Anthropogenic and natural disturbance lead to differing patterns of gene flow in the Rocky Mountain tailed frog, Ascaphus montanus

Understanding the influence of habitat alteration on population structure and persistence is critical for effective conservation strategies. Timber harvest and wildfire are two of the most prevalent disturbances across temperate forests, yet the long-term effects of these two forces on population connectivity have rarely been studied. We studied populations of the Rocky Mountain tailed frog (Ascaphus montanus) across landscapes that have experienced either timber harvest or broad scale fires. Rocky Mountain tailed frogs generally require forested habitat, and are a species of concern in managed forests. We used landscape genetic techniques to test the explanatory power of alternative paths of connectivity across both burned and harvested forests and identified topographical and climatic variables that significantly influenced gene flow. We found that timber harvest and fire led to different patterns of genetic connectivity. Widespread terrestrial gene flow was maintained across previously burned areas and connectivity was primarily limited by solar radiation. In contrast, gene flow among populations in managed forests followed riparian corridors, presumably to avoid the loss of cover due to timber harvest. Gene flow along riparian corridors in Rocky Mountain tailed frogs differs from a population of the closely related coastal tailed frog (Ascaphus truei), for which gene flow appears to occur primarily over land. These results suggest management of Rocky Mountain tailed frogs should focus on maintaining riparian buffer zones, which may be less effective for the coastal species. Thus, consideration of landscape differences is essential even for conservation of closely related, morphologically similar species.     

Status and recent trends of the great bustard (Otis tarda) population in the Iberian peninsula

We present an updated estimate of the great bustard population in the Iberian peninsula. The sum of the most recent counts was 20,243, and the estimated total, 24,490 birds. During the last two decades bustards increased at five intensive study areas, remained stable at two, and declined or became extinct at eight. These population changes were determined by both variations in breeding success at each area and migration of birds between areas. Our results suggest that the population is probably concentrating at high quality areas, and disappearing from poor quality ones. We also identified 29 areas where bustards have become extinct during the last four decades, mostly due to hunting and agricultural transformations. In conclusion, although the population is apparently not decreasing at present, the concentration trend described could lead to a higher species’ vulnerability, a loss of genetic diversity, and a further isolation of marginal groups. Strict conservation measures should thus be directed at preventing the decrease of the number of extant leks.     

Minimum viable population and conservation status of the Atlantic Forest spiny rat Trinomys eliasi

A population viability analysis (PVA) was conducted to assess the minimum viable population (MVP) of the Atlantic Forest spiny rat Trinomys eliasi, a species threatened by habitat loss and restricted geographical distribution. Objectives were to suggest quasi-extinction thresholds, estimate minimum areas of suitable habitat (MASH) and MVPs, and compare results with the species’ current status. The computer package VORTEX was used. The model predicted sizes of 200 animals to achieve demographic stability, but buffering declines in genetic variability required populations of 2000 animals. Estimated MASHs were approximately 250 and 2500 ha for demographic and genetic stability, respectively. Mortality rate and mean litter size were the most sensitive parameters to changes in model assumptions. The protection of known populations and the search for extant populations are the first steps in conservation. T. eliasi's issue could help protecting the coastal shrubland ecosystem of Rio de Janeiro state. Observing IUCN's criteria for listing threatened species, it is suggested that T. eliasi should be ranked as vulnerable in red lists.     

Assessing limitations on population growth in two critically endangered Acacia taxa

Flowering phenology, soil seedbank and the impact of fire, weeds and grazing were investigated in two rare Acacia taxa restricted to the agricultural district north of Perth Western Australia. Acacia aprica is known from six extant populations and one extinct population. Five of the extant populations are restricted to linear road reserves with the sixth population located in a small native vegetation remnant. Acacia cochlocarpa ssp. cochlocarpa is known from a single population also situated on a road verge. Size class structure, levels of canopy death and an absence of juveniles indicated that all populations are in decline. Flowering intensity and success varied between populations and years in A. aprica and between years in A. cochlocarpa ssp. cochlocarpa. Seed bank analysis indicated that seeds were patchily distributed while experimental fires demonstrated that such events could break seed dormancy and promote germination. Both taxa have similar germination physiologies and showed increased germination after seeds were exposed to heat. Following emergence, however, competition with annual weeds had a negative impact on seedling growth and survival in both taxa. While vertebrate grazing had some influence, weeds were the major inhibitory influence on recruitment. Reduced fire frequencies since fragmentation may be responsible for population decline in both taxa but other site specific factors such as weeds and grazing may affect the establishment of seedlings following fire.     

