Fundamentals of fungal molecular population genetic analyses

The last two decades have seen tremendous growth in the development and application of molecular methods in the analyses of fungal species and populations. In this paper, I provide an overview of the molecular techniques and the basic analytical tools used to address various fundamental population and evolutionary genetic questions in fungi. With increasing availability and decreasing cost, DNA sequencing is becoming a mainstream data acquisition method in fungal evolutionary genetic studies. However, other methods, especially those based on the polymerase chain reaction, remain powerful in addressing specific questions for certain groups of taxa. These developments are bringing fungal population and evolutionary genetics into mainstream ecology and evolutionary biology.     

Applications and techniques for non-invasive faecal genetics research in felid conservation

Non-invasive genetic techniques utilising DNA extracted from faeces hold great promise for felid conservation research. These methods can be used to establish species distributions, model habitat requirements, analyse diet, estimate abundance and population density, and form the basis for population, landscape and conservation genetic analyses. Due to the elusive nature of most felid species, non-invasive genetic methods have the potential to provide valuable data that cannot be obtained with traditional observational or capture techniques. Thus, these methods are particularly valuable for research and conservation of endangered felid species. Here, we review recent studies that use non-invasive faecal genetic techniques to survey or study wild felids; provide an overview of field, laboratory and analysis techniques; and offer suggestions on how future non-invasive genetic studies can be expanded or improved to more effectively support conservation.     

On the independent gene trees assumption in phylogenomic studies

Multilocus coalescent methods for inferring species trees or historical demographic parameters typically require the assumption that gene trees for sampled SNPs or DNA sequence loci are conditionally independent given their species tree. In practice, researchers have used different criteria to delimit “independent loci.” One criterion identifies sampled loci as being independent of each other if they undergo Mendelian independent assortment (IA criterion). O'Neill et al. (2013, Molecular Ecology, 22, 111–129) used this approach in their phylogeographic study of North American tiger salamander species complex. In two other studies, researchers developed a pair of related methods that employ an independent genealogies criterion (IG criterion), which considers the effects of population‐level recombination on correlations between the gene trees of intrachromosomal loci. Here, I explain these three methods, illustrate their use with example data, and evaluate their efficacies. I show that the IA approach is more conservative, is simpler to use and requires fewer assumptions than the IG approaches. However, IG approaches can identify much larger numbers of independent loci than the IA method, which, in turn, allows researchers to obtain more precise and accurate estimates of species trees and historical demographic parameters. A disadvantage of the IG methods is that they require an estimate of the population recombination rate. Despite their drawbacks, IA and IG approaches provide molecular ecologists with promising a priori methods for selecting SNPs or DNA sequence loci that likely meet the independence assumption in coalescent‐based phylogenomic studies.     

Forensic DNA analysis for animal protection and biodiversity conservation: A review

The use of DNA analysis in forensic investigations into animal persecution and biodiversity conservation is now commonplace and crimes such as illegal collection/smuggling, poaching, and illegal trade of protected species are increasingly being investigated using DNA based evidence in many countries. Using DNA analysis, it is possible to identify the species and geographical origin (i.e. population) of a forensic sample, and to also individualise the sample with high levels of probability. Despite extensive literature in animal species, there is unfortunately a serious lack of information on plant species, with only a handful of recent studies. In this review, I detail the applications and diverse forensic investigations that have been carried out to date whilst also highlighting recent developmental studies which offer forensic potential for many species in the future.     

基于高通量测序的植物群体基因组学研究进展

作为群体遗传学一种新的表现形式,群体基因组学是将基因组概念和技术与群体遗传学理论体系相结合,通过覆盖全基因组范围内的多态位点的分布式样推测位点特异性效应和全基因组效应,从而提升人们对微进化的理解。近年来,随着第二代高通量测序技术的出现和改进,完成基因组测序的植物种类迅速增加,大规模的重测序也随之开展。与此同时,在一些尚未完成基因组测序的植物物种中,也开展了一些平行测序。这些重测序和平行测序极大地促进了群体基因组学的发展,加深了人们对相关植物种群在基因组水平上的遗传多样性、连锁不平衡水平、选择作用、群体历史及复杂性状的分子机理等群体基因组学方面的认识。本文简要介绍了群体基因组学的概念、研究方法等,重点综述了基于高通量测序的植物群体基因组学的研究动态,展望了植物群体基因组学的发展前景并讨论了存在的问题,以期为相关研究提供借鉴和参考。     

