A new species of Paracapillaria (Nematoda: Capillariidae) from the intestine of the toad Duttaphrynus melanostictus (Anura) from the Malayan Peninsula

A new species of parasitic nematode, Paracapillaria malayensis n. sp. (Capillariidae), is described from the small intestine of the toad Duttaphrynus melanostictus imported from the Malayan Peninsula to the Czech Republic. The new species differs from the only other congeneric species, Paracapillaria spratti, mainly in the shape and structure of the spicular proximal end (with a lobular rim), smaller eggs (45-51 x 21-24 microm), longer spicule (336 microm), and the number (37-38) of stichocytes in gravid females; whereas P. spratti parasitizes frogs of the Microhylidae in Papua New Guinea, P. malayensis is a parasite of Bufonidae in the Malayan Peninsula. Other Paracapillaria spp. are parasites of fishes, birds, or mammals and they mostly differ from P. malayensis in the structure of eggs and some other morphological features.     

Molecular identification of blood meal sources of ticks (Acari,Ixodidae) using cytochrome b gene as a genetic marker

Blood meal analysis (BMA) from ticks allows for the identification of natural hosts of ticks (Acari: Ixodidae). The aim of this study is to identify the blood meal sources of field collected on-host ticks using PCR analysis. DNA of four genera of ticks was isolated and their cytochrome b (Cyt b) gene was amplified to identify host blood meals. A phylogenetic tree was constructed based on data of Cyt b sequences using Neighbor Joining (NJ) and Maximum Parsimony (MP) analysis using MEGA 5.05 for the clustering of hosts of tick species. Twenty out of 27 samples showed maximum similarity (99%) with GenBank sequences through a Basic Local Alignment Search Tool (BLAST) while 7 samples only showed a similarity range of between 91–98%. The phylogenetic trees showed that the blood meal samples were derived from small rodents (Leopoldamyssabanus, Rattustiomanicus and Sundamysmuelleri), shrews (Tupaiaglis) and mammals (Tapirusindicus and Prionailurusbengalensis), supported by 82–88% bootstrap values. In this study, Cyt b gene as a molecular target produced reliable results and was very significant for the effective identification of ticks’ blood meal. The assay can be used as a tool for identifying unknown blood meals of field collected on-host ticks.     

Morphological evolution and ecological diversification of the forest-dwelling poppies (Papaveraceae: Chelidonioideae) as deduced from a molecular phylogeny of the ITS region

Sequences of the ITS region of nrDNA were analyzed for the seven genera of Papaveraceae subf. Chelidonioideae s.str. Three major clades can be recognized. These are 1.Chelidonium/Hylomecon/Stylophorum, 2.Eomecon/Sanguinaria, and 3.Bocconia/Macleaya. The monophyly of genera in the first of these three clades is doubtful, and clades two and three are sister to each other. Use of the ITS phylogeny of the subfamily to trace its morphological and ecological evolution shows that morphological change is concentrated in theBocconia/Macleaya clade, and probably related to the evolution of wind-pollination from insect-pollination in these two genera after habitat shift.     

Sequence-related amplified polymorphism (SRAP) marker as a new method for identification of endophytic fungi from Taxus

A total of 20 endophytic fungi stains were classified into four groups using traditional morphological identification method, and were studied for genetic diversity by sequence-related amplified polymorphism (SRAP) technique. Genomic DNA (deoxyribonucleic acid) of these strains was extracted with CTAB method. SRAP analysis was done with 24 pairs of primers. All strains could be uniquely distinguished with 584 bands and 446 polymorphism bands which generated 76.4% of polymorphic ratio. Unweighted pair-group method with arithmetical averages cluster analysis enabled construction of a dendrogram for estimating genetic distances between different strains. All strains, which were just divided into four groups by traditional morphology identification, were clustered into four major groups at GS = 0.603 and further separated into eight sub-groups at GS = 0.921. Dendrogram also revealed a large genetic variation in 20 strains; different primer combinations allowed them distinctly distinguished one from others with relatively low genetic similarity. The results show that the SRAP technology is more efficient than traditional morphology identification. It is found that SRAP markers could more really reflect the genetic diversity of endophytic fungi strains from Taxus, and also could be used as a method for identification of endophytic fungi from Taxus. It also suggests that SRAP can be used to establish foundation for further screening of taxol-producing endophytic fungi strains which can produce high levels of paclitaxel.     

