Conceptual analysis of methods applied to assessment of diversity within and distance between populations with asexual or mixed mode of reproduction

Measures of diversity within populations, and distance between populations, are compared for organisms with an asexual or mixed mode of reproduction. Examples are drawn from studies of plant pathogenic fungi based on binary traits including presence/absence of DNA bands or virulence/avirulence to differential hosts. Commonly used measures of population diversity or genetic distance consider either genotype frequencies or allele frequencies. Kosman's diversity and distance measures are the most suitable for populations with an asexual or mixed mode of reproduction, because by considering genetic patterns of all individuals they take into account not just the genotype frequencies but also the genetic similarities between genotypes in the populations. The Kosman distance and diversity measures for populations can be calculated using different measures of dissimilarity between individuals (the simple mismatch, Jaccard and Dice coefficients of dissimilarity). Kosman's distances based on the simple mismatch and Jaccard dissimilarities are metrics. Comparisons of diversity indices for hypothetical examples as well as for actual data sets are presented to demonstrate that inferences from diversity analysis of populations can be driven by techniques of diversity and distance assessments and not only data driven.     

竹叶眼子菜居群遗传多样性和克隆结构

采用ISSR技术对长江中游南岸豹澥湖和大冶湖不同生境中的竹叶眼子菜(Potamogeton malaianus)居群的遗传多样性及克隆结构进行了研究.结果表明,在两居群的106株个体中,利用6条ISSR引物共得到40条符合3/N标准并无连锁不平衡的清晰位点,竹叶眼子菜具有较高的遗传多样性,其多态位点百分率为75.0％,Shannon多样性指数为0.3736, 两居群的遗传分化很小.竹叶眼子菜的克隆多样性很高(D=0.9917),两居群间的克隆分化很大,不具有共有的基因型.竹叶眼子菜的基株分布为游击型构型,位于湖心的大冶居群克隆距离(3.0～31.5 m)明显地大于湖岸豹澥居群(2.4～6.7 m).     

genclone: a computer program to analyse genotypic data,test for clonality and describe spatial clonal organization

genclone 1.0 is designed for studying clonality and its spatial components using genotype data with molecular markers from haploid or diploid organisms. genclone 1.0 performs the following tasks. (i) discriminates distinct multilocus genotypes (MLGs), and uses permutation and resampling approaches to test for the reliability of sets of loci and sampling units for estimating genotypic and genetic diversity (a procedure also useful for nonclonal organisms); (ii) computes statistics to test for clonal propagation or clonal identity of replicates; (iii) computes various indices describing genotypic diversity; and (iv) summarizes the spatial organization of MLGs with adapted spatial autocorrelation methods and clonal subrange estimates.     

Assessing genetic diversity in clonal organisms: low diversity or low resolution? Combining power and cost efficiency in selecting markers

The increasing use of molecular tools to study populations of clonal organisms leads us to question whether the low polymorphism found in many studies reflects limited genetic diversity in populations or the limitations of the markers used. Here we used microsatellite datasets for two sea grass species to provide a combinatory statistic, combined with a likelihood approach to estimate the probability of identical multilocus genotypes (MLGs) to be shared by distinct individuals, in order to ascertain the efficiency of the markers used and to optimize cost-efficiently the choice of markers to use for deriving unbiased estimates of genetic diversity. These results strongly indicate that conclusions from studies on clonal organisms derived using markers showing low polymorphism, including microsatellites, should be reassessed using appropriate polymorphic markers.     

Geographic patterns of clonal diversity in the earth mite species Penthaleus major with particular emphasis on species margins

Patterns of clonal diversity in parthenogenetic species can be influenced by occasional sexual reproduction, selection due to environmental heterogeneity, and genetic drift. Here we investigate geographic patterns of clonal diversity in the obligate parthenogenetic earth mite species, Penthaleus major, in southeastern Australia. Spatial autocorrelation analysis indicated a relationship between geographic distance and clonal diversity at the sites. However, there was no evidence for an association between distance and clonal composition or the frequency of the two most common clones. Regression analyses indicated that clonal diversity and the frequency of one of the common clonal groups were associated with latitude and/or climatic variables, whereas the other common group was not associated with any of those variables. In four transects (each 250-400 km in length) extending from the center of the distribution of P. major to its margins, there was a consistent and continuous decline in clonal diversity as the margin was approached but no evidence that one clonal group predominated at the margin. This argues against the importance of a general-purpose genotype being favored at margins and suggests that either selection acts to decrease local diversity at sites near the margin or that diversity declines due to drift at these sites.     

