Journal of Threatened Taxa | www.threatenedtaxa.org | 26 September 2026 | 18(9): 29729–29735

 

ISSN 0974-7907 (Online) | ISSN 0974-7893 (Print) 

https://doi.org/10.11609/jott.10249.18.9.29729-29735

#10249 | Received 11 November 2025 | Final received 09 June 2026| Finally accepted 05 September 2026

 

 

DNA Barcode based species delineation within Freshwater Garfish Xenentodon cancila (Hamilton, 1822) (Beloniformes: Belonidae) reveals the existence of multiple lineages under single nominal taxon

 

Ranjit More 1 , Jiwan Sarwade 2   , A. Shabnam 3  & K.P. Dinesh 4  

 

1 Department of Zoology, Pratapsinh Mohite-Patil Mahavidyala, Near Girdhardas Devi Highschool, Pune Road Tal Karmala, Solapur District, Maharashtra 413203, India.

2 Department of Zoology, Arts, Science and Commerce College Indapur, Pune-Solapur Road (NH 65), Indapur, Pune, Maharashtra 413106, India.

3,4 Zoological Survey of India, Western Regional Centre, Vidya Nagar, Sector-29, P.C.N.T. (PO), Rawet Road, Akurdi, Pune, Maharashtra 411044, India.

1 zoologistranjit@gmail.com (corresponding author),2 j.sarwade@rediffmail.com, 3 a.shabnam1312@gmail.com, 4 kpdinesh.zsi@gmail.com

 

 

 

Editor: Mandar Paingankar, Government Science College, Gadchiroli, India.          Date of publication: 26 September 2026 (online & print)

 

Citation: More, R., Jiwan Sarwade, A. Shabnam & K.P. Dinesh (2026). DNA Barcode based species delineation within Freshwater Garfish Xenentodon cancila (Hamilton, 1822) (Beloniformes: Belonidae) reveals the existence of multiple lineages under single nominal taxon. Journal of Threatened Taxa 18(9): 29729–29735. https://doi.org/10.11609/jott.10249.18.9.29729-29735

  

Copyright: © More et al. 2026. Creative Commons Attribution 4.0 International License. JoTT allows unrestricted use, reproduction, and distribution of this article in any medium by providing adequate credit to the author(s) and the source of publication.

 

Funding: None.

 

Competing interests: The authors declare no competing interests.

 

Author details: Ranjit More is Assistant Professor and Head, Department of Zoology, Pratapsinh Mohite-Patil Mahavidyalaya, Karmala, Solapur (MS) 413203, India, working as a fisheries biologist specializing in taxonomy, ecology and conservation of freshwater ichthyofauna. Jiwan Sarwade is Principal, Arts, Science and Commerce College, Indapur, Pune, Maharashtra, India. A. Shabnam is a as Research Associate at Zoological Survey of India and works on cockroach taxonomy with expertise in DNA barcoding and molecular phylogenetics. K.P. Dinesh is an amphibian taxonomist with expertise in DNA barcoding and molecular phylogenetics and scientist at the Zoological Survey of India.

 

Author contributions: RM and JS designed the experiment. RM conducted the practical work. AS and KPD collected and analyzed the molecular data. RM, AS, and KPD contributed to manuscript writing. All authors have read and approved the final manuscript.

 

Acknowledgements: RM and JS thank the principal and head of the Department of Zoology, Modern College, Pune, and Arts, Science and Commerce College, Indapur. RM is grateful to SARTHI for financial support during the study period. The Maharashtra State Biodiversity Board for granting permission to access biological resources (No. MSBB/Research/CR 622/615/2019-2020) is duly acknowledged. AS and KPD thank the director, Zoological Survey of India, Kolkata, and the officer-in-charge, ZSI WRC, Pune, for their support. We acknowledge Sahil Shikalgar, research fellow, ZSI, WRC, Pune for help in photography.

