Achariya Rangsiruji

Using Phylogenetic and Coalescent Methods to Understand the Species Diversity in the Cladia aggregata Complex (Ascomycota, Lecanorales)

The Cladia aggregata complex is one of the phenotypically most variable groups in lichenized fungi, making species determination difficult and resulting in different classifications accepting between one to eight species. Multi-locus DNA sequence data provide an avenue to test species delimitation scenarios using genealogical and coalescent methods, employing gene and species trees. Here we tested species delimitation in the complex using molecular data of four loci (nuITS and IGS rDNA, protein-coding GAPDH and Mcm-7), including 474 newly generated sequences. Using a combination of ML and Bayesian gene tree topologies, species tree inferences, coalescent-based species delimitation, and examination of phenotypic variation we assessed the circumscription of lineages. We propose that results from our analyses support a 12 species delimitation scenario, suggesting that there is a high level of species diversity in the complex. Morphological and chemical characters often do not characterize lineages but show some degree of plasticity within at least some of the clades. However, clades can often be characterized by a combination of several phenotypical characters. In contrast to the amount of homoplasy in the morphological characters, the data set exhibits some geographical patterns with putative species having distribution patterns, such as austral, Australasian or being endemic to Australia, New Zealand or Tasmania.

Phenotypic disparity and adaptive radiation in the genus Cladia (Lecanorales, Ascomycota)

Phylogenetic relationships of the genera Cladia, Heterodea and Ramalinora were reconstructed using a combined dataset of ribosomal nuclear ITS and LSU and mitochondrial SSU, and protein-coding Mcm7 DNA sequences. Maximum likelihood and Bayesian analyses strongly supported a monophyletic group in which the species of the foliose genus Heterodea and the crustose genus Ramalinora were nested within the fruticose genus Cladia. Alternative hypothesis testing rejected an independent status of Ramalinora. We tested the hypothesis that an adaptive radiation led to the morphological disparity found in the Cladia clade. Gamma-statistics indicated a significantly disproportional clustering of origins of extant lineages at the base of the Cladia clade and lineage-through-time plots were also consistent with the hypothesis of an adaptive radiation at the base of the Cladia clade. Ancestral-range reconstructions supported an origin of Cladia and the three major lineages within Cladia in Australia. On the basis of these results, we propose an evolutionary hypothesis for the genus. The results suggest that processes of adaptive radiation of the ancestor of Cladia in Australia led to the morphological disparity in the extant taxa, and that the broad distribution of some extant species is due to subsequent long-distance dispersal.

Isolation and characterisation of a novel Podoviridae-phage infecting Weissella cibaria N 22 from Nham, a Thai fermented pork sausage

A novel Podoviridae lactic acid bacteria (LAB) phage from Nham, a Thai fermented pork sausage, is reported. From a total of 36 samples, 41 isolates of LAB were obtained and employed as hosts for the isolation of phages. From these LAB, only one phage, designated Φ 22, was isolated. The lactic acid bacterial isolate named N 22, sensitive to phage Φ 22 infection was identified by an API 50 CHL kit and N 22s complete sequence of the 16S rDNA sequence. BLASTN analysis of the 16S rDNA sequence revealed a 99% similarity to the 16S rDNA sequence of Weissella cibaria in the GenBank database. Electron micrographs indicated that the phage head was icosahedral with head size and tail length of 92 × 50 nm and 27 nm, respectively. On the basis of the morphology, this phage belongs to the family Podoviridae. Host-range determination revealed that the phage Φ 22 was not capable of infecting the other 40 isolates of LAB and referenced Weissella strains used. A one-step growth experiment showed that the latent period and burst size were estimated at 110 min and 55 phage particles/infected cell, respectively. Furthermore, the phage was infective over a wide range of pH (pH 5.0-8.0) and the D time of Φ 22 was calculated as 88 s at 70 °C and 15s at 80 °C. Phage titers decreased below the detection limit (20 PFU/ml) after heating for more than 60s at 80 °C, or 20s at 90 °C or less than 10s at 100 °C. The results from the study of Nham revealed that Φ 22 was active against the potential starter culture (W. cibaria N 22) for Nham fermentation. Phage infection could adversely affect the fermentation process of Nham by delaying acidification when using W. cibaria N 22 as a starter. However, the results from a sensory test revealed that the panelists did not detect any defects in the final products. This is the first report on the isolation of W. cibaria phage.

