{"paper_id":"1b2d8e42-2825-4c49-8583-ce656af2a7da","body_text":"Assembly and analysis of the complete mitochondrial genome of the Bogotá Robber Frog, Pristimantis bogotensis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Assembly and analysis of the complete mitochondrial genome of the Bogotá Robber Frog, Pristimantis bogotensis Kevin S. Jaramillo, Andrew J. Crawford This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9153070/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background Mitochondrial genomes are widely used in evolutionary studies due to their maternal inheritance, high mutation rate, and utility for inferring phylogeographic and phylogenetic relationships. The Neotropical frog genus, Pristimantis (Anura: Craugastoridae), with over 620 described species, represents the most species-rich anuran genera in the world, yet no mitogenome assemblies are available from this group. Here we use long-read DNA sequencing to assemble the complete mitogenome of Pristimantis bogotensis , a high-elevation Andean frog endemic to the Eastern Cordillera of Colombia. This mitogenomic characterization represents a valuable resource within this group of direct-developing anurans. Methods and results The complete mitochondrial genome of P. bogotensis was sequenced using Oxford Nanopore Technologies and assembled de novo . The mitogenome assembly was unusually large at 19,565 bp, yet exhibited the structure typical of vertebrate mitochondrial genomes, with a GC content of 40.2% and containing 13 protein-coding genes, 2 rRNA genes, 1 light-strand origin of replication, 1 heavy-strand origin of replication, 1 D-loop and 22 tRNA genes. Notably, the tRNA-Cys lacked the D-arm. A phylogenetic analysis of previously published complete and partial anuran mitogenomes confirmed the placement of P. bogotensis within the Pristimantis genus and support the loss of the D-arm in tRNA-Cys as a potential synapomorphy is this speciose clade. Conclusion To our knowledge, this is the first complete mitochondrial genome reported for the family Craugastoridae, and represents an important resource for phylogeography, metabarcoding, and phylogenetic studies in frogs, with direct applications in conservation and taxonomy. Craugastoridae Long-read sequencing Mitogenomics Molecular phylogenetics Molecular evolution Oxford Nanopore Technology Figures Figure 1 Figure 2 Figure 3 Introduction Mitochondrial genomes have been widely utilized in evolutionary biology due to their compact structure, maternal inheritance, and high mutation rate [ 1 ]. These features make them valuable markers for phylogenetics, phylogeography, and species identification and delimitation [ 2 , 3 ]. Vertebrate mitochondrial genomes typically encode 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), a light-strand origin of replication, and a control region (containing the D-loop and a heavy-strand origin of replication) [ 4 ]. This conserved genomic architecture provides a robust framework for comparative evolutionary studies across vertebrate lineages. Although mitochondrial genomes have traditionally been characterized using Sanger sequencing and more recently with next-gen short-read sequencing approaches, these methods may present limitations in resolving repetitive regions and structural duplications, such as those found in mitochondrial control regions [ 5 ]. Short-read sequencing may result in repetitive DNA sequences being collapsed upon in-silico assembly and genic length being underestimated. The implementation of long-read sequencing represents more than a technical improvement, as it enables the detection of structural variation that refines our understanding of genome evolution and organization [ 6 , 7 ]. In parallel, advances in long-read assembly algorithms have further enhanced the accurate reconstruction of repetitive sequences and structural features, often resulting in larger and more structurally accurate assemblies [ 2 , 8 ], highlighting how modern sequencing approaches can reveal previously hidden genomic features and provide new insights into molecular evolution in animals, including structural evolution of vertebrate mitochondrial genomes. Pristimantis is the most diverse genus of amphibians, with over 620 species distributed throughout Central and South America [ 9 ]. Striking new species are continually being discovered and described, even near major urban centers. Despite its high taxonomic diversity, the availability of complete mitochondrial genome data for this genus is nil. Pristimantis bogotensis is an Andean frog endemic to the Eastern Cordillera of Colombia, inhabiting high-elevation environments where extreme climatic conditions pose significant adaptive challenges [ 10 ]. Understanding its mitochondrial genome may offer new insights into the molecular evolution and phylogenetics of the genus, and provide bioinformatic resources for DNA-based inventories, resolving taxonomic uncertainties and improving conservation planning for this hyper-diverse genus. Previous phylogenetic studies of Pristimantis have relied mainly on a handful of mitochondrial and nuclear genes [ 9 ] without incorporating whole mitogenomes, despite the potential for more genes as well as structural variations to provide additional synapomorphies [ 11 ]. Here we DNA sequence the mitogenome using third-generation technology and then assemble and annotate the first complete mitogenome of a Craugastoridae family. Materials and Methods Sample collection, DNA extraction and ONT sequencing A single specimen of P. bogotensis was collected in September of 2024 from Fómeque, Cundinamarca, Colombia (4.563183°, -73.835816°), at 3100 m elevation. The specimen was fixed and deposited in the amphibian collection of the Museo de Historia Natural C.J. Marinkelle at the Universidad de los Andes, under accession number ANDES-A 5749. Before fixing the specimen, muscle and liver tissues were removed, flash-frozen in liquid nitrogen, and stored promptly at -80°C. Genomic DNA of the samples was isolated using the Monarch Genomic DNA Purification Kit (New England Biolabs). The quality and concentration of extracted DNA were assessed using a Nanodrop and Qubit 4.0 fluorometer. Sequencing libraries were prepared using the Ligation Sequencing Kit V14 SQK-LSK114 from Oxford Nanopore Technologies (ONT), and raw sequencing data were generated using the ONT PromethION platform, using R10.4.1 cells. This process was made by GenCore at Universidad de los Andes, with a total of three independent sequencings, each in a different cell. Mitogenome assembly and annotation ONT reads (57.8 Gb) obtained were base-called using Dorado/0.8.2 ( https://github.com/nanoporetech/dorado ) , and adaptors were removed with Porechop/1.0 ( https://github.com/rrwick/Porechop ). To extract similar contigs, we filtered reads using the mitochondrial genome assembly of Phyllobates terribilis (selected as the closest complete mitochondrial genome to P. bogotensis ) like a mitogenome map (GenBank accession: NC037380.1), using Minimap2/2.24 [ 12 ]. High-quality reads obtained (Q ≥ 10) were filtered using SAMtools/1.16.1 [ 13 ] and converted to FASTQ format for de novo assembly with Flye/2.9.3 [ 8 ]. The assembly was polished with Medaka/2.0.1 ( https://github.com/nanoporetech/medaka ). The assembled mitochondrial genome was annotated using MITOS2/2.1.9 into Proksee [ 14 , 15 ]. A circular genome map was generated with Proksee [ 15 ]. The consensus genome sequence has been deposited in GenBank under accession number [PX779192]. tRNA secondary structure prediction To confirm structural features of the tRNA genes annotated with MITOS2, we used the RNAfold web server ( http://rna.tbi.univie.ac.at ) from the ViennaRNA Package, which help us to predict secondary structures of ribosomal genes in the P. bogotensis mitogenome. The inferred structures were compared to the canonical vertebrate mitochondrial tRNA model [ 16 ] to identify conserved domains (acceptor stem, anticodon stem, TΨC stem) as well as deviations. Phylogenetic analyses Mitochondrial genomes of anuran species closely related to Pristimantis were retrieved from National Center for Biotechnology Information (NCBI). Taxon sampling was based on the phylogenetic framework for Neobatrachia [ 17 ], selecting taxa with available complete mitochondrial genomes, and including the two available partial mitogenomes of Pristimantis species to represent the genus, as no complete mitochondrial genomes were previously available. The 13 mitochondrial protein-coding genes were extracted and concatenated into a single dataset. Sequences were aligned using MAFFT/7.526 ( https://github.com/GSLBiotech/mafft ). A maximum likelihood (ML) phylogenetic tree was inferred using IQ-TREE/2.2.0 [ 18 ] with 1,000 ultrafast bootstrap replicates and 1,000 SH-aLRT replicates to evaluate statistical support for each node, and the Bayesian Information Criterion (BIC) was used to determine the optimal