Biometric and genetic differences in kelabau ( Osteochilus spp.) as revealed using cytochrome c oxidase subunit 1

Background: Kelabau ( Osteochilus spp.) is a freshwater fish commonly found in the rivers of Riau, Indonesia. Researchers believe that these are Osteochilus kelabau; however, accurate taxonomic determination of these fish in Riau waters has not been made. The purpose of this study was to facilitate the identification of the kelabau based on its morphology and genetics using biometric and cytochrome c oxidase subunit 1 ( CO1) analyses, respectively. Methods: Fish samples were collected from the Siak, Kampar and Rokan rivers in Riau Province, Indonesia. The DNA of 90 fish was extracted from the caudal fins using a DNA extraction kit, after which it was amplified using primers Fish-F1 and Fish-R1. Sequencing was conducted by Applied Biosystems Macrogen Korea, and the DNA sequences were then edited and aligned using MEGA v. 7. All samples were BLAST-searched for identification using the National Center for Biotechnology Information and BOLD System. Phylogenetic trees were constructed, and the similarity index was calculated using accession numbers AP011385.1 and KC631202.1 in GenBank. Results: Analysis of the consensus barcode sequence for 86 species revealed a high percentage of barcode matches (96%–97% in GenBank and 96.6%–96.76% in the BOLD System). The nucleotide distance between groups of kelabau from the different rivers based on the Kimura 2-parameter model gave the following results: 0.05% between groups from the Siak and Kampar rivers, 0.09% between those from the Siak and Rokan rivers and 0.05% between those from the Kampar and Rokan rivers. The nucleotide distance between the groups in the Siak (0.09%), Kampar (0.00%) and Rokan (0.10%) Rivers indicated that the kelabau in those rivers were related to each other. Conclusions: Based on the results of the research data using CO1 and biometric analyses, the kelabau were confirmed to be O. melanopleurus.


Introduction
Kelabau are ancient fish belonging to genus Osteochilus of family Cyprinidaes. Kelabau fish are distributed throughout Thailand, Vietnam, Peninsular Malaysia, Borneo and Sumatra 1,2 . In Sumatra Island in Indonesia, these fish is commonly found in the Siak, Kampar and Rokan rivers in Riau Province 3,4 .
According to local fishers in Riau, kelabau are divided into two types on the basis of morphology; although, there is no detailed information about these fish types. The local fishers said that the first type of kelabau fish is the fish found in this study. The morphological characteristic of this fish is brownish body, with brighter bottom, dark hazy blotches present above the pectoral fins, and the size ranges from 15 to 2,778.93 gr. The second type of fish is larger sized and yellowish color. However, during the study time, from 2017 to 2018, this type was not found. Thus, a study was needed to identify the species using morphological and molecular methods to determine these types in the Siak, Rokan and Kampar rivers. Identification of any species using morphological traits can be difficult and can lead to errors 5 ; therefore, owing to morphological similarities among Osteochilus spp., molecular markers, such as DNA barcodes, are important to identify the fish species uses a specific sequence region (i.e. cytochrome c oxidase subunit 1 (CO1)) to identify a species and is a technique that can identify taxonomic units as well as biodiversity for determining species of several organisms 5-9 . Unlike molecular phylogeny used to determine relationships among species, the purpose of DNA barcoding is to identify unknown or undetermined species into phylogeny 10 . The common mitochondrial (mt) DNA region used as a barcode in protists and animals comprises 600 bp. In addition, CO1 is one of the genetic markers used to identify insects, birds, primates and fish to species [11][12][13] . MtDNA CO1 is selected as a target in DNA barcoding because it is a highly conserved site. This method has advantages over the morphological identification approach in that it is fast, reliable and it can be used for all types of samples because it uses a single gene along with mutations in the nucleotides to acknowledge the taxonomic features of each species 13 .
The study on DNA barcoding for freshwater fish has been widely practiced in various countries, including Nigeria 14 , India 15 , Philippines 16 , Canada 9 and Indonesia 5,17-19 . The method has been successfully validated for the taxonomic status within Rasbora in Lake Laut Tawar 20 ; Anguillidae in Aceh waters 21 ; Ornamental fish from Peat lands 5 ; Channidae in Peninsular Malaysia, Sarawak, Sumatera, Borneo, Myanmar, Vietnam, India, Germany, Singapore and the United Kingdom 22 and Cichlidae in northeastern Nigeria 23 ; therefore, it can be used to equally successfully validate the taxonomic unit of the kelabau using its morphology supported by molecular data. This information is crucial for designing a remedial course of action about the conservation strategy for this species in the Siak, Kampar and Rokan rivers in Riau Province, Indonesia.

