Metabarcoding of Fish Larvae in the Merbok River Reveals Species Diversity and Distribution Along its Mangrove Environment

Abstract

The Merbok River (north-west of Peninsular Malaysia) is a mangrove estuary that provides habitat for over 100 species of fish, which are economically and ecologically important. Threats such as habitat loss and overfishing are becoming a great concern for fisheries conservation and management. The identification of larval fish in this estuarine system is important to complement information on the adults. This is because the data could inform the spawning behaviour, reproductive biology, selection of nursery grounds and migration route of fish. Such information is invaluable for fisheries and aquatic environmental monitoring, and thus for their conservation and management. However, identifying fish larvae is a challenging task based only on morphology and even traditional DNA barcoding. To address this, DNA metabarcoding was utilised to detect the diversity of fish in the Merbok River. To complete the study, the fish larvae were collected at six sampling sites of the river. The extracted larval DNA was amplified for the Cytochrome Oxidase subunit 1 (COI) and 12S ribosomal RNA (12S rRNA) genes based on the metabarcoding approach using shotgun sequencing on the next-generation sequencing (NGS) Illumina MiSeq platform. Eighty-nine species from 65 genera and 41 families were detected, with Oryzias javanicus, Oryzias dancena, Lutjanus argentimaculatus and Lutjanus malabaricus among the most common species. The lower diversity observed from previous morphological studies is suggested to be mainly due to seasonal variation over the sampling period between the two methods and limited 12S rRNA sequences in current databases. The metabarcode data and a validation Sanger sequencing step using 15 species-specific primer pairs detected three species in common: Oryzias javanicus, Decapterus maruadsi and Pennahia macrocephalus. Several discrepancies observed between the two molecular approaches could be attributed to contaminants during sampling and DNA extraction, which could mask the presence of target species, especially when DNA from the contaminants is more abundant than the target organisms. In conclusion, this rapid and cost-effective identification method using DNA metabarcoding allowed the detection of numerous fish species from bulk larval samples in the Merbok River. This method can be applied to other sites and other organisms of interest.

Keywords: DNA metabarcoding; Fish larvae; Mangrove estuary; Merbok River; Next-generation sequencing.

Fig. 1.

Merbok River with six sampling stations, divided into three zones: upper [St1: Lalang River (5°42'00.1"N 100°30'17.2"E), St2: Semeling River (5°41'14.0"N 100°28'41.6"E)], middle [St3.1: Keluang River (5°39'17.8"N 100°26'45.0"E) and St3.2: Teluk Wang River (5°38'00.9"N 100°25'56.6"E)] and lower [St4.1: Gelam River (5°38'38.4"N 100°25'00.0"E) and St4.2: Terus River (5°38'11.2"N 100°23'52.4"E)].

Fig. 2.

The relative abundance of fish larvae species detected among sampling sites (St1, St2, St3 and ST4) based on the number of scaffolds reads of the COI and 12S rRNA genes.

Fig. 3.

Abundances of non-target DNA reads.

Fig. 4.

Bray-Curtis similarity plotted by two-dimensional non-metric multidimensional scaling (NMDS) showing the similarity in larval fish species diversity among all four stations of the Merbok River identified using the COI and 12S rRNA genes.