Accepted author version posted online: 29 January 2020 Sequencing and Characterisation of Complete Mitochondrial DNA Genome for Trigonopoma pauciperforatum (Cypriniformes: Cyprinidae: Danioninae) with Phylogenetic Consideration 1 Chung Hung Hui*, 1Leonard Lim Whye Kit, 2Liao Yunshi, 2Tommy Lam Tsai Yuk and 1 Chong Yee Ling 1 Faculty of Resource and Technology, Universiti Malaysia Sarawak, Kota Samarahan 94300 Sarawak, Malaysia 2 School of Public Health, The University of Hong Kong, Hong Kong *Corresponding author: hhchung@unimas.my Running title: mitochondrial DNA genome of Trigonopoma pauciperforatum Abstract. The Trigonopoma pauciperforatum or the redstripe rasbora is a cyprinid commonly found in marshes and swampy areas with slight acidic tannin-stained water in the tropics. In this study, the complete mitogenome sequence of T. pauciperforatum was first amplified in two parts using two pairs of overlapping primers and then sequenced. The size of the mitogenome is 16,707 bp, encompassing 22 transfer RNA genes, 13 protein-coding genes, two ribosomal RNA genes and a putative control region. Identical gene organisation was detected between this species and other family members. The heavy strand accommodates 28 genes while the light strand houses the remaining nine genes. Most protein-coding genes utilize ATG as start codon except for COI gene which uses GTG instead. The terminal associated sequence (TAS), central conserved sequence block (CSB-F, CSB-D and CSB-E) as well as variable sequence block (CSB-1, CSB-2 and CSB-3) are conserved in the control region. The maximum likelihood phylogenetic tree revealed the divergence of T. pauciperforatum from the basal region of the major clade, where its evolutionary relationships with Boraras maculatus, Rasbora cephalotaenia and R. daniconius are poorly resolved as suggested by the low bootstrap values. This work contributes towards the genetic resource enrichment for peat swamp conservation and comprehensive in-depth comparisons across other phylogenetic researches done on the Rasbora-related genus. Keywords: Trigonopoma pauciperforatum, mitogenome, gene arrangement, light strand origin, phylogenetic analysis INTRODUCTION The redstripe rasbora (Trigonopoma pauciperforatum) (Weber & de Beaufort, 1916) is grouped under the subfamiliy Danioninae in the Cyprinidae family. It has the distinctive thick striking red neon stripe which aligns in parallel to its spine, starting from the side of its jaw, crossing upper part of the eye and up till before its tail fin (Weber & de Beaufort, 1916). Its greyish brown streamline body are equipped with smoke-grey fins, white belly as well as a fork-shaped caudal fin tail (Ward, 2003). Females have bigger bellies than 1 males in general because this species is egg-spawning fish. This red-striped rasbora fish can be found abundantly in school around stagnant fresh waters (rivers, drainages, lakes and streams) of South East Asia, including Peninsular Malaysia, Sarawak and Sumatra (Ward, 2003). The type locality of this species is Sumatra. Their natural habitat has heavily grown and overhanging vegetation with minimal lighting. The diet of this fish is mainly made up of zooplankton, larvae and insects. Adult fish can grow up to the length of >6 centimetre (Ward, 2003). The T. pauciperforatum is a popular ornamental aquarium fish often mistaken for the Glowlight Tetra (Hemigrammus erythrozonus) (Durbin, 1909) due to their high morphological similarities but they are distinguishable by the much brighter red stripe and the absence of adipose fin in the Redline Rasbora (Durbin, 1909; Weber & de Beaufort, 1916; Ward, 2003). Due to their extremely selective breeding behaviour, breeding them in aquarium conditions is not easy and the success rate is higher when they are placed in school of 6 to 10 (Ward, 2003). Adult females scatter their eggs all over overgrown vegetation before the adult males are stimulated release sperms to fertilise the eggs during the action of tailing the