Variation of Nuclear DNA Content in the Biflorus Species of Lonchocarpus (Leguminosae)

This represents the first study of nuclear DNA content in alarge sample (135 spp.) from a tropical arboreal genus, in whicha large proportion of the species were examined (42 spp., 31.1%).Somatic chromosome numbers and 4C-DNA values for 51 taxa ofLonchocarpus are reported. All taxa were diploid with 2 n =22,but their DNA content ranged from 1.92 to 2.86 pg 4C nucleus,corresponding to a 48.95% variation in genome size. In the 74collections studied, no correlation was observed between DNAcontent and habitat altitude. Variation in nuclear DNA contentwas analysed at the level of genus, subgenus, section and subsection.Variation in genome size was also studied within some species,either among widely separated populations or among differentintraspecific taxa. Very little variation in genome size wasdetected between populations, subspecies, and varieties of thesame species. The taxonomic implications of variation in nuclearDNA content are discussed.Copyright 2000 Annals of Botany Company Lonchocarpus (Leguminosae), DNA content, chromosome number.     

Substantial Genome Size Variation in Taraxacum stenocephalum (Asteraceae, Lactuceae)

There are only a few exceptions to the rule that polyploidy in Taraxacum is associated with agamospermy. One of them is the sexual, tetraploid species Taraxacum stenocephalum. Incidentally, remarkable variation in karyology was found in this species. The present study aims to confirm this variation by an extensive screen of nuclear DNA content. Individuals from two large populations in the Lesser and Greater Caucasus, Georgia were analyzed using flow cytometry to ascertain intraspecific nuclear DNA content variation. Across the whole data set comprising all 159 individuals, a 1.223-fold difference was detected based on propidium iodide (PI) analyses. To verify this finding, we compared flow-cytometric data obtained using DAPI (4′,6-diamidino-2-phenylindole) and PI staining using a representative subset of individuals. This comparison revealed a 1.194-fold difference in DNA content for DAPI and a 1.219-fold difference for PI. Mean nuclear genome size in absolute terms (2C value ± s.d.) was estimated at 4.38?±?0.21 pg, ranging from 4.01 pg to 4.89 pg, despite the invariable chromosome counts of 2n?=?32. A regression analysis comparing the datasets for DAPI and PI staining found a strong correlation between data obtained by the DAPI and PI dyes (R?=?0.976; P?=?0.0001). Simultaneous high-resolution flow-cytometric analyses also proved the accuracy of our findings. We discuss possible sources of these large differences in DNA content within Taraxacum stenocephalum. Further research is needed to identify the source of this remarkable variation.     

Chloroplast and nuclear DNA studies in a few members of the <Emphasis Type="Italic">Brassica oleracea</Emphasis> L. group using PCR-RFLP and ISSR-PCR markers: a population genetic analysis

A population genetic analysis of chloroplast and nuclear DNA was performed covering nine wild populations of Brassica oleracea. Three members of the n = 9 group, all close to B. oleracea, Brassica alboglabra Bailey, Brassica bourgeaui (Webb) O. Kuntze and Brassica montana Pourret, were also studied to better understand their relationship with B. oleracea. Chloroplast DNA was analysed using the PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism) method. The ISSR-PCR (inter-simple sequence repeat - polymerase chain reaction) technique was adopted to study nuclear DNA. Twelve primer pairs of chloroplast DNA showed very good amplification. The amplified product of each primer pair, digested by three restriction enzymes, revealed no variation of cpDNA among the taxa studied. This indicates they may have the same chloroplast genotype. Seven selected ISSR primers have detected genetic variation, both within and among the populations/taxa surveyed. The information obtained on the intra- and inter-populational genetic diversity of wild populations of B. oleracea neatly defined the individual plants. It could provide important guidelines for backing management and conservation strategies in this species. The study confirms a close relationship between B. alboglabra, B. bourgeaui and B. montana, which is parallel to their morphological similitude.     

