Development of microsatellite markers from an ectomycorrhizal fungus,Laccaria amethystina,by a dual‐suppression‐PCR technique

Laccaria amethystina is a major early successional ectomycorrhizal fungus. We isolated 10 polymorphic loci from L. amethystina using a dual‐suppression‐PCR (polymerase chain reaction) technique. These loci provided microsatellite markers with polymorphism of two to 10 alleles per locus. The observed and expected heterozygosities ranged from 0.136 to 0.545 and 0.206 to 0.877, respectively.     

Beech roots are simultaneously colonized by multiple genets of the ectomycorrhizal fungus Laccaria amethystina clustered in two genetic groups

In this study, we characterize and compare the genetic structure of aboveground and belowground populations of the ectomycorrhizal fungus Laccaria amethystina in an unmanaged mixed beech forest. Fruiting bodies and mycorrhizas of L. amethystina were mapped and collected in four plots in the ?wi?tokrzyskie Mountains (Poland). A total of 563 fruiting bodies and 394 mycorrhizas were successfully genotyped using the rDNA IGS1 (intergenic spacer) and seven simple sequence repeat markers. We identified two different genetic clusters of L. amethystina in all of the plots, suggesting that a process of sympatric isolation may be occurring at a local scale. The proportion of individuals belonging to each cluster was similar among plots aboveground while it significantly differed belowground. Predominance of a given cluster could be explained by distinct host preferences or by priority effects and competition among genets. Both aboveground and belowground populations consisted of many intermingling small genets. Consequently, host trees were simultaneously colonized by many L. amethystina genets that may show different ecophysiological abilities. Our data showed that several genets may last for at least 1 year belowground and sustain into the next season. Ectomycorrhizal species reproducing by means of spores can form highly diverse and persistent belowground genets that may provide the host tree with higher resilience in a changing environment and enhance ecosystem performance.     

Temporal persistence and spatial distribution of an American inoculant strain of the ectomycorrhizal basidiomycete Laccaria bicolor in a French forest plantation

Selected strains of ectomycorrhizal fungi, such as the basidiomycete Laccaria bicolor , are currently being used as inoculants in nurseries to improve growth of forest trees after outplanting. Information is needed on the survival of these introduced strains in forests and their impact on indigenous biodiversity. Dissemination and persistence of an American strain, L. bicolor S238N, were studied 10 years after outplanting in a Douglas fir plantation located at Saint-Brisson (Morvan, France). About 430 Laccaria spp. sporophores were collected over 3 years. Inheritance of nuclear ribosomal DNA, as well as RAPD markers, was characterized in L. bicolor S238N, using a haploid progeny set of 91 monokaryons. More than 50 markers were identified (19 heterozygous and 33 homozygous or cytoplasmic markers), which unambiguously confirmed that the introduced strain was still present in the inoculated plots. Neither selfing ( P < 0.0008) nor introgression with indigenous strains was detected although in vitro interfertility between the American strain and indigenous L. bicolor was identified. No ingress of the introduced genet into adjacent uninoculated plots colonized by various local Laccaria genets was detected. It is proposed that the spatial distributions identified have developed through mycelial propagation of the introduced strain and intraspecific competition with native genets. Although longer-term data is still lacking, the stability of the inoculant strain and the limited disturbance to indigenous populations described support large-scale nursery production of this host-fungal combination.     

Isolation and characterization of five microsatellite loci in an ectomycorrhizal fungus Laccaria laccata

Laccaria laccata is an early successional ectomycorrhizal fungus. We isolated five polymorphic microsatellite loci from L. laccata using a dual‐suppression polymerase chain reaction technique. The number of alleles per locus ranged from three to six. The observed and expected heterozygosities ranged from 0.269 to 0.462, and 0.249 to 0.775, respectively. These microsatellite markers would be valuable molecular tools for population genetic studies of L. laccata.     

