An annotated checklist of fishes from Bhutan

Acknowledgements: The authors are grateful to the Rufford Small Grant Foundation and the Royal University of Bhutan for providing financial support for the study. We acknowledge all the forestry staff who provided assistance during the ichthyofaunal survey. The first record of freshwater fish from Bhutan was by McClelland (1839) who reported Balitora brucei Gray and Schizothorax richardsonii Gray from the 1835 Boutan (sic) expedition of William Griffith. Five fish species were subsequently recorded from Bhutan by Day (1889) as part of a larger work on the fishes of South Asia. The most comprehensive listings of the freshwater fishes of Bhutan till date by Dubey (1978), Dhendup & Boyd (1994), and Petr (1999) record 47 species, of which eight are alien. In a study of the fish diversity in the Bumdeling Wildlife Sanctuary, Bhattarai & Thinley (2005) recorded 52 species; with Dema (2007) adding Triplophysa stoliczkai (Steindachner) to the list of fish species found in Bhutan. In comparison to the 520 species reported from the eastern Himalayan region (Allen et al. 2010), 213 from the Arunachal Pradesh in northeastern India (Bagra et al. 2009), and 296 species from northeastern India (Vishwanath et al. 2007) the number of fish species currently known from Bhutan is a gross underestimate. The paucity of information on the fish fauna of Bhutan is mainly attributed to the very poor representation of material in collections and the conservation regulations that prohibit fishing without a permit, making collection of fresh material difficult. The major drainage systems of Bhutan from the east to west are the Dangmechhu and Mangdechhu (Manas), Punatsangchhu (Sankosh), Wangchhu (Raidak) and Amochhu (Toorsa), with all these rivers debouching into the Brahmaputra River in India. In the higher elevations, these rivers support very low fish diversity due to their low productivity (Dubey 1978). There are also lakes in Bhutan, with Brown Trout (Salmo trutta fario Linnaeus) introduced in high altitude lakes (e.g., Nobtshonapata located at 4092m in Haa District and Tshophu at 4405m in Paro District) and Common Carp (Cyprinus carpio Linnaeus) in the lower altitude lakes (e.g., Hokotsho in Punakha and Ada in Wangduephodrang). Given our poor knowledge of Bhutanese ichthyofaunal diversity and the imminent threats to aquatic biodiversity arising from large hydropower Abstract: We examined 66 species of fishes during an ichthyofaunal diversity survey of Bhutan in 2011 and provide an annotated checklist of the freshwater fish species known to occur in Bhutan. A total of 91 species are now known from Bhutan, but this is clearly a gross underestimate of the true freshwater fish diversity of the country. This highlights the need for more extensive freshwater fish surveys to be carried out in the country.

The first record of freshwater fish from Bhutan was by McClelland (1839) who reported Balitora brucei Gray and Schizothorax richardsonii Gray from the 1835 Boutan (sic) expedition of William Griffith.Five fish species were subsequently recorded from Bhutan by Day (1889) as part of a larger work on the fishes of South Asia.The most comprehensive listings of the freshwater fishes of Bhutan till date by Dubey (1978), Dhendup & Boyd (1994), and Petr (1999) record 47 species, of which eight are alien.In a study of the fish diversity in the Bumdeling Wildlife Sanctuary, Bhattarai & Thinley (2005) recorded 52 species; with Dema (2007) adding Triplophysa stoliczkai (Steindachner) to the list of fish species found in Bhutan.
In comparison to the 520 species reported from the eastern Himalayan region (Allen et al. 2010), 213 from the Arunachal Pradesh in northeastern India (Bagra et al. 2009), and 296 species from northeastern India (Vishwanath et al. 2007) the number of fish species currently known from Bhutan is a gross underestimate.The paucity of information on the fish fauna of Bhutan is mainly attributed to the very poor representation of material in collections and the conservation regulations that prohibit fishing without a permit, making collection of fresh material difficult.
The major drainage systems of Bhutan from the east to west are the Dangmechhu and Mangdechhu (Manas), Punatsangchhu (Sankosh), Wangchhu (Raidak) and Amochhu (Toorsa), with all these rivers debouching into the Brahmaputra River in India.In the higher elevations, these rivers support very low fish diversity due to their low productivity (Dubey 1978).There are also lakes in Bhutan, with Brown Trout (Salmo trutta fario Linnaeus) introduced in high altitude lakes (e.g., Nobtshonapata located at 4092m in Haa District and Tshophu at 4405m in Paro District) and Common Carp (Cyprinus carpio Linnaeus) in the lower altitude lakes (e.g., Hokotsho in Punakha and Ada in Wangduephodrang).
Given our poor knowledge of Bhutanese ichthyofaunal diversity and the imminent threats to aquatic biodiversity arising from large hydropower projects planned in all the major rivers of Bhutan, an assessment of freshwater fish diversity in Bhutan is not only timely, but also necessary.In this study, based on a rapid assessment of ichthyofaunal diversity in selected water bodies of Bhutan, we present a list of 91 fishes that are now known to occur in Bhutan.

