Study on the Amynthas (Kinberg, 1867) earthworm (Megascolecidae: Oligochaeta) diversity through DNA barcoding from Northeast India

Abstract

To study species diversity of Amynthas (Kinberg, 1867) earthworm from Northeast India using mitochondrial CO1 gene by DNA sequencing. Earthworms were collected from different habitats of Northeast states of India. Morphological identification upto the genus was performed. Genomic DNA was extracted by CTAB method followed by PCR amplification of mtDNA CO1 gene. The amplicons obtained were sequenced by AB3500 Genetic Analyzers. A total of 2355 Amynthas species were collected from 361 different sampling sites from various habitats of north east India. Sequencing of mtDNA CO1 marker followed by matching with BOLD system database confirmed the presence of ten different species of Amynthas earthworms such as Amynthas alexandri, A. corticis, A.diffringens, A. gracilis, A. hawayanus, A. hupeiensis, A. incongruus, A. morrisi, A. papulosus and A. robustus. The DNA sequence was submitted to NCBI GenBank. Among them, A. corticis was the most widely distributed species found in six states, followed by A. diffringens found in five states. A. incongruus was confined to Mizoram only and is a new record for the northeast region of India. Four species reported from Nagaland (A. alexandri, A. diffringens, A. gracilis, A. morrisi) are new records for the state, A. diffringens was new to Meghalaya, and A. hawayanus, A. incongruus and A. populosus were new records for the state of Mizoram. A. hupeiensis was a new record for Assam. Based on the molecular analysis of mtDNA CO1 sequences, we have reported five newly recorded species for Nagaland, three for Mizoram and one each for Meghalaya and Assam states. Our results indicated the species richness of Amynthas earthworms. Many more species of Amynthas earthworms are yet to be contributed towards the species richness from Northeast India.