MONOPHYLY OF ANEUPLOID ASTRAGALUS (FABACEAE): EVIDENCE FROM NUCLEAR RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER SEQUENCES

Evolutionary relationships within Astragalus L. (Fabaceae) were inferred from nucleotide sequence variation in nuclear ribosomal DNA of both New World and Old World species. The internal transcribed spacer regions (ITS) of 18S–26S nuclear ribosomal DNA from representatives of 26 species of Astragalus, three species of Oxytropis DC., and two outgroup taxa were analyzed by polymerase chain reaction amplification and direct DNA sequencing. The length of the ITS 1 region within these taxa varied from 221 to 231 bp, while ITS 2 varied in length from 207 to 217 bp. Of the aligned, unambiguous positions, approximately 34% were variable in each spacer region. In pairwise comparisons among Astragalus species and outgroup taxa, sequence divergence at these sites ranged from 0 to 18.8% in ITS 1 and from 0 to 21.7% in ITS 2. Parsimony analyses of these sequences resulted in a well-resolved phylogeny that is highly concordant with previous cytogenetic and chloroplast DNA evidence for a major phylogenetic division in the genus. These data suggest that the New World aneuploid species of Astragalus form a monophyletic but morphologically cryptic group derived from euploid species of Old World (Eurasian) origin, which are consequently paraphyletic.     

Delineating closely related species with DNA barcodes for routine biological monitoring

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Molecular signatures of divergence and selection in closely related pine taxa

Efforts to detect loci under selection in plants have mostly focussed on single species. However, assuming that intraspecific divergence may lead to speciation, comparisons of genetic variation within and among recently diverged taxa can help to locate such genes. In this study, coalescent and outlier detection methods were used to assess nucleotide polymorphism and divergence at 79 nuclear gene fragments (1212 SNPs) in 16 populations (153 individuals) of the closely related, but phenotypically and ecologically distinct, pine taxa Pinus mugo, P. uliginosa and P. uncinata across their European distributions. Simultaneously, mitochondrial DNA markers, which are maternally inherited in pines and distributed by seeds at short geographic distance, were used to assess genetic relationships of the focal populations and taxa. The majority of nuclear loci showed homogenous patterns of variation between the taxa due to a high number of shared SNPs and haplotypes, similar levels of polymorphism, and low net divergence. However, against this common genetic background and an overall low population structure within taxa at mitochondrial markers, we identified several genes showing signatures of selection, accompanied by significant intra- and interspecific divergence. Our results indicate that loci involved in species divergence may be involved in intraspecific local adaptation.     

植物核DNA含量在全球尺度上的纬度变异式样及其气候适应意义——以菊科植物为例

核DNA含量是重要的生物学概念,涉及DNA C-值和基因组大小。前人有关植物核DNA含量在纬度梯度上的变异规律存在着矛盾的报道,而且多数将核DNA含量与纬度、海拔、气候等因素之间的关系描述成线性关系。核DNA含量是否具有环境适应上的意义,也还存在争议。先前有关核DNA含量与环境因素间关系的矛盾性报道可能与取样过小、地理范围过窄、研究对象遗传背景差异过大有关。如果对一个遗传背景相近的类群在全球范围内进行取样,核DNA含量会呈现有规律的纬度梯度变化,可能与大的气候因素之间存在非线性关系。菊科(Asteraceae)是被子植物的最大科,是一个广泛认可的自然分类群。为揭示全球空间尺度上植物核DNA含量在纬度梯度上的变异规律,以及这种变异是否具有环境适应意义,以菊科为对象开展了核DNA含量与纬度、生物气候因素关系的统计分析。从"植物DNA C-值数据库"检索到822种菊科植物的核DNA含量数据;在全球范围内,沿经度方向上设立10条样带,每条样带横跨15个经度,每条样带又均分成22个样块,每个样块纵跨7.5个纬度;其次,从"世界气候数据网站"下载1950—2000年时间段14个生物气候因子数据,应用Arc GIS 9.3获得每个样块14个生物气候因子的平均值;根据"全球生物多样性信息网站"记录,计算每个样块菊科植物平均的核DNA含量数据。为避免气候变量之间的多重共线性对数据分析的影响,应用主成分分析对数据进行了降维,发现最冷季度平均温度、最干季度雨量分别是第一、二主成分上荷载最大的因子,去除与它们相关性在-0.7至+0.7之间的其他气候因子后获得了最冷季度平均温度、最干季度雨量和最湿月份雨量三个变量用于进一步数据分析。结果发现,菊科植物在全球10个样带上的核DNA含量与纬度关系密切,与最冷季度平均温度、最干季度雨量和最湿月份雨量呈现极显著的单峰型的非线性关系,可以用二项式进行拟合。因此,全球空间尺度上植物核DNA含量沿着纬度梯度有规律性的非线性变化,这种变化具有很强的气候适应意义。     

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.     