Standardizing methods to address clonality in population studies

Although clonal species are dominant in many habitats, from unicellular organisms to plants and animals, ecological and particularly evolutionary studies on clonal species have been strongly limited by the difficulty in assessing the number, size and longevity of genetic individuals within a population. The development of molecular markers has allowed progress in this area, and although allozymes remain of limited use due to their typically low level of polymorphism, more polymorphic markers have been discovered during the last decades, supplying powerful tools to overcome the problem of clonality assessment. However, population genetics studies on clonal organisms lack a standardized framework to assess clonality, and to adapt conventional data analyses to account for the potential bias due to the possible replication of the same individuals in the sampling. Moreover, existing studies used a variety of indices to describe clonal diversity and structure such that comparison among studies is difficult at best. We emphasize the need for standardizing studies on clonal organisms, and particularly on clonal plants, in order to clarify the way clonality is taken into account in sampling designs and data analysis, and to allow further comparison of results reported in distinct studies. In order to provide a first step towards a standardized framework to address clonality in population studies, we review, on the basis of a thorough revision of the literature on population structure of clonal plants and of a complementary revision on other clonal organisms, the indices and statistics used so far to estimate genotypic or clonal diversity and to describe clonal structure in plants. We examine their advantages and weaknesses as well as various conceptual issues associated with statistical analyses of population genetics data on clonal organisms. We do so by testing them on results from simulations, as well as on two empirical data sets of microsatellites of the seagrasses Posidonia oceanica and Cymodocea nodosa. Finally, we also propose a selection of new indices and methods to estimate clonal diversity and describe clonal structure in a way that should facilitate comparison between future studies on clonal plants, most of which may be of interest for clonal organisms in general.     

Clonal and fine-scale genetic structure in populations of a restricted Korean endemic, Hosta jonesii (Liliaceae) and the implications for conservation

BACKGROUND AND AIMS: In plant populations the magnitude of spatial genetic structure of apparent individuals (including clonal ramets) can be different from that of sexual individuals (genets). Thus, distinguishing the effects of clonal versus sexual individuals in population genetic analyses could provide important insights for evolutionary biology and conservation. To investigate the effects of clonal spread on the fine-scale spatial genetic structure within plant populations, Hosta jonesii (Liliaceae), an endemic species to Korea, was chosen as a study species. METHODS: Using allozymes as genetic markers, spatial autocorrelation analysis of ramets and of genets was conducted to quantify the spatial scale of clonal spread and genotype distribution in two populations of H. jonesii. KEY RESULTS: Join-count statistics revealed that most clones are significantly aggregated at < 3-m interplant distance. Spatial autocorrelation analysis of all individuals resulted in significantly higher Moran's I values at 0-3-m interplant distance than analyses of population samples in which clones were excluded. However, significant fine-scale genetic structure was still observed when clones were excluded. CONCLUSIONS: These results suggest that clones enhance the magnitude of spatial autocorrelation due to localized clonal spread. The significant fine-scale genetic structure detected in samples excluding clones is consistent with the biological and ecological traits exhibited by H. jonesii including bee pollination and limited seed dispersal. For conservation purposes, genetic diversity would be maximized in local populations of H. jonesii by collecting or preserving individuals that are spaced at least 5 m apart.     

