New mitochondrial genomic resources for the freshwater fishes of the Sanaga river (Cameroon).

New mitochondrial genomic resources for the freshwater fishes of the Sanaga river (Cameroon). | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 27 January 2026 V1 Latest version Share on New mitochondrial genomic resources for the freshwater fishes of the Sanaga river (Cameroon). Authors : HERVE TJOMB 0009-0003-8745-2535 , Arnold Roger Bitja Nyom 0000-0002-8974-051X , Pierre Caminade , Leah Anne BECHE , Khalid Belkhir , Jean-Francois Agnese , and Nicolas Hubert 0000-0001-9248-3377 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.176950163.32847778/v1 Published Ecology and Evolution Version of record Peer review timeline 198 views 83 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The Sanaga River Basin in Cameroon harbors a rich and largely undocumented ichthyofaunal diversity. This study presents the first complete mitochondrial genome dataset for nine freshwater fish species from the basin, spanning four teleost orders: Siluriformes, Characiformes, Cypriniformes, and Cichliformes. A total of 39 specimens were sequenced using Illumina NovaSeq, and mitogenomes were assembled and annotated via a dedicated bioinformatic pipeline. Genome sizes ranged from 16,524 to 16,692 bp, with GC content between 42% and 47%. All mitogenomes exhibited conserved gene structure and order, while the control region (D-loop) showed notable size variation, consistent with patterns observed in other teleosts. Phylogenetic analyses based on 13 protein-coding genes and two rRNA genes revealed well-supported monophyletic clades for each species, confirming taxonomic assignments and validating morphological identifications. This work provides foundational genomic resources for Sanaga basin freshwater fishes and contributes to regional efforts in taxonomy, phylogeography, and conservation. New mitochondrial genomic resources for the freshwater fishes of the Sanaga river (Cameroon). Herve TJOMB 1,* , Arnold BITJA NYOM 2,3,* , Pierre CAMINADE 1 , Leah BECHE 4 , Khalid BELKHIR 1 , Jean-François AGNESE 1 , Nicolas HUBERT 1,* 1 ISEM, Univ Montpellier, CNRS, IRD, Montpellier, France. 2 Laboratory of Ecology and Sustainable Development, University of Ngaoundere, Faculty of Sciences, P.O. Box 454, Ngaoundere, Cameroon. 3 Laboratory of Ecosystems and Aquatic Resources ; National Higher School of Agronomy, Halieutics and Veterinary Medicine (ENSAHV) ex-Institute of fisheries and Aquatic sciences; University of Douala, PO Box 2701, Douala, Cameroon. 4 Centre d’Ingénierie Hydraulique, EDF Hydro, 4 allée du lac de Tignes, 73290 La Motte Servolex, France. *Corresponding authors: Nicolas HUBERT ( [email protected] ) Arnold BITJA NYOM ( [email protected] ) Herve TJOMB ( [email protected] ) Abstract The Sanaga River Basin in Cameroon harbors a rich and largely undocumented ichthyofaunal diversity. This study presents the first complete mitochondrial genome dataset for nine freshwater fish species from the basin, spanning four teleost orders: Siluriformes, Characiformes, Cypriniformes, and Cichliformes. A total of 39 specimens were sequenced using Illumina NovaSeq, and mitogenomes were assembled and annotated via a dedicated bioinformatic pipeline. Genome sizes ranged from 16,524 to 16,692 bp, with GC content between 42% and 47%. All mitogenomes exhibited conserved gene structure and order, while the control region (D-loop) showed notable size variation, consistent with patterns observed in other teleosts. Phylogenetic analyses based on 13 protein-coding genes and two rRNA genes revealed well-supported monophyletic clades for each species, confirming taxonomic assignments and validating morphological identifications. This work provides foundational genomic resources for Sanaga basin freshwater fishes and contributes to regional efforts in taxonomy, phylogeography, and conservation. Keywords Mitochondrial genome; Sanaga River; freshwater fishes; Phylogenetics, Siluriformes, Characiformes, Cypriniformes, Cichliformes. Introduction The Sanaga River is the largest river in Cameroon, extending over 900 km (Olivry, 1986) and comprising seven dams, three hydroelectric and four storages, along its course. Its ichthyofaunal diversity represents an exceptionally rich-yet genetically understudied and increasingly threatened-freshwater biodiversity. To date, published data on complete mitochondrial genomes of Cameroonian freshwater fishes remain scarce, with only a few studies available from neighboring drainage systems. Among these are the mitogenome of Chrysichthys nigrodigitatus (Lacépède, 1803) from coastal rivers near Bamusso (Kim et al., 2018), Clarias camerunensis Lönnberg, 1895 (De Alwis et al., 2023), and the recent works on Enteromius thysi (Trewavas, 1974) and