Introduction

Mudskippers are amphibious fishes with peculiar characteristics, living in muddy areas of mangroves1,2. The genus Boleophthalmus is known to be one of the most terrestrial among mudskippers, exhibiting locomotory, respiratory, vertebral adaptation and specializations that enable overland excursions lasting up to 14 h3,4. Despite being a crucial group of fish for evolutionary and ecological studies, only seventeen species are available in literature (Table 2), three of which belong to the genus Boleophthalmus5,6,7,8.

Mitogenome studies enable a wide range of investigations, including tandem repeats, phylogenetic analyses, gene rearrangements, gene overlap, analysis short or long intergenic regions9,10, also including loss of genes11,12,13,14,15. The configuration, structure, and organization of the mitochondrial genome in many bony fishes exhibit a similar structural conformity with 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs) and 2 ribosomal RNA (rRNAs) genes, and one control region16,17. However, some organisms present variation in the length of genes (resulting in long gene sequences) and sometimes repetitions may be found in portions of the genes, including in the protein-coding genes18, or in the control region19.

Several studies have used tools to conduct comparative analysis, aiming to understand the correlations in terms of GC contents percentages, presence of complete or short gene repetition, protein-coding and non-coding regions, as well as the consistency between different regions19,20, which provides relevant information on proportions of changes in gene rearrangements within each class or family. These studies have provided valuable insights into the proportions of gene rearrangements within each class or family11,21.

Variations in mitogenome length and genetic organization are often associated with various evolutionary events, such as nucleotide insertions and deletions in the control region22,23, as well as duplications followed by deletion in certain regions, particularly on tRNAs24. This features directly impact functional differences, considering that the distinct gene structure influences gene function25.

The analysis of complete mitogenomes offers significant advantages in studying vertebrates, and researchers can utilize the available data in public databases, such as the NCBI, to address phylogenetic questions11,21.

Mudskippers exhibit variations in individual characteristics, encompassing both genetic variations and environmental adaptations26,27,28. Therefore, through research utilizing these tools, various mechanisms and genetic information can still be uncovered. Studies already carried out with mudskipper mitogenomes generally describe their complete mitogenomes, except for some studies that, in addition to studying phylogenetic relationships, they bring insights on the genetic functioning of the group6,7,8,27,29,30.

In this study, we present for the first time, the complete mitochondrial genome of the B. dussumieri (Valenciennes 1837), a mudskipper species. The primary objective of the study was first to describe the complete mitochondrial genome and present the comparative analysis with available mudskipper mitogenomes. Additionally, we aimed to reconstruct the phylogenetic relationships among the seventeen mitogenomes of individuals within the family, based on the thirteen protein-coding genes. Furthermore, we implemented a repeat sequence analysis in the control region to assess the potential presence of gene repetitions as well as duplication in CR and certain gene/regions.

Results and discussions

Mitogenome organization, composition, and skewness

The complete mitochondrial genome of B. dussumieri is (GenBank accession XX) 16,685 bp of length, which is similar to other mudskippers mitogenomes (16,470–17,243 bp) (Tables 1 and 2). The mitochondrial genome and the structure were also typical of the mudskipper species and with highly conservative sites, comprising 37 mitochondrial genes (13 PCGs, 22 tRNAs, and 2 rRNAs) and one control region (CR) (Figs. 1 and 2). The mitogenome composition and features are presented in Tables 1 and 2, highlighting the characteristics of the mitogenome, including gene content and specific regions.

Table 1 Description of the mitogenome.
Full size table
Table 2 Composition and skewness.
Full size table
Figure 1
figure 1

The mitochondrial genome map of B. dussumieri is represented by different colors, adapted for colorblind individuals following Wong’s protocol (2011). Gene groups are visually distinguished by their respective colors. The credit for the fish image goes to G. Polgar.

Full size image
Figure 2
figure 2

Gene order of the seventeen mudskippers and three goby fishes.

