Ichthyofaunal diversity with relation to environmental variables in the snow-fed Tamor River of eastern Nepal

laboratory Bahadur Shrestha, field Asmit Limbu for Abstract: Tamor River in eastern Nepal supports diverse hill stream fishes. From winter, spring, summer, and autumn of 2020, we investigated the ichthyofaunal diversity with environmental variables in the snow-fed Tamor River covering four seasons (winter, spring, summer, and autumn) and field surveys were carried out in January, April, July, and October 2020. We used two cast nets of different sizes, one with a mesh size of 2 cm, 6 m diameter and 6 kg weight and another having 0.5 cm, 3 m diameter and 2 kg weight. In addition, monofilament gill nets with mesh sizes of 6, 8, and 10 were used for fish sampling. A total of 6,373 fish individuals representing 28 species belonging to three orders, seven families, and 16 genera were recorded. One-way permutational multivariate analysis of variance (perMANOVA) on the Non-metric Multidimensional Scaling (NMDS) showed no significant ( P >0.05) difference between winter, spring, and autumn season but summer season showed significant ( P <0.05) difference from winter, spring, and autumn seasons. Furthermore, one-way analysis of variance on redundancy analysis (RDA) vindicated that among the selected parameters, pH, air temperature and total hardness were the influencing factors ( P <0.05) to determine the fish community structure in Tamor River.

The spatial and temporal variations of the fish community structure in rivers and streams of eastern Nepal are poorly understood (Limbu et al. 2019;Adhikari et al. 2021). However, some of the important studies done in eastern Nepal's rivers and streams include (Shrestha 2009;Shrestha 2016;Shah 2016;Subba et al. 2017;Limbu & Prasad 2017Limbu et al. 2018Limbu et al. , 2019. Some outlook of the fisheries and fish ecological studies such as their diversity, spatial & seasonal distribution, and plenty in rivers of Nepal are needed (Mishra & Baniya 2017). To better understand, manage, and conserve (Ngor et al. 2018), and also to know the status (Limbu et al. 2019) of the fisheries, there is an urgent need to update the information on the spatial and temporal fish diversity, community structure and distribution patterns (Ngor et al. 2018).
Thus, the present study aimed to understand relationships among spatio-temporal variation in fish and environmental variables of Tamor River, to reduce the gap in the information and hence dilate the fish diversity profile of Nepal. The present study hypothesized that fish numbers in the Tamor River would be greater during the annual dry season when water current and volume are reduced. We also hypothesized that fish assemblage structure would vary between seasonal variation defined by environmental variables.

Study area
Tamor River lies in eastern Nepal, which begins around Kanchenjunga. The Tamor and the Arun join the Sunkoshi at Tribeni Ghat to form the giant SaptaKoshi which flows through Mahabharat range (Shrestha 2009). It lies in the latitude and longitude co-ordinates of 26.913 o N and 87.157 0 E respectively. The total length of this river is about 190 km with 5,817 km catchment area (Shrestha et al. 2009). The study area has connections with four districts, i.e., Taplejung, Panchthar, Terathum, and Dhankuta. Boulders, pebbles, sand, and gravels were the major characteristic features of this river.

Data collection, Identification and Preservation
Fish sampling was done in winter, spring, summer, and autumn (January, April, July, and October) of 2020. It started on the 15 th and continued to the 30 th of the selected months. We made 28 samples at seven stations, namely, (SA) Kabeli Dovan, (SB) Hewa Dovan, (SC) Nawa Khola Dovan, (SD) Chharuwa Dovan, (SE) Yakchana Ghat, (SF) Mulghat, and (SG) Triveni with fish sampling carried out between 0700 and 1100 h. We used two cast nets of different sizes, one with mesh size of 2 cm, 6 m diameter, and 6 kg weight and another with 0.5 cm mesh size, 3 m diameter, and 2 kg weight. Cast netting was carried out covering 150-200 m (Limbu et al. 2021) across each station and all possible habitats were covered. In addition, monofilament gill nets with mesh sizes of 6, 8, and 10 were used to capture the fish. In each station, nine gill nets were left late in the evening (1700-1800 h) and taken out early in the morning (0600-0700 h) in a sampling distance of 150-200 m.
The collected fish were photographed in a fresh condition and identified in the field and if not, then the voucher specimens were preserved in 10% formalin. After the photography, the remaining samples were returned to their own natural habitat from where they were captured. Fishes were identified with the help of standard literature (Talwar & Jhingran 1991; Jayaram 2010; Shrestha 2019) and other available standard literature. The environmental variables were examined during field visit following the standard methods of American Public Health Association (APHA 2012). Water temperature, dissolved Oxygen (DO), pH, total hardness, water velocity, conductivity, alkalinity, and free carbondioxide (CO). Water temperature ( 0 C) was measured with a digital thermometer by placing it in the water at a depth of 0.3 m. DO (mg/l) was measured by the Winkler titrimetric method. pH was measured using a pH meter 20192 J TT (HI 98107, HANNA Instrument). Total hardness (mg/l) was determined using EDTA titrimetric method. Water velocity (m/s) was measured by the float method with the help of a stop watch, small ball and measuring tape. Titration method was used to measure the alkalinity (mg/l). Free carbon dioxide (mg/l) was measured by the titrimetric method using phenolphthalein as an indicator.