females. Egg hatching occurs within 1 to 2 days post fertilisation and the fry can swim freely within 3 to 5 days (Ward, 2003). The lifespan of this fish ranges from 3 to 5 years with good care and maintenance under the following conditions: pH 6.2 to 7.0, 0 to 6-degree hardness and 22.7 to 26°C (Ward, 2003). The T. pauciperforatum was previously classified under the genus Rasbora. The Rasbora genus encompasses a large group of diversified freshwater fishes, making it the most species-enriched genus (87 species as of 2015) in the Cyprinidae family (Fricke et al., 2018). The classification of the Rasbora genus possesses complications as it is known as the catch-all group lacking synapomorphies or shared derived characters (Brittan, 1954; Kottelat & Vidthayanon, 1993; Liao et al., 2010; Tang et al, 2010). The eight Rasbora species complexes defined by Brittan (1954) had been revised recurrently over the years by various researchers (Kottelat & Vidthayanon, 1993; Siebert & Guiry, 1996; Kottelat, 2005; Liao et al., 2010) with some new genera being introduced and till now majority of them still hold firm on the Rasbora sensu lato concept by Brittan (1954) which encompasses all the new genera created. Yet, most of the Rasbora species lack the distinctive characters to form a monophyletic clade of its own both morphologically (Liao et al., 2010) and molecularly (mitochondrial COI, Cytb and nuclear RAG1) (Kusuma et al., 2016). The use of Rasbora species in genetic research is picking up its pace recently with the discovery of their potential as ecotoxicology models (Lim et al., 2018; Wijeyaratne & Pathiratne, 2006). To date, only nine Rasbora species (namely R. argyrotaenia, R. sumatrana, R. trilineata, R. aprotaenia, R. steineri, R. lateristriata, R. daniconius, R. borapetensis and R. cephalotaenia) and four other species previously classified under the Rasbora genus (Rasboroides vaterifloris, Trigonostigma heteromorpha, T. espei and Boraras maculatus) (Miya, 2009; Tang et al., 2010; Chang et al., 2013; Ho et al., 2014; Zhang et al., 2014; Kusuma & Kumazawa, 2015; Kusuma et al., 2017) had their mitochondrial genomic sequences published out of the total 87 species discovered thus far (Fricke et al., 2018), a mere 14.94%. The genus T. pauciperforatum resides in (Trigonopoma) contains only two species thus far, where its sole genus counterpart is T. gracile. To the best of our knowledge, T. pauciperforatum is the only species from this genus that have had its mitogenome sequenced and this accounts for the urgency to unravel more about the mitogenomes of its genus as well as natural habitat counterparts 2 in order to obtain a bigger picture of the genetic biodiversity in the peat swamp for conservation purposes (Chen et al., 2016; Sule et al., 2018). On the other hand, the phylogenetic data based on whole mitogenome sequences of this species provides opportunities for comprehensive comparison of the phylogenetic tree constructed based on morphologies (Liao et al., 2010). Thus, this study had shed light on the landscape of the complete mitochondrial genome of T. pauciperforatum beside further dissecting on the genetic contents and revealing the molecular phylogenetic relationship across 13 other closely related members of the Danioninae subfamily (from Rasbora genus and other species previously classified under Rasbora genus). This study also contributes towards the genetic resource enrichment for peat swamp conservation (Sule et al., 2018) and comprehensive in-depth comparisons across other phylogenetic researches (Liao et al., 2010; Kusuma et al., 2016) done on the Rasbora-related genus. MATERIALS AND METHODS Sampling and Genomic DNA Extraction The T. pauciperforatum specimen was collected from Matang River, Sarawak, Malaysia (1.5755° N, 110.2990° E) with the permit issued by Sarawak Forestry Department (permit number: NCCD.94047(Jld13)-178). Adult fish was sacrificed humanely using TricaneTM as anaesthetics with permission from Universiti Malaysia Sarawak