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Fulltext Large-scale collection of full-length cDNA and transcriptome analysis in Hevea brasiliensis

Makita Y, Ng KK, Veera Singham G, Kawashima M, Hirakawa H, Sato S, et al.

DNA Res, 2017 Apr 01;24(2):159-167.
PMID: 28431015 DOI: 10.1093/dnares/dsw056

Natural rubber has unique physical properties that cannot be replaced by products from other latex-producing plants or petrochemically produced synthetic rubbers. Rubber from Hevea brasiliensis is the main commercial source for this natural rubber that has a cis-polyisoprene configuration. For sustainable production of enough rubber to meet demand elucidation of the molecular mechanisms involved in the production of latex is vital. To this end, we firstly constructed rubber full-length cDNA libraries of RRIM 600 cultivar and sequenced around 20,000 clones by the Sanger method and over 15,000 contigs by Illumina sequencer. With these data, we updated around 5,500 gene structures and newly annotated around 9,500 transcription start sites. Second, to elucidate the rubber biosynthetic pathways and their transcriptional regulation, we carried out tissue- and cultivar-specific RNA-Seq analysis. By using our recently published genome sequence, we confirmed the expression patterns of the rubber biosynthetic genes. Our data suggest that the cytoplasmic mevalonate (MVA) pathway is the main route for isoprenoid biosynthesis in latex production. In addition to the well-studied polymerization factors, we suggest that rubber elongation factor 8 (REF8) is a candidate factor in cis-polyisoprene biosynthesis. We have also identified 39 transcription factors that may be key regulators in latex production. Expression profile analysis using two additional cultivars, RRIM 901 and PB 350, via an RNA-Seq approach revealed possible expression differences between a high latex-yielding cultivar and a disease-resistant cultivar.
Morphological divergence, reproductive isolating mechanism, and molecular phylogenetic relationships among Indonesia, Malaysia, and Japan populations of the Fejervarya limnocharis complex (Anura, Ranidae)

Djong TH, Matsui M, Kuramoto M, Belabut DM, Sen YH, Nishioka M, et al.

Zoolog Sci, 2007 Dec;24(12):1197-212.
PMID: 18271636 DOI: 10.2108/zsj.24.1197

In order to elucidate the taxonomic status of the Fejervarya limnocharis complex relative to Malaysia and Japan populations, morphological observations and molecular phylogenetic analysis were carried out using three populations from Indonesia (type locality), Malaysia, and Japan. In addition, we conducted histological and spermatogenic observations using hybrids among these populations. Principal component and cluster analyses demonstrated that these populations could be clearly separated from one another. Abnormal testes were found in the hybrids between the Japan and Indonesia populations and between the Japan and Malaysia populations, but testes of the controls and hybrids between the Malaysia and Indonesia populations were quite normal. The mean number of univalents per cell was 5.42, 4.58, and 0.20 in hybrids between the Indonesia and Japan populations, Malaysia and Japan populations, and Indonesia and Malaysia populations, respectively. Sequence divergences in 16S rRNA and Cyt b genes were 0-0.4% (xbar=0.2%) and 0.3-1.5% (xbar=1.0%), respectively, between the Malaysia and Indonesia populations, and 2.4-2.6% (xbar=2.5%) and 11.0-12.0% (xbar=11.5%) between the Japan population and F. limnocharis complex, including the Malaysia and Indonesia populations and F. multistriata from China. This study indicated that the Malaysia population and F. multistriata from China should be designated as a subspecies of topotypic F. limnocharis, and that the Japan population should be regarded as a distinct species.
Multiple invasions of the Ryukyu Archipelago by Oriental frogs of the subgenus Odorrana with phylogenetic reassessment of the related subgenera of the genus Rana

