Morphometric and Molecular Assessment of Tor putitora and Neolissochilus hexagonolepis Population in Jia Bhoroli river Ecotone Zone, Assam

The concept of an ecotone was first introduced by Clements in 1905 as a way to describe the boundary between two different biological communities. An ecotone is defined as “a zone of transition between adjacent ecological systems, with a set of characteristics determined by the strength of interaction between the adjacent ecological systems” (Holland, 1988). It represents a region characterized by a relatively swift transition, giving rise to a narrow ecological region that separates two distinct and relatively uniform community types (van der Maarel, 1990). The River Jia Bhoroli originates in the upper Himalayan range of Arunachal Pradesh at an elevation of 5400 m msl where it is known as Kameng, It then flows through the Sonitpur district of Assam before joining the mighty River Brahmaputra at Tezpur (elevation 69 m msl). The middle reaches of the Jia Bharali, from Bhalukpung to Balipara in Sonitpur district, provide excellent opportunities for sport fishing and rafting. Bhalukpung area in the River Jia Bhoroli is an ideal example of an ecotone, as it shifts from resembling a foothill river ecosystem to a river floodplain ecosystem. This transition results in a high level of fish diversity, due to the unique combination of the two contrasting ecosystems (Naiman et al., 2005).
       
Mahseer, a globally renowned sport and table fish, is considered as the National Heritage of India (Nautiyal, 2014). They are flow-loving cyprinids (Order: Cypriniformes, Family: Cyprinidae) characterized by large scales and can grow to be large-bodied carps with a maximum recorded weight of 54 kg. They are taxonomically partitioned into the genera Naziritor, Neolissochilus and Tor (Kottelat, 2013; Froese and Pauly, 2018; Eschmeyer et al., 2017). Only species of the genus Tor are considered as true mahseer (Desai, 2003) and this genus includes 16 valid species. These species are considered highly potamodromous, with upstream spawning migrations often over considerable distances being necessary for successful reproduction (Nautiyal et al., 2001 and 2008).
       
Golden Mahseer, Tor putitora, is a species of fish that is widely distributed in the Himalayan region and across South and Southeast Asia, from Afghanistan, Pakistan, India, Nepal, Bangladesh, Bhutan, Myanmar, Sri Lanka, Western Iran to Thailand. This fish is restricted to montane and sub-montane streams and rivers in the Indus, Ganga and Brahmaputra river basins and is found at an elevation of 70-1891 m above sea level and latitude of 8°N to 36°N. The species has been declared Endangered (EN) by the International Union for Conservation of Nature (IUCN) Red List due to severe threat from overfishing, loss of habitat, decline in quality of habitat resulting in loss of breeding grounds and from other anthropogenic effects such as construction of dams leading to blocking their migrations and affecting their breeding (Jha et al., 2018).
       
Chocolate mahseer, Neolissochilus hexagonolepis, is a freshwater fish that inhabits hill streams and rivers in India, Bangladesh, Nepal, Myanmar, Indonesia and China. In India, it is found in water bodies located at 50-2000 m above sea level in states such as Arunachal Pradesh, Assam, Bihar, Jharkhand, Meghalaya, Nagaland, Uttar Pradesh and West Bengal. It is an omnivorous, opportunistic feeder that feeds on green filamentous algae, aquatic insects, small fish, weeds and mollusks (Majhi et al., 2013). The species has been declared as Near Threatened (NT) as per IUCN Red List due to factors such as habitat loss, pollution and increasing water temperature in its inhabiting area (Arunachalam, 2010).
       
Previous researchers (Samways and Stewart, 1997; Attril and Rundle, 2002) have emphasized the function of ecotones in determining the biotic makeup of ecosystems, but there is a dearth of information regarding the role of ecotones in conserving threatened species. The lack of specific studies on Mahseer populations especially for T. putitora and N. hexagonolepis in ecotone zones makes the current study important and first of its kind. It aims to generate new information regarding the relationship between this fishes populations and their foothill-floodplain river interfaces ecotone environment in the Jia Bhoroli river. The results of this study will contribute to a better understanding of the ecology of this species and facilitate the development of informed conservation and management decisions.

