Nitrous Oxide Reductase (nosZ) Gene Fragments Differ between Native and Cultivated Michigan Soils

The effect of standard agricultural management on the genetic heterogeneity of nitrous oxide reductase (nosZ) fragments from denitrifying prokaryotes in native and cultivated soil was explored. Thirty-six soil cores were composited from each of the two soil management conditions. nosZ gene fragments were amplified from triplicate samples, and PCR products were cloned and screened by restriction fragment length polymorphism (RFLP). The total nosZ RFLP profiles increased in similarity with soil sample size until triplicate 3-g samples produced visually identical RFLP profiles for each treatment. Large differences in total nosZ profiles were observed between the native and cultivated soils. The fragments representing major groups of clones encountered at least twice and four randomly selected clones with unique RFLP patterns were sequenced to verify nosZ identity. The sequence diversity of nosZ clones from the cultivated field was higher, and only eight patterns were found in clone libraries from both soils among the 182 distinct nosZ RFLP patterns identified from the two soils. A group of clones that comprised 32% of all clones dominated the gene library of native soil, whereas many minor groups were observed in the gene library of cultivated soil. The 95% confidence intervals of the Chao1 nonparametric richness estimator for nosZ RFLP data did not overlap, indicating that the levels of species richness are significantly different in the two soils, the cultivated soil having higher diversity. Phylogenetic analysis of deduced amino acid sequences grouped the majority of nosZ clones into an interleaved Michigan soil cluster whose cultured members are α-Proteobacteria. Only four nosZ sequences from cultivated soil and one from the native soil were related to sequences found in γ-Proteobacteria. Sequences from the native field formed a distinct, closely related cluster (D_mean = 0.16) containing 91.6% of the native clones. Clones from the cultivated field were more distantly related to each other (D_mean = 0.26), and 65% were found outside of the cluster from the native soil, further indicating a difference in the two communities. Overall, there appears to be a relationship between use and richness, diversity, and the phylogenetic position of nosZ sequences, indicating that agricultural use of soil caused a shift to a more diverse denitrifying community.     

Population structure of pioneer specialist solitary bee Andrena vaga (Hymenoptera: Andrenidae) in central Europe: the effect of habitat fragmentation or evolutionary history?

Because patchiness of food sources or nesting opportunities frequently limits gene flow, specialists often exhibit distinct population structures in fragmented habitats. We studied the influence of habitat fragmentation on population structure in the solitary bee Andrena vaga, an early spring species that nests exclusively in sandy soil and feeds strictly on willows (Salix spp.). Because the homogenous habitat of the German floodplains, where the species was studied previously, resulted in the species’ weak population structure, we expected more structured populations in central Europe, where the sandy soils essential for nesting are highly fragmented. We analysed 387 females from 21 localities in the Czech Republic and Slovakia using nine microsatellite loci, and we inferred population structure using landscape genetics and Bayesian clustering methods. Contrary to our expectations, habitat fragmentation did not result in increased genetic isolation at the localities; however, two differentiated groups of localities, separated by a wide clinal zone of admixture, were detected within the study area. The observed pattern suggests that dispersive ability of A. vaga compensates the species dependence on unstable fragmented habitats. We propose that the population structure may mirror a secondary contact formed by the expansion of two populations that had been separated in the past. We emphasise the necessity of knowing the studied species’ population history before making conclusions concerning correlations between habitat and population structure, especially in areas of known suture zones created by the secondary contact of populations expanding from separate refugia.     

