The second internal transcribed spacer of nuclear ribosomal DNA as a tool for Latin American anopheline taxonomy - a critical review

Among the molecular markers commonly used for mosquito taxonomy, the internal transcribed spacer 2 (ITS2) of the ribosomal DNA is useful for distinguishing among closely-related species. Here we review 178 GenBank accession numbers matching ITS2 sequences of Latin American anophelines. Among those, we found 105 unique sequences corresponding to 35 species. Overall the ITS2 sequences distinguish anopheline species, however, information on intraspecific and geographic variations is scarce. Intraspecific variations ranged from 0.2% to 19% and our analysis indicates that misidentification and/or sequencing errors could be responsible for some of the high values of divergence. Research in Latin American malaria vector taxonomy profited from molecular data provided by single or few field capture mosquitoes. However we propose that caution should be taken and minimum requirements considered in the design of additional studies. Future studies in this field should consider that: (1) voucher specimens, assigned to the DNA sequences, need to be deposited in collections, (2) intraspecific variations should be thoroughly evaluated, (3) ITS2 and other molecular markers, considered as a group, will provide more reliable information, (4) biological data about vector populations are missing and should be prioritized, (5) the molecular markers are most powerful when coupled with traditional taxonomic tools.     

DNA barcoding in Tardigrada: the first case study on Macrobiotus macrocalix Bertolani & Rebecchi 1993 (Eutardigrada, Macrobiotidae)

Morphological and molecular studies on a tardigrade species have been carried out to verify the possibility of using a DNA barcoding approach for species identification in this phylum. Macrobiotus macrocalix Bertolani & Rebecchi, 1993 was chosen as the test species since it belongs to a group of species in which the taxonomy is quite problematic. Animals and eggs belonging to three Italian and one Swedish populations have been investigated. Both morphological and molecular analyses show that all the populations belong to the same species. The low genetic distances recorded among the studied populations (0.3-1.0%) and the high genetic distance (15.9-16.3%) between these populations and a closely related species confirm the possibility of identifying a specimen of this species by its cytochrome oxidase subunit I sequence. Data from other authors support our results indicating that DNA barcoding can be applied to tardigrades. With our protocols, we have obtained voucher specimens that enable us to show a correspondence between morphology and molecular data.     

Combining multiple autosomal introns for studying shallow phylogeny and taxonomy of Laurasiatherian mammals: Application to the tribe Bovini (Cetartiodactyla,Bovidae)

Mitochondrial sequences are widely used for species identification and for studying phylogenetic relationships among closely related species or populations of the same species. However, many studies of mammals have shown that the maternal history of the mitochondrial genome can be discordant with the true evolutionary history of the taxa. In such cases, the analyses of multiple nuclear genes can be more powerful for deciphering interspecific relationships.Here, we designed primers for amplifying 13 new exon-primed intron-crossing (EPIC) autosomal loci for studying shallow phylogeny and taxonomy of Laurasiatherian mammals. Three criteria were used for the selection of the markers: gene orthology, a PCR product length between 600 and 1200 nucleotides, and different chromosomal locations in the bovine genome. Positive PCRs were obtained from different species representing the orders Carnivora, Cetartiodactyla, Chiroptera, Perissodactyla and Pholidota.The newly developed markers were analyzed in a phylogenetic study of the tribe Bovini (the group containing domestic and wild cattle, bison, yak, African buffalo, Asian buffalo, and saola) based on 17 taxa and 18 nuclear genes, representing a total alignment of 13,095 nucleotides. The phylogenetic results were compared to those obtained from analyses of the complete mitochondrial genome and Y chromosomal genes. Our analyses support a basal divergence of the saola (Pseudoryx) and a sister-group relationship between yak and bison. These results contrast with recent molecular studies but are in better agreement with morphology. The comparison of pairwise nucleotide distances shows that our nuDNA dataset provides a good signal for identifying taxonomic levels, such as species, genera, subtribes, tribes and subfamilies, whereas the mtDNA genome fails because of mtDNA introgression and higher levels of homoplasy. Accordingly, we conclude that the genus Bison should be regarded as a synonym of Bos, with the European bison relegated to a subspecies rank within Bos bison. We compared our molecular dating estimates to the fossil record in order to propose a biogeographic scenario for the evolution of Bovini during the Neogene.     

