Molecular phylogenetic analysis of a novel strain from Neelipleona enriches Wolbachia diversity in soil biota

Eubacterial cellular endoparasites belonging to the genus Wolbachia (Rickettsiales) are extremely widespread symbionts of Arthropoda and Nematoda. Their ability to manipulate the reproductive behavior of the host is of particular importance to the fauna of the deep soil horizon, an environment in which parthenogenesis-inducing symbionts can play a crucial role in shaping population dynamics and speciation processes. In this study, three novel cases of infection in parthenogenetic Collembola (Parisotoma notabilis, Neelus murinus and Megalothorax minimus) are described. Sequences for molecular markers 16S rDNA and ftsZ were obtained for each species; their phylogenetic affinities with known Wolbachia supergroups were established using Bayesian inference. The analysis confirmed the presence of a Wolbachia strain belonging to the supergroup E, already reported from Folsomia candida and the Tullbergiidae, in the isotomid P. notabilis, while the Neelipleona M. minimus and N. murinus host a well differentiated strain which is phylogenetically distinct from supergroup E. Multiple events of Wolbachia infection in springtails as well as a richer diversity of the symbiont strains in soil arthropods were hereby confirmed.     

The western jewel butterfly (Hypochrysops halyaetus): factors affecting adult butterfly distribution within native Banksia bushland in an urban setting

Adult western jewel butterflies Hypochrysops halyaetus Hewitson were studied in the Koondoola Regional Bushland Reserve on the northern outskirts of the Perth metropolitan area, Australia, in 1999. The butterfly is myrmecophilous, and the southern (Perth) form is considered to be ‘Vulnerable’. Butterfly dispersal, distribution, population size and habitat preferences were estimated using mark-recapture techniques. Butterflies were capable of moving large distances, and were widespread within the reserve but with a low density; however, one small physical area (80 × 20 m) contained a colony with large numbers of individuals. This colony, and an adjacent area of bush, was subject to a detailed microdistribution study: 1158 butterflies were marked within it over a 25-day period. The small size of the microdistribution study area compromised statistical independence assumptions: spatial autocorrelation was tested for and corrected, where appropriate, using a Markov-chain Monte-Carlo approach. Sex differences were evident in spatial autocorrelation: female data were not spatially autocorrelated, whilst male data was. The latter probably reflects the impact of the perching mode of mate location employed by males. Butterflies appeared to prefer rather degraded bush typical of a post fire/disturbance regime with high densities of the host plant Jacksonia sternbergiana. Males were positively correlated with the proportion of bare ground, again probably relating to preferred perching sites for mate location. Females were negatively correlated with Conospremum stoechadis probably indicating dense ground cover in degraded areas and Xanthorrhoea preissii probably an indicator at the site of more mature vegetation. Reserve management to improve vegetation quality may threaten the persistence of the butterfly within the reserve, whilst controlled burning to create even quite small new areas of habitat in an urban situation may be difficult to achieve. The creation of suitable habitat may be possible using mechanical means, provided the action of fire is not an essential component of habitat creation. As the western jewel appears to prefer degraded vegetation, it is possible that there may be opportunities for this species’ expansion within the urban environment provided that its mutualistic ant partner is also present.     

Functional conservation units for the endangered Alcon Blue butterfly Maculinea alcon in Belgium (Lepidoptera: Lycaenidae)

To organize and prioritise species-specific conservation efforts, we delineate `functional conservation units' for the threatened Alcon Blue butterfly Maculinea alcon in Belgium. We used detailed distribution data on the butterfly, its host plant and its habitat, present-day population sizes and its mobility and colonization capacity to determine functional conservation units (FCUs) on different spatial scales: FCU-1, i.e., the 12 presently occupied habitat patches plus the area within a range of 500 m surrounding them (the maximum local movement distance, based on mark-release-recapture data), FCU-2, i.e., the areas within a range of 2 km around the occupied habitat patches (the maximum observed colonization capacity) and FCU-3, i.e., potential re-introduction sites (sites where M. alcon went extinct recently). We suggest different management and planning measures for each type of functional conservation unit and discuss translocation and re-introduction as `intensive care' conservation measures for this threatened and sedentary species.     