Comparative study reveals unique features of the mycobiota in peat soils samples from Japan and Scotland

We isolated filamentous fungi from soil samples of peat layers in Aomori and Oita Prefectures in Japan and Perth and Kinross district in Scotland by a serial dilution plate technique. The mycobiota in each peat soil showed some common and characteristic features. The abundance of fungal isolates (CFU/g) from peat soil was low: about 1/3 to 1/30 compared with evergreen or coniferous forests or cultivated soil. Trichoderma or Mucorales species were scarcely observed; these fungi occupied only 3% of the total number of colonies. On the other hand, fungi such as Conioscypha and Tolypocladium that are normally isolated rather rarely were encountered at a comparatively high rate. Acremonium guillematii and Tolypocladium cylindrosporum were recorded for the first time in Japan. Sterile fungi occupied 50% of the total number of isolates. The low abundance of fast-growing fungi enabled us to pick slow-growing fungi up easily from the isolation medium. It is interesting that species not previously described in Japan, or scarcely reported, were isolated commonly from both Japanese and Scottish samples. A peat soil sample is therefore an attractive source of untapped microbial resources.     

Occurrence and diversity of fungal entomopathogens in soils of low and high Arctic Greenland

Knowledge of the occurrence, distribution and diversity of pathogens of insects and arachnids (entomopathogens) in the Arctic is very limited. Climate change is expected to affect Arctic terrestrial arthropod communities and therefore also host–pathogen interactions, given that entomopathogens are present. We conducted a survey of fungal entomopathogens in soil samples collected at four localities in Greenland; two at low Arctic sites (Ritenbenk and Disko Island) and two at sites in the high Arctic (Zackenberg and Danmarkshavn). Fungi were isolated from soil samples using larvae of the insect species Galleria mellonella (Lepidoptera) and Tenebrio molitor (Coleoptera) as baits providing evidence that the fungal isolates were indeed entomopathogenic. Five fungal species (Ascomycota; Hypocreales) were found: Isaria fumosorosea Wize, Isaria farinosa (Holmsk.) Fr., Beauveria bassiana (Bals.) Vuill., Beauveria pseudobassiana Rehner and Humber and Tolypocladium inflatum W. Gams (syn.?=?T. niveum). I. farinosa was found at all four localities, while I. fumosorosea was detected in single samples at each of three localities including both high Arctic sites. Only the locality on Disko Island revealed B. bassiana, whereas B. pseudobassiana was isolated at the three other sites. T. inflatum was only found on Disko Island and only isolated with T. molitor as a bait insect. The results document that fungal entomopathogens are widely distributed in the soil environment in Greenland. Entomopathogens should therefore be included in future studies of arthropod ecology in the Arctic.     

Revisiting the host effect on ectomycorrhizal fungal communities: implications from host–fungal associations in relict Pseudotsuga japonica forests

Host identity is among the most important factors in structuring ectomycorrhizal (ECM) fungal communities. Both host–fungal coevolution and host shifts can account for the observed host effect, but their relative significance in ECM fungal communities is not well understood. To investigate these two host-related mechanisms, we used relict forests of Pseudotsuga japonica, which is an endangered endemic species in Japan. As with other Asian Pseudotsuga species, P. japonica has been isolated from North American Pseudotsuga spp. since the Oligocene and has evolved independently as a warm-temperate species. We collected 100 soil samples from four major localities in which P. japonica was mixed with other conifers and broadleaf trees. ECM tips in the soil samples were subjected to molecular analyses to identify both ECM fungi and host species. While 136 ECM fungal species were identified in total, their communities were significantly different between host groups, confirming the existence of the host effect on ECM fungal communities. None of the 68 ECM fungal species found on P. japonica belonged to Pseudotsuga-specific lineages (e.g., Rhizopogon and Suillus subgroups) that are common in North America. Most of ECM fungi on P. japonica were shared with other host fungi or phylogenetically close to known ECM fungi on other hosts in Asia. These results suggest that after migrating, Pseudotsuga-specific fungal lineages may have become extinct in small isolated populations in Japan. Instead, most of the ECM fungal symbionts on P. japonica likely originated from host shifts in the region.     