 

 

Abstract: The present correspondence provides the first DNA barcode-based evidence of multiple evolutionary lineages within the freshwater garfish Xenentodon cancila across South Asia. Taxonomically validated specimens from the Ujani Reservoir, Maharashtra, India, were analyzed with 77 publicly available mt COI sequences. Phylogenetic and species delimitation analyses revealed seven distinct clades within Xenentodon, substantially exceeding the currently recognized two species. The Ujani population formed a strongly supported monophyletic cluster with sequences from India, Bangladesh, and Korea, corresponding closely to the nominal X. cancila. Other geographically structured lineages from southern, northeastern, and central India, exhibit deep genetic divergence, indicating potential undescribed taxa. These findings highlight persistent taxonomic gaps within Xenentodon and emphasize the need for integrative revision combining molecular and morphological datasets.

 

Keywords: Cryptic diversity, freshwater biodiversity, garfish, integrative taxonomy, morphological datasets, phylogeny, polyphyletic, Ujani Reservoir.

 

 

 

INTRODUCTION

 

DNA barcoding is a widely used molecular tool, enabling rapid species identification and discovery of cryptic diversity in fishes (Hajibabaei et al. 2007; Stein et al. 2014; Rahman et al. 2019).  Although extensive DNA barcode datasets are available through global repositories such as BOLD (Ratnasingham & Hebert 2007) and GenBank, significant geographic gaps persist across Asia, resulting in taxonomic uncertainties in many Indian fish species.

The family Belonidae (order Beloniformes) comprises 47 species in 10 genera (Fricke et al. 2018). Among them, the southern and southeastern Asia endemic genus Xenentodon includes two recognized species, Xenentodon cancila (Hamilton, 1822) and X. canciloides (Bleeker, 1854).  X. cancila, originally described from the Ganga River, India, is widely distributed in India, Bangladesh, Cambodia, Malaysia, Nepal, Sri Lanka, Bhutan, Laos, Myanmar, Pakistan, Vietnam, Hawaii, Hong Kong, Indonesia, and Thailand (Hossain et al. 2013), while X. canciloides is restricted to Cambodia, Indonesia, Laos, Thailand, and Vietnam  (Froese and Pauly 2026).

The freshwater garfish X. cancila is an economically important fish in southern Asia and is widely traded in regional fisheries (Islam & Dutta 2018). Despite its broad distribution range and commercial significance, molecular studies have been limited and geographically fragmented (Thapliyal et al. 2013; Khedkar et al. 2014; Patil et al. 2018), resulting in its poorly resolved genetic identity. Against this backdrop, the present study examines the genetic identity of morphologically verified X. cancila specimens from Ujani Reservoir, Maharashtra, India, using mitochondrial Cytochrome C Oxidase subunit 1 (mt COI) barcodes to assess species monophyly of X. cancila through single-gene-based phylogenetic analysis. The phylogenetic pattern recovered are discussed and ascertained through ASAP species-delimitation analysis to delineate the potential existence of cryptic species within Xenentodon.

                  

 

MATERIALS AND METHODS

 

Specimens were collected and studied from the Ujani Reservoir, Maharashtra, India, during 2018–2021. The Identity of the specimens as Xenentodon cancila was confirmed by a taxonomic expert at the Zoological Survey of India. The studied specimens were deposited in the Zoology Museum, Modern College, Pune, and the Arts, Science and Commerce College, Indapur (ICASC/ZOO/105–106/2021–22). Fresh specimens were photographed (Image 1) before DNA-barcoding studies.

Genomic DNA was extracted from white muscle or fin clips using the DNeasy Blood & Tissue Kit (Qiagen), following the manufacturer’s protocol. DNA quality was assessed using a Qubit 2.0 fluorometer, and samples above 50 ng were amplified with universal mitochondrial Cytochrome C Oxidase subunit 1 (mt COI) primers. PCR was performed in a 25 µL reaction volume under the following thermal-cycling conditions: 95 ⁰C for three min; 35 cycles of 95 ⁰C for 30 s, 50 ⁰C for 30 s, 72 ⁰C for 45 s; final extension at 72 ⁰C for three min (Patil et al. 2018). Amplified ~700 bp COI fragments were bidirectionally sequenced at Barcode Biosciences, Bengaluru, India.