Coptodon camerunensis (Lönnberg, 1903) from the Nyong Basin (Kundu et al., 2022, 2023). This study provides the first genomic reference dataset for nine fish species from the Sanaga basin, based on the complete characterization of their mitochondrial genomes. These species were chosen for study because of their conservation status, commercial importance, or to compare to closely related species already sequenced. Among the study species, five are endemic to the Sanaga basin: Sanagia velifera Holly, 1926 classified as Near Threatened by the International Union for the Conservation of Nature (IUCN, 2025); Labeobarbus mbami (Holly, 1927) listed as Endangered; as well as Labeo sanagaensis Tshibwabwa, 1997; Labeo nunensis (Pellegrin, 1929) and Coptodon cameronensis (Holly, 1927), all listed as least-concern. Coptodon nyonganus (Thys van den Audenaerde, 1971), was also included to compare to the morphologically similar and closely related species to C. cameronensis originally described from the neighboring Nyong basin and present across southern Cameroon, Equatorial Guinea, and Gabon. Other species analyzed include Alestes macrophthalmus Günther, 1867, Schilbe mystus (Linnaeus 1758), and Brycinus macrolepidotus (Valenciennes, 1850), which hold substantial commercial value due to their abundance and importance for local fisheries and food security. Unlike the five taxa restricted to the Sanaga Basin, the four other species exhibit broad continental distributions across multiple ichthyogeographic provinces (Roberts, 1975), including the Nilo-Sudan, Upper and Lower Guinea, and Congo provinces, as well as the East Coast, Zambezi, and Quanza provinces for S. mystus . With the objective to improve the availability of mitochondrial genomic resources for this ichthyofauna, we performed Illumina sequencing of total genomic DNA to sequence complete mitochondrial genomes for these nine emblematic fish species from the Sanaga River. Material and methods Sampling and morphological identifications Specimens were collected from various sites along the Sanaga River and its main tributaries, encompassing the upper, middle and lower portions, of the watershed. In total, 39 specimens representing nine freshwater fish species ( Coptodon cameronensis, n = 4 ; Coptodon nyonganus, n = 1 ; Alestes macrophthalmus , n = 5 ; Brycinus macrolepidotus, n = 5 ; Labeo sanagaensis , n = 5 ; Labeo nunensis, n = 5 ; Labeobarbus mbami , n = 4 ; Sanagia velifera, n = 5; Schilbe mystus, n = 5) were sampled between 2023 and 2024 using gill nets and electrofishing (TableS1). All fish sampling campaigns were conducted under official permits granted by the Cameroonian Ministry of Scientific Research and Innovation (MINRESI), the Ministry of Forestry and Wildlife (MINFOF) for sampling within national parks and the Ministry of Environment, Nature Protection and Sustainable Development (MINEPDED) for the Prior Informed Consent (N° 166 -169/ MINRESI/ BOO/ COO/C10/C13; N° 2376/PRBS/MINFOF /SETAT/ SG/DFAP / SDVEF/SC/ENJ; N°00054/D/MINEPDED/CAN ) . Morphological identifications were performed following the ichthyological keys of Stiassny et al. (2007). DNA Extraction, Sequencing and Genome assembly DNA was extracted using the BioBasic kit, following the manufacturer’s protocol. DNA quality and concentration were assessed using NanoDrop spectrophotometry. Sequencing was performed on the Illumina NovaSeq platform, generating paired-end reads of 150 bp. Raw reads obtained from Illumina NovaSeq sequencing were processed using an automated pipeline dedicated to mitochondrial genome assembly and annotation. Initial assembly was performed using MEGAHIT v1.2.9 (Li et al., 2015). Mitochondrial scaffolds were identified using the FindMitoScaf module of MITOZ v2.0 (Meng et al., 2019) and annotated using the AnnotateReads module of MITOZ. Taxonomic validation of the sequences was conducted using BLASTn (Altschul et al., 1990). Overall pipeline quality was assessed with MultiQC v1.13 (Ewels et al., 2016). Circular mitogenomes were visualized using Chloroplot (Zheng et al., 2020). Phylogenetics reconstructions Phylogenetic analyses were conducted using concatenated datasets comprising the 13 protein-coding genes (PCGs) and two ribosomal RNA genes (12S and 16S) from the complete mitogenomes. The dataset included both the newly assembled sequences from this study and additional mitochondrial genomes publicly available in GenBank. Phylogenetic trees were reconstructed separately for each of the four orders represented in our species set, including Characiformes, Cichliformes, Cypriniformes and Siluriformes, using partitioned analyses as implemented in IQ-TREE v2.2.0 (Minh et al., 2020). The best-fit substitution model for each partition was automatically selected by ModelFinder (Kalyaanamoorthy