New data on the distribution of the Volturno spined loach Cobitis zanandreai (Teleostei: Cobitidae)

The Volturno Spined Loach Cobitis zanandreai is thought to be restricted to only two places in Italy, the Fondi Lake (Latium) and the Volturno river basin (Campania). In this study we carried out a review of the available literature and surveyed 37 sites of potential occurrence. We discovered seven previously unknown populations of C. zanandreai in the Liri-Garigliano and the Pontine plain watersheds, significantly expanding the known limits of its distribution. We found the invasive C. bilineata only in the Amaseno River. Our results agree with records from the 19th and 20th centuries, highlighting the importance of historical data in understanding current species distribution. Our findings provided a substantial distribution update that allow to assess the species conservation status more objectively.     

Biocoenotic and karyological characterization of four upspring critical taxa of the eastern Po Plain (North-Eastern Italy)

ABSTRACT

Four critical species of a damp site of the eastern Po Plain were examined from a karyological point of view, and their population density related to their status based on UICN categories. These taxa are: Armeria helodes Martini et Poldini, an endemic species to lower Friuli, with 2n=18; Gladiolus palustris Gaudin, a species of humid grasslands with a fluctuating water level, with 2n=60; Senecio doria L., a vulnerable species with 2n=40, and S.paludosus L. considered an Italian endangered species, with 2n=40. Some observations about the biotopes studied are also reported.     

Flower-visiting and pollen-carrying arthropods of Leucojum aestivum L. (Amaryllidaceae) in wild,reintroduced and ex situ populations

Leucojum aestivum L. is a wetland-dependent perennial geophyte occurring in Europe and western Asia. It is self-incompatible, with high level of fruit-set and seed-set. Yet, its pollinators are poorly known. Here, we present the most recent research on potential pollinators of L. aestivum. We collected data on flower-visiting and pollen-carrying arthropods in three populations of the species in the River Po plain (N-Italy), where L. aestivum occurs mainly in forests with Alnus glutinosa and Fraxinus sp. pl. close to rivers. We studied a wild population, a new reintroduced population and an ex situ population located at the Botanical Garden of the University of Pavia. Our study identified 18 arthropod taxa carrying L. aestivum pollen; the soft-winged flower beetle Dasytes plumbeus (Coleoptera: Melyridae) and the sac-spider Clubiona sp. pl. (Araneae: Clubionidae) were the most frequent visitors. Hymenoptera only occasionally visited L. aestivum flowers (e.g. the sweat bee Lasioglossum punctatissimum). D. plumbeus, the long-horned beetle Grammoptera ruficornis (Coleoptera: Cerambycidae), Clubiona sp. pl. and L. punctatissimum resulted in the taxa with the highest average abundance of pollen grains on their body suggesting a potential role in L. aestivum pollination. Differences in flower-visiting and pollen-carrying arthropods were observed between the three populations, with a decreasing taxonomic diversity of visiting species from the wild population to the ex situ population. Our results, based on direct observation in the field, are partially in contrast with a previous study on L. aestivum pollinators that suggested diurnal and nocturnal Lepidoptera and occasionally bees as main pollinators for the species.

     

Preferential utilization of organic and inorganic sources of phosphorus by wheat plant

On soils of low P supply organic P (Po) makes up a similar or even larger part in soil solution than inorganic P (Pi). The ability of wheat (Triticum aestivum L., cv. Star) plants to hydrolyze and absorb this Po in comparison to similar concentrations of Pi was studied. Four concentration levels of Pi and Po were obtained by extracting two soils with deionized water in a ratio of 1:1 and concentrating the resulting filtrate by freeze drying to different degrees. The concentration of Pi varied between 5 and 36 μM and Po between 3 and 22 μM. Wheat seedlings were grown in these solutions for 12 and 24 h and acid and alkaline phosphatase activity determined. The reduction of Po concentration in solution expressed on a root length basis gave the rate of Po hydrolysis and the reduction in concentration of Pi and Po gave the P inflow into the roots. No alkaline phosphatase activity was detected. The activity of wheat root acid phosphatase increased with Po concentration in solution. Phosphorus uptake was 2 to 6 fold higher from Pi than from Po at similar concentrations of both. The rate of uptake from Pi, the inflow, as well as the rate of hydrolysis of Po increased linearly with concentration but at similar concentration the inflow was 2 to 4 times higher than the rate of Po hydrolysis. Results suggest that plants can utilize Po after hydrolysis by phosphatase, but Pi is more important and preferentially used by plants; Po may be essential for plant nutrition especially in high P-fixing soils.     