Hybridization and population genetics of two macaque species in Sulawesi, Indonesia

This study investigates hybridization and population genetics of two species of macaque monkey in Sulawesi, Indonesia, using molecular markers from mitochondrial, autosomal, and Y-chromosome DNA. Hybridization is the interbreeding of individuals from different parental taxa that are distinguishable by one or more heritable characteristics. Because hybridization can affect population structure of the parental taxa, it is an important consideration for conservation management. On the Indonesian island of Sulawesi an explosive diversification of macaques has occurred; seven of 19 species in the genus Macaca live on this island. The contact zone of the subjects of this study, M. maura and M. tonkeana, is located at the base of the southwestern peninsula of Sulawesi. Land conversion in Sulawesi is occurring at an alarming pace; currently two species of Sulawesi macaque, one of which is M. maura, are classified as endangered species. Results of this study indicate that hybridization among M. maura and M. tonkeana has led to different distributions of molecular variation in mitochondrial DNA and nuclear DNA in the contact zone; mitochondrial DNA shows a sharp transition from M. maura to M. tonkeana haplotypes, but nuclear DNA from the parental taxa is homogenized in a narrow hybrid zone. Similarly, within M. maura divergent mitochondrial DNA haplotypes are geographically structured but population subdivision in the nuclear genome is low or absent. In M. tonkeana, mitochondrial DNA haplotypes are geographically structured and a high level of nuclear DNA population subdivision is present in this species. These results are largely consistent with a macaque behavioral paradigm of female philopatry and obligate male dispersal, suggest that introgression between M. maura and M. tonkeana is restricted to the hybrid zone, and delineate one conservation management unit in M. maura and at least two in M. tonkeana.     

Genetic variation and biogeography of the disjunct Vitis subg. Vitis (Vitaceae)

Aim Vitis subg. Vitis provides an example of a plant disjunction occurring in the Northern Hemisphere. It shows broad morphological variation but is assumed to be a species complex with limited genetic differentiation. Based on a comprehensive sampling of taxa and polymorphism in both chloroplast and nuclear DNA, we assessed genetic variation within this subgenus. Our aims were to clarify the relationships among species and to examine their historical biogeography. Location Asia, Europe, North America. Methods We analysed a total of 30 species and putative hybrids from subgenus Vitis and examined the infra‐specific variation in some species. Polymorphism in chloroplast DNA was assessed in trnL and trnH–psbA–trnK sequences (c. 2170 bp) and in 15 microsatellite loci. We also obtained nuclear data for size variation at 24 microsatellite loci. Phylogenetic inference was performed with Bayesian analyses. A maximum parsimony network was constructed to depict the evolutionary relationships among haplotypes, and microsatellite data were also subjected to hierarchical clustering analysis using the Ward distance. In addition, we assessed size homoplasy by sequencing both chloroplast and nuclear microsatellite loci. Results Chloroplast polymorphisms resolved subgenus Vitis as a monophyletic group with limited genetic variation. The ancestral haplotypes were found in Eurasia. American taxa all harboured derived haplotypes. Most of them formed a monophyletic group that did not include Vitis californica. The four main haplotypes in Vitis vinifera corresponded to two different origins. Nuclear microsatellites indicated that genetic variation was especially large in North America. Asian species exhibited a lower level of nuclear divergence and the European V. vinifera corresponded to a differentiated nuclear lineage. Main conclusions We obtained some evidence that subgenus Vitis has an Asian origin and then dispersed to Europe and North America. Geographic separation was followed by diversification, presumably during the Pleistocene, resulting in phylogeographic patterns similar to other biota. In contrast to chloroplast DNA, nuclear DNA shows a larger than expected genetic variation. Our molecular data also highlight the need to re‐examine certain aspects of the current subgeneric classification.     

Mining the mammalian genome for artiodactyl systematics

A total of 7,806 nucleotide positions derived from one mitochondrial and eight nuclear DNA segments were used to provide a robust phylogeny for members of the order Artiodactyla. Twenty-four artiodactyl and two cetacean species were included, and the horse (order Perissodactyla) was used as the outgroup. Limited rate heterogeneity was observed among the nuclear genes. The partition homogeneity tests indicated no conflicting signal among the nuclear genes fragments, so the sequence data were analyzed together and as separate loci. Analyses based on the individual nuclear DNA fragments and on 34 unique indels all produced phylogenies largely congruent with the topology from the combined data set. In sharp contrast to the nuclear DNA data, the mtDNA cytochrome b sequence data showed high levels of homoplasy, failed to produce a robust phylogeny, and were remarkably sensitive to taxon sampling. The nuclear DNA data clearly support the paraphyletic nature of the Artiodactyla. Additionally, the family Suidae is diphyletic, and the nonruminating pigs and peccaries (Suiformes) were the most basal cetartiodactyl group. The morphologically derived Ruminantia was always monophyletic; within this group, all taxa with paired bony structures on their skulls clustered together. The nuclear DNA data suggest that the Antilocaprinae account for a unique evolutionary lineage, the Cervidae and Bovidae are sister taxa, and the Giraffidae are more primitive.     