Correspondence between genet diversity and spatial distribution of above- and below-ground populations of the ectomycorrhizal fungus Hebeloma cylindrosporum

Population studies of ectomycorrhizal fungal species have largely relied upon fruit body (the reproductive organ) sampling. Analysis of the fruit bodies alone supposes that they reflect the present and spatial organization of all below-ground genets (mycorrhizas and extramatrical mycelia). The relation between fruit bodies and ectomycorrhizas was investigated for the basidiomycete agaric Hebeloma cylindrosporum in four Pinus pinaster stands in south-west France. Genet identification was based on the comparison of polymorphisms within a hypervariable segment of the ribosomal intergenic spacer amplified by polymerase chain reaction (PCR) using a H. cylindrosporum species-specific primer. Mycorrhizas were sorted from soil samples collected underneath patches of fruit bodies or patches where fruit bodies had or had not been observed during the years prior to mycorrhiza collection. On average 65% of the 1026 mycorrhizas collected underneath fruit bodies were formed by H. cylindrosporum, whereas only 2% of the 954 collected in places from where fruit bodies were absent were formed by this species. All genotypes identified above ground were also identified below ground. In patches where one genotype formed all or more than 90% of the fruit bodies, the same genotype formed all or a large majority of the mycorrhizas. In patches occupied by several different fruiting genotypes, additional nonfruiting ones could be present on the root systems. In all cases, the mycorrhizas of one genotype were found no more than 10-20 cm away from its corresponding fruit bodies, and fruit body disappearance at a given place was associated with the disappearance of the corresponding mycorrhizas within 1 year. Although there was not a strict coincidence between the total numbers of genets present below ground and of those forming fruit bodies, fruit body analysis for H. cylindrosporum appears to reflect both the genetic diversity and the spatial structure of its below-ground populations. The results obtained also illustrate the rapid turnover of ectomycorrhizal fungal species on the root systems in the absence of any obvious major disturbance of the ecosystem.     

Extensive gene flow over Europe and possible speciation over Eurasia in the ectomycorrhizal basidiomycete Laccaria amethystina complex

Biogeographical patterns and large-scale genetic structure have been little studied in ectomycorrhizal (EM) fungi, despite the ecological and economic importance of EM symbioses. We coupled population genetics and phylogenetic approaches to understand spatial structure in fungal populations on a continental scale. Using nine microsatellite markers, we characterized gene flow among 16 populations of the widespread EM basidiomycete Laccaria amethystina over Europe (i.e. over 2900 km). We also widened our scope to two additional populations from Japan (10(4) km away) and compared them with European populations through microsatellite markers and multilocus phylogenies, using three nuclear genes (NAR, G6PD and ribosomal DNA) and two mitochondrial ribosomal genes. European L. amethystina populations displayed limited differentiation (average F(ST) = 0.041) and very weak isolation by distance (IBD). This panmictic European pattern may result from effective aerial dispersal of spores, high genetic diversity in populations and mutualistic interactions with multiple hosts that all facilitate migration. The multilocus phylogeny based on nuclear genes confirmed that Japanese and European specimens were closely related but clustered on a geographical basis. By using microsatellite markers, we found that Japanese populations were strongly differentiated from the European populations (F(ST) = 0.416), more than expected by extrapolating the European pattern of IBD. Population structure analyses clearly separated the populations into two clusters, i.e. European and Japanese clusters. We discuss the possibility of IBD in a continuous population (considering some evidence for a ring species over the Northern Hemisphere) vs. an allopatric speciation over Eurasia, making L. amethystina a promising model of intercontinental species for future studies.     

High genetic diversity in a small population: the case of Chilean blue whales

It is generally assumed that species with low population sizes have lower genetic diversities than larger populations and vice versa. However, this would not be the case for long‐lived species with long generation times, and which populations have declined due to anthropogenic effects, such as the blue whale (Balaenoptera musculus). This species was intensively decimated globally to near extinction during the 20th century. Along the Chilean coast, it is estimated that at least 4288 blue whales were hunted from an apparently pre‐exploitation population size (k) of a maximum of 6200 individuals (Southeastern Pacific). Thus, here, we describe the mtDNA (control region) and nDNA (microsatellites) diversities of the Chilean blue whale aggregation site in order to verify the expectation of low genetic diversity in small populations. We then compare our findings with other blue whale aggregations in the Southern Hemisphere. Interestingly, although the estimated population size is small compared with the pre‐whaling era, there is still considerable genetic diversity, even after the population crash, both in mitochondrial (N = 46) and nuclear (N = 52) markers (H_d = 0.890 and H_o = 0.692, respectively). Our results suggest that this diversity could be a consequence of the long generation times and the relatively short period of time elapsed since the end of whaling, which has been observed in other heavily‐exploited whale populations. The genetic variability of blue whales on their southern Chile feeding grounds was similar to that found in other Southern Hemisphere blue whale feeding grounds. Our phylogenetic analysis of mtDNA haplotypes does not show extensive differentiation of populations among Southern Hemisphere blue whale feeding grounds. The present study suggests that although levels of genetic diversity are frequently used as estimators of population health, these parameters depend on the biology of the species and should be taken into account in a monitoring framework study to obtain a more complete picture of the conservation status of a population.     

