Characteristics and Evolutionary Relationships of Two Mitochondrial Genomes of Iguanodectes (Characiformes, Iguanodectidae)

TL;DR

This study analyzes the mitochondrial genomes of two freshwater fish species to confirm their distinct evolutionary status and provide data for conservation.

Contribution

The study provides the first complete mitochondrial genomes for Iguanodectes species and confirms their taxonomic distinction using molecular data.

Findings

I. geisleri and I. adujai form a strongly supported monophyletic clade but are distinct evolutionary lineages.

Genomic structure, base composition, and codon usage differ between the two species despite morphological similarities.

The study confirms the taxonomic status of I. geisleri and I. adujai using mitochondrial genomic data.

Abstract

The complete mitochondrial genomes of I. geisleri and I. adujai were obtained using high-throughput sequencing, assembly, and annotation. The mitochondrial genomes of both fish species exhibited typical structural characteristics of vertebrate mitochondria. Concurrently, species-specific differences were observed in genomic structures and codon usage. Further, the ML and BI phylogenetic analyses of a concatenated dataset of 13 PCGs demonstrated that I. geisleri and I. adujai form a strongly supported monophyletic clade, with the two species being sister taxa. However, they were also determined to constitute independent evolutionary lineages, confirming their taxonomic status as distinct species. This study supplements the fundamental mitochondrial genomic data for I. geisleri and I. adujai, provides a reliable basis for the molecular identification of species within the genus Iguanodectes, and offers scientific support for genetic evolutionary research on Characiformes fishes, the conservation of freshwater fish resources in South America, and precise species delineation in the ornamental fish trade.

Iguanodectes geisleri and I. adujai are freshwater fish from South America. Their taxonomic status and phylogenetic relationships are uncertain due to limited molecular data. High-throughput sequencing was applied to obtain and annotate for the first time the complete mitochondrial genomes of I. geisleri and I. adujai to clarify their phylogenetic positions. Mitochondrial genome sequences of 73 Characoidei species were retrieved from GenBank, with Gyrinocheilus aymonieri and Microphysogobio alticorpus designated as outgroups. Phylogenetic trees were constructed using a mitochondrial protein-coding gene dataset and Maximum Likelihood and Bayesian Inference methods. The complete mitochondrial genome measured 16,774 and 16,802 bp, respectively. Both genomes exhibited highly conserved structures. Despite morphological similarities and a close phylogenetic relationship, differences were detected in genomic structure, base composition, codon usage bias, and the control region between the two species. The two species comprise a strongly supported monophyletic clade and are sister species but represent distinct, independent branches. I. geisleri and I. adujai have been recognized as distinct species based on morphological differences, and this study provides molecular confirmation of their separate taxonomic status. The study provides molecular data for the taxonomic identification of fishes of the genus, Iguanodectes, and foundational mitochondrial genomic data for Characiformes. The study advances research on the genetic evolution of this group and resource conservation.

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1. Introduction

The mitochondrial genome encodes key components of cellular energy metabolism and serves as a critical resource for evolutionary and genetic studies [1]. Mitochondria synthesize ATP through oxidative phosphorylation, generating heat during cellular respiration. This process accounts for more than 95% of cellular ATP, sustaining essential biochemical and physiological functions [2]. Mitochondrial DNA (mtDNA) is a small, typically circular molecule, approximately 16 kb in size, which has unique and irreplaceable value in the study of maternal lineage, population history, species kinship, forensic identification and ancient DNA analysis [3].

Additionally, mitochondrial genes evolve five to ten times faster than nuclear genes, and their nucleotide sequences are easily obtainable, making them widely applicable for determining phylogenetic relationships among species [4]. Furthermore, genes encoding oxidative phosphorylation components in mitochondria may have evolved under natural selection, with distinct adaptive evolutionary characteristics in mammals, birds, and fish [5]. However, individual mitochondrial genes often provide limited phylogenetic information and may not accurately depict the true phylogenetic relationships among species [6]. In contrast, multigene datasets, such as complete mitochondrial genomes, offer more comprehensive genetic information, which enables a more robust reconstruction of species phylogenies [7].