Animal Ethics Committee (reference number: UNIMAS/TNC(PI)-04.01/06-09(17)). The muscle tissues were harvested from the fish body before subjecting to storage in 95% ethanol. The genomic DNA was extracted using CTAB method (Thomas et al., 2010). Primers Design, Long-PCR Amplification and DNA Sequencing A total of two pairs of primers were designed based on the multiple alignment outcomes from the complete mitochondrial genome of four closely related Rasbora species including R. argyrotaenia, R. sumatrana, R. trilineata and R. aprotaenia. The primer pairs (Table 1) were designed to amplify two large fragments from the mitochondrial genome with overlapping of at least 2 kb at both ends of fragments to ensure good sequencing reads. The complete mitochondrial genome of T. pauciperforatum was assembled by joining the two large amplicon fragments and trimming overlapping sequences. Long-Polymerase Chain Reaction (Long-PCR) was conducted using Bio-Rad T-100 Thermal Cycler in 20 μL total reaction volume encompassing 0.4 μL 10 μM forward and reverse primer each, 1.6 μL 2.5mM dNTP, 2.0 μL 10X PCR buffer (with Mg2+), 2.5 U high-fidelity Taq polymerase, 14.6 μL nuclease-free water and 0.8 μL genomic DNA extract orchestrated under conditions: one cycle of pre-denaturation at 94°C for 2 min, followed by 35 cycles of denaturation, annealing and extension at 94°C (30 s), primerspecific temperature (30 s) and 72°C (5 min) respectively and a final extension cycle at 72°C for 5 min. Agarose gel electrophoresis was performed to size separate the amplicons on 1% agarose gel for visualization under UV light. PCR purification was done prior to pair-ended short-read DNA sequencing on Illumina HiSeq 4000 System. Sequencing reads are quality-checked, adaptor-trimmed using cutadapt (Martin, 2011) 3 and assembled into the complete genome sequences using de novo assembler SPAdes (Bankevich et al., 2012). Mitochondrial Genome Characterisation and Gene Analysis The mitochondrial genome map was constructed using MitoFish (Iwasaki et al., 2013) (http://mitofish.aori.u-tokyo.ac.jp/annotation/input.html). Using MEGA 7.0 (Kumar et al., 2016), the protein-coding genes were subjected to translation into amino acid sequences to amend truncated or premature stop codons to ensure their functionalities. The codon usage was determined using MEGA 7.0 (Kumar et al., 2016) whereas the nucleotide composition was calculated using DNA nucleotide counter (Heracle BioSoft, 2014). All anti-codons of tRNA genes were identified using default search mode of the tRNA-scan SE v. 2.0 software (Lowe & Chan, 2016) (http://lowelab.ucsc.edu/cgi-bin/tRNAscanSE2.cgi). The L-strand origin (OL) determined thru sequence homology was then subjected to secondary structure visualisation using RNA structure 6.0 (Reuter & Mathews, 2010). All DNA sequences forming the complete mitochondrial genome was deposited into the GenBank database via the Sequin software (http://www.ncbi.nlm.nih.gov/Sequin/). Phylogenetic Tree Construction The raw data for phylogenetic analysis was collected from GenBank database which includes 13 other closely related members of the Danioninae subfamily (from Rasbora genus and other species previously classified under Rasbora genus) with complete mitochondrial genomic DNA available publicly; Acheilognathus typus and Danio rerio were selected as the outgroup. A total of 12 protein-coding genes (except for ND6 due to its high heterogeneity (Miya & Nishida, 2000) were concatenated to one single fasta format entry for each species to be analysed by first conducting multiple sequence alignment using clustal w in MEGA 7.0. A model test was performed using MEGA 7.0 prior to phylogenetic tree construction and the best suited model determined, the GTR+G (General Time Reversible model with Gamma distributed rates among sites) was employed via Maximum Likelihood (ML) analysis with bootstrap of 1000 replicates. The resultant phylogenetic tree was viewed using FigTree v1.4.2. RESULTS AND DISCUSSION