Evolutionary relationships among 12 species belonging to three genera of the family Microhylidae in Papua New Guinea revealed by allozyme analysis

To elucidate the potential of electrophoretic analysis for understanding relationships among microhylid frogs in Papua New Guinea, an allozyme analysis was conducted. A total of 119 individuals from nine species of Cophixalus, two species of Sphenophryne and one species of Barygenys, all of which belong to the family Microhylidae, were studied. Fourteen enzymes extracted from skeletal muscles and livers were analyzed by starch-gel electrophoresis. These enzymes were encoded by genes at 20 loci. There were 2-15 phenotypes produced by 2-12 alleles at these loci. The mean proportion of heterozygous loci per individual, mean proportion of polymorphic loci per population, and mean number of alleles per locus in 12 species were 6.1%, 17.1% and 1.17a on average, respectively. The NJ and ML trees constructed from Nei's genetic distances showed that the genus Sphenophryne can be distinguished biochemically from Cophixalus and Barygenys, and that the species groups of Cophixalus, which are similar in external morphology, can be divided biochemically into several species.     

Allozyme and RAPD analysis of the genetic diversity and geographic variation in wild populations of the American chestnut (Fagaceae)

Genetic variation among 12 populations of the American chestnut (Castanea dentata) was investigated. Population genetic parameters estimated from allozyme variation suggest that C. dentata at both the population and species level has narrow genetic diversity as compared to other species in the genus. Average expected heterozygosity was relatively low for the population collected in the Black Rock Mountain State Park, Georgia (He = 0.096 +/- 0.035), and high for the population in east central Alabama (He = 0.196 +/- 0.048). Partitioning of the genetic diversity based on 18 isozyme loci showed that ~10% of the allozyme diversity resided among populations. Cluster analysis using unweighted pair-group method using arithmetric averages of Rogers' genetic distance and principal components analysis based on allele frequencies of both isozyme and RAPD loci revealed four groups: the southernmost population, south-central Appalachian populations, north-central Appalachian populations, and northern Appalachian populations. Based on results presented in this study, a conservation strategy and several recommendations related to the backcross breeding aimed at restoring C. dentata are discussed.     

Allozyme diversity in Pinus virginiana (Pinaceae): intraspecific and interspecific comparisons

Two of the four members of subsection Contortae of the genus Pinus occur in the southeastern United States: Pinus virginiana, which ranges throughout the southern and central Appalachian Mountains, and P. clausa, which is restricted to Florida and southern Alabama. We examined allozyme variation within P. virginiana and genetic relationships between this species and the two varieties of P. clausa (var. clausa and var. immuginata). P. virginiana maintains more genetic diversity at both the species (Hes = 0.139) and population (Hep = 0.128) levels than the other three species in the subsection, which may reflect the combination of its widespread distribution and the absence of cone serotiny. Genetic differentiation among populations in P. virginiana was relatively low (GST = 0.053), but significant contrasts in allozyme frequencies and genetic diversity were apparent between populations to the northwest vs. outheast of the Appalachian Mountains. These regional differences likely resulted initially from historical processes that occurred during the Pleistocene and early Holocene, and have been reinforced by modern selective pressures and barriers to gene flow. The mean genetic distance between populations of P. virginiana and P. clausa (D = 0.071) was greater than that between populations of the two varieties of P. clausa (D = 0.012), which suggests that the two varieties diverged at some point after the separation of the two species.     

Esterase Allozymes of Soybean Cyst Nematode,Heterodera glycines,from China,Japan, and the United States

Individual females from 19 populations of Heterodera glycines from China, Japan, and the United States were analyzed for esterase allozyme polymorphism. Eight esterase electrophoretic phenotypes were resolved. Four putative loci, est-1, est-2, est-3, and est-4, were identified, having one, one, two, and one allele, respectively. The four loci expressed six genotypes in the four Chinese populations. Loci est-2, est-3, and est-4 were identified in five Japanese populations and expressed five genotypes, whereas only loci est-2 and est-3 were identified in 10 populations from the United States and expressed four genotypes. Putative alleles at each locus were defined as characters for data analysis. Phylogenetic analysis using parsimony (PAUP) was utilized to determine relationships among the 19 populations. More loci and alleles in populations from China and phylogenetic similarities among populations from Japan and the United States are consistent with a founder effect resulting from dissemination of progenitor H. glycines from China to Japan and subsequent introductions of founder populations from Japan to the United States.     