Ethics
The study population was collected and sampled according to the guidelines on the use of living organisms for research from the Laboratory of the Faculty of Fisheries and Marine, Riau University, Indonesia 24 Sampling sites and collection A total of 90 kelabau (30 fish from each river) were collected from the Siak, Kampar and Rokan rivers ( Figure 1). Fish were caught using a gill net 3 m deep and 20 m long with a 12.7-cm mesh. The gill nets were installed in the river water close to the riverbank and remained for 24 h from 08:00 to 08:00 the following day. The fish collected were counted using hand-counter and cleaned using freshwater. A number of 50-mm caudal fin tissue samples were taken using a sterile scissors and preserved in ethanol, after which a photo of each fish sample was taken for documentation using a digital camera.
All samples were preserved in 3-kg sample bags which were labeled according to site location, date and serial number. Before preservation, the fish samples were injected with 10% formalin. The fish samples were then transported to the laboratory for further evaluation. The morphologies of the collected fish were identified up to species level using the identification book produced by the Indonesian Institute of Sciences ichthyology museum 1,25 . The fish morphologies observed were length, color, shape of scales, mouth shape, barbels, number of fins and special marks on the body.

Biometrics
Biometric analyses were used to measure morphological characteristics in this study 1 . This tool is considered conventional for identifying organisms. Molecular identification using CO1 gene sequences has been supported for providing additional organism classification.
DNA isolation and amplification DNA was extracted using the spin-column method from the gSYNC DNA Extrusion Kit (Geneaid Catalogue No. GS 300, Taiwan). The extracted DNA was then transferred to a 1X Tris-borate ethylenediaminetetraacetic acid (TBE) solution with a 1.5% agarose gel and Pegreen gel dye (PEQLAB Biotechnologies GmbH, Erlangen, Germany) 19 . The quantity of DNA was visualized with the help of a GeneQuant Spectrophotometer by adding 78 μL nuclease-free water in a cuvette along with 2 μL DNA. The DNA was then amplified using the universal primer

Amendments from Version 2
In this new version of the manuscript we have revised as follows: 1. We have added three citations in the discussion -"35,36" become "35 to 39". 2. In paragraph three in the discussion we have added reasons for limited variation and a discussion of data Table 3. 3. Dr Windarti was added as a co-author for her work in improving the manuscript Any further responses from the reviewers can be found at the end of the article REVISED Fish-F1(5'-TCA-ACC-AAC-CAC-AAA-GAC-ATT-GGC-AC-3') and Fish-R1 (5'-TAG-ACT-TCT-GGG-TGG-CCA-AAG-AAT-CA-3') with a target of 707 bp and 655 bp 26 , respectively. The amplification thermocycling conditions as follow: the PCR condition using pra PCR (94°C for 5 min), 35 cycles of denaturation (94°C for 30 s), annealing (56.6°C for 30 s) and extension (72°C for 30 s), followed by post-PCR extension (72°C for 5 min) and hold (4°C for 5 min). PCR results were analyzed using 1.5% agarose gel at 100 V to assess the bands, and only the clear products were sent to Applied Biosystems Macrogen Korea for sequencing.

Controlling molecular samples and sequence quality
The PCR amplicon was 707 bp, which implied that no sequence of DNA was derived from mtDNA nuclear mitochondrial DNA segments (NUMTs), because a NUMT barely reaches 600 bp 9 .
The selected CO1 sequences were entered into GenBank and the BOLD System databases to compare the alignment of nucleotide sequences and 99%-100% values with that with no insertions/deletions. All sequences were aligned using ClaustalW with MEGA v.7 27 .

