easier to estimate seed than pollen migration. Three-locus nuclear-dicytoplasmic data only increase the conditions allowing seed migration estimates; however, the additional dicytonuclear disequilibria allow more accurate estimates of both forms of gene flow. Estimates and their confidence limits for simulated data sets confirm that two-locus data with paternal cytoplasmic inheritance provide better estimates than those with maternal inheritance, while three-locus dicytonuclear data with three modes of inheritance generally provide the most reliable estimates for both types of gene flow. Similar results are obtained for hybrid zones receiving pollen and seed flow from two source populations. An estimation program is available upon request.     

Polyploidy induces centromere association

Many species exhibit polyploidy. The presence of more than one diploid set of similar chromosomes in polyploids can affect the assortment of homologous chromosomes, resulting in unbalanced gametes. Therefore, a mechanism is required to ensure the correct assortment and segregation of chromosomes for gamete formation. Ploidy has been shown to affect gene expression. We present in this study an example of a major effect on a phenotype induced by ploidy within the Triticeae. We demonstrate that centromeres associate early during anther development in polyploid species. In contrast, centromeres in diploid species only associate at the onset of meiotic prophase. We propose that this mechanism provides a potential route by which chromosomes can start to be sorted before meiosis in polyploids. This explains previous reports indicating that meiotic prophase is shorter in polyploids than in their diploid progenitors. Even artificial polyploids exhibit this phenotype, suggesting that the mechanism must be present in diploids, but only expressed in the presence of more than one diploid set of chromosomes.     

Higher rate of tissue regeneration in polyploid asexual versus diploid sexual freshwater snails

Characterizing phenotypic differences between sexual and asexual organisms is a critical step towards understanding why sexual reproduction is so common. Because asexuals are often polyploid, understanding how ploidy influences phenotype is directly relevant to the study of sex and will provide key insights into the evolution of ploidy-level variation. The well-established association between genome size and cell cycle duration, evidence for a link between genome size and tissue regeneration rate and the growing body of research showing that ploidy influences growth rate and gene expression led us to hypothesize that healing and tissue regeneration might be affected by ploidy-level variation. We evaluated this hypothesis by measuring the rate of regeneration of antenna tissue of Potamopyrgus antipodarum, a New Zealand snail characterized by frequent coexistence between diploid sexuals and polyploid asexuals. Antennae of triploid and presumptive tetraploid asexuals regenerated more rapidly than the antennae of diploid sexuals, but regeneration rate did not differ between triploids and tetraploids. These results suggest either that ploidy elevation has nonlinear positive effects on tissue regeneration and/or that factors associated directly with reproductive mode affect regeneration rate more than ploidy level. The results of this study also indicate that the lower ploidy of sexual P. antipodarum is unlikely to confer advantages associated with more rapid regeneration.     

Allopolyploid origin and population genetics of the rare orchid Spiranthes diluvialis

The process of becoming and the attributes of being polyploid play a major role in the development and maintenance of genetic variation in allopolyploid species. A genetic survey employing protein electrophoresis on 12 populations of S. diluvialis, as well as on populations of eight congeneric species, was conducted to assess the putative allopolyploid origin of S. diluvialis and to determine the genetic variability within and among populations. Genetic identity values indicated S. diluvialis was more similar to S. magnicamporum (0.619) and S. romanzoffiana (0.727) than to any of the other congeneric species assayed. Similar to most allopolyploids, S. diluvialis showed high levels of fixed, or nearly fixed, heterozygosity and a high percentage of polymorphic loci (57.1-71.4%). The mean number of alleles per polymorphic locus in populations of S. diluvialis (2.6-3.3), however, was similar to mean values for both animal-pollinated, outcrossing, diploid species, and geographically restricted, diploid species (2.6 and 2.5, respectively). Genetic divergence among populations (mean Fst = 0.083) was low, leading to relatively high estimates of interpopulational gene flow (mean Nm = 5.41). Thus, each population harbors most of the genetic variability found within the species. The genetic variation observed within S. diluvialis supports the occurrence of at least two separate hybridization events giving rise to S. diluvialis.     

Nonrandom mating in pikas Ochotona princeps: evidence for inbreeding between individuals of intermediate relatedness

The extent and effect of inbreeding in natural populations remain largely undetermined. Pikas Ochotona princeps have been considered a likely candidate for close inbreeding in natural populations due to observations of frequent juvenile philopatry (colonization of natal home range or neighbouring home range) and high levels of spatial overlap and social tolerance between neighbouring individuals of the opposite sex. A 4-year investigation of inbreeding in pikas, however, has revealed that dispersal and mating patterns are uncoupled in this species, i.e. explained by different hypotheses. DNA fingerprinting analysis revealed that band-sharing scores between mated pairs, identified via parentage analysis, were not commensurate with band-sharing among known first-order relatives, but were similar to scores for a small sample of known second-order relatives (i.e. half-siblings, grandparent-grandchild pairs). Band-sharing scores between mated pairs were then compared with those between potential mated pairs within the population to assess whether mating was random or nonrandom with respect to genetic similarity. The results of Monte Carlo randomization tests show that pikas mated with individuals with intermediate genetic similarity in greater proportion than would be expected by chance. These data suggest mate choice in pikas may be based upon intermediate levels of relatedness.