The mitochondrial DNA of fish, similar to that of other vertebrates, is a closed, circular, double-stranded DNA molecule comprising a heavy (H) strand and a light (L) strand. Moreover, it is capable of autonomous replication, transcription, and translation [8]. Typically, fish mitochondrial genomes encode a conserved set of 37 genes categorized into three groups: two ribosomal RNA (rRNA) genes, 13 protein-coding genes (PCGs), and 22 transfer RNA (tRNA) genes.

I. geisleri belongs to the order Characiformes of the family Iguanodectidae. It is mainly distributed in the Negro River of the Brazilian Amazon Basin and the upper Río Linoko River system of Venezuela, where it inhabits shallow, fast-flowing freshwater environments with dense aquatic vegetation. The species is characterized by conspicuous lateral coloration, including a prominent red stripe accompanied by green and black markings [9].

The order Characiformes represents one of the most diverse and ecologically significant groups of freshwater fishes, comprising approximately 2000 species distributed across the Neotropical region and Africa [10]. This order exhibits remarkable morphological and ecological diversity, ranging from small-bodied tetras and piranhas to large predatory species such as tigerfishes and trahiras. Characiformes is currently classified into 24 families, with the vast majority of diversity concentrated in the suborder Characoidei, which includes well-known families such as Characidae (tetras and piranhas), Serrasalmidae (pacus and silver dollars), and Lebiasinidae (pencilfishes) [11]. Despite their economic importance in the ornamental fish trade and as food resources, the phylogenetic relationships among characiform families remain incompletely resolved.

I. adujai and I. geisleri both belong to the subfamily Characiformes and the family Iguanodectidae, and their distributions roughly overlap. Due to their similar body shape and the presence of a red lateral stripe, the two species are frequently confused in both field identification and the ornamental fish trade. I. adujai has a light-yellow body with a bright red neon stripe extending from the upper part of the gill to the upper part of the caudal peduncle. The core morphological difference between the two is that I. geisleri has obvious fluorescent green and black stripes below the red sideline and each fin has a red tone; however, I. adujai does not have these characteristics [12].

Both species are popular ornamental fish with significant economic value. At present, the taxonomic relationships among Characoidei are unclear, and the number of reported Iguanodectidae species is limited, making it difficult to fully understand the species diversity of this suborder. Therefore, an in-depth comparison of the mitochondrial genome characteristics and genetic relationships between I. geisleri and I. adujai provides a molecular basis for the classification and identification of the genus Iguanodectes and can also be used to supplement information on the Characiformes order.

In the current study, high-throughput sequencing technology was used to sequence, assemble, and annotate the complete mitochondrial genomes of I. geisleri and I. adujai. The genomic structure, nucleotide composition, codon usage patterns, and selection pressure characteristics were examined. In addition, phylogenetic trees were constructed to determine their taxonomic status. These findings provide molecular evidence for species identification within the genus Iguanodectes and supplement the fundamental mitochondrial genomic data for Characiformes and offer scientific support for genetic evolutionary studies, resource conservation, and precise species delineation with respect to the ornamental fish trade.

2. Materials and Methods

2.1. Sample Collection, Identification, and Sequencing

Specimens of I. geisleri and I. adujai were obtained from the Fangcun Flowers, Birds, Fishes, and Insects Market (Guangzhou, China) in September 2025. Voucher specimens were deposited in the Zoology Laboratory, School of Life Sciences, Nanjing Forestry University, under the catalogue numbers PV440321 (I. geisleri) and PV765882 (I. adujai). Fresh tissue samples were immediately preserved in absolute ethanol and stored at −20 °C in the Zoology Laboratory of the School of Life Sciences, Nanjing Forestry University, for subsequent analysis. Total genomic DNA was extracted from dorsal muscle tissue using the DNAiso reagent (Takara Biomedical Technology Co., Ltd., Beijing, China). To verify the accuracy of the morphological identification, mitochondrial DNA from both species was amplified using polymerase chain reaction (PCR) with universal 16S primers [13]. Subsequently, 150 bp insert-size libraries were constructed, and high-throughput sequencing was conducted by the Nanjing Qingke Biological Company (Nanjing, China).