Data analysis
Blasting of CO1 by NCBI (GenBank) and BOLD System (online) The entire nucleotide sequence obtained from the sequence chromatogram was assembled using DNA Baser Assembler, aligned and then analyzed using MEGA 7. It was further aligned (multiple alignments) using the reference NCBI GenBank accession numbers AP011385.1 and KC631202.1. Similarly, the percentages of CO1 sequences were blasted using NCBI Blast and BOLD Systems databases.

Nucleotide variations
Nucleotide variations among samples were analyzed using dnaSP v.5 28 . The parameters of these calculations were haplotype number, variable site, parsimony site, haplotype diversity and nucleotide diversity.

Phylogenetic tree
Phylogenetic trees were estimated using all samples from the three populations and calculated according to the Tamura-Nei model 29 using MEGA 7 27 .

Nucleotide distance
The distance among the nucleotide bases of the mtDNA CO1s was analyzed using the Kimura 2-parameter model 30 . The nucleotide distances between and within the populations were examined according to the model based on the similarity of frequencies and ratios of transition to transversion (Ti:Tv) using MEGA 7 27 .

Morphological identification
The morphological traits of all kelabau used in this study matched those of O. melanopleurus. We used the important morphological traits to identify these fish according to Kottelat et al. 1 . The morphological characteristics measurement of O. melanopleurus showed that the fish have 16-19 branched dorsal rays, the number of scales was ranged from 10.5 to 12.5 in between dorsal origin and lateral line, the number of circum peduncu-lar rows of scale was ranged from 20 to 24 and lips covered with folds and plicae and there was no hard tubule at the tip of the mouth (Figure 2a). This species has one pair of barbels at above and one pair at bottom, dark hazy blotches near above of the pectoral fins. The body is brownish, with the bottom brighter than the top and the type of steroid scales ( Figure 2b). Raw biometric data are available on OSF 31 . as ~707 bp.

Genetic analysis
A sequence amplified by Fish-F1 primer was successfully identified in 86 of 90 samples of mtDNA fish. The base length of the CO1 nucleotide obtained from the formulation process and electrophoresis (Figure 3) Based on genetic analysis using the Tamura-Nei model, there was an unequal distribution of all nucleotides with the following frequencies: adenine (A), 26.73%; thymine (T), 30.44%; cytosine (C), 25.93% and guanine (G), 16.90% ( Table 1). The rates ratio between transition and transversion was 10.257 purines and 1.915 pyrimidines, and the overall transition and transversion bias were R= 2.499 based on Tamura-Nei model 32 . The pattern of nucleotide distribution of A, T, C and G was T > A > C > G, but the carps which cultivated in India have the following pattern of nucleotide distribution: T > C > A > G 33 .
The nucleotide distances between nucleotide bases within the groups indicated that the values of the nucleotide base sequences within the fish population were 0.0009, 0.0000 and 0.0010 in the Siak, Kampar and Rokan rivers, respectively. The evolutionary distance between the nucleotides of the groups had a comparative difference in the nucleotide sequences of 0.0005 for fish from the Siak and Kampar rivers, of 0.0009 in those from the Siak and Rokan rivers and of 0.0005 for those from the Rokan and Kampar rivers. Based on the nucleotide distance, the fish were identified as being from the same species (0.06%) ( Table 2).
In the phylogenetic tree consisted of two major groups ( Figure 4)