Ancient population genomics and the study of evolution

Recently, the study of ancient DNA (aDNA) has been greatly enhanced by the development of second-generation DNA sequencing technologies and targeted enrichment strategies. These developments have allowed the recovery of several complete ancient genomes, a result that would have been considered virtually impossible only a decade ago. Prior to these developments, aDNA research was largely focused on the recovery of short DNA sequences and their use in the study of phylogenetic relationships, molecular rates, species identification and population structure. However, it is now possible to sequence a large number of modern and ancient complete genomes from a single species and thereby study the genomic patterns of evolutionary change over time. Such a study would herald the beginnings of ancient population genomics and its use in the study of evolution. Species that are amenable to such large-scale studies warrant increased research effort. We report here progress on a population genomic study of the Adélie penguin (Pygoscelis adeliae). This species is ideally suited to ancient population genomic research because both modern and ancient samples are abundant in the permafrost conditions of Antarctica. This species will enable us to directly address many of the fundamental questions in ecology and evolution.     

Nuclear locus divergence at the early stages of speciation in the Orchard Oriole complex

As two lineages diverge from one another, mitochondrial DNA should evolve fixed differences more rapidly than nuclear DNA due to its smaller effective population size and faster mutation rate. As a consequence, molecular systematists have focused on the criteria of reciprocal monophyly in mitochondrial DNA for delimiting species boundaries. However, mitochondrial gene trees do not necessarily reflect the evolutionary history of the taxa in question, and even mitochondrial loci are not expected to be reciprocally monophyletic when the speciation event happened very recently. The goal of this study was to examine mitochondrial paraphyly within the Orchard Oriole complex, which is composed of Icterus spurius (Orchard Oriole) and Icterus fuertesi (Fuertes' Oriole). We increased the geographic sampling, added four nuclear loci, and used a range of population genetic and coalescent methods to examine the divergence between the taxa. With increased taxon sampling, we found evidence of clear structure between the taxa for mitochondrial DNA. However, nuclear loci showed little evidence of population structure, indicating a very recent divergence between I. spurius and I. fuertesi. Another goal was to examine the genetic variation within each taxon to look for evidence of a past founder event within the I. fuertesi lineage. Based on the high amounts of genetic variation for all nuclear loci, we found no evidence of such an event – thus, we found no support for the possible founding of I. fuertesi through a change in migratory behavior, followed by peripheral isolates speciation. Our results demonstrate that these two taxa are in the earliest stages of speciation, at a point when they have fixed differences in plumage color that are not reflected in monophyly of the mitochondrial or nuclear DNA markers in this study. This very recent divergence makes them ideal for continued studies of species boundaries and the earliest stages of speciation.     

Identifying species of moths (Lepidoptera) from Baihua Mountain,Beijing, China,using DNA barcodes

DNA barcoding has become a promising means for the identification of organisms of all life‐history stages. Currently, distance‐based and tree‐based methods are most widely used to define species boundaries and uncover cryptic species. However, there is no universal threshold of genetic distance values that can be used to distinguish taxonomic groups. Alternatively, DNA barcoding can deploy a “character‐based” method, whereby species are identified through the discrete nucleotide substitutions. Our research focuses on the delimitation of moth species using DNA‐barcoding methods. We analyzed 393 Lepidopteran specimens belonging to 80 morphologically recognized species with a standard cytochrome c oxidase subunit I (COI) sequencing approach, and deployed tree‐based, distance‐based, and diagnostic character‐based methods to identify the taxa. The tree‐based method divided the 393 specimens into 79 taxa (species), and the distance‐based method divided them into 84 taxa (species). Although the diagnostic character‐based method found only 39 so‐identifiable species in the 80 species, with a reduction in sample size the accuracy rate substantially improved. For example, in the Arctiidae subset, all 12 species had diagnostics characteristics. Compared with traditional morphological method, molecular taxonomy performed well. All three methods enable the rapid delimitation of species, although they have different characteristics and different strengths. The tree‐based and distance‐based methods can be used for accurate species identification and biodiversity studies in large data sets, while the character‐based method performs well in small data sets and can also be used as the foundation of species‐specific biochips.     

