Betta siamorientalis , a new species of bubble-nest building fighting fish ( Teleostei : Osphronemidae ) from eastern Thailand

Betta siamorientalis sp. n., a new species of fighting fish belonging to the Betta splendens group, inhabiting four provinces in the eastern Thailand, is described. The combination of slender body shape with reddish vertical bars on the black opercle and an apparent black body distinguishes the new species from other congeners especially B. imbellis. The blacker appearance relative to B. imbellis is due to a higher proportion of black body area relative to that occupied by blue/green iridescence of the scales. Based on morphological similarity and fin colour pattern and vertical bar colour on the opercle, the new species appears to be more closely related to B. imbellis and B. splendens than other members of the B. splendens group.


Introduction
Based on behaviourial and morphological characters and details of head and body patterns, Witte & Schmidt (1992) have classified betta fighting fishes into 15 species groups (see also tan & ng, 2005), one of which is the Betta splendens group.The term species group is used here for an assemblage of species sharing diagnostic characters.Members of the B. splendens group are bubble-nest building bettas with the following characters: small head, often brightly coloured body, iris of eye with iridescent green or blue patches, body slender, head with parallel opercles when viewed dorsally, red or brown anal and caudal fin rays contrasting with iridescent interradial membrane (especially in male) (Witte & Schmidt, 1992).
These members are B. splendens Regan, 1910;B. smaragdina LadigeS, 1972;B. imbellis LadigeS,1975;B. stiktos tan & ng, 2005; and the newly identified B. mahachaiensis KoWaSupat, panijpan, RuenWongSa & SRiWattanaRothai, 2012).It is well recognised that the native habitats for wild B. smaragdina are in the northeast and Laos PDR, those for B. imbellis are in south of Thailand extending to peninsular Malaysia.Betta splendens is distributed more or less countrywide from the north to central to the upper southern peninsula and also from east to west of Thailand and beyond.Betta mahachaiensis has been found confined to the Samut Sakhon province, west of Bangkok (KoWaSupat et al.,2012).Since recorded history, Thais have reared these fighting fish and bred them, especially, B. splendens, for both fighting and ornamental purposes (Smith, 1927(Smith, , 1945;;monviSeS et al., 2009).However, there are only a few reports on the distribution of the bubble-nest building fighting fish in Thailand or nearby countries.For example, LeRt panich & aRanyavaLai (2007) had identified the fish from the eastern Thailand all as B. splendens based mainly on the presence of reddish bars on the opercle.Our ongoing extensive fieldwork since 2007 on Betta distribution all over Thailand has now covered sixtyseven provinces (as of 2012) of the total seventy-seven including all seven provinces in eastern Thailand and some bordering Cambodia and Laos PDR.It has been noted by us and others that the betta fighting fish in the Chachoengsao province and some other eastern provinces possess different characteristics to other known members of the B. splendens group (authors′ personal communication).Locals say such unique characteristics have been known for generations, but the red bars on the opercle have persuaded them to believe the fish to be B. splendens.This fish has been called black imbellis by those familiar with it due to its black opercle and apparently black body.This black imbellis has a general appearance quite similar to the fish caught from Cambodia in Seam Reap by Kühne (2008), who named it Betta cf.imbellis, and similar to the Betta sp. in the Priay Khmang village by LinKe (2009).SchäfeR (2009) also received from Vietnam some traded fish and identified it as Betta cf.stiktos based on preserved specimens of the fish for comparison.However, upon later comparative work with live wild-caught specimens from the type-locality, he then changed the name to Betta cf.imbellis and called it Vietnam black emphasising the paucity of blue/green iridescence on the black body.
Upon in-depth analysis, our data suggested that the fish was a new species, at least those found in four provinces of eastern Thailand: Chachoengsao, Sa Kaeo, Prachin Buri and Chonburi (near border of Chachoengsao) and also at one site in Cambodia across the border from Sa Kaeo (KoWaSupat & panijpan, 2012; LinKe 2012).The aim of this paper is to formally describe this so-called black imbellis.