Raw sequence files were manually checked for quality to be used for further analysis. Additionally, 77 sequences of Belonidae were downloaded from GenBank, cleaned, and aligned in MEGA X (Kumar et al. 2018). Maximum--Likelihood (ML) analysis was performed with 83 sequences (Table 1) in the IQ-TREE v1.6.12 web server (Trifinopoulos et al. 2016), with 1,000 ultrafast bootstraps under the GTR+F+I+G4 model selected by Bayesian information criterion (BIC). The obtained tree was visualized in FigTree v1.4. The same dataset was subjected to species-delimitation analysis using the Assemble Species by Automatic Partitioning (ASAP) method (Puillandre et al. 2021). Sequences from this study were submitted to GenBank under accessions OM865416–OM865419.

 

 

RESULTS AND DISCUSSION

 

The mt COI phylogenetic analysis of Belonidae revealed multiple lineages within Xenentodon, although with low bootstrap support indicating polyphyly of the genus (Image 2). However, X. cancila sequence from the Ujani Reservoir formed monophyletic clade with sequences from India, Bangladesh, and Korea. Overall, seven distinct clades were recovered, far beyond the two currently recognized Xenentodon species. Species delimitation using ASAP (Image 3) corroborated the phylogenetic pattern, supporting the existence of multiple independently evolving lineages.

Although mt COI inference has limited utility for resolving deeper phylogenetic relationships, it remains valuable marker for detecting lineage diversity and identifying taxonomic hypotheses for further taxonomic revisions (Kalawate et al. 2022). Notably, most publicly available Xenentodon sequences are from India but lack associated voucher-specimen data, limiting taxonomic assignment with confidence.

Type locality of X. cancila is the Ganga River, India (Hamilton 1822), whereas that of X. canciloides is the Kapuas River, Indonesia (Bleeker 1854) (Image 4). Considering the proximity of sequences origin location and respective type localities tentatively Clade I can be assigned to X. cancila (sequences from India and Bangladesh) (Images 2 & 3) and Clade III to X. canciloides (sequences from Thailand) tentatively (Images 2 & 3). The Remaining lineages corresponding to Clade II (from Maharashtra, India), Clade IV (from Kerala and Tamil Nadu, India), Clade V (from Manipur, India), Clade VI (from Uttarakhand, India), and Clade VII (from Arunachal Pradesh, West Bengal, India) (Images 2 & 3) likely represent undescribed species, warranting ‘Integrative Taxonomic Studies’ with broader sampling from respective localities and taxonomic revision of the group.

 

 

CONCLUSION

 

The genetic heterogeneity among populations suggests that current knowledge of species distribution and diversity is far from complete. This brings into attention multiple lineages and the need to revisit and re-evaluate riverine and watershed-based biogeographic patterns of Xenentodon species across southern Asia, primarily in India and Bangladesh, to better understand the diversity, distribution, evolutionary relationships, and ecology.

 

Data availability statement

All relevant data supporting the findings of this study are included within the manuscript. No additional datasets were generated or analyzed during the current study.

 

Table 1. Sequences details of 600 bps fragment of mt COI gene data used in maximum likelihood IQ tree construction.