DNA Barcoding to Improve the Taxonomy of the Afrotropical Hoverflies (Insecta: Diptera: Syrphidae)

The identification of Afrotropical hoverflies is very difficult because of limited recent taxonomic revisions and the lack of comprehensive identification keys. In order to assist in their identification, and to improve the taxonomy of this group, we constructed a reference dataset of 513 COI barcodes of 90 of the more common nominal species from Ghana, Togo, Benin and Nigeria (W Africa) and added ten publically available COI barcodes from nine nominal Afrotropical species to this (total: 523 COI barcodes; 98 nominal species; 26 genera). The identification accuracy of this dataset was evaluated with three methods (K2P distance-based, Neighbor-Joining (NJ) / Maximum Likelihood (ML) analysis, and using SpeciesIdentifier). Results of the three methods were highly congruent and showed a high identification success. Nine species pairs showed a low (< 0.03) mean interspecific K2P distance that resulted in several incorrect identifications. A high (> 0.03) maximum intraspecific K2P distance was observed in eight species and barcodes of these species not always formed single clusters in the NJ / ML analayses which may indicate the occurrence of cryptic species. Optimal K2P thresholds to differentiate intra- from interspecific K2P divergence were highly different among the three subfamilies (Eristalinae: 0.037, Syrphinae: 0.06, Microdontinae: 0.007–0.02), and among the different general suggesting that optimal thresholds are better defined at the genus level. In addition to providing an alternative identification tool, our study indicates that DNA barcoding improves the taxonomy of Afrotropical hoverflies by selecting (groups of) taxa that deserve further taxonomic study, and by attributing the unknown sex to species for which only one of the sexes is known.     

基于线粒体CO 1基因序列的DNA条形码在鲤科鲌属鱼类物种鉴定中的应用

本研究探讨了线粒体CO1基因作为DNA条形码对鲌属鱼类进行物种鉴定的可行性。研究中获得了鲌属4种鱼类共32个个体长度为816bp的CO1基因序列。利用MEGA软件计算鲌属鱼类种间及种内遗传距离,利用邻接法、最大简约法、最大似然法和Bayesian方法分别构建分子系统树。结果显示,鲌属鱼类的种间遗传距离显著大于种内遗传距离。在系统树中,鲌属鱼类每一物种的个体分别形成各自独立的分支。基于CO1基因的DNA条形码在识别鲌属鱼类物种方面和传统形态学基本一致,而且该基因可以探讨鲌属鱼类种间的系统发育关系。本研究表明以CO1基因作为鲌属鱼类DNA条形码进行物种鉴定具有一定的可行性。     

VIP Barcoding: composition vector‐based software for rapid species identification based on DNA barcoding

Species identification based on short sequences of DNA markers, that is, DNA barcoding, has emerged as an integral part of modern taxonomy. However, software for the analysis of large and multilocus barcoding data sets is scarce. The Basic Local Alignment Search Tool (BLAST) is currently the fastest tool capable of handling large databases (e.g. >5000 sequences), but its accuracy is a concern and has been criticized for its local optimization. However, current more accurate software requires sequence alignment or complex calculations, which are time‐consuming when dealing with large data sets during data preprocessing or during the search stage. Therefore, it is imperative to develop a practical program for both accurate and scalable species identification for DNA barcoding. In this context, we present VIP Barcoding: a user‐friendly software in graphical user interface for rapid DNA barcoding. It adopts a hybrid, two‐stage algorithm. First, an alignment‐free composition vector (CV) method is utilized to reduce searching space by screening a reference database. The alignment‐based K2P distance nearest‐neighbour method is then employed to analyse the smaller data set generated in the first stage. In comparison with other software, we demonstrate that VIP Barcoding has (i) higher accuracy than Blastn and several alignment‐free methods and (ii) higher scalability than alignment‐based distance methods and character‐based methods. These results suggest that this platform is able to deal with both large‐scale and multilocus barcoding data with accuracy and can contribute to DNA barcoding for modern taxonomy. VIP Barcoding is free and available at http://msl.sls.cuhk.edu.hk/vipbarcoding/ .     

