04_Muchlisin-1.indd UDC 597.551.2:57.06(594.41) TAXONOMIC DIVERSITY OF THE GENUS TOR (CYPRINIDAE) FROM ACEH WATERS IN INDONESIA BASED ON CYTOCHROME OXIDASE SUB-UNIT I (COI) GENE Z. A. Muchlisin1,2*, N. Fadli1,2, A. S. Batubara3, F. M. Nur1, M. Irham1,2, A. A. Muhammadar1, D. Efi zon4, Roza Elvyra5, M. N. Siti-Azizah6 1Faculty of Marine and Fisheries, Universitas Syiah Kuala, Banda Aceh 23111, Indonesia 2Marine and Fisheries Research Center, Universitas Syiah Kuala, Banda Sceh 23111, Indonesia Faculty of Mathematics and Natural Sciences, Universitas Negeri Medan, Medan, Indonesia 4Faculty of Fisheries and Marine Sciences, Universitas Riau, Pekanbaru, Indonesia 5Faculty of Mathematic and Natural sciences, Universitas Riau, Pekanbaru, Indonesia 6Institute Marine Biotechnology, Universiti Malaysia Terengganu, Terengganu, Malaysia *Corresponding author E-mail: muchlisinza@unsyiah.ac.id Z. A. Muchlisin (https://orcid.org/0000-0002-0858-1853) N. Fadli (https://orcid.org/0000-0001-6845-1383) A. S. Batubara (https://orcid.org/0000-0001-8788-9567) F. M. Nur (https://orcid.org/0000-0001-9142-0630) M. Irham (https://orcid.org/0000-0002-2639-6723) A. A. Muhammadar (https://orcid.org/0000-0002-8086-0099) D. Efi zon (https://orcid.org/0000-0003-3667-7796) Roza Elvyra (https://orcid.org/0000-0002-9250-3611) Taxonomic Diversity of the Genus Tor (Cyprinidae) from Aceh Waters in Indonesia Based on Cytochrome Oxidase Sub-Unit I (COI) Gene.  Muchlisin, Z. A., Fadli, N., Batubara, A. S., Nur, F., Irham, M. M., Muhammadar, A. A., Efi zon, D., Roza Elvyra, Siti-Azizah, M. N. — Th e mahseer or keureling, members of the genus Tor J. E. Gray, 1833 are the commercial freshwater fi sh. It has potency to be cultivated commercially. Presently, no studies have been conducted on the molecular taxonomy of these fi sh. Hence, the objectives of the present study were to complement this morphological identifi cation using the DNA barcoding gene, cytochrome oxidase subunit I (COI). Samples were obtained from seven areas of Aceh Province, namely; Aceh Besar, Aceh Barat, Nagan Raya, Aceh Selatan, Aceh Tenggara, Gayo Lues and Pidie Districts. A total of 140 fi sh samples have been collected during the sampling, of these 37 samples have been successfully sequenced. Based on the results of the sequencing data and blasting to NCBI data, only two species of Tor occur in Aceh waters: T. tambra (Valenciennes, 1842) and T. tambroides (Bleeker, 1854); while T. soro (Valenciennes, 1842) and T. douronensis (Valenciennes, 1842) were not validated. K e y w o r d s : threatened fi sh species, freshwater fi sh, genetic, mahseer Zoodiversity, 56(3): 195–202, 2022 DOI 10.15407/zoo2022.03.195 196 Z. A. Muchlisin, N. Fadli, A. S. Batubara, F. M. Nur1, M. Irham, A. A. Muhammadar et al. Introduction At least 114 species freshwater fi shes were reported in the biodiverse Indonesian region of the Aceh Province (Muchlisin & Siti-Azizah, 2009). Of them, 15 species are highly valued commercially. Th e members of the genus Tor J. E.Gray, 1833 collectively referred to as the keureling, among the local people (Muchlisin 2013), or oft en having a vernacular name mahseer, are included on this list. Based on morphological variations, three taxa of keureling, Tor soro (Valenciennes, 1842), T. tambra (Valenciennes, 1842), and T. tambroides (Bleeker, 1854) were reported to occur in the Aceh waters. Th ese morphologically identifi ed species were recorded in the Alas River region (Southeast Aceh), Batee Iliek River (Biureun), Montala River (Aceh Besar), Meurebo River and Woyla River (Aceh Barat) and Nagan River (Muchlisin & Siti-Azizah, 2009; Muchlisin 2010). However, it was suspected that the number of Tor species in Aceh waters was not accurate, because the identifi cation was only