Ribosomal DNA ITS Research Articles

Antimicrobial Activity of Necklace Orchids is Phylogenetically Clustered and can be Predicted With a Biological Response Method.

Necklace orchids (Coelogyninae, Epidendroideae) have been used in traditional medicine practices for centuries. Previous studies on a subset of unrelated orchid species utilized in these traditional practices revealed they possessed antimicrobial, anti-inflammatory, and anti-oxidant activity, providing experimental proof for their medicinal properties. To date however none of these species have been investigated ethno-botanically in a phylogenetic context. This study carried out comparative bioprospecting for a group of wild orchids using EBDCS (the Economic Botany Data Collection Standards) organ targeted and biological response methods. The traditional medicinal use of necklace orchids was recorded from books and journals published between 1984 and 2016. Two orchids, Coelogyne cristata and Coelogyne fimbriata, were selected, cultivated both indoors and outdoors, and the antimicrobial properties on extracts from their leaves and pseudobulbs tested against a selection of human pathogens. A molecular phylogeny of Coelogyninae based on nuclear ribosomal ITS and plastid matK DNA sequences obtained from 148 species was reconstructed with Maximum Likelihood (ML) using RAxML, Maximum Parsimony (MP) using PAUP and Bayesian Inference using MrBayes. Bioprospecting comparison of EBDCS and biological response was carried out using customized R scripts. Ethanolic extracts obtained from leaves of C. fimbriata inhibited growth of Bacillus cereus, Staphylococcus aureus, and Yersinia enterocolitica, confirming the antimicrobial properties of these extracts. Leaf extracts were found to have slightly stronger antimicrobial properties for plants cultivated outdoors than indoors. These differences were not found to be statistically significant though. Three hot nodes with high potency for antimicrobial activities were detected with the EBDCS organ targeted classification method, and eight hot nodes were detected with the biological response classification method. The biological response classification method is thus a more effective tool in finding hot nodes amongst clades of species with high medicinal potential.

Species Diversity of Pin Nematodes (Paratylenchus spp.) from Potato Growing Regions of Southern Alberta, Canada.

Pin nematodes (Paratylenchus spp.) are polyphagous parasitic species with a wide host range and geographical distribution; their diversity is unknown in the potato growing region of Alberta, Canada. The present study aims to provide morphological and molecular characterization of three pin nematode species, namely P. neoprojectus, P. tateae, and a new species, Paratylenchus enigmaticus sp. nov. All of them were recovered from the potato growing region of southern Alberta. The nematodes were isolated using the sieving and flotation-centrifugation method, and their morphology was assessed by light microscopy. Molecular characterization was performed using partial 18S, D2–D3 expansion domains of the 28S and ITS ribosomal genes. This study is the first report of molecular characterization of P. tateae and P. neoprojectus, being new records from southern Alberta, and two Spanish populations of P. tateae comprising the first report of this species in Europe. The phylogenetic analysis of the 18S, D2–D3 expansion domains of the 28S and ITS ribosomal DNA regions underscores the importance of using molecular data for accurate species identification and clarifies the status of P. nanus type B and P. sheri. Moreover, our findings will be useful to determine the impact of pin nematodes on potato production in future field research.

Gerricola queenslandensis n. g., n. sp., a new monorchiid trematode from the eastern Australian coast and its life cycle partially elucidated.