Development and characterization of microsatellite markers for Berberis thunbergii (Berberidaceae)

? Premise of the study: Microsatellite markers were isolated and characterized in Berberis thunbergii, an invasive and ornamental shrub in the eastern United States, to assess genetic diversity among populations and potentially identify horticultural cultivars. ? Methods and Results: A total of 12 loci were identified for the species. Eight of the loci were polymorphic and were screened in 24 individuals from two native (Tochigi and Ibaraki prefectures, Japan) and one invasive (Connecticut, USA) population and 21 horticultural cultivars. The number of alleles per locus ranged from three to seven, and observed heterozygosity ranged from 0.048 to 0.636. ? Conclusions: These new markers will provide tools for examining genetic relatedness of B. thunbergii plants in the native and invasive range, including phylogeographic studies and assessment of rapid evolution in the invasive range. These markers may also provide tools for examining hybridization with other related species in the invasive range.     

Patterns of hybridization and introgression between invasive Ulmus pumila (Ulmaceae) and native U. rubra

Natural hybridization between introduced species and their native congeners occurs frequently and can create serious conservation concerns. Ulmus pumila (Siberian elm) is an introduced Asian elm species that has naturalized in the United States and is now considered invasive in 41 states. Red elm (U. rubra), a native to the eastern United States, often occurs in sympatry with Siberian elm, and the two species are thought to hybridize. Here, we genetically characterized reference populations of the two elm species to identify species-specific microsatellite alleles. These markers were used to classify individuals in putative hybrid zones as parental species or hybrids, assess the extent of hybridization, and track patterns of introgression. We identified nine U. rubra, 32 U. pumila, and 51 hybrid individuals in our hybrid zones. Of the 51 hybrids, 35 were classified as first-generation hybrids and 16 as backcrosses. The majority of the backcrosses (88%) were introgressed toward U. pumila. Our classification of genotypes was consistent whether we used manual classification, principal coordinate analyses or Bayesian clustering. We observed greater genetic diversity and new combination of alleles in the hybrids. Our study indicates widespread hybridization between U. pumila and U. rubra and an asymmetric pattern of introgression toward U. pumila.     

Genetic variation and evidence of hybridization in the genus Rhus (Anacardiaceae)

Rhus michauxii, a rare plant species endemic to the southeastern United States, was previously known only from central North Carolina and one site in Georgia. An additional site, which is now believed to represent the largest known concentration of R. michauxii, was recently discovered at Ft. Pickett near Blackstone, Virginia. Morphological characteristics in several of the Ft. Pickett Rhus populations appear to be intermediate between R. michauxii and the widespread R. glabra, a closely related congener that co-occurs at Ft. Pickett. Although morphological evidence of hybridization between R. michauxii and R. glabra in North Carolina has been provided previously, genetic marker data are lacking. In the present study we examined levels of allozyme variation at 11 polymorphic loci within and among seven populations of R. michauxii, one population of R. glabra, and four putative hybrid populations at Ft. Pickett. Overall, R. michauxii had typical levels of within-population genetic variation when compared to other species with similar life-history characteristics. In contrast, the proportion of genetic variation among populations (G(ST)) was considerably lower than expected. Finally, R. michauxii and R. glabra appear to have a fixed allelic difference at the Idh2 locus. This enabled us to confirm hybridization in all four of the putative hybrid populations and one of the R. michauxii populations.     