Matsui M, Shimada T, Ota H, Tanaka-Ueno T

Mol Phylogenet Evol, 2005 Dec;37(3):733-42.
PMID: 15964212

The genus Rana, notably diversified in Oriental regions from China to Southeast Asia, includes a group of cascade frogs assigned to subgenera Odorrana and Eburana. Among them, R. ishikawae and the R. narina complex represent the northernmost members occurring from Taiwan to the Ryukyu Archipelago of Japan. Relationships of these frogs with the continental members, as well as the history of their invasions to islands, have been unclear. The taxonomic status of Odorrana and related genera varies among authors and no phylogenetic reassessment has been done. Using partial sequences of mitochondrial 12S and 16S rRNA genes, we estimated phylogenetic relationships among 17 species of the section Hylarana including Odorrana and Eburana, and related species from the Ryukyus, Taiwan, China, Thailand, Malaysia, and Indonesia. We estimate that (1) Odorrana is monophyletic and encompasses species of Eburana and R. hosii, which is now placed in Chalcorana, (2) the ancestor of R. ishikawae separated from other Rana in the middle to late Miocene prior to its entry to the Ryukyu Archipelago, (3) the ancestor of the R. narina complex later diversified in continental Asia, and invaded the Ryukyu Archipelago through Taiwan, (4) the R. narina complex attained its current distribution within the Ryukyus through niche segregations, and (5) vicariance of R. hosii between Malay Peninsula and Borneo occurred much later than the divergence events in the R. narina complex. Current subgeneric classification of Rana, at least of Southeast Asian members, requires full reassessment in the light of phylogenetic relationships.
Fulltext Ongoing declines for the world's amphibians in the face of emerging threats

Luedtke JA, Chanson J, Neam K, Hobin L, Maciel AO, Catenazzi A, et al.

Nature, 2023 Oct;622(7982):308-314.
PMID: 37794184 DOI: 10.1038/s41586-023-06578-4

Systematic assessments of species extinction risk at regular intervals are necessary for informing conservation action1,2. Ongoing developments in taxonomy, threatening processes and research further underscore the need for reassessment3,4. Here we report the findings of the second Global Amphibian Assessment, evaluating 8,011 species for the International Union for Conservation of Nature Red List of Threatened Species. We find that amphibians are the most threatened vertebrate class (40.7% of species are globally threatened). The updated Red List Index shows that the status of amphibians is deteriorating globally, particularly for salamanders and in the Neotropics. Disease and habitat loss drove 91% of status deteriorations between 1980 and 2004. Ongoing and projected climate change effects are now of increasing concern, driving 39% of status deteriorations since 2004, followed by habitat loss (37%). Although signs of species recoveries incentivize immediate conservation action, scaled-up investment is urgently needed to reverse the current trends.
Phylogenetic relationships of megophryid frogs of the genus Leptobrachium (Amphibia, Anura) as revealed by mtDNA gene sequences

Matsui M, Hamidy A, Murphy RW, Khonsue W, Yambun P, Shimada T, et al.

Mol Phylogenet Evol, 2010 Jul;56(1):259-72.
PMID: 20302957 DOI: 10.1016/j.ympev.2010.03.014

By investigating genealogical relationships, we estimated the phylogenetic history and biogeography in the megophryid genus Leptobrachium (sensu lato, including Vibrissaphora) from southern China, Indochina, Thailand and the Sundaland. The genealogical relationships among the 30 named and unnamed taxa were estimated using 2009 bp of sequences from the mitochondrial DNA genes 12S rRNA, tRNA(val), and 16S rRNA using maximum parsimony, maximum likelihood, and Bayesian inference methods. The genus Leptobrachium was a well-supported monophyletic group that contained two major clades. One clade had three subclades primarily from disjunct regions including Borneo, Peninsular Malaysia and Java, and Thailand. The Bornean subclade included one species each from the Philippines and Sumatra. The other major clade consisted of two subclades, one from Indochina and the other from southern China (Vibrissaphora). Divergence times estimated an old evolutionary history of each subclade, one that could not be explained by the geohistory of Southeast Asian major landmasses.
Fulltext RNA-Seq analysis provides insights for understanding photoautotrophic polyhydroxyalkanoate production in recombinant Synechocystis Sp