Resource availability, matrix quality, microclimate, and spatial pattern as predictors of patch use by the Karner blue butterfly

Determination of which aspects of habitat quality and habitat spatial arrangement best account for variation in a species’ distribution can guide management for organisms such as the Karner blue butterfly (Lycaeides melissa samuelis), a federally endangered subspecies inhabiting savannas of Midwest and Eastern United States. We examined the extent to which three sets of predictors, (1) larval host plant (Lupinus perennis, wild lupine) availability, (2) characteristics of the matrix surrounding host plant patches, and (3) factors affecting a patch’s thermal environment, accounted for variation in lupine patch use by Karner blues at Indiana Dunes National Lakeshore, Indiana and Fort McCoy, Wisconsin, USA. Each predictor set accounted for 7-13% of variation in patch occupancy by Karner blues at both sites and in larval feeding activity among patches at Indiana Dunes. Patch area, an indicator of host plant availability, was an exception, accounting for 30% of variation in patch occupancy at Indiana Dunes. Spatially structured patterns of patch use across the landscape accounted for 9-16% of variation in patch use and explained more variation in larval feeding activity than did spatial autocorrelation between neighboring patches. Because of this broader spatial trend across sites, a given management action may be more effective in promoting patch use in some portions of the landscape than in others. Spatial trend, resource availability, matrix quality, and microclimate, in general, accounted for similar amounts of variation in patch use and each should be incorporated into habitat management planning for the Karner blue butterfly.     

Molecular markers and ex situ conservation of the European elms (Ulmus spp.)

The enormous losses suffered by the European elms during recent Dutch elm disease outbreaks led to concern over the conservation of elm genetic resources, and the subsequent establishment of a series of ex situ collections. However, as ex situ collections are inevitably finite in size, some consideration needs to be given to selecting which samples to include in them. To contribute towards this process for European ex situ elm collections we have undertaken genetic studies on a Europe-wide sample of 535 individuals. A major aim has been to use genetic markers to clarify the identification of samples to ensure that the ex situ collections contain a representative spread of taxonomic diversity. This is important given the paucity of mature elms in the landscape due to Dutch elm disease. The lack of mature material (critical for identification) compounds identification problems in what was already a taxonomically difficult group. Our data (derived from random amplified polymorphic DNA and inter-simple sequence repeats) have provided a useful supplement to morphology in undertaking such sample identifications. The molecular data served to highlight mis-identified samples and led to extensive revisions of sample identities within individual countries. Our results were less useful in detecting regional intra-specific genetic structure, and do not provide sufficient information for prioritising within-species sample selections.     

Dynamics of early wolf and cougar eradication efforts in Montana: implications for conservation

Bounty records from Montana (1902-1930) were used to better understand spatial and temporal response of wolves (Canis lupus) and cougars (Puma concolor) to an eradication program. Number of payments for wolves declined from a high of 4116 in 1903 to 0 by 1928. Numbers of cougar pelts fluctuated, but generally declined from 177 in 1908 to 2 in 1930. An inverse spatial relationship existed between density of payments for wolves and cougars. Payments for wolves were greatest in the Prairie ecoregion, while payments for cougars were greatest in the Montane. The ratio of pup to adult wolves also was greatest in the Prairie ecoregion and least in the montane. If distribution and frequency of bounty payments were a reflection of the distribution and abundance of wolves, populations were highest and most productive in areas where they currently do not occur.     

