Communication Journal of Threatened Taxa | www.threatenedtaxa.org | 26 December 2013 | 5(17): 5214–5227 Western Ghats Special Series 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 ISSN Online 0974–7907 Print 0974–7893 OPEN ACCESS Unmesh Katwate 1, Mandar S. Paingankar 2, Shrikant Jadhav 3 & Neelesh Dahanukar 4 1 Bombay Natural History Society (BNHS), Hornbill House, Opposite Lion Gate, Shaheed Bhagat Singh Road, Mumbai, Maharashtra 400001, India 2,4 Systemaics, Ecology & Conservaion Laboratory, Zoo Outreach Organizaion (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 4 Indian insitute of Science Educaion 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) Abstract: Pethia setnai is an endemic and threatened freshwater ish of the Western Ghats of India. It has a restricted distribuion in the west lowing rivers in the states of Maharashtra, Goa and Karnataka. We clarify the phylogeneic posiion of Pethia setnai, provide osteological details of topotypic material, and morphometric data of specimens from Maharashtra, Goa and Karnataka. We also provide details on micro-level distribuion, habitat and threats to the species in its naive range. Keywords: Biometrics, Punius setnai, threatened, topotype. DOI: htp://dx.doi.org/10.11609/JoTT.o3857.5214-27 | ZooBank: urn:lsid:zoobank.org:pub:4F75DBB8-C599-402B-A9D1-C63D5CDD9DE7 Editor: Anonymity requested. Date of publicaion: 26 December 2013 (online & print) Manuscript details: Ms # o3857 | Received 21 November 2013 | Final received 17 December 2013 | Finally accepted 18 December 2013 Citaion: Katwate, U., M.S. Paingankar, S. Jadhav & N. Dahanukar (2013). Phylogeneic posiion and osteology of Pethia setnai (Chhapgar & Sane, 1992), an endemic barb (Teleostei: Cyprinidae) of the Western Ghats, India, with notes on its distribuion and threats. Journal of Threatened Taxa 5(17): 5214–5227; htp://dx.doi. org/10.11609/JoTT.o3857.5214-27 Copyright: © Katwate et al. 2013. Creaive Commons Atribuion 3.0 Unported License. JoTT allows unrestricted use of this aricle in any medium, reproducion and distribuion by providing adequate credit to the authors and the source of publicaion. Funding: Neelesh Dahanukar is a DST-INSPIRE Faculty Fellow. This study was parially supported by DST-INSPIRE Research Grant [IFA12-LSBM-21], Government of India to Neelesh Dahanukar and CEPF-ATREE Western Ghats Small Grant (CEPF-ATREE-WGhats/SGP/WGSG186 – BNHS_FISHES) to Unmesh Katwate. Compeing interests: Declared none. Funding source had no role in study design, data collecion, results interpretaion and manuscript wriing. Author Contribuion: UK, MP and ND collected fresh specimens. UK, SJ and ND studied museum specimens. UK, MP, SJ and ND performed morphometry. UK performed osteology. MP and ND performed geneic analysis. UK, MP, SJ and ND wrote the manuscript. Acknowledgements, Author Details: see end of this aricle. This aricle forms part of a special series on the Western Ghats of India, disseminaing the results of work supported by the Criical Ecosystem Partnership Fund (CEPF), a joint iniiaive of l’Agence Française de Développement, Conservaion Internaional, the European Commission, the Global Environment Facility, the Government of Japan, the MacArthur Foundaion and the World Bank. A fundamental goal of CEPF is to ensure civil society is engaged in biodiversity conservaion. Implementaion of the CEPF investment program in the Western Ghats is led and coordinated by the Ashoka Trust for Research in Ecology and the Environment (ATREE). 5214 Phylogeneic posiion and osteology of Pethia setnai INTRODUCTION Two major hurdles in conservaion of threatened and endemic species, especially those that occur in biodiversity hotspots is the Wallacean shorfall (arising from the incomplete informaion regarding the distribuion of species) and Linnean shorfall (several species sill not formally described) (Bini et al. 2006). The Western Ghats region, part of the Western GhatsSri Lanka biodiversity hotspot is no excepion to this, as it has already been suggested that the freshwater ish fauna (especially endemic and threatened species) of this region is poorly known with regard to their taxonomy and distribuion (Dahanukar et al. 2011; Raghavan et al. 2012). One reason for this gap in knowledge regarding geographical distribuion is the lack of detailed descripions and reliable geneic data, which can ulimately lead to misideniicaions. As a