Dibesh Bikram Karmacharya , Prajwol Manandhar, Sulochana Manandhar, Adarsh M Sherchan, Jyoti Joshi, Hemanta K Chaudhary, Bimala Dhakal, Rajindra Napit, Bishwo Shrestha, Pragun G Rajbhandari, Ajit Poudel , Saman M Pradhan, Ashok Chaudhary, Dhiraj Puri, Seily Shrestha , Smriti Khadka , Deepak Risal, Nilu Basnyat, Nikita Pradhan, Jessie A Moravek , Pablo Cardinale, David Philipp, Julie E Claussen, David Gillette , Sushan M Shakya, Genuine Prajapati, Laura Bertola 

Although over 180 freshwater fish species have been reported from Nepal, little is known of their ecology and distribution. This information is needed because their diversity may be threatened by developments like hydropower constructions. We conducted Nepal’s first environmental DNA (eDNA) based fish biodiversity assessment in two major river systems- Karnali River (KR), which is still pristine and Trishuli River (TR) with numerous hydropower plants. The eDNA was concentrated by filtering (0.45 μm pore size) two liters of water collected at different sampling points in each study site. A total of 224 eDNA samples (KR=162 and TR= 62) were collected, from which fish species was identified by 12S rRNA metabarcording approach utilizing Illumina sequencing platform. Alpha and beta diversity of species between two sites were compared. Also, in KR site, fish (N=795) were caught, and identified using COI based DNA barcoding- building Nepal’s first fish DNA reference database. Field sampling identified 21 species through morphology and DNA barcoding, where Barilius spp. and Schizothorax spp. were the most abundant. From 244 eDNA samples, 24 Operational Taxonomic Units (OTUs) were identified in TR and 46 in KR with 19 being common to both sites, 27 being unique in KR, and five in TR only. Most fishes were of Cypriniformes and Siluriformes orders, with Barilius spp. and Schizothorax spp. being the most abundant. Long distance migratory fish (Tor spp, Neolissochilus hexagonolepis) and non-native fish (Cyprinus carpio, Hypophthalmichthys molitrix, Gymnocorymbuster netzi, Ctenopharyngo donidella, Clarias gariepinus) were identified in eDNA samples as well. Alpha diversity in TR was significantly lower than in KR. High beta diversity between the two sites indicated low similarity in fish diversity between the TR and KR. This study demonstrated the utility of eDNA as a non-invasive technique for biodiversity assessment which is particularly useful in areas like Nepal with scarce data on fish species distribution.