Developing EST-SSR markers to study molecular diversity in Liriope and Ophiopogon

Twenty-two EST-SSR loci for further genetic analysis of Liriope and Ophiopogon species were developed by analyzing of 545 ESTs in Ophiopogon japonicus cDNA library and used to assess genetic diversity and population structure in 68 Liriope and Ophiopogon accessions (Liriope platyphylla, Liriope spicata, O. japonicus, and Ophiopogon jaburan). Among the 22 SSR loci, the di-nucleotide motif was the most prevalent, with a ratio of 56%, and AG/GA was the most common among di-nucleotide repeats, accounting for 42%. The polymorphic analysis showed a broad genetic background with an average genetic diversity index of 0.46. A model-based STRUCTURE analysis revealed the presence of three subpopulations, which was basically consistent with clustering based on the genetic distance. These newly developed EST-SSR markers will be useful for further studies such as taxonomy and molecular breeding studies and will further enhance our scientific understanding of the population genetic and conservation of Liriope and Ophiopogon.     

Phytochemical constituents and chemotaxonomic study of Polygonatum prattii Baker

Fifteen compounds were obtained from Polygonatum prattii Baker, which could be divided into four steroids, nine flavonoids, one lignanoid and one acidamide. The chemotaxonomic significance of P. prattii and the some other species among Asparagaceae was also discussed. As a result, Polygonatum may have close relationships with Liriope, Disporopsis, Ophiopogon and Aspidistra. Additionally, homoisoflavanones and steroids can be recognized as typical biomarkers for the above genus.     

New species of Peliosanthes,Rohdea and Tupistra (Asparagaceae) from Laos and Vietnam

We describe and illustrate four new species, Peliosanthes choriandra, P. tatianae and Tupistra orlovii from central to northern Laos, and Rohdea filosa from northern Vietnam. These are all very local in distribution and endemic to the respective countries. We also report new localities and their ecological conditions for five other species of Peliosanthes (P. argenteostriata, P. hirsuta, P. irinae, P. micrantha and P. nivea) recently described from Laos and/or Vietnam. Furthermore, Peliosanthes nivea is recorded as new to the flora of Laos.     

Karyotype analysis of medicinal plant <Emphasis Type="Italic">Liriope spicata</Emphasis> var. <Emphasis Type="Italic">prolifera</Emphasis> (Liliaceae)

Karyotype of Liriope spicata var. prolifera, a Chinese endemic species, was described in detail for the first time. Its proto-variety L. spicata was also investigated for comparison. The basic chromosome number of these two species was x = 18. L. spicata var. prolifera, recorded as triploid 2n = 54, consisted of 30 metacentric chromosomes and 24 submetacentric chromosomes. Only one chromosome of the 11th group had a secondary constriction with a satellite in the short arm. L. Spicata was tetraploid 2n = 72 and consisted of four sets of 6 submetacentric chromosomes and 12 metacentric chromosomes without visible satellites. This paper provides further available data on Liriope chromosomes, and also indicates that L. spicata var. prolifera and L. spicata are probably separate species.     

The Functional Significance of Black-Pigmented Leaves: Photosynthesis,Photoprotection and Productivity in Ophiopogon planiscapus ‘Nigrescens’

Black pigmented leaves are common among horticultural cultivars, yet are extremely rare across natural plant populations. We hypothesised that black pigmentation would disadvantage a plant by reducing photosynthesis and therefore shoot productivity, but that this trait might also confer protective benefits by shielding chloroplasts against photo-oxidative stress. CO₂ assimilation, chlorophyll a fluorescence, shoot biomass, and pigment concentrations were compared for near isogenic green- and black-leafed Ophiopogon planiscapus ‘Nigrescens’. The black leaves had lower maximum CO₂ assimilation rates, higher light saturation points and higher quantum efficiencies of photosystem II (PSII) than green leaves. Under saturating light, PSII photochemistry was inactivated less and recovered more completely in the black leaves. In full sunlight, green plants branched more abundantly and accumulated shoot biomass quicker than the black plants; in the shade, productivities of the two morphs were comparable. The data indicate a light-screening, photoprotective role of foliar anthocyanins. However, limitations to photosynthetic carbon assimilation are relatively small, insufficient to explain the natural scarcity of black-leafed plants.     

Diversity of cultivable bacteria associated with fruiting bodies of wild Himalayan Cantharellus spp.