Bromus fasciculatus presl--a third diploid progenitor of Bromus section Genea allopolyploids (Poaceae)

Allozyme variation of ten heterozymes of seven enzymes among five accessions of a rare diploid Bromus fasciculatus was analysed with the use of PAGE and compared with that for six other species of section Genea of the genus Bromus. Allozymes charasteristic for diploids B. tectorum and B. fasciculatus are combined in fixed heterozygous phenotypes of tetraploid B. rubens. Fixed heterozygous phenotypes of tetraploid B. madritensis combine one allozyme of B. fasciculatus with another of diploid B. sterilis at each of the loci studied. Of the three diploids studied, only B. sterilis fits well for a role of a genome donor for the polyploid B. diandrus-rigidus complex. Bromus fasciculatus thus appears to be a diploid ancestor for the two tetraploids of section Genea, B. rubens and B. madritensis.     

Recent hybridization between Taenia asiatica and Taenia saginata

Five Taenia tapeworms collected from humans in Tibetan Plateau, Sichuan, China, where three species of human Taenia are sympatrically endemic, were examined for the mitochondrial cox1 gene and two nuclear genes, ef1 and elp. Phylogenetic analyses of these genes revealed that two adult worms showed nuclear-mitochondrial discordance, suggesting that they originated from hybridization between Taenia saginata and Taenia asiatica. One of two worms had T. asiatica-type mtDNA, whereas another worm had T. saginata-type mtDNA, indicating that reciprocal hybridization between T. saginata and T. asiatica could occur. The worm having T. asiatica-type mtDNA was heterozygous at both nuclear loci with T. saginata-type alleles and T. asiatica-type alleles. In another worm, the ef1 locus was heterozygous with a T. saginata-type alleles and T. asiatica-type alleles, while the elp locus was homozygous with T. saginata-type alleles. Self-fertilization is the main reproductive method of the genus Taenia. Since self-fertilization represents a type of inbreeding, each locus in the offspring would become homozygous over generations with genetic drift. The fact that some nuclear loci are still heterozygous means that hybridization might have occurred recently. Hybridization between T. asiatica and T. saginata is probably an ongoing event in many areas in which they are sympatrically endemic.     

THE MITOCHONDRIAL AND NUCLEAR GENETIC HOMOGENEITY OF THE PHENOTYPICALLY DIVERSE DARWIN'S GROUND FINCHES

The most extensively studied group of Darwin's finches is the genus Geospiza, the ground finches, and yet little is known about the evolutionary history and genetic relationships of these birds. Studies using either allozyme or morphological data have been unable to resolve relationships between the six species and numerous populations of ground finches. In this paper we report the results of a study using mitochondrial control region and nuclear internal transcribed spacer (ITS) 1 sequence data. The differentiation of the ground finch species based on morphological data is not reflected in either mitochondrial or nuclear DNA sequence phylogenies. Furthermore, there is little concordance between the mitochondrial haplotypes and ITS alleles found within individuals. We suggest that the absence of species-specific lineages can be attributed to ongoing hybridization involving all six species of Geospiza. There are no long term selective pressures against hybridization within this genus, and therefore a genetically homogenous genus may be maintained indefinitely. Hybridization has apparently played a role in the adaptive radiation of Darwin's finches.     