Discussion
Overall, the morphological traits and DNA barcoding showed that the majority of, if not all, kelabau fish in the three rivers at     The lack nucleotide diversity of O. melanopleurus from the three rivers was likely to be caused by limited opportunities for kelabau migration, so that the genetic exchanges with other populations are very small 36 ; moreover, the lack nucleotide diversity is believed to be caused by inbreeding, and overfishing 5, 40 . Variation in nucleotide diversity of the fish from the sampling areas is different. In the Kampar River, the fish was sampled from the relatively narrow area and it may cause the lowness of the nucleotide diversity. The diversity was 0 and it means that the nucleotide of the fish samples was almost identic. The low nucleotide diversity may occur as the habitat of the fish is very suitable and the fish may not face environmental related problem that trigger any genetic changing 41 . However the nucleotid diversity of the fish from the Siak and Rokan River was higher than Kampar River. Siak and Rokan Rivers have relatively low water quality that was caused mainly by anthropogenic activities. Changing in water quality may trigger the fish to adapt with that environmental condition and it may cause changes in genetic variation. Freeland et al. 41 stated that fish population with high genetic variation may be able to face problems related to environmental changing    The identity of a species was derived using the morphological identification method to distinguish between species or individuals 45,46 . Basically, the genetic identification of a species can be done using mtDNA CO1, a more effective approach than using rRNA 5,33 . The nucleotide locus and mutations were used as references to conduct DNA barcoding in all fish samples 13 . Previous studies have identified several species using DNA bar-coding, such as ornamental fish of wetlands 5 , wetland fish larvae 46 , rainbow fish 47 , Cyprinidae fish 48 , salmon and trout 7 and freshwater fish 9,19 . Furthermore, the phylogenetics of CO1 sequences can effectively show congeneric and confamilial species 9 .
The phylogenetic trees could describe the line of biological evolution from species or organisms with a different ancestry 49 . Nonetheless, the results of all these species did not show a 100% undistinguishable identity. The branch length between species leading to a gap in the pairwise distance distribution is referred to as the barcoding gap in CO1 23 . Intra-species relationships were quite high in all samples, which confirmed that kelabau melanopleurus) were native in the three rivers and could to adapt to changes in environmental conditions 40 .
Moreover, the existence of inter-nucleotide patterns and distances between A, T, C and G in the chromosomes showed the characteristics and genetic signs that distinguished each of the individuals, even though they belong to the same species 50 . This is reinforced by referencing the phylogenetic tree made using the neighbor-joining model.
The identification of fish species is normally conducted using morphological characteristics such as dorsal fins, pelvic fins, pectoral fins, anal fins, linea lateral fins, upper linea lateral fins, lower linea lateral fins, around body fins, and caudal peduncle fins; however, in this study, we used 12 morphological traits as described by the classification system of Kottelat et al. 1 . These results supported the classification using biometric data that all fish in the three rivers were O. melanopleurus. The morphological characteristics were consistent with the species having a relatively large body with a standard length of 119-560 mm, lips covered with folds and plicae, no tubercles on the snout, a pair of maxillary barbels, and a pair of lower jaw barbels. The body is brownish, with the bottom brighter than the top. Dark hazy blotches near above of the pectoral fins, which is a special trait of O. melanopleurus.
However, this method can be difficult, and molecular identification is necessary. In particular, using mtDNA CO1 was an effective approach 9,17 . The results from nucleotide distance data based on the Kimura 2-parameter model indicated that the nucleotide distance among the fish was short in intraspecific species using mtDNA CO1 40 , which was supported by data showing that the percentage identity in O. melanopleurus species ranged between 96% and 97%. The Kelabau fish from the three sample sites were identified as O. melanopleurus by percentage identity, supported by an E-value of 0.0 and a 99%-100% query cover. The p-value indicated that the BLASTN results contained no errors. Besides, the low nucleotide distance values (<3%-5%) among the samples of O. melanopleurus from the Siak, Kampar and Rokan rivers, indicated that all samples were monophyletic.

Conclusion
Based on our findings, we concluded that 86 of the 90 samples of kelabau from the Siak, Kampar and Rokan rivers in Riau were O. melanopleurus, as revealed by their morphological traits and the molecular analyses.