Fine-scale structure of populations of the ectomycorrhizal fungus Hebeloma cylindrosporum in coastal sand dune forest ecosystems

The basidiomycete mushroom Hebeloma cylindrosporum is a frequently found pioneer ectomycorrhizal species naturally associated with Pinus pinaster trees growing in coastal sand dune ecosystems along the Atlantic south-west coast of France. The genotypic diversity and spatial structure of three populations of this fungal species have been studied. At each site the basidiocarps were mapped, sampled and propagated as pure mycelial cultures. For each of the isolates, we have studied polymorphisms in the mitochondrial genome, polymorphisms at two different nuclear loci and also fingerprints produced with a multicopy DNA probe. The comparison of the different polymorphisms obtained, with each of the four molecular methods used, allowed the identification of several of the different genets present in each site. In two of the studied sites most of the basidiocarps, which often occurred as dense patches of 10–30 in 1 m² or less, were of a unique genotype, suggesting the below-ground mycelia to be of a small size (from 50 cm² to approx. 7 m² for the larger mycelia) and that the root system of a single Pinus tree can host several genets of the same symbiotic fungus. In the two sites, which were studied again after a 3-year interval, none of the genotypes identified in the first year of sampling was re-identified 3 years later. These results contrast with those reported for other species of soilborne homobasidiomycete species, either ectomycorrhizal, parasitic or saprophytic, showing mostly large clones resulting from the vegetative growth and from persistence of below-ground mycelia. Sexual reproduction through meiospore dispersal seems to play a key role in the structuring of the populations of H. cylindrosporum. Mycelia associated with the root systems seem to be replaced after 1 or a few years, during which basidiocarp differentiation takes place. As opposed to the few other studied ectomycorrhizal species, H. cylindrosporum has the characteristics of ruderal species, with a short life-span adapted to pioneer situations, e.g. to nutrient-poor and unstable sandy soils of coastal sand dunes.     

Effects of repeated harvesting of forest residues on the ectomycorrhizal community in a Swedish spruce forest

The use of biofuels has been proposed as one possible substitute for fossil fuels, which contribute substantially to the increase in [CO₂] in the atmosphere. However, increased harvesting of forest residues for biofuel might affect the availability of base cations, P and N, as well as the development, community dynamics and function of ectomycorrhizas. This in turn might influence nutrient uptake and tree growth. In this study we investigated the effects of repeated forest residue harvesting on ectomycorrhizal species colonizing spruce roots in the humus layer of a 35-yr-old forest. Harvesting significantly decreased the thickness of the humus layer as well as decreasing the numbers of ectomycorrhizal root tips both per metre root length and per unit humus volume. Changes in mycorrhizal community structure were studied by ITS typing with the use of PCR–RFLP analysis. In total, 19 different ITS types were found on two different sampling occasions (autumn and spring); 11 of these were common to both samplings. Nine of the ITS types were identified to at least the genus level by comparison with RFLP patterns of identified fruiting bodies or axenic cultures. Five species, Cortinarius sp. 2, Thelephora terrestris (Ehrenb.) Fr., Lactarius theiogalus (Bull.:Fr.) S. F. Gray s.st. Neuhoff, Tylospora fibrillosa Donk and Tö-96-12, occurred on over 5% of the total sampled root tips. Together these five types colonized 63% of the mycorrhizas screened. A similarity index assessment showed no shift in mycorrhizal community structure as a result of harvesting. Our findings suggest that the repeated removal of forest residues might have a strong effect on the quantity and development of ectomycorrhizal roots in the organic horizon, but little effect on the species composition of the community.     