The cultivable bacteria associated with fruiting bodies of different Cantharellus species collected from the forests of the north-western Himalayan region were studied. Repetitive extragenic palindromic (REP) and BOX-PCR fingerprinting analyses revealed that different strains were distributed within Cantharellus species. The number of bacteria varied from 0.5 to 1.1?×?10³?CFU/g of fresh tissue for different fruiting bodies. Thirty different operational taxonomic units (OTUs) were found among the bacteria analyzed. 16S rRNA sequence analysis revealed that most of the bacteria associated with different Cantharellus species were Gammaproteobacteria belong to the genera Hafnia, Enterobacter, Ewingella, Rahnella, Stenotrophomonas, and Pseudomonas, and γ-proteobacterium, followed by Betaproteobacteria (Alcaligenes) and Firmicutes, (Bacillus). The most common bacteria associated with the majority of Cantharellus species were Hafnia and Stenotrophomonas species.     

Hard ticks (Acari,Ixodidae) of Croatia

The present paper is based on original and literature data. In Croatia the first studies on the occurrence of ixodid species were made about 80 years ago. The number of tick species recorded in Croatia considerably increased during the 1950s, 60s, 70s and 80s of the past century. A total of 21 species of hard tick belonging to 5 genera have been recorded in Croatia. Ixodes is the best represented genus, with seven species recorded. Haemaphysalis is represented by six species, followed by Rhipicephalus with four species. Dermacentor and Hyalomma are represented by two species each. The ticks were collected on 47 different host species. Eleven tick species were collected on Bos taurus and Ovis aries, followed by Capra hircus and Equus caballus with 8 species and Canis lupus familiaris with 6 species. On the remaining 42 host species one, two or three tick species were collected. The most widespread tick is Ixodes ricinus which was found on 25 different host species.     

Experimental investigations on the host range of Sarcocystis podarcicolubris

Lizards experimentally infected with S. podarcicolubris sarcocysts were fed to different species of the family Colubridae. Only species of the genus Coluber proved to be suitable definitive hosts and passed sporocysts in their feces. No sporo- cysts were shed by the species of the genus Elaphe, Coronella and Malpolon that were tested. Fecal sporocysts from different species of the genus Coluber were orally administered to numerous lizards of the genera Lacerta, Podarcis and Algyroides. In all experimental lizards, except the controls, sarcocysts of S. podarcicolubris developed.     

Analysis of the specificity of three root-feeders towards grasses in coastal dunes

Among the root-feeding nematodes that accumulate in the rhizosphere of grasses in European dunes, the genus Pratylenchus is of special relevance given its diversity and distribution. Although different species of Pratylenchus have been reported in dune grasses, the specificity towards dune plants, a fundamental aspect of the biology of the species, has hitherto not been studied. Two inoculation experiments using different combinations of grasses and nematodes were performed. The multiplication and the effect on plant growth of P. dunensis and P. brzeskii, two species which only occur in dune areas was compared with that of P. penetrans, a broad host-range species. The three Pratylenchus spp. could multiply under all hosts; however, there was a clear host-dependent response. The species-specific response observed might account for the shift of Pratylenchus spp. detected in the field. Although, a negative effect on the growth of A. arenaria was demonstrated for the three nematode species, different densities were needed to observe the same effects in plant biomass which point at nematode-specific tolerance. While the typical dune species needed very high densities to produce damage, P. penetrans needed very few specimens. The results obtained indicate that species with similar feeding adaptations show very different multiplication abilities on co-occurring hosts, an aspect that is usually overlooked for belowground herbivores in natural systems. The obtained results might suggest a coevolutionary relationship between specific nematode species and Ammophila arenaria.     

Diversity of Wolbachia in Natural Populations of Spider Mites (genus Tetranychus): Evidence for Complex Infection History and Disequilibrium Distribution

Wolbachia are endosymbiotic bacteria that commonly infect arthropods and cause reproductive disorders in host. Within several Tetranychus species, Wolbachia have been detected and shown to affect their reproduction. However, little is known about their transmission and distribution patterns in natural populations of Tetranychus species. Here, we used multilocus sequence typing to confirm Wolbachia infection status and examined the relationship between Wolbachia infection status and host phylogeny, mitochondrial diversity, and geographical range in five Tetranychus species (Tetranychus truncatus, Tetranychus urticae, Tetranychus pueraricola, Tetranychus phaselus, and Tetranychus kanzawai) from 21 populations in China. The prevalence of Wolbachia within the five Tetranychus species ranged from 31.4 to 100 %, and the strains were remarkably diverse. Together, these observations indicate that Wolbachia was introduced to these populations on multiple separate occasions. As in other arthropods, the same Tetranychus species can accommodate very different strains, and identical Wolbachia occasionally infect different species. These observations suggest that Wolbachia are transmitted both vertically and horizontally. Horizontally, transmission is probably mediated by the host plants. The distribution patterns of Wolbachia were quite different among populations of the same species, suggesting that the dynamics of Wolbachia in nature may be affected by ecological and other factors.     