Materials and methods
Fighting fish specimens were preserved in 70% ethanol for long-term storage.The left side of all specimens was used for morphometric measurements point to point using digital Vernier caliper readings to the nearest 0.1 mm according to the method of SchindLeR & Schmidt (2006) and tan & ng (2005), both modi-fied from Witte & Schmidt (1992).Ratios are given in percent standard length or percent head length.The terminology and general format of description is based on Witte & Schmidt (1992), tan & KotteLat (1998) and tan & ng (2005).Meristic counts were carried out as described by Witte & Schmidt (1992) except for the number of predorsal scales, which were counted continuously following ng & KotteLat (1994).The phylogenetic species concept is used in this study (cRacRaft, 1989;see tan & ng, 2005).
All statistical analyses were performed using SPSS Version 20.To avoid undesirable effects of using ratios in statistical analyses, the logarithms of these ratios (LGRAT) were used (atchLey & andeRSon, 1978).The LGRATs of each trait of each species were tested for normality using the Shapiro-Wilk test.For nonnormal traits, the Kruskal-Wallis one-way ANOVA together with Mann-Whitney U test were used to compare means.Otherwise, the Levene′s test was used to test for homogeneity of variances.If the variances were equal, the one-way ANOVA together with the Tukey post-hoc test were used to compare means.Conversely, the Welch one-way ANOVA and the Games-Howell post-hoc test were used.Significant level was adjusted by the Bonferroni correction.
Specimens examined have been deposited at the Thailand Natural History Museum, Pathum Thani (THNHM) and also kept in the authors' own collection for the Thailand Betta Project (TBP).Abbreviations used are SL -standard length, HL -head length, and TL -total length.

Comparative material
The following materials were compared for morphometric and meristic characteristics as well as general appearance and body shape.
Betta imbellis caught from thirteen provinces of southern Thailand, one province of Malaysia (type locality), and one province of Vietnam Description.General body shape, appearance and colour pattern are illustrated in Figs. 1 -3.Head small, short (head length 28.0 -34.0 %SL), eyes large (orbit diameter 23.7 -31.6 %HL); body slender (body depth at dorsal-fin origin 23.6 -32.1 %SL); opercles parallel when head viewed dorsally; dorsal fin positioned after mid-body (predorsal length 57.9 -67.1 %SL); caudal fin posterior margin rounded; dorsal fin distal margin rounded; pelvic fins falcate; anal fin pointed and most of male distal tip extended to half or more of caudal fin length (30 out of total 40).Meristic and morphometric data are summarised in Tables 1 and 2. Maximum known size is 32.7 mm SL (THNHM -F -01825).
Colouration.See Fig. 2 for colouration in live specimens.Male with dark-brown to black head, with iridescent yellowish-green to bluish-green scales; opercles with double parallel reddish vertical bars (a vertical bar on the edge of opercle redder than the inner one nearer to the base); opercular membrane dark-brown to black with red patches; eyes with iridescent yellowishgreen to bluish-green patches at bottom and posterior regions; body background dark-brown to black with the iridescent yellowish-green to bluish-green scales: high ratio (greater than 60%) of body background (dark-brown to black) relative to scale iridescence (yellowish-green to bluish-green) in most specimens making it appear dark; dorsal fin rays dark-brown to black contrasting with iridescent yellowish-green to bluish-green interradial membranes, and at least proximal two-thirds having black transverse bars; caudal and anal fin rays red-brown to black contrasting with iridescent yellowish-green to bluish-green interradial membranes; caudal fin with distal red crescent and thin black edge; caudal fin interradial membranes with

Results
Betta siamorientalis sp.n.Figs 1 -3; Tables 1,2 Holotype.THNHM -F -01540 ( 36 small black spots in some specimens; posterior anal fin with a small red patch at distal tip (some rays have red colour near the end, especially, those near the distal tip); anal fin interradial membranes with small black dots in some specimens; red-brown to black and red from proximal pelvic fin base to the white tip; pectoral fins hyaline.Female less colourful (see Fig. 3): head and body yellowish to light-brown with iridescent yellowish-green to bluish-green scales; opercles pale gold with two faint reddish vertical bars; throat with chin-bar; body with distinct upper central stripe nearly parallel to central stripe, at level of eye; caudal-peduncle black spot present; dorsal, caudal, anal, and pelvic fin rays yellowish with faint iridescent green to bluishgreen interradial membranes; caudal fin with distal pale red crescent margin and pale thin black edge, or yellowish on both margin and edge; distal half of pos-   Distribution.Based on our extensive survey of fighting fish in sixty-seven (total of seventy-seven as of 2012) provinces, the wild B. siamorientalis sp.n. is currently found in only 4 provinces in Thailand: Chachoengsao, Sa Kaeo, Prachin Buri, and Chon Buri    Etymology.The name siamorientalis is adopted as an allusion to the type locality of the new species collected in this study.It is derived from Siam, the former name of Thailand, and orientalis, a Latin version for east, the region of the country where the fish is located.