Proportion of individuals with anti-Batrachochytrium dendrobatidis skin bacteria is associated with population persistence in the frog Rana muscosa

The emergence of the infectious disease chytridiomycosis is a major factor responsible for amphibian extinctions in pristine habitats. However, some populations coexist with Batrachochytrium dendrobatidis (Bd), the agent of chytridiomycosis, and others go extinct when Bd arrives. Variation of pathogen severity among populations may be explained by differences of antimicrobial skin peptides and anti-Bd skin bacteria. Previous work showed that a population of the frog Rana sierrae had a high proportion of individuals with at least one species of cultured anti-Bd skin bacteria and was coexisting with Bd for more than 6 years. A population of the closely related sister species Rana muscosa had a significantly lower proportion of individuals with anti-Bd bacteria, and the following year, it went extinct due to Bd. We extended previous work to include another sampling of the R. sierrae population coexisting with Bd and found that, although the anti-Bd bacterial community somewhat differed, both populations had a high proportion of individuals with antifungal bacteria. We also included a population of R. muscosa that was naïve of Bd and predicted to go extinct once Bd emerged since that was the fate of neighboring populations. However, the naïve R. muscosa population had a high proportion of antifungal bacteria, which suggested that the population might survive if Bd emerged. Two years later, the population was surviving with Bd endemic. Variation in the proportion of individuals with anti-Bd skin bacteria appears to explain why some populations persist when Bd emerges in a population.     

Soil feedback effects to the foredune grass Ammophila arenaria by endoparasitic root-feeding nematodes and whole soil communities

In coastal foredunes, the grass Ammophila arenaria develops a soil community that contributes to die-back and replacement by later successional plant species. Root-feeding nematodes and pathogenic soil microorganisms are involved in this negative feedback. Regular burial by wind-blown beach sand results in vigorous growth of A. arenaria, probably because of enabling a temporary escape from negative soil feedback. Here, we examine the role of root-feeding nematodes as compared to the whole soil community in causing negative feedback to A. arenaria. We performed a 3-year sand burial experiment in the field and every year we determined the feedback of different soil communities to plant growth in growth chamber bioassays.In the field, we established A. arenaria in tubes with beach sand, added three endoparasitic root-feeding nematode species (Meloidogyne maritima, Heterodera arenaria and Pratylenchus penetrans) or root zone soil to the plants, and created series of ceased and continued sand burial. During three subsequent years, plant biomass was measured and numbers of nematodes were counted. Every year, bioassays were performed with the field soils and biomass of seed-grown A. arenaria plants was measured to determine the strength of feedback of the established soil communities to the plant.In the field, addition of root zone soil had a negative effect on biomass of buried plants. In the bioassays, addition of root zone soil also reduced the biomass of newly planted seedlings, however, only in the case when the field plants had not been buried with beach sand. Addition of the three endoparasitic root-feeding nematodes did not influence plant biomass in the field and in the bioassays. Our results strongly suggest that the negative feedback to A. arenaria is not due to the combination of the three endoparasitic nematodes, but to other components in the soil community, or their interactions with the nematodes.     

Quantification of Wautersia [Ralstonia] basilensis in the mycorrhizosphere of Pinus thunbergii Parl. and its effect on mycorrhizal formation