Mitochondrial DNA Genome Structure The size of the complete mitochondrial genome of T. pauciperforatum is 16,707 bp with the inclusion of 22 tRNA genes, 13 protein-coding genes, two rRNA genes and a control region (Figure 1; Table 2). The complete mitochondrial genome sequence was deposited in the GenBank database with the assigned accession number MK034301. The heavy strand (H-strand) of the mitochondrion carries a total of 28 genes whereas the remaining are housed on the light strand (L-strand). All 4 overlaps detected from the entire mitochondrial genome are found on the H-strand. The greatest overlap (7 bp) was observed in both between genes ATP8 and ATP6 as well as between genes ND4L and 4 ND4. The lengthiest intergenic spacer (34 bp) was detected between genes tRNAAsn and tRNACys. The overall A+T content of the mitochondrial genome (60.0%) is much greater than G+C content (40.0%) (Table 3) which is similar to Cobitis lutheri, R. borapetensis and R. steineri (Cui et al., 2013; Zhang et al., 2014; Chang et al., 2013). The A+T content of protein-coding genes (60.6%) and control region (66.5%) differ by a slight 5.9%. Interestingly, the overall base composition of the entire mitochondrial genome and overall protein-coding genes did not deviate much from each other: 34.0% for A, 25.2% for C, 14.8% for G, 26% for T in terms of overall genome; 33.7% for A, 25.9% for C, 13.4% for G, 26.9% for T in total of 13 protein-coding genes. Protein-Coding Gene Features The gene group that made up almost 68.3% of the entire T. pauciperforatum mitochondrial genome is none other than the protein-coding gene group with a total of 11,412 bp coverage over 13 genes. With the translation capacity of up to 3801 amino acids, the protein-coding gene group incorporates genes with size ranging between 165 bp (ATP8) and 1830 bp (ND5). All three overlaps found in this group are located on the H-strand. The start codon usage of all 12 protein-coding genes are generally ATG, except for the GTG which is found exclusively in COI gene. These phenomena can be seen commonly occurring in Brama japonica, R. steineri, R. trilineata, R. argyrotaenia, R. borapetensis, R. aprotaenia and R. lateristriata (Chen et al, 2016; Chang et al., 2012; Kusuma et al., 2017; Ho et al., 2014; Zhang et al., 2014; Kusuma & Kumazawa, 2015). Looking at the termination codon usage, TAA is used by ND1, COI, ATP8, ND4L, ND5 and Cytb; TAG is utilized by ND6; whereas the others (ND2, COII, ATP6, COIII, ND3 and ND4) terminate with incomplete codons. This stop codon pattern is similar as seen in R. steineri (Chang et al., 2013). However, the termination codon usage is slightly varied across B. japonica, R. trilineata, R. argyrotaenia, R. borapetensis, R. aprotaenia and R. lateristriata (Chen et al, 2016; Kusuma et al., 2017; Ho et al., 2014; Zhang et al., 2014; Kusuma & Kumazawa, 2015) and this dissimilarity is deemed typical among the vertebrate mitogenomes (Ojala et al., 1981). The base composition of all protein-coding genes is depicted in Table 3. Transfer and Ribosomal RNA Gene Features Out of the 22 tRNA genes identifies in this study, 63.6% (14) of them are encoded by Hstrand while L-strand is responsible for encoding the other 8 tRNA genes. The anticodons of all tRNA genes are highly conserved across other fish metagenome such as R. borapetensis and B. japonica (Zhang et al., 2014; Chen et al., 2016). The 22 tRNA genes made up nucleotide length of 1552 bp with A+T content of 57.1%, the tRNAAla topped the group with A+T content of 69.2% whereas the tRNAThr bottomed the list with A+T content of 48.6%. Occupying a sum of 15.7% (2624 bp) of the entire mitochondrial genome of T. pauciperforatum, both rRNA genes (12S rRNA and 16S rRNA) are 71 bp apart on the H-strand with tRNAVal gene sandwiched in between them. The A+T content of 16S rRNA gene (58.1%) is slightly greater than that of 12S rRNA gene (54.2%), both contributing 5 to the overall total rRNA A+T content of 56.6% and base composition as displayed in