大中型兽类种群数量估算的研究进展

大中型兽类种群数量的估算是动物生态学中重要的基本问题, 受到研究者、管理者和公众的共同关注。国际上从20世纪中期开始研究该问题, 已出现了多种研究方法和相应案例, 且还在快速发展, 但世界各地仍有很多物种的种群数量尚未知晓。在我国, 从20世纪80年代开始调查大中型兽类种群数量, 取得了重要进展, 也还有很多物种的种群数量尚不清楚。因此, 有必要归纳国际上种群数量估算的研究进展, 同时, 总结国内研究的现状、优势和趋势, 供研究者参考。本文首先选择估算大中型兽类种群数量的原理、数据来源和模型这3个要素归纳出简明的研究框架, 将现有的多种方法置于其中予以阐述。在该框架下, 根据估算原理分为4大类方法, 为距离取样法、标志重捕法、基于遇见率法和遥感影像直接计数法。针对每一大类方法, 论述其基本原理模型和模型假设, 说明能实现该原理的相应数据来源(视觉观测、红外相机拍摄、DNA微卫星识别、卫星定位跟踪、声音监测或遥感影像)的特点及如何实现该原理, 评价其适用性及优缺点, 并选择其中具有可比性的方法予以比较评价。其次, 参照该研究框架, 总结我国的研究现状, 分析未来发展的优势和趋势: 我国的红外相机数据积累充分, 可以发展以此为数据源的距离取样法、标志重捕法和基于遇见率法; 发展以粪便样品为数据来源的距离取样法和粪便DNA标志重捕法; 相比地面调查数据, 获取高分辨率遥感影像数据更容易, 尽量以此估算符合适用条件的大中型兽类的种群数量。最后, 本文提出了适用于我国大中型兽类种群数量的估算方法的选择流程, 供研究者参考。     

Mitochondrial COI gene transfers to the nuclear genome of Dendroctonus valens and its implications

Mitochondrial gene transfer to the nuclear genome could affect the accuracy of results in population genetics and evolutionary studies using mitochondrial gene markers. In a population genetics study of the red turpentine beetle (Dendroctonus valens), an invasive species in China, we found numerous ambiguous sites existing in the Cytochrome Oxidase I (COI) gene sequences obtained directly from polymerase chain reaction (PCR) products amplified from total genomic DNA using universal primers. By comparing the profiles of restriction endonuclease digestions and the sequences of PCR products amplified from mitochondrial DNA and nuclear DNA of the same individuals, we confirmed it was a phenomenon of mitochondrial gene transfer to the nuclear genome. Large numbers of COI pseudogenes were detected in this species. According to different levels of condon position bias and phylogenetic analysis, these should have originated from independent integration events. The impact of nuclear mitochondrial DNA sequences on population genetics analyses was discussed.     

The habitat sampling and analysis paradigm has limited value in animal conservation: A prequel

In this essay, I make the case that our studies of wildlife and habitat are largely decoupled from any meaningful relationship to the distribution of the study species. The field that we broadly classify as wildlife–habitat relationships is characterized by an increasing number of studies that gather additional data on phenomena that are already well studied. I offer that unless we make changes to the fundamental aspect of study design, our studies will fail to advance conservation of species. The current habitat sampling and analysis paradigm involves identification of a convenient study area, drawing samples from the usual list of parameters, conducting a series of statistical analyses, comparing findings to other studies, and justifying publication by extrapolating findings to some unspecified larger area. Recommendations for management are usually vague and are seldom tested for efficacy. Most of our habitat studies have little relevance to the target species with regard to viability. Attempts to translate the “best scientific information” into a set of management guidelines for a species produce one size fits all documents. I describe how we usually compromise our studies well before data collection by failing to establish a cogent framework for sampling from an ecologically meaningful unit of a population, but rather sample based on funding priorities and convenience. Specifying the sampling universe for a species sets the stage for properly establishing the sampling frame. Although we always have a target population, that target is often the result of personal, political, or administrative interest, but has little to do with biological reality. I review various intraspecies levels that could be a focus for study, including subspecies and especially ecotypes. Although making assumptions about our study species and habitat parameters is a necessary step, carrying forward untested assumptions from previous studies and failing to test new ones substantially negates the application of research results to meaningful management actions. I include recommendations for enhancing studies of wildlife and habitat with the intent of altering the current norm of wildlife–habitat studies. © 2012 The Wildlife Society.     