The bacterium Wautersia [Ralstonia] basilensis has been shown to enhance the mycorrhizal symbiosis between Suillus granulatus and Pinus thunbergii (Japanese black pine). However, no information is available about this bacterium under field conditions. The objectives of this study were to detect W. basilensis in bulk and mycorhizosphere soils in a Japanese pine plantation in the Tottori Sand Dunes, determine the density of W. basilensis in soil, and determine the optimal cell density of W. basilensis for mycorrhizal formation in pine seedlings. We designed and validated 16S rRNA gene-targeted specific primers for detection and quantification of W. basilensis. SYBR Green I real-time PCR assay was used. A standard curve relating cultured W. basilensis cell density (10³-10⁸ cells ml⁻¹) to amplification of DNA showed a strong linear relationship (R = 0.9968). The specificity of the reaction was confirmed by analyzing DNA melting curves and sequencing of the amplicon. The average cell density of W. basilensis was >4.8 × 10⁷ cells g⁻¹ of soil in the mycorrhizosphere and 7.0 × 10⁶ cells g⁻¹ in the bulk soil. We evaluated the W. basilensis cell density required for mycorrhizal formation using an in vitro microcosm with various inoculum densities ranging from 10² to 10⁷ cells g⁻¹ soil (10⁴-10⁹ cells ml⁻¹). Cell densities of W. basilensis of >10⁶ cells g⁻¹ of soil were required to stimulate mycorrhizal formation. In vivo and in vitro experiments showed that W. basilensis was sufficiently abundant to enhance mycorrhizal formation in the mycorrhizosphere of Japanese black pine sampled from the Tottori Sand Dunes.     

Effects of Medicago sativa, Taraxacum officinale and Bromus sterilis on the density and diversity of Collembola in grassy arable fallows of different ages

The density and diversity of Collembola of nine grassy arable fallows of different ages were investigated in a factorial design with the factors ‘plant species’ (legume: Medicago sativa, herb: Taraxacum officinale, grass: Bromus sterilis) and ‘age class’ (2-3, 6-8 and 12-15 years) including the random effect ‘site’ (1-9). In May 2008, four plots were selected randomly at each fallow. Within each plot five M. sativa, T. officinale and B. sterilis plants were extracted with their associated soil using steel cylinders. The material from each plant species was used for extraction of Collembola and for determination of environmental parameters. Thus, the new aspect of the present study compared to other field studies investigating the relationships between plant and Collembolan communities is the focus on the “micro-scale”, investigating the Collembolan communities of the soil associated with single plants.We found that species richness and density of total and euedaphic Collembola were significantly higher in B. sterilis than in T. officinale samples with the M. sativa samples being intermediate. Fine-root feeding euedaphic Collembola particularly benefited from the higher amount of fine roots in B. sterilis samples. We also discovered that the age of the fallows had no significant influence on the number of Collembolan species and the density of the Collembolan groups. Notably, however, species of the epedaphic genera Lepidocyrtus and Sminthurinus were associated with the 12-15 year-old fallows and presumably benefited from the high number of plant species in the old fallows. Finally, canonical correspondence analysis (CCA) indicated that the soil organic matter content and the microbial biomass, both potential food sources for many Collembolan species, were important structuring forces for the Collembolan communities.     

Density and distribution of the endemic Seram cockatoo Cacatua moluccensis in relation to land use patterns

The endemic Seram cockatoo, Cacatua moluccensis, was placed on Appendix I of CITES in response to declining trade statistics but in the absence of population data. We conducted population surveys and collected data on habitat structure at seven sites on Seram in 1998. Cockatoo densities ranged from 0.93 to 17.25 birds/km² and averaged 7.9 birds/km² across sites. We classified habitat into three types but found that cockatoo densities did not correspond closely to habitat differences across sites. Cockatoo abundance was significantly associated with presence of potential nest trees and strangling figs. Analysis of forest cover and landuse indicates that while most of the island is still covered in lowland forest, only 14% of these forests are protected and almost half the island is classified as logging concession. Conservation recommendations include better information and enforcement of laws protecting Seram cockatoos and resolution of boundary conflicts between parks and logging concessions.     

Host status of 10 fungal isolates for two nematode species, Filenchus misellus and Aphelenchus avenae

The classification of nematodes in the family Tylenchidae into plant parasites, plant associates or fungal-feeders for community analyses, have been much discussed by nematode ecologists. For an appropriate classification, fungal-feeding habits in the family need to be studied. To evaluate the host status of 10 fungal isolates for Filenchus misellus (Tylenchidae) and Aphelenchus avenae (Aphelenchida, Aphelenchidae), population growth rates, body length and width and sex ratios of the nematodes were measured after 40-day culture on fungal colonies at 25 °C. For F. misellus, the fungi determined as good hosts were two Basidiomycota fungi (Agaricus bisporus, Coprinus cinereus), three Ascomycota fungi (Chaetomium cochlioides, Chaetomium funicola, Chaetomium globosum) and a plant-pathogenic fungus (Rhizoctonia solani) on the basis of nematode population growth rate and female body length. Interestingly Pleurotus ostreatus, known as a predaceous fungus for the other nematodes, was also a good host for F. misellus. While, for A. avenae, good hosts were four plant-pathogenic fungi (Fusarium oxysporum f. sp. conglutinans, F. oxysporum f. sp. cucumerinum, Pythium ultimum, R. solani) and A. bisporus. A. avenae was trapped and preyed upon by Pleurotus hyphae. In F. misellus, males were 7-21% of adults, but the ratio did not correlate significantly with the population growth rate. In A. avenae, no male occurred. Differences in habitat preference between Filenchus and Aphelenchus were explained on the basis of the host status and habitat preferences of the tested fungi.     