Lau NS, Foong CP, Kurihara Y, Sudesh K, Matsui M

PLoS One, 2014;9(1):e86368.
PMID: 24466058 DOI: 10.1371/journal.pone.0086368

The photosynthetic cyanobacterium, Synechocystis sp. strain 6803, is a potential platform for the production of various chemicals and biofuels. In this study, direct photosynthetic production of a biopolymer, polyhydroxyalkanoate (PHA), in genetically engineered Synechocystis sp. achieved as high as 14 wt%. This is the highest production reported in Synechocystis sp. under photoautotrophic cultivation conditions without the addition of a carbon source. The addition of acetate increased PHA accumulation to 41 wt%, and this value is comparable to the highest production obtained with cyanobacteria. Transcriptome analysis by RNA-seq coupled with real-time PCR was performed to understand the global changes in transcript levels of cells subjected to conditions suitable for photoautotrophic PHA biosynthesis. There was lower expression of most PHA synthesis-related genes in recombinant Synechocystis sp. with higher PHA accumulation suggesting that the concentration of these enzymes is not the limiting factor to achieving high PHA accumulation. In order to cope with the higher PHA production, cells may utilize enhanced photosynthesis to drive the product formation. Results from this study suggest that the total flux of carbon is the possible driving force for the biosynthesis of PHA and the polymerizing enzyme, PHA synthase, is not the only critical factor affecting PHA-synthesis. Knowledge of the regulation or control points of the biopolymer production pathways will facilitate the further use of cyanobacteria for biotechnological applications.
Resurrection of Staurois parvus from S. tuberilinguis from Borneo (Amphibia, Ranidae)

Matsui M, Mohamed M, Shimada T, Sudin A

Zoolog Sci, 2007 Jan;24(1):101-6.
PMID: 17409723

Two forms of Staurois that are differentiated by body size occur parapatrically in the Crocker Range, Sabah, Borneo. Analyses of a total of 1,499 bp of the mitochondrial cytochrome b, 12S rRNA, and 16S rRNA genes revealed that the two forms could be completely split genetically. The two forms could be also clearly differentiated morphologically, not only by snout-vent length but also by the relative sizes of snout, eye, and finger disk. Comparisons of the two forms with all known species of the genus revealed the large and small forms to be S. tuberilinguis and S. parvus, respectively. The latter species has long been synonymized with the former, but we here consider them to represent different species.
Systematics of Calliophis intestinalis with the Resurrection of Calliophis nigrotaeniatus (Elapidae, Serpentes)

Fukuyama I, Vogel G, Matsui M, Eto K, Munir M, Hossman MY, et al.

Zoolog Sci, 2020 Dec;37(6):586-594.
PMID: 33269875 DOI: 10.2108/zs200100

The red-bellied form of Calliophis intestinalis (Laurenti, 1768) sensu lato was originally reported from Pahang, west Malaysia. To determine the taxonomic status of this form, we examined the type specimens of Elaps sumatranus Lidth De Jeude, 1890, Calliophis intestinalis everetti (Boulenger, 1896), and Callophis furcatus var. nigrotaeniatus Peters, 1863. The results indicated that the red-bellied form of C. intestinalis should be named as Calliophis nigrotaeniatus comb. nov., whose valid species status was based on morphological and molecular analyses. We designate a lectotype and redescribe the species, which is genetically close to Calliophis bilineatus (Peters, 1881) from the Philippines, and is clearly distinguishable from other congeners by possessing a pair of gray or dark blue lateral stripes and by being bright red on the ventrum. Elaps sumatranus and C. i. everetti are relegated to subjective junior synonyms of C. nigrotaeniatus.
Taxonomic relationships of Ansonia anotis Inger, Tan, and Yambun, 2001 and Pedostibes maculatus (Mocquard, 1890), with a description of a new genus (Amphibia, Bufonidae)

Matsui M, Yambun P, Sudin A

Zoolog Sci, 2007 Nov;24(11):1159-66.
PMID: 18348617 DOI: 10.2108/zsj.24.1159

Examination of types and recently collected specimens revealed that Ansonia anotis Inger, Tan, and Yambun, 2001 and Pedostibes maculatus (Mocquard, 1890), both described from Kinabalu, Sabah, Malaysia, are hardly differentiated morphologically. Analyses of a total of 2,427 bp of the 12S rRNA, tRNA(val), and 16S mitochondrial rRNA genes revealed that the two species are very close genetically. Thus A. anotis is regarded as conspecific and is synonymized with P. maculatus. Genetically, this species proved to form a lineage distinct from other bufonids from Southeast Asia, including species of Ansonia and Pedostibes. Because the species has also some unique morphological traits different from known bufonid genera, we propose to establish a new genus for Nectophryne maculata Mocquard, 1890.
Fulltext The rubber tree genome shows expansion of gene family associated with rubber biosynthesis

Lau NS, Makita Y, Kawashima M, Taylor TD, Kondo S, Othman AS, et al.