Study area and sampling
 
During the present study, fish samples were collected from the wild stocks in Bhalukpong area (27°0'56"N 92°38'46"E) of the Jia Bhoroli river which falls near the Nameri National Park (Fig 1). The samples were collected with the help of local fishermen using various fishing methods (gill nets, angling and cast nets) from January to December 2021, following the guidelines of the National Biodiversity Authority of India (Biological Diversity Act, 2002). The fish specimens were identified using standard taxonomic keys (Vishwanath and Nebeshwar, 2009; Kottelat, 2013). Fish that weighed less than 140 g were counted, photographed and then released back into the water, while the remaining samples were brought to the laboratory for further morphometric, meristic and molecular analysis. These samples were deposited in the Fish Museum of the Department of Aquatic Environment Management, College of Fisheries, Assam Agricultural University, Raha, Nagaon, Assam (COFAAUMU 01-55). The population abundance was calculated as:
 


 
Water quality parameters
 
Water samples were collected from Bhalukpong of River Jia Bhoroli from January to December, 2021. Some of the physical parameters like depth and surface water temperature (Mercury thermometer) were determined on the spot. Other parameters like, dissolved oxygen, pH, total alkalinity, total hardness of the water samples was analyzed in the laboratory following APHA (2017).
 
DNA extraction and barcoding
 
Further, for molecular analysis, fin and muscle tissue samples were dissected from the mahseer specimens and stored in 100% ethanol. The extraction of total DNA from the ethanol-preserved tissue was carried out according to the standard DNA barcoding methods for fish as described by (Ward et al., 2005). A 655 bp region from the 5' end of the COI gene was amplified using the primers (Table 1) outlined by Ward et al., (2005) through a Polymerase Chain Reaction (PCR) with a total volume of 50 μl. The thermal cycling conditions consisted of an initial step of 2 minutes at 95°C followed by 35 cycles of denaturing (94°C, 30 seconds), annealing (54°C, 30 seconds) and extension (72°C, 1 minute) with a final extension of 10 minutes at 72°C. The PCR products were screened for success on a 1.0% agarose gel. The sequencing of the PCR products was outsourced to Eurofin India, Pvt. Ltd. The sequences were then manually edited, aligned and proofread using Clustal W in MEGA 10.0 software and submitted to GenBank (NCBI).
 

 
Statistical analysis
 
All the analysis was performed in Microsoft Excel 2016. The results are displayed either in range or Mean±SD based on applicability. Correlation analysis was done between Total Length and other external features.

Odum and Barrett, (1971) defined an ecotone as an area where two or more different ecosystems overlap and interact. They described it as a transition zone between communities, such as between a forest and grassland or between a soft bottom and hard bottom marine community. Odum also referred to the ecotone as a “junction zone or tension belt” that contains a unique set of organisms from the overlapping communities, as well as organisms that are specific to and often restricted to the ecotone itself. Mahseer, a globally renowned sport and table fish, is considered the national heritage of India (Nautiyal, 2014). T. putitora and N. hexagonolepis are two threatened Mahseer fishes which need to be conserved (Jha et al., 2018; Arunachalam, 2010). Even though there are multiple reports of assessment of these species in different rivers and other ecosystems. But no specific study has been conducted in an ecotone zone of a river. Thus, the current study is the first of its kind to study of two important threatened in a foothill-floodplain river interfaces ecotone zone.
       
