Identifying PCR primers to facilitate molecular phylogenetics in Caddisflies(Trichoptera)

The molecular phylogenetics of the Lepidoptera （butterflies and moths） is well studied, but that of Trichoptera （caddisflies）, the sister clade of Lepidoptera, is less studied. The PCR primer libraries developed for lepidopteran phylogenetics might work in Trichoptera. DNA from 8 caddisfly species （Asynarchus nigriculus （Banks, 1908）, Grammotaulius lorettae Denning, 1941, Hesperophylax occidentalis （Banks, 1908）, Limnephilus externus Hagen, 1861, Limnephilus picturatus McLachlan, 1875, Limnephilus secludens Banks, 1914, Limnephilus sublunatus Provancher, 1877 and Agrypnia deflata （Milne, 1931）） was used to screen for amplification. 107 primer pairs for 45 nuclear and 3 mitochondrial genes were tested. Primers for 1 new gene （40S ribosomalprotein $2 （RPS2）） and 8 genes previously used in Trichopteran phylogenetics were recovered （16S rRNA, 18S rRNA, carbamoyl-phosphate synthetase （CAD）, cytoehrome oxidase I （CO1）, cytochrome oxidase 11 （COIl）, elongation factor-1 alpha （EF-1 alpha）, isoeitrate dehydrogenase （IDH）, and RNA polymerase-II （POL-I1））. New primer pairs extended the genomic region sampled for many genes. Evolution rates among loci varied by 2 orders of magnitude. Differences among evolution rates and modes of inheritance offer flexible tools for resolving phylogenetic questions and examining genome evolution in the Trichoptera. Screening libraries of PCR primers is a useful approach for identifying PCR primers in related taxa with limited molecular genetic resources.

Molecular Phylogeny of the Genus Gloydius(Serpentes: Crotalinae)

Based on two mitochondrial genes (cyt b, ND4) and one nuclear gene (c-mos), we explored the relationships within the Asian pit viper genus Gloydius. In total, 23 samples representing 10 species were analyzed. All phylogenetic analyses support a monophyletic Gloydius with two major clades, one comprising G. brevicaudus, G. blomhoffii, and G. ussuriensis with the sister clade consisting of G. intermedius, G. saxatilis, G. halys and G. shedaoensis. The relationships among the three montane species G. strauchi, G. qinlingensis and G. liupanensis, as well as the two monophyletic groups, are unstable, and discussed. Divergence date estimation indicates that Gloydius lineage formed 15 Ma and diversification of the genus occurred at 9.89 Ma. Issues regarding the taxonomy of this genus are discussed where necessary.

A novel pathogen Fusarium cuneirostrum causing common bean(Phaseolus vulgaris)root rot in China

Several fungal pathogens cause root rot of common bean,among which Fusarium spp.are the most common pathogens causing Fusarium root rot(FRR)worldwide.FRR has been becoming an increasingly severe disease of common bean in China,but the species of Fusarium spp.have remained unclear.Thus,this study was performed to identify the pathogen causing common bean root rot in Liangcheng County,Inner Mongolia,China.Nineteen Fusarium-like isolates were obtained after pathogen isolation and purification.The pathogenicity test indicated that eight isolates caused severe disease symptoms on common bean,while 11 other isolates were not pathogenic.The eight pathogenic isolates,FCL1–FCL8,were identified as Fusarium cuneirostrum by morphological characterization and phylogenetic analysis using partial sequences of EF-1α,ITS,28S,and IGS regions.Host range test showed that the representative F.cuneirostrum isolate FCL3 was also pathogenic to mung bean,while not pathogenic to adzuki bean,chickpea,cowpea,faba bean,pea,and soybean.Moreover,50 common bean and 50 mung bean cultivars were screened for resistance to FRR,and seven highly resistant or resistant cultivars of common bean were identified,while no resistant cultivars of mung bean were screened.This study revealed that F.cuneirostrum was one of common bean FRR pathogens in Inner Mongolia and it could induce mung bean root rot as well.To our knowledge,this is the first report of F.cuneirostrum causing FRR of common bean in China.