Full size image

The base composition of the complete mitochondrial genome of B. dussumieri was determined to be A = 29.68%; C = 27.90%; T = 26.87% and G = 15.53%, respectively. In the mitogenome composition, we observed the GC and AT content, as well as the GC skew + and GC skew region. The overall GC and A + T content for the entire genome were calculated to be 43.43% and 56.54%, respectively, which were similar to the concatenated composition of the PCGs, tRNA and rRNA. Furthermore, the nucleotide composition of all mitochondrial genes exhibited a bias, with A + T content notably higher (56.54%) than the GC content (43.43%). This pattern of nucleotide composition is consistent with other mudskippers mitogenomes, and no significant differences were observed among them (Table 2). Furthermore, to access weather there were biased or not in the nucleotide composition, the GC and AT skew were measured for the whole mitogenome, as well as for the PCGs, ribosomal genes, and the control region. The GC and AT skew trends of all analyzed genes and control region are presented in (Table 2). Generally, most of the fish mitogenome present a clear bias in the nucleotide composition, which is the case of this study, where the GC skew was notably negative as most of the teleost fish31,32,33.

The GC and AT skew followed the same conventional preference of most mitogenomes, with AT skew positive, except for PCGs, and GC skew negative, except for tRNAs and CR, that varies from negative to positive in some mitogenomes. The AT skews of whole mitogenome ranged from − 0.0 (Periophthalmus novaeguineaensis) to 0.065 (Boleophthalmus.sp. JZ-2015), rRNAs 0.22 (P. minutus) to 0.254 (Boleophthalmus pectinirostris) whereas AT skew of the tRNAs range from 0.009 (Periophthalmus novemradiatus) to 0.039 (Scartelaos gigas), respectively (Fig. 3). These preferences in the sequenced mitogenomes are like other teleost mitogenomes6,7,8.

Figure 3
figure 3

Composition and skewness. The brown bars represent the mitogenome repeat regions and the black line represents the size of the genes.

Full size image

PCGs, amino acid composition and codon usage

The PCG size of each mudskipper mitogenome ranged from 11,399 bp (B. dussumieri) to 11.713 bp (P. novaeguineaensis) (Supplementary Table S1). We compared seventeen mudskippers mitogenomes, and among these almost all PCG encoded in the H-strand, except for ND6 that encoded in the L-strand, as typical of most vertebrate mitogenomes10. Overall, all PCGs showed the same configuration with ATG as start codon with exception of the COI gene that started with GTG codon. The preferences for codon termination of five PCGs (NADH-1, NADH-2, COI, NADH-4L, and NADH-5) were TAA and three (ATP8, NADH-3 and NADH-6) terminated with TAG (Table 2). Some protein coding genes had an incomplete start and stop codon as in most fish mitogenomes34,35. For example, NADH-4, COII, CYT B gene, had an incomplete nucleotide T- whereas ATP6 and COIII had incomplete TA- codon as stop codon (Table 1). These features are in accordance with most mudskipper fishes36. However, we found one unusual start and stop codon for most fishes (CCT/AAC) in ATP8 gene of Periophthalmus novemradiatus species. The CCT codon, codes the Proaline amino acid whereas the AAC codes Asparagine amino acid.

Curiously, P. novaeguineaensis presented very peculiar codon preferences, totally different from others with only ten (ATG) as start codon, common GTG for COI, and three non-common codons (ATA for ATP6 a, TTA for ATP6 b, TTC and ACT for NADH5-0 and NADH5-1, respectively). In contrast, the codon termination was also different from most mudskippers, presenting only four genes with TAA, as stop codon in the (NADH-2, COI, NADH-4L and NADH-6) genes and six different stop codons, AGG for (NADH-1), a common TAG stop codon for (ATP8 and NADH-3), ACC for ATP 6 a, GTT for ATP 6 b, CTT and CTC for NADH5-0 and NADH5-1, respectively.

The relative Synonyms codon usage (RSCU) is shown in (Fig. 4). The use of RSCU was biased for both two and six- fold degenerate codons. In overall, 5556 codons were analysed with exclusion of stop codons for B. dussumieri mitochondrial genome.

Figure 4
figure 4

Relative synonymous codon usage in Boleophthalmus dussumieri mitogenome. Graphs from left to right represents Ala, Cys, Asp, Gln, Phe, Gly, His, Ile, Lys, Leu1, Leu2, Met, Asn, Pro, Gln, Arg, Ser1, Ser2, Thr, Val, Trp, Tvr.