Water quality parameters of River Jia Bhorali at Bhalukpong during the sampling period are given in Table 2. The study site is a flowing stream characterized by a moderate current and an average width of 60 m and depth of 1.5 m. The riverbed is composed of boulders (5%), cobbles (35%), coarse gravels (40%) and fine gravels (20%) according to the Indian Standard Soil Classification System (Ranjan and Rao, 2000). The riparian zone is made up of shrubs, bushes and small trees like Ziziphus mauritiana in the terrestrial part and pools and riffles in the aquatic part. The banks of the river are covered by the dense forest of Nameri National Park.
 

       
Water quality in the study area was found to be intermediate between that of foothill river water and floodplain river water when compared to previous reports of this river (Khound et al., 2012; Singh et al., 2020). The continuous input of organic materials, such as plant litter, into the river may be a primary factor in this classification and the river continuum concept provides a suitable framework for its description. However, the riparian flora and fauna require further study.
       
The current study thoroughly examined and evaluated each collected specimen. The general body shape of identified T. putitora and N. hexagonolepis can be seen in Fig 2 (a and b). The body colour of fresh specimens T. putitora were greenish and silvery on the side of the body, but turns reddish yellow or golden on the anal and pectoral fins. Mouth with lower jaw slightly shorter than the upper jaw, caudal fin deeply forked. The dorsal fin ray of the specimens found to be 11 (2/9); pectoral fin ray was 14-15, anal fin ray 7, pelvic fin ray count 8-9 while caudal fin ray count was 20. Lateral line scale found to vary between 25-27; scales above lateral line 4.5 and below lateral line 2.5 (Table 3 and 4). Based on this, the derived fin formula of T. putitora is D11(2/9)P14-15V8-9A7(2/7)LLS25-27. The standard length (SL) of T. putitora ranges 234-219 mm. A positive correlation was observed between total length and all the external body parts in T. putitora (Table 5). In relation to TL, the highly correlated body parameters were FL (0.983), SL (0.932), VFL (0.906) and GL (0.905).
 

 

 

       
The scales of N. hexagonolepis were hexagonal in shape as its name derived from it, snout length was less than that of the body depth and body color was dark brown in freshly collected specimens. The specimens were found to have 26-32 lateral line scales, 3.5-4.5 scales above lateral line and 2.5 scales below lateral line. The dorsal fin ray of the specimens found to be 11 (2/9); pectoral fin ray was 14-15, anal fin ray 7, pelvic fin ray count 8-9 while caudal fin ray count was 18-21. Lateral line scale found to be 25; scales above lateral line 3.5 and below lateral line 2.5 (Table 3 and 4). Based on this, the derived fin formula of N. hexagonolepisis D11(2/9)P14-15V8-9A7(2/7)LLS25. The standard length (SL) of N. hexagonolepis ranged from 211-256 mm. Positive correlation was observed between total length and the external body parts of N. hexagonolepis except ED, LJL, GL, GW, DFH and DoM. The highly correlated body parameters in relation to TL were VFL (0.910), DaE (0.898), HD (0.895) and PFL (0.895).
       
The morphometric as well as meristic counts of T. putitora and N. hexagonolepis specimens recorded during the present study were found to be similar as described by previous authors (Vishwanath et al., 2011; Langer et al., 2013 and Laskar et al., 2013). The positive correlation between total length and other external morphological features observed during the present study indicates isometric growth pattern of T. putitora in natural condition. Langer et al., (2013) also reported similar morphometric relationship in Golden Mahseer from Jhajjar stream, J and K, India.
 
DNA barcoding
 
Identification of the Mahseer species were confirmed through DNA barcoding, the generated mitogenome sequences were submitted to NCBI and accession numbers were obtained (Table 5). The construction of a phylogenetic tree using maximum likelihood and neighbor-joining algorithms showed that the COI gene sequences of the specimens were closely related to those of N. hexagonolepis and T. putitora from other parts of the world (Fig 3). The overall genetic distance calculated within the species was 0.002 and 0.01 for T. putitora and N. hexagonolepis, respectively.
 