Molecular Phylogeny of the “True Citrus Fruit Trees” Group (Aurantioideae, Rutaceae) as Inferred from Chloroplast DNA Sequence

The genus Citrus L. has a long controversial taxonomy history, and a well-resolved molecular phylogeny of the ＂true citrus fruit trees＂ group in the future will provide new information for advancing breeding techniques and developing better conservation strategies. In the present study, three cpDNA fragments （TrnL-TrnF, PsbH-PetB, and TrnS-TrnG） of 30 genotypes chosen from the six genera of the ＂true citrus fruit trees＂ group were analyzed. A molecular phylogenetic tree of the ＂true citrus fruit trees＂ group ＂~as reconstructed based on plastid DNA sequences. The results confirmed that the ＂true citrus fruit trees＂ group was monophyletic, and thereby the group was divided into genera as previously suggested based on morphological characters. The cpDNA data also suggested that Poncirus might be the first genus separated from the other five genera in the group. The genus Fortunella were of hybrid origin and Citrus might be as its putative paternal parent. The genera Microcitrus, Eremocitrus, and Clymenia were possibly monophyletic and their common ancestor might branch out from Citrus. Furthermore, the phylogenetic relationships within the Citrus genus were discussed.

Molecular phylogenetic analysis of sulfate-reducing bacteria from deep sediment layers of the tropical West Pacific warm pool

The diversity of sulfate-reducing bacteria （SRB） from deep layers of deep-sea sediments [ more than 2 m bsf （below seafloor） ] of two sites （WO1 -3 and WPO1 -4） in a tropical West Pacific warm pool region was characterized by using molecular phylogenetic analysis. The results of culture-independent samples demonstrated that the dominant clones from both sites were related to Grampositive spore forming genus, Desulfotomaculum, which accounted for 36.8% of all the sequencing clones from Site WP01 - 3 and 62.8% from Site WP01 -4. However, the other SRB group which was generally reported to be predominant in the deep-sea sediments of other regions, δ- subclass of the proteobacteria was found to be in very low percentages. Therefore, it could be speculated that there existed a unique chemical environment in the deep-sea sediment of this warm pool region. When comparing the Desulfotomaculum sp. related sequences from both sites, it was revealed that though the Desulfotomaculum-like sequences from Site WP01 -3 were more diverse than those from Site WP01 -4, all these sequences from both sites showed high similarity and formed a new phylogenetically homogeneous cluster in the Desulfotomaculum genus which had never been reported before. Successful enrichment of SRB was only achieved from samples of Site WP01 -4 and the sequence analysis of culture-dependent samples further confirmed the dominance of Desulfotomaculum genus. But Desulfotomaculum-related sequences from culture-dependent and culture-independent samples belonged to two different clusters respectively. This difference showed the choice of cultivation to the microorganisms.

Molecular cloning,expression pattern and phylogenetic analysis of Guanine nucleotide exchange factor Vav2 in lamprey,Lampetra japonica

The Guanine nucleotide exchange factor Vav2（Vav2） is a member of the Vav family that serves as an important regulators for the Rho family of Ras-related GTPases. In the current study, an ortholog（Lj-Vav2） of Vav2 was identified in the lamprey（Lampetra japonica）. To elucidate the phylogenetic relationship of Vav2, the metazoan genome databases were analyzed to mine the ortholog of Vav. It was found that Vav2 genes were only existed in vertebrates and Lj-Vav2 was the original one found in agnathans. The evolutionary dynamics of conserved motifs of Vav2 were explored using combined amino acid sequence as markers, and it is revealed that the Calponin homology（CH） domain, Dbl-homologous（DH） domain, Pleckstrin homology（PH） domain, Cysteine-rich（C1）domains, Src homology 3（SH3） domains and Src homology 2（SH2） domain were conserved throughout the Vav2 gene family in vertebrates during gene evolution. Relative quantitative real-time PCR analysis showed that the LjVav2 was distributed in the heart, kidney, supraneural myeloid body, liver, gill and lymphocyte-like cells. The LjVav2 was found to be expressed in these tissues, and the level of which was upregulated in lymphocyte-like cells after the animal was stimulated with LPS. These results indicated that the Lj-Vav2 might be involved in the immune response of lymphocyte-like cells in lamprey. Meanwhile, our findings provided a foundation for further investigation of the function of Lj-Vav2 in the primary vertebrate.