 

Accessions

Name in GenBank

Locality as per GenBank

References

1

OM865416.1

Xenentodon cancila

Ujani reservoir, Maharashtra, India

This study

2

OM865417.1

X. cancila

Ujani reservoir, Maharashtra, India

This study

3

OM865418.1

X. cancila

Ujani reservoir, Maharashtra, India

This study

4

OM865419.1

X. cancila

Ujani reservoir, Maharashtra, India

This study

5

MH087053.1

X. cancila

Bangladesh

Unpublished

6

MK359874.1

X. cancila

Korea

Unpublished

7

KF742433.1

X. cancila

Maharashtra, India

Unpublished

8

MK359936.1

X. cancila

Korea

Unpublished

9

MK359971.1

X. cancila

Korea

Unpublished

10

MK572631.1

X. cancila

Moulvibazar, Sylhet, Bangladesh

Rahman et al. 2019

11

MK814506.1

X. cancila

Rajasthan, India

Unpublished

12

MK814492.1

X. cancila

Rajasthan, India

Unpublished

13

JX983511.1

X. cancila

Narmada river, Dindori, MP, India

Khedkar et al. 2014

14

JX983513.1

X. cancila

Narmada river, Dindori, MP, India

Khedkar et al. 2014

15

KX399218.1

X. cancila

Tripura, India

Unpublished

16

KX399217.1

X. cancila

Tripura, India

Unpublished

17

KX399216.1

X. cancila

Tripura, India

Unpublished

18

JX983512.1

X. cancila

Narmada river, Dindori, MP, India

Khedkar et al. 2014

19

KU685520.1

X. cancila

Tripura, India

Unpublished

20

MK814522.1

X. cancila

Rajasthan, India

Unpublished

21

KX399222.1

X. cancila

Tripura, India

Unpublished

22

KU685522.1

X. cancila

Tripura, India

Unpublished

23

KX399223.1

X. cancila

Tripura, India

Unpublished

24

KX399221.1

X. cancila

Tripura, India

Unpublished

25

KU685524.1

X. cancila

Tripura, India

Unpublished

26

KX399220.1

X. cancila

Tripura, India

Unpublished

27

KX399219.1

X. cancila

Tripura, India

Unpublished

28

KU685521.1

X. cancila

Tripura, India

Unpublished

29

KU685523.1

X. cancila

Tripura, India

Unpublished

30

JX260995.1

X. cancila

Maharashtra, India

Unpublished

31

MK572630.1

X. cancila

Bangladesh

Rahman et al. 2019

32

MK572629.1

X. cancila

Bangladesh

Rahman et al. 2019

33

KX946829.1

X. cancila

Maharashtra, India

Patil et al. 2018

34

KX946828.1

X. cancila

Maharashtra, India

Patil et al. 2018

35

MK049515.1

X. cancila

Thailand

Panprommin et al. 2019

36

MK049514.1

X. cancila

Thailand

Panprommin et al. 2019

37

MK049516.1

X. cancila

Thailand

Panprommin et al. 2019

38

KU569073.1

Xenentodon sp.

South Africa

Unpublished

39

MK348178.1

X. cancila

Ashtamudi lake, Kerala, India

Unpublished

40

MH377838.1

X. cancila

Kerala, India

Unpublished

41

MH377837.1

X. cancila

Kerala, India

Unpublished

42

MH377836.1

X. cancila

Kerala, India

Unpublished

43

MK348194.1

X. cancila

Ashtamudi lake, Kerala, India

Unpublished

44

MK348176.1

X. cancila

Ashtamudi lake, Kerala, India

Unpublished

45

KX826913.1

X. cancila

Tamil Nadu, India

Unpublished

46

MG923408.1

X. cancila

Vembanad Lake, Kerala, India

Unpublished

47

MG923407.1

X. cancila

Vembanad Lake, Kerala, India

Unpublished

48

MK348177.1

X. cancila

Ashtamudi lake, Kerala, India

Unpublished

49

MG923406.1

X. cancila

Vembanad Lake, Kerala, India

Unpublished

50

EF607569.1

S. strongylura

China

Unpublished

51

KF715035.1

Tylosurus gavialoides

Philippines

Unpublished

52

EF607566.1

Strongylura leiura

China

Zhang 2011

53

FJ237566.1

Strongylura leiura

India

Lakra et al. 2011

54

GU674374.1

Tylosurus crocodilus

Indonesia

Unpublished

55

GU674427.1

Ablennes sp.