Modelling populations of long-lived birds of prey for conservation: A study of imperial eagles (Aquila heliaca) in Kazakhstan

Eagle populations worldwide are in decline and their demography is generally poorly understood. We use novel sensitivity analysis of stochastic simulation models to analyse the demography of the worlds highest-density and longest-studied population of eastern imperial eagles (Aquila heliaca), at the Naurzum National Nature Reserve in Kazakhstan. Single variable perturbation (a simple elasticity-type analysis) showed that population growth was most sensitive to changes in adult survival but provided no information on how interactions between parameters may influence population growth. Multiple-variable perturbations (a more comprehensive elasticity-type analysis) suggested that population growth is relatively more sensitive to adult survival than is indicated by single-variable perturbation but also that when adult survival is within a biologically reasonable range, other parameters are still highly consequential to model outputs. For Naurzum’s imperial eagles, and for other structured populations of vertebrates, effective conservation and management likely requires an approach that addresses the importance of simultaneous variation in multiple vital rates including both survivorship and reproductive output.     

Ex situ genetic conservation of endangered Vatica guangxiensis (Dipterocarpaceae) in China

RAPD polymorphisms were applied to check the efficiency of ex situ genetic conservation of endangered Vatica guangxiensis X. L. Mo. endemic to southwestern China. Low level of genetic variation was revealed in three remaining natural populations. Twenty random primers, each with 10 base pairs, generated 231 bands with 53.68% being polymorphic, and with an average of 32.46% being polymorphic in each natural population. Strong population differentiation was revealed by AMOVA (analysis of molecular variance) and Gst value was 0.3764. The population ML ex situ conserved in the Xishuangbanna Tropical Botanical Garden contained an intermediate genetic variation compared with natural populations, with 30.74% bands being polymorphic. Of the total 231 bands generated in V. guangxiensis, 204 bands were also detected in population ML, indicating that 88.31% of the total genetic variations of this species were conserved in ex situ population. If only the alleles with moderate to high frequency (P>0.05) were considered, 204 out of 209 bands (97.61%) occurred in ex situ population ML. RAPD analysis also detected one exclusive band in natural population NS, and five in natural population NP, three of these exclusive bands were generated in every samples of natural population (NP), and other three had moderate to high frequencies. While none of these exclusive bands were detected in ex situ conserved population ML. Our conclusions are that the ex situ conserved population ML contains representative genetic variation to maintain long-term survival and evolutionary process of V. guangxiensis, and that more extensive ex situ sampling in natural population NS and NP is needed to conserve more exclusive alleles in ex situ population. The tropical area in the Botanical Garden would play a more important role in the ex situ conservation of rare and endangered plants.     

Population genetic structure and the conservation of isolated populations of Acacia raddiana in the Negev Desert

There is much concern over the high mortality of many populations of Acacia raddiana, a keystone tree species in the Negev desert of Israel. We used random amplified polymorphic DNA (RAPD) to assess patterns of genetic variation within and among 12 populations of A. raddiana from the Arava (Syrian-African Rift) valley and western Negev. A high level of genetic polymorphism was recorded within populations. An analysis of molecular variance (AMOVA) showed that about 59.4% of total genetic variance occurred among populations, which is considerably greater population differentiation than that recorded for other outbreeding species. Cluster and principal coordinates analyses and AMOVA indicate that the western Negev and Arava valley populations are highly differentiated. We suggest that there may have been two invasions of A. raddiana into Israel: one across the northern Sinai/Gaza Strip area into the western Negev, with some plants reaching the Dead Sea and a second invasion across the southern part of the Sinai peninsula, or even from Saudi Arabia, up to the Arava valley. From the conservation point of view, each population should be conserved separately because they are genetically highly differentiated and loss of any one population would lead to a dramatic loss of genetic variation. The mixing of genetically distinct populations may give rise to outbreeding depression (particularly because of GXE interactions). An obvious first step to the maintenance of this species' genetic diversity is the separate management of the western Negev and Arava valley populations because of their different evolutionary histories.     