Materials and Methods
We divided a topographical map of Bhutan into different grids and used convenience sampling to assess the ichthyofaunal diversity in each grid.Focus was given to subtropical (below 1000m) rivers under the assumption that the diversity is higher in warmer water bodies (Fig. 1).Sampling was carried out in both the monsoon and post-monsoon seasons to cover both migratory and residential species.We sampled with cast nets wherever possible, and conducted searches by hand for species with cryptic behaviour.Basic parameters of water like pH, dissolved oxygen, and temperature were recorded at the sampling sites.Geographical coordinates and altitudes were also recorded.
Morphometric data were measured to a tenth of a millimetre using a dial calliper.We used existing literature ( Nebeshwar et al. 2012) to identify the species and sought experts' advice wherever possible.For uncertain species, voucher specimens were prepared after euthanizing the fishes with 0.0001% clove oil solution and treating in 10% formalin (Li & Li 2007).After 1-2 weeks of fixation in formalin, the specimens were transferred to 70% alcohol.The specimens were catalogued and preserved as voucher specimens in the laboratory collection of the College of Natural Resources (CNR).

Results and Discussion
We collected and examined 66 species of fishes from different regions of Bhutan.In order to avoid repeated collections and support scientific studies in future, 135 specimens comprising 45 species were catalogued and preserved as voucher specimens.For small species that could not be tagged easily, digital photographs were taken and preserved without catalogue numbers.Here, we provide a list of 91 fish species confirmed as occurring in Bhutan.However, species from Dubey  Dubey (1978), Dhendup & Boyd (1994), Petr (1999) We examined a specimen of Heteropneustes fossilis Bloch in a private aquarium at Phuntsholing that was reportedly collected from river Toorsa.Similarly, the only specimen of Channa punctata Bloch that we examined was collected from the crocodile farm at Phuntsholing.Some non-native species were recorded during the survey.Onchorhynchus mykiss Walbaum was introduced in a hatchery at Haa in 2007 (Norbu 2013).It has not been released in the rivers of Bhutan yet, but its escape in the river nearby is suspected.Oreochromis mossambica Peters was collected from the crocodile farm at Phuntsholing; this species was probably introduced to Bhutan in the mid 1990s (Ram Bahadur, pers.comm.).
Clarias gariepinus (Burchell) is another introduced species found in a sewage system and river Toorsa at Phuntsholing.It was introduced through "tshethar" practice (a compassionate act of releasing live fish in river water to prevent killings, mostly practiced by Buddhist monks).Even the elderly people with religious inclination, other than the monks, buy live fishes from across the border town and release them in the river nearby.This method of introducing alien aquatic organisms has also been reported elsewhere (Ng & Tan 2010).The live fishes sold in the border towns of India adjoining Bhutan for such practices are imported from Bangladesh via Kolkata.Unless the people are educated on the ecological consequences of such practices, this may serve as a major avenue of the introduction of alien species into the freshwaters of Bhutan and will become a major threat to conservation of native species in the country.

Conclusion
This study records 91 fish species from Bhutan.As the survey was carried out mainly in the monsoon season, a more extensive sampling regime both temporally (covering the inter-monsoon period) and geographically (in other areas of the country not sampled during this survey) is necessary to gain a better understanding of the freshwater fish diversity of Bhutan.
Although Bhutan pursues conservation of native fish species fervently, this is hampered by the poor state of knowledge of the diversity.Compared to the number of species reported from adjoining areas, the list of species presented here is clearly a gross underestimate of the freshwater fish diversity of Bhutan.The rapid development sweeping over Bhutan and the planned mega-hydropower projects to harness 10,000 MW by the year 2020 (RGOB 2010) is expected to have a significant impact on the biodiversity of the country, making it imperative that a further understanding of Bhutan's ichthyofaunal diversity is critically needed.

Figure 1 .
Figure 1.Map of study area Likewise, a juvenile specimen of Macrognathus morehensis Arunkumar & Tombi was collected from River Manas during the monsoon.The description of Garra species from the region (Viswanath et al. 2007; Nebeshwar et al. 2012) are inadequate for the diagnosis of Garra species found in Bhutan.Similarly, the taxonomic keys provided by Viswanath et al. (2007) and Jayaram (2009) are inadequate in resolving the taxonomy of the catfishes known from Bhutan.