substitution model (mtVer+R4). Xenopus laevis was employed as outgroup. The tree was visualized using iTOL/v6. ( https://itol.embl.de ). Results and discussion The mitochondrial genome assembly of P. bogotensis was 19,565 bp and encodes the standard number of genes in the vertebrate mitogenome of 13 PCGs, 22 tRNAs, 2 rRNAs, 1 light-strand origin of replication (O L ), and 1 control region (containing 1 heavy-strand origin of replication (O H ) and 1 D-loop), with a GC content of 40.2% (Table 1 ). The gene order is identical to the ancestral vertebrate mitochondrial arrangement [ 4 , 6 ], exhibiting no gene rearrangements (Fig. 1 ), although two intergenic spacers of 99 bp and 51 bp were identified between ND1–tRNA(Leu2) and COX3–ATP6, respectively. All PCGs and structural RNAs are encoded on the heavy strand, except for nad6 and eight tRNAs (tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser, tRNA-Glu, tRNA-Pro), consistent with the conserved strand asymmetry observed across anuran mitogenomes [ 7 ]. The relatively large size of the P. bogotensis mitogenome likely reflects the improved recovery of repetitive regions through long-read ONT sequencing, which allows more accurate reconstruction of structural features, contributing to the increasing detection of larger mitogenomes [ 3 , 5 ]. Such improved structural resolution may provide a more realistic view of mitochondrial genome organization and variation [ 6 , 14 ]. Table 1 Genic location and boundaries of genes in the mitochondrial genome assembly of P ristimantis bogotensis , including the name of the gene, its specific location in the sequence, length in base pairs, the strand (H for heavy and L for light), the adjacent intergenic regions, the start and stop codons, and the DNA sequence of the corresponding anticodon for the transfer RNA (tRNA) genes. Gene Sequence location Length in bp Strand Intergenic region length in bp Start/stop codons Anticodon ATP6 1-629 629 H -10 ATG/TAA - ATP8 620–784 165 H 0 ATG/TAA - tRNA (Lys) 785–853 69 H -3 - UUU COX2 851–1538 688 H 1 ATG/TAA - tRNA (Asp) 1540–1607 68 H 1 - GUC tRNA (Ser2) 1609–1679 71 L -9 - UGA COX1 1671–3224 1554 H 1 GTG/TAG - tRNA (Tyr) 3226–3292 67 L 0 - GUA tRNA (Cys) 3293–3348 56 L -1 - GCA Light strand Origin of replication 3348–3371 24 H 2 - - tRNA (Asn) 3374–3446 73 L 0 - GUU tRNA (Ala) 3447–3515 69 L 0 - UGC tRNA (Trp) 3516–3583 68 H 9 - UCA ND2 3593–4613 1021 H 0 ATG/TAA - tRNA (Met) 4614–4684 71 H -1 - CAU tRNA (Gln) 4684–4754 71 L -1 - UUG tRNA (Ile) 4754–4824 71 H 6 - GAU ND1 4831–5666 836 H 99 ATG/TAA - tRNA (Leu2) 5766–5838 73 H 8 - UAA 16S ribosomal RNA 5847–7372 1526 H 2 - - tRNA (Val) 7375–7442 68 H -3 - UAC 12S ribosomal RNA 7440–8363 924 H 0 - - tRNA (Phe) 8364–8431 68 H -1 - GAA tRNA (Pro) 8431–8498 68 L -1 - UGG tRNA (Thr) 8498–8567 70 H 1 - UGU tRNA (Leu1) 8569–8640 72 H 17 - UAG Heavy strand Origin of replication 8658–8889 232 L 0 - - D-loop 8890–12997 4109 L 0 - - CYTB 12998–14141 1144 H 5 ATG/TAA - tRNA (Glu) 14147–14215 69 L 0 - UUC ND6 14216–14707 492 L -17 ATG/TAA - ND5 14691–16481 1791 H 4 ATG/TAA - tRNA (Ser1) 16486–16549 64 H 0 - GCU tRNA (His) 16550–16616 67 H 4 - GUG ND4 16621–17980 1360 H -7 ATG/TAA - ND4L 17974–18273 300 H 1 ATG/TAA - tRNA (Arg) 18275–18342 68 H 0 - UCG ND3 18343–18658 316 H 2 ATG/TAA - tRNA (Gly) 18661–18729 69 H -1 - UCC COX3 18729–19514 786 H 51 ATG/TAA - A striking feature of the P. bogotensis mitogenome is the absence of the Dihydrouridine arm (D-arm) in the mitochondrial tRNA-Cys gene, a structural deletion previously reported in Pristimantis [ 11 ] based on Sanger sequencing. This loss, confirmed here using long-read sequencing, eliminates a conserved four-base-pair stem structure, typical in most mitogenomes in vertebrates (Fig. 2 ). Comparative secondary structure analysis of the mitochondrial tRNA-Cys gene across the three Pristimantis species included in this study ( P. bogotensis, P. thymelensis , and P. fenestratus ) revealed a consistent absence of the D-arm, corroborating the structural findings [ 11 ] and supporting its potential use as a synapomorphy of the genus. The D-arm loss underscores the potential utility of tRNA structural evolution in resolving deep phylogenetic relationships, where homoplasy in base substitutions complicates phylogenetic resolution [ 11 , 16 ]. However, with over 620 species in the genus, it remains to be seen whether these observations will hold across major clades within Pristimantis . The mitochondrial genome of P. bogotensis also revealed a loss of D-arm in the tRNA-Ser1 gene, forming a simplified loop instead of the canonical cloverleaf structure (Fig. 2 ). This D-arm loss has been reported in other Neobatrachian frogs, including Zhangixalus schlegelii and Z. dennysi [ 19 ], and Buergeria buergeri [ 20 ]. This phenomenon is not exclusive to anurans; for example among molluscs, the scaphopod Pictodentalium vernedei exhibits similar D-arm truncations in tRNA-Ser1 [ 21 ], as do the trematodes, Diplodiscus japonicus and D. mehari [ 22 ]. The parallel loss of the D-arm in distantly related groups implies biochemical flexibility in the tRNA-Ser1 molecule [ 23 , 24 ]. Our molecular phylogeny based on all mitochondrial PCG’s recovered a well-resolved topology with strong statistical support across nodes (Fig. 3 ). The ingroup was well supported relative to the outgroup, Xenopus laevis , while representatives of Ranidae ( Rana kukunoris & R. temporaria ) were recovered as an early-diverging clade. Within Neobatrachia, species of the family Craugastoridae formed a strongly supported monophyletic group, where P. bogotensis clustered closely with P. thymelensis and P. fenestratus , confirming its phylogenetic placement within the genus [ 9 ]. The family Aromobatidae represented by Anomaloglossus blanci and the dendrobatid, Phyllobates terribilis , were recovered as sister lineages, consistent with previous phylogenomic studies [ 17 ]. Additionally, the family Bufonidae was also strongly supported as a monophyletic group, including Bufo gargarizans , B. stejnegeri , and Rhinella marina . The strong statistical support for most nodes reflect previous findings in amphibian phylogenomics and confirm the phylogenetic position of P. bogotensis within a well-supported clade of congeneric species. The phylogenetic results obtained here highlights the robustness of mitogenomic data for clarifying evolutionary relationships within Neobatrachia as these results are consistent with previous studies employing nuclear genome-wide data for amphibian phylogenetics [ 17 , 25 ]. Conclusion This study presents the first complete mitochondrial genome of P. bogotensis , filling a significant gap in anuran mitogenomics. The application of ONT long-read sequencing enabled the recovery of structurally complex regions and may contribute to a more accurate representation of mitochondrial genome architecture in vertebrates. By expanding mitochondrial genome datasets and integrating comparative approaches, this research enhances our understanding of the evolutionary history of P. bogotensis . Future studies should explore mitogenome variations across populations and congeneric species, as well as their implications in adaptations. Pristimantis bogotensis demonstrates that the retention of a stable mitochondrial gene order alongside tRNA structural plasticity underscores a dynamic in that while gene arrangement remains conserved, tRNA architecture tolerates major changes. Declarations Ethical Approval The CICUA (Institutional Animal Care and Use Committee) of the Universidad de los Andes approved this study (C.FUA_24 − 009). The individual was collected under the umbrella permit No. PR.6.2015.2182 to the Universidad de los Andes by the National Environmental Licensing Authority (ANLA) of Colombia. Competing interests The authors have no relevant financial or non-financial interests to disclose. Funding This research was funded by the Faculty of Sciences at the Universidad de los Andes under the Proyecto semilla to KSJ and AJC, and the grant in basic research (INV-2021-128-2323) to AJC. Author Contribution Laboratory work, mitochondrial genome assembly, annotation, and bioinformatic analyses were performed by KSJ. Library preparation and sequencing were conducted by the GenCore Facility at Universidad de los Andes. KSJ wrote the first draft of the manuscript. KSJ and AJC contributed to supervise, edit, revise, and improve the manuscript. Both authors read and approved the final manuscript. Acknowledgement We thank Ronald Díaz for his invaluable support in the logistics and collection of the frog, Victor Araújo and Angie Tovar for their guidance and detailed explanations during the DNA extraction process, GenCore at the Universidad de los Andes for genome sequencing using Nanopore Technology (ONT), which allowed the generation of fundamental data for this study. Finally, we would like to thank the Faculty of Sciences at the Universidad de los Andes for the financial