Of over 250 species of Monorchiidae Odhner, 1911, just four are known from gerreid fishes. Here, we report adult specimens of a new species infecting Gerres oyena (Forsskål) and Gerres subfasciatus Cuvier from off Heron Island and North Stradbroke Island, Queensland, Australia. The species is morphologically most similar to the concept of Lasiotocus Looss, 1907, which currently comprises eight species, in the possession of an unspined genital atrium, bipartite terminal organ, round oral sucker and unlobed ovary. However, phylogenetic analyses of the 28S ribosomal DNA gene region shows the species to be distantly related to the two sequenced species of Lasiotocus - Lasiotocus mulli (Stossich, 1883) Odhner, 1911 and Lasiotocus trachinoti Overstreet & Brown, 1970 - and that it clearly requires a distinct genus; thus, we propose Gerricola queenslandensis n. g., n. sp. Morphologically, G. queenslandensis n. g., n. sp. differs significantly from L. mulli and L. trachinoti only in the possession of distinctly longer caeca, which terminate in the post-testicular region, and in the absence of a distinct gap in the terminal organ spines. The remaining species of Lasiotocus possess caeca that also terminate in the post-testicular region, which might warrant their transfer to Gerricola n. g. However, doubt about their monophyly due to a combination of significant morphological variation, a lack of information on some features and infection of a wide range of hosts, lead us to retain these taxa as species of Lasiotocus until molecular sequence data are available to better inform their phylogenetic and taxonomic positions. Sporocysts and cercariae of G. queenslandensis n. g., n. sp. were found in a lucinid bivalve, Codakia paytenorum (Iredale), from Heron Island. Sexual adult and intramolluscan stages were genetically matched with the ITS2 ribosomal DNA and cox1 mitochondrial DNA regions. This is the second record of the Lucinidae as a first intermediate host for the Monorchiidae. Additionally, we report sporocysts and cercariae of another monorchiid infection in a tellinid bivalve, Jactellina clathrata (Deshayes), from Heron Island. Molecular sequence data for this species do not match any sequenced species and phylogenetic analyses do not suggest any generic position.

Crittendenia gen. nov., a new lichenicolous lineage in the Agaricostilbomycetes (Pucciniomycotina), and a review of the biology, phylogeny and classification of lichenicolous heterobasidiomycetes

AbstractThe lichenicolous ‘heterobasidiomycetes’ belong in the Tremellomycetes (Agaricomycotina) and in the Pucciniomycotina. In this paper, we provide an introduction and review of these lichenicolous taxa, focusing on recent studies and novelties of their classification, phylogeny and evolution. Lichen-inhabiting fungi in the Pucciniomycotina are represented by only a small number of species included in the genera Chionosphaera, Cyphobasidium and Lichenozyma. The phylogenetic position of the lichenicolous representatives of Chionosphaera has, however, never been investigated by molecular methods. Phylogenetic analyses using the nuclear SSU, ITS, and LSU ribosomal DNA markers reveal that the lichenicolous members of Chionosphaera form a monophyletic group in the Pucciniomycotina, distinct from Chionosphaera and outside the Chionosphaeraceae. The new genus Crittendenia is described to accommodate these lichen-inhabiting species. Crittendenia is characterized by minute synnemata-like basidiomata, the presence of clamp connections and aseptate tubular basidia from which 4–7 spores discharge passively, often in groups. Crittendenia, Cyphobasidium and Lichenozyma are the only lichenicolous lineages known so far in the Pucciniomycotina, whereas Chionosphaera does not include any lichenicolous taxa.

Post-COVID mucormycosis: Ascertainment of the pathological diagnostic approach.

Post-COVID mucormycosis: Ascertainment of the pathological diagnostic approach.

A note on systematic position of Indian species of Cyathocline (Compositae)

The Genus Cyathocline has been merged with Blumea following molecular phylogenetic evidence (Plastid trnL-F and nuclear ribosomal DNA ITS sequences data). While the type species, C. purpurea has been transferred to Blumea, other species have not been transferred. In light of this, new combinations are made here for C. lutea & C. manilaliana. C. purpurea var. alba and C. purpurea var. bicolor are synonymized under Blumea purpurea. B. manilaliana is reported here the first time from Maharashtra state.

