Description of eight new mitochondrial genomes for the genus Neoarius and phylogenetic considerations for the family Ariidae (Siluriformes)

The genus Neoarius, known as marine catfish, is a group of the family Ariidae, composed of 10 species found in Oceania. None of the species in this genus have their mitochondrial genome described, which is highly valuable in phylogenetic and molecular evolution studies. For the present work, eight species from the Neoarius genus were selected: Neoarius utarus, Neoarius midgleyi, Neoarius graeffei, Neoarius leptaspis, Neoarius berenyi, Neoarius paucus, Neoarius pectoralis, and Neoarius aff. graeffei. DNA sequences of the eight species were obtained through the NCBI Sequence Read Archive (SRA) database, and the mitochondrial genomes were assembled using the NOVOplasty tool on the Galaxy platform, subsequently annotated with the MitoAnnotator tool. We then utilized the protein-coding genes from the mitogenomes to estimate the phylogenetic relationships within the group, including seven additional mitogenomes available in the NCBI. In all species, the mitochondrial genomes presented 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 1 D-loop.


Introduction
Neoarius is a genus of catfish belonging to the family Ariidae within the order Siluriformes.Currently, it comprises 11 described species distributed in Australia and New Guinea, with conservation status ranging from "least concern" to "vulnerable" according to the International Union for Conservation of Nature Red List (IUCN) [1].

Description of eight new mitochondrial genomes for the genus Neoarius and phylogenetic considerations for the family Ariidae (Siluriformes)
freshwater environments [2].The reproductive period for these species typically starts in spring, around September, and extends until the end of summer, in February.Notably, N. graeffei employs a rare reproductive strategy known as mouthbrooding, where the eggs are incubated in the mouth of the adult individual until they mature enough to be independent [3].
The catfish of the Ariidae family have a wide distribution across the globe, being found in various regions and countries such as Brazil, Australia, and New Guinea.They inhabit coastal, estuarine, and large river regions in both tropical and temperate areas.The majority of species have a coastal distribution, but there are also exclusively marine species found at various depths, as well as species occurring in freshwater environments.In species from marine habitats, males exhibit a unique behavior of mouthbrooding, where they incubate the eggs in their mouths.This characteristic sets them apart within the Ariidae family.Currently, the family comprises 156 species distributed among 30 genera.
The mitochondrial genome, also known as the mitogenome, consists of a circular extrachromosomal DNA molecule present in the mitochondria.In eukaryotic organisms, the mitochondrial genome has an average size of 16-17 kbp and contains highly conserved genes.In vertebrates, there are a total of 37 genes, including 13 protein-coding genes (PCGs), two rRNA genes, and 22 tRNA genes necessary for the translation of proteins encoded by mitochondrial DNA, in addition to the control region known as the D-loop.Comparing mitochondrial genomes from different groups allows for evolutionary and phylogenetic studies, as there is significant similarity among mitogenomes in closely related taxa.This similarity enables researchers to trace the evolutionary history and relationships between species and understand their genetic diversification over time [4].
The mitochondrial genome in fishes serves various purposes, such as phylogenetic reconstruction, phylogeography, population migration observation, geographic distribution analysis, genetic diversity analysis among distinct populations, examination of gene order variations, haplotype variations, and gene flow patterns.Therefore, conducting studies involving the mitochondrial genome in fishes, as well as other vertebrate groups, is of great importance [4].
The objective of this study is to describe the mitochondrial genome of eight out of the 11 existing species within the Neoarius genus, as none of the species currently have their mitogenome characterized.To achieve this, we assembled the mitogenomes of Neoarius berenyi, Neoarius utarus, Neoarius midgleyi, Neoarius graeffei, Neoarius utarus, Neoarius leptaspis, Neoarius paucus, and Neoarius aff.graeffei.Additionally, we conducted a phylogenetic analysis using the PCGs from the assembled mitogenomes, providing new insights into the interspecific phylogenetic relationships within Neoarius.

Methods
For the completion of this study, DNA samples from Neoarius species were collected from muscular tissue and sequenced by Iridian Genomics In platform HiSeq x Ten of Illumina.The resulting sequence data were provided by Dr. Ricardo Betancur through Sequence Read Archive (SRA) files hosted on NCBI (Table 1).These files were imported into the Galaxy Europe platform [5].Subsequently, we conducted mitochondrial genome assembly using NOVOplasty v4.2 with a K-mer size of 39 [6].We utilized sequences from the cytochrome B oxidase gene of the respective species available on GenBank (Table 1) as seeds for the assembly.The circularized sequences were then annotated using the MitoAnnotator software on the MitoFish server [7].Finally, a comparative analysis was conducted using BLAST among all Neoarius, Arius, Hypostomus, and Occidentarius mitogenomes included in our study, along with our assembly of N. graeffei, using the BRIG software [8].
For phylogenetic reconstruction, we manually extracted all 13 PCGs from the eight Neoarius mitogenomes, as well as the PCGs from ten other Siluriformes species available on GenBank (Supplementary Material 1).Individual PCGs were aligned using MEGA 11 software [9] with the MUSCLE algorithm [10].Subsequently, alignments were concatenated using SequenceMatrix v1.7.8 software [11].Phylogeny was generated using IQ-TREE web server software [12], with parameters of 10,000 replicates of Ultrafast Bootstrap iterations and replicates.Tree visualization was performed using the Interactive Tree of Life (IToL) online tool [13].