Microbiological Survey of Adirondack Lakes with Various pH Values

Nine high-altitude oligotrophic Adirondack lakes in upstate New York having water of pH 4.3 to 7.0 were surveyed for total bacterial numbers and possible adaptation of the microbial communities to environmental pH. The number of heterotrophic bacteria from water samples recoverable on standard plate count agar were low (10¹ to 10³ per ml) for most of the lakes. Acridine orange direct counts were approximately two orders of magnitude higher than plate counts for each lake. Sediment aerobic heterotrophs recovered on standard plate count agar ranged from 1.4 × 10⁴ to 1.3 × 10⁶ per g of sediment. Direct epifluorescence counts of bacteria in sediment samples ranged from 3.0 × 10⁶ to 1.4 × 10⁷ per g. Low density values were consistent with the oligotrophic nature of all the lakes surveyed. There were no apparent differences in numbers of bacteria originally isolated at pH 5.0 and pH 7.0 between circumneutral lakes (pH > 6.0) and acidic lakes (pH < 5.0). Approximately 1,200 isolates were recultured over a range of pH from 3.0 to 7.0. Regardless of the original isolation pH (pH 5.0 or pH 7.0), less than 10% of the isolates grew at pH < 5.0. Those originally isolated at pH 5.0 also grew at pH 6.0 and 7.0. Those originally isolated at pH 7.0 preferred pH 7.0, with 98% able to grow at pH 6.0 and 44% able to grow at pH 5.0. A chi-square contingency test clearly showed (P < 0.005) that two distinct heterotrophic populations had been originally isolated at pH 5.0 and pH 7.0, although there is undoubtedly some overlap between the two populations.     

Soil Drying As a Model for the Action of Stress Factors on Natural Bacterial Populations

The drying of soil samples reduced the abundance (especially of predominant species) and the diversity of bacteria isolated from these samples, making easier the isolation of rare bacterial species. Some bacterial species that were minor before soil drying became dominant in dried soil samples. In general, soil drying allowed the diversity of soil bacteria to be determined more adequately. The bacteria that were isolated from dried soil samples turned out to be resistant to gamma radiation (with LD₉₀ = 2.8–4.6 kGy) and desiccation. It is concluded that soil drying may serve as a model for the action of stress factors on natural bacterial populations. The hypothesis that periodic desiccation was the primary cause of formation of bacterial radioresistance in nature is discussed.     

Coexistence of similar genotypes of Daphnia magna in intermittent populations: response to thermal stress

We investigated the diversity and thermal response of a fitness related trait, juvenile growth rate, in seasonal population samples of Daphnia magna from two temperate ponds. Both populations were intermittent, i.e. they disappeared from the water body and recolonized seasonally by hatching from resting eggs in the sediment.
Temporally isolated clones of Daphnia magna showed the typical asymmetric response for growth rate with temperature and a sharp decline after the maximum response at 26°C (TMR). There was no evidence for genetically adapted seasonal groups. Despite significant genetic variation among clones and for phenotypic plasticity (G×E interactions without genetic correlations), seasonal groups of clones showed no shift in TMR and mean temperature reaction norms were similar among groups and both populations. Heritabilities remained similar among temperatures despite a large increase in genetic variance at stressfully high temperatures of 29°C and 32°C, due to simultaneous increase in environmental variance. Further, heritabilities remained high among sample periods and were not eroded during several months of asexual reproduction.
Regular diapause, an intrinsic feature of intermittent Daphnia populations, may replace the need for physiological temperature adaptation and promote maintenance of diversity through phenotypic similarity by reducing the time over which competitive interactions occur. Such populations are unlikely to be directly affected by elevated temperatures. They have a large potential for phenotypic plasticity as their TMR is higher than the temperature normally encountered.     

Genetic Variability at the Esterase-6 Locus in Natural Populations of DROSOPHILA SIMULANS in Relation to Environmental Heterogeneity

The allozymic variation at the esterase-6 locus was examined in fourteen samples of natural populations of Drosophila simulans collected in two different localities. The samples were collected from different species of fruits in the two localities and from banana baits located some meters apart from the fruits. The analysis was done by sequential electrophoresis, using varied gel concentrations and buffers. By this technique, we detected 27 alleles at the esterase-6 locus, where only five alleles were detected by the usual method. The results obtained suggest that habitat choice alone may not be a selective factor, but may be selective in combination with other environmental factors.     