Rare Specimens Yield DNA to Confirm Additional Members of Tribe Stifftieae (Stifftioideae, Asteraceae) on the Guiana Highlands Tepuis

Eight of the ten genera of the Guiana Highlands shrubby Mutisieae were sequenced for the ITS and ETS of the nuclear ribosomal DNA. Results from phylogenetic analyses of the sequence data are congruent with earlier reports that support two independent introductions from different lineages of the basal Asteraceae resulting in the diversification of these taxa in the Guiana Highlands. The two clades segregated according to corolla symmetry. The five genera with bilabiate corollas, Achnopogon, Duidaea, Eurydochus, Gongylolepis and Neblinaea, were supported with members of subfamily Stifftioideae whereas those with actinomorphic corollas, Chimantaea, Stenopadus and Stomatochaeta, constitute a monophyletic group sister to Wunderlichia and strongly supported in the Wunderlichioideae. Resolution, but not statistical support, was found for relationships within the Guiana Highlands shrubby Mutisieae.

Asymmetric hybridization of roach Rutilus rutilus and common bream Abramis brama in controlled backcrosses: Genetic and morphological patterns

In most cases in natural environments, hybrids of roach Rutilus rutilus L. and bream Abramis brama L. possess mitochondrial DNA of bream. Presumably, the genetic basis for unidirectional hybridization of roach and bream is the high level of divergence in the genes of the mitochondrial electron transport chain (cytochrome b and cytochrome c oxidase subunits I, III). Disruption of the interaction of the products of these genes leads to nuclear-cytoplasmic incompatibility of alien genomes, what is shown in a decrease of viability and developmental abnormalities in hybrids. In the present work we studied the viability and morphology of hybrid underyearlings obtained by crossing of hybrid females of first generation (RA and AR) with males of roach R. rutilus and bream A. brama. The method of genotyping (ITS1 ribosomal DNA, cytochrome b mtDNA) and comparative analysis of the complex of 23 plastic and meristic characteristics of backcrossed hybrids are used. All progenies showed an increase in morphological variability compared to parental species and F1 hybrids. In progenies with introgression of mtDNA, a violation of associations between traits and the formation of transgressive phenotypes that go beyond the parental populations were found. In RAA backcrosses (combining mtDNA of R. rutilus and nuclear genome of A. brama) a decrease in viability with impaired recovery of external traits of bream was found. Conversely, ARR backcrosses (combining mtDNA of A. brama and the nuclear genome of R. rutilus) have a high viability and completely restore the morphotype of roach, which indicates the stable development of hybrids when they include alien genetic material. The differences in viability and morphology between backcrossed hybrids with the mtDNA of R. rutilus and A. brama evidence varying degrees of nuclear-cytoplasmic compatibility of the genomes of roach and bream. The complete interaction between mitochondrial and nuclear DNA from different species (wild-type-like) happens in direction of introgression mtDNA of A. brama, the less polymorphic of the two parental genomes. In the direction of introgression of highly polymorphic mtDNA of R. rutilus the formation of a reproductive barrier occurs. Our results show that the main reason for the asymmetry of hybridization of R. rutilus and A. brama is unequal rates of mitochondrial evolution and the priority of the introgression of mtDNA belongs a species with a lower rate of changes in mtDNA.

Five novel and highly efficient endophytic fungi isolated from Huperzia serrata expressing huperzine A for the treatment of Alzheimer’s disease

Huperzine A (Hup A) is an important drug for treating Alzheimer's disease (AD) and mainly extracted from the Huperzia serrata (Thunb.) Trevis. (Lycopodiaceae) (HS). Nevertheless, the content of Hup A in HS is very low of 0.007% with growing circle of 8 to 10 years, and the chemical synthesis of Hup A still has some insurmountable limitations in the industrialized production. So, the available resources of Hup A for clinical treatment of AD are scarce. The purpose of this work was to construct a biosynthesis platform based on the endophytic fungi from HS. In this work, five endophytic fungi Mucor racemosus NSH-D, Mucor fragilis NSY-1, Fusarium verticillioides NSH-5, Fusarium oxysporum NSG-1, and Trichoderma harzianum NSW-V were firstly found and isolated from the Chinese folk medicine HS, which were identified according to their morphological characteristics and nuclear ribosomal DNA ITS sequences. The highest efficient fungus could effectively biosynthesize Hup A in a liquid culture of 319.8 ± 0.17 mg/L which were 112 times higher than that of other reported conventional endophytic fungi. Moreover, these fungi with higher hereditary stability could possess the initial expressing ability of Hup A after 40 generations, and the expressed Hup A from these biosynthesis systems has prior physicochemical properties, a better inhibition activity of acetylcholinesterase and a lower cytotoxicity compared with the listed active pharmaceutical ingredients (APIs) of Hup A. These results provide promising alternative resources for producing Hup A at an industrial scale by biosynthesis, and it may also shed light on millions of AD patients. KEY POINTS: • Five novel endophytic fungi with high stability could highly express prior Hup A Graphical abstract.