Eye colour as a genetic marker for fertility and fecundity of Triatoma infestans (Klug, 1834) Hemiptera,Reduviidae, Triatominae

Eye colour of Triatoma infestans is controlled at a single autosomal locus, with black-eye as the dominant gene and red-eye as the recessive. Inheritance of these characters follows a classical Mendelian system, enabling eye colour to be used as a marker for studies of mating frequency. We found no significant differences in oviposition rates and egg hatching rates irrespective of parental phenotypes. Different mating schedules between red-eye and black-eye parents showed that eye colour did not affect mating competence. Females mated with a single male or with different males together or in succession produced similar numbers of fertile eggs, with the eye colour of the offspring reflecting exposure to the different males. We conclude that although a single mating can provide sufficient sperm for the whole reproductive life of the female, multiple matings can result in balanced assortative sperm usage from the spermatheca.     

Morphological and molecular differentiation of Parastrigea (Trematoda: Strigeidae) from Mexico,with the description of a new species

Parastrigea plataleae n. sp. (Digenea: Strigeidae) is described from the intestine of the roseate spoonbill Platalea ajaja (Threskiornithidae) from four localities on the Pacific coast of Mexico. The new species is mainly distinguished from the other 18 described species of Parastrigea based on the ratio of its hindbody length to forebody length. A principal component analysis (PCA) of 16 morphometric traits for 15 specimens of P. plataleae n. sp., five of Parastrigea cincta and 11 of Parastrigea diovadena previously recorded in Mexico, clearly shows three clusters, which correspond to the three species. DNA sequences of the internal transcribed spacers (ITSs) of ribosomal DNA and the mitochondrial gene cytochrome c oxidase subunit I (cox 1) were used to corroborate this morphological distinction. The genetic divergence estimated among P. plataleae n. sp., P. cincta and P. diovadena ranged from 0.5 to 1.48% for ITSs and from 9.31 to 11.47% for cox 1. Maximum parsimony (MP) and maximum likelihood (ML) analyses were performed on the combined datasets (ITSs + cox 1) and on each dataset alone. All of the phylogenetic analyses indicated that the specimens from the roseate spoonbill represent a clade with strong bootstrap support. The morphological evidence and the genetic divergence in combination with the reciprocal monophyly in all of the phylogenetic trees support the hypothesis that the digeneans found in the intestines of roseate spoonbills represent a new species.     

Morphological and genetic description of Octopus insularis, a new cryptic species in the Octopus vulgaris complex (Cephalopoda: Octopodidae) from the tropical southwestern Atlantic

A medium-sized Octopus species is described based on materialcollected in shallow equatorial waters around the oceanic islandsof Fernando de Noronha Archipelago, Rocas Atoll, St Peter andSt Paul Archipelago and the mainland of northeastern Brazil.The new species, Octopus insularis, is described morphologically,and also characterized by the large mitochondrial subunit ribosomalRNA gene (mt 16S rDNA). The new species has relatively shortand stout arms, rugose reddish brown skin in preserved specimens,8 to 11 gill lamellae on the outer demibranchs, small ligula,characteristic symmetrical radula, spermatophore and beak, smalleggs and high fecundity (213,000 oocytes under 1.5 mm diameter).The habitats and skin patterns of living animals are brieflydescribed. The new species differs both morphologically andgenetically from Octopus vulgaris in the Mediterranean, Venezuelaand southern Brazil. (Received 10 November 2006; accepted 19 October 2007)     

Morphological and molecular description of a new species of Isospora (Apicomplexa) from a New Holland honeyeater (Phylidonyris novaehollandiae)

A new Isospora species is described from New Holland honeyeaters (Phylidonyris novaehollandiae). Sporulated oocysts (n = 25) were characterised as subspheroidal, 29–32 × 28–31 (29.8 × 29.4); length/width (L/W) ratio 1.01–1.02 (1.01). Wall bi-layered, 1.3–1.6 (1.5) thick, outer layer smooth, c.2/3 of total thickness. Micropyle and oocyst residuum absent, but usually two polar granules are present. Sporocysts (n = 25) ovoidal, 18–19 × 12–14 (18.4 × 12.3); L/W ratio 1.42–1.53 (1.50). Stieda body present, flattened, c.0.5 deep × 2.5 wide; sub-Stieda present, rounded, c.2.5 deep × 3.5 wide; para-Stieda body absent; sporocyst residuum present, usually a distinctly irregular body consisting of numerous small granules that appear to be membrane-bound. Sporozoites vermiform, with robust anterior and posterior refractile bodies. Molecular characterization was conducted at the 18S and 28S ribosomal RNA and the mitochondrial (mt) cytochrome oxidase (COI) loci. Phylogenetic analysis of genomic 18S and mt COI sequences indicated that Isospora phylidonyrisae n. sp. was genetically similar to Isospora coronoideae, isolated from an Australian raven (Corvus coronoides) in Western Australia, with a 99.3% and 98.4% homology, respectively. The 28S rRNA sequence was most similar to Isospora anthochaerae (KF766053) and Isospora manorinae (KT224381), both with a 98.2% genetic similarity. Based on morphological and genetic data, this isolate is a new species of Isospora, which is named Isospora phylidonyrisae n. sp. after its host.     

