Carboxylic acid reductases (CARs) are attracting burgeoning attention as biocatalysts for organic synthesis of aldehydes and their follow-up products from economic carboxylic acid precursors. The CAR enzyme class as a whole, however, is still poorly understood. To date, relatively few CAR sequences have been reported, especially from fungal sources. Here, we sought to increase the diversity of the CAR enzyme class. Six new CAR sequences from the white-rot fungus Pycnoporus cinnabarinus were identified from genome-wide mining. Genome and gene clustering analysis suggests that these PcCAR enzymes play different natural roles in Basidiomycete systems, compared to their type II Ascomycete counterparts. The cDNA sequences of all six Pccar genes were deduced and analysis of their corresponding amino acid sequence showed that they encode for proteins of similar properties that possess a conserved modular functional tri-domain arrangement. Phylogenetic analyses showed that all PcCAR enzymes cluster together with the other type IV CARs. One candidate, PcCAR4, was cloned and over-expressed recombinantly in Escherichia coli. Subsequent biotransformation-based screening with a panel of structurally-diverse carboxylic acid substrates suggest that PcCAR4 possessed a more pronounced substrate specificity compared to previously reported CARs, preferring to reduce sterically-rigid carboxylic acids such as benzoic acid. These findings thus present a new functionally-distinct member of the CAR enzyme class.
Two subspecies of cynomolgus macaques (Macaca fascicularis) are alleged to co-exist in the Philippines, M. f. philippensis in the north and M. f. fascicularis in the south. However, genetic differences between the cynomolgus macaques in the two regions have never been studied to document the propriety of their subspecies status. We genotyped samples of cynomolgus macaques from Batangas in southwestern Luzon and Zamboanga in southwestern Mindanao for 15 short tandem repeat (STR) loci and sequenced an 835 bp fragment of the mtDNA of these animals. The STR genotypes were compared with those of cynomolgus macaques from southern Sumatra, Singapore, Mauritius and Cambodia, and the mtDNA sequences of both Philippine populations were compared with those of cynomolgus macaques from southern Sumatra, Indonesia and Sarawak, Malaysia. We conducted STRUCTURE and PCA analyses based on the STRs and constructed a median joining network based on the mtDNA sequences. The Philippine population from Batangas exhibited much less genetic diversity and greater genetic divergence from all other populations, including the Philippine population from Zamboanga. Sequences from both Batangas and Zamboanga were most closely related to two different mtDNA haplotypes from Sarawak from which they are apparently derived. Those from Zamboanga were more recently derived than those from Batangas, consistent with their later arrival in the Philippines. However, clustering analyses do not support a sufficient genetic distinction of cynomolgus macaques from Batangas from other regional populations assigned to subspecies M. f. fascicularis to warrant the subspecies distinction M. f. philippensis.
The population history of Aboriginal Australians remains largely uncharacterized. Here we generate high-coverage genomes for 83 Aboriginal Australians (speakers of Pama-Nyungan languages) and 25 Papuans from the New Guinea Highlands. We find that Papuan and Aboriginal Australian ancestors diversified 25-40 thousand years ago (kya), suggesting pre-Holocene population structure in the ancient continent of Sahul (Australia, New Guinea and Tasmania). However, all of the studied Aboriginal Australians descend from a single founding population that differentiated ~10-32 kya. We infer a population expansion in northeast Australia during the Holocene epoch (past 10,000 years) associated with limited gene flow from this region to the rest of Australia, consistent with the spread of the Pama-Nyungan languages. We estimate that Aboriginal Australians and Papuans diverged from Eurasians 51-72 kya, following a single out-of-Africa dispersal, and subsequently admixed with archaic populations. Finally, we report evidence of selection in Aboriginal Australians potentially associated with living in the desert.