2.2. Genome Assembly and Annotation

The DNA samples were subjected to PE150 sequencing (paired-end 150 bp, generating two 150 bp reads per fragment) using the Illumina HiSeq X Ten platform. Subsequently, quality control was performed on the sequencing data, ultimately retaining sample datasets with valid reads exceeding 10 GB [14]. For the valid reads of each sample, de novo assembly was conducted using SPAdes v3.0.0 software to obtain complete mitochondrial genome sequences [15]. The FASTA files of the mitochondrial genome sequences of I. geisleri and I. adujai were imported into MITOS for initial annotation [16]. The sequences of the PCGs and rRNA genes were then compared to those of related species and manually corrected using Geneious 2025 [17].

2.3. Sequence Analysis

To comprehensively investigate the mitochondrial genome structure of I. geisleri and I. adujai, PhyloSuite v1.2.3 was utilized to calculate the AT content, AT-skew, GC-skew, and relative synonymous codon usage (RSCU) of both species. The results were visualized using the same software [18]. In evolutionary analysis, the ratio of nonsynonymous (Ka) to synonymous (Ks) mutations (Ka/Ks) is usually used to determine whether there is selective pressure on a gene [19]. Furthermore, to analyze the genomic evolutionary characteristics in detail, the Ka/Ks ratio was calculated using MEGA 11 and DNASP 6 to estimate evolutionary rates [20]. The results were visualized using Origin 64 to examine the differences in selective pressure among the genes [21].

2.4. Phylogenetic Analysis

To clarify the taxonomic status of I. geisleri and I. adujai, the mitochondrial genome sequences of 73 Characoidei fish species with two outgroup species, Gyrinocheilus aymonieri (NC_008672.1) and Microphysogobio alticorpus (NC_021451.1), were downloaded from the NCBI database for phylogenetic tree reconstruction (Table S1). For PCG dataset analysis, multiple sequence alignment was conducted using MAFFT v7.313 and Gblocks 0.91b was used to eliminate gaps and ambiguous regions from the alignment [22]. The aligned sequences of the individual genes were concatenated using SequenceMatrix to form a combined dataset comprising all 13 PCGs [23]. The optimal partitioning scheme and corresponding evolutionary models were determined using ModelFinder [24]. Phylogenetic analyses were conducted using PhyloSuite v1.2.1. A Maximum Likelihood (ML) tree was constructed using IQ-TREE v1.6.8, and branch support was assessed using 50,000 bootstrap replicates [25]. Concurrently, Bayesian Inference (BI) analysis was conducted using MrBayes v3.2.6, running four parallel Markov chains for 20 million generations, sampling every 1000 generations. Convergence was considered to be achieved when the effective sample size exceeded 200 and the average standard deviation of the split frequencies fell below 0.01. The first 25% of samples were discarded as burn-in, and a consensus tree was constructed from the remaining samples to calculate the posterior probabilities [26]. Finally, phylogenetic trees were visualized and optimized using iTOL (https://itol.embl.de/) (accessed on 25 August 2025) [27].

3. Results

3.1. Mitochondrial Genome Structure

The mitochondrial genomes of both I. geisleri and I. adujai exhibited a typical circular structure (Figure 1), with total lengths of 16,774 and 16,802 bp and overall GC contents of 43.0% and 44.5%, respectively. Each mitochondrial genome contained 13 PCGs, 22 tRNA genes, two rRNA genes, and one non-coding control region (D-loop). The gene order of both mitochondrial genomes follows the typical vertebrate mitochondrial gene arrangement without any rearrangement events.

