Arunachal Pradesh, one of the best tropical rain forest areas within Eastern Himalayan biodiversity hotspot is expected to have a rich earthworm diversity. Therefore, the present study was undertaken to document the diversity of earthworms in Arunachal Pradesh from field collection and existing literature. Earthworm specimens were collected by random sampling method by digging and hand sorting method from different habitat in Arunachal Pradesh, northeast India. Conventional methods of identification based on morpho-anatomical characters was performed. Molecular characterization, the genomic DNA was extracted followed by PCR amplification by mtDNA CO1 gene. The PCR product obtained was subjected to the Sanger sequencing by AB3500 Genetic Analyzers (ThermoFisher). Twelve earthworm taxa were identified using conventional and molecular methods of identification under eight families. Out of which, four families were previously recorded and two families viz., Rhinodrilidae and Eudrilidae are new to the existing record. Out of the 12 earthworm species, seven species are new record from the state, such as Amynthas corticis, A. gracilis, Bimastos parvus, Dichogaster modigliani, Eudrilus eugeniae, Octochaetona beatrix and Pontoscolex corethrurus. Among the identified species, A. gracilis was the most widely distributed species followed by A. corticis and Drawida nepalensis. With these seven new additions, the valid species of earthworms of Arunachal Pradesh comes to 46 species. The CO1 gene sequence of newly recorded species from Arunachal Pradesh was submitted to NCBI GenBank. Our findings enriched the diversity of earthworm species in Arunachal Pradesh by contributing two new families i.e. Rhinodrilidae and Eudrilidae and seven new species record such as Amynthas corticis, A. gracilis, Bimastos parvus, Dichogaster modigliani, Eudrilus eugeniae, Octochaetona beatrix and Pontoscolex corethrurus. Thus, the total earthworm diversity in Arunachal Pradesh has become 46, the second-highest record of all northeastern Indian states with a high potential of the new addition.

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.