中华猕猴桃和美味猕猴桃自然居群遗传结构及其种间杂交渐渗

 利用9对SSR引物对中华猕猴桃（Actinidia chinensis）和美味猕猴桃（A. deliciosa）两近缘种的5个同域分布复合体和各自1个非同域分布居群进行了遗传多样性、居群遗传结构的分析以及种间杂交渐渗的探讨。结果表明：1）两物种共有等位基因比例高达81.13%,物种特有等位基因较少（中华猕猴桃：13.27%,美味猕猴桃：5.61%）,但共享等位基因表型频率在两近缘种间存在差异,而且与各同域复合体中两物种样本的交错程度或间距存在关联;2）两种猕猴桃均具有极高遗传多样性,美味猕猴桃的遗传多样性（H_o＝0 .749, PIC＝0.818）都略高于中华猕猴桃（H_o＝0.686,PIC＝0.799）;3）两猕 猴桃物种均具有较低的Nei’s居群遗传分化度,但AMOVA分析结果揭示种内异域居群间（F_ST=0.091 5）和同域复合体种间（F_ST=0.111 5）均存在一定程度的遗传分化;中华猕猴桃居群遗传分化（G_ST=0.086; F_ST=0.212 1）高于美味猕猴桃（G_ST= 0.080;F_ST=0.142 0）;4）同域分布复合体两物种间的遗传分化（G_ST=0.020）低于物种内异域居群间的遗传分化（中华猕猴桃：G_ST=0.086; 美味猕猴桃：G_ST=0.080）,同域复合体物种间的基因流（N_m＝7.89 -29.75）远远高于 同种异域居群间（中华猕猴桃：N_m =2.663; 美味猕猴桃：N_m=2.880）; 5）居群UPGMA聚类揭示在同一地域的居群优先聚类,个体聚类结果显示多数个体聚在各自居群组内,但各地理居群并不按地理距离的远近聚类,这与Mantel相关性检测所揭示的居群间遗传距离与地理距离没有显著性相关的结果一致。进一步分析表明两种猕猴桃的遗传多样性和居群遗传结构不仅受其广域分布、远交、晚期分化等生活史特性的影响,同时还与猕猴桃的染色体基数高 (x=29)、倍性复杂和种间杂交等因素密切相关,其中两种猕猴桃的共享祖先多态性和同域分布种间杂交基因渗透对两猕猴桃的居群遗传结构产生了重要影响。     

Biochemical Polymorphisms and Genetic Relationships in Rodents of the Genera Oryzomys and Oligoryzomys (Sigmodontinae) from Brazil

An electrophoretic analysis of 12 allozyme systems (14 loci, 40 alleles) was performed in the rodent genera Oryzomys and Oligoryzomys, in order to determine the levels of genetic variability within and among populations and species. One hundred and fifty-five individuals from 16 populations of Oryzomys russatus, Or. angouya, Oligoryzomys flavescens, and O1. nigripes species were trapped in nine Brazilian localities. Genetic divergence among populations, as well as the interpopulational gene diversity, was higher in Oryzomys than in Oligoryzomys. The dendrogram of the phenetic relationships among the 16 populations of these four species displays three clusters: the first one joins the O1. flavescens populations with those of O1. nigripes; the second groups the populations of Or. russatus, and the last assembles the populations of Or. angouya. The genetic parameters analyzed reveal that the species belonging to the genus Oryzomys are genetically more structured than those of Oligoryzomys, the latter presenting lower levels of intrademe genetic differentiation (G(ST)')     

How many species are in the genus Batrachuperus? A phylogeographical analysis of the stream salamanders (family Hynobiidae) from southwestern China

Phylogeographical analysis of DNA sequence data has been routinely used to test species boundaries using the monophyly criterion; however, a complementary criterion, reproductive isolation, is often ignored. We used a combination of phylogenetic and population genetic approaches to determine species boundaries among stream salamanders in the genus Batrachuperus . First, cytochrome b sequence data from 174 Batrachuperus individuals, sampled from 78 populations, were used to reconstruct historical relationships within the genus. Second, allozyme data for 14 presumptive nuclear loci, from 463 individuals sampled from 60 populations, were collected and analysed to assess population similarity or disparity, as well as potential reproductive isolation. The DNA sequence data grouped all populations into seven major monophyletic groups, and the allozyme data provided evidence for reproductive isolation among four of the seven groups, thereby supporting the species status of these groups. The allozyme data suggested that two of the other groups share the same gene pool, and therefore belong to a single species. Finally, the allozyme data revealed two reproductively isolated units within the seventh group, which we suggest represents a case of 'budding speciation' based on the DNA gene tree. In total, seven species of the genus Batrachuperus were defined, two of which were previously unknown. The phylogeographical analysis also revealed that vicariance events might have dominated the evolutionary history of this group, but the speciation events might precede the formation of the existing mountain topology. This study demonstrates the importance of including frequency data from multiple nuclear gene loci in determining species boundaries.     

Low levels of allozyme differentiation betweenPyxidanthera (pyxie-moss) taxa (Diapensiaceae)

TwoPyxidanthera morphs of questionable taxonomic rank are described in the eastern United States. We analyzed leaf samples from ninePyxidanthera populations (four of each morph and one with intermediate morphology) for 13 allozyme loci. Our results do not support differentiation of the two morphs at the species level. Mean genetic identity among populations was high ( = 0.97), and typical of that found for conspecific populations. The proportion of total genetic diversity found among populations was low (G_ST = 0.079). Several low frequency alleles were confined to each of the morphs, being found in some of the populations of each morph, but not all.     

