Population Genetic Structure of a Widespread Bat-Pollinated Columnar Cactus

Bats are the main pollinators and seed dispersers of Stenocereus thurberi, a xenogamous columnar cactus of northwestern Mexico and a good model to illustrate spatial dynamics of gene flow in long-lived species. Previous studies in this cactus showed differences among populations in the type and abundance of pollinators, and in the timing of flowering and fruiting. In this study we analyzed genetic variability and population differentiation among populations. We used three primers of ISSR to analyze within and among populations genetic variation from eight widely separated populations of S. thurberi in Sonora, Mexico. Sixty-six out of 99 of the ISSR bands (P = 66.7%) were polymorphic. Total heterozygosity for all populations sampled revealed high genetic diversity (H_sp = 0.207, H_BT = 0.224). The AMOVA showed that most of the genetic variation was within populations (80.5%). At the species level, estimates of population differentiation, θ = 0.175 and θ^B = 0.194, indicated moderate gene flow among populations. The absence of a significant correlation between genetic and geographic distances indicated little isolation by geographic distance. The large genetic variation and diversity found in S. thurberi is consistent with its open reproductive system and the high mobility of bats, a major pollinator. However, small changes in number or kind of pollinators and seed dispersal agents, in the directionality of migratory routes, and/or in the timing of flowering and fruiting among populations, can critically affect gene flow dynamics.     

The structural and functional connectivity of the grassland plant Lychnis flos-cuculi

Understanding the relationship between structural and functional connectivity is essential for successful restoration and conservation management, particularly in intensely managed agricultural landscapes. We evaluated the relationship between structural and functional connectivity of the wetland plant Lychnis flos-cuculi in a fragmented agricultural landscape using landscape genetic and network approaches. First, we studied the effect of structural connectivity, such as geographic distance and various landscape elements (forest, agricultural land, settlements and ditch verges), on gene flow among populations as a measurement of functional connectivity. Second, we examined the effect of structural graph-theoretic connectivity measures on gene flow among populations and on genetic diversity within populations of L. flos-cuculi. Among landscape elements, forests hindered gene flow in L. flos-cuculi, whereas gene flow was independent of geographic distance. Among the structural graph-theoretic connectivity variables, only intrapopulation connectivity, which was based on population size, had a significant positive effect on gene flow, that is, more gene flow took place among larger populations. Unexpectedly, interpopulation connectivity of populations, which takes into account the spatial location and distance among populations, did not influence gene flow in L. flos-cuculi. However, higher observed heterozygosity and lower inbreeding was observed in populations characterised by higher structural interpopulation connectivity. This finding shows that a spatially coherent network of populations is significant for maintaining the genetic diversity of populations. Nevertheless, lack of significant relationships between gene flow and most of the structural connectivity measures suggests that structural connectivity does not necessarily correspond to functional connectivity.     

Inter-Simple Sequence Repeat Data Reveals High Genetic Diversity in Wild Populations of the Narrowly Distributed Endemic Lilium regale in the Minjiang River Valley of China

Lilium regale E.H. Wilson is endemic to a narrow geographic area in the Minjiang River valley in southwestern China, and is considered an important germplasm for breeding commercially valuable lily varieties, due to its vigorous growth, resistance to diseases and tolerance for low moisture. We analyzed the genetic diversity of eight populations of L. regale sampled across the entire natural distribution range of the species using Inter-Simple Sequence Repeat markers. The genetic diversity (expected heterozygosity= 0.3356) was higher than those reported for other narrowly distributed endemic plants. The levels of inbreeding (F _st = 0.1897) were low, and most of the genetic variability was found to be within (80.91%) than amongpopulations (19.09%). An indirect estimate of historical levels of gene flow (N _m =1.0678) indicated high levels of gene flow among populations. The eight analyzed populations clustered into three genetically distinct groups. Based on these results, we recommend conservation of large populations representing these three genetically distinct groups.     