Cosmopolitan versus cryptic meiofaunal polychaete species: an approach to a molecular taxonomy

Polychaete taxonomy is characterised by a high number of apparently cosmopolitan species. Detection of subtle but diagnostic ultrastructural differences and – in recent years – investigations at the molecular level have revealed that many of these "species" are actually complexes of morphologically identical or almost identical cryptic species. To disregard their existence would lead to an underestimation of global meiofauna diversity and undermine the value of many scientific studies. Therefore, we strongly recommend that they be given formal taxonomic recognition, beyond their published presentation as "operational taxonomic units", "types" or by alphabetic or numerical designators. Since there are neither generally accepted practical procedures nor any established consensus regarding the application of genetic data in taxonomy, we here provide examples of, and suggestions for, the treatment of meiofaunal species that are distinguished exclusively by molecular data, e.g. by genetic distance values, cluster analyses, diagnostic (= autapomorphic) DNA fragments from DNA fingerprinting procedures (RAPD) and/or DNA sequence differences (e.g. of ITS 2). Although no holotype material may be available because the molecular procedures require the preparation of entire specimens, practical taxonomic problems can be overcome and the recommendations of the Zoological Code of Nomenclature satisfied, by adopting the following procedures: (1) deposition of band-patterns of an individual obtained with the primers used to find diagnostic markers; (2) deposition of DNA in ethanol of one syntype individual; (3) deposition of fixed specimens (syntypes) from the locus typicus. Electronic Publication     

Testing the reliability of genetic methods of species identification via simulation

Although genetic methods of species identification, especially DNA barcoding, are strongly debated, tests of these methods have been restricted to a few empirical cases for pragmatic reasons. Here we use simulation to test the performance of methods based on sequence comparison (BLAST and genetic distance) and tree topology over a wide range of evolutionary scenarios. Sequences were simulated on a range of gene trees spanning almost three orders of magnitude in tree depth and in coalescent depth; that is, deep or shallow trees with deep or shallow coalescences. When the query's conspecific sequences were included in the reference alignment, the rate of positive identification was related to the degree to which different species were genetically differentiated. The BLAST, distance, and liberal tree-based methods returned higher rates of correct identification than did the strict tree-based requirement that the query was within, but not sister to, a single-species clade. Under this more conservative approach, ambiguous outcomes occurred in inverse proportion to the number of reference sequences per species. When the query's conspecific sequences were not in the reference alignment, only the strict tree-based approach was relatively immune to making false-positive identifications. Thresholds affected the rates at which false-positive identifications were made when the query's species was unrepresented in the reference alignment but did not otherwise influence outcomes. A conservative approach using the strict tree-based method should be used initially in large-scale identification systems, with effort made to maximize sequence sampling within species. Once the genetic variation within a taxonomic group is well characterized and the taxonomy resolved, then the choice of method used should be dictated by considerations of computational efficiency. The requirement for extensive genetic sampling may render these techniques inappropriate in some circumstances.     

Phylogenetic Inference with Weighted Codon Evolutionary Distances

We develop a new approach to estimate a matrix of pairwise evolutionary distances from a codon-based alignment based on a codon evolutionary model. The method first computes a standard distance matrix for each of the three codon positions. Then these three distance matrices are weighted according to an estimate of the global evolutionary rate of each codon position and averaged into a unique distance matrix. Using a large set of both real and simulated codon-based alignments of nucleotide sequences, we show that this approach leads to distance matrices that have a significantly better treelikeness compared to those obtained by standard nucleotide evolutionary distances. We also propose an alternative weighting to eliminate the part of the noise often associated with some codon positions, particularly the third position, which is known to induce a fast evolutionary rate. Simulation results show that fast distance-based tree reconstruction algorithms on distance matrices based on this codon position weighting can lead to phylogenetic trees that are at least as accurate as, if not better, than those inferred by maximum likelihood. Finally, a well-known multigene dataset composed of eight yeast species and 106 codon-based alignments is reanalyzed and shows that our codon evolutionary distances allow building a phylogenetic tree which is similar to those obtained by non-distance-based methods (e.g., maximum parsimony and maximum likelihood) and also significantly improved compared to standard nucleotide evolutionary distance estimates.     