Full size image

The Codon degeneracy patterns indicates that most amino acids (14) Glu, Met, Tyr, Asp, Lys, Ile, Ser1, Leu2, Cys, Trp and Phe used a combination of two fold degenerated codon and eight amino acids (Pro, Thr, Leu1, Arg, Ala, Ser2, Val and Gly used four codon combination to encode with a duplication of Leucine and Serine, both with two fold degenerated codon (Fig. 4). These results were also observed in the literature13,33. The most used amino acid was Leu (13.75%), Pro (9.78%), Ser (8.48%) and Thr (7.51%) and the least used were Glu (2.89%), Trp (2.72%), Asp (2.32%), Arg (2.27%) and Cys (1.94), respectively (Fig. 5). The values appear to be similar, but the amino acid types are different from other studies10,37,38,39. All the detailed information about the amino acid composition and codon usage are in the Figs. 4 and 5.

Figure 5
figure 5

Amino acid composition of the Boleophthalmus dussumieri mitogenome.

Full size image

Ribosomal and transfer RNAs genes

Gene size showed similar values among all mudskippers. The 12S rRNAs gene presented a size of 948 bp in a group that presented values between 947 and 956 bp, while for the 16S rRNAs gene, the size of the genes varied from 1683 bp belonging to (Parapocryptes serperaster) to 1699 bp (B. dussumieri) (Supplementary Table S1). In general, the tRNAs genes were very conserved compared to some fish species35,37,38. We found 14 tRNAs out of 22 encoding the H-strand and eight (trnA, trnC, trnE, trnN, trnP, trnQ, trnS2, and trnY) in L-strand.

The structure of almost all tRNAs were typical clover structure. Although most of them presented a secondary structure similar to other species with four arms and a central loop. Some of those tRNAs (trnM, trnS1, trnE, trnH, trnW, trnT, trnN, trnF, trnV, trnL) presented a variation in certain arms with presence of small loops (Fig. 6). The entire length of tRNA of the B. dussumieri was 1556 bp and the length of 22 individual tRNAs gene ranged from 65 to 76 bp. This value was between the length range of the other mudskippers analysed. (Supplementary Table S1).

Figure 6
figure 6

Secondary structures of tRNA in B. dussumieri mitogenome. The gene names of each tRNA are stated above each secondary structure.

Full size image

Gene rearrangement, repeated region, and concerted evolution

The organization of almost all vertebrate animals generally have their mitogenome structurally conserved with one set gene copy, no intron or long intergenic spacer and single CR40,41. Nevertheless, gene rearrangement in mitogenome of vertebrates are deeply explained using different models including shuffling, translocation, and inversion42, but most common is duplication and deletion model approaches43,44. The CR of this study followed typical rearrangement located between the tRNA-Pro and tRNA-Phe gene, as most vertebrates mitogenomes10,45.

We analysed seventeen mudskippers’ species and nine of them presented duplication in their mitogenome specially in the control region. The duplication was found in three different regions, most of them, in a non-coding gene, the control region (Boleophtalmus boddarti, B. pectinirostris, Boleophthalmus sp JZ-2015, Oxuderces dentatus, S. gigas); three in the PCGs, ATP6, NADH-5 gene (P. novaeguineaensis), ATP8 (P. novemradiatus and P. minutus); and one ribosomal gene, the 12S rRNA gene duplication in the Periophthalmodon schlosseri. Our study shows two species with the OH region duplicated, P. serperaster with four OH region, with the sequence length ranging from 78 to 320 bp, and S. gigas with one duplication involving 31 bp and 316 bp, respectively (Table 4 and Fig. 3). Furthermore, our results also presented one species (P. serperaster) with both CR duplication and one coding-region duplication, the NADH-1 gene.

So how can these duplication mechanisms in mudskipper be explained? Several models are suggested to be involved in fish mitogenome rearrangements10. In this study the conformity of the gene did not show any gene reverse or abnormal gene exchange and because this model is so rare in fish mitogenome, we consider that is not appropriate to explain the rearrangement in the mudskipper mitogenome44. The second most studied model for mitogenome rearrangements is the TDNL and DRRL models. In the majority of cases, these models offer explanations for instances of gene clustering based on their site of encoding. (L- or H-chain coding)46,47.