used the morphological characters as the presence and size of the median lobe of lower lip. Th e keureling fi sh has great potential in the aquaculture industry due to the economic demand as compared to other freshwater fi sh species in Aceh, Indonesia. Th erefore, it is the major target of anglers using various fi shing methods, including destructive fi shing practices. Th is has led to a drastic population declines of members of the genus Tor in its natural habitat, not only in Indonesia but almost throughout its range worldwide. Several species of Tor have been listed in the Endangered Category of the IUCN Red List fairly early on (Raghavan & Ali, 2011; Jha et al., 2018; Pinder et al., 2018). Kottelat et al. (1993) and Singh (2007) attributed the decline and threatened extinction of mahseer due to overfi shing, pollution, and environmental damage. To address this situation, a comprehensive information is critical encompassing various aspects of the genus. Several aspects of the mahseer from Aceh waters have been studied, for example the bio-ecology (Muchlisin et al., 2015 a), parasites infestation (Muchlisin et al., 2014, 2015 c), and nutritions requirement (Muchlisin et al., 2016 a, b; Muchlisin et al., 2017 a, b) However, information on its genetic diversity is not available. Genetic profi le information is crucial in relation to planning a better fi sheries management strategy and aquaculture development, for example in crossbreeding program. Th e cytochrome oxidase sub-unit I (COI) mitochondrial gene commonly applied for DNA barcoding was used for identifi cation of Tor species in the Aceh waters. DNA barcoding is one of the most reliable ways today to validate the taxonomic status of living organisms including fi sh (Tautz et al., 2003; Muchlisin et al., 2013). Th is genetic approach have been recognized for their usefulness in species identifi cation (Prioli et al., 2002; Muchlisin et al., 2012), monitoring fi sheries resources (Menezes et al., 2006), aquaculture program (Barriga- Sosa et al., 2004), especially for selective breeding. Th ere have been several studies on DNA barcoding of the Mahseer. Th ese include investigations of two and fi ve species of Indian mahseer using COI gene (Sati et al., 2013), and Laskar et al. (2018), respectively, and the disentangling the taxonomy of the mahseers from Malaysian waters has been reviewed by Walton et al. (2017). Understanding the taxonomic status as well as species and genetic diversity are critical to the conservation and protection of a species (or a group of), particularly of an endangered one. However, so far the taxonomy of the genus Tor is still contentious. Although record of species diversity in Aceh waters is available, but this has been based on morphological identifi cation with its associated diffi culties for this genus (Muchlisin & Siti-Azizah, 2009; Muchlisin et al., 2015 b) Presently, the genetic diversity of the mahseer from Aceh waters, Indonesia has not been reported. Hence, the objective of the present study was to elucidate the taxonomic diversity of the genus Tor and genetic diversity within each taxon in the Aceh waters, Indonesia with the DNA barcoding method. Materials and methods S a m p l i n g A total of 140 fi sh samples preliminarily identifi ed to belong to four taxa of Tor have collected during the sampling at seven regions within Aceh Province namely; Aceh Besar, Aceh Barat, Nagan Raya, Aceh Selatan, Aceh Tenggara, Gayo Lues and Pidie during July to August 2016 (table 1 and table 2). Of them, 37 samples were successfully sequenced. Specifi c sampling sites were determined based on information from local anglers; the specimens were caught using casting nets and gill nets. Some samples also were purchased from local