Molecular systematics,phylogeography, and the effects of Pleistocene glaciation in the painted turtle (Chrysemys picta) complex

Abstract.— The painted turtle, Chrysemys picta , is currently recognized as a continentally distributed polytypic species, ranging across North America from southern Canada to extreme northern Mexico. We analyzed variation in the rapidly evolving mitochondrial control region (CR) in 241 turtles from 117 localities across this range to examine whether the painted turtle represents a continentally distributed species based on molecular analysis. We found strong support for the novel hypothesis that C. p. dorsalis is the sister group to all remaining Chrysemys , with the remaining Chrysemys falling into a single, extremely wide-ranging and genetically undifferentiated species. Given our goal of an evolu-tionarily accurate taxonomy, we propose that two evolutionary lineages be recognized as species within Chrysemys : C. dorsalis (Agassiz 1857) in the southern Mississippi drainage region, and C. picta (Schneider 1783) from the rest of the range of the genus. Neither molecular nor recent morphological analyses argue for the hybrid origin of C. p. marginata as previously proposed. Within C. picta , we find evidence of at least two independent range expansions into previously glaciated regions of North America, one into New England and the other into the upper Midwest. We further find evidence of a massive extinction/recolonization event across the Great Plains/Rocky Mountain region encompassing over half the continental United States. The timing and extent of this colonization is consistent with a recently proposed regional aridification as the Laurentide ice sheets receded approximately 14,000 years ago, and we tentatively propose this paleoclimatological event as a major factor shaping genetic variation in Chrysemys .     

A comprehensive and validated molecular taxonomy of beaked whales, family Ziphiidae

DNA sequences from orthologous loci can provide universal characters for taxonomic identification. Molecular taxonomy is of particular value for groups in which distinctive morphological features are difficult to observe or compare. To assist in species identification for the little known family Ziphiidae (beaked whales), we compiled a reference database of mitochondrial DNA (mtDNA) control region (437 bp) and cytochrome b (384 bp) sequences for all 21 described species in this group. This mtDNA database is complemented by a nuclear database of actin intron sequences (925 bp) for 17 of the 21 species. All reference sequences were derived from specimens validated by diagnostic skeletal material or other documentation, and included four holotypes. Phylogenetic analyses of mtDNA sequences confirmed the genetic distinctiveness of all beaked whale species currently recognized. Both mitochondrial loci were well suited for species identification, with reference sequences for all known ziphiids forming robust species-specific clades in phylogenetic reconstructions. The majority of species were also distinguished by nuclear alleles. Phylogenetic comparison of sequence data from "test" specimens to these reference databases resulted in three major taxonomic discoveries involving animals previously misclassified from morphology. Based on our experience with this family and the order Cetacea as a whole, we suggest that a molecular taxonomy should consider the following components: comprehensiveness, validation, locus sensitivity, genetic distinctiveness and exclusivity, concordance, and universal accessibility and curation.     

The power and perils of 'molecular taxonomy': a case study of eyeless and endangered Cicurina (Araneae: Dictynidae) from Texas caves

Rapid development in karst-rich regions of the US state of Texas has prompted the listing of four Cicurina species (Araneae, Dictynidae) as US Federally Endangered. A major constraint in the management of these taxa is the extreme rarity of adult specimens, which are required for accurate species identification. We report a first attempt at using mitochondrial DNA (mtDNA) sequences to accurately identify immature Cicurina specimens. This identification is founded on a phylogenetic framework that is anchored by identified adult and/or topotypic specimens. Analysis of approximately 1 kb of cytochrome oxidase subunit I (CO1) mtDNA data for over 100 samples results in a phylogenetic tree that includes a large number of distinctive, easily recognizable, tip clades. These tip clades almost always correspond to a priori species hypotheses, and show nonoverlapping patterns of sequence divergence, making it possible to place species names on a number of immature specimens. Three cases of inconsistency between recovered tip clades and a priori species hypotheses suggest possible introgression between cave-dwelling Cicurina, or alternatively, species synonymy. Although species determination is not possible in these instances, the inconsistencies point to areas of taxonomic ambiguity that require further study. Our molecular phylogenetic sample is largest for the Federally Endangered C. madla. These data suggest that C. madla occurs in more than twice the number of caves as previously reported, and indicate the possible synonymy of C. madla with C. vespera, which is also Federally Endangered. Network analyses reveal considerable genetic divergence and structuring across caves in this species. Although the use of DNA sequences to identify previously 'unidentifiable' specimens illustrates the potential power of molecular data in taxonomy, many other aspects of the same dataset speak to the necessity of a balanced taxonomic approach.     

