Endosymbiotic bacteria nodulating a new endemic lupine Lupinus mariae-josephi from alkaline soils in Eastern Spain represent a new lineage within the Bradyrhizobium genus

Lupinus mariae-josephi is a recently described endemic Lupinus species from a small area in Eastern Spain where it thrives in soils with active lime and high pH. The L. mariae-josephi root symbionts were shown to be very slow-growing bacteria with different phenotypic and symbiotic characteristics from those of Bradyrhizobium strains nodulating other Lupinus. Their phylogenetic status was examined by multilocus sequence analyses of four housekeeping genes (16S rRNA, glnII, recA, and atpD) and showed the existence of a distinct evolutionary lineage for L. mariae-josephi that also included Bradyrhizobium jicamae. Within this lineage, the tested isolates clustered in three different sub-groups that might correspond to novel sister Bradyrhizobium species. These core gene analyses consistently showed that all the endosymbiotic bacteria isolated from other Lupinus species of the Iberian Peninsula were related to strains of the B. canariense or B. japonicum lineages and were separate from the L. mariae-josephi isolates. Phylogenetic analysis based on nodC symbiotic gene sequences showed that L. mariae-josephi bacteria also constituted a new symbiotic lineage distant from those previously defined in the genus Bradyrhizobium. In contrast, the nodC genes of isolates from other Lupinus spp. from the Iberian Peninsula were again clearly related to the B. canariense and B. japonicum bv. genistearum lineages. Speciation of L. mariae-josephi bradyrhizobia may result from the colonization of a singular habitat by their unique legume host.

Lupinus mariae-josphi, a new lupin endemic of soils with active lime and high pH in South Eastern Spain, is nodulated by a new bacterial lineage within Bradyrhizobium genus

Lupinus mariae-josephi is a recently described species (Pascual, 2004) able to grow in soils with high pH and active lime content in the Valencia province (Spain). L. mariae-josephi endosymbionts are extremely slowgrowing bacteria with genetic and symbiotic characteristics that differentiate them from Bradyrhizobium strains nodulating Lupinus spp. native of the Iberian Peninsula and adapted to grow in acid soils. Cross-inoculation experiments revealed that all the endosymbiotic isolates from L. mariae-josephi tested are legume-host selective and are unable to nodulate species such as L. angustifolius, and L. luteus. In contrast, Bradyrhizobium strains from Lupinus spp. tested were able to nodulate L. mariae-josephi, although the nodules fixed nitrogen inefficiently. Phylogenetic analysis was performed with housekeeping genes (rrn, glnII, recA, atpD) and nodulation gene nodC. Housekeeping gene phylogeny revealed that L. mariae-josephi rhizobia form a strongly supported monophyletic group within Bradyrhizobium genus. This cluster also includes B. jicamae and certain strains of B. elkanii. Contrarily, isolates from other Lupinus spp. native of the Iberian Peninsula were grouped mainly within B. canariense and two B. japonicum lineages. Phylogenetic analysis of L. mariae-josephi isolates based on the nodC symbiotic gene defined a solid clade close to isolates from Algerian Retama spp. and to fast-growing rhizobia.

Census, reproductive biology, and germination of Astragalus gines-lopezii (Fabaceae), a narrow and endangered endemic species of SW Spain

Astragalus gines-lopezii Talavera, Podlech, Devesa & F.M.Vázquez (Fabaceae) is a threatened endemic species with a distribution restricted to a very small area in Badajoz Province (Extremadura Region, SW Spain) and only 2 populations are known. This species was catalogued in the ?Endangered? category in the 2008 Red List and the 2010 Threatened Spanish Vascular Flora List. Despite its status as an endangered species, at present very little is known about the distribution, census, and reproductive biology of this species. In this study we have carried out an exhaustive census of A. gines-lopezii , and we have evaluated the production of flowers, fruits, and seeds and the existence or not of intra- and interpopulation variability in seed germination. Results have highlighted the high reproductive capacity of this species on the basis of a high production of flowers, fruits, and seeds. Mechanical scarification of seeds was effective for increasing germination. Thus, initial germination (22%?60%) was increased to 97%?99% when seeds were rubbed with sandpapers. A high intra- and interpopulation variability in seed germination was found in this species. A. gines-lopezii produces seeds with different degrees of physical dormancy, varying this grade among different individuals within a population.

Census, reproductive biology, and germination of Astragalus gines-lopezii (Fabaceae),a narrow and endangered endemic species of SW Spain.

Astragalus gines-lopezii Talavera, Podlech, Devesa & F.M.Vazquez (Fabaceae) is a threatened endemic species with a distribution restricted to a very small area in Badajoz Province (Extremadura Region, SW Spain) and only 2 populations are known.This species was catalogued in the "Endangered" category in the 2008 Red List and the 2010 Threatened Spanish Vascular Flora List. Despite its status as an endangered species, at present very little is known about the distribution, census, and reproductive biology of this species. In this study we have carried out anexhaustive census of A. gines-lopezii, and we have evaluated the production of flowers, fruits, and seeds and the existence or not of intra- and interpopulation variability in seed germination. Results have highlighted the high reproductive capacity of this species on the basis of a high production of flowers, fruits, and seeds. Mechanical scarification of seeds was effective for increasing germination. Thus, initial germination (22%-60%) was increased to 97%-99% when seeds were rubbed with sandpapers. A high intra- and interpopulation variability in seed germination was found in this species. A. gines-lopezii produces seeds with different degrees of physical dormancy, varying this grade among different individuals within a population.