Incomplete estimates of genetic diversity within species: Implications for DNA barcoding

DNA barcoding has greatly accelerated the pace of specimen identification to the species level, as well as species delineation. Whereas the application of DNA barcoding to the matching of unknown specimens to known species is straightforward, its use for species delimitation is more controversial, as species discovery hinges critically on present levels of haplotype diversity, as well as patterning of standing genetic variation that exists within and between species. Typical sample sizes for molecular biodiversity assessment using DNA barcodes range from 5 to 10 individuals per species. However, required levels that are necessary to fully gauge haplotype variation at the species level are presumed to be strongly taxon‐specific. Importantly, little attention has been paid to determining appropriate specimen sample sizes that are necessary to reveal the majority of intraspecific haplotype variation within any one species. In this paper, we present a brief outline of the current literature and methods on intraspecific sample size estimation for the assessment of COI DNA barcode haplotype sampling completeness. The importance of adequate sample sizes for studies of molecular biodiversity is stressed, with application to a variety of metazoan taxa, through reviewing foundational statistical and population genetic models, with specific application to ray‐finned fishes (Chordata: Actinopterygii). Finally, promising avenues for further research in this area are highlighted.     

A non-invasive technique for rapid extraction of DNA from fish scales

DNA markers are being increasingly used in studies related to population genetics and conservation biology of endangered species. DNA isolation for such studies requires a source of biological material that is easy to collect, non-bulky and reliable. Further, the sampling strategies based on non-invasive procedures are desirable, especially for the endangered fish species. In view of above, a rapid DNA extraction method from fish scales has been developed with the use of a modified lysis buffer that require about 2 hr duration. This methodology is non-invasive, less expensive and reproducible with high efficiency of DNA recovery. The DNA extracted by this technique, have been found suitable for performing restriction enzyme digestion and PCR amplification. Therefore, the present DNA extraction procedure can be used as an alternative technique in population genetic studies pertaining to endangered fish species. The technique was also found equally effective for DNA isolation from fresh, dried and ethanol preserved scales.     

Perspective: gene divergence, population divergence, and the variance in coalescence time in phylogeographic studies

Molecular methods as applied to the biogeography of single species (phylogeography) or multiple codistributed species (comparative phylogeography) have been productively and extensively used to elucidate common historical features in the diversification of the Earth's biota. However, only recently have methods for estimating population divergence times or their confidence limits while taking into account the critical effects of genetic polymorphism in ancestral species become available, and earlier methods for doing so are underutilized. We review models that address the crucial distinction between the gene divergence, the parameter that is typically recovered in molecular phylogeographic studies, and the population divergence, which is in most cases the parameter of interest and will almost always postdate the gene divergence. Assuming that population sizes of ancestral species are distributed similarly to those of extant species, we show that phylogeographic studies in vertebrates suggest that divergence of alleles in ancestral species can comprise from less than 10% to over 50% of the total divergence between sister species, suggesting that the problem of ancestral polymorphism in dating population divergence can be substantial. The variance in the number of substitutions (among loci for a given species or among species for a given gene) resulting from the stochastic nature of DNA change is generally smaller than the variance due to substitutions along allelic lines whose coalescence times vary due to genetic drift in the ancestral population. Whereas the former variance can be reduced by further DNA sequencing at a single locus, the latter cannot. Contrary to phylogeographic intuition, dating population divergence times when allelic lines have achieved reciprocal monophyly is in some ways more challenging than when allelic lines have not achieved monophyly, because in the former case critical data on ancestral population size provided by residual ancestral polymorphism is lost. In the former case differences in coalescence time between species pairs can in principle be explained entirely by differences in ancestral population size without resorting to explanations involving differences in divergence time. Furthermore, the confidence limits on population divergence times are severely underestimated when those for number of substitutions per site in the DNA sequences examined are used as a proxy. This uncertainty highlights the importance of multilocus data in estimating population divergence times; multilocus data can in principle distinguish differences in coalescence time (T) resulting from differences in population divergence time and differences in T due to differences in ancestral population sizes and will reduce the confidence limits on the estimates. We analyze the contribution of ancestral population size (theta) to T and the effect of uncertainty in theta on estimates of population divergence (tau) for single loci under reciprocal monophyly using a simple Bayesian extension of Takahata and Satta's and Yang's recent coalescent methods. The confidence limits on tau decrease when the range over which ancestral population size theta is assumed to be distributed decreases and when tau increases; they generally exclude zero when tau/(4Ne) > 1. We also apply a maximum-likelihood method to several single and multilocus data sets. With multilocus data, the criterion for excluding tau = 0 is roughly that l tau/(4Ne) > 1, where l is the number of loci. Our analyses corroborate recent suggestions that increasing the number of loci is critical to decreasing the uncertainty in estimates of population divergence time.     