support granted under the ‘Projecto Semilla’, which made this research possible. Data Availability The complete mitochondrial genome sequence has been deposited in GenBank [PX779192]. References Ladoukakis ED, Zouros E (2017) Evolution and inheritance of animal mitochondrial DNA: rules and exceptions. J Biol Res (Thessaloniki) 24(2):1–7 Dowling DK, Wolff JN (2023) Evolutionary genetics of the mitochondrial genome: insights from Drosophila . Genetics 224(3):1–27 Zhang C, Zhang K, Peng Y, Zhou J, Liu Y, Liu B (2022) Novel gene rearrangement in the mitochondrial genome of three garra and insights into the phylogenetic relationships of Labeoninae. Front Genet 13:1–14 Boore JL (1999) Animal mitochondrial genomes. Nucleic Acids Res 27(8):1767–1780 Amarasinghe SL, Su S, Dong X, Zappia L, Ritchie ME, Gouil Q (2020) Opportunities and challenges in long-read sequencing data analysis. Genome Biol 21(1):1–16 Formenti G, Rhie A, Balacco J, Haase B, Mountcastle J, Fedrigo O, Brown S, Capodiferro MR, Al-Ajli FO, Ambrosini R et al (2021) Complete vertebrate mitogenomes reveal widespread repeats and gene duplications. Genome Biol 22(1):1–22 Zhang P, Liang D, Mao R-L, Hillis DM, Wake DB, Cannatella DC (2013) Efficient sequencing of anuran mtDNAs and a mitogenomic exploration of the phylogeny and evolution of frogs. Mol Biol Evol 30(8):1899–1915 Kolmogorov M, Yuan J, Lin Y, Pevsner PA (2019) Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol 37(5):540–546 Pinto-Sánchez NR, Ibañez R, Madriñan S, Sanjur OI, Berminham E, Crawford AJ (2012) The Great American Biotic Interchange in frogs: Multiple and early colonization of Central America by the South American genus Pristimantis (Anura: Craugastoridae). Mol Phylogenet Evol 62(3):954–972 Carvajalino-Fernández JM, Bonilla-Gomez MA, Giraldo-Gutierréz L, Navas CA (2021) Freeze tolerance in neotropical frogs: an intrageneric comparison using Pristimantis species of high elevation and medium elevation. J Trop Ecol 37(3):118–125 Crawford AJ, Ryan MJ, Jaramillo CA (2010) A new species of Pristimantis (Anura: Strabomantidae) from the Pacific coast of the Darien Province, Panama, with a molecular analysis of its phylogenetic position. Herpetologica 66(2):192–206 Li H (2018) Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34(18):3094–3100 Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics 25(16):2078–2079 Donath A, Jühling F, Al-Arab M, Bernhart SH, Reinhardt F, Stadler PF, Middendorf M, Bernt M (2019) Improved annotation of protein-coding genes boundaries in metazoan mitochondrial genomes. Nucleic Acids Res 47(20):10543–10552 Grant JR, Enns E, Marinier E, Mandal A, Herman EK, Chen CY, Graham M, Van-Domselaar G, Stothard P (2023) Proksee: in-depth characterization and visualization of bacterial genomes. Nucleic Acids Res 51(1):484–492 Macey JR, Larson A, Ananjeva NB, Fang Z, Papenfuss TJ (1997) Two novel gene orders and the role of light-strand replication in rearrangement of the vertebrate mitochondrial ge-nome. Mol Biol Evol 14(1):91–104 Feng Y, Blackburn DC, Liang D, Hillis DM, Wake DB, Cannatella DC, Zhang P (2017) Phylogenomics reveals rapid, simultaneous diversification of three major clades of Gondwanan frogs at the Cretaceous–Paleogene boundary. Proc Natl Acad Sci U S A 114(29):1–7 Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, Von-Haeseler A, Lanfear R (2020) IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol 37(5):1530–1534 Jiang L, Song W, Liu Y, Zhang Y, Liu J (2023) Characterization and phylogenetic implications of the complete mitogenomes of two species in the genus Zhangixalus (Anura: Rhacophoridae). Asian Herpetol Res 14(3):191–211 Sano N, Kurabayashi A, Fujii T, Yonekawa H, Sumida M (2004) Complete nucleotide sequence and gene rearrangement of the mitochondrial genome of the bell-ring frog, Buergeria buergeri (family Rhacophoridae). Genes Genet Syst 79(3):151–163 Zhang T, Wang Y, Song H (2023) The complete mitochondrial genome and gene arrangement of the enigmatic scaphopod Pictodentalium vernedei . Genes 14(210):1–12 An Q, Qiu YY, Lou Y, Jiang Y, Qiu HY, Zhang ZH, Li B, Zhang AH, Wei W, Chen YY, Gao JF, Wang CR (2022) Characterization of the complete mitochondrial genomes of Diplodiscus japonicus and Diplodiscus mehari (Trematoda: Diplodiscidae): Comparison with the members of the superfamily Paramphistomoidea and phylogenetic implication. Int J Parasitol Parasites Wildl 19:9–17 Kuhle B, Hirschi M, Doerfel LK, Lander GC, Schimmel P (2022) Structural basis for shape-selective recognition and aminoacylation of a D-armless human mitochondrial tRNA. Nat Commun 13(5100):1–12 Pons J, Bover P, Bidegaray-Batista L, Arnedo MA (2019) Arm-less mitochondrial tRNAs conserved for over 30 millions of years in spiders. BMC Genomics 20(665):1–16 Hime PM, Lemmon AR, Lemmon ECM, Prendini E, Brown JM, Thomson RC, Kratovil JD, Noonan BP, Pyron RA, Peloso PLV et al (2021) Phylogenomics reveals ancient gene tree discordance in the amphibian Tree of Life. Syst Biol 70:49–66 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 01 Apr, 2026 Reviews received at journal 01 Apr, 2026 Reviews received at journal 30 Mar, 2026 Reviews received at journal 28 Mar, 2026 Reviewers agreed at journal 23 Mar, 2026 Reviewers agreed at journal 21 Mar, 2026 Reviewers agreed at journal 20 Mar, 2026 Reviewers invited by journal 19 Mar, 2026 Editor assigned by journal 18 Mar, 2026 Submission checks completed at journal 18 Mar, 2026 First submitted to journal 17 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-9153070\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Short Report\",\"associatedPublications\":[],\"authors\":[{\"id\":610531716,\"identity\":\"f341d432-babe-45f6-b2c3-3ce2e933e4d7\",\"order_by\":0,\"name\":\"Kevin S. Jaramillo\",\"email\":\"data:image/png;base64,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\",\"orcid\":\"\",\"institution\":\"Museo de Historia Natural C.J. Marinkelle, Universidad de los Andes\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Kevin\",\"middleName\":\"S.\",\"lastName\":\"Jaramillo\",\"suffix\":\"\"},{\"id\":610531717,\"identity\":\"a05e67d0-e5e5-4426-bd46-8b12cf121dfc\",\"order_by\":1,\"name\":\"Andrew J. Crawford\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Museo de Historia Natural C.J. Marinkelle, Universidad de los Andes\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Andrew\",\"middleName\":\"J.\",\"lastName\":\"Crawford\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2026-03-18 00:38:17\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-9153070/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-9153070/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":105283698,\"identity\":\"2da014e2-80e1-4e77-9597-cdb928094db4\",\"added_by\":\"auto\",\"created_at\":\"2026-03-24 10:43:19\",\"extension\":\"jpeg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":228193,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eCircular map of the mitochondrial genome assembly of \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003ePristimantis bogotensis\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e.\\u003c/strong\\u003eGenes are color-coded according to functional categories indicated in the accompanying legend. Genes located on the heavy strand (H-strand) are shown on the outer side of the thick gray circle, while those on the light strand (L-strand) are shown on the inner side. The inner grey histogram represents the GC content across the mitogenome, highlighting regional variation in base composition. The map was generated using Proksee. Photo of \\u003cem\\u003eP. bogotensis\\u003c/em\\u003e voucher ANDES-A 5749 by Ronald A. Díaz-Flórez.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage1.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9153070/v1/813f98b675afd5ee79c9c5da.jpeg\"},{\"id\":105283693,\"identity\":\"004a9cc7-7769-493c-a78b-d766139977eb\",\"added_by\":\"auto\",\"created_at\":\"2026-03-24 10:43:16\",\"extension\":\"jpeg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":240427,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eInferred secondary structure of all tRNAs in \\u003cem\\u003ePristimantis bogotensis\\u003c/em\\u003emitochondrial genome: tRNAs typically adopt a \\\"cloverleaf\\\" secondary structure, with four characteristic arms: acceptor, TΨC, anticodon, and D-arm (dihydrouridine). Note the shape of tRNAs with structural peculiarities. tRNA-Cys (cysteine) lacks a D-arm, a potential molecular synapomorphy of the genus \\u003cem\\u003ePristimantis\\u003c/em\\u003e, while tRNA-Ser1 (serine) has a truncated D-arm.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage2.