Identifying and Distinguishing Sibling Species in the Tetrahymena pyriformis Complex (Ciliophora, Oligohymenophorea) Using PCR/RFLP Analysis of Nuclear Ribosomal DNA

ABSTRACT We describe a riboprinting strategy for identifying and distinguishing among sibling species in the Tetrahymena pyriformis complex. It involves use of the polymerase chain reaction to amplify a large segment of the nuclear ribosomal DNA and internal transcribed spacers, and digestion of this DNA with restriction enzymes. Unique restriction fragment length patterns or haplotypes were then used to distinguish species into: (1) six taxa that were identifiable to the species level, (2) eight taxa that were separated into four pairs, and (3) a group of eight taxa that were identical to each other. The latter result indicates that a more variable molecule is needed to distinguish the most closely related species in the complex. There was no intraspecific variation between two strains from one species ( Tetrahymena thermophila ) nor among multiple isoiates from another species ( Tetrahymena empidokyrea ). This approach provides an alternative to traditional techniques for identifying T. pyriformis species that require living reference specimens and/or that reveal high levels of intraspecific variation.     

Assessing Nuclear and Mitochondrial DNA Sequence Variation Within Steinernema (Rhabditida: Steinernematidae)

DNA sequence analysis was used to characterize the nuclear ribosomal DNA ITS1 region and a portion of the COII and 16S rDNA genes of the mitochondrial genome from Steinernema entomopathogenic nematodes. Nuclear ITS1 nucleotide divergence among seven Steinernema spp. ranged from 6 to 22%, and mtDNA divergence among five species ranged from 12 to 20%. No intraspecific variation was observed among three S. feltiae strains. Phylogenetic analysis of both nuclear and mitochondrial DNA sequences confirms the existing morphological relationships of several Steinernema species. Both the rDNA ITS1 and mtDNA sequences were useful for resolving relationships among Steinernema taxa.     

Host-specific differentiation among populations of Venturia inaequalis causing scab on apple,pyracantha and loquat

Patterns of multilocus DNA sequence variation within and between closely related taxa can provide insights into the history of divergence. Here, we report on DNA polymorphism and divergence at six nuclear loci in globally distributed samples of the ascomycete Venturia inaequalis, responsible for scab on apple, loquat, and pyracantha. Isolates from different hosts were differentiated but did not form diagnosable distinct phylogenetic species. Parameters of an Isolation-with-Migration model estimated from the data suggested that the large amount of variation shared among groups more likely resulted from recent splitting than from extensive genetic exchanges. Inferred levels of gene flow among groups were low and more concentrated toward recent times, and we identified two potentially recent one-off shifters from apple and pyracantha to loquat. These findings support a scenario of recent divergence in allopatry followed by introgression through secondary contact, with groups from loquat and pyracantha being the most recently differentiated.     

AFLP and RAPD analyses of genetic diversity of wild and/or weedy Guizotia (Asteraceae) from Ethiopia

Amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) markers were used to provide estimates of the comparative genetic variation within and among populations of various Guizotia taxa with the goal of conserving and utilizing their genetic diversity. The percentage of polymorphic loci (P(S)) ranged from 28.5%-90% (AFLP) and 85.6%-99.6% (RAPD). The overall gene diversity estimate () has shown slight variation among taxa ranging from 0.32-0.37 (AFLP) and from 0.22 to 0.28 (RAPD). The within population diversity of "Chelelu" and "Ketcha" was found to be unexpectedly high. Both parameters used to estimate population differentiation (G(ST) and F(ST)) revealed the highest population differentiation G. zavattarii in followed by G. arborescens. Genetic variation among populations within a taxon was highly significant for all the five taxa as revealed by AMOVA (P<0.0001). The need for immediate conservation activities for G. arborescens and G. zavattarii, and factors that contribute to the existing genetic variability and population genetic structures are discussed.     