Remarks.
There is only one indigenous species in the catch sites.It should be noted that although most specimens of B. siamorientalis sp.n. have reddish vertical bars on the black opercle, the bar colour can vary, for example, reddish to pale red, greenish-silvery or just silvery or no colour in some populations.That B. siamorientalis sp.n. is a new species and not a hybrid is supported by the results from DNA barcoding using a mitochondrial gene (cytochrome c oxidase I or COI) and a the nuclear gene (internal transcribed spacer 1 or ferent from B. splendens.As an important step toward distinguishing the black imbellis fish from the others, our extensive survey covering all eastern provinces of Thailand (including those on the Thai border shared also noticed the distinct characters of this fish, including teeRaWat utapong (personal communication) who has remarked that the fish from Kabin Buri district (Prachin Buri province) could possibly be quite dif- Vertebrate Zoology n 62 (3) 2012 addition to the bubble-nest builders above, the mouthbrooder in eastern Thailand, B. prima (KotteLat, 1994) and the southern brooder B. pallida (SchindLeR & Schmidt, 2004) in the southern peninsula also share similar characters.It is thus plausible that B. siamori entalis sp.n. and B. imbellis have evolved from a common ancestor(s).However, we cannot explain the fact that no mouth-brooders have been found in the upper part of the Thai pennisula from Petchaburi to upper Chumporn in spite of its present connectivity to the more extreme southern areas (south of Chumporn province) where mouth-brooders are common.
Regarding the phylogenetic relationship between congeners, previous results from DNA barcoding of bubble-nest builder bettas using cytochrome c oxidase (COI) and 16S mitochondrial gene (SRiWattanaRothai et al., 2010)  At this juncture, we wish to address the value of the above short DNA sequences in complementing other characters for species identification.The COI DNA for barcoding or other short DNA sequences as tools for species differentiation and identification, have proved to be useful (hebeRt et al., 2003), albeit not perfect in this role (KRiShnamuRthy & fRanciS, 2012).Nevertheless these sequences are not just of any short pieces of DNA but ones judiciously selected for the purpose (RatnaSingham & hebeRt, 2007).Some re-with Cambodia), yields morphological results which show B. siamorientalis sp.n. to be a new species member of the B. splendens group.Even though the morphometric and meristic data seem to show only slight differences in some of these characters (Tables 1, 2), our statistical analysis, nevertheless, reveals a significantly higher caudal peduncle depth in B. siamorien talis sp.n. when compared to that of B. splendens.The lack of clear differences in morphometric and meristic characters is consistent with tan & ng (2005) statement that it is rather difficult to use meristic characteristics and morphometric data to differentiate species within members of the same group due to the rather conservative nature of these characters (see also tan & tan, 1996).The overall results, however, suggest that B. siamorientalis sp.n. is more closely related to B. imbellis than B. splendens despite the red vertical bars on the opercle.This is further supported by our DNA barcoding results (in preparation).