CLONAL STRUCTURE,GENOTYPIC DIVERSITY,AND SEED PRODUCTION IN POPULATIONS OF FILIPENDULA RUBRA (ROSACEAE) FROM THE NORTHCENTRAL UNITED STATES

Queen of the prairie, Filipendula rubra (Rosaceae), is a clonal plant species inhabiting calcareous fens and wet meadows of the northcentral United States. F. rubra reproduces asexually by underground rhizomes and sexually by seed. While many studies have explored genotype diversity in clonal species with limited sexual reproduction, fewer have been conducted on clonal species with the potential for extensive sexual reproduction. We studied the relationship between the extent of sexual reproduction and genotype diversity. Although genotype diversity in F. rubra was double that reported by others for 27 nearly obligate clonal plant species, it was still quite low. For 25 populations studied, the mean number of genotypes was 5.5 (range = 1–15; SE = 0.964) and the average proportion of distinguishable genotypes was 0.38 (range = 0.03–1.00; SE = 0.07). The production of viable seed was quite variable among populations (mean proportion of viable seeds = 0.242; range = 0.002–0.565; SE = 0.04). Considering that some inflorescences can produce over 5,000 seeds, the potential for recruitment of sexually produced individuals is very large. No correlation was found between seed production and genotype diversity as was expected in a self-incompatible species in which one-third of the populations possessed a single genotype. It was hypothesized that the low genotype diversity found in numerous populations may be due to competition limiting recruitment of new seedlings.     

Genetic variation, its assessment and implications to the conservation of seagrasses

In a study of the widespread Australian endemic seagrass Posidonia australis , allozyme analysis identified a wide range in population genetic structure assessed using the multilocus genotype diversity statistic ( D _G). Values of D _G between zero and one were obtained; however, RAPD analysis generally detected higher levels of diversity, where D _G values were all greater than 0.5 ( D _G = 0.67 – 1). Some populations were allozymically monomorphic using allozyme analysis yet were highly polymorphic using RAPD analysis. The differences observed between methods, particularly among allozymically uniform populations, demonstrate the importance of choosing an appropriate method when assessing genotypic diversity. Different methods may reflect different historical aspects of population processes where allozymes reflect broader-scale gene flow and population establishment and DNA fingerprinting methods such as RAPDs may reflect fine-scale local recruitment events and shorter-term population processes. Using either method alone, particularly in genotypically depauperate organisms such as seagrasses and other clonal organisms, will be problematic in assessing their population genetic potential, a parameter being used by conservation managers to decide upon management strategies in rare and endangered organisms. It is recommended that the impact of disturbance assessed using genotypic diversity measures requires more than one technique to provide the most appropriate information for designing subsequent conservation strategies.     

Genetic diversity and population structure of Elytrigia pycnantha (Godr.) (Triticeae) in Mont Saint-Michel Bay using microsatellite markers

During the last decade, an invasive wheatgrass species (Elytrigia pycnantha) has colonized the low salt marshes of the Mont Saint Michel Bay resulting in an accelerated change in the vegetation. This study was conducted using microgeographical genetic diversity in order to understand the genetic structure of this invasive and clonal species. Genetic variation and population structure of fifteen populations collected in high and low marsh habitats around the Bay were analyzed using five microsatellite loci. Because E. pycnantha is an allohexaploid, the application of standard genetic diversity statistics was not possible; we chose to summarize genetic diversity using statistics calculated from banding phenotypes. The mean number of alleles per locus was 10.2, the mean number of different alleles per sample was 6.87. The mean number of allelic phenotypes across all populations was 7.21. The mean value of genetic diversity for the species, calculated as the average number of alleles by which pairs of individuals differ, was H's = 1.91 and H't = 2.04. Little genetic differentiation among populations was detected (0.067). The association between pairwise genetic differentiation and geographic distances exhibited no evidence for isolation by distance. A geographical pattern of population differentiation, where a single population GI was clearly separated from the remaining population groups (considered as a metapopulation), was revealed by principal component analysis (PCA), and we propose that this is because GI represents a new genotype.     

