Complete mitochondrial genome of four Scleromystax barbatus (Siluriformes: Callichthyidae) populationsAbstract
The complete mitogenome of four Scleromystax barbatus populations distributed along the species natural range was reconstructed, comprising the first study with this genus and the second with Corydoradinae specimens sampled from their natural environment and deposited in permanent collections. The mitogenome of S. barbatus is a circular molecule of 16,694 base pairs (bp) comprising 37 genes, 22 of which are tRNA, two are rRNA, 13 are protein-coding genes and one control region (D-loop). An 18-nucleotide insertion sequence was found between the ATPase subunit 6 and COIII genes. Most genes are encoded on the heavy strand, while the ND6 and eight tRNAs are found on the light strand. Phylogenetic analyses using other available Callichthyidae mitogenomes confirmed the monophyly of Callichthyinae and Corydoradinae and indicate that S. barbatus populations form a separate and more closely related branch of Corydoras nattereri + Corydoras paleatus.
Keywords: Markers; Mitochondrial DNA; Molecular identification; Phylogenetic analysis
Resumo
O mitogenoma completo de quatro populações de Scleromystax barbatus coletadas ao longo da área de distribuição natural foi reconstruído, compreendendo o primeiro estudo com este gênero e o segundo com espécimes de Corydoradinae amostrados em seu ambiente natural e depositados em coleções permanentes. O mitogenoma de S. barbatus é uma molécula circular de 16.694 pares de bases (pb) compreendendo 37 genes, dos quais 22 são tRNA, dois são rRNA, 13 são genes codificadores de proteínas e uma região controle (D-loop). Uma sequência de inserção de 18 nucleotídeos foi encontrada entre a subunidade 6 da ATPase e os genes COIII. A maioria dos genes é codificada na fita pesada, enquanto o ND6 e oito tRNAs são encontrados na fita leve. Análises filogenéticas usando outros mitogenomas de Callichthyidae disponíveis confirmaram a monofilia de Callichthyinae e Corydoradinae e indicam que as populações de S. barbatus formam um ramo separado e mais intimamente relacionado a Corydoras nattereri + Corydoras paleatus.
Palavras chave: Análises filogenéticas; DNA Mitocondrial; Identificação molecular; Marcadores
INTRODUCTION
Corydoradinae is a subfamily of Callichthyidae, one of the largest monophyletic family of catfish widely distributed in the Neotropical region (Reis, 2003, 1998). Currently, this subfamily is composed of three genera and 207 valid species (Britto, 2003; Tencatt et al., 2022a; Fricke et al., 2023). Nevertheless, despite its richness, the main studies concerning Corydoradinae are focused on the description of new species, especially in Corydoras Lacepède, 1803 (Rocha et al., 2022; Tencatt et al., 2022b). Some attempts, however, have been performed to resolve phylogenetic relationships between groups (Britto, 2003; Shimabukuro-Dias et al., 2004; Alexandrou et al., 2011; Marburger et al., 2018).
Among Corydoradinae members, Scleromystax Günther, 1864 exhibits the lowest diversity and is restricted to Atlantic Rainforest coastal rivers (Britto, 2003; Britto et al., 2016). This genus was revalidated by Britto (2003), including three species previously belonging to Corydoras. Subsequently, two Scleromystax species were described (Britto, Reis, 2005; Britto et al., 2016), and one species previously included in Aspidoras Ihering, 1907 has been recently reclassified to Scleromystax (Tencatt et al., 2022a), bringing the total number of known species to six for the genus.