Table 3: 35.9% for A, 23.7% for C, 19.6% for G and 20.7% for T. Non-Coding Region Excluding the light strand origin and control region, the other non-coding regions are relatively miniature from 1 to 11 bp. The light strand origin (OL) and the control region are the two large non-coding regions to be highlighted among the 16 non-coding regions identified. The light strand origin was located between tRNAAsn and tRNACys in the T. pauciperforatum mitochondrial genome. This 37 bp region has the stem-loop secondary structure forming capability with the allocation of 11 complementary nucleotide pairs contributing to the stem whilst the loop conformation takes up to 15 nucleotides arranged in closed circle (Figure 2). The largest non-coding region of the T. pauciperforatum mitochondrial genome, the control region, has A+T content of 66.5%, depicting higher A+T content than that of the overall mitogenome (60.0%), which was similarly detected in mitogenome of B. japonica (Chen et al., 2016). On the side note, the base composition of this control region is as below: 34.0% for A, 20.9% for C, 12.6% for G and 32.5% for T respectively as shown in Table 3. Besides, the terminal associated sequence (TAS), central conserved sequence block (CSB-F, CSB-D and CSB-E) as well as variable sequence block (CSB-1, CSB-2 and CSB-3) were all traced within the control region of this species. Phylogenetic Relationship Analysis A maximum likelihood tree was constructed to unravel the phylogenetic relationship of T. pauciperforatum and its closely related species with the whole mitogenome now available (Figure 3). The R. aprotaenia, R. lateristriata, R. sumatrana and R. steineri form a distinctive cluster with bootstrap value of 100%. Besides, the T. heteromorpha and T. espei pair as well as the R. argyrotaenia and R. borapetensis pair also scored 100% bootstrap possibilities which also in agreement to the findings by Kusuma & Kumazawa (2015) as well as Kusuma et al. (2017). T. pauciperforatum diverged from the basal region of the major clade, where its evolutionary relationships with B. maculatus, R. cephalotaenia and R. daniconius are poorly resolved as suggested by the low bootstrap values there. The phylogeny is rooted (indicated by the dashed line) by the outgroups Acheilognathus typus and Danio rerio. Comparing to the morphology based phylogenetic tree constructed by Liao et al. (2010) on 29 species of Rasbora with 41 morphological characters investigated, some distinctive dissimilarities were observed. For instances, R. lateristriata, R. cephalotaenia and R. trilineata were found to share the same clade when characterized morphologically (Liao et al., 2010) but that is not the case in this study. The T. pauciperforatum reside on the same clade as T. heteromorpha and R. vaterifloris when scored morphologically but in this study all three of them are located far apart. Some comparisons across the results of these two trees are not possible yet due to the absence of some species in both analysis. R. borapetensis was observed to be closely related to R. rubrodorsalis and both of them formed clade with R. cf. beauforti and R. semilineata (Liao et al., 2010) whereas in this study, R. borapetensis is closely related to R. argyrotaenia in which R. argyrotaenia was not included in the analysis by Liao et al. (2010). T. pauciperforatum was discovered as the closest neighbour to its only genus counterpart, T. gracile beside 6 sharing the clade with other members like B. brigittae, Rasbosoma spilocerca and Horadandia atukorali which four of them were not included in this study because of the lack of the whole mitogenome sequences (Liao et al., 2010). Another comparison of phylogenetic tree was done to that from Kusuma et al. (2016) and the input sequences used are COI, Cytb, RAG1 and opsin gene sequences. One of the similarities detected is that R. lateristriata was grouped closely with R. aprotaenia and R. sumatrana. The grouping of R. borapetensis and R. agryrotaenia inside the