Genetic uniformity characterizes the invasive spread of water hyacinth (Eichhornia crassipes), a clonal aquatic plant

Aquatic plant invasions are often associated with long‐distance dispersal of vegetative propagules and prolific clonal reproduction. These reproductive features combined with genetic bottlenecks have the potential to severely limit genetic diversity in invasive populations. To investigate this question we conducted a global scale population genetic survey using amplified fragment length polymorphism markers of the world’s most successful aquatic plant invader –Eichhornia crassipes (water hyacinth). We sampled 1140 ramets from 54 populations from the native (South America) and introduced range (Asia, Africa, Europe, North America, Central America and the Caribbean). Although we detected 49 clones, introduced populations exhibited very low genetic diversity and little differentiation compared with those from the native range, and ～80% of introduced populations were composed of a single clone. A widespread clone (‘W’) detected in two Peruvian populations accounted for 70.9% of the individuals sampled and dominated in 74.5% of the introduced populations. However, samples from Bangladesh and Indonesia were composed of different genotypes, implicating multiple introductions to the introduced range. Nine of 47 introduced populations contained clonal diversity suggesting that sexual recruitment occurs in some invasive sites where environmental conditions favour seedling establishment. The global patterns of genetic diversity in E. crassipes likely result from severe genetic bottlenecks during colonization and prolific clonal propagation. The prevalence of the ‘W’ genotype throughout the invasive range may be explained by stochastic sampling, or possibly because of pre‐adaptation of the ‘W’ genotype to tolerate low temperatures.     

Long‐lived larch clones may conserve adaptations that could restrict treeline migration in northern Siberia

The occurrence of refugia beyond the arctic treeline and genetic adaptation therein play a crucial role of largely unknown effect size. While refugia have potential for rapidly colonizing the tundra under global warming, the taxa may be maladapted to the new environmental conditions. Understanding the genetic composition and age of refugia is thus crucial for predicting any migration response. Here, we genotype 194 larch individuals from an ~1.8 km² area in northcentral Siberia on the southern Taimyr Peninsula by applying an assay of 16 nuclear microsatellite markers. For estimating the age of clonal individuals, we counted tree rings at sections along branches to establish a lateral growth rate that was then combined with geographic distance. Findings reveal that the predominant reproduction type is clonal (58.76%) by short distance spreading of ramets. One outlier of clones 1 km apart could have been dispersed by reindeer. In clonal groups and within individuals, we find that somatic mutations accumulate with geographic distance. Clonal groups of two or more individuals are observed. Clonal age estimates regularly suggest individuals as old as 2,200 years, which coincides with a major environmental change that forced a treeline retreat in the region. We conclude that individuals with clonal growth mode were naturally selected as it lowers the likely risk of extinction under a harsh environment. We discuss this legacy from the past that might now be a maladaptation and hinder expansion under currently strongly increasing temperatures.     

珠芽蓼种群克隆多样性及克隆结构的初步研究

珠芽蓼(Polygonum viviparum)是青藏高原东缘广泛分布的克隆植物,具有有性和无性(根状茎和珠芽)两种生殖方式。该研究采用RAPD技术对分布于不同海拔的珠芽蓼7个自然种群进行了克隆结构和克隆多样性(是单克隆种群还是多克隆种群)以及克隆多样性与海拔因子之间的相关性研究,为了解高山克隆植物对环境的适应性策略及揭示克隆植物的繁殖和分布特点提供科学依据。研究结果表明:1)采用13条RAPD引物对珠芽蓼7个种群共140个样本进行扩增分析,共扩增到117个位点,其中多态性位点84个,多态位点百分率PPL达到71.79%,检测到43个基因型,且全部为局限型基因型;2)与Ellstrand和Roose(1987)总结的克隆植物的克隆多样性平均值相比(PD=0.17,D=0.62),珠芽蓼种群克隆多样性水平稍高,Simpson指数平均为0.639,基因型比率PD平均为0.307;3)克隆结构分析表明,珠芽蓼种群内克隆之间的镶嵌明显,这可能与珠芽蓼过渡型的克隆构型有关。研究中珠芽蓼种群的构型有游击型、密集型以及这两者之间的过渡类型;4)采用SPSS软件对珠芽蓼种群的克隆多样性与海拔高度进行相关性分析,结果显示它们之间并无明显的相关性。     

