Phylogenetic position and osteology of Pethia setnai ( Chhapgar & Sane , 1992 ) , an endemic barb ( Teleostei : Cyprinidae ) of the Western Ghats , India , with notes on its distribution and threats

1 Bombay Natural History Society (BNHS), Hornbill House, Opposite Lion Gate, Shaheed Bhagat Singh Road, Mumbai, Maharashtra 400001, India 2,4 Systematics, Ecology & Conservation Laboratory, Zoo Outreach Organization (ZOO), 96 Kumudham Nagar, Villankurichi Road, Coimbatore, Tamil Nadu 641035, India 3 Zoological Survey of India (ZSI), Western Regional Center, Akurdi, Pune, Maharashtra 411044, India 4Indian institute of Science Education and Research (IISER), Dr. Homi Bhabha Road, Pashan, Pune, Maharashtra 411008, India 1 theunmesh@gmail.com, 2 mandarpaingankar@gmail.com, 3 shrikantj123@yahoo.com 4 n.dahanukar@iiserpune.ac.in (corresponding author)


INTRODUCTION
Two major hurdles in conservation of threatened and endemic species, especially those that occur in biodiversity hotspots is the Wallacean shortfall (arising from the incomplete information regarding the distribution of species) and Linnean shortfall (several species still not formally described) (Bini et al. 2006).The Western Ghats region, part of the Western Ghats-Sri Lanka biodiversity hotspot is no exception to this, as it has already been suggested that the freshwater fish fauna (especially endemic and threatened species) of this region is poorly known with regard to their taxonomy and distribution (Dahanukar et al. 2011;Raghavan et al. 2012).One reason for this gap in knowledge regarding geographical distribution is the lack of detailed descriptions and reliable genetic data, which can ultimately lead to misidentifications.As a result, genetic sequences from topotypic material and detailed morphometric data will not only help in understanding the systematics of the species, but also provide reliable identification criteria.
In a recent review of the South Asian barbs previously placed under the genus Puntius, Pethiyagoda et al. (2012) tentatively placed Puntius setnai Chhapgar & Sane, 1992 in the genus Pethia based on the data available in the original description of the species.The tentative placement was probably due to the limited information on the morphology, osteology and/or genetic data of the species.In the current study, we provide genetic information of Pethia setnai from topotypic material, osteological details to confirm the placement of the species under Pethia, and reliable genetic and morphometric data for accurate identification of the species.We also provide information on the distribution, habitat and threats to the species.

Morphometry
Measurements were taken point to point using dial calipers to the nearest 0.01mm and then rounded to 0.1mm.Subunits of the body are presented as percent of standard length (SL), and subunits of the head are presented as percent of head length (HL).All pored scales were counted when reporting the lateral line scales.Methods for taking counts and measurements follow Kullander (2008) and Pethiyagoda et al. (2012).

Osteology
Two specimens (BNHS FWF 55 and BNHS FWF 70) were cleared and stained using the procedure described by Potthoff (1984).Osteological nomenclature follow Conway (2011) and descriptions of osteology follow Pethiyagoda et al. (2012) so as to allow easy comparison  with other related taxa.Osteological illustrations were made from images captured by a digital camera fitted on stereo-zoom light microscope (Leica S8 APO, USA).
We used the cytb gene dataset of South Asian cyprinid fishes by Pethiyagoda et al. (2012), Raghavan et al. (2013a) and additional sequences of Pethia, Dawkinsia and Haludaria available from NCBI GenBank (http://www.ncbi.nlm.nih.gov/).Accession numbers for the sequences used for the analysis are provided in Fig. 1.Gene sequences were aligned using MUSCLE (Edgar 2004).Phylogeny was performed using the freeware MEGA 5 (Tamura et al. 2011).Best fit model for nucleotide substitution was selected from 24 models available in MEGA 5 based on minimum Akaike Information Criterion (AIC) value (Posada & Crandall 2001).Tamura & Nei (1993) nucleotide substitution model including invariant sites and a Gamma parameter was obtained as a best fit model (TN93+G+I, AIC = 12894.38, lnL = -6328.70).This model was then used for constructing a phylogenetic tree using the maximum likelihood method.Reliability of the phylogenetic tree was estimated using bootstrap values run for 1000 iterations.

Phylogenetic position
Pethia setnai forms a monophyletic group (Fig. 1) with the genus Pethia sensu stricto as defined by Pethiyagoda et al. (2012).The genetic divergence in the cytb gene between the topotypic material collected from Sanguem, Goa and additional material collected from Terekhol River at Madkhol, Maharashtra, was 0.325%, while the divergence between the two Terekhol specimens as well as two Sanguem specimens was 0.2%.Because of the very low genetic divergence, the two populations have to be considered as genetically similar.We could not collect fresh specimens of the species for genetic analysis from Karnataka State, and as a result, future studies are essential to establish the extent of genetic variation in different known populations of the species.Nevertheless, as we are providing the genetic information of the topotypic material, future studies can have a more reliable comparative material.

Type material information
Holotype and six paratypes of Pethia setnai collected from Sanguem, Goa, by S.R. Sane on 1.iii.1985are available in good condition in the museum collection of ZSI Kolkata under the accession numbers FF2766 (Image 1) and FF2767 (Image 2).Chhapgar & Sane (1992) described the species based on 25 specimens (the holotype and 24 paratypes) with the note that "… holotype and some of the paratypes will be deposited with the Zoological Survey of India, Calcutta".The whereabouts of remaining 18 paratypes is not known.Interestingly, 26 specimens of P. setnai are in the museum collection of BHNS under the accession numbers BNHS FWF 17-42 (see Materials examined).However, whether some of the specimens from BNHS constitute the missing paratypes could not be deciphered.