same clade is the other similar scenario observed and the only difference is that in the tree constructed by Kusuma et al. (2016), R. dusonensis was found to be related closer to R. agryotaenia than R. borapetensis. The tree from Kusuma et al. (2016) depicted a strong clade with members like T. pauciperforatum, T. gracile, Kottelatia brittani, B. merah and R. kalbarensis, with B. merah being the closest to T. pauciperforatum. However, due to the absence of mitogenome sequences from the abovementioned species that shares the same clade with T. pauciperforatum, this analysis cannot be conducted in this study. CONCLUSION The complete mitogenome of T. pauciperforatum has been unravelled with the completion of the sequencing and characterization process. Besides, this study had also revealed the close molecular phylogenetic relationship between this species and 13 other closely related members of the Danioninae subfamily (from Rasbora genus and other species previously classified under Rasbora genus). This study also serves as an enrichment towards the complete mitochondrial genome count within the Trigonopoma genus in terms of evolution and conservation genetics. REFERENCES Bankevich, A., Nurk, S., Antipov, D., Gurevich, A. A., Dvorkin, M., Kulikov, A. S., Lesin, V. M., Nikolenko, S. I., Pham, S., Prjibelski, A. D., Pyshkin, A. V., Sirotkin, A. V., Vyahhi, N., Tesler, G., Alekseyev, M. A., & Pevzner, P. A. (2012). SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. Journal of Computational Biology, 19(5), 455-477. Brittan, M. R. (1954). A revision of the Indo-Malayan fresh-water fish genus Rasbora. Manila, Philippines: Monographs of the Institute of Science and Technology. p. 224. Chang, C. H., Tsai, C. L., & Jang-Liaw, N. H. (2013). Complete mitochondrial genome of the Chinese rasbora Rasbora steineri (Teleostei, Cyprinidae). Mitochondrial DNA, 24(3), 183-185. Chen, F., Ma, H., Ma, C., Zhang, H., Zhao, M., Meng, Y., Wei, H., & Ma, L. (2016). Sequencing and characterization of mitochondrial DNA genome for Brama japonica (Perciformes: Bramidae) with phylogenetic considerations. Biochemical Systematics and Ecology, 68, 109-118. Cui, J., Xu, J., Li, Q., Wang, K., Xu, P., & Sun, X. (2013). The complete mitochondrial genome of Cobitis lutheri (Cypriniformes: Cobitidae: Cobitis). Mitochondrial DNA, 26(6), 875-876. 7 Durbin, M. L. (1909). Reports on the expedition to British Guiana of the Indiana University and the Carnegie Museum, 1908. Report No. 2: A new genus and twelve new species of tetragonopterid characins. Annals of the Carnegie Museum, 6(1), 55-72. Fricke, R., Eschmeyer, W. N. & R. van der Laan (eds) (2018). Catalog of fishes: genera, species, references. (http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatmain.a sp). Electronic version accessed 02 Oct 2018. Heracle BioSoft. (2014). DNA nucleotide counter. Retrieved September 11, 2018, from http://www.dnabaser.com/download/DNA-Counter/index.html. Ho, C. W., Liu, M. Y., & Chen, M. H. (2014). Complete mitochondrial genome of Rasbora trilineata (Cypriniformes, Cyprinidae). Mitochondrial DNA, 27(3), 1755-1757. Iwasaki, W., Fukunaga, T., Isagozawa, R., Yamada, K., Maeda, Y., Satoh, T. P., Sado, T., Mabuchi, K., Takeshima, H., Miya, M., & Nishida, M. (2013). MitoFish and MitoAnnotator: A mitochondrial genome database of fish with an accurate annotation pipeline. Molecular Biology and Evolution, 30, 2531-2540. Kottelat, M. (2005). Rasbora notura, a new species of cypinid fish from the Malay Peninsula. Ichthyological Exploration of Freshwaters, 16, 265-270. Kottelat, M., & Vidthayanon, C. (1993). Boraras micros, a new genus and species of minute freshwater fish from Thailand (Teleostei: Cyprinidae). Ichthyological Exploration of Freshwaters, 4, 161-176. Kumar, S., Stecher, G., & Tamura, K. (2016). MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33(7), 1870-1874. Kusuma, W. E., & Kumazawa, Y. (2015). Complete mitochondrial genome sequences of two Indonesian rasboras (Rasbora aprotaenia