Molecular evidence for multiple origins of woodiness and a New World biogeographic connection of the Macaronesian Island endemic Pericallis (Asteraceae: Senecioneae)

The prevalence of woody species in oceanic islands has attracted the attention of evolutionary biologists for more than a century. We used a phylogeny based on sequences of the internal-transcribed spacer region of nuclear ribosomal DNA to trace the evolution of woodiness in Pericallis (Asteraceae: Senecioneae), a genus endemic to the Macaronesian archipelagos of the Azores, Madeira, and Canaries. Our results show that woodiness in Pericallis originated independently at least twice in these islands, further weakening some previous hypotheses concerning the value of this character for tracing the continental ancestry of island endemics. The same data suggest that the origin of woodiness is correlated with ecological shifts from open to species-rich habitats and that the ancestor of Pericallis was an herbaceous species adapted to marginal habitats of the laurel forest. Our results also support Pericallis as closely related to New World genera of the tribe Senecioneae.

Emergence of FY*Anull in a Plasmodium vivax-endemic region of Papua New Guinea

In Papua New Guinea (PNG), numerous blood group polymorphisms and hemoglobinopathies characterize the human population. Human genetic polymorphisms of this nature are common in malarious regions, and all four human malaria parasites are holoendemic below 1500 meters in PNG. At this elevation, a prominent condition characterizing Melanesians is α+-thalassemia. Interestingly, recent epidemiological surveys have demonstrated that α+-thalassemia is associated with increased susceptibility to uncomplicated malaria among young children. It is further proposed that α+-thalassemia may facilitate so-called “benign” Plasmodium vivax infection to act later in life as a “natural vaccine” against severe Plasmodium falciparum malaria. Here, in a P. vivax-endemic region of PNG where the resident Abelam-speaking population is characterized by a frequency of α+-thalassemia ≥0.98, we have discovered the mutation responsible for erythrocyte Duffy antigen-negativity (Fy[a−b−]) on the FY*A allele. In this study population there were 23 heterozygous and no homozygous individuals bearing this new allele (allele frequency, 23/1062 = 0.022). Flow cytometric analysis illustrated a 2-fold difference in erythroid-specific Fy-antigen expression between heterozygous (FY*A/FY*Anull) and homozygous (FY*A/FY*A) individuals, suggesting a gene-dosage effect. In further comparisons, we observed a higher prevalence of P. vivax infection in FY*A/FY*A (83/508 = 0.163) compared with FY*A/FY*Anull (2/23 = 0.087) individuals (odds ratio = 2.05, 95% confidence interval = 0.47–8.91). Emergence of FY*Anull in this population suggests that P. vivax is involved in selection of this erythroid polymorphism. This mutation would ultimately compromise α+-thalassemia/P. vivax-mediated protection against severe P. falciparum malaria.

Molecular evidence for an African origin of the Hawaiian endemic Hesperomannia (Asteraceae)

Identification of the progenitors of plants endemic to oceanic islands often is complicated by extreme morphological divergence between island and continental taxa. This is especially true for the Hawaiian Islands, which are 3,900 km from any continental source. We examine the origin of Hesperomannia, a genus of three species endemic to Hawaii that always have been placed in the tribe Mutisieae of the sunflower family. Phylogenetic analyses of representatives from all tribes in this family using the chloroplast gene ndhF (where ndhF is the ND5 protein of chloroplast NADH dehydrogenase) indicate that Hesperomannia belongs to the tribe Vernonieae. Phylogenetic comparisons within the Vernonieae using sequences of both ndhF and the internal transcribed spacer regions of nuclear ribosomal DNA reveal that Hesperomannia is sister to African species of Vernonia. Long-distance dispersal northeastward from Africa to southeast Asia and across the many Pacific Ocean island chains is the most likely explanation for this unusual biogeographic connection. The 17- to 26-million-year divergence time between African Vernonia and Hesperomannia estimated by the DNA sequences predates the age of the eight existing Hawaiian Islands. These estimates are consistent with an hypothesis that the progenitor of Hesperomannia arrived at one of the low islands of the Hawaiian-Emperor chain between the late Oligocene and mid-Miocene when these islands were above sea level. Subsequent to its arrival the southeast Pacific island chains served as steppingstones for dispersal to the existing Hawaiian Islands.