These models are not suitable to explain the rearrangements or the duplication observed in the present study. Therefore, we consider that the model that best suits the redundant rearrangements observed in mudskippers is the tandem duplication-random model (TDRL), mainly because it is related to the repetitions and duplications that are found in some portions of the mitogenome48,49. For example, further we explain the rearrangement found in the Boleophthalmus group, where repetition and duplication events of the motif region were very frequent, associated with the presence of intergenic spacer and incorrect initiation or termination of certain genes, followed by random deletions of part of these fragments specially in the control region50.

Gene duplication in the Boleophthalmus genus

The genus Boleopthalmus was represented by four species, B. dussumieri (this study), B. boddarti, Boleopthalmus pectinirostris and Boleophthalmus. sp. The first was considered the basal species and thus being the first to be analysed and further identified the motif fragment in their mitogenome, however no duplication was found. On the other hand, the B. boddarti presented 131 bp of the motif sequence, which was duplicated 2.4 times, whereas the other two species (B. pectinirostris and Boleophthalmus sp.) had this region repeated 5 times, meaning that there was a systematic increasing of the region (Fig. 3). This phenomenon is common in most fish mitogenomes34, however there are other different gene organization the fish mitochondrial genome10. All the repeats were found in the 5′–3′ directions. The alignment of the motif region of all Boleophthalmus was clearly similar showing that this duplication evolved in concert51. In addition, studies show that, when the motif region is considered homologues, the phenomena is therefore considered concerted evolution52,53.

Intergenic spacers, overlapping and evidence of TDRL/DMNL models

Intergenic spacers (IGS) are non-coding regions that are found between genes and are typically found in Metazoa and most vertebrate animals and in most cases serve as a transcription promotor mitogenomes50,54 and, they have influence on the growth rate of some invertebrate animals55. They are important signal for evolutionary studies and even species delimitation56,57. Besides IGS event, there is also segments of gene overlapping in mitochondrial genome45.

In the newly sequenced mitogenome of B. dussumieri there were 17 intergenic spacer regions with a total sequence length of 899 bp (Table 3). Four of these regions were considered long IGS with the sequence length ranging from 14 bp, located between COX III and tRNA-Gly, 51 bp between tRNA-Val and r16S; 117 bp between ATP6/COX III on the H-strand and one longest IGS of 495 bp located between tRNA-Pro/OH on the L-strand. Large intergenic spacer has been reported in many vertebrate mitogenomes58,59. Besides those, all of IGS had a length ranging from (1–8 bp). To explain this phenomenon, three main evolutionary mechanisms of IGS origin in mitogenomes have been discussed in previous studies, the tandem duplication/random loss (TDRL) model and slipped-strand mispairing and the dimer-mitogenome and nonrandom loss (DMNL) when there is similarity in mitogenome rearrangements10,60,61.

Table 3 Intergenic spacer and overlap regions.
Full size table

To understand how rearrangements in intergenic spacing occurred in mudskippers, it is important to observe the location of gene spacing. In this study, the gene spacing with high values occurred in the same position (tRNA-Pro/OH) except for Periophthalmus cantonensis which had its spacing in the tRNA Leu1/ND5_0 gene which probably did not follow the same rearrangement mechanism. Another important aspect tobe observed is the variation in the sizes of base pairs involved. This phenomenon is common in vertebrate mitogenomes32. Looking at these variations in the mitogenome rearrangements, it raises questions about how these phenomena occur and why there are similarities in its positions? The DMNL model supports dimerism in the vertebrate mitogenome, a fact that may condition this conformity. In fact, dimeric conformity in the fish mitogenome have already been observed60. Anguilliformes fish is a group is fish where this scenario was deeply elucidated.

In addition to IGSs, seven overlapping sequences were also observed in B. dussumieri mitogenome, ranging from (1–7 bp) (Table 2). The total length of the sequence was (24 bp) that was divided in to two main region ATP8/ATP6 (7 bp) and NADH-4L/NADH-4 (7 bp), both on the H-strand. These two overlapping regions, both being 7 bp of length, are very common in fish mitogenomes, including mudskippers (Table 3)31,62.