Potential distribution modelling, niche characterization and conservation status assessment using GIS tools: a case study of Iberian Copris species

Dung beetle populations, in decline, play a critical ecological role in extensive pasture ecosystems by recycling organic matter; thus the importance of their conservation status. Presence data available for Copris hispanus (L.) and Copris lunaris (L.) (Coleoptera, Scarabaeidae) in Comunidad de Madrid (CM), and BIOMAPPER, a GIS-based tool, was used to model their environmental niches. The so derived potential distributions of both species were used to exemplify the utility of this kind of methodologies in conservation assessment, as well as its capacity to describe the potential sympatry between two or more species. Both species, distributed along a Dry-Mediterranean to Wet-Alpine environmental conditions gradient, overlap in areas of moderate temperatures and mean annual precipitations in the north of CM. Copris are poorly conserved in the existing protected sites network, but protection provided by new sites included in the future Natura 2000 Network will improve the general conservation status of these species in CM.     

The conservation status of the spectacled petrel Procellaria conspicillata

The spectacled petrel Procellaria conspicillata is listed as critically endangered due to its small population size and ongoing mortality on long-lines. Spectacled petrels were counted in 2004, repeating a census made in 1999 at their sole breeding locality, Inaccessible Island. The 2004 survey took place earlier in the breeding season than the previous count, allowing for more robust estimates of burrow occupancy. During early incubation, birds responded to call playback at 69% of burrow entrances, but birds in at least 8% of burrows remained silent. Birds in shallow burrows were less likely to respond to playback than were those in deep burrows. Two repeat trials at 100 marked nests showed that at least 61% of apparently ‘unoccupied’ burrows were occupied on subsequent checks, resulting in an overall occupancy estimate of 91%. Occupancy was equally high in peripheral colonies. The apparent spatial extent of colonies increased slightly from 1999, and the estimate of total burrow numbers increased by 50%, from 5900 burrows in 1999 to 8900 in 2004. Validation surveys indicated that burrow numbers were underestimated (84 ± 3%) to the same extent as that in 1999 (85 ± 4%), and repeat checks of one colony where all nests were marked showed that even careful counts underestimated actual numbers of burrows by up to 10%. This suggests there are some 11-12,000 burrows, and assuming 90% occupancy, the adult population is likely to be at least 20,000 birds. The population has increased over the last five years, continuing the apparent recovery from a very small population size in the early 20th century. Despite this increase, demographic models indicate that the population remains at risk from relatively small increases in mortality, if mortality is determined primarily by fishing effort. Mitigation of long-line mortality remains the key conservation goal for this species.     

The leaves of Pelargonium sidoides may substitute for its roots in the treatment of bacterial infections

Pelargonium sidoides (Geraniaceae) is a medicinal plant used for the treatment of various infections including tuberculosis and bronchitis. Traditionally, only the roots of the plant are used for medicinal formulations in the Eastern Cape. In addition to traditional uses, there is a great demand for the plant for export, therefore, the rate of its harvesting and the number of its collector has increased in recent years. This has led to a drastic drop in the population of the species in the wild. A method that could achieve the sustainable harvesting of P. sidoides would be to substitute its shoot for the roots in medicinal formulations. A comparative study of the antibacterial activities of the leaves and the roots of the plant was conducted on the materials collected from three different populations. Generally, both the shoot and the roots of P. sidoides have antimicrobial properties. There was no significant observable difference between the MIC of extracts from both parts. The choice of the root by the traditional healers over the leaves may be arbitrary or due to easy collection. The leaves of P. sidoides may, as well, substitute for its roots in medicinal formulations especially in the treatment of bacterial diseases like tuberculosis and bronchitis.     

Ecology of the native and introduced crayfishes Austropotamobius pallipes and Procambarus clarkii in southern Spain and implications for conservation of the native species

The former and present distribution of white clawed crayfish (Austropotamobius pallipes) in the province of Granada (southern Spain) is studied. Before 1980 it was widely distributed but at present only 16 populations exist. The decline is related to the presence of the freshwater red-swamp crayfish (Procambarus clarkii), an American species, vector of the aphanomycosis disease, introduced to the Iberian Peninsula in 1974 and now widely distributed in the watercourses and marshes of southern Spain. To establish an appropriate conservation policy for A. pallipes at its southernmost distribution limit, we studied watercourses from two river basins, Genil and Guadiana Menor, (tributaries of the Guadalquivir River). P. clarkii inhabits the medium to lower reaches of these two river basins (with its upper limit at 820 m a.s.l.). The distribution of this species was best explained by the effect of three of the 12 analyzed variables: altitude, water-current and minimum winter temperatures. From our results, the repopulation of the native crayfish is almost impossible in those reaches inhabited by P. clarkii. However, based on the habitat selection study, it is clear that upper reaches are unsuitable for the red-swamp crayfish, where the native white-clawed crayfish may have greater survival possibilities, and these sites can be used for future restocking projects.     