Morphological and Molecular Characterization of a New Trichuris Species (Nematoda- Trichuridae), and Phylogenetic Relationships of Trichuris Species of Cricetid Rodents from Argentina

Populations of Trichuris spp. isolated from six species of sigmodontine rodents from Argentina were analyzed based on morphological characteristics and ITS2 (rDNA) region sequences. Molecular data provided an opportunity to discuss the phylogenetic relationships among the Trichuris spp. from Noth and South America (mainly from Argentina). Trichuris specimens were identified morphologically as Trichuris pardinasi, T. navonae, Trichuris sp. and Trichuris new species, described in this paper. Sequences analyzed by Maximum Parsimony, Maximum Likelihood and Bayesian inference methods showed four main clades corresponding with the four different species regardless of geographical origin and host species. These four species from sigmodontine rodents clustered together and separated from Trichuris species isolated from murine and arvicoline rodents (outgroup). Different genetic lineages observed among Trichuris species from sigmodontine rodents which supported the proposal of a new species. Moreover, host distribution showed correspondence with the different tribes within the subfamily Sigmodontinae.     

Phylogeny of the StH genome species in Triticeae (Poaceae): Evidence from chloroplast trnL-F and mitochondria COXII intron sequences

The StH genome species in Triticeae exhibit different morphological variations and extensive geographic distribution. To estimate the phylogenetic relationship of the StH genome species in Triticeae, mitochondria COXII intron and chloroplast trnL-F sequences of 16 StH genome species were analyzed with those of four Pseudoroegneria species (St) and four Hordeum species (H). Sequence diversity and genealogical analysis suggested that (1) the trnL-F and COXII sequence may evolve faster in the polyploid species than in the diploids; (2) the COXII intron has a high evolutionary rate compared to trnL-F sequence and would provide potentially useful phylogenetic analysis in the StH genome species; (3) different Pseudoroegneria species might serve as the maternal donor during the polyploid speciation of the StH genome species; (4) phylogenetic relationships of the StH genome species may be not linked with the inter-continental disjunction between Eurasian and North American.     

RAPD and FAME analyses of Astragalus species growing in eastern Anatolia region of Turkey

Fatty acid (FAs) and RAPD profiles were used to examine phenotypic and genetic relationships between eight Astragalus species including Astragalus maximus Willd. var. maximus, Astragalus coadunatus Hub. Mor. & Chamb., Astragalus kurdicus Boiss. var. kurdicus, Astragalus lagurus Willd, Astragalus christianus L., Astragalus cicer L., Astragalus atrocarpus Champ & Matthews and Astragalus onobrychioides Bieb., which were wildly growing in eastern Anatolia region of Turkey. All of the eight Astragalus species tested in this study were separated based on the presence and composition of 45 different FAs. Four of the Astragalus species including A. coadunatus, A. lagurus, A. christianus, and A. atrocarpus were rich in terms of FA contents containing at least 22–31 different FAs. The relative proportions of two fatty acids, 16:0, and 18:1:ω8c were higher in these four Astragalus species. The remaining species have limited number of FAs with unique FAMEs profiles. Six of the 10 decamer primers examined were selected to find out genetic polymorphism in Astragalus species. A total of 98 polymorphic bands were observed, ranging in size from 250 bp to 3000 bp. The RAPD results suggested that A. atrocarpus, A. onobrychioides and A. kurdicus are closely related and completely different from the other species. Six genetically distinct groups were found among the species of Astragalus. High genetic variations among Astragalus species growing wildly in eastern Anatolia region of Turkey may imply the differences in their origins. The results in the present study suggested that both RAPD and FA analyses are useful for differentiation of Astragalus species.     