Trogia venenata (Agaricales), a novel poisonous species which has caused hundreds of deaths in southwestern China

About 400 unexplained sudden deaths have occurred in Yunnan Province in southwestern China during summers over the last 35?years. Extensive field investigations revealed that a fungus called ??little white mushroom?? or ??nail-like mushroom?? by the local people could be one of the chief causes. Fieldwork showed that this fungus is a common saprophytic species growing on rotten wood in southwestern, central, and northwestern Yunnan in subtropical mountainous areas between 1,700 and 3,000?m altitude. Morphological and molecular phylogenetic analyses revealed that it is a new species of the genus Trogia, closely related to the palaeotropical T. infundibuliformis and its allies. Accordingly, T. venenata is described and illustrated.     

Host genetic variation strongly influences the microbiome structure and function in fungal fruiting‐bodies

Despite increasing knowledge on host‐associated microbiomes, little is known about mechanisms underlying fungus‐microbiome interactions. This study aimed to examine the relative importance of host genetic, geographic and environmental variations in structuring fungus‐associated microbiomes. We analyzed the taxonomic composition and function of microbiomes inhabiting fungal fruiting‐bodies in relation to host genetic variation, soil pH and geographic distance between samples. For this, we sequenced the metagenomes of 40 fruiting‐bodies collected from six fairy rings (i.e., genets) of a saprotrophic fungus Marasmius oreades. Our analyses revealed that fine genetic variations between host fungi could strongly affect their associated microbiome, explaining, respectively, 25% and 37% of the variation in microbiome structure and function, whereas geographic distance and soil pH remained of secondary importance. These results, together with the smaller genome size of fungi compared to other eukaryotes, suggest that fruiting‐bodies are suitable for further genome‐centric studies on host–microbiome interactions.     

Genetic diversity of endangered Manglietia patungensis assessed by inter simple sequence repeat and sequence-related amplified polymorphism markers

Manglietia patungensis Hu is an endangered plant native to China. Knowledge of its genetic diversity and structure would aid its conservation. This study assessed nine natural populations of M. patungensis using two methods: inter simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) markers. Using 10 ISSR primer pairs, 334 bands were generated, and 10 SRAP primer pairs generated 276 bands. The percent of polymorphic bands (91.32% and 93.48%), Nei's genetic diversity (0.3448 and 0.3323), and Shannon's information index (0.5075 and 0.4935) revealed a high level of genetic diversity at the species level. Total heterozygosity was 0.3439 by ISSR and 0.3281 by SRAP. The mean heterozygosity was 0.2323 by ISSR and 0.2521 by SRAP. The coefficient of genetic differentiation among natural populations was 0.3245 by ISSR and 0.2316 by SRAP. These data indicated higher levels of genetic diversity of M. patungensis within, rather than among, populations. Estimates of gene flow among natural populations were 1.0411 and 1.0589, which implied a certain amount of gene exchange among populations. A Mantel test revealed no significant correlation between genetic and geographic distance. ISSR and SRAP markers are both effective for genetic diversity research in M. Patungensis. Based on these results, conservation of M. patungensis should be performed both in situ and ex situ.     

EFFECTIVE POPULATION SIZE IN A CONTINUOUSLY DISTRIBUTED POPULATION

An individual-based simulation model was created to examine genetic variability, time until fixation and spatial genetic structure in a continuously distributed population. Previous mathematical models for continuously distributed populations have the difficulty that the assumption of independent reproduction and independent dispersal of offspring cause clumped spatial distribution and thus violate an assumption of random spatial distribution. In this study, this problem is avoided by considering the dispersal behavior of offspring. The simulation results showed that the inbreeding effective population size estimated by the rate of decrease of heterozygosity during the first 15 generations corresponds to the neighborhood size calculated by the standard deviation of the dispersal distance (σ_T). This inbreeding effective population size does not greatly change with the area of simulation when the densities and σ_T are the same. However, the inbreeding effective population size estimated by heterozygosity using the first 500 generations is larger than the neighborhood size calculated by the dispersal distance and increases with the area of simulation with the same densities. The variance effective population size, estimated by time until fixation of alleles, increases with dispersal distance (σ_T) and with the area of simulation given the same densities. The inbreeding effective population size and variance effective population size were smaller than the actual population size unless σ_T is sufficiently large (2 σ_T > approximate L/2, where L is a side of the simulation square).     