Data analysis
One-way analysis of variance (ANOVA) was used for temperature, pH, dissolved oxygen, hardness and water velocity to calculate the existence of any differences between space and time spectrum. A post-hoc Tukey HSD test was used to test which means were significantly different at a 0.05 level of probability (Spjøtvoll & Stoline 1973). The diversity of the fish assemblage was quantified in the first step of data processing, and then statistical comparison was performed (Appendix I). Fish abundance data were subjected to various diversity indices (Shannon, Simpson, an evenness). All three diversity indices were generated using data from the four seasons (in each season seven samples were made, SA-SG) and seven stations (in each station four samples were made, winter, spring, summer, and autumn), and were used directly in the analysis (Yan et al. 2010) for each fish community sample according to Magurran (1988). Shannon diversity index (Shannon & Weaver 1963) considers both the number of species and the distribution of individuals among species. The Shannon diversity was calculated by following formula: where S is the total number of species and Pi is the relative cover of i th of species. All of the sample (28) was used in the multivariate analysis, and no species or environmental variables were excluded (Appendix I & II). Collected fish abundance and determined environmental variables were used directly in the multivariate analysis (Yan et al. 2010;Hossain et al. 2012;Vieira et al. 2020) One-way permutational multivariate analysis of variance (perMANOVA) (Clarke 1993) was used to test the significant difference among the spatial and temporal scales of the collected fish data. To visualize the major contributing species both to space and time, similarity percentage (SIMPER) (Clarke 1993) analysis was performed.
Detrended correspondence analysis (DCA) (Hill & Gouch 1983) was used to investigate the relationship between fish community structure and environmental variables. The eigen value (0.13) and axis length (1.17) obtained from DCA suggested that the linear model associated with RDA was more applicable. Therefore, a direct multivariate ordination method (Legendre & Legendrem 1998) based on a linear response of species to environmental gradients was applied. In addition, using non-metric multi-dimensional scaling analysis (NMDS), the relationships between assemblages from each station and seasons are graphically depicted (Clarke & Warwick 2001).

J TT
diversity study reported 30 species in Tamor River (Shrestha 2009). The diversity in terms of number (28 species) observed in the present study was nine species greater than Swar & Shrestha (1998), seven species greater than Swar & Upadhaya (1998). It's possible that this is due to the preceding report's limited scope of research. Furthermore, the species diversity may be influenced by fishing gear selectivity and survey efforts. As a result, the current investigation identified a greater number of fish species. But the present study reported two species lower than Shrestha et al. (2009). It might be due to riparian loss, deforestation, river corridor engineering, dams and water diversion, aquatic habitat loss and fragmentation (Dudgeon et al. 2006;Limbu et al. 2021). Ongoing road development, microhydropower generation, poisonous herbicide use, illegal electro-fishing, deforestation, and water diversion are all found to be major threats to the current fish species of Nepal's hillside rivers and streams, according to Limbu et al. (2021) and Adhikari et al. (2021). Garra nasuta, Botia Dario, Schistura rupecula, Schistura multifaciata, and Pseudecheneis crossicauda, according to local fishermen, have suffered a serious drop in population and are not detected in our collection. The most abundant and species-rich order and family, respectively, were Cypriniformes and Cyprinidae. This is  (2007) also stated that the majority of the fish in the river belong to the Cypriniformes order, which includes 2,422 species of freshwater fish.
The present study reported two mahseer fishes (Tor spp.) representing an iconic genus of large-bodied species of the Cyprinidae family. Throughout southern and southeastern Asia, these species are revered for their religious and cultural significance (Pinder et al. 2019). Despite their economic and cultural importance, Tor fishes have seen their riverine habitats damaged by anthropogenic activities such as hydroelectric dam construction and exploitation, putting their survival in jeopardy. Furthermore, conservation attempts have been hampered by the fact that the genus' expertise is primarily bent toward aquaculture with significant knowledge gaps on their taphonomy (Bhatt & Pandit 2016;Pinder et al. 2019). The IUCN Red List has classified Tor putitora as an 'Endangered' species, whereas Tor tor has been classified as 'Data Deficient' (Image 1, 2). Urbanization, poaching, overfishing, and ecological changes in the natural environment's physical, chemical, and biological qualities, according to local fishermen and consent authority, have severely reduced the population of these species in their native habitat. As a result, the conservation of these species is critical.

Diversity status
The Shannon diversity index considers the richness and proportion of each species, while the Evenness and Dominance indices represent the sample's relative number of individuals and the proportion of common species, respectively (Hossain et al. 2012). Highest Shannon diversity index (2.88) was found at station SB and in summer (3.01) whereas lowest (2.63) was found at SE and in winter (2.56). In contrast, highest Simpson dominance index value was observed at station SA, SB, and SC (0.932, 0.93, 0.93) and in summer (0.94) whereas lowest value was observed at, SG (0.908) and in winter (0.90). Similarly, highest value of evenness index was observed at SB (0.69) and in summer (0.65) whereas lowest value of evenness index was observed at SG and in winter (0.62) ( Table 4 & 5). According to Hossain et al. (2012), a high Shannon diversity index is associated with a small number of individuals, whereas a low Shannon diversity index is associated with a large number of individuals. A biodiversity index attempts to classify the diversity of a sample (Magurran 1988) and is easily affected by the number of specimens, sampling size, and ecological factors (Leonard et al. 2006).

Fish community structure vs. environmental variables
The result obtained after the redundancy analysis