Species-Specific Antimonial Sensitivity in Leishmania Is Driven by Post-Transcriptional Regulation of AQP1

Leishmania is a digenetic protozoan parasite causing leishmaniasis in humans. The different clinical forms of leishmaniasis are caused by more than twenty species of Leishmania that are transmitted by nearly thirty species of phlebotomine sand flies. Pentavalent antimonials (such as Pentostam or Glucantime) are the first line drugs for treating leishmaniasis. Recent studies suggest that pentavalent antimony (Sb(V)) acts as a pro-drug, which is converted to the more active trivalent form (Sb(III)). However, sensitivity to trivalent antimony varies among different Leishmania species. In general, Leishmania species causing cutaneous leishmaniasis (CL) are more sensitive to Sb(III) than the species responsible for visceral leishmaniasis (VL). Leishmania aquaglyceroporin (AQP1) facilitates the adventitious passage of antimonite down a concentration gradient. In this study, we show that Leishmania species causing CL accumulate more antimonite, and therefore exhibit higher sensitivity to antimonials, than the species responsible for VL. This species-specific differential sensitivity to antimonite is directly proportional to the expression levels of AQP1 mRNA. We show that the stability of AQP1 mRNA in different Leishmania species is regulated by their respective 3’-untranslated regions. The differential regulation of AQP1 mRNA explains the distinct antimonial sensitivity of each species.     

Analysis of Yeast Flora Associated with Grape Sour Rot and of the Chemical Disease Markers

The frequency and the density of the species associated with grape sour rot in different cultivars were determined. The most frequent species in the rotten grapes, Candida krusei, Kloeckera apiculata, and Metschnikowia pulcherrima, and a less frequent species, Issatchenkia occidentalis, when inoculated with Saccharomycopsis crataegensis were able to induce in vitro the symptoms of the disease. The gas chromatographic determination of the volatile compounds in the headspace was used to evaluate the metabolic role of the different species associated with the disease. These analyses made it possible to presume that, whereas some species, such as Candida krusei and Hanseniaspora uvarum, can be considered responsible for these modifications and in particular for the ethyl acetate production, others, such as Saccharomycopsis crataegensis, can promote the development of the former species.     

Molecular identification of seven Sarcocystis species in red deer (Cervus elaphus) from Lithuania

The diaphragm muscles of 77 free-ranging red deer (Cervus elaphus) were examined for Sarcocystis species in Lithuania. Sarcocysts were detected in 61 out of 77 (79.2%) animals investigated. A total of 60 isolated sarcocysts were identified to species using subunit I of cytochrome c oxidase (cox1) sequence analysis. Overall, seven species, S. entzerothi, S. hjorti, S. iberica, S. linearis, S. pilosa, S. truncata and S. venatoria, were confirmed in Lithuanian red deer. Sarcocystis entzerothi was reported in red deer for the first time. Previously this species was shown to use sika deer as well as roe deer and fallow deer as an intermediate host. Based on cox1, with the addition of the current data, altogether 13 Sarcocystis species have so far been shown to use red deer as an intermediate host. Species detected in red deer demonstrated considerable differences in intraspecific genetic variation at cox1. Genetic distances between different samples of S. hjorti and S. linearis were calculated using principal coordinates analysis (PCoA), implying molecular divergence of same Sarcocystis species using different hosts in the same geographical area and divergence of those employing same intermediate host species from different areas.     

Identification of Seed Gall Nematodes of Agronomic and Regulatory Concern with PCR-RFLP of ITS1

A molecular analysis of eight described species of seed gall nematode, along with six undescribed isolates from different hosts, has revealed a strong association between nucleotide sequence polymorphism and host status. Each anguinid nematode associated with a unique host produced a unique PCR-RFLP pattern for the ITS1 region. Anguina species that had been synonymized in the past, Anguina agrostis, A. funesta, and A. wevelli (Afrina wevelli), were readily discriminated. Two undescribed species from northern New South Wales and southeastern South Australia, reported to be vectors of Rathyaibacter toxicus in the disease called ''''floodplain staggers,'''' were differentiated by a single restriction enzyme, and both could be separated easily from A. funesta, the vector of R. toxicus in annual ryegrass toxicity. Other species differentiated in this study include A. agropyronifloris, A. graminis, A. microlaenae, A. pacificae, and undescribed species from host species Dactylis glomerata, Agrostis avenacea, Polypogon monospeliensis, Stipa sp., Astrebla pectinata, and Holcus lanatus. Phylogenetic analysis of the ITS1 region suggests that considerable anguinid genetic diversification has accompanied specialization on different host species.     

Genetic differentiation in Indonesian and French rats of the subgenus Rattus

The allozyme polymorphism at 28 loci was examined in four Indonesian species of Rattus (R. diardii, R. tiomanicus, R. argentiventer and R. exulans) and in two French species (R. rattus and R. norvegicus). R. diardii was the most polymorphic species (H = 0.089) and R. tiomanicus and R. exulans the least polymorphic (H = 0.026). The six species were found to differ from one another by 2–7 diagnostic loci. The use of allozyme characters for systematic identification is discussed. Genetic relationships between the six species were established using two different methods. In both cases, the results were different from those published earlier. These results are discussed in view of the representatives of the loci investigated.