Status assessment of the Indus River dolphin, Platanista gangetica minor, March-April 2001

A survey was conducted in March and April 2001, to assess the status of the Indus River dolphin, Platanista gangetica minor, throughout its present range. A total of 1535 km of survey effort was conducted, consisting of 1375 km of the Indus River main channel, 136 km of Indus River secondary channels, and 24 km of the Panjnad River, a tributary of the Indus. The effective range of the Indus dolphin has declined by 80% since 1870. The sum of best group size estimates produced an abundance estimate of 965 dolphins. Extrapolation of encounter rates to un-surveyed channels and application of a correction factor to account for missed dolphins indicates that the metapopulation may number approximately 1200 individuals. Dolphins occur in five subpopulations separated by irrigation barrages. A pronounced increase in dolphin abundance and encounter rate was observed in each subsequent downstream subpopulation (except the last). The three largest subpopulations were between Chashma and Taunsa Barrages (84 dolphins; 0.28/km), Taunsa and Guddu Barrages (259 dolphins; 0.74/km) and Guddu and Sukkur Barrages (602 dolphins; 3.60/km). Reasons suggested for the high encounter rate between Guddu and Sukkur Barrages, include high carrying capacity, low levels of anthropogenic threat, effective conservation, and augmentation of the subpopulation by downstream migration of dolphins from upstream.     

Herbicides, weeds and endangered species: management of bitou bush (Chrysanthemoides monilifera ssp. rotundata) with glyphosate and impacts on the endangered shrub, Pimelea spicata

Environmental weed invasion threatens the biodiversity of native species. Unfortunately, managing these weeds may also affect biodiversity adversely. A recent example occurred when glyphosate, a herbicide used to control the highly invasive weed, bitou bush (Chrysanthemoides monilifera ssp. rotundata), accidentally drifted over a small population of an endangered shrub, Pimelea spicata. Following concerns that the affected population would not recover and, thereby, cause the local extinction of P. spicata, we conducted a series of glasshouse and field experiments to explore the impacts of glyphosate on this endangered species. Seedlings and young plants of P. spicata, in which the tap root was undeveloped, were killed by a single application of glyphosate. Older plants with a well developed tap root also died back initially, but about 50% of individuals re-sprouted. This re-growth was associated with a significant decrease in tap root diameter, implying that further disturbance, including repeated treatment with glyphosate, would kill plants by impairing their potential for recovery. Unlike some sclerophyllous native shrubs, the tolerance of P. spicata to glyphosate was limited, even when its growth was slowed artificially by limiting water availability. Winter applications of glyphosate to manage infestations of bitou bush will impact adversely on populations of P. spicata and may also affect the other rare and endangered species whose survival is threatened by this species, even though some natives are unaffected by the herbicide. Protecting native biodiversity from bitou bush will involve sustainable weed management that minimises impacts on non-target native species.     

Growth promoting effects of corn (Zea mays) bacterial isolates under greenhouse and field conditions