Sci Rep, 2016 06 24;6:28594.
PMID: 27339202 DOI: 10.1038/srep28594

Hevea brasiliensis Muell. Arg, a member of the family Euphorbiaceae, is the sole natural resource exploited for commercial production of high-quality natural rubber. The properties of natural rubber latex are almost irreplaceable by synthetic counterparts for many industrial applications. A paucity of knowledge on the molecular mechanisms of rubber biosynthesis in high yield traits still persists. Here we report the comprehensive genome-wide analysis of the widely planted H. brasiliensis clone, RRIM 600. The genome was assembled based on ~155-fold combined coverage with Illumina and PacBio sequence data and has a total length of 1.55 Gb with 72.5% comprising repetitive DNA sequences. A total of 84,440 high-confidence protein-coding genes were predicted. Comparative genomic analysis revealed strong synteny between H. brasiliensis and other Euphorbiaceae genomes. Our data suggest that H. brasiliensis's capacity to produce high levels of latex can be attributed to the expansion of rubber biosynthesis-related genes in its genome and the high expression of these genes in latex. Using cap analysis gene expression data, we illustrate the tissue-specific transcription profiles of rubber biosynthesis-related genes, revealing alternative means of transcriptional regulation. Our study adds to the understanding of H. brasiliensis biology and provides valuable genomic resources for future agronomic-related improvement of the rubber tree.
Thermo-Responsive Polymer-siRNA Conjugates Enabling Artificial Control of Gene Silencing around Body Temperature

Honda Y, Onodera S, Takemoto H, Harun NFC, Nomoto T, Matsui M, et al.

Pharm Res, 2023 Jan;40(1):157-165.
PMID: 36307662 DOI: 10.1007/s11095-022-03414-8

PURPOSE: Controlling small interfering RNA (siRNA) activity by external stimuli is useful to exert a selective therapeutic effect at the target site. This study aims to develop a technology to control siRNA activity in a thermo-responsive manner, which can be utilized even at temperatures close to body temperature.
METHODS: siRNA was conjugated with a thermo-responsive copolymer that was synthesized by copolymerization of N-isopropylacrylamide (NIPAAm) and hydrophilic N,N-dimethylacrylamide (DMAA) to permit thermally controlled interaction between siRNA and an intracellular gene silencing-related protein by utilizing the coil-to-globule phase transition of the copolymer. The composition of the copolymer was fine-tuned to obtain lower critical solution temperature (LCST) around body temperature, and the phase transition behavior was evaluated. The cellular uptake and gene silencing efficiency of the copolymer-siRNA conjugates were then investigated in cultured cells.
RESULTS: The siRNA conjugated with the copolymer with LCST of 38.0°C exhibited ~ 11.5 nm of the hydrodynamic diameter at 37°C and ~ 9.8 nm of the diameter at 41°C, indicating the coil-globule transition above the LCST. In line with this LCST behavior, its cellular uptake and gene silencing efficiency were enhanced when the temperature was increased from 37°C to 41°C.
CONCLUSION: By fine-tuning the LCST behavior of the copolymer that was conjugated with siRNA, siRNA activity could be controlled in a thermo-responsive manner around the body temperature. This technique may offer a promising approach to induce therapeutic effects of siRNA selectively in the target site even in the in vivo conditions.
Three new species of Leptolalax from Thailand (Amphibia, Anura, Megophryidae)

Matsui M

Zoolog Sci, 2006 Sep;23(9):821-30.
PMID: 17043405

Three new megophryid species, Leptolalax melanoleucus, L. fuliginosus, and L. solus, are described from southwestern and southern Thailand on the bases of acoustic and morphological characteristics. Leptolalax melanoleucus and L. fuliginosus are similar to L. pelodytoides from northern Thailand, but differ from it completely in advertisement call characteristics and ventral color. Leptolalax solus is similar to L. heteropus from peninsular Malaysia, but differs from it by advertisement call, as well as by some body proportions. The distributional pattern of Leptolalax within Thailand is discussed.
Two New Ansonia from Mountains of Borneo (Anura, Bufonidae)