Fine-scale population genetic structure in a fission-fusion society

Nonrandom patterns of mating and dispersal create fine-scale genetic structure in natural populations — especially of social mammals — with important evolutionary and conservation genetic consequences. Such structure is well-characterized for typical mammalian societies; that is, societies where social group composition is stable, dispersal is male-biased, and males form permanent breeding associations in just one or a few social groups over the course of their lives. However, genetic structure is not well understood for social mammals that differ from this pattern, including elephants. In elephant societies, social groups fission and fuse, and males never form permanent breeding associations with female groups. Here, we combine 33 years of behavioural observations with genetic information for 545 African elephants ( Loxodonta africana ), to investigate how mating and dispersal behaviours structure genetic variation between social groups and across age classes. We found that, like most social mammals, female matrilocality in elephants creates co-ancestry within core social groups and significant genetic differentiation between groups (Φ_ST = 0.058). However, unlike typical social mammals, male elephants do not bias reproduction towards a limited subset of social groups, and instead breed randomly across the population. As a result, reproductively dominant males mediate gene flow between core groups, which creates cohorts of similar-aged paternal relatives across the population. Because poaching tends to eliminate the oldest elephants from populations, illegal hunting and poaching are likely to erode fine-scale genetic structure. We discuss our results and their evolutionary and conservation genetic implications in the context of other social mammals.     

Genetic diversity is retained in a bottlenecked Cinereous Vulture population in Turkey

Vulture populations worldwide have suffered precipitous declines in recent decades. The Cinereous Vulture Aegypius monachus, a highly philopatric scavenger distributed across southern Europe and the central Asian plateau, is threatened in many parts of its range. Turkey holds the second largest population of this species in the Western Palaearctic, but there has been no research on its genetic structure and the possible implications of this structure for the future of the species. Here we report nuclear diversity and relatedness determined by short tandem repeat genotyping of 81 individuals from the four largest colonies. Our results demonstrated no significant genetic structuring, suggesting a single panmictic metapopulation connected by frequent dispersal. Furthermore, we show that the study population has retained moderate levels of genetic diversity, despite passing through a recent demographic bottleneck. We estimated the effective population size to be 112 individuals (95% confidence interval 74–201). Our results imply that the observed lack of increase in population size since the 1990s has not been caused by lowered fitness due to genetic inbreeding but rather by increased mortality via demographic processes. In the short term, we suggest that conservation efforts should treat the Turkish subpopulations as a single management unit and aim to increase population size through effective protection, especially during the breeding season.     

High genetic variation in leopards indicates large and long-term stable effective population size

In this paper we employ recently developed statistical and molecular tools to analyse the population history of the Tanzanian leopard (Panthera pardus), a large solitary felid. Because of their solitary lifestyle little is known of their past or present population dynamics. Eighty-one individuals were scored at 18 microsatellite loci. Overall, levels of heterozygosity were high (0.77 +/- 0.03), with a small heterozygote deficiency (0.06 +/- 0.03). Effective population size (Ne) was calculated to be 38 000-48 000. A Ne:N ratio of 0.42 (average from four cat studies) gives a present population size of about 100 000 leopards in Tanzania. Four different bottleneck tests indicated that this population has been large and stable for a minimum of several thousand years. FST values were low and no significant genetic structuring of the population could be detected. This concurs well with the large migration values (Nm) obtained (>3.3 individuals/generation). Our analysis reveals that ecological factors (e.g. disease), which are known to have had major impact on other carnivore populations, are unlikely to have impacted strongly on the population dynamics of Tanzanian leopards. The explanation may be found in their solitary life-style, their often nonconfrontational behaviour toward interspecific competitors, or that any bottlenecks have been of limited size, localized, or too short to have affected genetic variation to any measurable degree. Since the genetic structuring is weak, gene flow is not restricted to within protected areas. Local loss of genetic variation is therefore not of immediate concern.     

Extremely low effective population sizes, genetic structuring and reduced genetic diversity in a threatened bumblebee species, Bombus sylvarum (Hymenoptera: Apidae)

Habitat fragmentation may severely affect survival of social insect populations as the number of nests per population, not the number of individuals, represents population size, hence they may be particularly prone to loss of genetic diversity. Erosion of genetic diversity may be particularly significant among social Hymenoptera such as bumblebees (Bombus spp.), as this group may be susceptible to diploid male production, a suggested direct cost of inbreeding. Here, for the first time, we assess genetic diversity and population structuring of a threatened bumblebee species (Bombus sylvarum) which exists in highly fragmented habitat (rather than oceanic) islands. Effective population sizes, estimated from identified sisterhoods, were very low (range 21-72) suggesting that isolated populations will be vulnerable to loss of genetic variation through drift. Evidence of significant genetic structuring between populations (theta = 0.084) was found, but evidence of a bottleneck was detected in only one population. Comparison across highly fragmented UK populations and a continental population (where this species is more widespread) revealed significant differences in allelic richness attributable to a high degree of genetic diversity in the continental population. While not directly related to population size, this is perhaps explained by the high degree of isolation between UK populations relative to continental populations. We suggest that populations now existing on isolated habitat islands were probably linked by stepping-stone populations prior to recent habitat loss.     