Potential of MALDI-TOF MS-based proteomic fingerprinting for species identification of Cnidaria across classes,species, regions and developmental stages

Morphological identification of cnidarian species can be difficult throughout all life stages due to the lack of distinct morphological characters. Moreover, in some cnidarian taxa genetic markers are not fully informative, and in these cases combinations of different markers or additional morphological verifications may be required. Proteomic fingerprinting based on MALDI-TOF mass spectra was previously shown to provide reliable species identification in different metazoans including some cnidarian taxa. For the first time, we tested the method across four cnidarian classes (Staurozoa, Scyphozoa, Anthozoa, Hydrozoa) and included different scyphozoan life-history stages (polyp, ephyra, medusa) in our dataset. Our results revealed reliable species identification based on MALDI-TOF mass spectra across all taxa with species-specific clusters for all 23 analysed species. In addition, proteomic fingerprinting was successful for distinguishing developmental stages, still by retaining a species specific signal. Furthermore, we identified the impact of different salinities in different regions (North Sea and Baltic Sea) on proteomic fingerprints to be negligible. In conclusion, the effects of environmental factors and developmental stages on proteomic fingerprints seem to be low in cnidarians. This would allow using reference libraries built up entirely of adult or cultured cnidarian specimens for the identification of their juvenile stages or specimens from different geographic regions in future biodiversity assessment studies.     

A morphometric and genetic framework for the genus Gazella de Blainville, 1816 (Ruminantia: Bovidae) with special focus on Arabian and Levantine mountain gazelles

Gazella is one of the most species‐rich genera within horned ruminants. Despite overall similarity in body size and morphology, gazelles show variability in coloration and horn morphology. Unfortunately, however, species differentiation based on these characters, or on discrete skull characters, is very difficult due to high intraspecific variability. Furthermore, most species have fragmented and allopatric distributions, so that species boundaries were hard to define in the past. Mitochondrial DNA sequences have proven useful for investigating gazelle taxonomy in recent years, but especially for old museum material, i.e. type specimens, destructive sampling is often impossible. We provide a comprehensive morphometric framework for the genus Gazella based on linear skull measurements reconciled with results from molecular phylogenetic analysis based on the largest dataset available so far. In particular for males, the skull morphology shows interspecific differences concurrent with DNA data and provides a reliable tool for species identification. Based on morphometric data we synonymize G. karamii with G. marica, and confirm the identification of the G. arabica and G. a. rueppelli type skulls from analyses of mitochondrial DNA sequences. © 2013 The Linnean Society of London     

江西新岗山森林昆虫种内遗传距离研究

DNA条形码目前广泛用于昆虫多样性研究。本研究采用DNA条形码（即线粒体细胞色素c氧化酶亚基I基因COI 5′端）,通过比较所获分子分类操作单元（Molecular operational taxonomic units,MOTU）的种内遗传距离,探究DNA条形码在亚热带森林（位于我国江西省新岗山）不同昆虫类群中的物种鉴定和界定效用。数据分析中结合数据库比对信息,采用jMOTU、ABGD、bPTP、GMYC 这4种物种界定方法获得MOTU,从而开展种内遗传距离分析。本研究共挑选出479个昆虫样本,获得475条COI序列,经NCBI、BOLD在线数据库比对属于6个目,与形态初步划分一致;物种界定分析获得288个MOTU,其中鳞翅目最多,达85个,膜翅目、双翅目、半翅目、鞘翅目次之,分别为80、74、21和20个,直翅目最少,仅8个。膜翅目和双翅目的种内遗传距离均值及标准偏差较大（膜翅目：0.89%±0.87%;双翅目：0.73%±0.58%）,鳞翅目的最小（0.28%±0.20%）。研究表明：不同昆虫类群的种内遗传距离虽然整体在一定范围,但仍然存在一定的差异,因此不能笼统地依靠遗传距离的距离阈值进行物种划分;现有数据库需要补充足够的昆虫物种信息,才能提升物种鉴定效率。本研究丰富了亚热带森林昆虫分子数据库,同时也为进一步探索基于分子分类学开展昆虫多样性研究提供了基础数据和参考。     