Scleromystax barbatus (Quoy & Gaimard, 1824) is the type species of the genus and exhibits the widest distribution among Scleromystax representatives, extending to the coastal rivers of the Atlantic Rainforest, from the Paraíba do Sul River, in the State of Rio de Janeiro, to Itapocu River, in the State of Santa Catarina (Reis, 2003; Menezes et al., 2007). The species presents an accentuated sexual dimorphism. Adult males present odontoids in the lateral region of the head, spines on the dorsal fin, and elongated pectoral fins (Britto, 2003). It also inhabits shallow environments with weak to moderate currents and substrates consisting of clay or sandy bottoms, moving in small shoals or alone in clear stream and river waters. Its diet is omnivorous, and its food is obtained by exploring the bottom in search of small invertebrates, plant fragments and algae (Aranha et al., 1998; Gonçalves, Braga, 2013). Molecular genetic variation within S. barbatus populations of southern Brazil indicate that this species is an interesting model for phylogeographic studies (Tschá et al., 2017).
The rapid evolution of mitochondrial DNA and its small size make this an important tool in evolutionary genetics, molecular ecology, species identification and conservation biology (Harrison, 1989; Ma et al., 2012; Zhang et al., 2022). Complete mitochondrial genome sequences are becoming an increasingly important apparatus in the study of fish phylogenies due to advances in sequencing techniques and biological data analyses (Betancur-R et al., 2017; Rocha-Reis et al., 2020). With regard to Corydoradinae, however, studies are scarce and concentrate only on Corydoras (Moreira et al., 2016; Liu et al., 2019b; Sun et al., 2022). In this context, the present study aims to sequence, assemble, and annotate the complete mitogenome of four S. barbatus populations, providing an important addition to future phylogenetic Corydoradinae studies as the first mitogenome from a Scleromystax member.
MATERIAL AND METHODS
Specimen samplings. Individuals from four Scleromystax barbatus populations were collected by electrofishing and trawling along their known distribution between 2019 and 2021 (Tab. 1). In the field, individuals were anesthetized and sacrificed. Tissue samples (pieces of muscle and pectoral fin) were removed from each individual and preserved in 100% alcohol. The specimens were then fixed in 4% formalin and transferred to 70% alcohol in the laboratory. All specimens were deposited in the ichthyological collection at the Museu de Historia Natural Capão da Imbuia, Curitiba (MHNCI).
Sequencing and mitogenome assembly. Genomic DNA was extracted using the saline method and the purity was checked on a 1% agarose gel and on NanoVue. The extracted DNA was sequenced using the DNBSeq sequencing platform (BGI Genomics), which generated 150pb paired-end sequences. The quality of the clean data was verified using FASTQC. We then used the GetOrganelle v. 1.7.6.1 (Jin et al., 2020) for the de novo assembly of the mitogenome, using Corydoras Genbank sequences as a reference (Tab. 2). We checked genome circularity using the Bandage v. 0.9.0 software (Wick et al., 2015). Annotation was performed using the MitoAnnotator tool (http://mitofish.aori.utokyo.ac.jp/annotation/input.html), included in the Mitofish Database v. 3.73 (Iwasaki et al., 2013).
Phylogenetic analysis. Phylogenetic analyses were performed using 13 PCGs (protein-coding genes), including four mitogenomes from S. barbatus and some related Callichthyidae species obtained at Mitofish and Genbank (Tab. 2). All species were aligned using the MAFFT v. 7.017 software (Katoh et al., 2002). The tree was constructed using Mr. Bayes v. 3.2 (Ronquist, Huelsenbeck, 2003).
Code used in the analysis of Scleromystax barbatus populations, locality, river, and coordinates where the collections were carried out.
Species used from Genbank and the present study and their respective accession and source numbers.
RESULTS
The mitochondrial genome of the four Scleromystax barbatus populations is a circular molecule 16,694 base pairs (bp) in length. It consists of 37 genes, 22 of which are tRNA, two are rRNA, 13 are protein-coding genes, and one is a control region (D-loop). An 18-nucleotide insertion sequence was found between the ATPase subunit 6 and COIII genes. Most of the genes are encoded on the heavy strand, while ND6 and eight tRNAs are found on the light strand. All protein-coding genes use ATG start codons, except for COI, which uses GTG. Seven protein-coding genes end with the complete stop codon, five of which end with TAA (ATP8, ATP6, ND4L, ND5 and ND6), one with TAG (ND1) and one with AGG (COI), while the remaining protein-coding genes are terminated with incomplete stop codons (ND2, COII, COIII., ND3, ND4, Cyt b). Fig. 1 and Tab. 3 depict the mitogenome annotation of the AR population, which is the closest to the S. barbatus type-locality (Reis et al., 2003). A summary of the other populations is reported in Fig. S1 and Tab. S2. The nucleotide composition is summarized in Tab. 4.