Clonal diversity of Clintonia udensis Trautv. et Mey. populations and its correlation with ecological factors

The clonal diversity of Clintonia udensis Trautv.et Mey.was detected by ISSR markers among 16 populations,and its correlation with ecological factors was analyzed as well in this work.Results showed that individuals(clonal ramets)per genotype were 1.12 and 1.149 at population and species levels,respectively,and that the 16 populations were all multiclonal.The detected genotypes were localized,without exception,within populations but demonstrated relatively high clonal differentiation among populations.The clonal diversity of the studied populations was high,with the average Simpson's index of 0.975,while the genets showed a clonal architecture of"guerilla".The population genetic diversities revealed by genet were consistent with those by ramet,further confirming their genetic differentiation among populations.And its genotype diversity within populations probably resulted largely from the frequent seedling regeneration and self-compatibility.In addition,the correlation analysis further revealed that,among the ecological factors,Simpson's index of C.udensis had a significant positive correlation(P<0.05)with pH values in the soil but not others.     

Spatial genotypical diversity of <Emphasis Type="Italic">Sesleria albicans</Emphasis> (Poaceae) in a dry grassland community

Spatial genotypical diversity of Sesleria albicans Kit. ex Schultes was studied in a dry grassland community by isozyme analysis. The aim was to identify the genetical individuals within the studied population and to assess the species’ clonal growth parameters. Vegetative mobility and branching intensity were measured in field for the sake of the correct interpretation of the results. Five isozyme systems were analyzed and interpreted (MDH, MNR, 6-PGDH, SkDH, LAP). Altogether, 98 distinct isozyme profiles were identified within an area of 2 m². Average genotype identity rapidly decreased with distance. Several very remote ramets (more than 1 m) belonging to the same genet were identified. The longest distance between ramets of the same genotype was 153 cm. With average annual rhizome increment of 13.59 mm assessed for the studied population the age of genet with the most remote ramets exceeded 56 years by a bi-directional growth and 112 years by a growth in single direction. Number of daughter tillers produced by a tiller per year was 0.67 (branching intensity, median) and the median of tiller life span was 2.5 years. The high genotypical diversity of S. albicans in the studied population could be a result of both, regular and continuous seed production with subsequent seedling recruitment and long life span of genets.     

GENOTYPE‐BY‐TEMPERATURE INTERACTIONS MAY HELP TO MAINTAIN CLONAL DIVERSITY IN ASTERIONELLA FORMOSA (BACILLARIOPHYCEAE)

Marine and freshwater phytoplankton populations often show large clonal diversity, which is in disagreement with clonal selection of the most vigorous genotype(s). Temporal fluctuation in selection pressures in variable environments is a leading explanation for maintenance of such genetic diversity. To test the influence of temperature as a selection force in continually (seasonally) changing aquatic systems we carried out reaction norms experiments on co‐occurring clonal genotypes of a ubiquitous diatom species, Asterionella formosa Hassall, across an environmentally relevant range of temperatures. We report within population genetic diversity and extensive diversity in genotype‐specific reaction norms in growth rates and cell size traits. Our results showed genotype by environment interactions, indicating that no genotype could outgrow all others across all temperature environments. Subsequently, we constructed a model to simulate the relative proportion of each genotype in a hypothetical population based on genotype and temperature‐specific population growth rates. This model was run with different seasonal temperature patterns. Our modeling exercise showed a succession of two to several genotypes becoming numerically dominant depending on the underlying temperature pattern. The results suggest that (temperature) context dependent fitness may contribute to the maintenance of genetic diversity in isolated populations of clonally reproducing microorganisms in temporally variable environments.     