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9153070/v1/ab0420575e19cab8c1415459.jpeg\"},{\"id\":105283671,\"identity\":\"10d3ed8a-a685-450e-aff4-e30eed4470c0\",\"added_by\":\"auto\",\"created_at\":\"2026-03-24 10:43:09\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":189396,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMaximum likelihood phylogenetic tree of \\u003cem\\u003ePristimantis bogotensis\\u003c/em\\u003e and 17 additional Neobatrachia taxa based on complete and partial mitogenomes available in NCBI as of 2025. Tree rooted with \\u003cem\\u003eXenopus laevis\\u003c/em\\u003e. The tree was inferred from a concatenated alignment of 13 protein-coding genes from complete and nearly complete mitochondrial genomes, under the substitution model (mtVer+R4). Bootstrap support values are indicated at the nodes. Codes in parentheses next to each taxon name correspond to the respective GenBank accession number for each mitogenome. The scale bar represents the number of substitutions per site. See Methods for details.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9153070/v1/5a39881ef00e8f4409cb39d6.png\"},{\"id\":105283751,\"identity\":\"5560e6c5-d9dc-4675-b54f-417a2e9de6fb\",\"added_by\":\"auto\",\"created_at\":\"2026-03-24 10:43:27\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1372265,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9153070/v1/c3133dc7-1cfb-4ad5-b754-c64ccd3c76a7.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Assembly and analysis of the complete mitochondrial genome of the Bogotá Robber Frog, Pristimantis bogotensis\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eMitochondrial genomes have been widely utilized in evolutionary biology due to their compact structure, maternal inheritance, and high mutation rate [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. These features make them valuable markers for phylogenetics, phylogeography, and species identification and delimitation [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. Vertebrate mitochondrial genomes typically encode 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), a light-strand origin of replication, and a control region (containing the D-loop and a heavy-strand origin of replication) [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. This conserved genomic architecture provides a robust framework for comparative evolutionary studies across vertebrate lineages.\\u003c/p\\u003e \\u003cp\\u003eAlthough mitochondrial genomes have traditionally been characterized using Sanger sequencing and more recently with next-gen short-read sequencing approaches, these methods may present limitations in resolving repetitive regions and structural duplications, such as those found in mitochondrial control regions [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. Short-read sequencing may result in repetitive DNA sequences being collapsed upon \\u003cem\\u003ein-silico\\u003c/em\\u003e assembly and genic length being underestimated. The implementation of long-read sequencing represents more than a technical improvement, as it enables the detection of structural variation that refines our understanding of genome evolution and organization [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. In parallel, advances in long-read assembly algorithms have further enhanced the accurate reconstruction of repetitive sequences and structural features, often resulting in larger and more structurally accurate assemblies [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e], highlighting how modern sequencing approaches can reveal previously hidden genomic features and provide new insights into molecular evolution in animals, including structural evolution of vertebrate mitochondrial genomes.\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003ePristimantis\\u003c/em\\u003e is the most diverse genus of amphibians, with over 620 species distributed throughout Central and South America [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. Striking new species are continually being discovered and described, even near major urban centers. Despite its high taxonomic diversity, the availability of complete mitochondrial genome data for this genus is nil. \\u003cem\\u003ePristimantis bogotensis\\u003c/em\\u003e is an Andean frog endemic to the Eastern Cordillera of Colombia, inhabiting high-elevation environments where extreme climatic conditions pose significant adaptive challenges [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. Understanding its mitochondrial genome may offer new insights into the molecular evolution and phylogenetics of the genus, and provide bioinformatic resources for DNA-based inventories, resolving taxonomic uncertainties and improving conservation planning for this hyper-diverse genus.\\u003c/p\\u003e \\u003cp\\u003ePrevious phylogenetic studies of \\u003cem\\u003ePristimantis\\u003c/em\\u003e have relied mainly on a handful of mitochondrial and nuclear genes [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e] without incorporating whole mitogenomes, despite the potential for more genes as well as structural variations to provide additional synapomorphies [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. Here we DNA sequence the mitogenome using third-generation technology and then assemble and annotate the first complete mitogenome of a Craugastoridae family.\\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eSample collection, DNA extraction and ONT sequencing\\u003c/h2\\u003e \\u003cp\\u003eA single specimen of \\u003cem\\u003eP. bogotensis\\u003c/em\\u003e was collected in September of 2024 from F\\u0026oacute;meque, Cundinamarca, Colombia (4.563183\\u0026deg;, -73.835816\\u0026deg;), at 3100 m elevation. The specimen was fixed and deposited in the amphibian collection of the \\u003cem\\u003eMuseo de Historia Natural C.J. Marinkelle\\u003c/em\\u003e at the Universidad de los Andes, under accession number ANDES-A 5749. Before fixing the specimen, muscle and liver tissues were removed, flash-frozen in liquid nitrogen, and stored promptly at -80\\u0026deg;C. Genomic DNA of the samples was isolated using the Monarch Genomic DNA Purification Kit (New England Biolabs). The quality and concentration of extracted DNA were assessed using a Nanodrop and Qubit 4.0 fluorometer. Sequencing libraries were prepared using the Ligation Sequencing Kit V14 SQK-LSK114 from Oxford Nanopore Technologies (ONT), and raw sequencing data were generated using the ONT PromethION platform, using R10.4.1 cells. This process was made by GenCore at Universidad de los Andes, with a total of three independent sequencings, each in a different cell.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eMitogenome assembly and annotation\\u003c/h3\\u003e\\n\\u003cp\\u003eONT reads (57.8 Gb) obtained were base-called using Dorado/0.8.2 (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://github.com/nanoporetech/dorado\\u003c/span\\u003e\\u003cspan address=\\\"https://github.com/nanoporetech/dorado\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003e)\\u003c/span\\u003e, and adaptors were removed with Porechop/1.0 (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://github.com/rrwick/Porechop\\u003c/span\\u003e\\u003cspan address=\\\"https://github.com/rrwick/Porechop\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003e).\\u003c/span\\u003e To extract similar contigs, we filtered reads using the mitochondrial genome assembly of \\u003cem\\u003ePhyllobates terribilis\\u003c/em\\u003e (selected as the closest complete mitochondrial genome to \\u003cem\\u003eP. bogotensis\\u003c/em\\u003e) like a mitogenome map (GenBank accession: NC037380.1), using Minimap2/2.24 [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]. High-quality reads obtained (Q\\u0026thinsp;\\u0026ge;\\u0026thinsp;10) were filtered using SAMtools/1.16.1 [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e] and converted to FASTQ format for de novo assembly with Flye/2.9.3 [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. The assembly was polished with Medaka/2.0.1 (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://github.com/nanoporetech/medaka\\u003c/span\\u003e\\u003cspan address=\\\"https://github.com/nanoporetech/medaka\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003e).\\u003c/span\\u003e The assembled mitochondrial genome was annotated using MITOS2/2.1.9 into Proksee [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. A circular genome map was generated with Proksee [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. The consensus genome sequence has been deposited in GenBank under accession number [PX779192].\\u003c/p\\u003e\\n\\u003ch3\\u003etRNA secondary structure prediction\\u003c/h3\\u003e\\n\\u003cp\\u003eTo confirm structural features of the tRNA genes annotated with MITOS2, we used the RNAfold web server (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://rna.tbi.univie.ac.at\\u003c/span\\u003e\\u003cspan address=\\\"http://rna.tbi.univie.ac.at\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003e)\\u003c/span\\u003e from the ViennaRNA Package, which help us to predict secondary structures of ribosomal genes in the \\u003cem\\u003eP. bogotensis\\u003c/em\\u003e mitogenome. The inferred structures were compared to the canonical vertebrate mitochondrial tRNA model [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e] to identify conserved domains (acceptor stem, anticodon stem, TΨC stem) as well as deviations.