Genetic structure of the alpine newt, Mesotriton alpestris (Salamandridae,Caudata), in the southern limit of its distribution: Implications for conservation

Tissue samples of Mesotriton alpestris veluchiensis were collected from 11 localities in Greece, and the sequences of two mitochondrial genes (cytochrome b and 16S rRNA), as well as frequencies of 18 allozyme loci, were used in order to describe levels and patterns of genetic variation, identify possible evolutionary units, and reveal aspects of their conservation status. Two major lineages, displaying considerable amount of genetic differences, were supported by both analyses. These lineages, which have been geographically separated since the middle Pleistocene, constitute separate Evolutionary Significant Units (ESUs) and correspond to populations from the Greek mainland and Peloponnisos, respectively. The particularly high inter-population differentiation within each region implies long-term isolation in fragmented habitats, while severe bottlenecking is proposed to have resulted in the observed lack of heterozygotes in the majority of populations. Conservational implications are also discussed, particularly in relation to environmental factors and human activities, which seem to have contributed to the genetic impoverishment of the most marginal populations studied.     

Population isolation rather than ecological variation explains the genetic structure of endangered myrmecophilous butterfly Phengaris (=Maculinea) arion

Genetic variation of the globally threatened obligatorily myrmecophilous Large Blue butterfly Phengaris (Maculinea) arion (Lepidoptera) was studied, using six microsatellite markers, in a country where its decline is dramatic (Poland). Material was collected on 13 sites showing considerable ecological variation as far as biotope, larval food plant and host ants of the butterfly were concerned. Genetic variability, estimated in terms of number of alleles and heterozygosity, was the lowest in the most isolated populations. However on sites localized in areas where suitable biotopes were extensive and interconnected, P. arion still held relatively high genetic diversity. Pairwise F _ST values indicated small and moderate differentiation among samples (F _ST = 0.01–0.15), with the exceptions of two isolated localities (0.20). We did not find clear evidence of isolation by distance. The presence of four or five genetic clusters was indicated. Analysis of the membership of each individual to each cluster showed that the vast majority of individuals from three isolated populations were clustered in three separate genetic groups. The most distinct population was the one, which had been found to be specialized towards Myrmica lobicornis in previous studies. Individuals from the remaining populations could not be clustered in separate genetic groups, however some dominance of different clusters in geographical regions was observed. Some portion of the population’s genetic variability could be explained by geographical distribution, however the percentage of variation, explaining the differences between two main regions (S and NE Poland), was very low. We conclude that the main factor shaping the current genetic structure of P. arion in Poland is the recent isolation of populations related to habitat fragmentation but local ecological specializations may be also a potential factor. Therefore the necessity of activities aiming to halt the further reduction of genetic variability, as well as the monitoring of priority populations (e.g. those belonging to unique host races), should be emphasized in future action plans in Central Europe.     

Intraspecific VariabiIity in Acrobeloides nanus (de Man) Anderson (Nematoda, Cephalobidae) and a Note on External Morphology

Uniparental free-living nematodes, Acrobeloides nanus (de Man, 1880) Anderson, 1968, were isolated from the raw humus layer at two coniferous forest sites in central Sweden. Intraspecific variability in progeny from single females cultured both on agar and in soil was studied. Females from field populations were much shorter than those from laboratory cultures and significant differences in length between cultured specimens from the two sites were found. No significant difference in length of animals from comparable cultures on agar and in soil was obtained. The substrate had some effect on the tail shape, but not on the labial probolae shape. No significant differences in taxonomic characters between populations from the two localities were observed. Enzyme analyses by horizontal starch gel electrophoresis showed qualitative differences in isozyme patterns of malate dehydrogenase (MDH) and superoxide dismutase (SOD) in populations from the two sites, while the esterase patterns were unstable with time and less suitable for population analysis.     