Chloroplast phylogeography of Iris dichotoma (Iridaceae), a widespread herbaceous species in East Asia

Both a complex topography and climate change have huge impacts on the distribution and genetic structure of extant species. Due to the lack of relevant molecular research, little is definitively known about the phylogeography of herbaceous plants in East Asia. Here we investigate the genetic diversity, population structure and historical population dynamics of Iris dichotoma Pallas, a widespread perennial herbaceous species in northeastern and northern China. Twenty-nine populations, totalling 297 individuals, were sampled throughout the Chinese distributional range of I. dichotoma. The combined sequences of six chloroplast DNA fragments (petA-psbE, rps18-clpp, psbJ-petA, trnD-trnT, rps16 and ndhA) were used to identify 13 haplotypes, of which six were private ones restricted in a single population. Genetic differentiation among I. dichotoma populations, enabled us to infer potential refugia during the glacial period in the Yinshan Mountains–Yanshan Mountains, where high levels of haplotype and nucleotide diversity were detected. The results of a neutral test and mismatch distribution analysis both indicated that I. dichotoma underwent a recent population expansion. In East Asia, postglacial environmental and climatic changes appear to have promoted genetic diversification not only in better-studied woody species, but also in herbaceous ones like I. dichotoma. Future studies of more herbaceous plant species are needed to obtain better insight into how modern temperate biodiversity has developed in East Asia.     

PCR-based assays of mendelian polymorphisms from anonymous single-copy nuclear DNA: techniques and applications for population genetics

This paper outlines a PCR-based approach for population genetics that offers several advantages over conventional Southern blotting methods for revealing restriction-fragment-length polymorphisms (RFLPs) in nuclear DNA. Primers are constructed from clones isolated from a nuclear DNA library, and these primers subsequently are employed in in vitro syntheses of homologous regions. Amplified products are then screened directly for RFLPs by using gel-staining procedures. Population applications for this PCR-based approach, including potential strengths and weaknesses, are exemplified by two RFLP data sets generated to estimate (a) male-mediated gene flow in the green turtle (Chelonia mydas) and (b) geographic population genetic structure in the American oyster (Crassostrea virginica). Restriction assays of amplified products from 14 or 15 independent primer pairs in each species revealed polymorphisms at several loci that proved highly informative in the population genetic analyses. In general, the Mendelian polymorphisms produced by this PCR-based approach will provide useful genetic markers for population studies, particularly in situations where simpler and less expensive allozyme methods have failed, for whatever reason, to provide adequate information.      

Classification of Nucleotide Sequences Using Support Vector Machines

Species identification is one of the most important issues in biological studies. Due to recent increases in the amount of genomic information available and the development of DNA sequencing technologies, the applicability of using DNA sequences to identify species (commonly referred to as “DNA barcoding”) is being tested in many areas. Several methods have been suggested to identify species using DNA sequences, including similarity scores, analysis of phylogenetic and population genetic information, and detection of species-specific sequence patterns. Although these methods have demonstrated good performance under a range of circumstances, they also have limitations, as they are subject to loss of information, require intensive computation and are sensitive to model mis-specification, and can be difficult to evaluate in terms of the significance of identification. Here, we suggest a new DNA barcoding method in which support vector machine (SVM) procedures are adopted. Our new method is nonparametric and thus is expected to be robust for a wide range of evolutionary scenarios as well as multilocus analyses. Furthermore, we describe bootstrap procedures that can be used to test the significances of species identifications. We implemented a novel conversion technique for transforming sequence data to real-valued vectors, and therefore, bootstrap procedures can be easily combined with our SVM approach. In this study, we present the results of simulation studies and empirical data analyses to demonstrate the performance of our method and discuss its properties.     