Based on our survey, the distribution of B. siam orientalis sp.n. in Thailand is apparently restricted to certain areas in the upper eastern region, namely, Chachoengsao, Chon Buri (only in areas bordering Chachoengsao), Prachin Buri, Sa Kaeo.The fish is also found at Banteay Meanchey, a Cambodian province near Sa Kaeo.We have not found representatives of this group as yet in lower eastern provinces namely Rayong, Chantaburi, and Trat, as well as Koh Kong of Cambodia (close to Trat province of Thailand).Nevertheless, the bubble-nest builder B. splendens is generally found in these latter areas.Recent reports on the findings of the fish from Cambodia (Kühne, 2008;LinKe, 2009) andVietnam (SchäfeR, 2009) suggest that at present the fish may be inhabiting certain regions of eastern Thailand through parts of Cambodia that connect the southern region of Vietnam.However, B. imbellis, which is closely similar to B. siamorien talis sp.n., is found in the southernmost provinces of Peninsular Thailand through to the upper part of peninsular Malaysia.In an attempt to explain the similarities, palaeogeographical evidence is called upon here.During the most recent ice age with lowered sea level, the land of southern peninsular Thailand is believed to have connected via a land mass, together with a major river system(s), to the land of the eastern Thailand, southern Cambodia, and Vietnam.Due to the rise of sea level after the ice age, the land mass is currently under water in the Gulf of Thailand thus separating the two land areas (dodSon et al., 1995;voRiS, 2000;SathiamuRthy & voRiS, 2006).This geographical evidence may at least partially explain that the same or very similar fish in the two places, separated by an expanse of sea water since the big thaw, still retain similarities; there are some visible differences due to the time that has elapsed since then for the divergence (allopatric speciation) in their new environments.In rientalis sp.n. are the descendants of the discarded B. imbellis (brought from the south) in recent past.When the new species status of B. siamorientalis sp.n. has been formally accepted, this knowledge will be disseminated for conservation and protection of this species.
cent publications (daSmahapatRa et al., 2010) may emphasize certain deficiencies in the barcoding methodology and the latter may not be the sole DNA-based species distinguishing tool for the future (tayLoR & haRRiS, 2012), it is still helpful in complementing morphological and other criteria.For example, the various commonly bred and traded betta fighting fish obtained by us in Thailand, be it as ornamentals or fighters, show vast differences in terms of colour, shape, fin, size, aggressiveness, etc., and yet the COI evidence indicates that they all belong to the same B. splendens wild type′s profile.This is consistent with the fact that breeders have used B. splendens mainly for selective breeding.Without the prior knowledge of how this vast variety of ornamentals and bred fighters have come about, these living things might be thought to be different species; and yet they prove to be nearly identical by DNA barcoding.
Here, we have another situation where B. siamo rientalis sp.n. and B. imbellis appearing not much dif ferent based on morphometric, meristic and other external criteria thus making distinction between the two difficult.We therefore have to resort to the DNA evi dence to further help us to decide whether the two are the same or different.From the COI barcoding (mito chondrial DNA) and ITS1 (nuclear DNA) data we are confident the two are different enough to be called different species.(The nuclear DNA can be derived from both maternal and parental lines, so it can show up a hybrid, which is not found here.)At present, B. imbellis and the black imbellis are known to locate very far apart geographically.Without any prior knowledge of their distant catch sites, even experienced persons presented with the two fish for the first time might perceive them to be identical or nearly so.Some might even mistake the black imbellis to be B. splendens.As mentioned above, there is a situation similar to the above in that in appearance B. stiktos and one type of B. smaragdina (type locality) appear very similar.We suggest that these two lines of complementary evidence above should be applied to determine whether B. stiktos is different enough to the nearest type locality B. smaragdina to be called a different species just as in the case of B. imbellis and B. siamorientalis sp.n.

(
only in areas close to Chachoengsao).The eastern region of Thailand harbouring this species is shown in Fig. 4. Betta siamorientalis sp.n. is also found in Banteay Meanchey, a province of Cambodia on the eastern border of Sa Kaeo province: this may indicate an even wider and farther distribution.It is worth mentioning that in the Chon Buri province, B. siamo rientalis sp.n. has been found only in Phanat Nikhom district which is next to Chachoengsao province, while in the eastern part of Chon Buri, that is connected to other provinces, only B. splendens has been found.Field notes.Betta siamorientalis sp.n. was found in the shallow freshwater marshes, grass fields, and paddy fields.It was also caught at the water edge (with dense vegetation) of ponds, lagoons, ditches, canals, and others water ways.Leersia hexandra (family fin with distal red crescent and thin black edge (vs.absence in B. smaragdina, B. stiktos, and B. maha chaiensis); absence of dark transverse bars on caudal fin (vs.presence in B. stiktos); posterior anal fin rays red with a small red patch at distal tip (vs.absence in B. smaragdina, B. stiktos, and B. mahachaiensis); redbrown to black and red from proximal pelvic fin base to the white tip (vs.absence in B. stiktos, and B. ma hachaiensis); absence of iridescent bluish-green front margin of pelvic fin (vs.presence in B. stiktos, and B. mahachaiensis).

