• Dwi Anggorowati Rahayu Department of Biology, Faculty of Mathematic and Natural Sciences, Universitas Negeri Surabaya, Indonesia
  • Sunu Kuntjoro Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Negeri Surabaya, Surabaya 60231, Indonesia
  • Widowati Budijastuti Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Negeri Surabaya, Surabaya 60231, Indonesia
  • Winarsih Winarsih Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Negeri Surabaya, Surabaya 60231, Indonesia
  • Reni Ambarwati Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Negeri Surabaya, Surabaya 60231, Indonesia
  • Endik Deni Nugroho Department of Biology Education, Faculty of Science Education, ITSNU, Pasuruan 60231, Indonesia
  • Abdul Basith Indonesian Genetic and Biodiversity Community, Malang 67118, Indonesia
  • Nia Kurniawan Department of Biology, Faculty of Mathematics and Natural Sciences, Brawijaya University, Malang 65145, Indonesia
  • Haryono Haryono Research Center for Biosystematics and Evolution, National Reserearch and Innovation Agency (BRIN), Cibinong 16911, Indonesia




DNA barcoding, morphology, Nemacheilus, phylogenetic, taxonomy


The identities of two local loaches, Nemacheilus chrysolaimos (Valenciennes, 1846) and N. fasciatus (Valenciennes, 1846) from six rivers, were obtained through a comprehensive examination of their morphology and molecular characteristics in Biltar Regency, East Java, Indonesia. Therefore, this study identified Nemacheilus spp. from Blitar based on morphology and partial sequence of COI. The meristic data obtained for N. chrysolaimos included DII. 7–8 (dorsal fin), AI. 3–5 (anal fin), PI. 9 (pectoral fin), VI. 6–7 (ventral fin), and C. 17 (caudal fin). On the other hand, N. fasciatus exhibited the following meristic data, namely D II 7–8 (dorsal fin), AI. 6 (anal fin), PI. 9–10 (pectoral fin), VI. 6–7 (ventral fin), and C. 17 (caudal fin). A significant difference was observed in the morphometric characteristics of N. fasciatus across various sampling sites, as determined by the Kruskal-Wallis Test. Furthermore, the nucleotide base composition sequences of Nemacheilus spp. consisted of Thiamine (T), Cytosine (C), Adenine (A), and Guanine (G) with a mean of 29.565%, 32.023%, 23.88%, and 16.244%. Maximum Likelihood (ML) and Minimum Evolution (ME) phylogenetic analysis was also conducted using the Kimura 2 Parameter model to establish two major clades on Nemacheilus spp. and one out-group significantly different from the Nemacheilus spp. The results showed that these major clades exhibited a close relationship at 100% bootstrap support and were grouped under the genus Nemacheilus. The study on Nemacheilus spp. from the Blitar locality differentiated COI sequences between N. fasciatus and N. chrysolaimos. Additionally, N. chrysolaimos, as inferred from reference sequences, was identified as the ancestral species to N. chrysolaimos MZB 26540 and MZB 26539. ABGD analyses, employing a prior maximal distance of 0.025, also indicated the separation of these species into distinct partitions. The integration of morphology and genetic data for Nemacheilus spp. should provide valuable insights for future genetic population studies and conservation initiatives.


Download data is not yet available.


Ahmed MD, Datta SK, Zhilik AA. 2020. Molecular diversity of freshwater fishes of Bangladesh assessed by DNA barcoding. Bangladesh J Zool 48(1): 1-19.

Ath-thar MHF, Ambarwati A, Soelistyowati DT, Kristanto AH. 2018. Keragaman genotipe dan fenotipe ikan uceng Nemacheilus Fasciatus (Valenciennes, 1846) asal Bogor, Temanggung, dan Blitar. J Ris Akuakultur 13(1): 1-10.

Bleeker P. 1853. Diagnostische beschrijvingen van nieuwe of weinig bekende vischsoorten van Sumatra. [Diagnostic descriptions of new or little-known fish species from Sumatra]. Tiental V–X. Natuurk Tijdschr Ned.-Indië 4: 243-302.