result, geneic sequences from topotypic material and detailed morphometric data will not only help in understanding the systemaics of the species, but also provide reliable ideniicaion criteria. In a recent review of the South Asian barbs previously placed under the genus Punius, Pethiyagoda et al. (2012) tentaively placed Punius setnai Chhapgar & Sane, 1992 in the genus Pethia based on the data available in the original descripion of the species. The tentaive placement was probably due to the limited informaion on the morphology, osteology and/or geneic data of the species. In the current study, we provide geneic informaion of Pethia setnai from topotypic material, osteological details to conirm the placement of the species under Pethia, and reliable geneic and morphometric data for accurate ideniicaion of the species. We also provide informaion on the distribuion, habitat and threats to the species. MATERIALS AND METHODS Study site Topotypes of Pethia setnai were collected from Salaulim River, a tributary of Zuari River in Sanguem (15.234⁰N & 74.182⁰E, 19m), Goa. Addiional material was also collected from Terekhol River at Madkhol Village (15.935⁰N & 73.910⁰E, 43m), Maharashtra (Fig. 1). Collecions were done responsibly following the guidelines set by IUCN (2008). Material collected in the present study is deposited in the museum of Bombay Natural History Society (BNHS), Mumbai; Wildlife Informaion Liaison Development (WILD), Coimbatore, Katwate et al. and the Zoological Survey of India, Western Regional Center (ZSI-WRC), Pune. 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 reporing the lateral line scales. Methods for taking counts and measurements follow Kullander (2008) and Pethiyagoda et al. (2012). Materials examined Pethia setnai: 9 exs., BNHS FWF 53, 63 to 70, 10.viii.2013, collected from Sanguem, Goa, by U. Katwate, M. Paingankar and N. Dahanukar; 3 exs., WILD13-PIS-043 to 045, 10.viii.2013, collected from Sanguem, Goa, by U. Katwate, M. Paingankar and N. Dahanukar; 2 exs., ZSI-WRC P/3567, 10.viii.2013, collected from Sanguem, Goa, by U. Katwate, M. Paingankar and N. Dahanukar; 9 exs., BNHS FWF 54 to 62, 12.vi.2013, collected from Terekhol River at Madkhol, Maharashtra, by U. Katwate and N. Dahanukar; 3 exs., WILD-13PIS-046 to 48, 12.vi.2013, collected from Terekhol River at Madkhol, Maharashtra, by Unmesh Katwate and Neelesh Dahanukar; 2 exs., ZSI-WRC P/3568, 12.vi.2013, collected from Terekhol River at Madkhol, Maharashtra, by U. Katwate and N. Dahanukar; 1 ex., ZSI-WRC P/3572, 5.ii.1971, collected from Kaneri River at Gund, District Karwar (Mysore), by B.S. Lamba and Party; 3 exs., ZSIWRC P/3571, 17.ii.1971, collected from Ramanguli, District Karwar (Mysore), by B. S. Lamba and Party; 26 exs. BNHS FWF 17-42, collected from Sanguem, Goa by S.R. Sane. Photographs examined Pethia setnai: Holotype, ZSI Kolkata FF2766, 01.iii.1985, collected from Sanguem, Goa, by S.R. Sane; Paratypes, 6 exs., ZSI Kolkata FF2767, 01.iii.1985, collected from Sanguem, Goa, by S.R. Sane. Pethia narayani: Syntypes, 2 exs., ZSI Kolkata F12180/1, collected from Cauvery River, Coorg, by C.R.N. Rao. Osteology Two specimens (BNHS FWF 55 and BNHS FWF 70) were cleared and stained using the procedure described by Pothof (1984). Osteological nomenclature follow Conway (2011) and descripions of osteology follow Pethiyagoda et al. (2012) so as to allow easy comparison Journal of Threatened Taxa | www.threatenedtaxa.org | 26 December 2013 | 5(17): 5214–5227 5215 Phylogeneic posiion and osteology of Pethia setnai 64 99 Katwate et al. AY708247 Pethia nigrofasciatus 99 EU604677 Pethia nigrofasciatus JF793612 Pethia nigrofasciatus 99 EU604673 Pethia bandula AY708244 Pethia bandula 52 99 JF793597 Pethia bandula EU604674 Pethia reval EU604675 Pethia cumingii 88 98 EU604676 Pethia cumingii 92 AY708249 Pethia cumingii 86 EU604679 Pethia ticto 99 EU604678 Pethia melanomaculatus AY708250 Pethia ticto KC696520 Pethia ticto 97 83 99 JQ795476 Pethia ticto 65 JQ795475 Pethia ticto EU241459 Pethia phutunio 55 JQ795488 Pethia conchonius 52 JQ795486 Pethia conchonius 83 AY004751 Pethia conchonius 99 JF793624 Pethia conchonius 92 EU241452 Pethia conchonius 50 KF977529 Pethia setnai Sanguem (Topotypic material) KF977530 Pethia setnai Sanguem (Topotypic material) 99 KF977531 Pethia setnai Terekhol 68 KF977532 Pethia setnai Terekhol EU241456 Pethia gelius JF793598 Puntius bimaculatus JF793620 