Molecular systematics analysis of Lymantria dispar based on 18S rRNA and cox1 mtDNA sequence data

The complete sequence of the 18S ribosomal RNA gene （18S rRNA） from Lymantria dispar was cloned and analysed here. 18S rRNA and mitochondrial cytochrome c oxidasel （coxl） gene sequences of Lymantria dispar were compared with homologous sequences of other nine insects from different orders. Analytic results showed that 18S rRNA of these insects had two conserved domains and the second domain was an even more conserved region. The phylogenetic trees based on the full-length sequence and the second domain fragment of 18S rRNA as well as sequence of coxl from different orders indicated that Lepidoptera and Trichoptera, which belongs to Amphiesmenoptera, had a closer phylogenetic relationship and fewer differences were observed comparing with traditional taxonomic results.

Phylogenetic, phylogeographic and divergence time analysis of Anopheles subpictus species complex using ITS2 and COI sequences

Objective:To address the phylogenetic and phylogeographic relationship between different lineages of Anopheles(An.)subpictus species complex in most parts of the Asian continent by maximum utilization of Internal Transcriber Spacer 2(ITS2)and cytochrome C oxidase I(COI)sequences deposited at the GenBank.Methods:Seventy-five ITS2,210 COI and 26 concatenated sequences available in the NCBI database were used.Phylogenetic analysis was performed using Bayesian likelihood trees,whereas median-joining haplotype networks and time-scale divergence trees were generated for phylogeographic analysis.Genetic diversity indices and genetic differentiation were also calculated.Results:Two genetically divergent molecular forms of An.subpictus species complex corresponding to sibling species A and B are established.Species A evolved around 37-82 million years ago in Sri Lanka,India,and the Netherlands,and species B evolved around 22-79 million years ago in Sri Lanka,India,and Myanmar.Vietnam,Thailand,and Cambodia have two molecular forms:one is phylogenetically similar to species B.Other forms differ from species A and B and evolved recently in the above mentioned countries,Indonesia and the Philippines.Genetic subdivision among Sri Lanka,India,and the Netherlands is almost absent.A substantial genetic differentiation was obtained for some populations due to isolation by large geographical distances.Genetic diversity indices reveal the presence of a long-established stable mosquito population,at mutation-drift equilibrium,regardless of population fluctuations.Conclusions:An.subpictus species complex consists of more than two genetically divergent molecular forms.Species A is highly divergent from the rest.Sri Lanka and India contain only species A and B.

Leptobrachium bompu(Amphibia,Anura,Megophryidae)Discovered in Upper Medog,Tibet,China with Descriptions of its Tadpoles,Advertisement Calls and Systematic Position

Leptobrachium bompu Sondhi and Ohler, 2011 was described based on a single specimen collected from Eaglenest in southern slope of Himalaya（holotype： No. KA0001/200905）. In April, 2014, seven adults and three tadpoles of Leptobrachium bompu were collected from upper Medog, Tibet, China since most the morphological characters of the adult frogs matched the species Leptobrachium bompu, while the tadpoles mentioned above were determined as the same species as the adults by molecular systematics analysis. Three types of iris coloration were found in the seven adult specimens and two types of spiraculum in the three tadpoles. Advertisement calls usually had 6-8 notes and the fundamental frequency ranged from 1076 to 1466 Hz. All the specimens collected at upper Medog were clustered as one lineage with very low genetic variation and located at the base of the phylogenetic tree of the genus Leptobrachium.

The various substrates of Usnea aurantiaco-atra and its algal sources in the Fildes Peninsula, Antarctica

The lichen species Usnea aurantiaco-atra （Jacq.） Bory is the most dominant vegetation on the Fildes Peninsula, Antarctica. Most individuals grow on rocks, and some are found with mosses. During the 27th and 28th Chinese National Antarctic Research expeditions of the Great Wall Station, U. aurantiaco-atra was observed growing on the lichen thallus of Umbilicaria antarctica Frey ＆ I.M. Lamb, or on wood, which indicated that Usnea aurantiaco-atra could grow on various substrates. The diversities of the symbionts in U. aurantiaco-atra collected in the Fildes Peninsula were investigated using ITS rDNA sequences. The results showed that the sequences from mycobionts of U. aurantiaco-atra growing on various substrates did not exhibit significant differences. All photobionts in this lichen species were the green algae Trebouxia jamesii （Hildreth ＆ Ahmadjian） Gartner. The identical sequences from the photobionts of both Umbilicaria antarctica and Usnea aurantiaco-atra indicated there was an algae pool in this area and different mycobionts could obtain their algal partners from this pool. The variety of substrates for U. aurantiaco-atra suggested its photobiont could be obtained from a mature lichen thallus by vegetative propagation; from other lichen thalli （e.g. Umbilicaria antarctica）; or from the surroundings. This study will promote understanding of the distribution of photobionts and the process of lichenization.