The Inhibition of Human Tumor Cell Proliferation by RNase Pol,a Member of the RNase T1 Family,from Pleurotus ostreatus

RNase Po1 is a guanylic acid-specific ribonuclease (a RNase T1 family RNase) from Pleurotus ostreatus. We determined the cDNA sequence encoding RNase Po1 and expressed RNase Po1 in Escherichia coli. A comparison of the enzymatic properties of RNase Po1 and RNase T1 indicated that the optimum temperature for RNase Po1 activity was 20 °C higher than that for RNase T1. An MTT assay indicated that RNase Po1 inhibits the proliferation of human neuroblastoma cells (IMR-32 and SK-N-SH) and human leukemia cells (Jurkat and HL-60). Furthermore, Hoechst 33342 staining showed morphological changes in HL-60 cells due to RNase Po1, and flow cytometry indicated the appearance of a sub-G1 cell population. The extent of these changes was dependent on the concentration of RNase Pol. We suggest that RNase Po1 induces apoptosis in tumor cells.     

Isolation of microsatellite loci in the codling moth,Cydia pomonella (Lepidoptera: Tortricidae)

Cydia pomonella L. is an important insect pest of pome fruits worldwide. We have isolated and characterized 17 microsatellite loci from the enriched genomic libraries constructed using a biotin/streptavidin capture protocol. Among these loci, 11 were scored polymorphic when 50 individuals from a laboratory population were screened. Their alleles numbered two to four, with the observed heterozygosity (H_O) ranging from 0.114 to 0.404. Successful amplification was obtained for all these loci when the designed primers were tested, showing the promise of use in genetic mapping and population studies.     

Genetic Structure of the Population of Alternaria solani in Brazil

Understanding the genetic structure of the population of Alternaria solani (AS) is an important component of epidemiological studies of early blight, a severe disease that affects potato (Po) and tomato (To) worldwide. Up to 150 isolates obtained from both hosts were analysed with RAPD and AFLP markers to estimate the amount and distribution of genetic variability of AS in Brazil. Using RAPD, gene diversity (h = 0.20) and scaled indices of diversity of Shannon (H′ = 0.66) and Stoddart and Taylor’s (G = 0.31) for the Po population were higher than those of the To (h = 0.07, H′ = 0.34, G = 0.17). For AFLP, the statistics for the Po (h = 0.17, H′ = 0.86, G = 0.49) and To (h = 0.17, H′ = 0.85, G = 0.36) populations were similar. For each RAPD and AFLP locus, the allele frequency for the overall population ranged from 0.006 to 0.988, and 0.007 to 0.993, respectively. Genetic differentiation was high (G_ST = 0.41 and θ = 0.59) and moderately high (G_ST = 0.23 and θ = 0.37) when estimated with RAPD and AFLP, respectively. Based on cluster analyses, there was strong evidence of association of pathogen haplotypes with host species. The null hypothesis of random association of alleles was rejected in the analysis of both RAPD (I_A = 13.1, P < 0.001) and AFLP (I_A = 2.2, P < 0.001) markers. The average number of migrants was estimated to be around one and two individuals per generation, using RAPD and AFLP, respectively. There was no correlation between genetic distance and geographical origin of AS haplotypes for RAPD (r = ?0.07, P = 0.84) and AFLP (r = ?0.03, P = 0.70). The AS population is clonal with high genetic variability, and there is genetic differentiation between the populations that affect To and Po.     

Phylogenetic position of a threatened stag beetle, Lucanus datunensis (Coleoptera: Lucanidae) in Taiwan and implications for conservation

Among nine endemic Lucanus beetles in Taiwan, L. datunensis is the island’s smallest and most threatened species. It currently exists as only one population located in tall grasslands of Mt. Datun in the Yangmingshan National Park. Given the isolated population, unique subtropical grassland, and the threats resulting from human activities, L. datunensis raises immediate conservation concern for its long-term survival. Phylogenies reconstructed from combined mitochondrial cytochrome oxidase subunit 1 (1310 bps) and nuclear wingless (436 bps) genes were resolved and placed L. datunensis as a phylogenetically distinct species sister to L. fortunei from China. All 13 examined individuals of L. datunensis shared just one mitochondrial haplotype suggesting extremely low mitochondrial DNA diversity and a small effective population size. L. datunensis and morphologically closest L. miwai were distantly related and appear to have evolved in parallel the life history traits of a small body size and diurnal mate-searching behavior. We hypothesize that these habitat-associated characters are convergent adaptations that have evolved in response to shifts from forests to grasslands.     