The spider suborder Mesothelae, containing a single extant family Liphistiidae, represents a species-poor and ancient lineage. These are conspicuous spiders that primitively retain a segmented abdomen and appendage-like spinnerets. While their classification history is nearly devoid of phylogenetic hypotheses, we here revise liphistiid genus level taxonomy based on original sampling throughout their Asian range, and on the evidence from a novel molecular phylogeny. By combining morphological and natural history evidence with phylogenetic relationships in the companion paper, we provide strong support for the monophyly of Liphistiidae, and the two subfamilies Liphistiinae and Heptathelinae. While the former only contains Liphistius Schiödte, 1849, a genus distributed in Indonesia (Sumatra), Laos, Malaysia, Myanmar, Thailand, we recognize and diagnose seven heptatheline genera, all but three removed from the synonymy of Heptathela: i) Ganthela Xu & Kuntner, gen. n. with the type species Ganthelayundingensis Xu, sp. n. is known from Fujian and Jiangxi, China; ii) a rediagnosed Heptathela Kishida, 1923 is confined to the Japanese islands (Kyushu and Okinawa); iii) Qiongthela Xu & Kuntner, gen. n. with the type species Qiongthelabaishensis Xu, sp. n. is distributed disjunctly in Hainan, China and Vietnam; iv) Ryuthela Haupt, 1983 is confined to the Ryukyu archipelago (Japan); v) Sinothela Haupt, 2003 inhabits Chinese areas north of Yangtze; vi) Songthela Ono, 2000 inhabits southwest China and northern Vietnam; and vii) Vinathela Ono, 2000 (Abcathela Ono, 2000, syn. n.; Nanthela Haupt, 2003, syn. n.) is known from southeast China and Vietnam.
Conservation of plant species plays a vital role in preventing the loss of valuable plant resources. The success of conservation depends on the correct identification and characterization of plant species. Bamboo is one of the most important plants with multiple uses that have contributed to the economy and socio-economy of many people in rural areas. It is under the subfamily of Bambusoideae that includes both woody and herbaceous bamboo. Conventionally, like other plants, bamboo has been classified dependently based on morphological characteristics. However, morphological identification leads to difficulties and misclassification of bamboo species due to their infrequent flowering behaviour and peculiar reproductive biology. Since then, molecular markers have been introduced to overcome the problems associated with bamboo taxonomy and phylogeny. This paper provides an overview of the diverse, predominantly molecular techniques used to assess and determine the genetic diversity of bamboo species.
An amylopullulanase of the thermophilic Anoxybacillus sp. SK3-4 (ApuASK) was purified to homogeneity and characterized. Though amylopullulanases larger than 200 kDa are rare, the molecular mass of purified ApuASK appears to be approximately 225 kDa, on both SDS-PAGE analyses and native-PAGE analyses. ApuASK was stable between pH 6.0 and pH 8.0 and exhibited optimal activity at pH 7.5. The optimal temperature for ApuASK enzyme activity was 60 °C, and it retained 54% of its total activity for 240 min at 65 °C. ApuASK reacts with pullulan, starch, glycogen, and dextrin, yielding glucose, maltose, and maltotriose. Interestingly, most of the previously described amylopullulanases are unable to produce glucose and maltose from these substrates. Thus, ApuASK is a novel, high molecular-mass amylopullulanase able to produce glucose, maltose, and maltotriose from pullulan and starch. Based on whole genome sequencing data, ApuASK appeared to be the largest protein present in Anoxybacillus sp. SK3-4. The α-amylase catalytic domain present in all of the amylase superfamily members is present in ApuASK, located between the cyclodextrin (CD)-pullulan-degrading N-terminus and the α-amylase catalytic C-terminus (amyC) domains. In addition, the existence of a S-layer homology (SLH) domain indicates that ApuASK might function as a cell-anchoring enzyme and be important for carbohydrate utilization in a streaming hot spring.