 

       
The COI gene sequence of T. putitora and N. hexagonolepis was compared to other sequences from various locations around the world available in the NCBI GenBank database. The blast alignment showed that the specimen was 98% identical to the other sequences of T. putitora and N. hexagonolepis, which, according to Ward et al., (2005) confirms their identity as the species with 98-99% resemblance. The mitogenome sequences of the Mahseer species generated from the River Jia Bhoroli, Brahmaputra drainage is the first such kind of information. Phylogenetic analysis supports the close evolutionary relationship between the species, with a highest bootstrap value of 100. Despite the geographical distance, the COI gene sequences of the compared species are genetically similar, suggesting conservation throughout the evolutionary process. Based on both morphological and phylogenetic analyses from COI gene sequences, it is concluded that the barb species are T. putitora and N. hexagonolepis.
 
Population
 
The survey carried out over the course of one year studied a total of 1026 nos. of N. hexagonolepis and 937nos. of T. putitora in the Bhalukpong region of Jia Bhoroli river with the density of N. hexagonolepis (with a CPUE of 4.3-12.6) higher in the study area compared to T. putitora (with a CPUE of 4.1-10.3). Both species showed the highest density during March and the lowest in January. The current study found a high abundance of adult as well as young individuals of both fish species in this area. The riffles and pools provide excellent habitats for young and small fish (de Moraris et al., 1995). The reproductive success of these populations is highly sensitive to any modifications of this ecotone zone (Schiemer and Zalewski, 1992). The fish fauna in this ecotone zone was found to be rich and diverse and these ecotones likely play an important role as a diversified food source for the fish (Khajuria et al., 2014). The higher abundance of fish in the study area indicates favorable conditions for these fish in this ecotone.
       
According to the IUCN, T. putitora and N. hexagonolepis are classified as endangered and near threatened, respectively (Jha et al., 2018; Arunachalam, 2010). The major reasons of this classification are rise in water temperature, overfishing, loss of habitat, decline in quality of habitat resulting in loss of breeding grounds and from other anthropogenic effects (Sarma et al., 2022). Hence, urgent efforts for conservation of these fish species are need of the hour. Past studies suggested that ecotones may act as hotspots for speciation, making them valuable areas for conservation investment as they can serve as centers for biodiversity. Chapman et al., (1996) claimed ecotones as refugia for endangered fishes as populations in ecotones are often pre-adapted to changing environments and may be more resilient to environmental changes, such as climate change and invasions by non-native species. As ecotones are small in size yet rich in biodiversity, conserving these areas as in-situ conservation may be a cost-effective strategy for maintaining biodiversity (Karl, 2017).

In conclusion, the findings of this study suggest that the ecotone zones of the Jia Bhoroli river are a refuge for threatened Mahseer population as it provides food and shelter. Both T. putitora and N. hexagonolepis are in need of conservation efforts as their populations are declining due to various anthropogenic activities. Conserving Bhalukpong as designated ecotone sites in Jia Bhoroli river as fish sanctuaries can give a big boost to restoration efforts of these threatened fish species. Further research with all the other factors of ecology should be conducted for better understanding of importance of ecotones for conservation of threatened fish species.

The authors are thankful to Dr. B. Kalita, Dean, College of Fisheries, Assam Agricultural University, Raha, Nagaon; Dr. S.K. Bhagabati, HoD, Dept. of AEM, College of Fisheries, AAU, Raha and Mr. Rupam Jyoti Nath, YP-I, College of Fisheries, AAU, Raha  for their help and support during the study. The authors also acknowledge help rendered by Dr. R. S. Haldar and Mr. Bhupendra Singh, DCFR, Bhimtal.

ICAR- Directorate of Coldwater Fisheries Research sponsored network project ‘Species and stock validation of Mahseer species of genus Tor and Neolissochilus from western and eastern Himalayan region of India for its propagation and conservation’ (Project id 681/19-20 dated 21/3/2020).

All the authors declare no potential conflict of interest.