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

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

Molecular analysis of the nuclear organellar genotype of somatic hybrid plants between tomato (Lycopersicon esculentum) and Lycopersicon chilense

Summary Somatic hybrid plants were recovered following fusion of leaf mesophyll protoplasts isolated from tomato (Lycopersicon esculentum) cultivar UC82 with protoplasts isolated from suspension cultured cells of L. chilense, LA 1959. Iodoacetate was used to select against the growth of unfused tomato protoplasts. Two somatic hybrids were recovered in a population of 16 regenerants. No tomato regenerants were recovered; all of the non-hybrid regenerants were L. chilense. The L. chilense protoplast regenerants were tetraploid. The hybrid nature of the plants was verified using species-specific restriction fragment length polymorphisms for the nuclear, chloroplast and mitochondrial genomes. The somatic hybrids had inherited the chloroplast DNA of the tomato parent, and portions of the mitochondrial DNA of the L. chilense parent. The somatic hybrids formed flowers and developed seedless fruit.     

Changes and availability of P fractions following 65 years of P application to a calcareous soil in a Mediterranean climate

The fate and availability of P derived from granular fertilisers in an alkaline Calcarosol soil were examined in a 65-year field trial in a semi-arid environment (annual rainfall 325 mm). Sequential P fractionation was conducted in the soils collected from the trial plots receiving 0–12 kg P ha⁻¹crop⁻¹, and the rhizosphere soil after growing wheat (Triticum aestivum L. cv. Yitpi) and chickpea (Cicer arietinum L. cv. Genesis 836) for one or two 60-day cycles in the glasshouse. Increasing long-term P application rate over 65 years significantly increased all inorganic P (Pi) fractions except HCl–Pi. By contrast, P application did not affect or tended to decrease organic P (Po) fractions. Increasing P application also increased Olsen-P and resin-P but decreased the P buffer capacity and sorption maxima. Residual P, Pi and Po fractions accounted for an average of 32, 16 and 52% of total P, respectively. All soil P fractions including residual P in the rhizosphere soil declined following 60-day growth of either wheat or chickpea. The decreases were greater in soils with a history of high P application than low P. An exception was water-extractable Po, which increased following plant growth. Changes in various P fractions in the rhizosphere followed the same pattern for both plant species. Biomass production and P uptake of the plants grown in the glasshouse correlated positively with the residual P and inorganic fractions (except HCl–Pi) but negatively with Po in the H₂O-, NaOH- and H₂SO₄-fractions of the original soils. The results suggest that the long-term application of fertiliser P to the calcareous sandy soil built up residual P and non-labile Pi fractions, but these P fractions are potentially available to crops.     

Isolation and characterization of eight microsatellite loci from <Emphasis Type="Italic">Lycorma delicatula</Emphasis> (White) (Hemiptera: Fulgoridae) for population genetic analysis in Korea

Lycorma delicatula (White) is native to China but is becoming an important insect pest in Korea. Polymorphic DNA markers like microsatellites are widely used for characterizing dispersal patterns and capacity of invasive insect pests which can contribute to designing effective management of the species. To facilitate such population genetic studies of L. delicatula in Korea, we isolated and characterized eight microsatellite loci for L. delicatula using a hybridization-biotin enrichment method. We further used these novel microsatellite loci to determine population genetic parameters for 33 L. delicatula specimens collected from Cheonan, South Korea where outbreaks of this species were first reported in Korea. The number of alleles per locus ranged from three to ten, with an average of 6.25. The mean expected (H _E) and observed heterozygosities (H _O) were 0.575 and 0.626, respectively. The eight loci showed no deviation from Hardy–Weinberg equilibrium according to the adjusted significance threshold (P = 0.00625), and there was no linkage disequilibrium between each pair of these eight markers. Bayesian cluster analysis using the program structure revealed no evidence of genetic structuring in L. delicatula samples from Cheonan. These new microsatellite markers will be widely applicable to future ecological genetic studies of L. delicatula, including assessment of the level of gene flow and genetic connectivity among populations that are necessary for effective management and monitoring of the species.     

Distribution map of Ambrosia artemisiifolia L. (Asteraceae) in Italy

The spread of the invasive and allergenic Ambrosia artemisiifolia L. in Italy was analysed and mapped using distribution data from a wide range of sources. Ambrosia artemisiifolia occupies 1057 floristic quadrants which are mostly distributed in the Po plain. The distribution obtained represents the basis to implement urgent management strategies.     