The value of bat-boxes in the conservation of Pipistrellus pygmaeus in wetland rice paddies

Wetlands with rice paddies are key habitats in the conservation of biodiversity in the Mediterranean Region and are potentially suitable habitats for foraging bats, since they provide food (insects) and drinking places; nevertheless, many wetlands lack natural roosting sites. A bat-box program designed to ascertain bat-box preferences was initiated in 1999 in the Ebro Delta (NE Spain), one of the most important wetlands in Europe. A total of 69 bat-boxes of two types (single and double compartment) were placed on three supports (trees, houses, and posts) facing east or west. Pipistrellus pygmaeus occupancy rates and the number of individuals per box were monitored on 16 occasions from July 2000 to February 2004. Bat-box preferences were only detected during the breeding season. Bat abundance was higher in east-facing boxes, in double-compartment boxes, and in boxes placed on posts and houses. Boxes on natural supports (trees) were avoided. Bat-box occupancy rates were higher during the breeding season (95.6%, spring-summer) due to the formation of maternity colonies. The number of individuals in bat-boxes during the breeding season increased as the study period progressed (from summer 2000 to summer 2003), suggesting a high degree of acceptance by maternity colonies of these alternative locations. Occupancy rates observed were the highest ever reported in bat-box scientific literature. This study highlights the role of bat-box programs as useful alternative management tools for the conservation of bat populations in highly productive wetland habitats where few natural roost sites are available.     

The conservation status of Townsend's shearwater Puffinus auricularis auricularis

Townsend's shearwater (Puffinus auricularis auricularis) is an endangered seabird endemic to the Revillagigedo Archipelago. It nested on Socorro, Clarion, and San Benedicto Islands. It was extirpated by the Barcena volcano on San Benedicto in 1952, and there are no recent indications of nesting. Introduced mammals—pigs and rabbits—preyed on them and destroyed habitat at Clarion; shearwaters were extirpated by 1988, and no breeding attempts have been reported since. Our results confirm that Socorro holds the last breeding grounds. We found breeding colonies above 800 m and a minimum population of 1100 individuals. This represents a significant reduction in distribution and population size. Intensive cat predation at Socorro could potentially kill ca. 350 females per season, and sheep progressively destroy nesting areas. Population projections suggest that demographic instability could occur in less than 100 years under severe predation and habitat degradation. Only low predation rates would allow population persistence for more than 150 years in spite of a declining population. Thus, the immediate eradication of all introduced mammals is necessary to prevent the extinction of this seabird.     

Population baseline data for monitoring genetic diversity loss for 2010: A case study for Brassica species in the UK

The Convention on Biological Diversity and the subsequent International Treaty on Plant Genetic Resources for Food and Agriculture have proved a watershed in plant genetic resources (PGR) conservation and consequent initiatives have set various PGR conservation targets to be met by 2010. The aim of our paper is to develop ideas and issues concerning the monitoring of natural genetic resources: particularly in terms of developing a baseline from which to measure levels of genetic diversity. Three species of Brassica (B. nigra, B. oleracea, B. rapa) found wild in the UK were assessed for levels of genetic diversity using AFLP. The relationship between genetic distribution and ecogeographic distribution was considered for each species to determine patterns that may be useful in formulating conservation strategies. Genetic distance between populations of B. nigra and B. rapa were correlated to geographic distance. Levels of genetic polymorphism in B. oleracea were correlated to soil pH while in B. rapa they were correlated to soil coarseness. In terms of PGR conservation these findings may suggest an emphasis toward in situ conservation of a selection of disparate populations would be appropriate where possible as such adaptations may be lost in ex situ collections.     

