Identification of selected world’s smallest fish species of the genus Paedocypris (cyprinidae) and population genetics of the Paedocypris progenetica through molecular techniques from Malaysia / Norjasmin Hussin

The accurate identification techniques of fish species are crucial to biodiversity conservation, fisheries management, and international aquarium trade. Globally, the genus Paedocypris is the smallest fish species and was declared as near threatened by the IUCN (2020) due to anthropogenic activities, including logging, urbanisation, and industrialization. Thus, this research aimed to identify the Paedocypris specimens in Malaysia into distinct species through conventional and molecular approaches as well as to elucidate the population structure of selected Paedocypris spp. A total of 16 morphometric parameters and six chromatophore patterns of Paedocypris specimens collected from three peat swamps in Malaysia (Selangor, Perak, and Sarawak) were measured on 66 out of 103 specimens. The analyses involved for morphological study were univariate analysis of variance (ANOVA) and multivariate discriminant function analysis (DFA). The species identification of specimens was further analysed using the cytochrome c oxidase I (COI) gene that performed on 51 out of the 66 Paedocypris specimens using standard DNA barcoding, phylogenetic analyses, and divergence time estimation of mitochondrial COI gene. Later, P. progenetica was selected and assessed using population genetics with newly developed D-loop primer and COI gene as it was detected in two out of three locations in Malaysia. The morphological studies have successfully classified and identified two Paedocypris species as Paedocypris progenetica and Paedocypris micromegethes by using DFA and chromatophore patterns. Nine out of 16 significant characteristics were found as the highest characters loading in Function 1 and Function 2 are PDL (predorsal length) and HL (head length) with small wilk’s lambda (0.185) in DFA. Further confirmation on Paedocypris species identification using DNA barcoding approach applying Neighbour-Joining and Maximum Likelihood methods, recorded high bootstrap values (n > 95%) and highly congruent with the morphological findings revealing three clades within Paedocypris species namely P. progenetica and P. micromegethes known to occur in Malaysia. A barcoding gap of 3% was apparent in the whole data set resulting in conspecies distances ranging from 4.93% to 5.44%. By contrast, congeneric distances varied between 4.28% and 7.71%, showing genetic variation. Based on divergence time estimation, the P. carbunculus was diverged earlier compared to P. micromegethes followed by P. progenetica. The divergence time between P. carbunculus from Sumatera showed the earliest divergence during the middle of Jurassic, followed by P. progenetica (24.5 Mya) and P. micromegethes (7.5 Mya) during the early Oligocene and Miocene. The population structure assessment on P. progenetica found they were divided into two clades in each marker namely Clade I (Perak) and II (Selangor) which found moderate haplotype diversity (h=0.668) and nucleotide diversity (nD=0.220) from COI analysis showing moderate genetic variation between Selangor and Perak. Thus, this reflects the possibility of past geological events and further supported by D-loop marker analysis where moderate genetic variation was also seen (h=0.628, nD=0.597). The population structure was supported with a high value of fixation index (Fst) between two populations for both markers (COI=1.30262, D-loop=0.70694). The mtDNA markers showed past population expansion through Tajima’s, Fu Fs’, and Bayesian skyline plot during the Oligocene. Therefore, the findings of morphological and molecular analyses successfully identified P. progenetica and P. micromegethes in Peninsular Malaysia and Sarawak respectively and may contribute to facilitate the conservation management of P. progenetica in Malaysia.