Puntius titteya JQ795453 Haludaria fasciata EU241458 Haludaria melanampyx 99 JF793606 Haludaria fasciata 57 99 AY708262 Haludaria fasciata JF793616 Systomus sarana 82 99 JF793614 Systomus timbiri 72 JF793610 Systomus sp WHT8836 76 80 JF793609 Systomus martenstyni JF793613 Systomus pleurotaenia 99 JX975487 Dawkinsia filamentosa JF793617 Dawkinsia singhala JF793618 Dawkinsia srilankensis JX049981 Dawkinsia tambraparniei JX975490 Dawkinsia tambraparniei 99 99 EU241450 Dawkinsia arulius JX975488 Dawkinsia rohani JF793607 Dawkinsia filamentosa 99 99 JX975489 Dawkinsia exclamatio 99 JX470424 Sahyadria chalakkudiensis 66 99 JX311437 Sahyadria chalakkudiensis JX481182 Sahyadria chalakkudiensis 99 JX470431 Sahyadria denisonii JX470431 Sahyadria denisonii 99 99 GQ247559 Sahyadria denisonii 96 88 JX470421 Sahyadria denisonii GQ247558 Sahyadria denisonii JX470426 Sahyadria denisonii 99 99 JX470426 Sahyadria denisonii 98 JX470426 Sahyadria denisonii JF793600 Puntius thermalis 99 54 JF793601 Puntius chola JF793619 Puntius sophore JF793611 Puntius mahecola JF793604 Puntius kelumi JF793603 Puntius cf layardii 76 98 JF793605 Puntius dorsalis JF793623 Barbonymus schwanenfeldii JF793596 Osteochilichthys nashii 72 JF793608 Hypselobarbus jerdoni 73 JF793625 Hypselobarbus curmuca 56 JF793622 Tor khudree JF793594 Labeo dussumieri JF793592 Garra ceylonensis JF793593 Garra mullya 0.05 Figure 1. Phylogeneic posiion of Pethia setnai. Maximum likelihood tree based on mitochondrial cytb gene. Bootstrap support with more than 50% based on 1000 iteraions for maximum likelihood. Garra species are used as outgroup. 5216 Journal of Threatened Taxa | www.threatenedtaxa.org | 26 December 2013 | 5(17): 5214–5227 Phylogeneic posiion and osteology of Pethia setnai Katwate et al. with other related taxa. Osteological illustraions were made from images captured by a digital camera ited on stereo-zoom light microscope (Leica S8 APO, USA). of the phylogeneic tree was esimated using bootstrap values run for 1000 iteraions. Geneic analysis Gills were taken from four fresh specimens (BNHS FWF 53, BNHS FWF 54, WILD-13-PIS-043 and WILD13-PIS-046) and preserved in absolute ethanol. The issue was digested at 600C for two hours using the STE bufer (0.1M NaCl, 0.05M Tris-HCl, 0.01M EDTA, 1%SDS) with 15µl Proteinase K (20mg/ml) per 500ml of STE bufer. DNA was extracted using convenional phenolchloroform method and re-suspended in TE (10mM Tris, 1mM EDTA, pH 8) bufer. Polymerase chain reacion was performed to amplify cytochrome b (cytb) gene using primer pair L14724 (5’-GACTTGAAAAACCACCGTTG-3’) and H15915 (5’-CTCCGATCTCCGGATTACAAGAC-3’) (Chen et al. 2007). PCR reacion was performed in a 25µl reacion volume containing 5µl of template DNA (~200ng), 5µl of 10X reacion bufer (100 mM Tris pH 9.0, 500 mM KCl, 15 mM MgCl2, 0.1% Gelain), 3µl of 25mM MgCl2, 1µl of 10mM dNTPs, 1µl of each primer, 0.5µl Taq polymerase (2.5 units) and 8.5µl nuclease free water. The thermal proile was 10 minutes at 950C, and 35 cycles of 1 minute at 940C, 1 minute at 520C and 2 min at 720C, followed by extension of 10 min at 720C. Ampliied DNA fragments were puriied using the Wizard Gel and PCR clean up system (Promega, USA). The puriied PCR products were sequenced using BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, USA) and ABI prism 3730 sequencer (Applied Biosystems, USA). Sequences were analyzed by BLAST tool (Altschul et al. 1990). All sequences used in the paper have been deposited in GenBank. We used the cytb gene dataset of South Asian cyprinid ishes by Pethiyagoda et al. (2012), Raghavan et al. (2013a) and addiional sequences of Pethia, Dawkinsia and Haludaria available from NCBI GenBank (htp://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 it model for nucleoide subsituion was selected from 24 models available in MEGA 5 based on minimum Akaike Informaion Criterion (AIC) value (Posada & Crandall 2001). Tamura & Nei (1993) nucleoide subsituion model including invariant sites and a Gamma parameter was obtained as a best it model (TN93+G+I, AIC = 12894.38, lnL = -6328.70). This model was then used for construcing a phylogeneic tree using the maximum likelihood method. Reliability RESULTS AND DISCUSSION Before it was formally described, Pethia setnai collected from Ponda, Goa, was irst reported as Pethia nigrofasciata (Günther, 1868) by Yazdani (1977). Chhapgar & Sane (1980) criicized this report, ater