Inferring habitat-suitability areas with ecological modelling techniques and GIS: A contribution to assess the conservation status of Vipera latastei

Some snakes are highly vulnerable to extinction due to several life history traits. However, the elusive behavior and secretive habits of some widespread species constrain the collection of demographic and ecological data necessary for the identification of extinction-prone species. In this scenario, the enhancement of ecological modelling techniques in Geographical Information Systems (GIS) is providing researchers with robust tools to apply to such species. This study has identified the environmental factors that limit the current distribution of Vipera latastei, a species with secretive behavior, and has evaluated how human activities affect its current conservation status, identifying areas of best habitat suitability in the Iberian Peninsula. Ecological-niche factor analysis (ENFA) indicated low marginality (0.299) and high tolerance (0.887) scores, suggesting strong tendency for the species to live in average conditions throughout the study area and to inhabit any of the environmental conditions. The analysis also revealed that this viper tends to select particular Mediterranean habitats, although topographic factors (altitude and slope) were the major environmental constraints for the Iberian distribution pattern of the species. The presence of other parapatric viper species in the north of the Iberian Peninsula (V. aspis and V. seoanei) and two human-related variables (landscape transformation and human density) also had a negative relation with the occurrence of V. latastei. All factors can explain its absence in northern Iberia and its fragmented distribution as currently is found mostly in mountains and relatively undisturbed low-altitude areas. The historical destruction and alteration of natural Mediterranean habitats and several life-history traits of the species contribute to its vulnerability to extinction. The ENFA analysis proved to be an outstanding method to evaluate the factors that limit the distribution range of secretive and widespread species such as V. latastei, updating evaluation of their conservation status.     

Non-pathogenic Fusarium strains protect the seedlings of Lepidium sativum from Pythium ultimum

Two root-colonizing Fusarium strains, Ls-F-in-4-1 and Rs-F-in-11, isolated from roots of Brassicaceae plants, induced the resistance in Lepidium sativum seedlings against Pythium ultimum. These strains caused an increase in the content of benzyl isothiocyanate, and of its precursor glucotropaeolin, in the roots of the host plants. The increased isothiocyanate content is one of the factors contributing to the resistance of L. sativum against P. ultimum. To be transformed into the fungitoxic compound benzyl isothiocyanate, glucotropaeolin has to be hydrolyzed by myrosinase, which can be produced either by plants or microorganisms. The Fusarium strain Ls-F-in-4-1 has a myrosinase activity but the strain Rs-F-in-11 has not. These results suggest that both strains are able to trigger the metabolic pathway leading to benzyl isothiocyanate production in the plant. In the case of the myrosinase-negative strain Rs-F-in-11, hydrolyzation into isothiocyanate is only due to the myrosinase activity of the plant, and in the other case, the myrosinase produced by the strain Ls-F-in-11 also would contribute to the production of isothiocyanate. This paper reports a new mode of action of non-pathogenic Fusarium strains in controlling P. ultimum.     

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.     

An alternative to dolphin oil as a fish attractant in the Ganges River system: conservation of the Ganges River dolphin

Oil extracted from blubber of the Ganges River dolphin, Platanista gangetica, is used as a fish attractant in India and Bangladesh. This oil fishery is associated with the mortality of hundreds of dolphins every year. Oil from fish scraps was field tested for 121 days during 1997-1999 in the Ganges at Patna and was a very effective alternative to dolphin oil as a fish attractant. Fishing with fish oil resulted in higher catches and greater income per person per hour (Rs. 17.8±12.5) than fishing with dolphin oil (Rs. 11.6±9.3). However, there is no statistically significant difference between the results from fishing with dolphin and fish oil. Oil from fish scraps is inexpensive and can be made readily available to all the fishermen. Its widespread use would help in conserving the endangered Ganges River dolphin.