Ethnobotanical study of the sages used in traditional Valencian medicine and as essential oil: characterization of an endemic Salvia and its contribution to local development

Seven wild and cultivated Salvia species and two Phlomis species, used traditionally in Valencian medicine to treat a variety of external and internal ailments, were studied. New ethnobotanical data are provided, obtained from semistructured interviews with 34 people in the Valencian area. A seasonal characterization of the essential oil of a wild sage, Salvia blancoana Webb & Heldr. subsp. mariolensis Figuerola, by GC-FID and GC-MS was carried out as a means to ensure quality control of endemic traditional species such as this one, which has been commercialized by local industries. A comparison with the essential oil of Salvia lavandulifolia Vahl subsp.lavandulifolia allowed inclusion of the wild sage within the commercial 'Spanish sage' oil.

Histological analysis of gallbladder diseases in relation to opisthorchiasis in endemic areas of Thailand

Chronic gallbladder disease frequently accompanies infection with the liver fluke, Opisthorchis viverrini, in Northeast Thailand. However, the pathology and pathogenesis of the gallbladder disease have not been described. Accordingly, gallbladder specimens from 187 consecutive patients who had undergone cholecystectomy at a referral hospital in an endemic area in Thailand were histologically characterized in relation to O. viverrini infection. The infection was assessed by the presence of parasite eggs in the bile and/or antibody response to the liver fluke. The average level of parasite-specific IgG was significantly higher in patients with Opisthorchis eggs in the bile than those without (P < 0.001). The main histopathologic features of the gallbladder included inflammation, mucosal atrophy/or hyperplasia, goblet cell metaplasia, mucous gland hyperplasia, Rokitansky-Aschoff sinus formation, dysplasia and fibrosis. The fibrosis was strongly associated with elevated levels of Opisthorchis-specific antibody (P < 0.001) but not with the presence of parasite eggs. Other pathologic features did not vary in frequency or severity with parasitological status. Our results show that severe fibrosis of the gallbladder is a more common histologic feature of cholecystitis among those with O. viverrini infection compared to those without infection. The close relationship between parasite-specific IgG and severe fibrosis suggests that specific immune response to the parasite play an important role in the pathogenesis of the fibrotic change. (C) 2003 Elsevier B.V. All rights reserved.

Simple In Vitro Assay for Determining the Sensitivity of Plasmodium vivax Isolates from Fresh Human Blood to Antimalarials in Areas where P. vivax Is Endemic

The aim of this study was to develop a simple, field-practical, and effective in vitro method for determining the sensitivity of fresh erythrocytic Plasmodium vivax isolates to a range of antimalarials. The method used is a modification of the standard World Health Organization (WHO) microtest for determination of P.falciparum drug sensitivity. The WHO method was modified by removing leukocytes and using a growth medium supplemented with AB(+) serum. We successfully carried out 34 in vitro drug assays on 39 P. vivax isolates collected from the Mae Sod malaria clinic, Tak Province, Thailand. The mean percentage of parasites maturing to schizonts (six or more merozoites) in control wells was 66.5% +/- 5.9% (standard deviation). This level of growth in the control wells enabled rapid microscopic determination (5 min per isolate per drug) of the MICs of chloroquine, dihydroartemisinin, WR238605 (tafenoquine), and sulfadoxine. P. vivax was relatively sensitive to chloroquine (MIC = 160 ng/ml, 50% inhibitory concentration [IC50] = 49.8 ng/ml) and dihydroartemisinin (MIC = 0.5 ng/ml, IC50 = 0.47 ng/ml). The poor response of P. vivax to both tafenoquine (MIC = 14,000 ng/ml, IC50 = 9,739 ng/ml) and sulfadoxine (MIC = 500,000 ng/ml, IC50 = 249,000 ng/ml) was due to the slow action of these drugs and the innate resistance of P. vivax to sulfadoxine. The in vitro assay developed in our study should be useful both for assessing the antimalarial sensitivity of P. vivax populations and for screening new antimalarials in the absence of long-term P. vivax cultures.

Endemic infection of the amphibian chytrid fungus in a frog community post-decline

The chytrid fungus Batrachochytrium dendrobatidis has been implicated in the decline and extinction of numerous frog species worldwide. In Queensland, Australia, it has been proposed as the cause of the decline or apparent extinction of at least 14 high-elevation rainforest frog species. One of these, Taudactylus eungellensis, disappeared from rainforest streams in Eungella National Park in 1985-1986, but a few remnant populations were subsequently discovered. Here, we report the analysis of B. dendrobatidis infections in toe tips of T. eungellensis and sympatric species collected in a mark-recapture study between 1994 and 1998. This longitudinal study of the fungus in individually marked frogs sheds new light on the effect of this threatening infectious process in field, as distinct from laboratory, conditions. We found a seasonal peak of infection in the cooler months, with no evidence of interannual variation. The overall prevalence of infection was 18% in T. eungellensis and 28% in Litoria wilcoxii/jungguy, a sympatric frog that appeared not to decline in 1985-1986. No infection was found in any of the other sympatric species. Most importantly, we found no consistent evidence of lower survival in T. eungellensis that were infected at the time of first capture, compared with uninfected individuals. These results refute the hypothesis that remnant populations of T. eungellensis recovered after a B. dendrobatidis epidemic because the pathogen had disappeared. They show that populations of T. eungellensis now persist with stable, endemic infections of B. dendrobatidis.