Bleeker P. 1854. Overzigt der ichthyologische fauna van Sumatra, met beschrijving van eenige nieuwe soorten. [Overview of the ichthyological fauna of Sumatra, with descriptions of some new species]. Natuurk Tijdschr Ned.-Indië 7: 49-108.

Bleeker P. 1859. Negende bijdrage tot de kennis der vischfauna van Banka. [Ninth contribution to the knowledge of the fish fauna of Bangka]. Natuurk Tijdschr Ned.-Indië 18: 359-78.

Bleeker P. 1862–63. Atlas ichthyologique des Indes Orientales Néêrlandaises, publié sous les auspices du Gouvernement colonial néêrlandais. Tome II. Siluroïdes, Chacoïdes et Hétérobranchoïdes. [Fish Atlas of the Dutch East Indies, published under the auspices of the Dutch Colonial Government. Volume II. Siluroids, Chacoids and Heterobranchoids]. Amsterdam: F. Muller, 112 pp., Pls. 49–101. [pp. 1–32 (November 1862), pp. 33–64 (January 1863), pp. 65–96 (April 1863), pp. 97–112 (September 1863), plates published 1862–1863, see Kottelat (2013b), p. 283].

Bleeker P. 1863a. Sur les genres de la famille des Cobitioïdes. [On the genera of the Cobitioid family]. Verslagen en Mededeelingen der Koninklijke Akademie van Wetenschappen, Afdeeling Natuurkunde 15: 32-44.

Bleeker P. 1863b. Atlas ichthyologique des Indes Orientales Néêrlandaises. Tome III. Cyprins. [Fish Atlas of the Dutch East Indies. Volume III. Cyprines]. Amsterdam: Müller.

Bleeker, P. 1860. De visschen van den Indischen Archipel beschreven en toegelicht. Deel 2. Ordo Cyprini, karpers. [The fish of the Indian Archipelago described and explained. Part 2. Ordo Cyprini, Carp.]. Acta Soc Regiae Sci Indo-Neêrl 7(2): 1-492.

Boratyn GM, Camacho C, Cooper PS, Coulouris G, Fong A, Ma N, Madden TL, Matten WT, McGinnis SD, Merezhuk Y, Raytselis Y, Sayers EW, Tao T, Ye J, Zaretskaya I. 2013. BLAST: a more efficient report with usability improvements. Nucleic Acids Res 41: 29-33.

Buhay JE. 2009. “COI-like” sequences are becoming problematic in molecular systematic and DNA barcoding studies. J Crustac Biol 29(1): 96-110.

Cope ED. 1871. Contribution to the ichthyology of the Lesser Antilles. Trans Am Philos Soc, New Series, 14(3): 445-83.

Cuvier G, Valenciennes A. 1846. Histoire naturelle des poissons. Paris: Tome dix-huitième. Bertrand.

Duvaud S, Gabella C, Lisacek F, Stockinger H, Ioannidis V, Durinx C. 2021. Expasy, the Swiss Bioinformatics Resource Portal, as designed by its users. Nucleic Acids Res. 49(W1): W216-W227.

Felsenstein J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783-91.

Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. 1994. DNA primers for amplification of mitochondrial Cytochrome C Oxidase Subunit I from diverse metazoan invertebrates. Mol Marine Biol Biotechnol 3(5): 294-99.

Froese R, Pauly D. (Eds.) 2022a. FishBase. Family Nemacheilidae - Brook loaches. [cited 2022 October 01]. Available from: https://www.fishbase.se/summary/FamilySummary.php?ID=692#famList_tab

Froese R, Pauly D. (Eds.). 2022b. FishBase. Nemacheilus Bleeker, 1863. [cited 2022 October 22]. Available from https://www.marinespecies.org/ aphia.php?p=taxdetails&id=154189

Gunbin K, Peshkin L, Popadin K, Annis S, Ackermann RR, Khrapko K. 2017. Data on the time of integration of the human mitochondrial pseudogenes (NUMTs) into the nuclear genome. Data Br 13: 536-44.

Haas TC, Blumand MJ, Hein DC. 2010. Morphological responses of a stream fish to water impoundment. Biol Lett 6: 803-06.