Population genetic structure and diversity of the endangered Cantabrian capercaillie

The Cantabrian capercaillie (Tetrao urogallus cantabricus) occupies the southwestern edge of the grouse family distribution range in Eurasia. It is endemic to the Cantabrian Mountains in northwestern Spain and is geographically isolated and separated from the neighboring population in the Pyrenees by a distance of 300 km. Over the last decades, the population has undergone a dramatic decline and is now threatened with extinction. This study presents the genetic analysis of the Cantabrian capercaillie population using non-invasive samples. We performed genotyping of 45 individuals using 20 microsatellites and a sex marker. The data highlight the need for using a large number of markers when considering fragmented small populations. Genetic diversity (H_E = 0.50) and average number of alleles (3.40) in the population were low. The population is fragmented into 2 clusters (F_ST = 0.113) that fit with areas on both sides of the transportation ways that divide its range. Both clusters exhibited additional heterozygote deficits. Geographical distance was negatively correlated with genetic relatedness (r = −0.44, P ≤ 0.001). The data show a recent decline in effective population size that can be related to an ongoing process of population reduction and fragmentation. Conservation actions should focus on the protection of local demes by maintaining a dense network of suitable patches to maximize reproductive output and the number of potential dispersers to reconnect the 2 subpopulations. © 2012 The Wildlife Society.     

High genetic diversity of the endangered Iberian three‐spined stickleback (Gasterosteus aculeatus) at the Mediterranean edge of its range

1. The three‐spined stickleback (Gasterosteus aculeatus) on the Iberian Peninsula is only distributed in freshwater habitats and has completely disappeared from most of its range, mainly as a consequence of habitat degradation and invasive fish introductions. Genetic investigations have shown that Mediterranean‐Adriatic sticklebacks constitute an evolutionarily significant unit. Here, we present the first genetic data for Iberian populations living on the southern edge of the stickleback’s range. We used microsatellite markers to study gene diversity, population structure and genetic demography of stickleback populations. 2. High genetic differentiation among collections yielded a model of four genetically homogeneous units related to geography. The observed pattern of isolation by distance resulted mainly from the hydrographical pattern and limited gene flow among rivers. Moreover, low levels of gene diversity, high isolation and recent bottleneck events, which have led to small or even critical effective population sizes in several locations, could be explained by additional recent anthropogenic fragmentation. 3. We defined at least four evolutionarily significant units threatened by habitat fragmentation in north‐eastern Iberian sticklebacks. Because they retain long evolutionary histories, these populations should be considered of high conservation priority, and urgent management measures should be implemented.     

Monitoring the persistence of Laccaria bicolor as an ectomycorrhizal symbiont of nursery-grown Douglas fir by PCR of the rDNA intergenic spacer

The large-scale inoculation of selected beneficial ectomycorrhizal fungi in forest nurseries has generated renewed interest in the ecology of these symbiotic fungi. However, information on the dissemination and persistence of introduced symbionts is scarce due to the limitation of the current identification methods. To identify ectomycorrhizal fungi on single root tips, we investigated the polymorphism of the PCR-amplified ribosomal DNA intergenic spacer (IGS) from a wide range of ectomycorrhizal fungi. To investigate the reliability of this molecular approach in large-scale surveys, the dissemination and persistence on Douglas fir seedlings of the introduced Laccaria bicolor S238N were assessed in a forest nursery in the Massif Central (France). Several hundred ectomycorrhizas and fruiting bodies were sampled from plots where control and L. bicolor inoculated-Douglas fir seedlings were grown for 1.5 years. PCR typing of mycorrhizas indicated that trees inoculated with L. bicolor S238N remained exclusively colonized by that isolate (or sexually derived isolates) for the entire test period. In contrast, control seedlings were infected by indigenous isolates of Laccaria laccata and Thelephora terrestris. The molecular evidence for the persistence of the introduced mycobiont despite the competition from indigenous isolates of the same species provides further illustration of the potential of exotic species for large-scale microbial application.