Genetic and leaf-trait variability of Vinca minor at ancient and recent localities in Central Europe

In Central Europe, Vinca minor has been planted for centuries as an ornamental, medicinal and ritual plant. We asked how variability in genetic and leaf traits of V. minor at ancient localities differs from that at recently established localities sampled mainly in the Czech Republic. Using selected ISSR primers, we obtained two clusters that correspond well with locality history. In the recent cluster, we identified a certain degree of genetic variability, whereas the ancient cluster exhibited none. We recorded significant differences in leaf shape between the clusters. Genotypes with narrow leaves were more characteristic of recent localities. Although the ancient cluster showed no variability in ISSR primers, it did show variability in leaf traits, indicating that some of the morphologically expressed genetic variability cannot be detected using ISSR primers. All samples were diploids (2n = 46), with an identical relative DNA content. Samples from ancient localities, such as deserted medieval settlements and castles, probably descended from a single or a few related clones. This supports the hypothesis concerning the allochthonous character of V. minor in the Czech Republic.     

GEOGRAPHIC GENETIC STRUCTURE IN THE FRANCISCANA DOLPHIN (PONTOPORIA BLAINVILLEI)

The franciscana, Pontoporia blainvillei , is endemic to the South Atlantic coast of South America. The species is of conservation concern because it suffers elevated mortality rates due to incidental captures in fishing nets, and perhaps it is one of the most threatened small cetaceans in this region. Previous morphological and genetic studies have suggested the existence of at least two distinct stocks to the north and south of Santa Catarina Island in Brazil. Fixed differences were found between a sample from Rio de Janeiro and one from Rio Grande do Sul, in southern Brazil. Using 94 sequences of the control region of mitochondrial DNA, we examined the genetic structure of the species. We found no shared haplotypes between Rio de Janeiro and samples from the southern range of the species distribution. However, a phylogenetic analysis suggests that the former population is diphyletic with respect to the southern samples. This suggests that the populations have not been isolated long enough to reach reciprocal monophyly. Furthermore, genetic differentiation is broadly consistent with a simple model of isolation by distance, that therefore appears as an alternative to a model of strict isolation of two stocks. The estimated levels of gene flow are higher among neighboring populations, and decrease as more distant localities are compared. Finally, the molecular data suggest that franciscanas have expanded in Rio de Janeiro.     

Nitrous oxide reductase (nosZ) gene fragments differ between native and cultivated Michigan soils

The effect of standard agricultural management on the genetic heterogeneity of nitrous oxide reductase (nosZ) fragments from denitrifying prokaryotes in native and cultivated soil was explored. Thirty-six soil cores were composited from each of the two soil management conditions. nosZ gene fragments were amplified from triplicate samples, and PCR products were cloned and screened by restriction fragment length polymorphism (RFLP). The total nosZ RFLP profiles increased in similarity with soil sample size until triplicate 3-g samples produced visually identical RFLP profiles for each treatment. Large differences in total nosZ profiles were observed between the native and cultivated soils. The fragments representing major groups of clones encountered at least twice and four randomly selected clones with unique RFLP patterns were sequenced to verify nosZ identity. The sequence diversity of nosZ clones from the cultivated field was higher, and only eight patterns were found in clone libraries from both soils among the 182 distinct nosZ RFLP patterns identified from the two soils. A group of clones that comprised 32% of all clones dominated the gene library of native soil, whereas many minor groups were observed in the gene library of cultivated soil. The 95% confidence intervals of the Chao1 nonparametric richness estimator for nosZ RFLP data did not overlap, indicating that the levels of species richness are significantly different in the two soils, the cultivated soil having higher diversity. Phylogenetic analysis of deduced amino acid sequences grouped the majority of nosZ clones into an interleaved Michigan soil cluster whose cultured members are alpha-Proteobacteria. Only four nosZ sequences from cultivated soil and one from the native soil were related to sequences found in gamma-Proteobacteria. Sequences from the native field formed a distinct, closely related cluster (D(mean) = 0.16) containing 91.6% of the native clones. Clones from the cultivated field were more distantly related to each other (D(mean) = 0.26), and 65% were found outside of the cluster from the native soil, further indicating a difference in the two communities. Overall, there appears to be a relationship between use and richness, diversity, and the phylogenetic position of nosZ sequences, indicating that agricultural use of soil caused a shift to a more diverse denitrifying community.     