First Report of Rhizoctonia solani AG 11 Causing Stem Rot of Phaseolus vulgaris in Idaho

In July 2018, bean plants (cv. Viva pink) at one location in south central Idaho were observed to have reduced growth with approximately 20% of the plants affected over 1 ha. Visual assessment showed that the below-ground parts of stems consistently displayed brown lesions up to 2 cm in length, which sometimes girdled the entire plant. To determine the causal agent, affected material (approximately 5 mm³) was surface disinfested in sodium hypochlorite (2%) for 30 s, rinsed twice in sterile water, and plated onto water agar medium amended with penicillin G (0.2 g/liter) and streptomycin sulfate (0.8 g/liter). After 3 days, colonies of Rhizoctonia solani were identified based on the presence of relatively broad (8 to 12 μm), septate hyphae with right-angle branching. Hyphal tips of R. solani colonies were transferred to potato dextrose agar (PDA) and grown for 2 weeks at 21°C. DNA was then extracted from hyphae taken from a single representative isolate (DB23). Anastomosis group (AG) was identified by ribosomal DNA ITS sequencing of isolate DB23. DNA was extracted and sequenced as previously described (Woodhall et al. 2013). The resulting sequence (GenBank no. MT256181) was over 99% identical with isolates previously identified as R. solani AG 11 (LC215403). To confirm pathogenicity, 2-week-old pinto bean plants (cv. Windbreaker) were each inoculated by placing a fully colonized 10-mm² plug taken from a 2-week-old PDA culture of isolate DB23 next to the stem 10 mm below the soil surface. Sterile PDA plugs were placed next to the stems of control plants. Each treatment was replicated five times. Plants were grown in a greenhouse at 21°C in potting compost (Scotts, Marysville, OH) under light for 16 h a day. After 5 weeks, each plant was assessed for disease using a previously described stem disease severity scale (Carling and Leiner 1990). No symptoms were present in the control plants. Plants inoculated with isolate DB23 had an average stem disease severity score of 2.6 (from a maximum of 4), and R. solani was consistently reisolated, thereby confirming Koch’s postulates. To our knowledge, this is the first report of R. solani AG 11 in Idaho on any host. Previously, R. solani AG 11 has been reported on Phaseolus vulgaris in Turkey (Eken and Demirci 2004) and on lily in Japan (Misawa et al. 2017). AG 11 has also been associated with soybeans in Arkansas (Ajayi-Oyetunde and Bradley 2017; Carling et al. 1994) and Illinois (Ajayi-Oyetunde and Bradley 2017). AG 11 has also been reported on rice in Arkansas (Carling et al. 1994; Spurlock et al. 2016) and sweet corn in New York (Ohkura et al. 2009). In western Australia, it was found on wheat and lupin, and these isolates were pathogenic to potatoes in controlled environment studies (Carling et al. 1994). Idaho is a major producer of wheat, potatoes, and dry beans; the presence of soilborne R. solani AG 11 also needs to be considered when diagnosing and managing Rhizoctonia diseases of these crops.

First Report of Anthracnose on Ctenanthe oppenheimiana Caused by Colletotrichum siamense in China.