Indonesia

Unpublished

56

MT323766.1

Ablennes hians

Gulf of Mexico

Unpublished

57

KC970513.1

Tylosurus acus

Philippines

Unpublished

58

MH377844.1

Tylosurus acus melanotus

India

Unpublished

59

GU702391.1

Tylosurus acus acus

Brazil

Unpublished

60

KY176687.1

Tylosurus acus imperialis

Turkey

Unpublished

61

GU225490.1

Strongylura timucu

Mexico

Unpublished

62

HQ937019.1

Strongylura marina

USA

April et al. 2011

63

KX245130.1

X. cancila

India

Unpublished

64

KX245129.1

X. cancila

India

Unpublished

65

JN965212.1

X. cancila

Dehradun, India

Thapliyal et al. 2013

66

FJ459540.1

X. cancila

India

Unpublished

67

FJ459539.1

X. cancila

India

Unpublished

68

FJ459538.1

X. cancila

India

Unpublished

69

FJ459541.1

X. cancila

India

Unpublished

70

MT812048.1

X. cancila

Arunachal Pradesh, India

Unpublished

71

MN096221.1

X. cancila

West Bengal, India

Unpublished

72

MT812049.1

X. cancila

Arunachal Pradesh, India

Unpublished

73

MT812050.1

X. cancila

Arunachal Pradesh, India

Unpublished

74

MT812052.1

X. cancila

Arunachal Pradesh, India

Unpublished

75

MT812051.1

X. cancila

Arunachal Pradesh, India

Unpublished

76

GU225484.1

Strongylura notata

Mexico

Unpublished

77

JQ843010.1

Platybelone argala

Trinidad and Tobago

Weigt et al. 2012

78

MF124076.1

Tylosurus choram

Israel

Unpublished

79

JF494139.1

Petalichthys capensis

South Africa

Unpublished

80

KJ768216.1

Belone belone

Portugal

Landi et al. 2011

81

KC500332.1

Belone belone

Turkey

Keskin & Atar 2014

82

MK572415.1

Oryzias dancena

Bangladesh

Rahman et al. 2019

83

MK572416.1

Oryzias dancena

Bangladesh

Rahman et al. 2019

 

 

FOR IMAGES - - CLICK HERE FOR FULL PDF

 

REFERENCES

 

April, J. et al. (2011). Genetic calibration of species diversity among North America’s freshwater fishes. Proceedings of the National Academy of Sciences 108(26): 10602–10607. https://doi.org/10.1073/pnas.1016437108

Bleeker, P. (1854). Vijfde bijdrage tot de kennis der ichthyologische fauna van Celebes. Natuurkundig Tijdschrift voor Nederlandsch Indië 7: 225–260.

Fricke, R. et al. (2018). Catalog of fishes: genera, species, references. California Academy of Sciences, San Francisco, CA, USA. http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatmain.asp. Accessed on 16.x.2025.

Froese, R. & D. Pauly (eds.) (2026). FishBase. www.fishbase.org. Accessed in vi.2026.

Hamilton, F. (1822). An account of the fishes found in the river Ganges and its branches, Vol. 1. Archibald Constable, Edinburgh.

Hajibabaei, M. et al. (2007). DNA barcoding: how it complements taxonomy, molecular phylogenetics and population genetics. Trends in Genetics 23(4): 167–172. https://doi.org/10.1016/j.tig.2007.02.001

Hossain, M.Y. et al. (2013). Life-history traits of the freshwater garfish Xenentodon cancila (Hamilton, 1822) (Belonidae) in the Ganges River, northwestern Bangladesh. Sains Malaysiana 42(9): 1207–1218. https://www.semanticscholar.org/paper/Life-history-traits-of-the-freshwater-garfish-1822)-Hossain-Jewel/af3b257c2386728d31c58e912dd307a34ed91698

Islam, M.S. & P. Dutta (2018). Food, feeding habit and reproductive biology of freshwater garfish (Xenentodon cancila) from south-western Bangladesh: implications to fishery management. Journal of Fisheries and Life Sciences 3(2): 26–33.