Data availability
Underlying data CO1 gene sequences and raw biometric data of Osteochilus melanopleurus from Riau rivers can be found on OSF. The raw CO1 sequence data were also deposited in Gen-Bank and can be found under sequential accession numbers MH430827-MH430854 and MH459085-MH459142. Grammatical and spelling mistakes throughout the paper have been revised. The introduction has been improved by adding information on the difference of the characteristics of two species of fish . As there are 2 types of fish kelabau (Osteochillus spp) that are named as "kelabau fish" by local people, information on the difference of those fishes was obtained by interviewing local fishermen. The presence of this information may be useful to give more explanation about the difference and the similarity of fish kelabau present in Riau in general. A phrase "The demand for it as …" until "… the population of these as well as many other has been deleted as it is not correlate with the theme of this manuscript. fish" Finally, the author has also added a quote in the following paragraph: "The study population was collected and sampled …" until "… of the Faculty of Fisheries and Marine Science, Riau .

University, Indonesia"
The discussion section has been improved by comparing the result of this study with more articles available. Thank you for your suggestions and we hope that our article will be approved.

Pallipuram Jayasankar
Fish Genetics and Biotechnology Division, ICAR-Central Institute of Freshwater Aquaculture, Bhubaneswar, Odisha, India I have gone through the revision/corrections effected by the authors in the manuscript "Biometric and I have gone through the revision/corrections effected by the authors in the manuscript "Biometric and genetic differences in kelabau ( spp.) as revealed using cytochrome c oxidase subunit 1". The Osteochilus manuscript has been improved as per my suggestions. I recommend to consider its indexing.
No competing interests were disclosed.

Competing Interests:
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
Author Response 08 Feb 2020 , Padjadjaran University, Indonesia, Sumedang, Indonesia Nur Asiah Dear Mr. Ja'afar Nuhu Ja'afar,, Thank you for your suggestions on our manuscript and we apologize for the late to response your suggestion because we need to discuss with other authors. Here we would like to inform that we already revised paragraph three by adding reasons for limited variation such as inbreeding within the species due to limited migration and we also added discussion about data on table 3.
Variation in nucleotide diversity of the fish from the sampling areas are different. In the Kampar River, the fish was sampled from the relatively narrow area and it may cause the lowness of the nucleotide diversity. The diversity was 0 and it means that the nucleotide of the fish samples was almost identical. Freeland ., (2011) stated that the low nucleotide diversity may occur as the et al habitat of the fish is very suitable and the fish may not face environmental related problem that trigger any genetic changing. However, the nucleotide diversity of the fish from the Siak and Rokan River, was higher than Kampar River. Siak and Rokan Rivers have relatively low water quality that was caused mainly by anthropogenic activities. Changing in water quality may trigger the fish to adapt with that environmental condition and it may cause changes in genetic variation. Freeland et ., (2011) stated that fish population with high genetic variation may be able to face problems al related to environmental changing.
Finally, we want to thank you very much for your suggestion on our manuscript and we hope that our manuscript will be approved.

Pallipuram Jayasankar
Fish Genetics and Biotechnology Division, ICAR-Central Institute of Freshwater Aquaculture, Bhubaneswar, Odisha, India DNA barcoding is an important and useful tool to ratify species status in circumstances where DNA barcoding is an important and useful tool to ratify species status in circumstances where morphology-based taxonomy fails or nearly-fails to perform unambiguous identification of species. The authors in the present study have taken up an important group of freshwater fish. There are many grammatical errors and some technical flaws in the manuscript. I felt one major drawback could be their total silence on the nominal species of . Of course using barcoding they have proved Osteochilus kelabu that the species is . However, they should have shown a phylogenetic comparison of O. melanopleurus these two nominal species to rule out confusion in identification. Morphological method followed does not appear to be robust. They are advised to use TRUSS MORPHOMETRICS which can be performed using user-friendly software nowadays. In the phylogenetic tree the majority of bootstrap values do not appear to be robust; hence it could reflect a spurious relationship.

Is the work clearly and accurately presented and does it cite the current literature? Partly
Is the study design appropriate and is the work technically sound? Partly

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes No competing interests were disclosed. Competing Interests: Reviewer Expertise: Fish Biology and Fisheries biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
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