Between 2005 and 2013 six severe pneumonia cases (all requiring mechanical ventilation, two fatal outcomes) caused by human adenovirus type 21 (HAdV-B21) were observed in Germany. So far, HAdV-B21 was mainly associated with non-severe upper and lower respiratory tract infections. However, a few highly virulent HAdV types, e.g. HAdV-B14p1, were previously associated with severe, fatal pneumonia. Complete genomic sequences of the German HAdV-B21 pneumonia isolates formed a single phylogenetic cluster with very high sequence identity (≥ 99.897%). Compared to the HAdV-B21 prototype (only 99.319% identity), all isolates had a unique 15 amino acid deletion and a 2 amino acid insertion in the RGD loop of the penton base which may affect binding to the secondary receptor on the host cells. Moreover, a recombinant E4 gene region derived of HAdV-B3 was identified by bootscan analysis. Thus, the highly virulent, pneumotropic HAdV-B21 was denominated as subtype 21a. Surprisingly, there was 99.963% identity with agent Y/SIBU97 (only 13.4 kb available in GenBank of the 35.4 kb genome) which was associated with 10 fatalities due to cardiopulmonary failure in Sarawak, Malaysia, in 1997. In conclusion, a HAdV-B21 subtype (21a) associated with severe pneumonia in Germany was phylogenetically linked to an adenovirus isolated in Malaysia.
A group of 31 rabies viruses (RABVs), recovered primarily from dogs, one deer and one human case, were collected from various areas in China between 1989 and 2006. Complete G gene sequences determined for these isolates indicated identities of nucleotide and amino acid sequences of >or=87% and 93.8%, respectively. Phylogenetic analysis of these and some additional Chinese isolates clearly supported the placement of all Chinese viruses in Lyssavirus genotype 1 and divided all Chinese isolates between four distinct groups (I-IV). Several variants identified within the most commonly encountered group I were distributed according to their geographical origins. A comparison of representative Chinese viruses with other isolates retrieved world-wide indicated a close evolutionary relationship between China group I and II viruses and those of Indonesia while China group III viruses formed an outlying branch to variants from Malaysia and Thailand. China group IV viruses were closely related to several vaccine strains. The predicted glycoprotein sequences of these RABVs variants are presented and discussed with respect to the utility of the anti-rabies biologicals currently employed in China.
A multi-locus approach was used to examine the DNA sequences of 10 nominal species of blackfly in the Simulium subgenus Gomphostilbia (Diptera: Simuliidae) in Malaysia. Molecular data were acquired from partial DNA sequences of the mitochondria-encoded cytochrome c oxidase subunit I (COI), 12S rRNA and 16S rRNA genes, and the nuclear-encoded 18S rRNA and 28S rRNA genes. No single gene, nor the concatenated gene set, resolved all species or all relationships. However, all morphologically established species were supported by at least one gene. The multi-locus sequence analysis revealed two distinct evolutionary lineages, conforming to the morphotaxonomically recognized Simulium asakoae and Simulium ceylonicum species groups.
Tilapia lake virus (TiLV) is a highly contagious viral pathogen that affects tilapia, a globally significant and affordable source of fish protein. To prevent the introduction and spread of TiLV and its impact, there is an urgent need for increased surveillance, improved biosecurity measures, and continuous development of effective diagnostic and rapid sequencing methods. In this study, we have developed a multiplexed RT-PCR assay that can amplify all ten complete genomic segments of TiLV from various sources of isolation. The amplicons generated using this approach were immediately subjected to real-time sequencing on the Nanopore system. By using this approach, we have recovered and assembled 10 TiLV genomes from total RNA extracted from naturally TiLV-infected tilapia fish, concentrated tilapia rearing water, and cell culture. Our phylogenetic analysis, consisting of more than 36 TiLV genomes from both newly sequenced and publicly available TiLV genomes, provides new insights into the high genetic diversity of TiLV. This work is an essential steppingstone towards integrating rapid and real-time Nanopore-based amplicon sequencing into routine genomic surveillance of TiLV, as well as future vaccine development.