Ctenanthe oppenheimiana (E. Morren) K. Schum. (Maranta oppenheimiana) is a shade-loving, monocotyledonous ornamental plant belonging to the family Marantaceae. The plant has attractive foliage and is used for decoration in a park and around offices building. Six million seedings were produced in China in 2017 (China Flower Association, 2018). Plants were seriously infected with a year-round foliar disease. The disease occurred in all planting areas and approximately 53.1% and 48.3% of the plants in Guangdong and Fujian provinces in China, March 2017, were infected and caused economic loss to control plant diseases with chemicals. The initial symptoms were observed after 1 month of planting and included small brown lesions that expanded to form large irregular yellow lesions at the tips or margins of the leaves in campus of Guangdong Ocean University. Diseased plants were collected in Zhanjiang city (110°30' E, 21°20' N), Gaozhou city Guangdong province and (117.42E, 25.3N) and Zhangping city Fujian province (117°42' E, 25°30' N). Seven single-spore cultures were recovered from symptomatic samples after they were disinfested with 1% NaOCl, and plated on potato sucrose agar (PSA) at 28 °C for one week. Three isolates were used for pathogenicity test, 200 μL conidial suspension (106 conidia ml-1) was dropped on the leaf sites with sterile needle-prick wound at 5-8 leaf stage. There were three replicates for each treatment and the experiment was repeated three times. Plants were placed in incubator at 25 ℃, with 80% humidity and a 12-h light/dark cycle. Control seedlings were treated identically except sterile water was placed on the wounds. Round brown necrotic lesions in the middle of the leaves grew in the inoculated wounded leaves 5 days after inoculation and brown necrotic lesions developed into large brown lesions of 2.2-3.6 cm 10 days after inoculation whilethere were no symptoms in the negative controls. Colletotrichum was re-isolated from the lesions in inoculated leaves fulfilling Koch's postulates. Colonies on PSA were grayish white with a regular border and a felty aerial mycelium that contained masses of orange conidia. No setae were observed in the PSA culture. Conidia were straight and hyaline with rounded ends measuring 10.0-15.0×5.0-6.0 μm (average =14.2×5.3 μm, n=50). Conidiophores hyaline, septate. Conidiogenous cells were hyaline, cylindric, and 13.2-22.5×4.5-5.5 µm (average =19.5×4.6 μm, n=50). Appressoria were cylindrical, 5.2-10.5 × 4.0-6.8 μm (average =7.5×5.2 μm, n=50). For molecular identification, the colony PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) was used to amplify the ITS of ribosomal DNA, glutamine synthetase (GS), calmodulin(CAL) and Apmat loci of three isolates using primer pairs of ITS4/ITS5, GSF1/GSR1, CL1C/CL2C and AM-F/AM-R (Liu et al. 2015). Phylogenetic tree derived from a neighbor-joining analysis of a concatenated alignment of ITS, GS, CAL and ApMAT sequences. The accession numbers of three isolates ZJCG, GZCG and FJCG used in this study were KP635210, MN133235, MN133236 for ITS, MT594433, MT594434, MT594435 for CAL, MT594436, MT594437, MT594438 for GS and MN133228, MN133229, MN133230 for ApMAT. The sequences of the three isolates were aligned with those of the related species in C. gloeosporioides complex (Liu et al. 2015). Analyses based on concatenated data sets of four genes showed that the sequences had high levels of identity to those of the C. siamense strains. According to both morphological and sequence analyses, the C. oppenheimiana pathogen was identified as C. siamense. There are two reports of foliar pathogens on C. oppenheimiana, Rhizoctonia solani and Pyricularia oryzae (Baiswar et al. 2010, Pappas and Vloutoglou, 1996). To our knowledge, this is the first report of anthracnose on C. oppenheimiana caused by C. siamense in China. Identification of the pathogen of this disease is essential for the development of effective and economical management practices.