Discussion
Betta siamorientalis sp.n. shares common features with other congeneric species in the B. splendens group (seeWitte & Schmidt (1992); tan & ng, 2005).However, upon close scrutiny of more than one hundred specimens from the four provinces of eastern Thailand and one province in Cambodia (next to the eastern border of Thailand), B. siamorientalis sp.n. can be well distinguished from other known members of the group: it has a slender body with a rather blackish colour due to a smaller proportion of area on the body for iridescent blue/green scales relative to the black body background.It differs from B. imbel lis and B. smaragdina in having reddish bars on the black opercle vs. green to bluish-green bars of the former and iridescent green scales on opercle of the latter.Nevertheless, it is rather difficult to differentiate between the less acclimatized male and female members of B. siamorientalis sp.n. due to the pale colour of the body.Most B. siamorientalis have a light reddish colour on the opercle edge, which may lead some people to mis-identify them as B. splendens.Indeed, this new species has previously been described as B. splendens by LeRtpanich & aRanyavaLai (2007), perhaps because of the authors′ overreliance on the red parallel bars on the opercle as a criterion while disregarding the body and fins which are similar to those of B. imbellis.On the other hand, Kühne (2008) named the so-called black imbellis from Cambodia as B. cf.imbellis probably due to its general body shape and fin colours similar to those of B. imbellis.Similarly, SchäfeR (2009) called his Vietnam black betta, B. cf.imbellis.We realize that there may be others who have Poaceae) and Eleocharis dulcis (family Cyperaceae) were common in such habitats.The habitat characteristics are quite similar to those of other members of B. splendens group (see Fig. 5).Betta siamorientalis sp.n. lives in still waters and uses water plants as shelter to set up safe territory for building the bubble-nest, courtship, and hatching of fry, the same preferences as other species of the B. splendens group.Other labyrinth fishes cohabiting with B. siamorientalis sp.n. are Trichopsis vittata, Trichopsis shalleri, Trichopsis pu mila, Trichopodus trichopterus, Anabas testudineus.Other non-labyrinth fishes sharing the same habitats are Lepi docephalichthys hasselti, Pangio anguillaris, Macro gnathus siamensis, and Monopterus albus.
showed that the species in the B. splend ens group are well separated phylogenetically into at least four main branches of B. splendens, B. imbellis, B. mahachaiensis, and B. smaragdina.Betta stiktos′s COI barcoding has now been done and shown to be different and yet very close to that of one type of B. smaragdina found in the Northeastern part of Thailand (in preparation).Our preliminary results based on both COI mitochondrial gene and internal transcribed spacer 1 (ITS1) nuclear gene point to B. siamorientalis sp.n. as well separated phylogenetically from the B. im bellis, albeit closely related to the latter than B. splend ens.It is well distinct from B. splendens and other congeners.Our DNA barcoding results (in preparation) also show that B. siamorientalis sp.n. have distinctly different maternal lines from other congeners: of the numerous B. siamorientalis sp.n. specimens studied so far none has the COI sequence identical or nearly identical to those of members of the B. splendens nor B. imbellis (see accession number of COI and ITS 1 gene in Remarks section).The results thus indicate that B. siamorientalis sp.n. is not a hybrid between B. imbellis and B. splendens.Another result from our barcoding work is that, despite opercular differences among specimens of the B. siamorientalis sp.n., all the above nuclear and mitochondrial DNA sequences remain remarkably identical and they all belong to this same species.Such opercular variation in terms of presence or absence of red bars is also observed in the large numbers of B. splendens studied by us.