Fertilizer costs are a major component of corn production. The use of biofertilizers may be one way of reducing production costs. In this study we present isolation and identification of three plant growth promoting bacteria that were identified as Enterobacter cloacae (CR1), Pseudomonas putida (CR7) and Stenotrophomonas maltophilia (CR3). All bacterial strains produced IAA in the presence of 100 mg l⁻¹ of tryptophan and antifungal metabolites to several soilborne pathogens. S. maltophilia and E. cloacae had broad spectrum activity against most Fusarium species. The only strain that was positive for nitrogen fixation was E. cloacae and it, and P. putida, were also positive for phosphate solubilization. These bacteria and the corn isolate Sphingobacterium canadense CR11, and known plant growth promoting bacterium Burkholderia phytofirmans E24 were used to inoculate corn seed to examine growth promotion of two lines of corn, varieties 39D82 and 39M27 under greenhouse conditions. When grown in sterilized sand varieties 39M27 and 39D82 showed significant increases in total dry weights of root and shoot of 10-20% and 13-28% and 17-32% and 21-31% respectively. Plants of the two varieties grown in soil collected from a corn field had respective increases in dry weights of root and shoot of 10-30% and 12-35% and 11-19% and 10-18%. In sand, a bacterial mixture was highly effective whereas in soil individual bacteria namely P. putida CR7 and E. cloacae CR1 gave the best results with 39M27 and 39D82 respectively. These isolates and another corn isolate, Azospirillum zeae N7, were tested in a sandy soil with a 55 and 110 kg ha⁻¹ of nitrogen fertility at the Delhi research Station of Agriculture and Agri-Food Canada over two years. Although out of seven bacterial treatments, no treatment provided a statistically significant yield increase over control plots but S. canadense CR11 and A. zeae N7 provided statistically significant yield increase as compared to other bacteria. The 110 kg rate of nitrogen provided significant yield increase compared to the 55 kg rate in both years.     

Distribution, status, and conservation needs of the golden-crowned sifaka (Propithecus tattersalli)

The golden-crowned sifaka is considered one of the world's most endangered primates. To evaluate the species' current conservation status we established its actual distribution and abundance, and assessed current threats that affect its survival. Study methods involved direct sifaka observations and interviews to local villagers. Propithecus tattersalli's distribution is restricted to the region comprised between the Loky and Manambato rivers. Throughout its range, the species is abundant and it can even be found in small forest fragments, although it was never observed at elevations above 700 m.a.s.l. Major threats to the species include habitat destruction through slash-and-burn agriculture, grass fires, wood and gold extraction, and poaching. Total population estimates range from 6100 to 10,000 individuals, with an effective population size of approximately 2500-4000 sifakas. Though sensitive to forest destruction, the species seems to be resilient to current levels of landscape fragmentation. We recommend the establishment of a system of protected areas throughout the Loky-Manambato region and the implementation of a “Flagship Species Program” utilizing the golden-crowned sifaka as a catalyst for a regional Conservation Management Plan.     

Changes in agricultural management drive the diversity of Burkholderia species isolated from soil on PCAT medium

In order to assess the diversity of culturable Burkholderia populations in rhizosphere and bulk soil and to evaluate how different agricultural management regimes and land use history affect this diversity, four treatments were evaluated: permanent grassland; grassland converted into maize monoculture; arable land and arable land converted into grassland. Burkholderia isolates obtained on PCAT medium were grouped in 47 clusters using 16S ribosomal RNA gene based PCR-DGGE combined with BOX genomic fingerprinting (DGGE-BOX). The distribution of the isolates in the DGGE-BOX clusters was used to calculate the Shannon diversity index per treatment. Interestingly, we observed that the Burkholderia diversity was affected by changes in the agricultural management, since the highest diversity was observed in permanent grassland and in continuous arable land. In addition, the diversity tended to be higher in the rhizosphere than in the corresponding bulk soil. The use of species abundance models indicated that rhizosphere communities had more even distributions than communities collected from the bulk soil. Identification of isolates revealed that only 2% of these belonged to the B. cepacia complex and that the majority was assigned to either (1) new Burkholderia species or (2) Burkholderia species that had originally been isolated from soil. Isolates classified as B. hospita, B. caledonica and Burkholderia sp. ‘LMG 22934’ and ‘LMG 22936’ were found mainly in the arable land, while isolates belonging to Burkholderia sp. ‘LMG 22929’ and B. phytofirmans were associated with the grassland area. Another potentially new Burkholderia species, ‘LMG 22932’, was found in both areas, in close association with the maize rhizosphere.     