青冈种群克隆多样性及其与环境因子的关系

 采用等位酶分析测定了6个青冈种群的克隆多样性。种群和种水平上每基因型含有的个体(克隆分株)数分别为1.270和1.508。青冈种群中克隆多样性较高,Simpson’s指数平均为0.9882,均匀度平均为0.7921。81.3％的基因型为局部分布,仅有1种(0.5％)基因型分布于75％以上的种群中。相关分析表明Simpson’s指数与总氮含量呈显著的负相关(P<0.05),与其他环境因子的相关性不显著。     

The importance of setting the right genetic distance threshold for identification of clones using amplified fragment length polymorphism: a case study with five species in the tropical plant genus Piper

Amplified fragment length polymorphism (AFLP) has been widely used for clone identification, but numerous studies have shown that clonemates do not always present identical AFLP fingerprints. Pairwise AFLP distances that distinguish known clones from nonclones have been used to identify a threshold genetic dissimilarity distance below which samples are considered to represent a single clone. Most studies to date have reported threshold values between 2% and 4%. Here, I determine the consistency of the clonal threshold across five species in the tropical plant genus Piper, and evaluate the sensitivity of genetic diversity indices and estimates of frequency of clonal reproduction to the threshold value selected. I sampled multiple ramets per individual from widely distributed plants for each of the five Piper species to set a threshold at the point where the error rate of clonal assignments was lowest. I then sampled all individuals of each shade‐tolerant species in a 1‐ha plot, and of each light‐demanding species in 25 × 35‐m plot, to estimate the frequency of asexual recruitment in natural populations using a series of different thresholds including the threshold set with the preliminary sampling. Clonal threshold values for the different species ranged from 0% to 5% AFLP genetic dissimilarity distance. To determine the sensitivity of estimates of clonal reproduction, I calculated several clonal diversity indexes for the natural populations of each of the five species guided by the range in clonal threshold values observed across the five Piper species. I show that small changes in the value of the clonal threshold can lead to very different conclusions regarding the level of clonal reproduction in natural populations.     

Tracing the geographic origin of the cosmopolitan parthenogenetic insect pest Liposcelis bostrychophila (Psocoptera: Liposcelididae)

The randomly amplified polymorphic DNA technique was used to trace the geographic origin of Liposcelis bostrychophila Badonnel populations in Australia from unknown geographic sources internationally. Haplotype (or clonal) diversity was high, with 474 unique haplotypes found from 616 individuals genotyped. Gene diversity estimates (0.10-0.28) and percent polymorphic loci (38.1-88.1%) were moderate to high for most populations. This resulted in genetic distance estimates that ranged from 0.04 to 0.26 and were significantly different for most pairwise population combinations. G ST values for all populations were also moderate (0.04-0.54) and again were significantly different for most pairwise population comparisons. Analysis of molecular variance revealed that the majority of variation was apportioned among individuals within populations regardless of the level at which they were grouped. Gene flow (Nm) was mostly low for all pairwise populations comparisons with an average Nm=1.8. A non-significant negative correlation between genetic distance and geographic distance was found for worldwide populations. In contrast, within Australian populations a significant positive correlation between genetic distance and geographic distance was detected. Genetic relationships explored using unweighted pair group method analysis and non-metric multidimensional scaling indicated a mixed pattern of genetic similarities among all populations. Multiple introductions, from a wide range of international source populations, have obscured the ability to accurately determine the geographic origin of L. bostrychophila in Australia.     

Evidence for a monophyletic origin of triploid clones of the Amazon molly, Poecilia formosa

Asexual reproduction in vertebrates is rare and generally considered an evolutionary dead end. Asexuality is often associated with polyploidy, and several hypotheses have been put forward to explain this relationship. So far, it remains unclear whether polyploidization in asexual organisms is a frequent or a rare event. Here we present a field study on the gynogenetic Amazon molly, Poecilia formosa. We used multilocus fingerprints and microsatellites to investigate the genetic diversity in 339 diploid and 55 triploid individuals and in 25 P. mexicana, its sexual host. Although multilocus DNA fingerprints found high clonal diversity in triploids, microsatellites revealed only two very similar clones in the triploids. Phylogenetic analysis of microsatellite data provided evidence for a monophyletic origin of the triploid clones of P. formosa. In addition, shared alleles within the triploid clones between the triploid and diploid genotypes and between asexual and sexual lineages indicate a recent origin of triploid clones in Poecilia formosa.