An online database on freshwater fish diversity and distribution in Mainland Southeast Asia

Mainland Southeast Asia, the region that includes Cambodia, Laos, Thailand, Vietnam and Peninsular Malaysia, is known for its high diversity of freshwater fishes (e.g., Myers et al. 2000; Dudgeon 2005; Kang et al. 2009). Recently, however, intensive exploitation pressures have been threatening this biodiversity. While some studies on fish fauna of the region have been made (e.g., Taki 1974; Rainboth 1996; Kottelat 2000; Vidthayanon and Premcharoen 2002), most have been of limited duration and geographical range, and the full extent of the effect of this exploitation on the fish diversity is not fully understood. The main obstacles standing in the way of a proper understanding of the threats to this biodiversity and determining the means to alleviate them have been hindered by several factors: one is a general shortage of trained scientists in the region proficient in fish taxonomy; another is that exchanges of the results of the taxonomical studies done by scientists in the region are few and far between. And finally there is a general lack of awareness of the significance of species diversity in ecosystems. Beginning in 2007, the Nagao Natural Environment Foundation (NEF), from Japan, has been working to improve this situation in the Mekong-Chao Phraya region, the results of which are presented in a new online database, ‘‘Fishes of Mainland Southeast Asia (FiMSEA)’’ (Fig. 1; URL: http://ffish.asia). Since the outset of the project, the NEF has worked in collaboration with a number of counterpart institutions in the region, namely: Can Tho University (Vietnam), The Inland Fisheries Research and Development Institute

Immunolocalization of allatostatin-like neuropeptides and their putative receptor in eyestalks of the tiger prawn, Penaeus monodon

Allatostatin (AST)-like immunoreactivity (IR) was localized in the eyestalk of Penaeus monodon by immunohistochemistry using four anti-AST antibodies. Depending on the antisera, AST-like immunoreactivity was detected in neuronal bodies of the lamina ganglionalis, cell bodies anterior to the medulla externa and cell bodies on the anterior and posterior of the medulla terminalis. Neuronal processes in neuropiles of the medulla externa, medulla terminalis, sinus gland and nerve fibers in the optic nerve were also recognized. No IR in cell bodies or in nerve fibers was found in the medulla interna. Strong AST-like immunoreactivity was found in hundreds of cells of the X organ. The localization of AST-like peptides suggests that they function as neurotransmitters and/or neuromodulators. Antiserum to the Drosophila AST receptor (Dar-2) recognized a single protein in P. monodon eyestalk protein extracts that was identical in size to that found in Drosophila protein extracts. Using this antiserum the putative P. monodon AST receptor was localized to the sinus gland in both juvenile and adult eyestalks. To our knowledge this is the first demonstration of a neuropeptide receptor localized to the crustacean sinus gland. This suggests that ASTs may function directly on the sinus gland as a neuromodulator. In juvenile eyestalks, the putative AST receptor was also localized to neuronal X organ cells of the medulla terminalis in males but not in females. The significance of this sex-specific receptor localization is unclear but emphasizes that ASTs function within the nervous system of the eyestalk.

Molecular identification of poisonous mushrooms using nuclear ITS region and peptide toxins: a retrospective study on fatal cases in Thailand

Cases of mushroom poisoning in Thailand have increased annually. During 2008 to 2014, the cases reported to the National Institute of Health included 57 deaths; at least 15 died after ingestion of amanitas, the most common lethal wild mushrooms inhabited. Hence, the aims of this study were to identify mushroom samples from nine clinically reported cases during the 7-year study period based on nuclear ITS sequence data and diagnose lethal peptide toxins using a reversed phase LC-MS method. Nucleotide similarity was identified using BLAST search of the NCBI database and the Barcode of Life Database (BOLD). Clade characterization was performed by maximum likelihood and Bayesian phylogenetic approaches. Based on BLAST and BOLD reference databases our results yielded high nucleotide similarities of poisonous mushroom samples to A. exitialis and A. fuliginea. Detailed phylogenetic analyses showed that all mushroom samples fall into their current classification. Detection of the peptide toxins revealed the presence of amatoxins and phallotoxins in A. exitialis and A. fuliginea. In addition, toxic α-amanitin was identified in a new provisional species, Amanita sp.1, with the highest toxin quantity. Molecular identification confirmed that the mushrooms ingested by the patients were members of the lethal amanitas in the sections Amanita and Phalloideae. In Thailand, the presence of A. exitialis was reported here for the first time and all three poisonous mushroom species provided new and informative data for clinical studies.

Diversity and phylogenetic survey of cyanobacterial lichens (Collematineae, Ascomycota) in mangrove forests of eastern Thailand

Abstract Peltigeralean lichenized fungi were surveyed in the mangroves of Trat Province at the eastern coast of Thailand. Eleven species were found belonging to the genera Coccocarpia, Lepidocollema, Leptogium and Physma. Among them, the genus Coccocarpia was most abundant and diverse. Lepidocollema wainioi was also recorded here from Thailand for the first time. Based on the current classification of the peltigeralean fungi the phylogenetic placement of the suborder Collematineae was evaluated using a combined dataset of nuclear ITS and mitochondrial SSU rDNA sequences. Phylogenetic analyses using maximum likelihood and Bayesian approaches revealed issues with the species delimitation in the genera Coccocarpia and Lepidocollema, where the data indicated non-monophyletic lineages. Additional studies with extended sampling will be necessary to address species boundaries in these lichens.