markets. Samples were morphologically identifi ed based on Kottelat et al. (1993), Walton et al. (2017) and Desrita et al. (2018). Species identifi cation was conducted based on the presence and size of median lobe of lower lip. S a m p l e c o l l e c t i o n a n d p r e p a r a t i o n A total of 5–15 specimens of each preliminarily identifi ed species of Tor were analysed for COI gene. Approximately 1 cm2 of caudal fi n tissue was taken from each specimen, placed into 2.0 ml tubes containing TNES-urea buff er, labeled and transported to the laboratory. Th ere, the tissues were further minced into small pieces in order to enhance lysis activity. Th e samples were preserved at least two weeks prior to DNA extraction. D N A e x t r a c t i o n Genomic DNA was isolated using Aqua Genomic DNA solution (Promega) following the manufacturer’s protocol. Successfully extracted DNA was determined by electrophoresis conducted 197Taxonomic Diversity of the Genus Tor (Cyprinidae) Based on Cytochrome Oxidase Sub-Unit I (COI) Gene on a 0.8 % agarose gel at 100 V for 45 minutes, stained by ethidium bromide and visualized on a gel documentation system (GENE FLASH, Syngene Bio-Imaging). The DNA extracts were kept at –20 oC prior to PCR amplification. P C R a m p l i f i c a t i o n Partial  sequences of the mitochondrial  COI gene were amplified using the primer pair developed by Ward et al. (2005); FishF1)5’-TCA ACC AAC CAC AAA GAC ATT GGC AC-3’) and FishR1-5’ (TAG ACT TCT GGG TGG CCA AAG AAT CA-3’). The 25 μL PCR reaction mix contained 17.65 μL of deionized water, 2.25 μL of 10X PCR buffer, 3.0 μL of MgCl2 (25 mM), 0.25 μL of each primer (0.01 mM), 0.5 μL of mixed dNTP (0.05 mM), 0.1 μL of Taq polymerase, and 1.0 μL of DNA template. Amplifications were performed using a Mastercycler® Eppendorf gradient thermal cycler (Brinkmann Instruments, Inc). The thermal regime consisted of an initial step of 2 minutes at 95 oC followed by 35 cycles of 30 seconds at 94 oC, 30 seconds at 54 oC, and 1 minute at 72 oC, and finally 10 minutes of final extension at 72 oC (Ward et al., 2005). the PCR product were purified (PCR Clean-up System, Promega) and were then sent for sequencing to First BASE Laboratories Sdn Bhd (Selangor, Malaysia) using an ABI3730XL Genetic Analyzer (Applied Biosystems), and an ABI PRISM BigDye terminator cycle sequencing kit v3.1 (Applied Biosystems). D a t a a n a l y s i s All obtained sequences were edited and aligned using MEGA 6.0 program (Tamura et al., 2013). Multiple sequence alignments were then performed on the edited sequences by Cluster W which is integrated into the MEGA 6.0 program. Nucleotide divergences among sequences were stimated based on Kimura 2-parameter (K2P) distances (Kimura, 1980)  and neighbor joining (NJ) tree was conducted using K2P molecular evolutionary model in MEGA 6.0 program. Branch supports were estimated using 1000 bootstrap replications.  Neolissochilus hendersoni (Accessing No. KT354858.1) sequence was used as outgroup. Results A final alignment of 655 bp of sequences among 37 individuals of Tor spp. from seven localities were obtained for the COI gene (table 1). Th e aligned sequences generated 9 sequences of T. tambra and 28 sequences of T. tambroides aft er BLAST analysis in the NCBI database with identical values ranging between of 98 % to 100 % (table 2). Th e higher number of samples were collected from Alas River (7 samples) consisting of T. tambra (4 samples) and T. tambroides (3 samples), while the least samples were recorded from Ulee Raket, Aceh Barat District where all sequences were T. tambroides. A total of 5 haplotypes, consisting of 4 haplotypes of the T. tambroides, and one haplotype of the T. tambra were generated from 37 samples. Haplotype number two T a b l e 