Species Diversity Of Bursaphelenchus Xylophilus And Molecular Systematics

The corticolous myxomycetes are special groups. The moist chamber cultures of bark can obtain the tiny species, which can enlarge the species diversity of myxomycetes. The researches of myxomycete species diversity were focused on the temperate, tropical, and boreal zones. In order to discuss the distribution pattern of corticolous myxomycetes throughout the world, it is necessary to carry out a research about the species diversity in subtropical forests. The study area was in the Tianmu Mountain National Natural Reserve, located in Lin’an City, northwest of Zhejiang Province, China. Four sampling sites (evergreen broadleaf forest, mixed evergreen/deciduous broadleaf forest, deciduous broadleaf forest, and coniferous forest) were chosen according to the vegetation types. At each site, five plots were sampled, and four trees were randomly selected from each plot. At each plot, a pair of bark samples was taken from each tree, one at 50 cm and the other at 150 cm above the ground. The same trees were recollected during the 3 sampling campaigns (October 2011, May 2012, and October 2012). A total of 480 bark samples were obtained, 1440 moist chambers cultures for myxomycetes. Approximately 79% of the data were recovered (1139 positive cultures, including the presence of myxomycete plasmodia or fruiting bodies) from all moist chamber cultures (1440 cultures), representing 42 species in 20 genera. Eight species (Comatricha elegans, Cribraria confusa, Licea pusilla, Cribraria microcarpa, Collaria arcyrionema, Licea biforis, Arcyria cinerea, and Clastoderma debaryanum) were abundant (exceeding 3% of all records), but about a third of all species were classified as rare. Species richness (S=33) and diversity (exp[H’]=16.60, S/G=1.74) of corticolous myxomycetes were the most diverse in the deciduous broadleaf forest. The species recorded from coniferous forest showed the lowest species richness (S=21) but the highest evenness (J’=0.91). The myxomycete biota in the deciduous broadleaf forest showed the lowest similarity with the mixed evergreen-deciduous broadleaf forest. The cluster analyses based on the Bray-Curtis similarity matrix indicated that corticolous myxomycete assemblages were distributed by a seasonal and annual pattern. Canonical correspondence analysis showed that season and pH were key factors in determining species distribution. Thus, the change of circumstances assumes that myxomycetes alter their distribution pattern according to environmental restriction.’DNA barcode’ provides rapid, accurate and convenient species identification. Myxomycetes can be difficult to identify because of limited morphological differentiation and cryptic speciation. We analyzed four potentially useful genes (cytochrome c oxidase 1 (COI), small subunit ribosomal RNA (SSU rRNA), elongation factor 1-alpha(EF-1α), and internal transcribed spaced (ITS) loci (including ITS1 and ITS2 subloci), for their effectiveness in DNA barcoding of myxomycetes. In total,144 sequences all representing 19 myxomycete species were assembled from newly sequenced samples and from GenBank. We calculated genetic distances and DNA barcoding gaps, and constructed phylogenetic trees individually in order to evaluate the candidate barcoding regions. The ITS locus, ITS1 and ITS2 subloci were amplified consistently only from the order Physarales (100%) and showed significant variation in their nucleotide contents and lengths, making them unsuitable as DNA barcode for Myxomycetes. Similarly, the high and random mutation rate of the third codon position with few informative sites made the EF-1α sequences unsuitable as DNA barcodes. COI and SSU rRNA could be amplified and sequenced from all tested species; however, the special RNA editing events of the COI gene meant that it could not be used to identify Myxomycetes. By contrast, the efficient of the identification of SSU rRNA (V1+V2) was better than other loci, and it was a good candidate DNA barcode for Myxomycetes. Further surveys and genetic analyses should to expand our knowledge of myxomycete biodiversity, and add additional loci for major group identification.Until now, the molecular phylogenetic analysis of myxomycetes has been based primarily on SSU rRNA and EF-1α genes. In order to enlarge the phylogenic resources and establish a natural classification system for the organisms, we examined phylogenetic relationships among myxomycete species using COI and SSU rRNA genes. Twenty new sequences were obtained, including 10 COI and 10 SSU rRNA sequences, were compared with sequences of related species from GenBank in order to construct phylogenic trees. Prior to this paper, available data of COI gene sequences was six species in GenBank. The analysis of the two data sets supported the modern phylogeny of myxomycetes:orders Liceales and Trichiales formed a sister group at the most basal clade, while orders Stemonitales and Physarales formed a close group, and order Echinosteliales was a sister group to Stemonitales and Physarales. However, the partial COI sequences were too conserved to resolve of the branches in orders Stemonitales and Physarales.The ITS2 locus is conserved at the level of secondary structure rather than primary sequences, and has been used revealing relationships at family or higher levels. We examined phylogenetic relationships among myxomycete species using ITS2 and SSU rRNA sequences to discuss the founction of ITS2 phylogeny. In total,56 ITS2 (44 species) and 39 SSU rRNA (28 species) sequences representing four orders (Liceales, Trichiales, Stemonitales, and Physarales) were assembled from newly sequenced samples and from GenBank. The ITS2 tree put the Liceales, Trichiales, and Stemonitales together as one group, at the base of Physarales. Thus, the ITS2 sequences were toot limited information to resolve the relationships within Myxomycetes. Instead, SSU rRNA could reflect the phylogeny of Myxomycetes:Stemonitales and Physarales formed a close group, Liceales and Trichiales formed a sister group at the basal clade. The SSU rRNA region was a good molecular marker for myxomycete phylogeny. Furthermore, the ITS2 RNA secondary structure supported the divergence of morphospecies in Didymium squamulosum.