PERMANENT GENETIC RESOURCES: Isolation, characterization and cross-species amplifications of microsatellite loci from Conradina (Lamiaceae)

We report the isolation of microsatellite loci from three species in the genus Conradina (Lamiaceae). To ensure their utility for multiple species, loci were screened for amplification and variability in all six Conradina species; 11 loci demonstrated high levels of amplification and polymorphism in most species. These 11 loci were characterized in 20 individuals from one population of Conradina brevifolia; alleles per locus ranged from five to 15, and observed heterozygosity ranged from 0.30 to 0.90. These microsatellites will be used to clarify species limits, detect interspecific hybridization, and understand the partitioning of genetic variation in each species of Conradina.     

猕猴桃属植物的cpSSR遗传多样性及其同域分布物种的杂交渐渗与同塑

同域分布的近缘物种常常发生杂交而导致种间基因渐渗, 从而对相关物种的自然居群遗传结构产生重要影响, 近缘种间的杂交渐渗已成为进化生物学和保护生物学关注的热点。本研究采用8对cpSSR引物对我国西部高原台地向中东部丘陵平原过渡地带同域重叠分布的猕猴桃属(Actinidia)7个物种的自然居群遗传多样性、居群遗传结构和同域分布种间遗传分化进行了检测。结果表明: (1)在6个多态性位点检测到18个等位基因形成的42个单倍型, 尽管各单倍型间显示了复杂的网状进化关系, 但还是具有明显的物种特异性; (2)各物种有丰富的cpSSR遗传多样性, 但种间存在较大差异, 绵毛猕猴桃(Actinidia fulvicoma var. lanata)的遗传多样性水平最高(P = 62.50%, h_T = 0.173, H_T = 0.897), 美味猕猴桃(A. deliciosa)的最低(P = 37.5％, h_T = 0.041, H_T = 0.516); (3)尽管不同物种的居群分化程度存在较大差异, 但种内居群间存在明显分化(G_ST为0.319–0.780, F_ST为0.401–0.695), 居群间的基因流不足(N_m为0.219–0.747<1); 其中以美味猕猴桃的居群遗传分化度最高(G_ST = 0.780, F_ST = 0.695); (4)遗传分化系数G_ST(unordered alleles)与N_ST(ordered alleles)无显著差异, 揭示本研究的大多数猕猴桃属物种不存在系统地理结构, 与用Mantel检验得出的居群遗传距离和地理距离不存在显著性相关的结果一致; (5)除了中华/美味猕猴桃复合体(A. chinensis / A. deliciosa complex)的湖北五峰(HW)和广西资源(GZ)两个同域复合居群外, 同域分布的物种间遗传分化强烈(F_ST为0.476–0.990), 与UPGMA聚类时多数居群按各自物种聚类的结果一致。进一步分析表明, 中华/美味猕猴桃复合体近缘种间存在明显的共祖多态性和杂交渐渗现象, 近缘种植株分布的交错程度以及是否存在亚居群结构对杂交渐渗存在着重要影响。亲缘关系较远的物种间杂交渐渗事件稀少, 但存在个别同塑事件。本研究结果有助于进一步了解猕猴桃属植物自然居群cpDNA的遗传特性和渐渗杂交进化模式, 为我国猕猴桃野生种质资源保育及可持续开发利用提供基础数据和科学依据。     

Genetic differentiation and detection of cryptic species in the genus Lepismachilis (Insecta: Microcoryphia) from the Western Mediterranean region