1 . Total sample and sample code of presumed species based on sampling locations. Th e presumed species was identifi ed based on the presence and size of median lobe of lower lip No Location Sampling site Samplecode Presumed taxa TotalT. tambra T. tambroides T. douronensis T. soro 1 Gayo Lues District Blang Kejeren River BLA – – 1 4 5 2 Aceh Tenggara District Alas River SAL 2 4 – 1 7 3 Aceh Besar District Leupung River SPL 4 – – – 4 Jreu River KJ – 4 – – 4 4 Nagan Raya District Nagan River KN – 4 – 1 5 5 Pidie District Geumpang River KGM – – – 3 3 6 Aceh Barat District Ulee Raket River UR 3 – – – 3 7 Aceh Selatan District Manggamat River KM 6 – – – 6 Total sample 15 12 1 9 37 198 Z. A. Muchlisin, N. Fadli, A. S. Batubara, F. M. Nur1, M. Irham, A. A. Muhammadar et al. belongs to T. tambroides and it was shared 15 samples from four different locations, namely; Nagan River, Leupung River, Geumpang River, and Jrue River. Haplotype number four is also belonging to T. tambroides and is common for 9 samples from two different locations of the Blangkejeren River, and Alas River. In addition, haplotype number five is belonging to T. tambra, this haplotype is shared by 9 samples from two different locations of the Blangkejeren River, and Alas River (table 3).Inter- specific variation calculated from 37 samples recovered two species (T. tambra and T. tambroides). The genetic distance between species was 3.1 % indicate these are the different species belonging in the same genus (table 4). The genetic distance value of the same species from different locations showed that the lowest genetic distance was found between T. tambra from Blangkejeren and T. tambra from Alas River (0.01 %), and the higher genetic distance occurred in the samples between T. tambroides from T a b l e 2 . Th e E-Value dan Identity of Tor samples from seven locations No. Region Sampling site Code Species E-Value Identity, % 1. Gayo Lues Blangkejeren River SOR_BLA_01 Tor tambra 0.0 99 SOR_BLA_07 Tor tambra 0.0 99 SOR_BLA_08 Tor tambra 0.0 99 SOR_BLA_06 Tor tambra 0.0 99 DUR_BLA_02 Tor tambra 0.0 100 2. Aceh Tenggara Alas River TAMB_SAL_01 Tor tambra 0.0 99 TAMB_SAL_02 Tor tambra 0.0 99 TAMB_SAL_03 Tor tambra 0.0 99 TAMB_SAL_04 Tor tambra 0.0 99 TAM_SAL_06 Tor tambroides 0.0 99 TAM_SAL_09 Tor tambroides 0.0 99 SOR_SAL_11 Tor tambroides 0.0 99 3. Aceh Besar Leupung River TAM_SPL_03 Tor tambroides 0.0 98 TAM_SPL_04 Tor tambroides 0.0 98 TAM_SPL_05 Tor tambroides 0.0 98 % TAM_SPL_02 Tor tambroides 0.0 98 Jreu River KR_KJ_01 Tor tambroides 0.0 98 KR_KJ_02 Tor tambroides 0.0 98 KR_KJ_06 Tor tambroides 0.0 98 KR_KJ_11 Tor tambroides 0.0 98 4. Nagan Raya Nagan River TAMB_KN_01 Tor tambroides 0.0 98 TAMB_KN_02 Tor tambroides 0.0 98 TAMB_KN_04 Tor tambroides 0.0 98 TAMB_KN_05 Tor tambroides 0.0 98 SORO_KN_02 Tor tambroides 0.0 98 5. Pidie Geumpang River SORO_KGM_06 Tor tambroides 0.0 98 SORO_KGM_01 Tor tambroides 0.0 98 SORO_KGM_07 Tor tambroides 0.0 98 6. Aceh Barat Ulee Raket River TAM_UR_08 Tor tambroides 0.0 98 TAM_UR_15 Tor tambroides 0.0 98 TAM_UR_14 Tor tambroides 0.0 98 7. Aceh Selatan Manggamat River TAM_KM_02 Tor tambroides 0.0 99 TAM_KM_04 Tor tambroides 0.0 99 TAM_KM_05 Tor tambroides 0.0 99 TAM_KM_08 Tor tambroides 0.0 99 TAM_KM_06 Tor tambroides 0.0 99 TAM_KM_13 Tor tambroides 0.0 99 N o t e : SOR = presumed taxa of Tor soro, TAM = presumed taxa of Tor tambra, TAMB = presumed taxa of Tor tambroides, DUR = presumed taxa of Tor douronensis 199Taxonomic Diversity of the Genus Tor (Cyprinidae) Based on Cytochrome Oxidase Sub-Unit I (COI) Gene Tor tambroides SORO KGM 01 Tor tambroides TAMB KN 05 Tor tambroides TAMB KN 04 Tor tambroides TAMB KN 02 Tor tambroides TAMB KN 01 Tor tambroides TAM SPL 05 Tor tambroides TAM SPL 04 Tor tambroides TAM SPL 03 Tor