Phylogeographic structure in mitochondrial DNA of a South-east Asian freshwater fish, Hemibagrus nemurus (Siluroidei; Bagridae) and Pleistocene sea-level changes on the Sunda shelf

We postulated that the biogeographical history of South-east Asia contributed to extensive admixture during Pleistocene low sea levels of genetic groups of an obligate freshwater fish (the river catfish, Hemibagrus nemurus) isolated during periods of high sea levels. During Pleistocene glacial maxima, the sea level was lower than at present and the islands of the Sunda shelf (Sumatra, Borneo and Java) and the Asian mainland were connected by lowlands traversed by rivers. Restriction fragment length polymorphisms in mitochondrial DNA were documented for 140 putative H. nemurus analysed from 13 sampling sites resulting in the definition of 35 haplotypes. The high level of haplotype differentiation (mean P × 100 = 2.22, SD = 1.33) indicates that the subdivision of the ancestral H. netnurus group was extensive and probably occurred early in the Pleistocene. The occurrence of some genetically divergent groups of the H. netnurus complex occurring in sympatry in widely separated locations supports the proposition that low sea levels aided the dispersion and mingling of genetic groups. Based on both genetic and morphological evidence, the main H. nemurus line gave rise to three regional groups: (1) a morphologically distinct ‘Indochinese’ group composed of two mtDNA clades overlapping in east peninsular Malaysia; (2) a ‘Sundaic’ group composed of various lineages of differing morphology and genetic identity; (3) a genetically distinct ‘Sarawak’ group in west Borneo, similar in morphology to the ‘Sundaic’ and ‘Indochinese’ groups, but including a small, golden colour morph as a distinct dade. The morphologically similar Sundaic forms from west Java, Sumatra and west Borneo show some degree of genetic divergence, but their phylogenetic relationships are poorly resolved. The most genetically and morphologically distinct Sundaic dade, assigned to H. hoevenii, colonized the Kapuas river (west Borneo), east Sumatra and south peninsular Malaysia. Contrary to our original hypothesis and present biogeographical theory, little exchange of genetic groups has apparently occurred between the mainland and the Sunda Islands during recent glaciations.     

Quantitative aspects of anin situ hybridization procedure for detecting mRNAs in cells using 96-well microplates

The universal quantitation of the DNA hybridization reaction has been a goal sought by many researchers. Part of this search has been the need to develop a rapid, sensitive, easy-to-perform, and quantitative method to measure the abundance of specific mRNAs directly within cells. Conventionally mRNA detection can be done by advanced quantitativein situ hybridization (ISH) using either image analysis or fluorescencein situ hybridization (FISH), or indirectly by extraction of mRNA from cells or tissue and using Northern blot or quantitative polymerase chain reaction (PCR). We examined the quantitative nature of probe binding to intracellular mRNA in a sensitive and easy-to-use nonisotopic method of ISH previously developed in our laboratories. The method is applicable to isolated primary cells or cells in culture. The procedural details are very simple, with cells being centrifuged into 96-well microplates, fixed with formalin, and pretreated with Triton X-100 and Nonidet P-40 before photobiotin-labeled cDNA probes are applied. Biotin from the hybridization of probe to target is detected using multiple applications of streptavidin and biotinylated alkaline phosphatase and visualized by thep-nitrophenyl phosphate conversion method. The quantitative parameters of the ISH procedure were determined by measuring the levels of expression of erythropoietin (EPO) mRNA and its translated protein in transfected COS-7 cells. There is a log-linear relationship between the levels of signal obtained in the ISH reaction in 96-well microplates and the EPO protein levels measured by enzyme-linked immunosorbent assay (ELISA). This demonstrated relationship is important in the standardization and use of these procedures to measure quantitatively mRNAs within cells.