Discovery of wild populations of Betta smaragdina Ladiges , 1972 ( Teleostei , Osphronemidae ) in a western province of Thailand

The bubble-nesting Betta smaragdina Ladiges, 1972 can be distinguished from the 4 other species of bubble-nesting Betta Bleeker, 1850 by being reportedly confined to Northeastern Thailand. We found large populations of fish in Western Thailand that closely resemble those from the Northeastern Region. The new populations inhabit a variety of places encompassing lakes, marshes, and streams. Morphological studies of Betta fish from the 2 well-separated regions, together with DNA analyses, show that the western and the northeastern populations are the same species.


Introduction
Freshwater fishes of the genus Betta Bleeker, 1850 are found in most of Southeast Asia countries, except in the Philippines and perhaps Myanmar.In Thailand, there are 5 species of wild bubble-nest building Betta (Panijpan et al. 2017) Ladiges, 1972 with populations confined to only 20 provinces in the northeastern Thailand (Fig. 1).Most of these Betta fishes have habitats confined to specific regions (Lertpanich and Aranyavalai 2007, Linke 2014, Goldstein 2015, Panijpan et al. 2017).One exception is B. splendens, which shares some habitats with other regional Betta species.These 5 species can be distinguished from one another morphologically, especially when the fully-grown males are in full aggressive display.Among these Thailand Betta fishes, cryptic species within B. smaragdina (Kowasupat et al. 2014) are the only ones with tens of bluish-green iridescent plates on the head region covering the opercles down to the lower part of the mouth region.These plates are usually well separated and can be very small.So far, wild Betta fish with this unique character have never been reported elsewhere outside the Northeastern Region (Linke 2014, Goldstein 2015).Here, we report western populations of Betta from 5 collection sites (covering ca 6 km 2 ) in Kanchanaburi Province of Thailand, and use morphological and DNA evidence to confirm that fish from western and northeastern Thailand belong to the same species.

Methods
Sample collection.The collection sites (Table 1) are relatively inaccessible areas near the Thailand-Myanmar border.All sites, except for K07, are located in areas less than 5 km away from the border (Fig. 2).We caught the fish using hand-held netted tools, transported them in plastic bags and put them in glass jars (10 cm × 10 cm × 20 cm) individually in the laboratory for observation and caring.Dead specimens were deposited at the Natural History Museum, Thailand.Photographs of the 5 collection sites are shown in Figure 3A-E.We observed the fish in waterbodies several kilometers downstream from the collections sites but fish density was lower than in the collection sites.
Observation of morphological characteristics.We identified fully grown fish following Tan and Ng's (2005) taxonomic key, which was modified from Witte and Schmidt (1992).We recorded body color patterns, iridescence of body scales, facial iridescence plates, fin shape, fin size, fin rays, and fin color while they fully display their aggressive behavior (Fig. 4).

Results
New records.Table 1.
Identification.We identified the new fish by comparing it with B. smaragdina from all parts of northeastern Thailand.Figure 4 shows the comparative photographs of B. smaragdina from Western and Northeastern regions.The western Betta fish are practically identical with B. sma- ragdina from the Upper Northeastern Region with only minor differences.Visible shared features.Kanchanaburi Betta fish shares a distinct feature in the head part, anterior to the gill openings, with the type-locality B. smaragdina from the northeastern Thailand.Both groups of fish have more than 30 well-separated bluish-green iridescent plates located in the ventral area of the head.The second shared feature is the caudal fin, having about 12 rays originating from the peduncle and splitting into 2 toward the posterior end of the fin.The third shared feature is 8 clear rows of very bright iridescent scales running laterally on both sides of the body.
Visible minor differences.Based on 5 male fish from each location, fully grown B. smaragdina (3.8-4.2 cm standard length) and Kanchanaburi fish (3.5-3.9 cm standard length) showed slight differences in the ratios of pelvic fins to body length, 0.43 + 0.03 for B. smaragdina and 0.32 + 0.03 for Kanchanaburi fish.To the naked eye, the body of B. smaragdina appeared more slender than Kanchanaburi fish, however, ratios of the broadest part of the body/standard length are 0.25 + 0.01 for B. smaragdina and 0.27 + 0.01 for Kanchanaburi fish.
DNA analysis.The DNA sequences from the mitochondrion and nucleus show the new fish to be practically identical to B. smaragdina from the type locality.COI, RAG1, and ITS1 sequences of the Kanchanaburi bettas, when compared to those from GenBank and our unpublished RAG1 data from all varieties of B. smaragdina (Table 2), show clearly that these Kanchanaburi fish belong to the same clade as B. smaragdina from the northern part of the northeastern Thailand.In particular, COI sequences of Kanchanaburi fish are practically identical (99.7-99.8%sequence identity) to that of a typical specimen of B. smaragdina from the Upper Northeastern Region while their RAG1 sequences present 99.4-99.7%identity.In addition, all informative sites in the RAG1 sequences from both western and northeastern B. smaragdina fish were indistinguishable.As for ITS1 sequences, the differences are 2-3 indels whose lengths are 1, 2 and 4 base pairs.The non-indel sites present 99.8-100% sequence identity.Figure 5 depicts the phylogenetic tree reconstructed from the combined DNA sequences (COI, RAG1, and ITS1).It is interesting that B. splendens, B. mahachaiensis, and B. siamorientalis have no cryptic species-populations phylogenetically separable by distinct DNA sequences.However, B. smaragdina has at least 4 cryptic species; only 2 of which were analyzed (Fig. 5).