Morphological and genetic differentiation of the acorn barnacle Tetraclita squamosa (Crustacea, Cirripedia) in East Asia and description of a new species of Tetraclita

The common intertidal barnacle Tetraclita squamosa occurs in two morphologically and genetically distinct forms in East Asia. The north-western Pacific form (Japan, Okinawa and Taiwan) has green parietes and the tergo-scutal flaps are black without any patterns. The south China form (Xiamen, Hong Kong) also has green parietes but the tergo-scutal flaps are black with two white spots on the tergal and scutal margin. Compared to the NW Pacific form, the south China form has a beaked tergum, a sharper tergal spur and cirrus I lacks serrulate type setae that have four rows of setules. The two forms differ by 15–16% in COI divergence, which is comparable to values for other congeneric barnacle species. The 12S rRNA and ITS1 sequences are also distinct between the two forms. Our results support the conclusion that the two forms are genetically differentiated species. We describe the NW Pacific form as a new species, Tetraclita pacifica . We are treating the other species as Tetraclita squamosa based on the fact that Pilsbry, in 1916, redescribed T. squamosa squamosa using samples collected from south China and the Philippines. Further studies are needed to confirm the identity and geographical distribution of the 'widely distributed' T. squamosa in the Indo-West Pacific.     

Internal transcribed spacer 2 amplicon as a molecular marker for identification of Peronospora parasitica (crucifer downy mildew)

AIMS: The purpose of the study was to characterize the internal transcribed spacer (ITS) regions of Peronospora parasitica (crucifer downy mildew) in order to evaluate their potential as molecular markers for pathogen identification. METHODS AND RESULTS: PCR amplification of ribosomal RNA gene block (rDNA) spacers (ITS1 and ITS2) performed in 44 P. parasitica isolates from different Brassica oleracea cultivars and distinct geographic origins, revealed no length polymorphisms. ITS restriction analysis with three endonucleases, confirmed by sequencing, showed no fragment length polymorphisms among isolates. Furthermore, ITS amplification with DNA isolated from infected host tissues also allowed the detection of the fungus in incompatible interactions. The combination of the universal ITS4 and ITS5 primers, for amplification of full ITS, with a new specific forward internal primer for ITS2 (PpITS2F), originates a P. parasitica specific amplicon, suitable for diagnosis. CONCLUSIONS: As ITS2 regions of P. parasitica, B. oleracea, other B. oleracea fungal pathogens and other Peronospora species are clearly distinct, a fast and reliable molecular identification method based on multiplex PCR amplification of full ITS and P. parasitica ITS2 is proposed for the diagnosis of crucifer downy mildew. SIGNIFICANCE AND IMPACT OF THE STUDY: The method can be applied to diagnose the disease in the absence of fungal reproductive structures, thus being useful to detect nonsporulating interactions, early stages of infection on seedlings, and infected young leaves packed in sealed plastic bags. Screening of seed stocks in sanitary control is also a major application of this diagnostic method.     

Morphological and phylogenetic studies of Cytospora (Valsaceae,Diaporthales) isolates from Chinese scholar tree,with description of a new species

Cytospora species are the most serious and widespread pathogens associated with canker disease on multiple plants. In this study, three species, i.e., Cytospora sophoricola, C. chrysosperma, and C. sophorae, which were isolated from Sophora in China, are described and illustrated based on their morphological characteristics and phylogenetic analyses. Cytospora sophoricola was distinguished clearly by its larger disc, multiple ostioles, cystic and multiple locules, and specific cultural characteristics, i.e., protruding fruiting bodies. Maximum parsimony and maximum likelihood analysis showed that it did not cluster with any known species of Cytospora, so it is described as a new species. Cytospora sophorae is a previously reported species from Sophora, which is redescribed based on new isolates and additional observations. Another species was identified as C. chrysosperma, which is reported for the first time on Sophora, so Papilionaceae is shown to be a new host family for C. chrysosperma. The morphological affinities of these species with related taxa are discussed, while the phylogenetic relationships of these species with other fungus in the genus Cytospora were elucidated based on their internal transcribed spacer (ITS) rRNA region sequences.     