Matsui M, Nishikawa K, Eto K, Hossman MY

Zoolog Sci, 2020 Feb;37(1):91-101.
PMID: 32068378 DOI: 10.2108/zs190078

Two lineages of stream toads in the genus Ansonia from Malaysian Borneo have long been suspected to be specifically distinct on the basis of molecular data. We assessed the taxonomic status of these lineages using morphological and additional genetic data. In mtDNA phylogeny, each lineage-one from Bario, Kelabit Highlands of Sarawak, the other from Mt. Mulu of Sarawak and the Crocker Range of Sabah-is separated from other congeners by large genetic distances, comparable with those observed between heterospecific species in the genus. These lineages are also morphologically distinguishable from other species, and are considered to represent valid, independently evolving species. We therefore describe them as A. kelabitensis sp. nov. and A. kanak sp. nov.
Two new species of fanged frogs from Peninsular Malaysia (Anura: Dicroglossidae)

Matsui M, Belabut DM, Ahmad N

Zootaxa, 2014;3881(1):75-93.
PMID: 25543621 DOI: 10.11646/zootaxa.3881.1.6

Taxonomic status of fanged frogs from the Peninsular Malaysia, previously assigned to Limnonectes kuhlii, is assessed using genetic and morphological approaches. Phylogenetic relationships inferred from sequences of the mitochondrial and nuclear genes revealed that the fanged frogs from the Peninsula form a monophyletic group and are clearly divergent from other species previously, or even now, assigned to L. kuhlii from Mainland Southeast Asia. In both mtDNA and nuDNA phylogeny, the Malay Peninsula clade diverges into two lineages, one from north (Larut Hill, Perak, and Hulu Terengganu, Terengganu) and another from south (Genting Highlands, Pahang, and Gombak, Selangor). These lineages are separated by large genetic distances, comparable with those observed between some other species of L. kuhlii-like frogs. Although the two lineages are very similar morphologically, they are distinguishable in several morphological traits and are considered heterospecific. We therefore describe them as L. utara sp. nov. and L. selatan sp. nov. These new species differ from all other species of kuhlii-like frogs from Mainland Southeast Asia by the surface of tibia, which is densely covered by large warts.
Unusually high genetic diversity in the Bornean Limnonectes kuhlii-like fanged frogs (Anura: Dicroglossidae)

Matsui M, Kuraishi N, Eto K, Hamidy A, Nishikawa K, Shimada T, et al.

Mol Phylogenet Evol, 2016 09;102:305-19.
PMID: 27374495 DOI: 10.1016/j.ympev.2016.06.009

A fanged frog Limnonectes kuhlii was once thought to be wide-ranging in Southeast Asia, but is now confined to its type locality Java through recent phylogenetic studies, which clarified heterospecific status of non-Javanese populations, and monophyly of Bornean populations. However, large genetic differences among Bornean populations suggest occurrence of cryptic species, which we test using dense geographic sampling. We estimated the phylogenetic relationships among samples of Bornean populations together with their putative relatives from the continental Southeast Asia, using 2517bp sequences of the 12S rRNA, tRNA(val), and 16S rRNA of mitochondrial DNA, and 2367bp sequences of the NCX1, POMC, and RAG1 of nuclear genes. In the mtDNA trees, Bornean L. kuhlii-like frogs formed a monophyletic group split into 18 species lineages including L. hikidai, with the deepest phylogenetic split separating L. cintalubang from the remaining species. Almost all of these lineages co-occur geographically, and two to three lineages were found syntopically in each locality. Co-occurrence of more than one lineage may be maintained by differential morphology and microhabitat selection. These syntopic lineages should be regarded as distinct species. Our results clearly indicate that taxonomic revision is urgent to clarify many evolutionary problems of Bornean L. kuhlii-like frogs.
Fulltext Whole genome amplification approach reveals novel polyhydroxyalkanoate synthases (PhaCs) from Japan Trench and Nankai Trough seawater

Foong CP, Lau NS, Deguchi S, Toyofuku T, Taylor TD, Sudesh K, et al.

BMC Microbiol, 2014;14:318.
PMID: 25539583 DOI: 10.1186/s12866-014-0318-z

Special features of the Japanese ocean include its ranges of latitude and depth. This study is the first to examine the diversity of Class I and II PHA synthases (PhaC) in DNA samples from pelagic seawater taken from the Japan Trench and Nankai Trough from a range of depths from 24 m to 5373 m. PhaC is the key enzyme in microorganisms that determines the types of monomer units that are polymerized into polyhydroxyalkanoate (PHA) and thus affects the physicochemical properties of this thermoplastic polymer. Complete putative PhaC sequences were determined via genome walking, and the activities of newly discovered PhaCs were evaluated in a heterologous host.

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