Hadiaty RK, Yamahira K. 2014. The loaches of the genus Nemacheilus (Teleostei: Nemacheilidae) in Sunda Islands, with an identification key. Indones J Ichthyol 14: 1-18.

Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symp Ser 41: 95-98.

Hebert PDN, Cywinska A, Ball SL, deWaard JR. 2003. Biological identifications through DNA barcodes. Proc R Soc B: Biol Sci 270(1512): 313-321.

Hebert PDN, Stoeckle MY, Zemlak TS, Francis CM. 2004. Identification of Birds through DNA Barcodes. PLoS Biol 2(10): e312.

Hendry AP, Taylor EB, McPhail JD. 2002. Adaptive divergence and the balance between selection and gene flow: lake and stream stickleback in the misty system. Evolution 56: 1199-1216.

Hubert N, Hanner R, Holm E, Mandrak NE, Taylor E, Burridge M, Zhang J. 2008. Identifying Canadian freshwater fishes through DNA barcodes. PLoS ONE 3(6): 19.e2490.

Hubert N, Lumbantobing D, Sholihah A, Dahrudin H, Busson F, Sauri S, Keith, P. 2019. Revisiting species boundaries and distribution ranges of Nemacheilus spp. (Cypriniformes: Nemacheilidae) and Rasbora spp. (Cypriniformes: Cyprinidae) in Java, Bali and Lombok through DNA barcodes: implications for conservation in a biodiversity hotspot. Conserv Genet 20: 517-29.

Kelley JL, Davies PM, Collin SP, Grierson PF. 2017. Morphological plasticity in a native freshwater fish from semiarid Australia in response to variable water flows. Ecol Evo 7(16): 6595-6605.

Kimura M. 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16: 111-120.

Knebelsberger T, Dunz AR, Neumann D, Geiger MF. 2015. Molecular diversity of Germany's freshwater fishes and lampreys assessed by DNA barcoding. Mol Ecol Resour 15(3): 562-72.

Kottelat M, Freyhof, J. 2007. Handbook of European freshwater fishes. Berlin: Kottelat, Cornol & Freyhof.

Kottelat M, Whitten AJ, Kartikasari SN, Wirjoatmodjo S. 1993. Freshwater fishes of Western Indonesia and Sulawesi. Hong Kong: Periplus.

Kottelat M. 1984. Revision of the Indonesian and Malaysian loaches of the subfamily Nemacheilinae. Japanese J Ichthyol 31: 225-60.

Kottelat M. 2013a. The fishes of the inland waters of Southeast Asia: a catalog and core bibliography of the fishes known to occur in freshwaters, mangroves and estuaries. Raffles Bull Zool Suppl 27: 1-663.

Kottelat M. 2013b. Dates of publication of Bleeker's Atlas ichthyologique and Poissons de Madagascar. Zootaxa 3681(3): 281-85.

Kottelat M. 2022. Nemacheilus pezidion, a new species of loach from southern Laos (Teleostei: Nemacheilidae). Zootaxa 5129(1): 92-104.

Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Bio Evol 35: 1547-49.

Kusuma RO, Dadlono MS, Kusum B, Syakuri H. 2021. Genetic Diversity of Stone loaches (Nemacheilus) in River of Banyumas Area based on Cytochrome Oxidase Subunit I (COI). Jurnal Perikanan 23(2): 89-94.

Lakra WS, Singh M, Goswami M, Gopalakrishnan A, Lal KK, Mohindra V, Sarkar UK, Punia PP, Singh KV, Bhatt JP, Ayyappan S. 2015. DNA Barcoding Indian Freshwater Fishes. Mitochondrial DNA A: DNA Mapp Seq Anal 27(6): 4510-17.

Lara A, Ponce De León JL, Rodriguez R, Casane D, Cote G, Bernatchez L, García-Machado ERIK. 2010. DNA barcoding of Cuban freshwater fishes: evidence for cryptic species and taxonomic conflicts. Mol Eco Resour 10(3): 421-30.

Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG. 2007. Clustal W and Clustal X version 2.0. Bioinformatics (Oxford, England), 23(21): 2947-48.

Nei M, Kumar S. 2000. Molecular evolution and phylogenetics. Oxford University Press, New York.

Nei M. 1972. Genetic distance between populations. Am Nat 106: 283-92.