First Report of Root Rot Caused by Pythium myriotylum of Onion in Idaho

In August 2017, onion plants (Allium cepa cv. Joaquin) were observed in one field in southwest Idaho with a high incidence of reduced bulb size. One area, approximately 3 ha in size, comprising approximately 20% of the total field, appeared to be affected. In this area, over half of the bulbs appeared smaller by at least 50% compared with nonaffected areas of the field. Closer inspection showed that the roots of the stunted bulbs were discolored and volume reduced. To determine the causal agent, root pieces, each 3 to 5 mm in length, sampled from symptomatic plants were surface sterilized (2% sodium hypochlorite) for 30 s and rinsed twice in sterile water prior to plating onto potato dextrose agar (PDA) amended with penicillin G (0.2 g/liter) and streptomycin sulfate (0.8 g/liter). After 5 days at 21°C in the dark, colonies were observed, and hyphal tips were transferred to fresh PDA plates to obtain pure cultures. After 7 days, suspect Pythium colonies were present that had abundant, white, aseptate hyphae up to 9 µm in width and sporangia. Species identity was confirmed through ribosomal DNA (rDNA) ITS sequencing from a representative isolate (designated OP1), as previously described (Woodhall et al. 2013). The resulting sequence (GenBank MT256180) was 99% identical to the sequence from Pythium myriotylum strain CBS 254.70 (GenBank AY598678). To confirm pathogenicity, onion seeds (cv. Vaquero) were planted in individual pots containing premium potting soil (Scotts, Marysville, OH). The plants were grown in a controlled environment chamber set at 21°C for 1 month until they were approximately 10 cm in height. Plants were watered as required. Plants were then inoculated with a plug of PDA (10 mm in diameter) that was either fully colonized with isolate OP1 or sterile in the case of the control treatment. Six plants were inoculated for each treatment. The plugs were placed adjacent to the main root mass at approximately 5 cm depth. Plants were grown for a further 18 days under the same conditions and then were destructively harvested and visually assessed for root symptoms. On all plants inoculated with isolate OP1, the roots had brown discoloration. The roots of the control plants appeared healthy with no discoloration. Root length was also significantly less (t test, P < 0.05) for the inoculated plants (mean = 18 mm) than for control plants (49 mm). Pythium spp. was reisolated from all inoculated plants, but no Pythium spp. was isolated from control plants. Several Pythium species can infect onion roots (Vincelli 2008). To our knowledge this is the first report of P. myriotylum affecting onions and in Idaho. P. myriotylum mainly occurs in warmer regions and has a wide host range including wheat, beans, and corn (Serrano et al. 2008; Wang et al. 2003). These crops are often grown in rotation with onion crops in southwest Idaho, and therefore growers may need to consider the potential presence of P. myriotylum in their fields.

Anopheles sundaicus complex and the presence of Anopheles epiroticus in Indonesia.

Anopheles sundaicus s.l. is an important malaria vector primarily found in coastal landscapes of western and central Indonesia. The species complex has a wide geographical distribution in South and Southeast Asia and exhibits ecological and behavioural variability over its range. Studies on understanding the distribution of different members in the complex and their bionomics related to malaria transmission might be important guiding more effective vector intervention strategies. Female An. sundaicus s.l. were collected from seven provinces, 12 locations in Indonesia representing Sumatra: North Sumatra, Bangka-Belitung, South Lampung, and Bengkulu; in Java: West Java; and the Lesser Sunda Islands: West Nusa Tenggara and East Nusa Tenggara provinces. Sequencing of ribosomal DNA ITS2 gene fragments and two mitochondrial DNA gene markers, COI and cytb, enabled molecular identification of morphologically indistinguishable members of the complex. Findings allowed inference on the distribution of the An. sundaicus s.l. present in Indonesia and further illustrate the phylogenetic relationships of An. epiroticus within the complex. A total of 370 An. sundaicus s.l specimens were analysed for the ITS2 fragment. The ITS2 sequence alignment revealed two consistent species-specific point mutations, a T>C transition at base 479 and a G>T transversion at base 538 that differentiated five haplotypes: TG, CG, TT, CT, and TY. The TG haplotype matched published An. epiroticus–indicative sequences from Thailand, Vietnam and peninsular Malaysia. The previously described insertion event (base 603) was observed in all identified specimens. Analysis of the COI and cytb genes revealed no consistent nucleotide variations that could definitively distinguish An. epiroticus from other members in the Sundaicus Complex. The findings indicate and support the existence of An. epiroticus in North Sumatra and Bangka-Belitung archipelago. Further studies are recommended to determine the full distributional extent of the Sundaicus complex in Indonesia and investigate the role of these species in malaria transmission.