tambroides TAM SPL 02 Tor tambroides SORO KN 02 Tor tambroides SORO KGM 06 Tor tambroides KR KJ 11 Tor tambroides KR KJ 06 Tor tambroides KR KJ 02 Tor tambroides KR KJ 01 Tor tambroides SORO KGM 07 Tor tambroides TAM UR 08 Tor tambroides TAM UR 14 Tor tambroides TAM UR 15 Clade 1 Tor tambroides SOR SAL 11 Tor tambroides TAM KM 02 Tor tambroides TAM KM 04 Tor tambroides TAM KM 05 Tor tambroides TAM KM 06 Tor tambroides TAM KM 08 Tor tambroides TAM KM 13 Tor tambroides TAM SAL 06 Tor tambroides TAM SAL 09 Clade 2 Tor tambra DUR BLA 02 Tor tambra SOR BLA 01 Tor tambra SOR BLA 06 Tor tambra SOR BLA 07 Tor tambra SOR BLA 08 Tor tambra TAMB SAL 01 Tor tambra TAMB SAL 02 Tor tambra TAMB SAL 03 Tor tambra TAMB SAL 04 Clade 3 Outgroup KT354858.1 Neolis s ochilus henders oni 99 99 99 87 99 62 0.002 the clade 1 (Geumpang River, Nagan River, Leupung River, Jreu River and Ulee Raket River) and T. tambroides from the clade 2 (Alas River and Manggamat River) had average genetic distance value of 2.4 %. The genetic three of 37 sequence samples were divided into three difference clades (fig. 1). Fig. 1. Th e Neighbor Joining tree for 37 sequences of Tor from seven locations in Aceh Province estimated using 1000 bootstrap replications. 200 Z. A. Muchlisin, N. Fadli, A. S. Batubara, F. M. Nur1, M. Irham, A. A. Muhammadar et al. Discussion A total of 37 samples were succesfl ully amplifi ed using 655 bp COI gene. Based on morphological characters, these samples were preliminarily identifi ed as T. soro (9 samples), T. douronensis (1 sample), T. brambroides (12 samples), and T. tambra (15 samples). However, the BLAST result performed with MEGA 6.0 recovered only two species: T. tambra and T. tambroides with the E-Value 0.0. Th is value defi nes the absence of errors or no bias during blasting process and therefore the data in Genbank and this study was fi t or suitable. In addition, the E-value of BLAST also shows the identity value in the two species ranged 98 % to 100 %, it means that these species have been well identifi ed genetically. Th e presumed taxa T. douronensis from Blang Keujeren River is actually T. tambra, while the presumed T. soro from Blang Kejeren River is T. tambra and presumed taxa T. soro from Nagan River and Geumpang River are T. tambroides. Th erefore, the presence and size of the median lobe of lower lip cannot be used as a sound character for identifi cation of Tor in Aceh waters. Th e genetic relationship was also analyzed in this study using the phylogenetic tree. Th e phylogenetic tree showed that the fi sh samples were divided into three diff erent clades, where the clade 1 is monophyletic consisting T. tambroides from Leupung River (SPL_05, 04, 03, and 02), Ulee Raket (UR_08, 14 and 15), Nagan River (KN_05, 04, 02, 01 and 02), Geumpang River (KGM_01, 06 and 07) and Jrue River (KJ_11, 06, 02 and 01). In the fi rst clade seen that T. tambroides from fi ve diff erent locations formed the same clade. Th ere is a close genetic relationship among T. tambroides samples, although the samples come from diff erent locations, it can be seen from the constructed of the phylogenetic tree where the bootstrap (1000x bootstrapping) value was higher than 99 %. Th ese values also strengthen the argument that the T. tambroides samples analyzed in this study are still from the same ancestor or monophyletic group, although the species T. tambroides from fi ve locations grouping into synapomorphic form a clade based its closest genetic relationship. A total of 19 samples of tambroides clustered to form a clade in the clade fi rst, where the higher samples incorporated in clade fi rst comes from Nagan River with 5 individuals, of the all samples are formed synapomorphic identical. T a b l e 3 . Haplotype number and