Multiple Data Sources Reveal a New Asian Leaf Litter Toad of Leptobrachella Smith, 1925 (Anura, Megophryidae) from Southwestern China

A new species of Leptobrachella is described from Sichuan Province and Chongqing Municipality,China.Molecular phylogenetic analyses based on mitochondrial and nuclear gene sequences indicated that the new species is genetically divergent from its congeners.It could be identified from its congeners by a combination of followings characters:body size of male 29.1-34.1 mm(n=14),female 34.1-34.9 mm(n=4);dorsal skin rough with large tubercles in size of humeral glands,without conical spines;fringes on toes narrow;ventral body basically floral white with deep grey pigments all over;dorsal body deep greyish brown with smoky black markings;iris gold above,gradually silver bellow;tibia-tarsal articulation reaches the level of the middle of the eye when leg being stretched forward;the main call type with dominant frequency4.08 ± 0.16 kHz(14.1-14.9℃),call duration 170.35± 15.19ms,the number of pulses for the first note in a call 3.50±0.89,and the number of pulses for the second note in a call 5.08±0.77.

Molecular phylogeny and taxonomy of Phlomoides(Lamiaceae subfamily Lamioideae)in China:Insights from molecular and morphological data

Phlomoides,with 150-170 species,is the second largest and perhaps most taxonomically challenging genus within the subfamily Lamioideae(Lamiaceae).With about 60 species,China is one of three major biodiversity centers of Phlomoides.Although some Phlomoides species from China have been included in previous molecular phylogenetic studies,a robust and broad phylogeny of this lineage has yet to be completed.Moreover,given the myriad new additions to the genus,the existing infrageneric classification needs to be evaluated and revised.Here,we combine molecular and morphological data to investigate relationships within Phlomoides,with a focus on Chinese species.We observed that plastid DNA sequences can resolve relationships within Phlomoides better than nuclear ribosomal internal and external transcribed spacer regions(nrITS and nrETS).Molecular phylogenetic analyses confirm the monophyly of Phlomoides,but most previously defined infrageneric groups are not monophyletic.In addition,morphological analysis demonstrates the significant taxonomic value of eight characters to the genus.Based on our molecular phylogenetic analyses and morphological data,we establish a novel section Notochaete within Phlomoides,and propose three new combinations as well as three new synonyms.This study presents the first molecular phylogenetic analyses of Phlomoides in which taxa representative of the entire genus are included,and highlights the phylogenetic and taxonomic value of several morphological characters from species of Phlomoides from China.Our study suggests that a taxonomic revision and reclassification for the entire genus is necessary in the future.

A New Species of the Genus Leptolalax(Anura: Megophryidae) from Northern Thailand

A new species of the genus Leptolalax is described from Doi Saket, Chiang Mai Province, northern Thailand. The new species is distinguished from its congeners by the following combination of characters： 1） adult size large （male SVL 45.8-52.5 mm）; 2） tympanum distinct; 3） skin of dorsal surface mostly smooth, with distinct tubercles on dorsal and lateral parts of the body; 4） ventrolateral glandular ridge distinct and complete; 5） lateral body and groin covered with dark brown or black spots; 6） throat, chest, and belly nearly immaculate white; and 7） bi-colored iris, upper one- third golden, and lower two-thirds gray. Molecular phylogenetic analysis of mitochondrial 16S rRNA further distin- guishes it from its congeners for which sequences are available.

An Integrative Taxonomy of Amphibians of Nepal:An Updated Status and Distribution

Amphibians are declining worldwide.At the same time,each year many species of amphibians are described,indicating that biological diversity on Earth remains poorly understood.This is especially true for mountainous regions,such as the Himalayas,which are also regarded as a biodiversity hotspot.However,it is most likely that the current diversity of amphibians in Nepal is highly underestimated.Amphibian inventories by the past researchers primarily used phenotypic characters as the basis for species identification.However,the application of molecular systematics methods to taxonomic studies has successfully uncovered the taxonomic status of several cryptic taxa.This study used both molecular(16 S rDNA sequences)and morphological comparisons and estimated the diversity of amphibians from Nepal.This study reports the occurrence of 38 species of amphibians based on both molecular and morphological comparisons.Among them,three species were recorded for the first time in Nepal.This study also invalids the taxonomic status of Sphaerotheca paschima.This study highlights the importa nce of integrative taxonomic approaches that help to resolve the current taxonomic ambiguities of Nepalese amphibians.