et al., 2017) prior to tree inference. Node support was assessed with 5,000 ultrafast bootstrap replicates (Hoang et al., 2018), providing robust and computationally efficient estimates of nodes’ statistical support. Model parameters were estimated under a shared set of branch lengths while allowing independent evolutionary rates across partitions. The resulting phylogenetic trees were visualized and annotated using FigTree v1.4.4 (Rambaut, 2014). Results & Discussion The complete mitochondrial genomes obtained for the 39 specimens from the nine fish species provide new genomic resources for West-Central African freshwater fishes. The assembled mitogenomes show typical lengths ranging from 16,524 to 16,692 bp, with a GC content between 42% and 47% (TableS1), values consistent with those reported in other teleosts species (Haÿ et al., 2025; Kundu et al., 2022, 2023; Yao et al., 2025) . Assembled mitogenomes exhibit the typical structure and gene content found in teleosts, including 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs), and a single control region (D-loop) (TableS2). Despite these species belong to distantly related lineages, gene order and length remain highly conserved across species. Minor variation in total genome length and GC composition, however, reflects lineage-specific differences. Alestidae (Alestes macrophthalmus and Brycinus macrolepidotus) possess the longest mitogenomes (up to 16,689 bp; 44–45 % GC) (Figure 1) while the mitogenomes in Cichlidae (Coptodon cameronensis and Coptodon nyonganus) are slightly shorter and GC-rich genomes (47 %) (Figure 2). Cyprinids mitogenomes (Labeo nunensis, L. sanagaensis, Labeobarbus mbami, and Sanagia velifera) show moderate variation (16,560–16,692 bp; 42–43 % GC) (Figure 3), and Schilbidae (Schilbe mystus) display the shortest genomes (16,524 bp; 45 % GC) (Figure 4). The control region (D-loop), in contrast, exhibits notable size variability, ranging from 859 bp in Coptodon nyonganus to 1,040 bp in Alestes macrophthalmus. Such variations are well-documented in teleost and are generally attributed to the accumulation of tandem repeats and insertion–deletion events within this non-coding region, which evolves rapidly under-relaxed selective constraints (Ma et al., 2015; Tan et al., 2025; Zhou et al., 2024) . Phylogenetic analyses based on complete mitochondrial genomes were conducted separately for the four teleost orders represented here (Characiformes, Cichliformes, Cypriniformes, and Siluriformes). Across all reconstructions, species consistently formed well-supported monophyletic clades, confirming their current taxonomic assignments and validating morphological identifications. Within Characiformes, Alestes macrophthalmus and Brycinus macrolepidotus were recovered in two distinct, strongly supported clades (Figure 5). In Cichliformes, Coptodon cameronensis and C. nyonganus each formed independent, well-supported lineages despite their morphological similarity (Figure 6), highlighting clear genetic divergence within the genus. In Cypriniformes, Labeo sanagaensis, L. nunensis, Labeobarbus mbami, and Sanagia velifera clustered into separate, well-supported lineages (Figure 7), congruent with the morphological identifications. Notably, Labeo species from the Sanaga basin formed a lineage distinct from Asian congeners (L. angra, L. catla, L. dussumieri, L. rajasthanicus, L. rohita) and from other African representatives (L. cylindricus, L. nasus, L. parvus). This pattern underscores the deep evolutionary divergence and long-term geographic isolation of the Sanaga Labeo lineage within Central Africa. In Siluriformes, all Schilbe mystus specimens clustered tightly within a single, well-supported lineage (Figure 8), clearly separated from other African schilbids such as Pareutropius debauwi. Overall, the mitochondrial phylogenies exhibit high resolution and strong node support, supporting the morphological identification underlying the present study. Conclusions This study provides a significant step forward in the development of mitochondrial resources for the freshwater fishes from the Sanaga River basin in Cameroon. A total of 39 complete mitochondrial genomes were assembled and annotated, representing nine species distributed across four orders including Siluriformes, Characiformes, Cypriniformes, and Cichliformes. For the first time, complete mitochondrial sequences are reported for the genera Sanagia , and Schilbe , which were previously absent from public genomic databases. These new genomic resources fill a critical gap in the molecular documentation of African freshwater fishes and provide valuable reference for future comparative and evolutionary studies. Overall, this work establishes the foundation for a regional mitochondrial inventory and