Genetic structure of Himalayan marmot (Marmota himalayana) population alongside the Qinghai–Tibet Railway

In order to verify the effect of social behavior and geographical isolation on the genetic structure of the Himalayan marmot (Marmota himalayana) population, we examined the genetic diversity of Himalayan marmots alongside the Qinghai–Tibet Railway using microsatellite markers. Eight microsatellite loci were used to examine 120 animals of 4 populations: Ulan (U), Delhi (D), Tuotuohe (T) and Ando (A). The results show that: (1) Himalayan marmots alongside the Qinghai–Tibet Railway are highly genetically diversified. The allele number (Na), effective allele number (Ne), observed heterozygosity (Ho), Nei’s expected heterozygosity (He) and polymorphism information content (PIC) of the total Himalayan marmot population were 4.75, 3.0332, 0.6990, 0.6672, 0.6102, respectively. (2) Himalayan marmots may be able to avoid inbreeding by a mechanism that will prevent the genetic diversity reduction caused by their social lifestyle. Heterozygote excess was observed at most loci. The inbreeding coefficients within the subpopulation (F_IS), in the total population (F_IT), the differentiation index of population (F_ST), and the gene flow (Nm) were ?0.2265, ?0.0477, 0.1458, and 1.4646, respectively. (3) The genetic differentiation of the Himalayan marmot population was in accordance with Wright’s “isolation by distance” theory. The Mantel test indicates that the correlation between genetic distance and geographic distance was significant (P < 0.05, r = 0.698). (4) Each of the four geographical populations had moderate differentiation. Both geographic distance and isolation could affect the population genetic structure of the Himalayan marmot. The maximum gene flow (3.5915), the smallest genetic differentiation index (0.0651), the lowest genetic distance (0.0700) and the highest genetic identity (0.9526) were all between the Ulan population and Delhi populations. (5) The cluster analysis, based on Nei’s standard genetic distance, showed that the populations of Delhi and Ulan were first merged in a cluster, and then Tuotuohe population was merged in the clustering. The Ando population was the last element in the clustering.     

Estimating inbreeding depression in natural plant populations using quantitative and molecular data

Inbreeding and inbreeding depression are important issues in the biology and conservation of natural plant and animal populations, primarily when subpopulation sizes are reduced due to habitat fragmentation. In this study, we propose a method for estimating inbreeding depression in progenies of natural plant populations, combining the estimation of the fixation index by codominant markers with the experimental evaluation of quantitative traits. Our technique estimates apparent inbreeding depression in structured natural populations using the linear regression of phenotypic means on the inbreeding coefficients estimated with codominant markers. This method was applied to data from 112 maternal progenies of 10 natural subpopulations of Eugenia dysenterica DC, a fruiting tree species from the Brazilian savanna (Cerrado). The results show that the proposed method was efficient at detecting the presence of inbreeding depression for seedling emergence and initial growth traits in the species. This corroborates the importance of maintaining high levels of heterozygosity for in situ conservation or genetic restoration of natural populations.     

Invasion genetics of <Emphasis Type="Italic">Senecio vulgaris</Emphasis>: loss of genetic diversity characterizes the invasion of a selfing annual,despite multiple introductions

Genetic variation in invasive populations is affected by a variety of processes including stochastic forces, multiple introductions, population dynamics and mating system. Here, we compare genetic diversity between native and invasive populations of the selfing, annual plant Senecio vulgaris to infer the relative importance of genetic bottlenecks, multiple introductions, post-introduction genetic drift and gene flow to genetic diversity in invasive populations. We scored multilocus genotypes at eight microsatellite loci from nine native European and 19 Chinese introduced populations and compared heterozygosity and number of alleles between continents. We inferred possible source populations for introduced populations by performing assignment analyses and evaluated the relative contributions of gene flow and genetic drift to genetic diversity based on correlations of pairwise genetic and geographic distance. Genetic diversity within Chinese populations was significantly reduced compared to European populations indicating genetic bottlenecks accompanying invasion. Assignment tests provided support for multiple introductions with populations from Central China and southwestern China descended from genotypes matching those from Switzerland and the UK, respectively. Genetic differentiation among populations in China and Europe was not correlated with geographic distance. However, European populations exhibited less variation in the relation between G _ST and geographical distance than populations in China. These results suggest that gene flow probably plays a more significant role in structuring genetic diversity in native populations, whereas genetic drift appears to predominate in introduced populations. High rates of selfing in Chinese populations may restrict opportunities for pollen-mediated gene flow. Repeated colonization-extinction cycles associated with ongoing invasion is likely to maintain low genetic diversity in Chinese populations.     