A Retrospective Approach to Testing the DNA Barcoding Method

A decade ago, DNA barcoding was proposed as a standardised method for identifying existing species and speeding the discovery of new species. Yet, despite its numerous successes across a range of taxa, its frequent failures have brought into question its accuracy as a short-cut taxonomic method. We use a retrospective approach, applying the method to the classification of New Zealand skinks as it stood in 1977 (primarily based upon morphological characters), and compare it to the current taxonomy reached using both morphological and molecular approaches. For the 1977 dataset, DNA barcoding had moderate-high success in identifying specimens (78-98%), and correctly flagging specimens that have since been confirmed as distinct taxa (77-100%). But most matching methods failed to detect the species complexes that were present in 1977. For the current dataset, there was moderate-high success in identifying specimens (53-99%). For both datasets, the capacity to discover new species was dependent on the methodological approach used. Species delimitation in New Zealand skinks was hindered by the absence of either a local or global barcoding gap, a result of recent speciation events and hybridisation. Whilst DNA barcoding is potentially useful for specimen identification and species discovery in New Zealand skinks, its error rate could hinder the progress of documenting biodiversity in this group. We suggest that integrated taxonomic approaches are more effective at discovering and describing biodiversity.     

Molecular Taxonomy of Phytopathogenic Fungi: A Case Study in Peronospora

Background

Inappropriate taxon definitions may have severe consequences in many areas. For instance, biologically sensible species delimitation of plant pathogens is crucial for measures such as plant protection or biological control and for comparative studies involving model organisms. However, delimiting species is challenging in the case of organisms for which often only molecular data are available, such as prokaryotes, fungi, and many unicellular eukaryotes. Even in the case of organisms with well-established morphological characteristics, molecular taxonomy is often necessary to emend current taxonomic concepts and to analyze DNA sequences directly sampled from the environment. Typically, for this purpose clustering approaches to delineate molecular operational taxonomic units have been applied using arbitrary choices regarding the distance threshold values, and the clustering algorithms.

Methodology

Here, we report on a clustering optimization method to establish a molecular taxonomy of Peronospora based on ITS nrDNA sequences. Peronospora is the largest genus within the downy mildews, which are obligate parasites of higher plants, and includes various economically important pathogens. The method determines the distance function and clustering setting that result in an optimal agreement with selected reference data. Optimization was based on both taxonomy-based and host-based reference information, yielding the same outcome. Resampling and permutation methods indicate that the method is robust regarding taxon sampling and errors in the reference data. Tests with newly obtained ITS sequences demonstrate the use of the re-classified dataset in molecular identification of downy mildews.

Conclusions

A corrected taxonomy is provided for all Peronospora ITS sequences contained in public databases. Clustering optimization appears to be broadly applicable in automated, sequence-based taxonomy. The method connects traditional and modern taxonomic disciplines by specifically addressing the issue of how to optimally account for both traditional species concepts and genetic divergence.     

Genetic markers for study of the anopheline vectors of human malaria

Human malaria is truly a disease of global proportions and is one of the most broadly distributed vector-borne infections. Anopheline mosquitoes are the exclusive vectors of human malaria. A handful of species predominate as the most notorious malaria vectors, but the species and forms involved in the transmission of human malaria world-wide are incredibly diverse. Many of the anophelines that vector malaria exist as members of species complexes that often contain vector and non-vector species. Additionally, single anopheline species often exhibit significant heterogeneity across the species' range. This phenotypic and genotypic plasticity exacerbates the difficulties in identification of vector populations and implementation of effective surveillance and control strategies. Polytene chromosome investigations were among the first to provide researchers with tangible genetic markers that could be used to differentiate between what are now recognised as species and chromosomal forms of anopheline mosquitoes. The advent of the polymerase chain reaction gave access to the molecular genetics of genomes and the techniques that followed have facilitated investigation of the genetics of individual specimens or population size samples. The variety and number of genetic markers available for the study of malaria vectors has literally exploded in the last 10 years. Markers have expanded from the 'traditional tools' to include a vast array of molecular markers. Contemporary markers range from what are now referred to as 'classical genetic markers' to methods used to detect and identify single nucleotide polymorphisms and finally to highly polymorphic markers. One of the greatest advantages of this wide variety of genetic markers is that researchers may choose to utilise any combination of markers or techniques to address multifaceted questions relating to malaria transmission. These molecular markers have proven useful in a wide variety of applications including molecular taxonomy, evolutionary systematics, population genetics, genetic mapping, and investigation of defined phenotypes.