Discussion
As mentioned earlier, there are other locations in Kanchanaburi Province where only B. splendens and no other Betta spp.are found.Betta smaragdina has so far been assigned solely as a northeastern Thailand fighting fish since its first description by Ladiges (1972).More recently, in the Western Region, which is well-separated  from the Northeast Region, B. smaragdina has been found in abundance over a relatively wide area covering various types of waterbodies, such as lakes, ponds, and streams.However, fish in these 2 regions live in water of different transparency.In the west, all collection sites have clear water that mostly originates from high elevations in mountains flowing down to lower altitudes (Fig. 1), whereas in the northeast, on the plateaus, turbid water is accumulated from rain and upwelling underground water.The morphological differences, for example, the pelvic fins and anal fin, are extremely minor between the fish from the 2 regions.In addition, their DNA sequences (COI, RAG1, and ITS1) are practically identical.If these fish had been translocated from one region to the other, the time of the separation of the fish from the 2 regions must not have been long enough for the fish to change its DNA sequences, but some minor morphological adaptations could have taken place.
Analyses of COI, RAG1, and ITS1 sequences of fish used for genetic identification of species (Hebert et al. 2003), show that they differ from those of B. mahachaiensis and any other Thailand bubble-nesting Betta species.Some features in western Betta, especially scale iridescence and body shape, may remind some people of B. mahachaiensis.However, as there is no indication of contamination by B. mahachaiensis DNA in our western fish, it is safe to say these morphological similarities between western B. smaragdina and B. mahachaiensis are homoplastic.
From our observation, western fish have thrived so well to populate these collection sites and beyond, where there are no other bubble-nesting species.Thus one could

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only speculate on how and when large populations of the same species have come to occupy different habitats in the Northeast and the West regions, which are separated by great distances.One proposal, that the fish of both regions emerged more or less simultaneously in the past, is not tenable because their DNA sequences are practically identical.Such coincidence is implausible.The northeastern B. smaragdina populations harbor at least 4 cryptic species, indicating the evolutionary time of over 5 million years.But there is only 1 of the 4 species in the Northeastern Region that is identical with the western Kanchanaburi fish.
The present sparse populations of Karen tribes, probably recent migrants from Myanmar, living near the sites do not know these tiny fish, which are considered by them to be too small to eat, nor do they have the game of fish fighting.These people are not the ones who brought the northeastern fish to the west.Moreover, these sites have been hidden from Thai and foreign aquarists because modern road transportation has been made possible only in the last 10 years.

Figure 1 .
Figure 1.Map showing locations of collection sites of Betta smaragdina in the Northeastern Region and Kanchanaburi Province, Thailand.

Figure 5 .
Figure 5. Phylogenetic tree reconstructed from analysis the combined DNA sequences (COI, RAG1, and ITS1).The number on each node represents the posterior probability supporting that clade.Upper and lower indicate northern and southern parts of the Thailand Northeast.

Table 1 .
New records of wild Western Thailand Betta smaragdina collected in this study.THNHM: Thailand Natural History Museum.
(Huelsenbeck and Ronquist 2001, Ronquist and Huelsenbeck 2003, Ronquist et al. 2012)uelsenbeck 2003, Ronquist et al. 2012)after partitioning them according to their locations (mitochondrion versus nucleus) and codon positions.Based on the parameters sampled from a preliminary posterior distribution, we adjusted the model used in the Bayesian inference until the marginal likelihoods could not be significantly improved.

Table 2 .
Locations of collection sites of Betta fish of Thailand used in the DNA analysis.ACNO: GenBank accession number.KP: Kanchanaburi Province.N: number of examined specimens.