Colonisation of seminal roots of wheat and barley by egg-parasitic nematophagous fungi and their effects on Gaeumannomyces graminis var. tritici and development of root-rot

This study provides evidence that egg-parasitic nematophagous fungi, Pochonia chlamydosporia, Pochonia rubescens and Lecanicillium lecanii, can also reduce root colonisation and root damage by a fungal pathogen. Interactions of nematophagous fungi with the take-all fungus, Gaeumannomyces graminis var. tritici (Ggt), and their influence on severity of the root disease it causes were studied in laboratory and pot experiments. In Petri dish experiments the three nematophagous fungi reduced colonisation of barley roots by Ggt and also reduced necrotic symptoms. On the contrary, root colonisation by nematophagous fungi was unaffected by Ggt. In growth tube experiments, the three nematophagous fungi again reduced Ggt root colonisation and increased effective root length of barley seedlings. This was true for both simultaneous and sequential inoculation of nematophagous fungi versus Ggt. In the pot experiments the inoculum of the tested fungi in soil was applied in the same pot, as a mixture or in layers, or in coupled pots used for wheat grown with a split-root system. The nematophagous fungi P. chlamydosporia (isolate 4624) and L. lecanii (isolate 4629), mixed with Ggt or in split root systems with the pathogen, promoted growth of wheat (i.e. increased shoot weight), although no disease reduction was found. In split root systems, lower levels of peroxidase activity were found in seedlings inoculated with Ggt in combination with the nematophagous isolates 4624 and 4629 than when the take-all fungus was applied alone.Our results show that nematophagous fungi reduce root colonisation by Ggt, root damage and stress induced senescence in Ggt-inoculated plants.     

Five years of simulated atmospheric nitrogen deposition have only subtle effects on the fate of newly synthesized carbon in Calluna vulgaris and Eriophorum vaginatum

To understand the implications of atmospheric nitrogen deposition on carbon turnover in peatlands, we conducted a ¹³C pulse labeling experiment on Calluna vulgaris and Eriophorum vaginatum already receiving long-term (5 years) amendments of 56 kg N ha⁻¹ y⁻¹ as ammonium or nitrate. We examined shoot tissue retention, net ecosystem respiration returns of the ¹³C pulse, and soil porewater DOC content under the two species. ¹³C fixation in Eriophorum leaves was enhanced with nitrogen addition and doubled with nitrate supply. This newly fixed C appeared to be relocated below-ground faster with nitrogen fertilization as respiration returns were unaffected by N inputs. By contrast, increases in ¹³C fixation were not observed in Calluna. Instead, net ecosystem respiration rates over Calluna increased with N fertilization. There was no significant label incorporation into DOC, suggesting a conservative strategy of peatland vegetation regarding allocation of C through root exudation. Greater concentrations of total DOC were identified with nitrate addition in Calluna. Given the long-term nature of the experiment and the high N inputs, the overall impacts of nitrogen amendments on the fate of recently synthesized C in Eriophorum and Calluna in this ombrotrophic peatland were surprisingly more moderate than originally hypothesized. This may be due to N being effectively retained within the bryophyte layer, thus limiting, and delaying the onset of, below-ground effects.     

Rapid identification and discrimination among Egyptian genotypes of Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti nodulating faba bean (Vicia faba L.) by analysis of nodC, ARDRA, and rDNA sequence analysis

Twenty-eight Rhizobium strains were isolated from the root nodules of faba bean (Vicia faba L.) collected from 11 governorates in Egypt. A majority of these strains (57%) were identified as Rhizobium leguminosarum bv. viciae (Rlv) based on analysis of a nodC gene fragment amplified using specific primers for these faba bean symbionts. The strains were characterized using a polyphasic approach, including nodulation pattern, tolerance to environmental stresses, and genetic diversity based on amplified ribosomal DNA-restriction analysis (ARDRA) of both 16S and 23S rDNA. Analysis of tolerance to environmental stresses revealed that some of these strains can survive in the presence of 1% NaCl and a majority of them survived well at 37 °C. ARDRA indicated that the strains could be divided into six 16S rDNA genotypes and five 23S rDNA genotypes. Sequence analysis of 16S rDNA indicated that 57% were Rlv, two strains were Rhizobium etli, one strain was taxonomically related to Rhizobium rubi, and a group of strains were most closely related to Sinorhizobium meliloti. Results of these studies indicate that genetically diverse rhizobial strains are capable of forming N₂-fixing symbiotic associations with faba bean and PCR done using nodC primers allows for the rapid identification of V. faba symbionts.