PCR and Culture Identification of Pathogenic Leptospira spp. from Coastal Soil in Leyte,Philippines, after a Storm Surge during Super Typhoon Haiyan (Yolanda)

Leptospirosis is a zoonosis caused by pathogenic Leptospira spp. Most of the outbreaks of leptospirosis occur after floods caused by heavy rain in countries where Leptospira spp. are endemic. It has been believed that the overflow of seawater rarely causes outbreaks of leptospirosis because the leptospires are killed by salt water. On 8 November 2013, a storm surge caused by Super Typhoon Haiyan (Yolanda) inundated the entire coastal areas of Tacloban and Palo in Leyte, Philippines. The present study was carried out in order to determine whether the environmental leptospires in soil were able to survive after the storm surge in the affected areas. We collected 23 wet soil samples along the coastal areas of Tacloban and Palo 2 months after the storm surge. The samples were suspended in HEPES buffer, and the supernatants were cultured in liquid or semisolid Korthof''s medium supplemented with five antimicrobial agents to inhibit the growth of contaminants. Leptospires were isolated from primary cultures of 22 out of 23 samples. The DNA of pathogenic Leptospira species was detected in 11 samples (47.8%) by analysis of flaB by nested PCR. Eventually, two pathogenic Leptospira strains were isolated and showed the highest 16S rRNA gene sequence similarity to Leptospira kmetyi. When these isolates were experimentally mixed with soil, they were found to survive in seawater for 4 days. These results show the possibility that leptospires living in soil survived after the storm surge. Our findings may serve as a warning that when seawater inundates the land during a storm surge or a tsunami, an outbreak of leptospirosis could occur in the disaster-stricken area.     

Looking for the Iberian lynx in central Spain: a needle in a haystack?

The Iberian lynx Lynx pardinus has suffered a dramatic reduction in its range throughout the Iberian Peninsula and at present is one of the most endangered mammals in the world. The latest studies report that, out of the 48 breeding areas that existed in 1990, only two populations are left in southern Spain. As a consequence, some of the formerly largest populations, such as Montes de Toledo (central Spain), are to all intents and purposes regarded as extinct. To determine the current distribution of Iberian lynx outside the two recognized populations, we surveyed five different areas where the species is considered extinct and collected 581 faeces for the genetic identification of the species. We identified 18 samples as belonging to Iberian lynx in four out of the five areas studied, providing clear evidence for the presence of lynx in central Spain. In some areas the species was detected repeatedly at different localities and on different dates, indicating a regular occurrence of an unknown number of individuals. The conservation implications of these results are discussed in terms of the genetic importance of the individuals found and future reintroductions of the species from an ongoing captive-breeding programme.     

Population Structure in the Roundtail Chub (Gila robusta Complex) of the Gila River Basin as Determined by Microsatellites: Evolutionary and Conservation Implications