Description of a New Species of the Asian Newt Genus Tylototriton sensu lato(Amphibia:Urodela:Salamandridae) from Southwest China

The newt genus Tylototriton sensu lato is widely distributed in Eastern,Southeastern and Southern Asia.Previous studies indicated that there still has been several cryptic species in the group.Here,we describe a new species of the genus from Guizhou Province,China.Phylogenetic analyses based on mitochondrial DNA supported that the new species was resolved as an independent clade nested into the Tylototriton sensu lato clade.On morphology,the new species could be distinguished from its congeners by a combination of the following characters:large body size(SVL 76.8-85.2 mm in male and 76.3-87.4 mm in female);head longer than wide;snout truncate in dorsal view;tail length longer than the snout-vent length in males;the distal digit ends,ventral digits,peripheral area of cloaca and the tail s lower margin are orange;relative length of toesⅢ> Ⅳ> Ⅱ> Ⅰ> Ⅴ;the distal tips of the limbs greatly overlapping when the fore and hind limbs are pressed along the trunk;fingertips reach the level beyond the snout when the forelimbs are stretched forward.

A New Species of the Gekko japonicus Group(Squamata:Gekkonidae)from Southwest China

A new species of the Gekko(Squa ma ta:Gekkonidae)is described from the border of Sichuan and Yunnan Province,southwest China,based on distinct morphological and molecular features.Gekko jinjiangensis sp.nov.is distinguished from congeners by a combination of the following characters:size small(SVL 50.2–61.6 mm,n=13);nares in contact with rostral;interorbital scales between anterior corners of the eyes 20–24;ventral scales between mental and cloacal slit 146–169;midbody scale rows 111–149;ventral scale rows 31–47;subdigital lamellae on first toe 8–11,on fourth toe 11–15;no webbing in the fingers and toes;with tubercles on uapper surface of fore and hind limbs;precloacal pores 4–5 in males;postcloacal unilateral tubercles 1–2;dorsal surface of body with 8–9 large greyish brown markings between nape and sacrum.In molecular analyses,the new species is sister to G.scabridus,but separated from them by approximately 9.9%–12.2%in genetic divergence as shown by a fragment of the partial mitochondrial ND2 gene.The new species is the highest Gekko with elevation range from 2000 to 2476 m.Further surveys are recommended to better understand the occurrence and population status of the new species.

Molecular phylogenetic relationship of Epinephelus based on sequences of mtDNA Cty b

The mtDNA Cyt b gene was sequenced partially for Variola louti of Serranidae,Epinephelinae and seven endemic species of groupers—Epinephelus awoara,E.brunneus,E.coioides,E.longispinis,E.sexfasciatus,E.spilotoceps and E.tauvina in China.The seven endemic species and other seven foreign species of groupers—E.aeneus,E.caninus,E.drummondhayi,E.haifensis,E.labriformis,E.marginatus and E.multinotatus from the GenBank were combined and analysed as ingroup,while Variola louti was used as outgroup.We compared the 420 bp sequences of Cyt b among the 15 species and constructed two types of molecular phylogenetic trees with maximum parsimony method(MP)and neighbor-joining method(NJ)respectively.The results were as follows:(1)As to the base composition of mtDNA Cyt b sequence(402 bp)of 14 species of Epinephelus,the content of(A+T)was 53.6%,higher than that of(G+C)(46.4%).The transition/transversion ratio was 4.78 with no mutation saturation.(2)The cluster relationships between E.awoara and E.sexfasciatus,E.coioides and E.tauvina,E.longispinis and E.spilotoceps were consistent with phenotypes in taxonomy.(3)In the phylogenetic tree,the species in the Atlantic Ocean were associated closely with those in the Pacific Ocean,which suggested that the Cyt b sequences of Epinephelus were highly conserved.This may be attributed to the coordinate evolution.(4)In wel1-bred mating or heredity management,mating Epinephelus of the same branch should be avoided.It is likely to be an effective way to mate the species of the Atlantic Ocean with those of the Pacific Ocean to improve the inheritance species.

Ultrastructure and phylogeny of Ustilago coicis

Ustilago coicis causes serious smut on Coix lacryma-jobi in Dayang Town,Jinyun County,Zhejiang Province of China.In this paper,ultrastructural assessments on fungus-host interactions and teliospore development are presented,and molecular phylogenetic analyses have been done to elucidate the phylogenetic placement of the taxon.Hyphal growth within infected tissues was both intracellular and intercellular and on the surface of fungus-host interaction,and the fungal cell wall and the invaginated host plasma membrane were separated by a sheath comprising two distinct layers between the fungal cell wall and the invaginated host plasma membrane.Ornamentation development of teliospore walls was unique as they appeared to be originated from the exosporium.In addition,internal transcribed spacer(ITS) and large subunit(LSU) sequence data showed that U.coicis is closely related to Ustilago trichophora which infects grass species of the genus Echinochloa(Poaceae).