Different species of the bristletail genus Lepismachilis were collected in 14 localities in Italy and Spain and an allozyme electrophoretic survey was carried out to estimate the degree of genetic variability and differentiation at intra- and interspecific levels. Four morphological species were initially identified (L osellai, L. y-signata, L. affinis, L. targionii), but the electrophoretic analysis demonstrated the presence of two additional species among the individuals of L. targionii (Lepismachilis spl and sp2). The validity of these species and their differentiation from L targionii were demonstrated by the fixation of alternative allelic patterns at several loci (7 in Lepismachilis spl and 8 in Lepismachilis sp2), coupled with fixed, previously undetected, morphological differences. In addition, Lepismachilis sp2 was sympatric with L. targionii in three collecting sites, where the fixation of alternative allelic patterns unequivocally demonstrated reproductive isolation. Genetic variability did not seem to be correlated with local ecological factors, and differences between species should rather be explained by different historical factors. Low levels of gene flow, estimated with two different indirect methods, were observed in L. targionii and L. y-signata, and were due to high levels of structuring among populations. Genetic differentiation among conspecific populations was not correlated to their geographical arrangement and the presence of loci fixed for different alleles among them suggested that stochastic factors (such as genetic drift) may have played a role in determining genetic differentiation of geographically isolated populations. Genetic divergence values indicated that the six species are well differentiated and allozyme profiles were diagnostic for all of them. On the other hand, allozyme data did not provide adequate information to resolve evolutionary relationships among the species, nor did they confirm the validity of the two subgenera (Lepismachilis and Berlesilis) in which the genus Lepismachilis is traditionally divided.     

COVARIATION OF SELFING RATES WITH PARENTAL GENE FIXATION INDICES WITHIN POPULATIONS OF MIMULUS GUTTATUS

Wright's gene fixation index F and two single-locus effective selfing rates—the selfing rate at loci with fixed alleles, and the selfing rate at loci without fixed alleles—were estimated in five populations of Mimulus guttatus. These two effective selfing rates describe the inbreeding observed at a single locus when both uniparental and biparental inbreeding are practiced. Estimates were made using progeny arrays assayed for six allozyme loci and two morphological loci exhibiting dominance. The average of the two selfing rates computed for subpopulations (ca. 10 m diameter) ranged from 24% to 59%, with a mean of 37%. When computed for populations (ca. 1 km diameter), average selfing rates were about 10% higher. In four populations, the selfing rate at loci with fixed alleles was higher than the selfing rate at loci without fixed alleles. Thus, the covariance of selfing with parental gene fixation was positive. In one of the populations, estimates for individual plants sampled along a transect gave positive correlations for selfing rates and for gene-fixation indices between adjacent plants. A highly positive correlation between selfing rate and gene fixation of individual plants was also observed. In another population, the covariance of selfing with gene fixation was higher for a locus causing leaf spots than for allozyme loci. This covariance is partially caused by 1) variation in homozygosity among neighborhoods and 2) biparental inbreeding within neighborhoods. The consequences of this covariance are discussed.     

Phylogeny of Cryptocercus species (Blattodea: Cryptocercidae) inferred from nuclear ribosomal DNA

The wood-feeding cockroaches of the genus Cryptocercus occur in temperate forests. Of the seven known species, five occur in the United States and two in Eurasia. Until 1997, all populations in the United States were considered a single species. Populations in the western United States were elevated to a species status based on variation in DNA sequence and morphology. In 1999, three new species were described from the eastern United States based on variation in chromosome number and mitochondrial DNA, bringing the number of species in the United States to five. The objective of this study was to determine if the DNA sequence of nuclear rRNA also signals the existence of four species in the eastern United States and to compare the inferred relationships with those proposed based on mitochondrial sequences. We obtained the DNA sequence from a portion of the 5.8S and 28S rRNA genes and the entire ITS2 region from 38 individuals and 30 additional clones to assess intraindividual, intraspecific, and interspecific variation. We found extensive sequence variation among the various species and little or no intraindividual and intraspecific variation. Phylogenetic analysis indicated the existence of monophyletic lineages among the eastern United States samples, which largely corresponded to the four species previously described. The inferred relationships were well-supported by bootstrap analysis and decay indices. Although the nuclear rRNA sequences resulted in a coherent phylogenetic tree, the ITS2 region contained many insertions and deletions, which may introduce homoplasy and ambiguity in alignment as more taxa are added to the data set.