Jayaram K.C. (1999). The freshwater fishes of the Indian region.  Narendra Publishing House, Delhi, 551 pp.

Kalawate, A.S. et al. (2022). Morphological characterization and mt DNA barcode of a tiger moth species, Asota ficus (Fabricius, 1775) (Lepidoptera: Noctuoidea: Erebidae: Aganainae) from India. Journal of Threatened Taxa 14(1): 20503–20510. https://doi.org/10.11609/jott.7638.14.1.20503-20510

Keskin, E. & H.H. Atar (2013). DNA barcoding commercially important fish species of Turkey. Molecular Ecology Resources 13(5): 788–797. https://doi.org/10.1111/1755-0998.12120

Khedkar, G.D. et al. (2014). DNA barcodes for the fishes of the Narmada, one of India’s longest rivers. PLoS ONE 9(7): e101460. https://doi.org/10.1371/journal.pone.0101460

Kumar, S. et al. (2018). MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution 35(6): 1547–1549. https://doi.org/10.1093/molbev/msy096

Lakra, W.S. et al. (2011). DNA barcoding Indian marine fishes. Molecular Ecology Resources 11(1): 60–71. https://doi.org/10.1111/j.1755-0998.2010.02894.x

Landi, M. et al. (2014). DNA barcoding for species assignment: the case of Mediterranean marine fishes. PLoS ONE 9(9): e106135. https://doi.org/10.1371/journal.pone.0106135

Panprommin, D. et al. (2019). DNA barcodes for the identification of species diversity in fish from Kwan Phayao, Thailand. Journal of Asia-Pacific Biodiversity 12(3): 382–389. https://doi.org/10.1016/j.japb.2019.05.003

Patil, T.S. et al. (2018). DNA barcode based delineation of freshwater fishes from northern Western Ghats of India, one of the world’s biodiversity hotspots. Biodiversity and Conservation 27(13): 3349–3371. https://doi.org/10.1007/s10531-018-1604-0

Puillandre, N. et al. (2021). ASAP: assemble species by automatic partitioning. Molecular Ecology Resources 21(2): 609–620. https://doi.org/10.1111/1755-0998.13281

Rahman, M.M. et al. (2019). Building a DNA barcode library for the freshwater fishes of Bangladesh. Scientific Reports 9: 9382. https://doi.org/10.1038/s41598-020-58810-0

Ratnasingham, S. & P.D.N. Hebert (2007). BOLD: The Barcode of Life Data System. Molecular Ecology Notes 7(3): 355–364. https://doi.org/10.1111/j.1471-8286.2007.01678.x

Stein, E.D. et al. (2014). Is DNA barcoding actually cheaper and faster than traditional morphological methods: results from a survey of freshwater bioassessment efforts in the United States? PLoS ONE 9(4): e95525. https://doi.org/10.1371/journal.pone.0095525

Thapliyal, M. et al. (2013). DNA barcoding of fishes from River Song, Dehradun, Uttarakhand using mitochondrial cytochrome-c Oxidase-I gene. Environment Conservation Journal 14(3): 113–121. https://doi.org/10.36953/ECJ.2013.14320

Trifinopoulos, J. et al. (2016). W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Research 44: W232–W235. https://doi.org/10.1093/nar/gkw256

Weigt, L.A. et al. (2012). Using DNA barcoding to assess Caribbean reef fish biodiversity: expanding taxonomic and geographic coverage. PLoS ONE 7(7): e41059. https://doi.org/10.1371/journal.pone.0041059

Zhang, J. (2011). Species identification of marine fishes in China with DNA barcoding. Evidence-Based Complementary and Alternative Medicine 2011: 978253. https://doi.org/10.1155/2011/978253