Morphometric and meristic data
Morphometric characters and meristics of the specimens collected for the present study from the type locality Sanguem, Goa; a newly discovered population from Terekhol River near Madkhol Village, Maharashtra and from Karwar, Karnataka are provided in Table 1 and  Table 2, respectively.Photographs specimens collected for the present study are provided in Images 3 and 4.

Osteology
Osteology of the specimen from the type locality is provided in Image 5. Gill rakers simple, acuminate (not   by Pethiyagoda et al. (2012).

Color variation
In life Pethia setnai has silvery-grey or dull golden color with three black transverse bands; anterior one situated between the dorsal profile and below the lateral line encompassing 3rd and 4th lateral line scales; middle band between the posterior half of the dorsal fin base and one scale below lateral line encompassing 9th to 11th lateral line scale; posterior band between the middle of end of dorsal fin base and caudal fin and the posterior half of the anal fin base (Image 4).Posterior most and anterior most bands are darker than the middle band (Image 4a).Dorsal, ventral and anal fins are bright red in certain specimens in their natural habitat (Image 4a) but the fin colors fade rapidly in captivity (Image 4b).Some

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specimens have indigo blue tinge on the dorsolateral area and therefore the species is also known as indigo barb among hobbyists.While, specimens with yellow fins are also known from this species we have not come across such specimens in our study.

Distribution
Based on the current collections as well as locality information deciphered from literature (Table 3), Pethia setnai is known to be distributed in the west flowing rivers of the Western Ghats in the states of Karnataka, Goa and Maharashtra between 12.66⁰N to 15.94⁰N latitude (Fig. 2).The species is currently known from seven fragmented populations, most of them reported from lower altitudes (Table 3) except the one from Agumbe, Karnataka located at 669m (Knight et al. 2012).
To our knowledge, there are no published and reliable records of Pethia setnai from the Cauvery River system, or any other east flowing rivers.Anecdotal reference to the presence of this species in the Cauvery, especially among the aquarium fish hobbyists, is as a result of the misidentification of Pethia setnai as Pethia narayani by Chhapgar & Sane (1980).While criticizing the report of Pethia nigrofasciata from Goa by Yazdani (1977), Chhapgar & Sane (1980) suggested that the species was Pethia narayani (Image 6), a species originally described from Cauvery River system in Coorg by Hora (1937).Mr. S.R. Sane who was active in the aquarium fish trade marketed the species occurring in Goa as Narayan's Barb until Chhapgar & Sane (1992) realized that they had overlooked the presence of osseous and serrated last unbranched dorsal fin ray in the specimens from Goa (as opposed to non-osseous and non-serrated last unbranched dorsal fin ray in Pethia narayani), and described Pethia setnai (B.F.Chhapgar, pers. comm. July 2013).Apart from having strongly osseous and serrated (vs.smooth articulated) last unbranched dorsal fin ray, P. setnai differs from P. narayani based on 8 (vs.9) branched dorsal fin rays, 5 (vs.6) branched anal fin rays and 19-20 (vs.22) lateral line scales.Because of the misleading common name 'Narayan's Barb' for P. setnai, it is likely that hobbyists have treated P. narayani from Cauvery River as P. setnai.
We failed to come across P. setnai during our repeated surveys in Mysore, Coorg and several other areas from Cauvery river system.As a result, unless a reliable report of P. setnai is available from Cauvery River system, backed up with genetic data, the species is considered as restricted to west flowing rivers of the Western Ghats based on Yadav (2003) and information presented in this paper.

Habitat
Chhapgar & Sane (1992) described Pethia setnai from clear streams of Sanguem and Ponda in Goa.However, they did not provide any information on the habitat in which the species was found.As part of the present study, fresh collection of P. setnai was made from the Salaulim River, a tributary of Zuari River near Sanguem, Goa (Image 7).The habitat consisted of a slow flowing, clear water stream with maximum substratum composed of sand and mud.Riparian cover was rich in

Threats
Based on IUCN Categories and Criteria (IUCN 2001), Dahanukar (2011) assessed Pethia setnai as a 'Vulnerable' species owing to its restricted distribution and on-going threats to habitat because of tourism, urbanization and agricultural pollution.The Goa populations are subjected to heavy mining operations in the headwaters, apart from the pressure from increasing tourism (Image 9).Habitats of the species in Terekhol River are severely threatened by organic wastes and sewage.Further, the species is also known to occur in aquarium trade under the common name Indigo Barb (Chhapgar & Sane 1992).Although no statistics is available on the extent of wild caught Pethia setnai in the trade, it has already been suggested that the unregulated aquarium trade is a plausible threat to the endemic and threatened species of freshwater fishes in India (Raghavan et al. 2013b).Further studies on the extent of collection and trade in P. setnai is therefore essential.

CONCLUSIONS
A comprehensive assessment of the status of freshwater fishes of Western Ghats for the IUCN Red List of Threatened Species revealed that little to no information exists regarding distribution, population status and life history of several threatened and endemic species (Dahanukar et al. 2011).This has led to renewed research on various aspects including taxonomic issues and those explaining detailed distribution of several threatened freshwater fishes of the Western Ghats (Ali et al. 2013a,b;Emmanuel et al. 2013;Katwate et al. 2012;Knight et al. 2013).We hope that the detailed biometric and molecular data of topotypic material of Pethia setnai will aid in the reliable identification of this threatened taxon, and the information on its distribution and threats will be helpful in directing future conservation efforts.

Figure 1 .
Figure 1.Phylogenetic position of Pethia setnai.Maximum likelihood tree based on mitochondrial cytb gene.Bootstrap support with more than 50% based on 1000 iterations for maximum likelihood.Garra species are used as outgroup.