Further, differences in the nucleotide composition and skewness were observed between the two species (Table 1). In I. geisleri, the base composition was A = 29.3%, T = 27.7%, G = 15.9%, and C = 27.1%, and the A+T content was higher than the G+C content. The AT-skew and GC-skew values were 0.03 and −0.262, respectively, indicating a distinct AT bias in the mitochondrial genome of I. geisleri. In I. adujai, the base composition was A = 29.0%, T = 26.5%, G = 16.2%, and C = 28.3%. Similarly, the A+T content exceeded the G+C content, and the AT-skew and GC-skew values were 0.045 and −0.273, respectively, also reflecting a pronounced AT bias in the mitochondrial genome of I. adujai.

Gene organization was largely conserved among species, with most genes sharing identical positions and lengths (Table 2). Minor length variations were detected in several regions, including a 10 bp difference in the 12S rRNA gene, 1–2 bp differences in several tRNA genes (e.g., tRNA-Ile and tRNA-Gly), and a 16 bp length difference in the control region (D-loop). Regarding start and stop codons, some genes shared the same initiation codons. For example, NAD1 was found to start with ATG in both the species, whereas COX1 was determined to use GTG as the start codon. However, differences were noted in the termination signals; the NAD1 gene ended with TAG in I. geisleri but with TAA in I. adujai. Additionally, variations were observed in the intergenic regions. For example, no nucleotides were present between tRNA-Asn and tRNA-Cys in I. geisleri, whereas a 32 bp intergenic spacer was identified in the same region in I. adujai.

3.2. PCGs and Codon Usage

Codon usage in the two Iguanodectidae species was strongly correlated with the AT bias of their mitochondrial genomes. High-frequency codons (RSCU > 1) predominantly ended with A or T, whereas low-frequency codons (RSCU < 1) mostly ended with G or C (Figure 2). However, specific codon usage patterns differed between the two species. In I. geisleri, the codon CGA (arginine) had the highest RSCU value of 2.26, whereas ACG (threonine) had the lowest RSCU (0.11). In total, 30 codons (46.88% of all non-terminating codons) were frequently used (RSCU > 1). In I. adujai, CGA was the most frequently used codon, with the highest RSCU of 2.15. In contrast, the AGA (arginine) codon was not used (RSCU = 0). In addition, 31 codons were used more than once, accounting for 48.44% of all non-termination codons, with one more high-frequency codon relative to the number in I. geisleri, encoding isoleucine AUU, and the RSCU was 1.02.

3.3. Analysis of Selective Pressure

To investigate the evolutionary dynamics of mitochondrial protein-coding genes, the ratio of Ka/Ks substitutions was calculated for 13 PCGs across 24 Characoidei species (Figure 3). COX1 showed the lowest median Ka/Ks value, approaching zero with minimal dispersion. COX2, COX3, and Cyt b exhibited median Ka/Ks values below 0.2, with relatively narrow interquartile ranges. ATP6, ATP8, NAD1, NAD3, NAD4, NAD4L, and NAD6 displayed median Ka/Ks values ranging from 0.2 to 0.5. NAD2 showed a median Ka/Ks value of approximately 0.6–0.8, with moderate dispersion. NAD5 exhibited the highest median Ka/Ks value, exceeding 1.0 (median: 1.20; range: 0.95–1.58 across different species comparisons), and also displayed the largest dispersion among all genes. In the pairwise comparison between I. geisleri and I. adujai, the Ka/Ks ratios for 12 of the 13 homologous protein-coding gene pairs were below 1.0, ranging from 0.01 (COX1) to 0.42 (NAD2). The NAD5 gene pair showed a Ka/Ks ratio of 1.15, representing the only instance where the ratio exceeded 1.0 in this species comparison.