Panprommin D, Soontornprasit K, Tuncharoen S, Pithakpol S, Keereelang J. 2019. DNA barcodes for the identification of species diversity in fish from Kwan Phayao, Thailand. J Asia-Pac Biodivers 12(3): 382-89.

Puillandre N, Lambert A, Brouillet S, Achaz G. 2012. ABGD, Automatic Barcode Gap Discovery for primary species delimitation. Mol Ecol 21(8): 1864-77.

Rahayu DA, Nugroho ED, Listyorini D. 2019. DNA Barcoding ikan introduksi khas telaga sari, Kabupaten Pasuruan. [DNA Barcoding of Introduced Fish Typical of Telaga Sari, Pasuruan Regency]. Biotropika 7(2): 51-62.

Ratnasingham S, Hebert PD. 2007. BOLD: the Barcode of Life Data System (http://www.barcodinglife.org). Mol Ecol Notes 7(3): 355-64.

Regan CT. 1911. The classification of the teleostean fishes of the order Ostariophysii. – 1. Cyprinoidea. Annals and Magazine of Natural History, Series 8, 8(43): 13-32.

Roberts TR. 1993. The freshwater fishes of Java, as observed by Kuhl and van Hasselt in 1820–23. Zoologische Verhandelingen 285: 1-94.

Saitou N, Nei M. 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Bio Evol 4: 406-25.

Sayers EW, Cavanaugh M, Clark K, Pruitt KD, Schoch CL, Sherry ST, Karsch-Mizrachi I. 2022. GenBank. Nucleic Acids Res 50(D1): D161-64.

Šlechtová V, Musilova Z, Tan HK, Kottelat M, Bohlen, J. 2021. One northward, one southward: Contrasting biogeographical history in two benthic freshwater fish genera across Southeast Asia (Teleostei: Cobitoidea: Nemacheilus, Pangio). Mol Phylogenet Evol 161: 107139.

Song H, Buhay JE, Whiting MF, Crandall KA. 2008. Many species in one: DNA barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are coamplified. PNAS 105(36): 13486-91.

Steinke D, Zemlak TS, Boutillier JA, Hebert PD. 2009. DNA barcoding of Pacific Canada’s fishes. Mar Biol 156(12): 2641-47.

van Hasselt, J.C. 1823. Uittreksel uit enn' brief van den Heer J. C. van Hasselt, aan den Heer C. J. Temminck, geschreven uit Tjecande, Residentie Bantam, den 29sten December 1822. [Extract from another letter from Mr. J. C. van Hasselt, to Mr. C. J. Temminck, written from Tjecande, Residentie Bantam, on 29 December 1822]. Algemeene Konst- en Letter-Bode voor het Jaar 1823, 2: 130-33.

van Hasselt, J.C. 1824. Extrait d'une seconde lettre sur les poissons de Java, écrite par M. van Hasselt à M. C.-J. Temminck, datée de Tjecande, résidence de Bantam, 29 décembre 1822 [Extract from a second letter on the fishes of Java, written by M. van Hasselt to M. C. -J. Temminck, date of Tjecande, residence of Bantam, December 29, 1822]. Bulletin Universel des Sciences et de l'Industrie, Section 2, Bulletin des Sciences Naturelles et de Géologie 2: 374-77.

Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PD. 2005. DNA barcoding Australia's fish species. Philos Trans R Soc Lond B Biol Sci 360(1462): 1847-57.

Wong EHK, Shivji MS, Hanner RH. 2009. Identifying sharks with DNA barcodes: Assessing the

utility of a nucleotide diagnostic approach.

Mol Eco.




How to Cite

Rahayu, D. A., Kuntjoro, S., Budijastuti, W., Winarsih, W., Ambarwati, R., Nugroho, E. D., Basith, A., Kurniawan, N., & Haryono, H. (2023). FURTHER STUDY ON TWO SPECIES OF LOACH FISHES (Cypriniformes: Nemacheilidae: Nemacheilus) BASED ON MORPHOLOGY AND MOLECULAR DATA. BIOTROPIA, 30(3), 329–345. https://doi.org/10.11598/btb.2023.30.2.1942