Population genetic characterization of the endangered dung beetle Copris tripartitus (Coleoptera: Scarabaeidae) using novel microsatellite markers

The dung beetle Copris tripartitus (Coleoptera: Scarabaeidae) has long been considered an endangered insect in South Korea; the detection of recent population increases leaves its endangered status uncertain. Population genetic analysis subsequent to development of molecular markers is essential for establishing proper conservation strategies. In this study, we developed ten microsatellite markers specifically for C. tripartitus. Sixty-eight individuals of C. tripartitus collected from six South Korean localities were genotyped to validate these markers and preliminarily assess population genetic characteristics. Per-locus observed number of alleles, observed heterozygosity (H_O), and expected heterozygosity (H_E) ranged from 5–12, 0.499–0.958, and 0.54–0.743, respectively. All populations showed higher H_O than H_E, negative values of inbreeding coefficient, and, overall, no sign of recent population bottlenecks (excluding one population, Seosan). This suggests that C. tripartitus did not suffer from genetic drift and inbreeding, which are typically severe in small, isolated populations. Nevertheless, detection of only one of the two gene pools in some populations and resultant genetic subdivision into two population groups may suggest that the population size is not enough to cover both gene pools. Thus, a more extended period of protection may be required to ensure higher genetic diversity of widespread populations and achieve the long-term conservation goal.     

Characterization of vitamin B12 compounds in the fruiting bodies of shiitake mushroom (Lentinula edodes) and bed logs after fruiting of the mushroom

This study determined the vitamin B₁₂ content in commercially available dried fruiting bodies of shiitake mushroom, Lentinula edodes. The vitamin B₁₂ contents in dried donko-type fruiting bodies with closed caps (5.61 ± 3.90 μg/100 g dry weight), did not significantly differ from those of dried koushin-type fruiting bodies with open caps (4.23 ± 2.42 μg/100 g dry weight). The bed logs after fruiting of the mushroom also contained the vitamin B₁₂ levels similar to that in the dried shiitake fruiting bodies. To determine whether the dried shiitake fruiting bodies and their bed logs contained vitamin B₁₂ or other corrinoid compounds that are inactive in humans, we purified corrinoid compounds using an immunoaffinity column and identified vitamin B₁₂ using vitamin B₁₂-dependent Escherichia coli 215 bioautograms and liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) chromatograms. Dried shiitake fruiting bodies rarely contained an unnatural corrinoid vitamin B₁₂[c-lactone] that is inactive in humans. Given that shiitake mushroom lacks the ability to synthesize vitamin B₁₂ de novo, the vitamin B₁₂ found in dried shiitake fruiting bodies must have been derived from the bed logs.     

Population Genetic Structure of the Endangered Kaiser’s Mountain Newt,Neurergus kaiseri (Amphibia: Salamandridae)

Species often exhibit different levels of genetic structuring correlated to their environment. However, understanding how environmental heterogeneity influences genetic variation is difficult because the effects of gene flow, drift and selection are confounded. We investigated the genetic variation and its ecological correlates in an endemic and critically endangered stream breeding mountain newt, Neurergus kaiseri, within its entire range in southwestern Iran. We identified two geographic regions based on phylogenetic relationships using Bayesian inference and maximum likelihood of 779 bp mtDNA (D-loop) in 111 individuals from ten of twelve known breeding populations. This analysis revealed a clear divergence between northern populations, located in more humid habitats at higher elevation, and southern populations, from drier habitats at lower elevations regions. From seven haplotypes found in these populations none was shared between the two regions. Analysis of molecular variance (AMOVA) of N. kaiseri indicates that 94.03% of sequence variation is distributed among newt populations and 5.97% within them. Moreover, a high degree of genetic subdivision, mainly attributable to the existence of significant variance among the two regions is shown (θ_CT = 0.94, P = 0.002). The positive and significant correlation between geographic and genetic distances (r = 0.61, P = 0.002) following controlling for environmental distance suggests an important influence of geographic divergence of the sites in shaping the genetic variation and may provide tools for a possible conservation based prioritization policy for the endangered species.     