frequencies, specimen I.D, contributing morph and location Haplotypes Number of sequences Specimen no. I.D Contributing morph Sampling Location 1 1 SORO KGM 01 Tor tambroides Geumpang River 2 15 TAMB KN 01, TAMB KN 02, TAMB KN 04, TAMB KN 05, SORO KN 02, TAM SPL 02, TAM SPL 03, TAM SPL 04, TAM SPL 05, SORO KGM 06, SORO KGM 07, KR KJ 01, KR KJ 02, KR KJ 06, KR KJ 11 Tor tambroides Nagan River, Leupung River, Geum- pang River, Jrue River 3 3 TAM UR 08, TAM UR 14, TAM UR 15 Tor tambroides Ulee Raket River 4 9 SOR SAL 11, TAM SAL 06, TAM SAL 09, TAM KM 02, TAM KM 04, TAM KM 05, TAM KM 06, TAM KM 08, TAM KM 13 Tor tambroides Blangkejeren River, Alas River 5 9 DUR BLA 02, SOR BLA 01, SOR BLA 06, SOR BLA 07, SOR BLA 08, TAMB SAL 01, TAMB SAL 02, TAMB SAL 03, TAMB SAL 04 Tor tambra Blangkejeren River, Alas River T a b e l 4 . Inter and intra-specifi c variation among valid taxa/species of Tor Species COI Inter-specifi c mean Th eta- prime mean Intra-specifi c min. Intra-specifi c max. Tor tambra n/a 0 0 0 Tor Tambroides 3.1 0.7 0 2.7 Neolissochilus hendersoni (outgroup) 3.8 4.5 0 0 0 201Taxonomic Diversity of the Genus Tor (Cyprinidae) Based on Cytochrome Oxidase Sub-Unit I (COI) Gene As seen in the fi rst clade, the T. tambroides in the second clade also comes from a diff erent location but their genetic relationships are formed synapomorphic identical with the bootstrap value of 99%. In addition, the T. tambroides on the second clade has 9 individuals come from two locations; Manggamat River (KM_ 02, 04, 05, 06, 08 and 13) and the Alas River (SAL_ 11, 06 and 09). Th e third clade was formed by T. tambra from two locations i. e. Blangkejeren River (BLA_ 02, 01, 06, 07 and 08), and Alas River (SAL_ 01, 02, 03 and 04). Th e T. tambra in the third clade also comes from a diff erent location but their genetic relationships are formed synapomorphic identical with the bootstrap value of 99 %. In the phylogenetic tree also form an outgroup clade. Th e out-group was used to determine the accuracy of genetic distance obtained from the constructed phylogenetic tree. Outgroup species used in this analysis was Neolissochilus hendersoni retrieved from GenBank (Accessing No. KT354858.1). Relationship of life organisms is determined by the genetic distance. Two species of Tor found in this study had a genetic distance of 3.1 %. It means that they are two valid distinct species. According to Hebert et al. (2003) that the organisms can be regarded as the same species if it has a genetic distance of less than 3 %. Th erefore, if the genetic distance is smaller or close to 0, it means an indication of the closer relationships, and vice versa. Tor genetic distance of T. tambra from Blangkejeren and the Alas River has a value of 0.0 %. Th is value indicates that there are no signifi cant diff erences (still from the same ancestor or monophyletic group) in genetic variability even though the location is diff erent. Th is is because in Blangkejeren River is a tributary of the Alas Rivers so, that both rivers are connected. Beside, tambroides samples from these rivers produces one shared haplotype. While the genetic distance T. tambroides of clade 1 (Geumpang River, Nagan River, Leupung River, Jreu River and Ulee Raket River) and clade 2 (Alas River and Manggamat River) has a genetic distance between 2.4–2.7 % with average value of 2.4 %. Th ese values also showed close genetic relationship or are still part of the same species that is T. tambroides. However, some of these rivers are not interconnected. Th e close genetic relationship between T. tambroides from some rivers is likely to happen in the past where these some rivers respectively interconnected (ancient rivers) (Tan et al., 2012). 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