and Rasbora lateristriata). Mitochondrial DNA Part A, 27(6), 4222-4223. Kusuma, W. E., Ratmuangkhwang, S., & Kumazawa, Y. (2016). Molecular phylogeny and historical biogeography of the Indonesian freshwater fish Rasbora lateristriata species complex (Actinopterygii: Cyprinidae): Cryptic species and west-to-east divergences. Molecular Phylogenetics and Evolution, 105, 212-223. Kusuma, W. E., Samuel, P. D., Wiadnya, D. G. R., Hariati, A. M., & Kumazawa, Y. (2017). Complete mitogenome sequence of Rasbora argyrotaenia (Actinopterygii: Cyprinidae). Mitochondrial DNA Part B, 2(2), 373-374. Liao, T. Y., Kullander, S. O., & Fang, F. (2010). Phylogenetic analysis of the genus Rasbora (Teleostei: Cyprinidae). Ichthyological Exploration of Freshwaters, 23, 3744. Lim, L. W. K., Tan, H. Y., Aminan, A. W., Jumaan, A. Q., Moktar, M. Z., Tan, S. Y., Balinu, C. P., Robert, A. V., Chung, H. H., & Sulaiman, B. (2018). Phylogenetic and expression of ATP-binding cassette transporter genes in Rasbora sarawakenesis. Pertanika Journal of Tropical Agricultural Science, 41(3), 1341-1354. Lowe, T.M. & Chan, P.P. (2016). tRNAscan-SE On-line: Search and Contextual Analysis of Transfer RNA Genes. Nucleic Acids Research 44: W54-57. Martin, M. (2011). Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. Journal, 17(1), 10-12. Miya, M. (2009). Whole mitochondrial genome sequences in Cypriniformes. Unpublished manuscript, Natural History Museum & Institute, Chiba, Japan. 8 Miya, M., & Nishida, M. (2000). Use of mitogenic information in teleostean molecular phylogenetics: A tree-based exploration under the maximum-parsimony optimality criterion. Molecular Phylogenetics and Evolution, 17, 437-455. Ojala, D., Montoya, J., & Attardi, G., (1981). tRNA punctuation model of RNA processing in human mitochondrial. Nature, 290, 470-474. Reuter, J. S., & Mathews, D. H. (2010). RNAstructure: Software for RNA secondary structure prediction and analysis. BMC Bioinformatics, 11,129. Siebert, D. J., & Guiry, S. (1996). Rasbora johannae (Teleostei: Cyprinidae), a new species of the R. trifasciata-complex from Kalimantan, Indonesia. Cybium, 20, 395404. Sule, H. A., Ismail, A., Amal, M. N. A., Zulkifli, S. Z., Roseli, M. F. M., & Shohaimi, S. (2018). Water quality influences on fish occurrence in peat swamp forest and its converted areas in North Selangor, Malaysia. Sains Malaysiana, 47(11), 2589-2600. Tang, K. L., Agnew, M. K., Hirt, M. V., Sado, T., Schneider, L. M., Freyhof, J., Sulaiman, Z., Swartz, E., Vidthayanon, C., Miya, M., Saitoh, K., Simons, A. M., Wood, R. M., & Mayden, R. L. (2010). Systematics of the subfamily Danioninae (Teleostei: Cypriniformes: Cyprinidae). Molecular Phylogenetics and Evolution, 57, 189-214. Thomas, J. C., Khoury, R., Neeley, C. K., Ann, M., Akroush, A. & Davies, E. C. (2010). A Fast CTAB Method of Human DNA Isolation for Polymerase Chain Reaction Applications. Biochemical Education, 25(4), 233-235. Ward, B. (2003). The aquarium fish surviving manual (8th Ed.). Hauppauge, NY: Quill Publishing Limited. Weber, M., & de Beaufort, L. F. (1916). The fishes of the Indo-Australian Archipelago. III. Ostariophysi: II Cyprinoidea, Apodes, Synbranchi. Leiden, Netherlands: E. J. Brill Ltd. p. 79. Wijeyaratne, W. M. D. N., & Pathiratne, A. (2006). Acetylcholinesterase inhibition and gill lesions in Rasbora caverii, an indigenous fish inhabiting rice field associated waterbodies in Sri Lanka. Ecotoxicology, 15(7), 609. Zhang, S., Cui, J., Li, C. Y., Mahboob, S., Al-Ghanim, K., Xu, P., & Sun, J. (2014). The complete mitochondrial genome of Rasbora borapetensis (Cypriniformes: Cyprinidae: Rasbora). Mitochondrial DNA, 27(2), 1-2. 9 Figure 1. Circular genome map of T. pauciperforatum. Genes encoded on heavy and light strand are depicted in outer and inner circle respectively. The inner ring displays the GC percent per every 5 bp where the darker lines represent higher GC percent. The size of the complete mitogenome of T. pauciperforatum is 16,707 bp with the contribution from 22 tRNA genes, 13 protein-coding genes, two rRNA genes and a control region. 