collecing this species from Sanguem, Goa, as well as examining the specimens studied by Yazdani (1977), and suggested that the species was Pethia narayani (Hora, 1937). However, subsequently, Chhapgar & Sane (1992) described the species as Punius setnai. Subsequently, Yazdani & Ghate (1994) provided further informaion on this species based on the specimens collected from Hosangadi in Karnataka. Phylogeneic posiion Pethia setnai forms a monophyleic group (Fig. 1) with the genus Pethia sensu stricto as deined by Pethiyagoda et al. (2012). The geneic divergence in the cytb gene between the topotypic material collected from Sanguem, Goa and addiional 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 geneic divergence, the two populaions have to be considered as geneically similar. We could not collect fresh specimens of the species for geneic analysis from Karnataka State, and as a result, future studies are essenial to establish the extent of geneic variaion in diferent known populaions of the species. Nevertheless, as we are providing the geneic informaion of the topotypic material, future studies can have a more reliable comparaive material. Taxonomy Pethia setnai (Chhapgar & Sane, 1992) Punius nigrofasciata (non Günther, 1868): Yazdani (1977) Punius narayani (non Hora, 1937): Chhapgar & Sane (1980) Punius setnai Chhapgar & Sane, 1992: Chhapgar & Sane (1992), Yazdani & Ghate (1994), Knight et al. (2012) Pethia setnai (Chhapgar & Sane, 1992): Pethiyagoda et al. (2012) Journal of Threatened Taxa | www.threatenedtaxa.org | 26 December 2013 | 5(17): 5214–5227 5217 Phylogeneic posiion and osteology of Pethia setnai Katwate et al. Type material informaion Holotype and six paratypes of Pethia setnai collected from Sanguem, Goa, by S.R. Sane on 1.iii.1985 are available in good condiion in the museum collecion 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, Calcuta”. The whereabouts of remaining 18 paratypes is not known. Interesingly, 26 specimens of P. setnai are in the museum collecion of BHNS under the accession numbers BNHS FWF 17-42 (see Materials examined). However, whether some of the specimens from BNHS consitute the missing paratypes could not be deciphered. Image 1. Holotype of Pethia setnai ZSI Kolkata FF 2766. Image 2. Paratypes of Pethia setnai ZSI Kolkata FF 2767 5218 Journal of Threatened Taxa | www.threatenedtaxa.org | 26 December 2013 | 5(17): 5214–5227 Phylogeneic posiion and osteology of Pethia setnai Katwate et al. Table 1. Morphometric characters and merisics of Pethia setnai collected from Sanguem, Madkhol and Karwar. Raw data for all the measurements are provided in Appendix A, B and C. Character Madkhol (n = 12)b Sanguem (n = 12)a Karwar (n = 4)c Mean (sd) Range Mean (sd) Range Mean (sd) Range Total length (mm) 42.4 (6.0) 34.0–50.4 29.7 (4.7) 25.1–42.2 Caudal in damaged Caudal in damaged Standard length (SL, mm) 32.6 (4.8) 26.3–39.7 23.0 (3.6) 19.2–32.4 34.0 (3.2) 30.7–37.3 29.9 (1.3) 27.7–32.4 29.3 (0.9) 27.7–31.0 30.0 (2.3) 28.2–33.1 % SL Head length (HL) Head depth 24.6 (0.9) 22.7–25.6 24.6 (1.1) 23.0–26.8 22.8 (1.0) 21.9–23.8 Head width 15.8 (0.5) 14.8–16.6 16.5 (0.4) 15.6–16.9 16.3 (0.2) 16.0–16.5 Body depth 40.5 (3.0) 35.8–44.4 35.3 (1.4) 33.4–38.6 40.9 (1.5) 39.1–42.3 Body width at dorsal in origin 16.4 (1.1) 14.6–17.7 16.0 (1.5) 12.6–18.2 14.5 (0.6) 13.7–14.9 Body width at anal in origin 11.9 (0.8) 9.9–12.5 12.5 (0.9) 11.2–14.9 9.1 (0.7) 8.3–9.9 Pre dorsal distance 52.5 (2.0) 47.1–54.8 52.0 (1.8) 49.9–56.6 51.8 (4.0) 46.1–55.4 Dorsal to hypural distance 47.4 (1.6) 43.2–50.1 47.7 (2.1) 43.1–49.9 52.2 (2.8) 48.8–55.0 Prepelvic distance 50.2 (2.7) 43.3–53.9 52.7 (2.3) 49.4–57.4 48.7 (1.4) 47.2–50.6 Preanal distance 72.3 (2.6) 66.7–76.3 71.5 (1.7) 69.2–74.2 71.0 (1.4) 69.8–72.6 Prepectoral distance 28.4 (1.4) 25.6–30.4 31.5 (1.3) 29.2–33.8 31.3 (2.3) 28.2–33.9 Dorsal in length 26.8 (1.7) 24.1–30.1 26.1 (1.2) 23.8–27.3 27.4 (2.5) 24.8–29.8 Dorsal in spine length 18.9 (1.0) 16.8–20.6 17.6 (1.0) 15.7–18.9 18.3 (2.2) 16.0–20.3 Length of dorsal in base 18.6 (1.4) 16.3–20.3 16.9 (2.5) 12.0–19.6 19.1 (1.4) 17.4–20.3 Pectoral in length 20.7 (3.6) 10.0–23.5 18.4 (1.4) 16.5–21.4 21.9 (3.5) 19.2–27.0 Anal in depth 18.8 (1.8) 15.2–21.6 18.6 (1.0) 17.1–20.3 19.6 (1.2) 18.6–21.3 Caudal peduncle length 17.6 (1.4) 15.6–19.9 19.3 (0.8) 18.3–21.0 16.0 (1.2) 15.0–17.6 Caudal peduncle depth 15.0 (0.4) 14.4–15.5 14.6 (1.0) 11.9–15.4 16.1 (0.5) 15.5–16.7 % HL a Head depth 82.4 (5.6) 69.9–92.2 83.9 (4.0) 77.8–90.8 76.5 (8.8) 66.3–84.2 Head width 52.8 (3.4) 46.9–58.3 56.2 (2.6) 51.5–60.5 54.7 (4.0) 50.0–58.4 Snout length 29.8 (2.2) 25.2–33.7 27.4 (1.8) 24.6–29.9 32.7 (2.4) 30.0–35.4 Eye diameter 30.2 (2.8) 26.7–37.1 34.1 (3.0) 27.1–37.6 30.8 (2.7) 28.7–34.6 Inter orbital width 34.4 (4.4) 21.6–39.6 30.2 (2.9) 26.2–35.8 37.0 (3.3) 33.9–41.6 BNHS FWF 63 to 70; WILD-13-PIS-044, 045; ZSI-WRC P/3567; b BNHS FWF 55 to 62; WILD-13-PIS-047, 48; ZSI-WRC P/3568; c ZSI-WRC P/3572 and P/3571 Morphometric and merisic data Morphometric characters and merisics of the specimens collected for the present study from the type locality Sanguem, Goa; a newly discovered populaion from Terekhol River near Madkhol Village, Maharashtra and from Karwar, Karnataka are provided in Table 1 and Table 2, respecively. Photographs specimens collected for the present study are provided in Images 3 and 4. Table 2. Range for merisics of Pethia setnai (n = 28). Merisics Lateral line scales Transverse scale rows Predorsal scales 19-21 (3½-4½)-1-(3-3½) 7-8 Prepelvic scales 10-11 Preanal scales 14-16 Dorsal in rays Osteology Osteology of the specimen from the type locality is provided in Image 5. Gill rakers simple, acuminate (not Range Pectoral in rays iiI 8 i 12/13 Pelvic in rays i7 Anal in rays iii 5 Journal of Threatened Taxa | www.threatenedtaxa.org | 26 December 2013 | 5(17): 5214–5227 5219 Phylogeneic posiion and osteology of Pethia setnai Katwate et al. Image 4. Pethia setnai showing color in life. (a) fresh specimen from Sanguem, Goa; and (b) live specimen from Terekhol River near Madkhol, Maharashtra. branched or laminate); post-epiphysial fontanelle absent (Image 5b); infraorbital 3 deep, parially overlapping the cheek and preoperculum (Image 5c); last unbranched dorsal-in ray sif, serrated (Image 5d); free uroneural absent (Image 5e); 4 supraneurals; 13 precaudal and 13 caudal vertebrae. Osteological details of P. setnai matches with the diagnosis of the genus Pethia provided Image 3. Pethia setnai collected in the present study. a - Male from Sanguem, Goa, b - Female from Sanguem, Goa; c - unsexed individual from Madkhol, Maharashtra. Table 3. Distribuion of Pethia setnai. Laitude and longitude data is approximated using Google Earth from the locality details available in the references. River system Laitude (0N) Longitude (0E) Alitude (m) State Locaion Reference Maharashtra Madkhol Terekhol 15.94 73.91 43 Current study Goa Ponda Zuari 15.40 74.00 18 Yazdani (1977) Goa Sanguem Zuari 15.23 74.18 19 Chhapgar & Sane (1992); Current study Karnataka Karwar Kalinadi 15.08 74.51 20 Current study Karnataka Karwar Kalinadi 15.79 74.58 20 Current study Karnataka Souparnika Souparnika 13.87 74.81 76 Knight et al. (2012) Karnataka Hosangadi Varahi 13.68 74.97 58 Yazdani & Ghate (1994) Karnataka Agumbe Sitanadi 13.50 75.09 669 Knight et al. (2012) Karnataka Gundia Kumaradhara and Nethravai river systems 12.73 75.66 340 Gururaja et al. (2007) Karnataka Kukke Subramanya Kumaradhara and Nethravai river systems 12.66 75.61 126 Knight et al. (2012) 5220 Journal of Threatened Taxa | www.threatenedtaxa.org | 26 December 2013 | 5(17): 5214–5227 Phylogeneic posiion and osteology of Pethia setnai Katwate et al. b d Ep Pls H6 e H5 H4 F So Sc H3 Pa c H2 H1 So IO5 Ph IO1 IO4 PU3 PU2 CC IO2 Pop 5mm IO3 Image 5. Pethia setnai topotypic material from Sanguem, Goa (BNHS FWF 70, 32.1mm SL: (a) cleared and stained specimen; (b) dorsal view of orbital region of cranium (F, frontal; Pa, parietal;; Sc, supraorbital sensory canal); (c) circumorbital series (So, supraorbital; IO1-5, infraorbitals 1-5; Pop, preopercle); (d) last unbranched dorsal-in ray and (e) caudal skeleton (CC, compound centrum; Ep, epural; H1-6, hypurals 1-6; Ph, parhypural; Pls, pleurostyle; PU2-3, preural centra 2-3). Photo credit: Unmesh Katwate. by Pethiyagoda et al. (2012). Color variaion In life Pethia setnai has silvery-grey or dull golden color with three black transverse bands; anterior one situated between the dorsal proile and below the lateral line encompassing 3rd and 4th lateral line scales; middle band between the posterior half of the dorsal in base and one scale below lateral line encompassing 9th to 11th lateral line scale; posterior band between the middle of end of dorsal in base and caudal in and the posterior half of the anal in base (Image 4). Posterior