Genetic variation in Ecballium elaterium (Cucurbitaceae): Breeding system and geographic distribution

Dioecy, the separation of sexes, has arisen independently many times in the course of angiosperm evolution. Avoidance of inbreeding is clearly involved in the evolution of dioecy, and as a consequence we predict that dioecious populations should maintain higher levels of genetic variation than closely related nondioecious populations. We tested that prediction by comparing allozymic variation in two closely related taxa, the monoecious and dioecious subspecies of the Mediterranean cucurbit, Ecballium elaterium. Thirteen polymorphic loci were screened for seeds sampled from 10 monoecious and 13 dioecious populations spanning the geographic ranges of the subspecies in Spain. The dioecious subspecies showed strikingly greater allelic diversity and heterozygosity than the monoecious subspecies. A hierarchical F-statistic analysis clearly demonstrated considerable genetic variation within populations for the dioecious populations, whereas for the monoecious populations almost all genetic variation resulted from differences among populations. The general pattern of homozygosity within monoecious populations suggests that they are highly inbred. In order to assess historical influences on current patterns of genetic variation, we conducted a genetic-distance analysis. The observed relationship between genetic distance and geographic distance between populations supports the hypothesis that the subspecies' current allopatric distributions on the Iberian Peninsula are the result of separate waves of colonization from the north (monoecious) and south (dioecious).     

Evidence against Barium in the Mushroom Trogia venenata as a Cause of Sudden Unexpected Deaths in Yunnan,China

This study examined barium concentrations in the mushroom Trogia venenata, the leading culprit for sudden unexpected deaths in Yunnan, southwest China. We found that barium concentrations in T. venenata from Yunnan were low and comparable to other foods, inconsistent with barium concentrations in this mushroom as a significant contributor to these deaths.     

Pleurotus and Agrocybe hemolysins,new proteins hypothetically involved in fungal fruiting

Novel hemolytic proteins, ostreolysin and aegerolysin, were purified from the fruiting bodies of the edible mushrooms Pleurotus ostreatus and Agrocybe aegerita. Both ostreolysin and aegerolysin have a molecular weight of about 16 kDa, have low isoelectric points of 5.0 and 4.85, are thermolabile, and hemolytic to bovine erythrocytes at nanomolar concentrations. Their activity is impaired by micromolar Hg²⁺ but not by membrane lipids and serum low-density lipoproteins (LDL). The sequence of respectively 50 and 10 N-terminal amino acid residues of ostreolysin and aegerolysin has been determined and found to be highly identical with a cDNA-derived amino acid sequence of putative Aa-Pri1 protein from the mushroom A. aegerita, Asp-hemolysin from Aspergillus fumigatus, and two bacterial hemolysin-like proteins expressed during sporulation. We found that ostreolysin is expressed during formation of primordia and fruiting bodies, which is in accord with previous finding that the Aa-Pri1 gene is specifically expressed during fruiting initiation. It is suggestive that the isolated hemolysins play an important role in initial phase of fungal fruiting.     

Development of twelve novel microsatellite markers in the medicinal mushroom Cordyceps militaris (Ascomycota: Clavicipitaceae) for its strain identification and genetic analysis

Cordyceps militaris (a caterpillar fungus), which belongs to the class Ascomycetes, has extensively been used for medicinal purposes in East Asia. Here, we isolated and characterized 12 microsatellite loci from the medicinal mushroom, C. militaris. Twenty‐nine individual samples were taken from a single locality in southwestern Korea and used to characterize the developed markers. The number of alleles of these loci ranged from two to 13 (mean allelic richness = 6.44). Observed heterozygosity and expected heterozygosity in the population ranged from 0.035 to 0.880 and from 0.035 to 0.886, respectively. Five of 12 loci significantly deviated from Hardy–Weinberg Equilibrium, most likely due to heterozygote deficiency caused by inbreeding. Tests of genotypic linkage disequilibrium between the 12 loci showed no significant association of alleles. These microsatellite markers will provide valuable tools for genetic analyses for the strain identification of C. militaris as well as for its resource conservation.     