The molecular characterization of Indonesian Gonystylus species

The proper identification of a species is a very important factor for establishment of management and conservation strategies. This study aimed to confirm the identification of Gonystylus spp. using DNA markers. Twenty-four leaf samples of nine species of ramin were extracted using the modified CTAB method to obtain the template DNAs. Four primers were used to amplify ITS ribosomal DNA (partial of ITS2) and three chloroplast non-coding regions sequences. DNA sequences were analyzed using BLAST online software to identify the species. The phylogenetic tree was constructed to confirm species relationship. Based on the DNA markers, one sample of Aquilaria was misidentified as Gonystylus, and the other 23 samples were clustered into seven groups i.e. G. bancanus, G. brunescens, G. stenosepalus, G. forbesii, G. consanguineus, G. lucidulus and an unspecified Gonystylus. The clustering of those samples based on molecular characters was in disagreement with clustering based on morphological characters. This finding suggests that molecular characterization should be conducted to confirm the identification of species prior to establishing conservation areas.

Symbiotic yeasts from the mycangium, larval gut and woody substrate of an African stag beetle Xiphodontus antilope (Coleoptera: Lucanidae).

Female stag beetles (Lucanidae) possess internal mycangia to maintain microbial cultures. Yeasts from these mycangia may help with larval nutrition in nutrient poor woody substrates, but only a few Lucanidae taxa have been studied and all reports originate from Europe and Asia. We identify the first mycangial yeasts of a South African endemic Lucanidae beetle, Xiphodontus antilope, using nuclear ribosomal RNA and ITS DNA sequence data. In addition we identified yeasts from the larval gut, fecal matter, frass and woody substrate surrounding larvae and pupae. The mycangium of X. antilope was confined to females and is structurally similar to all other Lucanidae. Unlike most Lucanidae that seemingly associate with single species of yeast, or whose mycangia contain yeast monocultures, three yeast species were commonly isolated from X. antilope. Scheffersomyces coipomoensis was the most numerically dominant species on most substrates and in most individuals, but a second, undescribed, Scheffersomyces species was present in high numbers. A third species, also undescribed and unrelated to Scheffersomyces, was recovered from all mycangia but could not be detected in the larval gut, fecal matter, frass or woody substrates. We confirm a close association of Scheffersomyces yeasts with Lucanidae globally, but other taxa may also be involved. We show that the predominant mycangial yeasts also form the predominant yeasts within the larval gut and the woody substrates around the larvae and pupae. This combined external and internal colonization by the same yeasts may provide enhanced opportunities for nutrient acquisition, but this needs validation in future studies.

On the identity of Goodyera rosulacea (Orchidaceae: Orchidoideae: Cranichideae: Goodyerinae)

Morphological studies based on the protologues, observations of herbarium specimens and field surveys suggest that Goodyera rosulacea is a synonym of G. brachystegia. According to genetic studies, accessions of G. rosulacea and G. brachystegia form a single clade in the phylogenetic trees obtained from analyses of the nuclear ribosomal DNA ITS region and four plastid regions (accD-psaI, matK, trnK-rps16 and trnL-F). This phylogenetic evidence further underpins the conclusion that the two names refer to one and the same species.

Widespread inspection and comparative analysis of ITS secondary structure conservation and covariation of Coccinellidae