Phylogenetic Analysis of Cynoglossidae in the Yangtze Estuary Based on Partial Sequence of Mitochondrial CO Ⅰ

To determine the role of mitochondrial COⅠgene in classification and identification of species,a total of 39 single individuals from nine species pertaining to two genera of Cynoglossidae in the Yangtze Estuary were barcoded by COⅠ,sequenced and compared with that of other Cynoglossidae species recorded in the Gen Bank. Total genomic DNA was extracted from each scale sample using the classic phenol / chloroform extraction method. Six hundred and fifty base pairs( bp)COⅠfragments were amplified using the primers ’i. e. ’ F1: 5’- TCA ACC AAC CAC AAA GAC ATT GGC AC- 3’,R1: 5’- TAG ACT TCT GGG TGG CCA AAG AAT CA- 3’. Every PCR amplification was performed in a total volume of 50 μL of PCR mixture. PCR products were purified and then sequenced in both forward and reverse directions using an ABI PRISMTM 3730 XL Automated Sequencer. DNA sequences were aligned with clustal W using default parameters. Base composition,variable and parsimony informative sites were determined using MEGA 5. 0. Neighbor- joining( NJ) and Maximum parsimony( MP) phylogenetic trees were constructed for COⅠhaplotypes( Kimura 2 Parameter substitution model,K2P; 1 000 bootstraps pseudoreplications) using MEGA 5. 0. Using the MEGA5. 0 software for statistical analysis,the averaged AT content was greater than the GC content( Tab. 2). The GC content of codon position 1 averaged 53. 8%( 51. 8%-57. 3%),which of position 2 for 42. 0%,and that of position 3 ranged from 28. 1% to 37. 8% in average of 32. 4%( Tab. 4). The transitional pairs( si) was slightly more than the transversional pairs( sv),and the ratio( R = si/sv) was 1. 45( Tab. 3). Analysis of the frequency of amino acids in COⅠgene encoding protein showed that the highest frequency of amino acid was leucine,and the lowest frequency of amino acid was tryptophan( Tab. 5). The average K2 P distances pairwise-species and within-species were 0. 191 and 0. 003,respectively( Tab. 6). The K2 P distance pairwise-species was 63. 7 times of that within-species. According to the MP and NJ trees for all 39 sequences,it demonstrated that Cynoglossinae in the Yangtze Estuary was a monophyletic group( Fig. 1). However,the phylogenetic relationships revealed by the COⅠsequence analyses were not consistent with those inferred from morphological classification. In contrast to morphological classification,Paraplagusis japonica of Rhinoplagusia and Cynoglossus robustus of Cynoglossoides were placed in a same clade with support of a high bootstrap value( Fig. 1). The species in the sub-genera of Areliscus clustered on an independent branch. But synonymic phenomena existed in the following two groups of species: C. abbreviatus and C. purpureomaculatus,C. lighti and C. joyner,with the pairwise-species of 0. 002 and 0. 007,respectively( Tab. 6). Our results highlight that the information from COⅠsequences not only can filter out the synonym of the same species,but also be able to be used to carry out effective identification for Cynoglossidae species,which further shows that mitochondrial COⅠis feasible as the classification barcode.

A multi-locus phylogenetic evaluation of Diaporthe (Phomopsis)

The genus Diaporthe(Phomopsis)includes important plant pathogenic fungi with wide host ranges and geographic distributions.In the present study,phylogenetic species recognition in Diaporthe is re-evaluated using a multi-locus phylogeny based on a combined data matrix of rDNA ITS,and partial sequences from the translation elongation factor 1-α(EF 1-α),βtubulin(TUB)and calmodulin(CAL)molecular markers.DNA sequences of available extype cultures have been included,providing a multi-locus backbone tree for future studies on Diaporthe.Four utilizable loci were analyzed individually and in combination,and ITS,EF 1-αand multi-locus phylogenetic trees are presented.The phylogenetic tree inferred by combined analysis of four loci provided the best resolution for species as compared to single gene analysis.Notes are provided for nine species previously known in Phomopsis that are transferred to Diaporthe in the present study.The unraveling of cryptic species complexes of Diaporthe based on Genealogical Concordance Phylogenetic Species Recognition(GCPSR)is emphasized.