We identified and characterized a new cosmocercid nematode species, Cosmocercoides wuyiensis n. sp., through microscopic examination and sequencing of the partial small ribosomal RNA gene (18S rDNA), internal transcribed spacer (ITS) and mitochondrial cytochrome c oxidase subunit 1 (COI) genes. The new species was isolated from the intestine of the Asiatic frog Amolops wuyiensis Liu and Hu, 1975 captured from four localities of the Anhui province in south-east China. Among the 25 recorded species of the Cosmocercoides genus, the morphology of C. wuyiensis n. sp. is closest to that of C. kiliwai and C. malayensis, which were isolated from various Mexican frog and Malaysian lizard species, respectively. However, C. wuyiensis n. sp. displayed several distinguishing features, such as small size of the male body, two spicules of unequal lengths in the male, small gubernaculum, pre-, ad- and post-cloacal caudal rosette papillae in the ratio of 18-24:2:6 and simple papillae in the ratio of 14:multiple:4, circle and number of punctation in each rosette at 1:11-16, sharply conical tail-end and the presence of lateral alae and somatic papillae in both sexes. BLAST and the phylogenetic analyses of the 18S rDNA and ITS sequences indicated that C. wuyiensis n. sp. belonged to the genus Cosmocercoides, while that of the COI gene sequence of C. wuyiensis n. sp. showed 16.36% nucleotide divergence with C. pulcher and 47.99% nucleotide divergence with C. qingtianensis. The morphological and molecular characterization of C. wuyiensis n. sp. provides new taxonomic data for this genus.
We found a uniquely colored dicroglossid frog of the genus Occidozyga from western Sarawak, East Malaysia. It is divergent from other congeners in morphology and mtDNA sequences. In a molecular phylogeny, this species is the sister lineage to the continental species O. lima and O. martensii with weak support. The species is small with SVL 16-18 mm in males and 18-19 mm in females, without dorsolateral fold but with transverse wrinkles on dorsum, tips of fingers lacking disks but of toes with disks, only first and second toes webbed to disks, and orange-brown dorsum with dark brown band. We thus describe it as a new species.
A new species of black fly, Simulium (Gomphostilbia) isanense, is described based on females, males, pupae and mature larvae from Thailand. This new species is placed in the Simulium epistum species-group of the subgenus Gomphostilbia Enderlein. It is characterized by the pupal gill with eight filaments arranged as 3+3+2 from dorsal to ventral, of which an inner filament of the ventral pair is slightly longer than its counter filament. Taxonomic notes are provided to distinguish this new species from S. (G.) angulistylum Takaoka & Davies from Peninsular Malaysia, and three other related species. The difference between this new species and S. (G.) angulistylum is supported by genetic distances using the mitochondrial COI gene.
We describe a new species of cascade frog of the genus Rana, from west Malaysia. Rana monjerai, new species is a medium-sized frog of the subgenus Odorrana (SVL of males, 38-43 mm; of one female, 75 mm), and is distinguished from all other members of this subgenus by the combination of: white lip stripe, dorsolateral fold, full web on the fourth toe, vomerine teeth, gular vocal pouch and relatively large tympanum in males, no dorsal marking, no clear light spots on rear of thigh, first finger subequal to second, finely tuberculated dorsum, and unpigmented ova. The significance of finding this species from peninsular Malaysia is discussed.
A new bufonid amphibian, belonging to a new monotypic genus, is described from the Andaman Islands, in the Bay of Bengal, Republic of India, based on unique external morphological and skeletal characters which are compared with those of known Oriental and other relevant bufonid genera. Blythophryne gen. n. is distinguished from other bufonid genera by its small adult size (mean SVL 24.02 mm), the presence of six presacral vertebrae, an absence of coccygeal expansions, presence of an elongated pair of parotoid glands, expanded discs at digit tips and phytotelmonous tadpoles that lack oral denticles. The taxonomic and phylogenetic position of the new taxon (that we named as Blythophryne beryet gen. et sp. n.) was ascertained by comparing its 12S and 16S partial genes with those of Oriental and other relevant bufonid lineages. Resulting molecular phylogeny supports the erection of a novel monotypic genus for this lineage from the Andaman Islands of India.
A new species of Philautus is described from western Sarawak. The new species was collected in lower montane forest in two national parks in Sarawak and recorded from another park. It differs from its congeners by a unique combination of morphological characters, including a long, acuminate snout, long legs, and comparatively extensive toe webbing. The advertisement call of the new species differs from all calls of other species that have been analyzed so far. Comparison of the mitochondrial 16S rRNA gene sequence corroborates its distinct specific status.