Cell patterning in branched and unbranched fruiting bodies of the cellular slime mold Polysphondylium pallidum

Cell patterning, the percentage of spores and stalk cells, was measured in branched and unbranched asexual fruiting bodies of Polysphondylium pallidum. Unlike D. discoideum, where small and large fruiting bodies are more stalky than average-sized fruiting bodies, the overall cell patterning was the same in branched and unbranched fruiting bodies of all sizes in P. pallidum. Light greatly increased the numbers of fruiting bodies in P. pallidum per unit area (or decreased aggregation territory size) so that most fruiting bodies formed in the light were small and unbranched. By contrast, light had little effect on the cell patterning of P. pallidum, although there was a slight increase in the percentage of stalk cells in the light compared to the dark. This indicates that the mechanisms governing light sensitivity of aggregation territory size and cell patterning have different components in P. pallidum. The accuracy of cell patterning of individual branches of branched fruiting bodies was so imprecise as to leave doubt that patterning is occurring at the branch level. Individual whorls of branched fruiting bodies had a greater percentage spores (90%) than whole fruiting bodies (78%) and the cell patterning was relatively imprecise. Only in whole fruiting bodies was the spore:stalk ratio highly correlated. These findings are consistent with cell pattern determination operating at the whole aggregate level, rather than at the individual whorl or branch level in P. pallidum.     

Increased inbreeding but not homozygosity in small populations of Sabatia angularis (Gentianaceae)

Understanding how the mating system varies with population size in plant populations is critical for understanding their genetic and demographic fates. We examined how the mating system, characterized by outcrossing rate, biparental inbreeding rate, and inbreeding coefficient, and genetic diversity varied with population size in natural populations of the biennial Sabatia angularis. We found a significant, positive relationship between outcrossing and population size. Selfing was as high as 40% in one small population but was only 7% in the largest population. Despite this pattern, observed heterozygosity did not vary with population size, and we suggest that selection against inbred individuals maintains observed heterozygosity in small populations. Consistent with this hypothesis, we found a trend of lower inbreeding coefficients in the maternal than progeny generation in all of the populations, and half of the populations exhibited significant excesses of adult heterozygosity. Moreover, genetic diversity was not related to population size and was similar across all populations examined. Our results suggest that the consequences of increased selfing for population fitness in S. angularis, a species that experiences significant inbreeding depression, will depend on the relative magnitude and consistency of inbreeding depression and the demographic cost of selection for outcrossed progeny in small populations.     

Autotetraploids of Vicia cracca show a higher allelic richness in natural populations and a higher seed set after artificial selfing than diploids

Background and Aims

Despite the great importance of autopolyploidy in the evolution of angiosperms, relatively little attention has been devoted to autopolyploids in natural polyploid systems. Several hypotheses have been proposed to explain why autopolyploids are so common and successful, for example increased genetic diversity and heterozygosity and the transition towards selfing. However, case studies on patterns of genetic diversity and on mating systems in autopolyploids are scarce. In this study allozymes were employed to investigate the origin, population genetic diversity and mating system in the contact zone between diploid and assumed autotetraploid cytotypes of Vicia cracca in Central Europe.

Methods

Four enzyme systems resolved in six putative loci were investigated in ten diploid, ten tetraploid and five mixed-ploidy populations. Genetic diversity and heterozygosity, partitioning of genetic diversity among populations and cytotypes, spatial genetic structure and fixed heterozygosity were analysed. These studies were supplemented by a pollination experiment and meiotic chromosome observation.

Key Results and Conclusions

Weak evidence of fixed heterozygosity, a low proportion of unique alleles and genetic variation between cytotypes similar to the variation among populations within cytotypes supported the autopolyploid origin of tetraploids, although no multivalent formation was observed. Tetraploids possessed more alleles than diploids and showed higher observed zygotic heterozygosity than diploids, but the observed gametic heterozygosity was similar to the value observed in diploids and smaller than expected under panmixis. Values of the inbreeding coefficient and differentiation among populations (ρ_ST) suggested that the breeding system in both cytotypes of V. cracca is mixed mating with prevailing outcrossing. The reduction in seed production of tetraploids after selfing was less than that in diploids. An absence of correlation between genetic and geographic distances and high differentiation among neighbouring tetraploid populations supports the secondary contact hypothesis with tetraploids of several independent origins in Central Europe. Nevertheless, the possibility of a recent in situ origin of tetraploids through a triploid bridge in some regions is also discussed.