most and anterior most bands are darker than the middle band (Image 4a). Dorsal, ventral and anal ins are bright red in certain specimens in their natural habitat (Image 4a) but the in colors fade rapidly in capivity (Image 4b). Some Journal of Threatened Taxa | www.threatenedtaxa.org | 26 December 2013 | 5(17): 5214–5227 5221 Phylogeneic posiion and osteology of Pethia setnai Katwate et al. O 22 N O 20 N 18ON O 16 N # Image 6. Syntypes of Pethia narayani ZSI Kolkata F 12180/1. # O 14 N # # # # O 12 N O 10 N Type locality Other records Western Ghats O 79 E 77OE 75OE 73OE O 71 E 69OE 8ON Figure 2. Distribuion of Pethia setnai. specimens have indigo blue inge on the dorsolateral area and therefore the species is also known as indigo barb among hobbyists. While, specimens with yellow ins are also known from this species we have not come across such specimens in our study. Distribuion Based on the current collecions as well as locality informaion deciphered from literature (Table 3), Pethia setnai is known to be distributed in the west lowing rivers of the Western Ghats in the states of Karnataka, Goa and Maharashtra between 12.66⁰N to 15.94⁰N laitude (Fig. 2). The species is currently known from seven fragmented populaions, most of them reported from lower alitudes (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 lowing rivers. Anecdotal reference to the presence of this species in the Cauvery, especially among the aquarium ish hobbyists, is as a result of the misideniicaion of Pethia setnai as Pethia narayani by Chhapgar & Sane (1980). While criicizing the report of Pethia nigrofasciata from Goa by Yazdani (1977), 5222 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 acive in the aquarium ish trade marketed the species occurring in Goa as Narayan’s Barb unil Chhapgar & Sane (1992) realized that they had overlooked the presence of osseous and serrated last unbranched dorsal in ray in the specimens from Goa (as opposed to non-osseous and non-serrated last unbranched dorsal in ray in Pethia narayani), and described Pethia setnai (B.F. Chhapgar, pers. comm. July 2013). Apart from having strongly osseous and serrated (vs. smooth ariculated) last unbranched dorsal in ray, P. setnai difers from P. narayani based on 8 (vs. 9) branched dorsal in rays, 5 (vs. 6) branched anal in 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 geneic data, the species is considered as restricted to west lowing rivers of the Western Ghats based on Yadav (2003) and informaion 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 informaion on the habitat in which the species was found. As part of the present study, fresh collecion 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 lowing, clear water stream with maximum substratum composed of sand and mud. Riparian cover was rich in Journal of Threatened Taxa | www.threatenedtaxa.org | 26 December 2013 | 5(17): 5214–5227 Phylogeneic posiion and osteology of Pethia setnai © Unmesh Katwate © Unmesh Katwate Katwate et al. vegetaion, and mostly dominated by Pundanus plant species. Pethia setnai was mostly found in small shoals with 7–8 individuals in each shoal. During each collecion atempt, maximum catch was dominated by females with one to two males in each shoal. Juveniles were observed to be conined at shallow depth (0.1–0.5 m), whereas adults were conined to greater depths (1–2 m). We also discovered a new populaion of P. setnai in the main river channel of Terekhol River at Madkhol Village (Image 8). In contrast with type locality, at Terekhol, the individuals of P. setnai were found in deep pools and fast lowing rapids (Image 8). Co-occurring species included Dawkinsia cf. ilamentosa, Haludaria pradhani, Devario aequipinnatus, Rasbora sp. and Salmophasia sp. Threats Based on IUCN Categories and Criteria (IUCN 2001), Dahanukar (2011) assessed Pethia setnai as a ‘Vulnerable’ species owing to its restricted distribuion and on-going threats to habitat because of tourism, urbanizaion and agricultural polluion. The Goa populaions are subjected to heavy mining operaions 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 staisics 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 ishes in India (Raghavan et al. 2013b). Further studies on the extent of collecion and trade in P. setnai is therefore essenial. Image 8. Habitat of Pethia setnai in Terekhol River at Madkhol Village, Sindhudurga District, Maharashtra [Image taken on 12.vi.2013] © Unmesh Katwate Image 7. Habitat in Zuari River near Sanguem, the type locality of Pethia setnai. [Image taken on 10.viii.2013] Image 9. Heavy mining operaion near type locality of Pethia setnai at Sanguem, Goa. A possible threat to Pethia setnai. [Image taken on 10.viii.2013] CONCLUSIONS A comprehensive assessment of the status of freshwater ishes of Western Ghats for the IUCN Red List of Threatened Species revealed that litle to no informaion exists regarding distribuion, populaion 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 distribuion of several threatened freshwater ishes 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 ideniicaion of this threatened taxon, and the informaion on its distribuion and threats will be helpful in direcing future conservaion eforts. 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Characters BNHS FWF 55 BNHS FWF 56 BNHS FWF 57 BNHS FWF 58 BNHS FWF 59 BNHS FWF 60 BNHS FWF 61 BNHS FWF 62 WILD13PIS047 WILD13PIS048 ZSIWRC P/3568 ZSIWRC P/3568 Total Length (mm) 42.20 32.17 30.30 32.74 26.15 27.53 25.18 25.10 30.47 29.11 29.13 26.56 Standard Length (mm) 32.44 25.34 23.79 25.20 20.37 20.94 19.17 19.79 23.81 22.26 22.63 20.15 Head length (mm) 8.97 7.48 6.91 7.11 6.01 6.09 5.95 5.94 7.12 6.42 6.85 5.83 Head depth (mm) 7.46 6.07 5.73 6.16 5.46 5.27 4.88 4.62 5.94 5.82 5.52 4.76 Head width (mm) 5.43 4.10 3.80 4.22 3.41 3.54 3.21 3.25 3.87 3.68 3.53 3.36 Snout length (mm) 2.55 1.93 1.73 2.01 1.64 1.82 1.54 2.00 1.98 1.78 1.96 1.74 Eye diameter (mm) 3.22 2.65 2.50 2.67 2.04 2.25 1.88 1.61 2.38 2.18 2.14 2.09 Inter orbital width (mm) 2.45 2.21 1.81 2.40 1.90 2.18 1.77 1.58 2.02 2.03 2.07 1.83 Body depth (mm) 12.51 9.12 8.23 9.20 7.04 7.18 6.92 6.73 7.96 8.07 7.94 6.98 Body width at dorsal in origin (mm) 5.89 4.14 3.90 4.32 3.35 3.45 2.42 2.68 3.73 3.80 3.53 3.35 Body width at anal in origin (mm) 4.83 3.08 2.84 3.23 2.57 2.64 2.15 2.26 2.93 2.84 2.80 2.52 Pre dorsal distance (mm) 16.58 13.07 12.07 12.62 10.53 10.88 10.85 10.48 11.87 11.8 12.05 10.26 Dorsal to hypural distance (mm) 15.97 12.64 11.67 12.54 9.93 9.78 8.26 8.99 11.02 10.66 10.68 9.97 Prepelvic distance (mm) 16.02 12.83 12.33 12.86 11.19 11.09 11.01 10.72 12.56 11.26 12.41 10.41 Preanal distance (mm) 23.55 18.32 16.75 17.71 14.38 15.53 13.94 14.6 16.59 15.41 16.40 14.11 Prepectoral distance (mm) 9.46 8.10 7.23 7.84 6.27 6.74 6.43 6.68 7.42 6.90 7.21 6.22 Dorsal in length (mm) 8.21 6.87 6.23 6.89 5.46 5.61 4.56 5.30 5.75 5.80 5.84 5.49 Dorsal in spine length (mm) 5.88 4.57 3.74 4.72 3.46 3.54 3.62 3.22 4.13 3.86 4.28 3.58 Length of dorsal in base (mm) 6.14 4.97 4.28 4.86 3.21 3.62 2.33 2.38 4.20 4.08 4.03 3.27 Pectoral in length (mm) 6.93 5.03 4.37 4.95 3.63 3.84 3.32 3.27 4.31 3.82 3.94 3.70 Anal in depth (mm) 6.23 5.14 4.33 5.11 3.73 3.76 3.31 3.39 4.21 4.18 4.23 3.80 Caudal peduncle length (mm) 6.47 4.64 4.53 4.85 4.06 4.09 4.02 3.74 4.42 4.16 4.15 4.01 Caudal peduncle depth (mm) 4.92 3.84 3.64 3.87 2.97 3.15 2.71 2.35 3.51 3.41 3.29 2.91 Journal of Threatened Taxa | www.threatenedtaxa.org | 26 December 2013 | 5(17): 5214–5227 5225 Phylogeneic posiion and osteology of Pethia setnai Katwate et al. Appendix B. Morphometry of specimens collected from Sanguem, Goa. BNHS FWF 63 BNHS FWF 64 BNHS FWF 65 BNHS FWF 66 BNHS FWF 67 BNHS FWF 68 BNHS FWF 69 BNHS FWF 70 WILD13PIS-044 WILD13PIS-045 ZSIWRC P/3567 Total Length (mm) 50.38 50.42 45.14 42.80 36.51 35.63 37.85 40.65 49.96 39.04 45.85 33.99 Standard Length (mm) 39.29 39.66 34.32 32.25 27.90 27.18 29.13 31.20 38.69 29.82 35.14 26.30 Characters ZSIWRC P/3567 Head length (mm) 10.90 12.04 10.08 9.97 8.06 7.87 9.06 9.47 11.14 9.03 10.53 8.53 Head depth (mm) 10.05 10.09 8.29 7.81 6.90 6.63 7.25 7.95 9.81 7.02 8.73 5.96 Head width (mm) 6.36 6.31 5.33 4.77 4.34 4.33 4.47 5.18 6.29 4.74 5.61 4.00 Snout length (mm) 3.54 3.65 2.87 2.94 2.34 2.38 3.05 2.39 3.29 2.63 3.31 2.44 Eye diameter (mm) 3.26 3.40 2.97 2.92 2.41 2.64 2.42 3.51 3.20 2.84 3.10 2.42 Inter orbital width (mm) 3.93 3.99 3.53 3.50 3.06 3.12 3.05 2.05 3.92 3.23 3.80 2.83 Body depth (mm) 17.34 17.61 14.19 12.98 10.91 11.11 10.78 11.17 16.72 11.69 15.36 9.62 Body width at dorsal in origin (mm) 6.95 7.01 5.62 4.94 4.90 4.31 4.44 4.88 6.85 4.74 5.97 3.83 Body width at anal in origin (mm) 4.90 4.95 4.12 3.89 3.17 3.32 3.40 3.09 4.82 3.69 4.37 2.88 Pre dorsal distance (mm) 20.48 21.73 17.60 17.32 14.59 14.80 15.12 14.69 