Molecular Identification, DNA Barcoding and Determination of Genetic Relationship of Killifish (Aplocheilus spp.) Present in Aththanagalu Oya, Sri Lanka

Abstract

Accurate identification of species is of paramount importance in conserving fish diversity due to practical limitations of recognizing closely related species based on morphological features. DNA barcoding is a rapid and reliable molecular tool widely used to identify organisms at species level. The present study was conducted to identify and determine genetic relationships of the Aplocheilus spp. present in Aththanagalu river basin, Sri Lanka using molecular tools. From 68 sampling sites along the river, Aplochielus spp. were collected and identified using morphological characteristics. Meristic characters of each fish sample were subjected to truss analysis by preparing principal component analysis (PCA) using PRIMER 5 software. Genomic DNA extracted from fresh muscle tissues of ten samples of killifish was subjected PCR targeting the COXI gene in mitochondrial genome using a universal primer pair, FishF1/FishR1. Ten PCR amplicons of the expected size (approximately 650 bp) were subjected to DNA sequencing. Eight successfully-sequenced samples were subjected to DNA homology search using BLAST, NCBI and phylogenetic analysis using Geneious 7.1.3 and MEGA 6 software. According to the results of morphological characteristics, 180 A. dayi and 143 A. parvus species were identified. Truss analysis based on morphological characteristics did not differentiate A. dayi and A. Parvus into separate clusters. Based on DNA homology search results, two A. dayi samples identified morphologically were highly homologous to A. werneri (with 83-94% identity and 75-77% query cover). Two other A. dayi samples and four A. parvus samples, identified morphologically were best matched with A. blockii voucher sample (with 90-97% identity and 80-98% query cover). By phylogenetic analysis, the two A. dayi samples which showed the highest DNA homology with A. werneri formed a separate cluster but indicated a closer relationship with A. werneri. The rest of the A. dayi and A. parvus samples which were highly homologous with A. blockii were clustered together and formed a monophyletic clade but showed a closer relationship to A. blockii. The findings revealed the ability of identifying A. parvus and A. blockii which is difficult to be differentiated by morphological characters by DNA barcoding targeting the COXI gene. Exact identification of A. dayi is not possible due to unavailability of sequence information of A. dayi. Closer genetic relationships of some A. dayi with A. werneri indicate possible hybrids due to interbreeding.
Keywords: DNA sequencing, Cytochrome c oxidase I gene, Phylogenetic tree, Polymerase chain reaction, Species identification