Ten microsatellite loci were characterized for 34 locations from roundtail chub (Gila robusta complex) to better resolve patterns of genetic variation among local populations in the lower Colorado River basin. This group has had a complex taxonomic history and previous molecular analyses failed to identify species diagnostic molecular markers. Our results supported previous molecular studies based on allozymes and DNA sequences, which found that most genetic variance was explained by differences among local populations. Samples from most localities were so divergent species-level diagnostic markers were not found. Some geographic samples were discordant with current taxonomy due to admixture or misidentification; therefore, additional morphological studies are necessary. Differences in spatial genetic structure were consistent with differences in connectivity of stream habitats, with the typically mainstem species, G. robusta, exhibiting greater genetic connectedness within the Gila River drainage. No species exhibited strong isolation by distance over the entire stream network, but the two species typically found in headwaters, G. nigra and G. intermedia, exhibited greater than expected genetic similarity between geographically proximate populations, and usually clustered with individuals from the same geographic location and/or sub-basin. These results highlight the significance of microevolutionary processes and importance of maintaining local populations to maximize evolutionary potential for this complex. Augmentation stocking as a conservation management strategy should only occur under extreme circumstances, and potential source populations should be geographically proximate stocks of the same species, especially for the headwater forms.     

Analysis of nifH Gene Pool Complexity in Soil and Litter at a Douglas Fir Forest Site in the Oregon Cascade Mountain Range

Nitrogen-fixing microbial populations in a Douglas fir forest on the western slope of the Oregon Cascade Mountain Range were analyzed. The complexity of the nifH gene pool (nifH is the marker gene which encodes nitrogenase reductase) was assessed by performing nested PCR with bulk DNA extracted from plant litter and soil. The restriction fragment length polymorphisms (RFLPs) of PCR products obtained from litter were reproducibly different than the RFLPs of PCR products obtained from the underlying soil. The characteristic differences were found during the entire sampling period between May and September. RFLP analyses of cloned nifH PCR products also revealed characteristic patterns for each sample type. Among 42 nifH clones obtained from a forest litter library nine different RFLP patterns were found, and among 64 nifH clones obtained from forest soil libraries 13 different patterns were found. Only two of the patterns were found in both the litter and the soil, indicating that there were major differences between the nitrogen-fixing microbial populations. A sequence analysis of clones representing the 20 distinct patterns revealed that 19 of the patterns had a proteobacterial origin. All of the nifH sequences obtained from the Douglas fir forest litter localized in a distinct phylogenetic cluster characterized by the nifH sequences of members of the genera Rhizobium, Sinorhizobium, and Azospirillum. The nifH sequences obtained from soil were found in two additional clusters, one characterized by sequences of members of the genera Bradyrhizobium, Azorhizobium, Herbaspirillum, and Thiobacillus and the other, represented by a single nifH clone, located between the gram-positive bacteria and the cyanobacteria. Our results revealed the distinctness of the nitrogen-fixing microbial populations in litter and soil in a Douglas fir forest; the differences may be related to special requirements for degradation and mineralization processes in the plant litter.     

Low heterogeneity in populations of the terrestrial earthworm,Metaphire peguana (Rosa, 1890), in Thailand,as revealed by analysis of mitochondrial DNA COI sequences and nuclear allozymes

The genetic variation of a widely distributed terrestrial earthworm species, Metaphire peguana (Rosa, 1890), was examined in Thailand using allozyme and mitochondrial (mt)DNA cytochrome oxidase subunit 1 (COI) sequence analyses. A total of 274 individuals were collected from 13 localities in Thailand and scored for 12 enzyme systems (18 presumptive allozyme loci) using horizontal starch gel electrophoresis. Fourteen of these presumed loci were found to be polymorphic. In addition, a 660 bp fragment of the COI mtDNA gene was amplified and sequenced from one representative individual for each of the 13 geographical regions. The expected heterozygosity (H_exp) was relatively low, ranging from 0.059–0.147 with an overall mean of 0.092 ± 0.02 and there was no significant isolation by distance pattern for the 13 populations (localities) across the entire study area (P-value > 0.05). Genetic divergences among the samples were low, with a low genetic distance (mean = 0.048) and uncorrected p-distance (mean = 0.02) indicating frequent gene flow among these populations in Thailand. The monophyly of M. peguana across these regions was supported by Neighbor joining, Maximum likelihood and Bayesian inference based phylogenetic analysis of the mtDNA COI sequences with no geographic location dependent pattern. This may be due to anthropogenic movement of M. peguana in soil or with plant scions throughout this region.