Microsatellites from the vairone Leuciscus souffia (Pisces: Cyprinidae) and their application to closely related species

Eleven microsatellites were isolated from the vairone Leuciscus souffia (Risso 1826), an endangered fish that inhabits river systems in and around the Alps in Europe. The level of genetic diversity was assessed in 29 individuals of the subspecies L. s. souffia, and their variability was further estimated in seven individuals of a different subspecies, L. s. muticellus. Eight of these microsatellite loci were also applied to seven closely related cyprinid species. Availability of the reported microsatellite loci will facilitate the investigation of population genetic structure of these species with applications for the development of conservation strategies and phylogeographical approaches.     

Mycorrhization helper bacteria: a case of specificity for altering ectomycorrhiza architecture but not ectomycorrhiza formation

Mycorrhization helper bacteria (MHB), isolated from phylogenetically distinct ectomycorrhizal symbioses involving Lactarius rufus, Laccaria bicolor or Suillus luteus, were tested for fungus specificity to enhance L. rufus–Pinus sylvestris or L. bicolor–P. sylvestris mycorrhiza formation. As MHB isolated from the L. rufus and S. luteus mycorrhiza were originally characterised using a microcosm system, we assessed their ability to enhance mycorrhiza formation in a glasshouse system in order to determine the extent to which MHB are system-specific. Paenibacillus sp. EJP73, an MHB for L. rufus in the microcosm, significantly enhanced L. bicolor mycorrhiza formation in the glasshouse, demonstrating that the MHB effect of this bacterium is neither fungus-specific nor limited to the original experimental system. Although the five MHB strains studied were unable to significantly enhance L. rufus mycorrhiza formation, two of them did have a significant effect on dichotomous short root branching by L. rufus. The effect was specific to Paenibacillus sp. EJP73 and Burkholderia sp. EJP67, the two strains isolated from L. rufus mycorrhiza, and was not associated with auxin production. Altered mycorrhiza architecture rather than absolute number of mycorrhizal roots may be an important previously overlooked parameter for defining MHB effects.     

Characterization of polymorphic microsatellite loci in the tropical ant‐plant Leonardoxa africana (Fabaceae: Caesalpinioideae)

Ten microsatellite loci were isolated from the African ant‐plant Leonardoxa africana (Fabaceae: Caesalpinioideae). They differentiate the two most divergent subspecies of this polytypic complex, L. a. africana and L. a. gracilicaulis, showing promise for the study of gene flow. In each of these two subspecies, high levels of within‐population variation were observed, with a number of alleles ranging from one to 10 in ssp. africana and from one to 7 in ssp. gracilicaulis, and heterozygosity from 0 to 0.933 in ssp. africana and to 0.867 in ssp. gracilicaulis. All loci amplified successfully in the two other subspecies of L. africana.     

Diversity of mitochondrial DNA of the endangered white-clawed crayfish (Austropotamobius italicus) in the Po River catchment

1. The strong contraction of the European populations of white‐clawed crayfish has stimulated research of its population genetics. As part of a conservation project to introduce and improve Italian populations of Austropotamobius italicus, the genetic diversity of 16 populations from 10 tributaries of the Po River drainage was evaluated by sequencing a 494 bp length of cytochrome oxidase subunit I (COI mtDNA). 2. The evolutionary tree topologies and the parsimony network of 26 haplotypes separated into two clusters, on the basis of high average genetic distance (6.51 ± 0.42%). This finding indicated the presence of distinct evolutionary lineages, other than different allopatric sources. 3. An ancestral haplotype (Nav) was identified and 13 haplotypes, found mainly as singletons, were independently connected to Nav by short branches with one mutation site. Unimodal mismatch distribution and the goodness‐of‐fit of the observed data reflected a model of sudden population expansion, after the last glacial bottleneck event. 4. A general moderate level of genetic variability (h = 0.22–0.7 and π = 0.045–0.27%) was highlighted within A. italicus populations inhabiting Alpine and Apennine tributaries of the Po River. Analysis of molecular variance (amova ) revealed significant genetic structuring across all hierarchical levels, indicating that a major proportion of genetic variance is structured among localities within drainages. Multiple adjacent localities, strictly connected by the Po River, shared the two most frequent haplotypes (Nav and Laz), while remote populations, far from the Po River inflow, move towards a mtDNA fixation. 5. The results of this study indicate that distinct populations should be treated as separate entities for management. The key implication of the data is that ad hoc measures for successful programmes of recovery and management strategies for A. italicus conservation, such as the phylogeographic study of haplotype distribution at a local scale and the genetic variability of populations used as stock breeders, should be considered.     