Morphological and genetic identification of the three pine pests of the genus Tomicus (Coleoptera, Scolytidae) in Europe

Abstract

• 1 Morphological characters were elaborated and part of the mitochondrial COI gene was sequenced in order to facilitate the determination of the three European pine bark beetles Tomicus piniperda, T. destruens and T. minor. The sequence information also provided the first information on the phylogenetic and phylogeographical relationships of these species.
• 2 Three hair rows were found on the antennal club of T. destruens between the second and third suture. Tomicus piniperda had only one row. Three different hair types were detected on the elytra – two hair types were found on T. piniperda, whereas the third hair type was only detected on the elytra of T. destruens.
• 3 The COI region (445 bp) revealed high sequence divergence among T. destruens, T. piniperda and T. minor. The three species proved to be monophyletic species with 16.98–19.23% sequence divergence. A phylogenetic approach placed T. minor and T. destruens as sister taxa, which contradicts the morphological findings.
• 4 European populations of T. piniperda shared two haplotypes, indicating a homogenous distribution of the genotypes. In the American populations only one of these European haplotypes was found. The Greek, Italian and Spanish T. destruens populations revealed three population‐specific haplotypes, indicating restricted gene flow.
• 5 Species‐specific primers were designed to allow a rapid and definitive determination of the two sibling Tomicus species by PCR.

     

ITS作为粒毛盘菌属DNA条形码的探索

以种内与种间差异以及PCR扩增和测序成功率为评价DNA条形码的重要指标,探索ITS序列作为粒毛盘菌属DNA条形码的可行性。结果表明,ITS成功区分了研究涉及的22个种,其PCR扩增和测序成功率为100%,该片段有望成为该属区分物种的DNA条形码。     

Dalnigrin, a neoflavonoid marker for the identification of Brazilian rosewood (Dalbergia nigra) in CITES enforcement

International trade in Brazilian rosewood, Dalbergia nigra (Vell.) Allemão ex Benth., is regulated by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). One problem in enforcing these regulations is the difficulty in distinguishing the wood of D. nigra from that of a closely-related but unregulated species, Dalbergia spruceana Benth. Using LC-MS to analyse methanol extracts of xylaria specimens, we identified a chemical marker for D. nigra heartwood, and determined its structure as the neoflavonoid 6-hydroxy-7-methoxy-4-(4-methoxyphenyl)-2H-1-benzopyran-2-one (4′-O-methylmelanettin; dalnigrin), using spectroscopic techniques. Dalnigrin was present in all nine available heartwood specimens of D. nigra, but it was not detected in extracts of 59 other heartwood samples representing 15 species of Dalbergia, including D. spruceana. Five other phenolic compounds were also isolated from D. nigra heartwood and similarly identified as the neoflavonoids 3′-hydroxymelanettin, melanettin, melannein and dalbergin, and the isoflavone caviunin. In extracts of D. spruceana heartwood, pseudobaptigenin was identified by LC-MS to be a major phenolic component that was not detected in wood extracts of D. nigra. We conclude that chemical analysis, in combination with anatomical investigation, can provide persuasive evidence to support the positive identification of untreated heartwood of D. nigra.     

Morphological and genetic diversity of the thermophilic cyanobacterium,Mastigocladus laminosus (Stigonematales,Cyanobacteria) from Japan and Myanmar

The morphology and phylogeny of 13 strains of a thermophilic cyanobacterium, Mastigocladus laminosus Cohn, isolated from hot springs in Japan and Myanmar were analyzed to determine taxonomy and biogeography. From the morphological observations of cell size, there were significant differences among strains. Phylogenetic analyses based on 16S rRNA gene sequences revealed two lineages: Lineages I and II. Lineage I consisted of strains collected in Japan and reference strains from a previous study (CCMEE 5329 and 5331, Hakone, Japan); Lineage II included all of the Myanmar strains and one Japanese strain, and was a novel lineage in phylogeographic studies on M. laminosus. Since strains in the Lineage II tended to have larger cells than those in the Lineage I, the morphological and phylogenetic lineages corresponded well with each other.