\\u003c/p\\u003e\\n\\u003ch3\\u003ePhylogenetic analyses\\u003c/h3\\u003e\\n\\u003cp\\u003eMitochondrial genomes of anuran species closely related to \\u003cem\\u003ePristimantis\\u003c/em\\u003e were retrieved from National Center for Biotechnology Information (NCBI). Taxon sampling was based on the phylogenetic framework for Neobatrachia [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e], selecting taxa with available complete mitochondrial genomes, and including the two available partial mitogenomes of \\u003cem\\u003ePristimantis\\u003c/em\\u003e species to represent the genus, as no complete mitochondrial genomes were previously available. The 13 mitochondrial protein-coding genes were extracted and concatenated into a single dataset. Sequences were aligned using MAFFT/7.526 (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://github.com/GSLBiotech/mafft\\u003c/span\\u003e\\u003cspan address=\\\"https://github.com/GSLBiotech/mafft\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003e).\\u003c/span\\u003e A maximum likelihood (ML) phylogenetic tree was inferred using IQ-TREE/2.2.0 [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e] with 1,000 ultrafast bootstrap replicates and 1,000 SH-aLRT replicates to evaluate statistical support for each node, and the Bayesian Information Criterion (BIC) was used to determine the optimal substitution model (mtVer+R4). \\u003cem\\u003eXenopus laevis\\u003c/em\\u003e was employed as outgroup. The tree was visualized using iTOL/v6. (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://itol.embl.de\\u003c/span\\u003e\\u003cspan address=\\\"https://itol.embl.de\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003e).\\u003c/span\\u003e\\u003c/p\\u003e\"},{\"header\":\"Results and discussion\",\"content\":\"\\u003cp\\u003eThe mitochondrial genome assembly of \\u003cem\\u003eP. bogotensis\\u003c/em\\u003e was 19,565 bp and encodes the standard number of genes in the vertebrate mitogenome of 13 PCGs, 22 tRNAs, 2 rRNAs, 1 light-strand origin of replication (O\\u003csub\\u003eL\\u003c/sub\\u003e), and 1 control region (containing 1 heavy-strand origin of replication (O\\u003csub\\u003eH\\u003c/sub\\u003e) and 1 D-loop), with a GC content of 40.2% (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). The gene order is identical to the ancestral vertebrate mitochondrial arrangement [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e], exhibiting no gene rearrangements (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e), although two intergenic spacers of 99 bp and 51 bp were identified between ND1\\u0026ndash;tRNA(Leu2) and COX3\\u0026ndash;ATP6, respectively. All PCGs and structural RNAs are encoded on the heavy strand, except for nad6 and eight tRNAs (tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser, tRNA-Glu, tRNA-Pro), consistent with the conserved strand asymmetry observed across anuran mitogenomes [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. The relatively large size of the \\u003cem\\u003eP. bogotensis\\u003c/em\\u003e mitogenome likely reflects the improved recovery of repetitive regions through long-read ONT sequencing, which allows more accurate reconstruction of structural features, contributing to the increasing detection of larger mitogenomes [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. Such improved structural resolution may provide a more realistic view of mitochondrial genome organization and variation [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eGenic location and boundaries of genes in the mitochondrial genome assembly of \\u003cem\\u003eP ristimantis bogotensis\\u003c/em\\u003e, including the name of the gene, its specific location in the sequence, length in base pairs, the strand (H for heavy and L for light), the adjacent intergenic regions, the start and stop codons, and the DNA sequence of the corresponding anticodon for the transfer RNA (tRNA) genes.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"7\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eGene\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eSequence location\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eLength in bp\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eStrand\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eIntergenic region length in bp\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eStart/stop codons\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eAnticodon\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eATP6\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1-629\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e629\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e-10\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eATG/TAA\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eATP8\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e620\\u0026ndash;784\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e165\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eATG/TAA\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (Lys)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e785\\u0026ndash;853\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e69\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e-3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eUUU\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCOX2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e851\\u0026ndash;1538\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e688\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eATG/TAA\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (Asp)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1540\\u0026ndash;1607\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e68\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eGUC\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (Ser2)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1609\\u0026ndash;1679\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e71\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eL\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e-9\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eUGA\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCOX1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1671\\u0026ndash;3224\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1554\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eGTG/TAG\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (Tyr)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3226\\u0026ndash;3292\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e67\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eL\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eGUA\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (Cys)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3293\\u0026ndash;3348\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e56\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eL\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e-1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eGCA\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eLight strand Origin of replication\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3348\\u0026ndash;3371\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e24\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (Asn)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3374\\u0026ndash;3446\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e73\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eL\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eGUU\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (Ala)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3447\\u0026ndash;3515\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e69\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eL\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eUGC\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (Trp)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3516\\u0026ndash;3583\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e68\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e9\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eUCA\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eND2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3593\\u0026ndash;4613\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1021\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eATG/TAA\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (Met)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e4614\\u0026ndash;4684\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e71\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