Fish diversity of a spring field in Hopong Town, Taunggyi District, Shan State, Myanmar (the Salween River Basin), with genetic comparisons to some “species endemic to Inle Lake”

Abstract Hopong, a small town in the Salween (Thanlwin) River Basin, Myanmar, is located 35 km northeast of Inle Lake, a famous ancient lake with numerous endemic fish species. We surveyed the fish fauna of a spring pond in Hopong in 2016, 2019 and 2020 and identified 25 species. Of these, seven, including Inlecyprisauropurpureus and Sawbwaresplendens, had been considered endemic to Inle Lake and at least three species were genetically unique. Eight were suspected or definite introduced species, including Oreochromisniloticus and Gambusiaaffinis. We were unable to identify a nemacheilid species of the genus Petruichthys, which would need a taxonomic examination. The Hopong area is being developed rapidly and, hence, it is crucial to conserve its native fish species and the freshwater ecosystems.


Introduction
Hopong is a small town in the Salween (Thanlwin) River Basin, situated 15 km east of Taunggyi, the capital of Shan State, Myanmar (Fig. 1). The town has several natural wetlands and springs. Danio ( Celestichthys) margaritatus, a minute cyprinid fish, is a symbolic fish species from this area (Roberts 2007, Conway et al. 2008. However, the fields of Hopong have been developed rapidly with extensive road construction and urbanisation (Roberts 2007). Hopong is about 35 km northeast of Inle Lake, a famous ancient lake with many endemic fish species (Annandale 1918, Kano et al. 2016, Win 2018. The Hopong area and Inle Lake are both in the Salween River Basin, so are close together geographically; however, they belong to different drainages separated by highlands and are ca. 400 km apart by river (Fig. 1). The endemism of fishes in Inle Lake has not been validated, because studies of the fish fauna of the middle-upper Salween Basin are insufficient. Thus, the ichthyofauna of the Hopong area is of biogeographical interest, especially in terms of comparison with that of Inle Lake, including the validation of species endemic to Inle Lake.
Generally, studies of freshwater fish biodiversity in Myanmar are insufficient; most are fragmentary reports on fauna of specific regions or new species. Annandale (1918) and Hora and Mukerji (1934) reported the fish fauna of Inle Lake and Shan State, respectively. Musikasinthorn (1998a) first reported Channa panaw from the Irrawaddy and Sittang River Basins, Myanmar. Britz (2003) Vidthayanon et al. (2005) provided a rough list of inland fish species of Myanmar. Data on the freshwater fish biodiversity of Myanmar are generally limited and more information is required.
Here, we report the results of a freshwater fish inventory for a spring field in Hopong conducted in 2016, 2019 and 2020. The list contains 25 species, including seven "species endemic to Inle Lake". We provide DNA barcoding (mitochondrial COI sequences) data for the species, which clarify their genetic uniqueness and should promote biogeographical research in this area.

Methods
Sampling was conducted nine times in Hopong from March 2016 to March 2020 (Fig. 2). Fish were collected with hand-nets, throwing nets and fish traps. Except for several individuals that were only photographed or recorded on-site and released, the collected specimens were photographed fresh (Kano and Nakajima 2014), fixed in 10% formalin and then transferred to 70% ethanol. The specimens and their tissue samples were catalogued and deposited at the Research Laboratory of Ichthyology, Department of Fishery Biology, Faculty of Fisheries, Kasetsart University, Bangkok, Thailand (RLIKU) and Kyoto University, Kyoto, Japan (tissue samples). All specimens were assigned IDs associated with the records of location (latitude, longitude and region name), collection date, DNA sequence accession numbers etc. The data were registered in the integrated Monsoon Asia "ffish.asia" online database of freshwater organism biodiversity (Watanabe et al. 2009, Kano et al. 2013) and can be retrieved at https://ffish.asia/Hopong2020.
The obtained sequences were aligned using MAFFT (Katoh and Standley 2013) at Unipro UGENE (Okonechnikov et al. 2012). Sequence data for the same or related species from Inle Lake and the surrounding rivers, as reported by Kano et al. (2016), were included in the analysis for comparison with the Hopong populations. To visualise the relationships amongst populations, haplotype networks, based on the TCS algorithm (statistical parsimony; Clement et al. 2000), were constructed for five genera endemic to this region-Inlecypris, Microrasbora, Sawbwa, Petruichthys and Physoschistura-using POPART (Leigh and Bryant 2015). The mean uncorrected sequence differences amongst different populations were calculated using MEGA 7 (Kumar et al. 2016).