Results and Discussion
We observed that the mitochondrial genomes have similar characteristics among them.All mitogenomes presented 22 tRNA genes, 13 PCGs, 2 rRNA genes, and a control region called the D-loop, which is consistent with other groups within the same family and even what is typically expected in vertebrates (Fig. 1) [4,[14][15][16].The size of the mitochondrial genomes was also similar, with 16,709 bp for N. berneyi, N. graeffei, N. leptaspis, N. paucus, and N. midgleyi, 16,702 bp for N. utarus, 16,710 bp for N. aff.graeffei RB-2021, and 16,711 bp for N. pectoralis (Supplementary Material 2).The complete mitochondrial genomes of N. berenyi, N. midgleyi, N. leptaspis, N. aff.graeffei, and N. paucus showed a CG composition of 45%, while N. utarus, N. pectoralis, and N. graeffei presented 44% of CG content.These calculations were performed using the geecee tool [17].The largest gene in the mitochondrial genome was ND5, occupying 5.48% of the entire mitogenome for all species, with a variation of 913 bp and 914 bp.
The phylogeny demonstrated a monophyletic grouping among Neoarius species, which could be separated into two distinct clades.One of these clades showed N. berneyi as the sister group to the clade formed by N. graeffei and N. utarus, while in the other clade, we observed, N. midgleyi as the first species to diverge, followed by N. pectoralis, and with N. leptaspis as the sister group to the clade containing N. paucus and N. aff.graeffei (Fig. 2).All internal branches of Neoarius showed high bootstrap values (>90).An interesting observation in our study was that the mitogenomes from the libraries identified as N. aff.graeffei and N. graeffei did not group together and, in fact, each of them fell into one of the two Neoarius subclades, indicating that both samples were extracted from different species within the genus.
Few studies have a complete phylogeny of the genus; however, from the limited number of species present in the phylogenetic relationships, we can make some comparisons with the phylogeny observed in the present study, as in the work of Betancur [18].In the work of Betancur [18], a phylogenetic reconstruction of the family Ariidae was conducted using molecular data, including the cytochrome b, ATP synthase subunit 6 and 8, 12S and 16S ribosomal genes, and the nuclear rag2 gene.It was observed that N. graeffei aligned closely to N. berenyi, but between these two species, it aligned with N. aff.graeffei.However, when considering the overall phylogenetic relationship, the grouping of these two species remains quite similar, with N. utarus included in the same clade [18].
Another work involving phylogenetic reconstruction of the Neoarius genus is the study by Barathkumar and Thangaraj [19].In the study by Barathkumar and Thangaraj [19], molecular data, specifically the cytochrome oxidase 1 gene, was used to conduct the phylogenetic reconstruction of the families Ariidae, Bagridae, and Plotosidae.Among the Neoarius genus, only N. midgleyi and N. graeffei were present in the study.The phylogenetic relationship revealed a grouping between these two species.Additionally, some species from the Arius genus were also included in the study, but there was no alignment of Neoarius and Arius in the same clade [19].

Conclusion
The mitochondrial genomes of the studied species showed many similarities in terms of size, composition, and nucleotide percentage.Despite their stable organization, the mitogenomes proved to be valuable in understanding the molecular evolution of these eight species belonging to the Neoarius genus.New studies using other sources of data, such as phylogenies based on nuclear loci or even the assembly of mitochondrial genomes from the remaining species, can be applied to fill possible gaps in the knowledge of the group's evolution and gain a better understanding of the speciation process.

Fig. 1 .
Fig. 1.Comparison of the eight mitochondrial genomes of the genus Neoarius with the mitochondrial genomes of the species used as outgroups in the phylogenetic reconstruction.The mitogenomes of the ingroup are located on the inner part of the circle, while the outgroup is located on the outgroup.

Fig. 2 .
Fig. 2. Phylogeny visualized in Interactive Tree of Life (IToL), with species forming the internal group (target group), with the eight species of Neoarius shown in red, followed by the genera Arius, which is the sister genus of Neoarius, and by the genus Occidentarius, being the most distant genus of the Ariidae family from Neoário.And shown in green are the siluriformes from other phylogenetically more distant families of Ariidae, being composed of the genera, Hypostomus, Kryptopterus, Phractocephalus, Silurus, Steindachneridion, and Rineloricaria.

Table 1 .
With all the access data for DNA sequences and seeds in NCBI that were used in the assembly of the mitochondrial genomes described in the present study https://doi.org/10.5808/gi.23059 2 / 5