Lemur catta in small forest fragments: Which variables best predict population viability?

Habitat fragmentation is an increasingly serious issue affecting primates in most regions where they are found today. Populations of Lemur catta (ring-tailed lemur) in Madagascar's south-central region are increasingly restricted to small, isolated forest fragments, surrounded by grasslands or small-scale agriculture. Our aim was to evaluate the potential for population viability of L. catta in nine forest fragments of varying sizes (2–46 ha, population range: 6–210 animals) in south-central Madagascar, using a set of comparative, quantitative ecological measures. We used Poisson regression models with a log link function to examine the effects of fragment size, within-fragment food availability, and abundance of matrix resources (food and water sources) on L. catta population sizes and juvenile recruitment. We found a strong association between overall population size and (a) fragment size and (b) abundance of key food resources Melia azedarach and Ficus spp. (per 100 m along transect lines). Juvenile recruitment was also associated with fragment size and abundance of the two above-mentioned food resources. When the largest population, an outlier, was removed from the analysis, again, the model containing fragment size and abundance of M. azedarach and Ficus spp. was the best fitting, but the model that best predicted juvenile recruitment contained only fragment size. While our results are useful for predicting population presence and possible persistence in these fragments, both the potential for male dispersal and the extent of human disturbance within most fragments play crucial roles regarding the likelihood of long-term L. catta survival. While seven of the nine fragments were reasonably protected from human disturbance, only three offered the strong potential for male dispersal, thus the long-term viability of many of these populations is highly uncertain.     

Genetic evaluation of marine biogeographical barriers: perspectives from two widespread Indo-Pacific snappers (Lutjanus kasmira and Lutjanus fulvus)

Aim In the Indo‐Pacific, the mass of islands of the Indonesian archipelago constitute a major biogeographical barrier (the Indo‐Pacific Barrier, IPB) separating the Pacific and Indian oceans. Evidence for other, more localized barriers include high rates of endemism at the Marquesas and other isolated peripheral islands in the Pacific. Here we use mitochondrial‐sequence comparisons to evaluate the efficacy of biogeographical barriers on populations of the snappers Lutjanus kasmira and Lutjanus fulvus across their natural ranges. Location Pacific and Indian oceans. Methods Mitochondrial cytochrome b sequence data were obtained from 370 individuals of L. kasmira and 203 individuals of L. fulvus collected from across each species’ range. Allele frequency data for two nuclear introns were collected from L. kasmira. Phylogenetic and population‐level analyses were used to determine patterns of population structure in these species and to identify barriers to dispersal. Results Lutjanus kasmira lacks genetic structure across the IPB and throughout 12,000 km of its central Indo‐Pacific range. In contrast, L. fulvus demonstrates high levels of population structure at all geographical scales. In both species, highly significant population structure results primarily from the phylogenetic distinctiveness of their Marquesas Islands populations (L. kasmira, d = 0.50–0.53%; L. fulvus, d = 0.87–1.50%). Coalescence analyses of the L. kasmira data indicate that populations at opposite ends of its range (western Indian Ocean and the Marquesas) are the oldest. Coalescence analyses for L. fulvus are less robust but also indicate colonization from the Indian to the Pacific Ocean. Main conclusions The IPB does not act as a biogeographical barrier to L. kasmira, and, in L. fulvus, its effects are no stronger than isolating mechanisms elsewhere. Both species demonstrate a strong genetic break at the Marquesas. Population divergence and high endemism in that archipelago may be a product of geographical isolation enhanced by oceanographic currents that limit gene flow to and from those islands, and adaptation to unusual ecological conditions. Lutjanus kasmira shows evidence of Pleistocene population expansion throughout the Indo‐central Pacific that originated in the western Indian Ocean rather than the Marquesas, further demonstrating a strong barrier at the latter location.     

Development of novel microsatellite markers for the grassland species Lolium multiflorum,Lolium perenne and Festuca pratensis

Twelve microsatellite markers were isolated from Lolium multiflorum. Allelic variability and cross‐species amplification were assessed on 16 individuals of each of the three grassland species L. multiflorum, Lolium perenne and Festuca pratensis. Cross‐species amplification success was 100% for L. perenne and 83% for F. pratensis. The number of alleles detected ranged from one to 14 with an average of 3.4. While three microsatellite loci were polymorphic in all three species, one marker produced species‐specific alleles in all three species. These microsatellite markers provide a valuable tool for population genetic studies within and among species of the Festuca–Lolium complex.