The genus Thelonectria(Nectriaceae,Hypocreales,Ascomycota)and closely related species with cylindrocarpon-like asexual states

The genus Thelonectria and closely related species with cylindrocarpon-like asexual states are a group of perithecial ascomycetes in the family Nectriaceae that occur as saprobes and in few cases as pathogens of hardwood trees,shrubs or other plants.Although a key component of forest ecosystems around the world,species relationships and distributions of these fungi are largely unknown.The objectives of this study were to:1)infer species rank phylogenetic relationships of the genus Thelonectria and closely related species with cylindrocarpon-like asexual states and test the monophyly of each of the groups studied;2)delimit taxa establishing taxon circumscriptions;3)resolve nomenclatural issues by identifying redundantly used names and synonyms;and 4)provide an updated outline to the genus,geographical distributions data and identification tools,specifically diagnostic keys and molecular data that can be used as molecular barcodes.The recovered consensus phylogeny resulted in a narrow circumscription of the genus Thelonectria,based on the type T.discophora,excluding one of the common species T.jungneri.According to the phylogenetic analyses,T.jungneri belongs in a segregate clade that should be recognized as a different genus.In the genus Thelonectria,a total of four new species and three new combinations are recognized.Additionally,three new genera,closely related to Thelonectria,are described to accommodate species displaying a morphological resemblance to those of Thelonectria:Cinnamomeonectria gen.nov.with C.cinnamomea as type species,Macronectria gen.nov.with M.jungneri as type species and including four additional newly described species,and Tumenectria gen.nov.with T.laetidisca as type species.

The phylogenetic relationships of introduced Aphelinus （Hymenoptera： Aphelinidae）, biological control agents of the Russian wheat aphid （Homoptera： Aphididae）

Several species of Aphelinus have been introduced to the US from the Old World for biological control of the Russian wheat aphid, Diuraphis noxia （Modvilko）. Reproductive incompatibility has been observed among populations collected from different geographic areas. We examined whether or not the reproductive incompatibility between strains of A. asychis was caused by distant phylogenetic relationships. Ribosomal DNA sequences of internal transcribed spacers 2 （ITS2） were analyzed in several species of Aphelinus collected from multiple sites of Europe and Asia. Yhe phylogenetic analysis showed that strains within the species A. albipodus and A. asychis are not monophyletic, and two clearly divergent clades were revealed among sequenced samples. Our results suggest that the reproductive incompatibility between three exotic strains ofA. asychis was more likely caused by divergence of phylogeny than by symbiotic bacteria.

Resolving the Mortierellaceae phylogeny through synthesis of multi-gene phylogenetics and phylogenomics

Early efforts to classify Mortierellaceae were based on macro-and micromorphology,but sequencing and phylogenetic studies with ribosomal DNA(rDNA)markers have demonstrated conflicting taxonomic groupings and polyphyletic genera.Although some taxonomic confusion in the family has been clarified,rDNA data alone is unable to resolve higher level phylogenetic relationships within Mortierellaceae.In this study,we applied two parallel approaches to resolve the Mortierel-laceae phylogeny:low coverage genome(LCG)sequencing and high-throughput,multiplexed targeted amplicon sequenc-ing to generate sequence data for multi-gene phylogenetics.We then combined our datasets to provide a well-supported genome-based phylogeny having broad sampling depth from the amplicon dataset.Resolving the Mortierellaceae phylogeny into monophyletic genera resulted in 13 genera,7 of which are newly proposed.Low-coverage genome sequencing proved to be a relatively cost-effective means of generating a high-confidence phylogeny.The multi-gene phylogenetics approach enabled much greater sampling depth and breadth than the LCG approach,but has limitations too.We present this work to resolve some of the taxonomic confusion and provide a genus-level framework to empower future studies on Mortierellaceae diversity and evolution.