Five out of the nine benzene-toulene-ethylbenzene-xylene (BTEX) tolerant bacteria that demonstrated high protease activity on skim milk agar were isolated. Among them, isolate 115b identified as Bacillus pumilus exhibited the highest protease production. The protease produced was stable in 25% (v/v) benzene and toluene and it was activated 1.7 and 2.5- fold by n-dodecane and n-tetradecane, respectively. The gene encoding the organic solvent tolerant protease was cloned and its nucleotide sequence determined. Sequence analysis revealed an open reading frame (ORF) of 1,149 bp that encoded a polypeptide of 383 amino acid residues. The polypeptide composed of 29 residues of signal peptide, a propeptide of 79 residues and a mature protein of 275 amino acids with a calculated molecular mass of 27,846 Da. This is the only report available to date on organic solvent tolerant protease from B. pumilus.
Strongyloides callosciureus n. sp. (Nematoda: Rhabditoidea), from Asian sciurids, is described based on morphology, morphometry, and the small and large subunit (SSU/LSU) ribosomal RNA gene (rDNA) sequences. This new species was collected from Pallas's squirrels (Callosciurus erythraeus) in the central part of mainland Japan (Honshu), which were originally introduced from Taiwan some decades ago, and plantain squirrels (Callosciurus notatus) imported from Malaysia as personal pets. For comparison, Strongyloides robustus Chandler, 1942 was collected from American red squirrels (Tamiasciurus hudsonicus) and southern flying squirrels (Glaucomys volans) imported from the United States as personal pets. The parasitic females found in North American and Asian sciurids shared some key morphological features such as the ovary running spirally around the gut, and the shapes of the stoma in the apical view and the tail. However, morphometric features of parasitic females in North American and Asian sciurids differed significantly from each other; the former was larger than the latter, and the relative position of the vulva to the whole body length from the mouth was different. The SSU/LSU rDNA sequences supported the division of sciurid Strongyloides isolates by geographical distribution of the host and morphological features, leading us to propose the erection of new species.
Minnows of the genus Phoxinus are common and an often highly abundant fish species in Palearctic freshwater habitats. Phoxinus species have a complex evolutionary history, phylogenetic relationships are not well understood and there are a number of unresolved taxonomic problems. There are currently 23 different mitochondrial genetic lineages identified in the genus Phoxinus, 13 of which are recognized as valid species. The taxonomic status of these lineages requires resolution, including the degree to which they can interbreed. Suitable nuclear molecular markers for studies of population divergence and interbreeding between morphotypes and mitochondrial lineages are lacking for Phoxinus species. Therefore, the authors developed a set of microsatellite markers using genomic information from Phoxinus lumaireul and tested their suitability for this and two related species, Phoxinus krkae and Phoxinus marsilii. Out of 16 microsatellite candidate loci isolated, 12 were found to be in Hardy-Weinberg equilibrium when tested on two P. lumaireul senso lato populations. Seven loci amplified across the three species, enabling the study of intraspecific genetic diversity and population structure within P. marsilii and P. krkae. The markers were able to clearly resolve differences among the three tested species, including the recently described P. krkae, and are therefore suitable for the detection of introgression and hybridization among populations consisting of mixtures of two or more of P. lumaireul s. l., P. marsilii and P. krkae.
An integrative taxonomic analysis of Subdoluseps herberti from southern Thailand and Peninsular Malaysia and S. samajaya from Sarawak, East Malaysia (Borneo) recovers the former as paraphyletic with respect to the latter. The analyses recover the three southernmost populations of S. herberti in Peninsular Malaysia as conspecific and the sister lineage of S. samajaya, whereas S. herberti from Thailand and northern Peninsular Malaysia constitute the sister species to S. samajaya plus the southern three Peninsular Malaysian populations. As such, the southern populations are described herein as S. malayana sp. nov. and all three species are referred to as the S. herberti group. Clade boundaries and breaks within this group on the Thai-Malay Peninsula occurring at the Isthmus of Kra, across the Kangar-Pattani line, and between the Thai-Malay Peninsula and Borneo are consistent with phylogeographic patterns of other Sundaic taxa. The discovery of S. malayana sp. nov. continues to underscore the fact that, despite the well-studied nature of the lizard fauna of Peninsular Malaysia, much of it still remains unrealized and for conservation efforts to move forward, field research followed by expeditiously revised taxonomies must continue.