The comparative analysis of the IGS and overlapping regions presented several variations in numbers, length, and locations within mudskippers. The number of IGS regions ranged from 13 to 17 with the total length varying from 418 (P. schloseri) to 1207 bp (Boleophthalmus sp.) which have the largest IGS (706 bp) located in the OH region (Table 3). Most of large IGS were located between tRNA-Pro and OH region, but only one (385 bp), belonging to P. novaeguineaensis, was located between tRNA-Leu1 and NADH5_0.

In overall, the overlapping length varied from 1 to 7 bp in most mitogenomes. However, two species of the genus Periophthalmus presented unusual overlapping, involving 20 bp (P. minutus and P. novaeguineaensiss). This may be due to the split/duplication of the ATP8 gene in these species. The study also presents some peculiarities involving the IGS of the OH regions. We found large IGS of the OH region ranging from 61 bp (Periophthalmus modestus), to 185 bp (B. dussumieri), and very large varying from 314 bp (B. boddarti) to 701 bp (B. pectinirostris) (Table 3).

Phylogenetic analysis

The phylogenetic relationship within mudskippers were determined using a combination of 13 protein-coding genes (PCGs) and 2 ribosomal genes from a total of 17 mudskipper species. Additionally, three species of gobies (Tridentiger bifasciatus, Tridentiger barbatus and Stiphodon alcedo) were included as outgroups. The evolutionary tree was constructed using the ML method. The resulting phylogenetic tree for the mudskippers positioned them as monophyletic group with a high support value (ML bootstrap = 100) (Fig. 7A). Several other studies have provided evidence for the monophyly of mudskippers using complete mitogenome analysis and analysis of the COI63. These studies have identified two major groups within the mudskippers. The first consists of all Periophthalmus species, which form a sister group of Periotphthalmodon, with high support values (> 90). The second group is comprised of two subclades. One subclade includes Boleophthalmus, which is sister group of Scatelaos, with a support value of >  = 90. The other subclade consists of Apocryptodon, Paraprocryptes, and Oxuderces, with bootstrap value > 50. Scartelaos is grouped together with the Periophthalmus clade. The Periophthalmus clade is considered monophyletic and there is high similarity (> 99%) of Scartelaos histophorus with Periophthalmus magnuspinnatus (GenBank accession numbers KT284931.1, KT357639.1), so the presence of S. histophorus (accession number JQ654459.1) in the Periophthalmus clade should be considered misidentification64. Therefore, the specie revalidation in the Genbank should be considered to avoid further taxonomic problems.

Figure 7
figure 7

(A) Phylogenetic relationships mudskippers species based on the maximum-likelihood methods from concatenated nucleotide sequences of the 13 PCGs regions. (B) Mean divergence times were estimated using a relaxed molecular clock model on a subset of mitochondrial genes. The colors indicate the genus group.

Full size image

The topology position of P. barbarus presented a different clustering in both trees. In the ML tree the taxon clustered together with P. minutus, P. modestus, P. novaeguineaensis and P. argentilineatus as a sister group, whereas in the IB the species was positioned out of the sister genus. The results presented in this study brings a clarification in relation to the separations of the groups within mudskippers.

Estimation of divergence times

In our phylogenetic analyses, the target species of the study, B. dussumieri, was correlated with other species of its genus and the genus Periophthalmus. Both the BEAST and ML analyses showed similar topologies in our simulations (Fig. 7A, B). The TMRCA estimated by BEAST demonstrated that the diversification of mudskippers from the Gobiidae family occurred approximately 28.72 million years ago (Ma) (Fig. 7B). Within the mudskippers, the genera Periophthalmus and Periophthalmodon were the first to diverge and diversify, dating back to around 19.52 Ma in the Early Miocene (Fig. 7B). This suggests that these genera represent the basal lineages within terrestrial gobies. The divergence between the clades of Boleophthalmus and Periophthalmus occurred around 24.42 Ma in the Early Miocene (Fig. 7B). Boleophthalmus dussumieri and its sister species within the genus are estimated to have diverged around 8.22 Ma during the Late Miocene (Fig. 7B). Comparing the divergence time of B. dussumieri with another sister genus, the estimated times were roughly 14.88 Ma for Exuderces, Apocryptodon, and Paraprocryptes, and 12.82 Ma for Scartelaos. These values are consistent with those recovered from other studies on closely related families, which are sister families of Mudskippers (Oxudercidae), including the subfamilies Amblyopinae Günther, 1861, Gobionellinae Bleeker, 1874, and Sicydiinae T.N. Gill, 186065,66. The minimum and maximum ages, with 95% Highest Posterior Density (HPD) for individual and group node ages, are shown in Table 4.