Nuclei purified from cauliflower mosaic virus-infected turnip leaves contain subgenomic, covalently closed circular cauliflower mosaic virus DNAs

Nuclei isolated from cauliflower mosaic virus (CaMV) infected turnip leaves contain subgenomic CaMV DNA species in addition to the genome length CaMV DNA. These subgenomic CaMV DNA species are present as covalently closed circles (form I), relaxed circles (form II) and linear (form III) molecules. The subgenomic form I DNA species range in size from about 10% of genome length to nearly genome length. These subgenomic DNA species appear in tissue infected with cloned CaMV DNA, indicating that they arise rapidly and have not accumulated in the virus population from serial propagation of CaMV. No specific region of the CaMV genome appears to be preferentially deleted to form the subgenomic CaMV DNA species. At least three distinct subgenomic species appear to accumulate preferentially in nuclei isolated from infected tissue. Two of these abundant subgenomic CaMV DNA species are form I and the other one is form III. Some of the subgenomic CaMV DNA species appear to be minichromosomes.     

Molecular Ecology of Rotifers: From Population Differentiation to Speciation

The development of cost-effective molecular tools allowing the amplification of minute amounts of DNA effectively opened the field of molecular ecology for rotifers. Here I review these techniques and the advances they have provided in the understanding of sibling species complexes, clonal structure, resting egg banks, population structure, phylogeographic patterns and phylogenetic relationships in rotifers. Most of the research to date has focused on the rotifer species complex Brachionus plicatilis. The use of DNA sequence and microsatellite variation, in the context of the background knowledge of life history, mating behaviour, and temporal population dynamics in these organisms have revolutionised our views into the processes shaping the genetic diversity in aquatic invertebrates. Rotifers have populations with a very high number of clones in genetic equilibrium. In temporary populations clonal selection is effective in eroding the number of clones. Rotifer populations are strongly differentiated genetically for neutral markers, even at small geographical scales, and exhibit deep phylogeographic structure which might reflect the impact of Pleistocene glaciations. Despite the high potential for dispersal afforded by resting eggs, rotifers display persistent historical colonisation effects, with gene flow effective only at a local scale and with marked isolation by distance. Instances of long-distance transcontinental migration resulting in successful colonisation have also been revealed. B. plicatilis is composed of a group of several ancient species and sympatry is common. Despite this, the presence of cosmopolitan species in this species complex cannot be discounted. I discuss future priorities and point out the main areas where our knowledge is still insufficient.     

The challenge of measuring trade-offs in human life history research

Life history theory has become a prominent framework in the evolutionary social sciences, and the concept of trade-offs, the cornerstone of life history theory in studies on non-human taxa, has likewise been widely adopted. Yet, human life history research often assumes trade-offs without demonstrating them. This is not surprising given the practical difficulties in measuring trade-offs in long-lived animals, like humans. Four main methods are used to demonstrate trade-offs: phenotypic correlations, experimental manipulations, genetic correlations and correlated responses to selection. Here, I discuss challenges with these methods along with potential solutions. For example, individual heterogeneity within a population in quality or access to resources can mask underling trade-offs, and this can be accounted for by careful experimental manipulation or proper statistical treatment of observational data. In general, trade-offs have proven more difficult than expected to measure, and evidence across species is mixed, but strong evidence exists in some cases. I use the key trade-off between reproduction and survival to exemplify methods, challenges and solutions, and review the mixed evidence for a cost of reproduction in humans. I conclude by providing directions for future research. Promising avenues are opening thanks to recent advances in quantitative genetic and genomic methods coupled with the availability of high-quality large-scale datasets on humans from different populations, allowing the study of the evolutionary implications of life history trade-offs in humans.