A multigene phylogeny reveals that Ochroconis belongs to the family Sympoventuriaceae (Venturiales,Dothideomycetes)

Ochroconis is a genus of ascomycete fungi that includes oligotrophic saprobes and some opportunistic species causing infections in vertebrates.The most important of these opportunists is the neurotropic species Ochroconis gallopava,which occurs in birds and occasionally in immunocompromised humans.Other Ochroconis species have been isolated from superficial infections of cats,dogs and fish.In their natural environment,these species are found in litter,soil,and on moist surfaces.Some thermophilic species have been isolated from hot springs,industrial effluents,and self-heated plant material.Although their ecology and epidemiology has been investigated,their classification within the ascomycetes is still unknown.Here,the phylogenetic placement of Ochroconis is investigated using a four-gene phylogeny(nuLSU,nuSSU,mtSSU and RPB2).The results show that Ochroconis and its recently described sister genus Verruconis belong to the order Venturiales(Dothideomycetes)and are nested within the Sympoventuriaceae,a family recently erected for a group of plant-saprobes or-pathogens sister to the Venturiaceae.They form a well-supported monophyletic group together with five species of the anamorphic genus Fusicladium isolated from leaf and needle litters.

Trapped on the Roof of the World:taxonomic diversity and evolutionary patterns of Tibetan Plateau endemic freshwater snails(Gastropoda:Lymnaeidae:Tibetoradix)

The high-elevation Tibetan Plateau(western China)is inhabited by a unique,though not particularly species-rich,community of organisms.We explored the species content and evolutionary history of the Tibetan Plateau endemic freshwater snail genus Tibetoradix.Phylogenetic relationships within the genus were reconstructed based on available sequence data.We used a single-rate Poisson Tree Processes approach for species delimitation and compared putative species-level clades with already described taxa.We found that the genus consists of at least 6 species,of which we described 4 as new to science.Shell and soft body morphology was examined and the radula in Tibetoradix was described for thefirst time.Based on ourfindings,the diversification of the genus did not result in a prominent morphological differentiation and a number of species can be regarded as morphologically cryptic.Single species found in different drainage areas indicate relatively good passive dispersal abilities of the snails.The allopatric distribution of the species could result from competitive exclusion between them.The absence of Tibetoradix spp.outside the Tibetan Plateau could be explained by a scenario of an“evolutionary trap”,where adaptations to high elevation conditions prevented the taxa from a successful colonization of lower elevations.

A large-scale phylogenetic revision of Roccellaceae(Arthoniales)reveals eight new genera

A two-locus phylogenetic hypothesis of the family Roccellaceae is presented based on data from the nuclear ribosomal large subunit(nucLSU)and the second largest subunit of RNA polymerase II(RPB2).This analysis includes 341 sequences(166 newly generated)and 180 specimens representing about 114 species.The genera Lecanactis,Roccellina,Schismatomma and Sigridea were found to be paraphyletic/polyphyletic.In order to make these groups monophyletic,the new genera Crocellina,Diromma,Gyrographa,Gyronactis,Ocellomma,Pseudoschismatomma,Psoronactis and Vigneronia are described.The genus Sagenidium is placed in synonymy with Lecanactis.The new species Enterographa incognita,Gyronactis asiatica and Lecanactis submollis are described.Several species of Opegrapha are transferred to the Roccellaceae.Sorediate morphs are recorded for the first time in the genus Syncesia.

New species and unexpected diversity of socially parasitic bees in the genus Inquilina Michener （Hymenoptera： Apoidea： Apidae）

Allodapine bees present particular problems for taxonomy due to a high level of morphological conservatism in adults, even between genera. However, this tribe of bees also presents a unique opportunity to explore the evolution of social parasitism because of the comparatively large number of origins of socially parasitic species. Morphological differences presented here, along with DNA sequence data and molecular phylogenetic analyses, indicate a much larger number of Australian social parasite species in the genus Inquilina than previously anticipated, and suggest that the final number of socially parasitic species may be considerable. We describe five new species and present sequence data that will help elucidate the delineation of further new species. Inquilina provides a unique opportunity to study the evolution of social parasitism in social insects, but further studies will need to encompass both population genetic and phylogenetic approaches.