20.25 15.60 18.92 14.01 Dorsal to hypural distance (mm) 18.91 18.41 16.53 14.91 13.99 13.06 13.94 13.49 18.27 14.26 16.66 12.57 Prepelvic distance (mm) 19.10 20.72 17.11 16.54 14.1 13.43 15.41 13.50 18.79 14.99 17.92 14.17 Preanal distance (mm) 27.98 29.28 24.59 24.24 19.83 19.36 21.41 20.81 28.05 20.90 25.99 20.07 Prepectoral distance (mm) 10.04 11.90 9.76 9.79 7.87 7.45 8.53 8.76 10.42 8.88 10.13 7.42 Dorsal in length (mm) 10.40 10.43 8.54 9.72 7.36 7.85 7.92 7.52 10.12 8.31 9.62 6.62 Dorsal in spine length (mm) 7.66 7.62 5.98 6.65 5.28 5.18 5.74 5.24 7.13 5.62 6.77 5.16 Length of dorsal in base (mm) 7.55 7.52 6.45 6.55 5.31 5.07 4.81 5.19 7.43 5.81 7.12 4.28 Pectoral in length (mm) 7.93 8.75 7.27 7.09 6.42 6.24 6.17 3.13 7.90 6.47 8.27 5.30 Anal in depth (mm) 7.43 6.58 6.63 6.98 5.42 5.33 5.16 4.73 7.44 6.21 7.04 4.65 Caudal peduncle length (mm) 6.26 6.62 5.96 5.82 5.27 4.94 5.80 5.10 6.02 5.81 6.14 4.55 Caudal peduncle depth (mm) 5.96 6.09 5.32 4.92 4.18 4.03 4.19 4.62 5.83 4.46 5.43 3.82 5226 Journal of Threatened Taxa | www.threatenedtaxa.org | 26 December 2013 | 5(17): 5214–5227 Phylogeneic posiion and osteology of Pethia setnai Katwate et al. Appendix C. Morphometry of specimens collected from Karwar, Karnataka. Characters ZSI-WRC P/3572 ZSI-WRC P/3571 ZSI-WRC P/3571 ZSI-WRC P/3571 Total Length (mm) Caudal in damaged Caudal in damaged Caudal in damaged Caudal in damaged Standard Length (mm) 37.28 36.1 31.76 30.73 Head length (mm) 11.33 11.95 8.95 8.68 Head depth (mm) 8.16 7.92 7.47 7.31 Head width (mm) 5.97 5.97 5.23 5 Snout length (mm) 4.01 4.03 2.84 2.6 Eye diameter (mm) 3.32 3.43 3.1 2.65 Inter orbital width (mm) 4.08 4.05 3.72 3.17 Body depth (mm) 15.66 14.6 13.42 12.01 Body width at dorsal in origin (mm) 5.55 4.93 4.73 4.45 Body width at anal in origin (mm) 3.11 3.38 3.16 2.65 Pre dorsal distance (mm) 19.94 20 16.54 14.16 Dorsal to hypural distance (mm) 20.51 17.6 16.22 16.57 Prepelvic distance (mm) 18.12 18.25 15.38 14.49 Preanal distance (mm) 26.8 26.21 22.18 21.46 Prepectoral distance (mm) 11.75 12.23 10 8.68 Dorsal in length (mm) 9.23 10.76 8.75 damaged Dorsal in spine length (mm) 5.96 6.69 6.44 damaged Length of dorsal in base (mm) 6.47 6.71 6.42 6.24 Pectoral in length (mm) 7.16 7.52 8.58 6.28 Anal in depth (mm) 7.1 6.98 5.92 6.55 Caudal peduncle length (mm) 5.6 6.34 5.1 4.67 Caudal peduncle depth (mm) 5.78 5.69 5.15 5.14 Author Details: Unmesh Katwate is a Zoology (Oceanography) postgraduate currently working as Scienist-A in BNHS. He works on conservaion, taxonomy and ecological aspects of freshwater ishes and amphibians of the Western Ghats. Mandar S. Paingankar is a molecular biologist and works on vector biology with an emphasis on host parasite interacions. He works on molecular phylogeny and systemaics of freshwater ishes as a hobby. Shrikant Jadhav is a scienist in Zoological Survey of India. He works on freshwater ish diversity and taxonomy. Neelesh Dahanukar works in ecology and evoluion with an emphasis on mathemaical and staisical analysis. He is also interested in taxonomy, distribuion paterns and molecular phylogeny of freshwater ish and amphibians. Acknowledgements: We are grateful to Abhijeet Gharat, Praik, Keerthi Krutha, Shreya Mulherkar, Saahil Acharya and Nikhil Modak for helping with the collecions. We thank Chetana Katwate for help in osteology and morphometric study. We thank Dr. Siby Philip for help with the phylogeneic analysis and Dr. B.F. Chhapgar for helpful discussions. We are grateful to Dr. Asad Rahmani, Director; Dr. Deepak Apte, COO; Rahul Khot, incharge Natural History Collecion department and Vithoba Hegde, senior ield assistant, for their help during study of the museum specimens and registraion of specimens in Bombay Natural History Society (BNHS), Mumbai. We are also grateful to Dr. Sanjay Molur, Founder Secretary; and Priyanka Iyer, curator of ish collecion, for holding our specimen vouchers in the museum collecion of Wildlife Informaion Liaison Development (WILD) Society, Coimbatore. We thank the Director and Oicer In-Charge (Collecions), Zoological Survey of India (ZSI), Kolkata for providing us the access to the type material of the species. We are also thankful to Dr. Pankaj Bhatnagar, Oicer-in-charge, Zoological Survey of India, Western Regional Center, Pune, for encouragement and support. Journal of Threatened Taxa | www.threatenedtaxa.org | 26 December 2013 | 5(17): 5214–5227 5227