ITS1 and ITS2 of ribosomal DNA (rDNA) of six subfamilies i.e. Epilachaninae, Chilocorinae, Coccinellinae, Coccidulinae, Scymininae Ortaliinae and Sticholotidinae were examined, aligned and conserved motifs in folding patterns of stable consensus secondary structures were assessed. RNAalifold and RNA fold algorithms applied by RNAz program to compute consensus secondary structures and MFE efficiently and derive a number of statistical scores i.e. SCI, Z-score and MPI. Assembled subsets in ITS1 (three sets) and ITS2 (ten sets) were significantly conserved. In this study, analysis of these consensus secondary structures shows that a motif sequence is remain conserved in all the species. Helices in subsets of ITS1 showed compensatory base involvement to maintain the stability and conservation of secondary structures during natural selection. Secondary structures of ITS2 found more conserved than ITS1. MP, BI and ML methods used to establish the evolutionary relationship, compared the topology significance on the basis of SCI and MPI in defined sets. Coccinellinae, Epilachaninae and Scymninae (all strains of species) were monophyletic whereas Coccidulinae, and Chilocorinae had inadequate sequence information. Of Ortaliinae and Sticholotidinae, yet no ITS sequences have been submitted. Thus, analysis of the structural folding patterns of ITS regions provided further prime information of species relatedness and inter-intra-specific variations.

An Empirical Analysis Rejects the Hybrid Speciation Hypothesis of a Crucial Kiwifruit Species, Despite Genomic Evidence of Frequent Interspecific Gene Flow in the Genus.

Hybrid speciation is an important way to generate species diversity. In general, however, interspecific hybridization is easily confused with the formation of hybrid species. Using the genomic resequencing data of the kiwifruit genus (Actinidia), at least ten species were documented recently as homoploid hybrid species, and thus a two-layer mode of species diversification has been proposed. As a crucial piece of evidence, Actinidia fulvicoma was identified as a hybrid derivative of Actinidia eriantha × Actinidia cylindrica, representing a rare case of hybrid species in kiwifruit that won the competition of ecological niches with one of its putative parental species, A. cylindrica. However, the hypothesized hybrid origin of A. fulvicoma is inconsistent with our specimen observations. Here, we present multiple lines of evidence to reject the hybrid speciation hypothesis for this species, despite genomic evidence for frequent interspecific gene flow. We collected the samples of A. fulvicoma in type locality and neighboring regions to contrast them with type specimen, and sequenced nuclear ribosomal DNA ITS, chloroplast trnL-trnF and mitochondrial nad2-i3, as well as four single-copy nuclear genes explored from kiwifruit genomes, to infer phylogenetic relationships among A. fulvicoma, its putative parental species, and their relatives. Our data definitely reveal that A. fulvicoma occupies an independent backbone lineage and it is not a hybrid. This study suggests that correct evolutionary applications on extensive surveys of the putative hybrid and its possible parents with strict criteria are necessary in the documentation of hybrid speciation to advance our understanding of the genomic basis of hybrid species.

&lt;p&gt;&lt;strong&gt;&lt;em&gt;Calanthe tsiana&lt;/em&gt;&lt;/strong&gt;&lt;strong&gt;, a new orchid species from China (Epidendroideae: Collabieae): evidence from morphological and molecular analyses&lt;/strong&gt;&lt;/p&gt;

A new orchid, Calanthe tsiana, is described from southeastern Yunnan, China, based on morphological and DNA evidence. In morphological comparisons, C. tsiana is similar to C. arisanensis, but it differs in having subspatulate petals and falcate-obovate lateral lobes of lip with one ridge on the disk. The phylogenetic analyses based on nuclear ribosomal ITS and plastid DNA (rbcL, matK and trnL-trnF) place C. tsiana with C. arisanensis as sister species and support C. tsiana as a new species.

Morphological and molecular characterization of Heterodera dunensis n. sp. (Nematoda: Heteroderidae) from Gran Canaria, Canary Islands.

Heterodera dunensis n. sp. from the coastal dunes of Gran Canaria, Canary Islands, is described. This new species belongs to the Schachtii group of Heterodera with ambifenestrate fenestration, presence of prominent bullae, and a strong underbridge of cysts. It is characterized by vermiform second-stage juveniles having a slightly offset, dome-shaped labial region with three annuli, four lateral lines, a relatively long stylet (27-31 µm), short tail (35-45 µm), and 46 to 51% of tail as hyaline portion. Males were not found in the type population. Phylogenetic trees inferred from D2-D3 of 28S, partial ITS, and 18S of ribosomal DNA and COI of mitochondrial DNA sequences indicate a position in the ‘Schachtii clade’.