A. Geographic location of the Scleromystax barbatus populations in coastal Atlantic Rainforest rivers. B. Male of S. barbatus. Photo by Caio Feltrin. C. Complete mitochondrial genome of S. barbatus from the AR population. Population codes in Tab. 1.
Annotation of the complete mitochondrial genome of a Scleromystax barbatus individual belonging to the Ariró River (AR) population.
Nucleotide composition of the four Scleromystax barbatus populations used in this study. %T (thymine percentage), %C (cytosine percentage), %A (adenine percentage), %G (guanine percentage), %GC content (guanine and cytosine percentages). Population codes in Tab. 1.
Phylogenetic analysis of 13 Corydoradinae and Hoplosternum littorale (Callichthyinae member) species as the outgroup based on the nucleotide sequences of 13 PCGs from the mitochondrial genome. Bootstrap values are shown next to nodes and the scale bar shows 0.03 changes. Population codes in Tab. 1.
Phylogenetic analyses using the mitochondrial genome of Callichthyidae species indicated that the Corydoradinae subfamily formed a well-separated Callichthyinae branch (Fig. 2). In addition, the validity and taxonomic identification of S. barbatus was also strengthened.
DISCUSSION
Phylogenetic analyses confirm the monophyly of the Callichthyidae subfamilies and among the Corydoradinae members whose mitogenome information is available. The Scleromystax barbatus populations are closer to the clade formed by Corydoras nattereri + Corydoras paleatus, similar to the findings reported by phylogenies with molecular (Shimabukuro-Dias et al., 2004; Alexandrou et al., 2011; Marburger et al., 2018) and morphological (Britto, 2003) markers.
The order and arrangement of the mitochondrial genomes of the evaluated S. barbatus populations were identical to each other and similar to other Siluriformes members (Saitoh et al., 2003; Moreira et al., 2016; Rocha-Reis et al., 2020; Zhang et al., 2022) with the ND6 gene and eight tRNAs found on the light strand. The size of the mitogenomes ranged from 16,614 to 16,694 bp, and the nucleotide composition was 41.6% G+C for all populations, very close to the percentages found in other Corydoradinae members (Moreira et al., 2016; Liu et al., 2019a,b; Chen et al., 2020; Xu et al., 2020; Zhang et al., 2022).
The four S. barbatus populations presented an 18-nucleotide sequence insertion between the ATPase subunit-6 and COIII genes, also found in other Callichthyidae members, with similar values noted for Corydoradinae members (Saitoh et al., 2003; Moreira et al., 2016) and higher values for Callicthyinae members (Parente et al., 2018), suggesting that this insertion may be a synapomorphy among Callichthyidae subfamilies.
This is the first report on the mitogenome of S. barbatus populations and the second study on Corydoradinae species using individuals sampled from their natural environment and deposited in permanent collections. In this context, this study fills an existing gap for the Neotropical region, furthering information on a genus restricted to coastal Atlantic Rainforest streams and may be useful for future studies on Callichthyidae population genetics and evolution.
ACKNOWLEDGEMENTS
We are grateful to Adriano Hauer, Axel M. Katz, Thiago T. Batista for the aid in field collections; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) through a doctoral scholarship to RHD and LEDLOG under VA and RFA supervision, respectively. This research was funded by Fundação Araucária – NAPIBioinformática, grant number agreement 033/2021 for massive DNA sequencing and molecular analysis.
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ADDITIONAL NOTES
Publication Dates
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Publication in this collection
13 Nov 2023 -
Date of issue
2023
History
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Received
07 Mar 2023 -
Accepted
21 Sept 2023