3.4. Phylogenetic Relationships Based on Complete Mitochondrial Genomes

Based on the connected nucleotide sequences of 13 protein coding genes from 38 representative taxonomic groups (including 36 Characoidei species and 2 outgroup species), the phylogenetic tree reconstructed using ML and BI methods is shown in Figure 4. The Iguanodectidae was recovered as a monophyletic clade with strong support (ML bootstrap = 100; BI posterior probability = 1.0). Within this clade, I. geisleri and I. adujai formed a strongly supported sister-group relationship (ML bootstrap = 100; BI posterior probability = 1.0), confirming their close genetic affinity. These 2 species were resolved as sister to Chalceus macrolepidotus (family Chalceidae), forming a well-supported clade that represents the Iguanodectinae (sensu lato). Phylogenetic trees reconstructed from the concatenated dataset of 13 PCGs across 75 taxa showed largely congruent topologies between ML and BI analyses, with generally high nodal support (Figures S1 and S2).

4. Discussion

The complete mitochondrial genomes of I. geisleri and I. adujai were 16,774 and 16,802 bp, respectively. Each genome contained a typical vertebrate mitochondrial complement: 13 PCGs, 22 tRNAs, two rRNAs (12S and 16S), and one D-loop. This is highly consistent with the genomic structures of closely related species in the order Characiformes, such as Colossoma macropomum, Hyphessobrycon heterorhabdus, and Nannostomus beckfordi [28,29,30], confirming the structural conservation of mitochondrial genomes in Neotropical freshwater fish. Moreover, both genomes exhibited an A+T bias, consistent with that reported for Serrasalmus [28], H. heterorhabdus [30], and Nannostomus spp. [29]. This observed A+T bias could have resulted from asymmetric mitochondrial replication and mutational bias, together with selective forces acting on the mtDNA [31].

In this study, D-loop identification relied on conserved genomic position, AT-rich composition, and cross-species motif conservation rather than direct sequence similarity to Iguanodectes references. As noted by Alvanou et al. [32], complete control region assembly can be difficult in some species; however, our high sequencing depth and careful verification of motif structure support the reliability of our annotation. Future studies using long-read sequencing could resolve any remaining uncertainties in D-loop boundaries.

The codon usage patterns observed in I. geisleri and I. adujai were strongly shaped by the pronounced AT bias of their mitochondrial genomes, a feature that has been widely reported in teleost mitochondrial DNA. The predominance of high-frequency codons ending in A or T, together with the underrepresentation of codons terminating in G or C, reflects mutational pressure associated with asymmetric replication and nucleotide compositional constraints in mitochondrial genomes [31]. Similar AT-driven codon usage biases have been documented in other Characiformes and freshwater teleost fishes, suggesting a conserved evolutionary pattern within this lineage [33,34,35]. The codon usage characteristics of I. geisleri and I. adujai support the view that mitochondrial genome evolution in Iguanodectidae is dominated by compositional bias, with species-specific codon usage differences providing supplementary molecular signals for distinguishing closely related taxa.

In terms of the evolutionary rate, the extremely low Ka/Ks ratios observed for COX1/COX3 reflect strong functional constraints associated with its essential role in oxidative phosphorylation, resulting in highly conserved amino-acid sequences [36]. In contrast, NADH dehydrogenase genes, such as NAD3, exhibit a relatively fast evolutionary rate, which may be associated with adaptive divergence in energy metabolism [37]. This is consistent with the selection pressure pattern for the genus Nannostomus [37]. Importantly, except for NAD5, all other gene pairs exhibited Ka/Ks ratios > 1, with no evidence of positive selection, confirming that mitochondrial gene evolution in these two Iguanodectidae species is predominantly governed by purifying selection, with gene-specific variation likely reflecting differences in functional importance and structural constraints [38].

NAD5 encodes a subunit of NADH dehydrogenase (Complex I), which plays a central role in the electron transport chain [39]. Unlike the core subunits of cytochrome c oxidase, where amino acid substitutions are highly constrained due to direct involvement in proton pumping and electron transfer, NAD5 occupies a more peripheral position in Complex I and may tolerate greater sequence variation without complete loss of function. In the pairwise comparison between I. geisleri and I. adujai, NAD5 was the only gene with Ka/Ks > 1 (1.15), suggesting potential functional divergence between these two congeneric species. Despite their morphological similarities, subtle differences in habitat preferences or metabolic requirements may have driven adaptive evolution in this energy metabolism gene.