10 Figure 2. The predicted secondary structure of light strand origin which is situated between tRNAAsn and tRNACys genes of R. T. pauciperforatum. The part of the tRNACys gene sequence is in the box. Figure 3. Phylogenetic tree of T. pauciperforatum with other Rasbora genus members and outgroups, based on 12 protein-coding genes (except ND6 gene) via the GTR+G (General Time Reversible model with Gamma distributed rates among sites) Maximum Likelihood (ML) analysis with bootstrap of 1000 replicates. The tree was rooted (represented by dashed line) by the outgroups Acheilognathus typus and Danio rerio. 11 Table 1. Primers used for the amplification of the T. pauciperforatum mitogenome. Primer name SF1 SR1 LF1 LR1 Primer sequence Tm (°C) GTGCTTCCTCTACACCAC TGATGTTGAGAAGGCTAC CCTATCTTACCGAGAAAG GAGGCCTTCCCATCTAGA 55.3 Amplification length (bp) 8923 48.6 9990 Table 2. Features of the whole T. pauciperforatum mitogenome. Gene tRNAPhe 12S rRNA tRNAVal 16S rRNA tRNALeu (UUA) ND1 tRNAIle tRNAGln tRNAMet ND2 tRNATrp tRNAAla tRNAAsn tRNACys tRNATyr COI tRNASer (UCA) tRNAAsp COII tRNALys ATP8 ATP6 COIII tRNAGly ND3 tRNAArg ND4L ND4 tRNAHis tRNASer (AGC) tRNALeu (CUA) ND5 ND6 tRNAGlu Cytb tRNAThr tRNAPro D-loop Position (5’-3’) Start End 1 70 1021 1092 2765 2841 3821 3961 3963 4032 5077 5218 5292 5391 5463 5465 7086 7088 7164 7855 7932 8090 8773 9558 9629 9978 10048 10338 11720 11789 11858 11931 14278 14347 14354 15495 15645 15646 69 1020 1091 2764 2839 3815 3892 3891 4031 5076 5148 5151 5220 5327 5393 7015 7016 7157 7854 7929 8096 8772 9557 9628 9977 10047 10344 11719 11788 11856 11930 13760 13757 14279 15490 15564 15576 16707 Codon Start Stopa Amino acid Anticodon Intergenic nucleotideb (bp) Strandc GAA 0 0 0 0 1 5 -2 1 0 0 2 1 34 1 1 0 1 6 0 2 -7 0 0 0 0 0 -7 0 0 1 0 -4 0 6 4 11 0 H H H H H H H L H H H L L L H H L H H H H H H H H H H H H H H H L L H H L - TAC TAA ATG TAA 325 GAT TTG CAT ATG T-- 348 TCA TGC GTT GCA GTA GTG TAA 517 TGA GTC ATG T-- 230 ATG ATG ATG TAA TATA- 55 227 261 ATG T-- 116 ATG ATG TAA TA- 99 460 TTT TCC TCG GTG GCT TAG ATG ATG TAA TAG 610 174 ATG TAA 379 TTC TGT TGG a TA- and T—indicate incomplete stop codons; b Numbers indicate interspaced nucleotides and negative numbers indicate overlapping nucleotides; c H and L indicate heavy or light strand respectively. 12 Table 3. The nucleotide base composition of all genes in the T. pauciperforatum mitogenome. Region A Protein-coding gene ND1 ND2 COI COII ATP8 ATP6 COIII ND3 ND4L ND4 ND5 ND6 Cytb Overall of protein-coding gene tRNA gene tRNAPhe tRNAVal tRNALeu (UUA) tRNAIle tRNAGln tRNAMet tRNATrp tRNAAla tRNAAsn tRNACys tRNATyr tRNASer (UCA) tRNAAsp tRNALys tRNAGly tRNAArg tRNAHis tRNASer (AGC) tRNALeu (CUA) tRNAGlu tRNAThr tRNAPro Overall of tRNA gene rRNA gene 12S rRNA 16S rRNA Overall of rRNA gene Control region Overall of the genome Base composition (%) C G T A + T content (%) 34.5 38.7 28.4 33.7 35.8 33.7 30.4 30.1 29.0 33.6 35.8 44.6 31.7 33.7 26.7 28.1 24.4 22.6 24.2 25.5 25.7 27.2 27.6 26.3 25.2 29.9 26.0 25.9 12.9 10.3 16.8 15.8 8.5 11.1 16.2 14.3 13.8 12.8 12.4 10.7 14.1 13.4 25.9 22.9 30.4 27.9 31.5 29.7 27.6 28.4 29.6 27.3 26.5 14.8 28.2 26.9 60.4 61.6 58.8 61.6 67.3 63.4 58.0 58.5 58.6 60.9 62.3 59.4 59.9 60.6 37.7 28.2 28.0 25.0 35.2 31.9 36.1 36.8 32.9 29.2 31.0 26.8 37.1 34.7 36.6 27.1 34.8 35.3 36.5 34.8 28.6 37.1 32.8 20.3 25.4 24.0 22.2 25.4 30.4 22.2 22.1 27.4 27.7 31.0 28.2 20.0 25.3 22.5 25.7 23.2 19.1 17.66 23.2 28.6 28.6 24.5 20.3 23.9 22.7 26.4 14.1 15.9 22.2 8.8 19.2 23.1 19.7 19.7 14.3 18.7 12.7 21.4 13.0 19.1 17.6 17.4 22.9 11.4 18.4 21.7 22.5 25.3 26.4 25.4 21.7 19.4 32.4 20.5 20.0 18.3 25.4 28.6 21.3 28.2 25.7 29.0 26.5 28.4 24.6 20.0 22.9 24.3 59.4 50.7 53.3 51.4 60.6 53.6 55.5 69.2 53.4 49.2 49.3 52.2 65.7 56.0 64.8 52.8 63.8 61.8 64.9 59.4 48.6 60.0 57.1 33.9 37.1 35.9 34.0 34.0 25.0 23.0 23.7 20.9 25.2 20.8 18.9 19.6 12.6 14.8 20.3 21.0 20.7 32.5 26.0 54.2 58.1 56.6 66.5 60.0 13