e-1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eCAU\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (Gln)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e4684\\u0026ndash;4754\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e71\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eL\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e-1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eUUG\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (Ile)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e4754\\u0026ndash;4824\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e71\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e6\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eGAU\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eND1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e4831\\u0026ndash;5666\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e836\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e99\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eATG/TAA\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (Leu2)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5766\\u0026ndash;5838\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e73\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e8\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eUAA\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e16S ribosomal RNA\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5847\\u0026ndash;7372\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1526\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (Val)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e7375\\u0026ndash;7442\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e68\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e-3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eUAC\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e12S ribosomal RNA\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e7440\\u0026ndash;8363\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e924\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (Phe)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e8364\\u0026ndash;8431\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e68\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e-1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eGAA\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (Pro)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e8431\\u0026ndash;8498\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e68\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eL\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e-1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eUGG\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (Thr)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e8498\\u0026ndash;8567\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e70\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eUGU\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (Leu1)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e8569\\u0026ndash;8640\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e72\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e17\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eUAG\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eHeavy strand Origin of replication\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e8658\\u0026ndash;8889\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e232\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eL\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eD-loop\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e8890\\u0026ndash;12997\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e4109\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eL\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCYTB\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e12998\\u0026ndash;14141\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1144\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eATG/TAA\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (Glu)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e14147\\u0026ndash;14215\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e69\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eL\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eUUC\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eND6\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e14216\\u0026ndash;14707\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e492\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eL\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e-17\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eATG/TAA\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eND5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e14691\\u0026ndash;16481\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1791\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e4\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eATG/TAA\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (Ser1)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e16486\\u0026ndash;16549\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e64\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eGCU\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (His)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e16550\\u0026ndash;16616\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e67\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e4\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eGUG\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eND4\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e16621\\u0026ndash;17980\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1360\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e-7\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eATG/TAA\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eND4L\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e17974\\u0026ndash;18273\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e300\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eATG/TAA\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (Arg)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e18275\\u0026ndash;18342\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e68\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eUCG\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eND3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e18343\\u0026ndash;18658\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e316\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eATG/TAA\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003etRNA (Gly)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e18661\\u0026ndash;18729\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e69\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e-1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eUCC\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCOX3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e18729\\u0026ndash;19514\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e786\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e51\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eATG/TAA\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eA striking feature of the \\u003cem\\u003eP. bogotensis\\u003c/em\\u003e mitogenome is the absence of the Dihydrouridine arm (D-arm) in the mitochondrial tRNA-Cys gene, a structural deletion previously reported in \\u003cem\\u003ePristimantis\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e] based on Sanger sequencing. This loss, confirmed here using long-read sequencing, eliminates a conserved four-base-pair stem structure, typical in most mitogenomes in vertebrates (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). Comparative secondary structure analysis of the mitochondrial tRNA-Cys gene across the three \\u003cem\\u003ePristimantis\\u003c/em\\u003e species included in this study (\\u003cem\\u003eP. bogotensis, P. thymelensis\\u003c/em\\u003e, and \\u003cem\\u003eP. fenestratus\\u003c/em\\u003e) revealed a consistent absence of the D-arm, corroborating the structural findings [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e] and supporting its potential use as a synapomorphy of the genus. The D-arm loss underscores the potential utility of tRNA structural evolution in resolving deep phylogenetic relationships, where homoplasy in base substitutions complicates phylogenetic resolution [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. However, with over 620 species in the genus, it remains to be seen whether these observations will hold across major clades within \\u003cem\\u003ePristimantis\\u003c/em\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe mitochondrial genome of \\u003cem\\u003eP. bogotensis\\u003c/em\\u003e also revealed a loss of D-arm in the tRNA-Ser1 