The phylogenetic tree constructed based on the concatenated dataset of 13 PCGs revealed that I. geisleri and I. adujai form a monophyletic group with high bootstrap support and consistently maintain a sister group relationship. This is consistent with their morphological similarities and overlapping distribution ranges, confirming their close affinity at the molecular level. This monophyletic group forms a sister clade with Chalceus macrolepidotus and is clearly differentiated from taxa such as the genus, Nannostomus (family Lebiasinidae), and genus, Brycon (family Bryconidae), supporting the independent taxonomic status of the genus Iguanodectes within the family Iguanodectidae. The topological structure identified in this study is also consistent with the conclusion proposed by Silva-Oliveira et al. in their research on Bryconops gracilis, which suggested that the family Iguanodectidae is distantly related to the family Lebiasinidae [40]. Furthermore, the distinct clade characteristics of Iguanodectes identified in the present study were similar to the unique clade pattern of Bryconops marabaixo reported by Silva-Oliveira et al. [40], supporting the independent evolutionary status of Iguanodectidae within the suborder Characoidei.

Revised information on closely related species and the taxonomic framework indicates that Iguanodectidae forms a relatively independent monophyletic clade with the genus Bryconops within the same order, and its family- and genus-level taxonomic status is jointly supported by morphological characteristics and geographical distribution data [41]. In addition, I. geisleri and I. adujai, closely related congeneric species, are both present in Colombian freshwater systems [42]. Further, they have the core diagnostic characteristics of Iguanodectes, such as a distinct lateral line on the body side and a horizontal mouth gape; however, they are classified as independent species due to differences in morphological details and local distribution ranges. No taxonomic synonym was found between these two species and other closely related species within the genus, further verifying their taxonomic status as independent and valid species within Iguanodectes in the phylogenetic framework. This also confirms the phylogenetic divergence between Iguanodectes and Bryconops within Iguanodectidae, as well as the monophyletic nature of these genera. The limitations of this study are mainly due to the use of only mitochondrial genome data and the limited range of group sampling. Future research should adopt the method of phylogenetic genomics, use nuclear gene data, and expand sampling to construct a more robust evolutionary tree, and integrate eco-geographic data to explore its adaptive evolution mechanism.

While our mitochondrial genome analysis supports the distinctiveness of I. geisleri and I. adujai, we acknowledge that definitive species delimitation ideally requires integrative evidence from multiple sources, including nuclear genetic markers, morphological data, and ecological information [43]. The mitochondrial genome represents a single maternally inherited locus and may not capture the complete speciation history or potential introgression events [44]. Future studies incorporating genome-wide nuclear markers (e.g., RAD-seq, ultraconserved elements) would provide a more robust framework for species boundary delineation in this genus.

5. Conclusions

The complete mitochondrial genomes of I. geisleri and I. adujai were obtained using high-throughput sequencing, assembly, and annotation. The mitochondrial genomes of both fish species exhibited typical structural characteristics of vertebrate mitochondria. Concurrently, species-specific differences were observed in genomic structures and codon usage. Further, the ML and BI phylogenetic analyses of a concatenated dataset of 13 PCGs demonstrated that I. geisleri and I. adujai form a strongly-supported monophyletic clade, with the two species being sister taxa. However, they were also determined to constitute independent evolutionary lineages, confirming their taxonomic status as distinct species. This study supplements the fundamental mitochondrial genomic data for I. geisleri and I. adujai, provides a reliable basis for the molecular identification of species within the genus Iguanodectes, and offers scientific support for genetic evolutionary research on Characiformes fishes, the conservation of freshwater fish resources in South America, and precise species delineation in ornamental fish trade.