Discussion
A total of eleven freshwater fish species have been reported from Hopong in previous papers (Hora and Mukerji 1934, Roberts 2007, Kullander et al. 2017). All of them, except for Barbus hexastichus (valid as Neolissochilus hexastichus), are judged to be included in the 25 species reported in this study. Hora and Mukerji (1934) reported Barbus hexastichus from Hopong as having two pairs of barbels, a light brown body colour and a black round The haplotype networks of five species groups ("species endemic to Inle Lake") generated with the TCS algorithm, based on COI regions (640 bp). Each bar on a branch corresponds to a single nucleotide substitution. Small dots represent hypothetical haplotypes. A Inlecypris auropurpureus obtained from Hopong and two other localities. B Microrasbora rubescens obtained from Hopong and two other localities. The Heho specimens were obtained from a local market and their original locality is unknown. C Physoschistura brunneana and P. rivulicola obtained from Hopong and two other localities. The Heho specimens were obtained from a local market and their original locality is unknown. D Sawbwa resplendens obtained from Hopong and Inle Lake. E Petruichthys brevis obtained from Hopong and two other localities and Petruichthys sp. obtained from Hopong.
Fish diversity of a spring field in Hopong Town, Taunggyi District, Shan ... spot at the base of the caudal fin. However, this record should be reconsidered since its main distribution range is in India (Talwar and Jhingran 1991). In fact, Neolissochilus nigrovittatus, which is distributed around Inle Lake (Talwar and Jhingran 1991, Kano et al. 2016), also fits the description of the fish by Hora and Mukerji (1934). Further survey and examination are necessary for the Neolissochilus species in this area.
Our results indicated that at least seven "species endemic to Inle Lake" (Annandale 1918, Kano et al. 2016, Win 2018 have wider distributions than previously thought, beyond the Inle Lake Basin (Table 1). Artificial introduction from Inle Lake cannot explain their wide distributions because of the genetic divergence of the Hopong populations from the Inle Lake populations, except for Sawbwa resplendens (only one DNA sample and details unknown) and Petruichthys brevis ( Fig. 3D and E). Therefore, Hopong shares fish fauna with the Inle Lake Basin, but the populations of several fish species in Hopong are genetically unique and require detailed taxonomic examinations.
Danio margaritatus is symbolic of Hopong (Roberts 2007 (2007) and Hary (2007). However, these sources are not formal scientific reports (website and local report, respectively) and, in fact, we could not access them. Thus, at present, it cannot be determined whether D. margaritatus is distributed only in the Hopong area or more widely. If the former is the case, conservation of D. margaritatus and its habitat, i.e. shallow ponds/wetlands with plants and clear water from springs ( Fig. 2B; Roberts 2007), is very important. Devario sondhii might also be endemic to the Hopong area; more information on its distribution is necessary to determine its conservation status.
Petruichthys sp. (Hopong) is genetically distinguishable from the sympatric P. brevis (Fig. 3  E). Morphologically, its body is smaller and the male is redder than that of P. brevis. Taxonomic examination of this fish is required.
The Hopong area is being developed rapidly (Roberts 2007). In addition, several invasive alien fish have become established, including Oreochromis niloticus and Gambusia affinis. This strongly suggests that the native freshwater fishes and other native freshwater organisms in Hopong are now severely threatened. As explained above, Hopong has a unique fish fauna that includes endemic species and species shared with Inle Lake, an unique ancient lake in Southeast Asia. Therefore, wetland and biodiversity conservation in Hopong are a high priority.
Leaf, Myanmar). This work was supported by the Sumitomo Foundation (the Grant for Environmental Research Project, 193271), JSPS KAKENHI (Grant Number JP26304007, JP19J23130, JP20HP8020) and the research grant of Kurita Water and Environment Foundation (18K013, 19K008).