gene, forming a simplified loop instead of the canonical cloverleaf structure (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). This D-arm loss has been reported in other Neobatrachian frogs, including \\u003cem\\u003eZhangixalus schlegelii\\u003c/em\\u003e and \\u003cem\\u003eZ. dennysi\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e], and \\u003cem\\u003eBuergeria buergeri\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. This phenomenon is not exclusive to anurans; for example among molluscs, the scaphopod \\u003cem\\u003ePictodentalium vernedei\\u003c/em\\u003e exhibits similar D-arm truncations in tRNA-Ser1 [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e], as do the trematodes, \\u003cem\\u003eDiplodiscus japonicus\\u003c/em\\u003e and \\u003cem\\u003eD. mehari\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. The parallel loss of the D-arm in distantly related groups implies biochemical flexibility in the tRNA-Ser1 molecule [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eOur molecular phylogeny based on all mitochondrial PCG\\u0026rsquo;s recovered a well-resolved topology with strong statistical support across nodes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). The ingroup was well supported relative to the outgroup, \\u003cem\\u003eXenopus laevis\\u003c/em\\u003e, while representatives of Ranidae (\\u003cem\\u003eRana kukunoris\\u003c/em\\u003e \\u0026amp; \\u003cem\\u003eR. temporaria\\u003c/em\\u003e) were recovered as an early-diverging clade. Within Neobatrachia, species of the family Craugastoridae formed a strongly supported monophyletic group, where \\u003cem\\u003eP. bogotensis\\u003c/em\\u003e clustered closely with \\u003cem\\u003eP. thymelensis\\u003c/em\\u003e and \\u003cem\\u003eP. fenestratus\\u003c/em\\u003e, confirming its phylogenetic placement within the genus [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. The family Aromobatidae represented by \\u003cem\\u003eAnomaloglossus blanci\\u003c/em\\u003e and the dendrobatid, \\u003cem\\u003ePhyllobates terribilis\\u003c/em\\u003e, were recovered as sister lineages, consistent with previous phylogenomic studies [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. Additionally, the family Bufonidae was also strongly supported as a monophyletic group, including \\u003cem\\u003eBufo gargarizans\\u003c/em\\u003e, \\u003cem\\u003eB. stejnegeri\\u003c/em\\u003e, and \\u003cem\\u003eRhinella marina\\u003c/em\\u003e.\\u003c/p\\u003e \\u003cp\\u003eThe strong statistical support for most nodes reflect previous findings in amphibian phylogenomics and confirm the phylogenetic position of \\u003cem\\u003eP. bogotensis\\u003c/em\\u003e within a well-supported clade of congeneric species. The phylogenetic results obtained here highlights the robustness of mitogenomic data for clarifying evolutionary relationships within Neobatrachia as these results are consistent with previous studies employing nuclear genome-wide data for amphibian phylogenetics [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eThis study presents the first complete mitochondrial genome of \\u003cem\\u003eP. bogotensis\\u003c/em\\u003e, filling a significant gap in anuran mitogenomics. The application of ONT long-read sequencing enabled the recovery of structurally complex regions and may contribute to a more accurate representation of mitochondrial genome architecture in vertebrates. By expanding mitochondrial genome datasets and integrating comparative approaches, this research enhances our understanding of the evolutionary history of \\u003cem\\u003eP. bogotensis\\u003c/em\\u003e. Future studies should explore mitogenome variations across populations and congeneric species, as well as their implications in adaptations. \\u003cem\\u003ePristimantis bogotensis\\u003c/em\\u003e demonstrates that the retention of a stable mitochondrial gene order alongside tRNA structural plasticity underscores a dynamic in that while gene arrangement remains conserved, tRNA architecture tolerates major changes.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e \\u003ch2\\u003eEthical Approval\\u003c/h2\\u003e \\u003cp\\u003eThe CICUA (Institutional Animal Care and Use Committee) of the Universidad de los Andes approved this study (C.FUA_24\\u0026thinsp;\\u0026minus;\\u0026thinsp;009). The individual was collected under the umbrella permit No. PR.6.2015.2182 to the Universidad de los Andes by the National Environmental Licensing Authority (ANLA) of Colombia.\\u003c/p\\u003e \\u003c/p\\u003e\\u003cp\\u003e \\u003ch2\\u003eCompeting interests\\u003c/h2\\u003e \\u003cp\\u003eThe authors have no relevant financial or non-financial interests to disclose.\\u003c/p\\u003e \\u003c/p\\u003e\\u003ch2\\u003eFunding\\u003c/h2\\u003e \\u003cp\\u003eThis research was funded by the Faculty of Sciences at the Universidad de los Andes under the \\u003cem\\u003eProyecto semilla\\u003c/em\\u003e to KSJ and AJC, and the grant in basic research (INV-2021-128-2323) to AJC.\\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eLaboratory work, mitochondrial genome assembly, annotation, and bioinformatic analyses were performed by KSJ. Library preparation and sequencing were conducted by the GenCore Facility at Universidad de los Andes. KSJ wrote the first draft of the manuscript. KSJ and AJC contributed to supervise, edit, revise, and improve the manuscript. Both authors read and approved the final manuscript.\\u003c/p\\u003e\\u003ch2\\u003eAcknowledgement\\u003c/h2\\u003e\\u003cp\\u003eWe thank Ronald D\\u0026iacute;az for his invaluable support in the logistics and collection of the frog, Victor Ara\\u0026uacute;jo and Angie Tovar for their guidance and detailed explanations during the DNA extraction process, GenCore at the Universidad de los Andes for genome sequencing using Nanopore Technology (ONT), which allowed the generation of fundamental data for this study. Finally, we would like to thank the Faculty of Sciences at the Universidad de los Andes for the financial support granted under the \\u0026lsquo;Projecto Semilla\\u0026rsquo;, which made this research possible.\\u003c/p\\u003e\\u003ch2\\u003eData Availability\\u003c/h2\\u003e\\u003cp\\u003eThe complete mitochondrial genome sequence has been deposited in GenBank [PX779192].\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eLadoukakis ED, Zouros E (2017) Evolution and inheritance of animal mitochondrial DNA: rules and exceptions. J Biol Res (Thessaloniki) 24(2):1\\u0026ndash;7\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDowling DK, Wolff JN (2023) Evolutionary genetics of the mitochondrial genome: insights from \\u003cem\\u003eDrosophila\\u003c/em\\u003e. Genetics 224(3):1\\u0026ndash;27\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhang C, Zhang K, Peng Y, Zhou J, Liu Y, Liu B (2022) Novel gene rearrangement in the mitochondrial genome of three garra and insights into the phylogenetic relationships of Labeoninae. Front Genet 13:1\\u0026ndash;14\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBoore JL (1999) Animal mitochondrial genomes. Nucleic Acids Res 27(8):1767\\u0026ndash;1780\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAmarasinghe SL, Su S, Dong X, Zappia L, Ritchie ME, Gouil Q (2020) Opportunities and challenges in long-read sequencing data analysis. Genome Biol 21(1):1\\u0026ndash;16\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eFormenti G, Rhie A, Balacco J, Haase B, Mountcastle J, Fedrigo O, Brown S, Capodiferro MR, Al-Ajli FO, Ambrosini R et al (2021) Complete vertebrate mitogenomes reveal widespread repeats and gene duplications. Genome Biol 22(1):1\\u0026ndash;22\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhang P, Liang D, Mao R-L, Hillis DM, Wake DB, Cannatella DC (2013) Efficient sequencing of anuran mtDNAs and a mitogenomic exploration of the phylogeny and evolution of frogs. Mol Biol Evol 30(8):1899\\u0026ndash;1915\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKolmogorov M, Yuan J, Lin Y, Pevsner PA (2019) Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol 37(5):540\\u0026ndash;546\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePinto-S\\u0026aacute;nchez NR, Iba\\u0026ntilde;ez R, Madri\\u0026ntilde;an S, Sanjur OI, Berminham E, Crawford AJ (2012) The Great American Biotic Interchange in frogs: Multiple and early colonization of Central America by the South American genus \\u003cem\\u003ePristimantis\\u003c/em\\u003e (Anura: Craugastoridae). Mol Phylogenet Evol 62(3):954\\u0026ndash;972\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eCarvajalino-Fern\\u0026aacute;ndez JM, Bonilla-Gomez MA, Giraldo-Gutierr\\u0026eacute;z L, Navas CA (2021) Freeze tolerance in neotropical frogs: an intrageneric comparison using \\u003cem\\u003ePristimantis\\u003c/em\\u003e species of high elevation and medium elevation. J Trop Ecol 37(3):118\\u0026ndash;125\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eCrawford AJ, Ryan MJ, Jaramillo CA (2010) A new species of \\u003cem\\u003ePristimantis\\u003c/em\\u003e (Anura: Strabomantidae) from the Pacific coast of the Darien Province, Panama, with a molecular analysis of its phylogenetic position. Herpetologica 66(2):192\\u0026ndash;206\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLi H (2018) Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34(18):3094\\u0026ndash;3100\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLi H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics 25(16):2078\\u0026ndash;2079\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDonath A, J\\u0026uuml;hling F, Al-Arab M, Bernhart SH, Reinhardt F, Stadler PF, Middendorf M, Bernt M (2019) Improved annotation of protein-coding genes boundaries in metazoan mitochondrial genomes. Nucleic Acids Res 47(20):10543\\u0026ndash;10552\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGrant JR, Enns E, Marinier E, Mandal A, Herman EK, Chen CY, Graham M, Van-Domselaar G, Stothard P (2023) Proksee: in-depth characterization and visualization of bacterial genomes. Nucleic Acids Res 51(1):484\\u0026ndash;492\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMacey JR, Larson A, Ananjeva NB, Fang Z, Papenfuss TJ (1997) Two novel gene orders and the role of light-strand replication in rearrangement of the vertebrate mitochondrial ge-nome. Mol Biol Evol 14(1):91\\u0026ndash;104\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eFeng Y, Blackburn DC, Liang D, Hillis DM, Wake DB, Cannatella DC, Zhang P (2017) Phylogenomics reveals rapid, simultaneous diversification of three major clades of Gondwanan frogs at the Cretaceous\\u0026ndash;Paleogene boundary. Proc Natl Acad Sci U S A 114(29):1\\u0026ndash;7\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMinh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, Von-Haeseler A, Lanfear R (2020) IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol 37(5):1530\\u0026ndash;1534\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJiang L, Song W, Liu Y, Zhang Y, Liu J (2023) Characterization and phylogenetic implications of the complete mitogenomes of two species in the genus \\u003cem\\u003eZhangixalus\\u003c/em\\u003e (Anura: Rhacophoridae). Asian Herpetol Res 14(3):191\\u0026ndash;211\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSano N, Kurabayashi A, Fujii T, Yonekawa H, Sumida M (2004) Complete nucleotide sequence and gene rearrangement of the mitochondrial genome of the bell-ring frog, \\u003cem\\u003eBuergeria buergeri\\u003c/em\\u003e (family Rhacophoridae). Genes Genet Syst 79(3):151\\u0026ndash;163\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhang T, Wang Y, Song H (2023) The complete mitochondrial genome and gene arrangement of the enigmatic scaphopod \\u003cem\\u003ePictodentalium vernedei\\u003c/em\\u003e. Genes 14(210):1\\u0026ndash;12\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAn Q, Qiu YY, Lou Y, Jiang Y, Qiu HY, Zhang ZH, Li B, Zhang AH, Wei W, Chen YY, Gao JF, Wang CR (2022) Characterization of the complete mitochondrial genomes of \\u003cem\\u003eDiplodiscus japonicus\\u003c/em\\u003e and \\u003cem\\u003eDiplodiscus mehari\\u003c/em\\u003e (Trematoda: Diplodiscidae): Comparison with the members of the superfamily Paramphistomoidea and phylogenetic implication. Int J Parasitol Parasites Wildl 19:9\\u0026ndash;17\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKuhle B, Hirschi M, Doerfel LK, Lander GC, Schimmel P (2022) Structural basis for shape-selective recognition and aminoacylation of a D-armless human mitochondrial tRNA. Nat Commun 13(5100):1\\u0026ndash;12\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePons J, Bover P, Bidegaray-Batista L, Arnedo MA (2019) Arm-less mitochondrial tRNAs conserved for over 30 millions of years in spiders. BMC Genomics 20(665):1\\u0026ndash;16\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHime PM, Lemmon AR, Lemmon ECM, Prendini E, Brown JM, Thomson RC, Kratovil JD, Noonan BP, Pyron RA, Peloso PLV et al (2021) Phylogenomics reveals ancient gene tree discordance in the amphibian Tree of Life. Syst Biol 70:49\\u0026ndash;66\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"molecular-biology-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"mole\",\"sideBox\":\"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)\",\"snPcode\":\"11033\",\"submissionUrl\":\"https://submission.nature.com/new-submission/11033/3\",\"title\":\"Molecular Biology Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"Craugastoridae, Long-read sequencing, Mitogenomics, Molecular phylogenetics, Molecular evolution, Oxford Nanopore Technology\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-9153070/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-9153070/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003eBackground\\u003c/h2\\u003e \\u003cp\\u003eMitochondrial genomes are widely used in evolutionary studies due to their maternal inheritance, high mutation rate, and utility for inferring phylogeographic and phylogenetic relationships. The Neotropical frog genus, \\u003cem\\u003ePristimantis\\u003c/em\\u003e (Anura: Craugastoridae), with over 620 described species, represents the most species-rich anuran genera in the world, yet no mitogenome assemblies are available from this group. Here we use long-read DNA sequencing to assemble the complete mitogenome of \\u003cem\\u003ePristimantis bogotensis\\u003c/em\\u003e, a high-elevation Andean frog endemic to the Eastern Cordillera of Colombia. This mitogenomic characterization represents a valuable resource within this group of direct-developing anurans.\\u003c/p\\u003e\\u003ch2\\u003eMethods and results\\u003c/h2\\u003e \\u003cp\\u003eThe complete mitochondrial genome of \\u003cem\\u003eP. bogotensis\\u003c/em\\u003e was sequenced using Oxford Nanopore Technologies and assembled \\u003cem\\u003ede novo\\u003c/em\\u003e. The mitogenome assembly was unusually large at 19,565 bp, yet exhibited the structure typical of vertebrate mitochondrial genomes, with a GC content of 40.2% and containing 13 protein-coding genes, 2 rRNA genes, 1 light-strand origin of replication, 1 heavy-strand origin of replication, 1 D-loop and 22 tRNA genes. Notably, the tRNA-Cys lacked the D-arm. A phylogenetic analysis of previously published complete and partial anuran mitogenomes confirmed the placement of \\u003cem\\u003eP. bogotensis\\u003c/em\\u003e within the \\u003cem\\u003ePristimantis\\u003c/em\\u003e genus and support the loss of the D-arm in tRNA-Cys as a potential synapomorphy is this speciose clade.\\u003c/p\\u003e\\u003ch2\\u003eConclusion\\u003c/h2\\u003e \\u003cp\\u003eTo our knowledge, this is the first complete mitochondrial genome reported for the family Craugastoridae, and represents an important resource for phylogeography, metabarcoding, and phylogenetic studies in frogs, with direct applications in conservation and taxonomy.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Assembly and analysis of the complete mitochondrial genome of the Bogotá Robber Frog, Pristimantis bogotensis\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-03-24 10:41:47\",\"doi\":\"10.21203/rs.3.rs-9153070/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2026-04-01T08:20:00+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-04-01T06:41:00+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-03-30T17:15:21+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-03-28T23:31:47+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"300730401762926812898095795036884604677\",\"date\":\"2026-03-23T08:54:36+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"34301647290396071756836791916800660081\",\"date\":\"2026-03-21T21:34:41+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"19964795701042751514065723254773202250\",\"date\":\"2026-03-20T06:24:51+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2026-03-19T15:44:05+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2026-03-18T10:26:46+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2026-03-18T10:26:26+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Molecular Biology Reports\",\"date\":\"2026-03-18T00:23:31+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"molecular-biology-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"mole\",\"sideBox\":\"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)\",\"snPcode\":\"11033\",\"submissionUrl\":\"https://submission.nature.com/new-submission/11033/3\",\"title\":\"Molecular Biology Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"67ee1ee9-b8ba-409b-bf93-eb60aa7bd441\",\"owner\":[],\"postedDate\":\"March 24th, 2026\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-05-16T18:23:20+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-03-24 10:41:47\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-9153070\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-9153070\",\"identity\":\"rs-9153070\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}