A new insight into the phylogeny of vascular cryptogams with special reference to Selaginella and Isoetes inferred from nuclear ITS/5.8S rDNA sequences

In order to provide a better understanding of the evolutionary history of vascular cryptogams, phylogenetic framework was developed based on ITS1, ITS2 and 5.8S rDNA sequences of 102 extant taxa of vascular cryptogams using Maximum Parsimony (MP) analysis. The analysis revealed high GC content in Isoetaceae (60.5 %) in comparison with Selaginellaceae (54.4 %) that was envisaged to be the result of variation in selection, mutational bias, and biased recombination-associated DNA repair within these two plant lineages during evolution. Transition/transversion ratio was observed to be 0.9 in Isoetaceae, 0.68 in Selaginellaceae and 0.57 among all the 102 taxa belonging to lycophytes and ferns. It is hypothesized that the lycophytes have been separated very early during evolution and therefore acquired independent line of evolution from the other plant lineages. Although Selaginellaceae and Isoetaceae are closely related ancient plant groups, pairwise sequence divergence of sampled taxa on the basis of transition and transversion, and disparity index values per site between sampled sequence pairs pointed towards the differential investment of natural selection process. These lead to high rate of nucleotide substitution within nuclear genome of Selaginellaceae with respect to Isoetaceae. MP phylogenetic tree identified Isoetes subinermis, Isoetes durieui and Salvia microphylla as separate group among the studied taxa due to high sequence variation within these species through the time of evolution. Our result interpreted the polyphyletic origin of ferns and provides valuable information regarding the lycophytes and their fern allies.     

A Method for Calibrating Molecular Clocks and Its Application to Animal Mitochondrial DNA

A generalized least-squares procedure is introduced for the calibration of molecular clocks and applied to the complete mitochondrial DNA sequences of 13 animal species. The proposed technique accounts for both nonindependence and heteroscedasticity of molecular-distance data, problems that have not been taken into to account in such analyses in the past. When sequence-identity data are transformed to account for multiple substitutions/site, the molecular divergence scales linearly with time, but with substantially more variation in the substitution rate than expected under a Poisson model. Significant levels of divergence are predicted at zero divergence time for most loci, suggesting high levels of site-specific heterozygosity among mtDNA molecules establishing in sister taxa. For nearly all loci, the baseline heterozygosity is lower and the substitution rate is higher in mammals relative to other animals. There is considerable variation in the evolutionary rate among loci but no compelling evidence that the average rate of mtDNA evolution is elevated with respect to that of nuclear DNA. Using the observed patterns of interspecific divergence, empirical estimates are derived for the mean coalescence times of organelles colonizing sister taxa.     

The relationship between flower size, inbreeding coefficient and inferred selfing rate in British Euphrasia species

The genus Euphrasia in Britain comprises a taxonomically complex group of self-compatible, morphologically similar, hemi-parasitic, annual plant species of high conservation importance. The 19 diploid and tetraploid taxa currently recognised show striking variation in flower size. The objective of this paper is to determine whether a relationship exists between flower size and breeding system within Euphrasia. Following a survey of flower size variation among the 19 taxa, seven diploid populations, encompassing a broad range of flower sizes, were selected for detailed study. Four nuclear microsatellite loci were used to estimate the inbreeding coefficient Fis within each population. Fis values varied from to 0.17-0.77 and showed a significant, negative correlation with flower size. These results are best explained as the consequence of variation in selfing rate among the Euphrasia populations, with selfing rate increasing as flower size decreases. The potential factors influencing breeding system evolution in Euphrasia are discussed, together with the role of autogamy in generating taxonomic complexity and facilitating lineage differentiation within the genus.     

Systematics and phylogeography of pocket gophers in the genera Cratogeomys and Pappogeomys.

Mitochondrial-DNA sequence data were analyzed from individuals sampled from 38 localities across the complete geographic range of the closely related pocket gopher genera Pappogeomys and Cratogeomys. Results of phylogenetic analysis of 1133 base pairs from the cytochrome b gene are consistent with past hypotheses of relationships among members of the castanops species group within the genus Cratogeomys. However, phylogeographic variation within the gymnurus species group of the genus Cratogeomys differs significantly from relationships reflected by current taxonomy. The data indicate that there are five geographically distinct clades within the gymnurus species group. Members of the two nominal species C. gymnurus and C. tylorhinus are scattered among these clades. The three peripherally isolated species, C. fumosus, C. neglectus, and C. zinseri, do not appear to be genetically distinct from other gymnurus species group taxa. An historical biogeographic hypothesis is proposed that will be tested using nuclear DNA data.