offers new perspectives for taxonomy, phylogeography, and the conservation of ichthyofaunal diversity in the Sanaga river. Acknowledgements This study would not have been possible without the financial support of the Nachtigal HydroPower Company (NHPC), to whom we express our sincere gratitude. We also acknowledge the institutional and logistical support of the Cameroonian authorities. In particular, we thank the Ministry of Scientific Research and Innovation (MINRESI), the Ministry of Forestry and Wildlife (MINFOF), and the Ministry of Environment, Nature Protection and Sustainable Development (MINEPDED) for their administrative assistance and research authorizations. Finally, we are grateful to the local fishermen of the Sanaga basin, whose field expertise and collaboration were instrumental to the success of the sampling campaigns. Author Contributions Conceptualization: N.H. and A.B.N. ; Field sampling and specimen identification: H.T. and A.B.N. ; Laboratory work: H.T., J‑F.A. and P.C. ; Genome assembly workflow: K.B. ; Genome assembly and phylogeny analyses: H.T. and N.H. ; Writing – original draft: H.T. ; Writing – review and editing: A.B.N., P.C., L.B., J‑F.A. and N.H. ; Resources and permits: A.B.N. and L.B. ; Supervision: N.H. and A.B.N. All authors contributed to the article and approved the final version of the manuscript. Conflicts of Interest The authors declare no conflicts of interest. Funding information This work was financially supported by the Nachtigal HydroPower Company (NHPC). Data Availability Statement The mitochondrial genomes generated in this study will be made publicly available in GenBank during the review process. Supporting Information includes TableS1 (specimen metadata and mitogenome statistics) and TableS2 (gene structure and orientation of mitochondrial genomes). 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Each genome includes 13 protein‑coding genes, 22 tRNA genes, 2 rRNA genes, and a control region (D‑loop). Arrows indicate gene orientation; colors denote functional categories. Figure 3. Circular mitochondrial genome maps of Labeo nunensis , Labeo sanagaensis , Labeobarbus mbami , and Sanagia velifera . Each genome includes 13 protein‑coding genes, 22 tRNA genes, 2 rRNA genes, and a control region (D‑loop). Gene orientation is shown by arrows, with colors indicating functional categories. Figure 4. Circular mitochondrial genome map of Schilbe mystus . The genome includes 13 protein‑coding genes, 22 tRNA genes, 2 rRNA genes, and a control region (D‑loop). Arrows indicate gene orientation; colors denote functional categories. Figure 5. Maximum‑likelihood phylogenetic tree of Characiformes, based on our samples (highlighted) and additional sequences retrieved from GenBank (accession numbers indicated). Figure 6. Maximum‑likelihood phylogenetic tree of Cichliformes, based on our samples (highlighted) and additional sequences retrieved from GenBank (accession numbers indicated). Figure 7. Maximum‑likelihood phylogenetic tree of Cypriniformes, based on our samples (highlighted) and additional sequences retrieved from GenBank (accession numbers indicated). Figure 8. Maximum‑likelihood phylogenetic tree of Siluriformes, based on our samples (highlighted) and additional sequences retrieved from GenBank (accession numbers indicated). Information & Authors Information Version history V1 Version 1 27 January 2026 Peer review timeline Published Ecology and Evolution Version of Record 8 May 2026 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords freshwater genetics molecular genetics vertebrate Authors Affiliations HERVE TJOMB 0009-0003-8745-2535 Institut des Sciences de l'Evolution de Montpellier View all articles by this author Arnold Roger Bitja Nyom 0000-0002-8974-051X Universite de Douala View all articles by this author Pierre Caminade Institut des Sciences de l'Evolution de Montpellier View all articles by this author Leah Anne BECHE Electricite de France SA View all articles by this author Khalid Belkhir Institut des Sciences de l'Evolution de Montpellier View all articles by this author Jean-Francois Agnese Institut des Sciences de l'Evolution de Montpellier View all articles by this author Nicolas Hubert 0000-0001-9248-3377 [email protected] Institut des Sciences de l'Evolution de Montpellier View all articles by this author Metrics & Citations Metrics Article Usage 198 views 83 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation HERVE TJOMB, Arnold Roger Bitja Nyom, Pierre Caminade, et al. New mitochondrial genomic resources for the freshwater fishes of the Sanaga river (Cameroon).. Authorea . 27 January 2026. DOI: https://doi.org/10.22541/au.176950163.32847778/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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