Molecular taxonomy and phylogenetic analysis of freshwater fish fauna within Malaysia with special emphasis on the Cyprinidae

Malaysian freshwater fish are significant economically and ecologically, yet the species making up this highly diverse fauna are going extinct at a greater rate than species can be identified and described using conventional systematic approaches. DNA barcoding based on nucleotide sequence of the mitochondrial gene cytochrome c oxidase subunit 1 (COI), is making a significant contribution to the identification and discovery of fish species worldwide, but has not been applied on any significant scale to Malaysian freshwater fish fauna. The first part of this thesis is devoted to assess the efficacy of DNA barcoding approach in identifying Malaysian freshwater fish species. A total of 181 COI sequences from 29 putative fish species were generated and compared with sequences from reference samples obtained in this study and records on BOLD and GenBank databases. Overall, the results show a moderate level of success of COI-based barcoding as only 132 of 181 COI sequences (73%) were assigned to those expected from field-based identification. However, the current study reveals that the relatively low success rate of COI barcoding is largely due to field based misidentification, presence of cryptic species and/or lack of appropriate reference sequences rather than any failure of the barcoding approach itself. The second part of the study focused on investigation of the phylogenetic relationships within local Cyprinidae family, which is the largest and most widely distributed family of Malaysian freshwater fish. The phylogenetic relationships and subfamily classifications within 16 Malaysian Cyprinidae species were investigated using concatenated sequence of four mitochondrial genes (COI, 16S rRNA, D-loop and Cyt-b). As 13 of 16 Malaysian Cyprinidae species in this study were from the subfamily Barbinae, the resultant phylogenetic trees based on maximum likelihood (ML) and Bayesian analyses show that this subfamily is a polyphyletic group. Further, phylogenetic relationships of the Malaysian Cyprinidae with 23 Cyprinidae species from Vietnam were studied based on concatenated sequence of three mitochondrial genes (16S rRNA, D-loop and Cyt-b). This comparative evaluation confirms the unreliability of the current subfamily concepts within the family, and reveals several taxonomic anomalies between the two fish faunas that will require further investigation using more extensive taxon, gene and geographic sampling.