Table 4 Confidence intervals of the diverge time scales.
Full size table

Conclusion

In this study we present the complete mitochondrial genome of the mudskipper species B. dusumieri with 16,685 bp of length and the first comparative mitogenome gathering all available mudskipper mitogenomes. Generally, the organization followed almost the same configuration as most fish mitogenomes. However, we found different peculiarities regarding gene rearrangement, long intergenic spacer, and duplication of gene fragments mostly on the CR but also in a coding gene and one ribosomal gene in the analysed mitogenomes. The phylogenetic reconstruction using 13 concatenated PCG positioned all mudskipper species as monophyletic group within the family Oxudercidae. The phylogenetic clustering recovered in this study are in accordance with other previous study. The time scale estimation demonstrated that within mudskippers, the Periophthalmus genus was the first to diverge in early Miocene and thus considered basal group. The complete mitogenome generated in this study is a source of genetic information for further molecular studies including conservation strategies.

Material and methods

Sample and DNA extraction

The mudskipper specimens used in this study were collected from the Bons Sinais estuary in Quelimane, Mozambique (Fig. 8). These samples belong to the B. dussumieri, which was recently identified for the first time on the cost of Mozambique67,68, an area that its occurrence is not quoted by IUCN. Upon collection, the fresh specimens were immediately placed in 96% ethanol and stored at – 20 °C. Voucher specimens were preserved in 10% formaldehyde and deposited at the Maputo Natural History Museum with the code MHNM.PIS.2021.0398.

Figure 8
figure 8

Sampling sites for B. dussumieri mudskipper specimens in the Bons Sinais estuary, Quelimane, Mozambique. The Shapes used were obtained from the Natural Earth website (https://www.naturalearthdata.com/, dataset available at 1:10 m, 1:50 m, and 1:110 million scales), and the maps were created using the QGIS software (https://qgis.org/en/site/, V 3.32).

Full size image

Muscle tissue was extracted from specimens and taken to the Evolution Laboratory at the Federal University of Pará, Brazil, for molecular analysis. One specimen was chosen for further analysis. Genomic DNA was extracted from the muscle tissue using a Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI—USA), following the manufacturer’s protocol. The purity and concentration of the DNA were assess using a spectrophotometer, and successful extraction was confirmed through 1% agarose gel electrophoresis. The DNA samples were then stored at – 20 °C.

Ethics declarations

All biological material used in this research came from an artisanal fishery and were exclusively for general fishery purpose. No live specimen was kept in captivity or manipulated. Therefore, no ethical approval was necessary.

PCR amplification and sequencing

Firstly, nineteen primers (Supplementary Table S2) were designed using Primer3 (https://academic.oup.com/nar/article/40/15/e115/1223759?login=true) implemented in GENEIOUS software based on the conserved regions of all available complete mitochondrial genome from the family, taking into consideration the closest species within the genus. To optimize the amplification of uncovered fragments, specific primers were redesigned.

The PCRs were run in a final volume of 15 μl containing 2.5 μl of dNTPs (1.25 mM), 1.5 μl of 10 × buffer solution, 0.7 μl of MgCl2 (50 mM), 0.5 μl of each primer (10 pmol/μl), 1.0 μl of total genomic DNA (100 ng/μl), 0.2 μl of Taq DNA polymerase (5 U/μl) and pure water to complete the final volume of the reaction following previous studies67,68. The amplification protocol consisted of initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 52–65 °C for 40 s, then extension at 72 °C for 45 s, followed by a final extension at 72 °C for 5 min. All positive reactions were sequenced using the Sanger method with the same primers used to amplify the fragment, with a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), according to the manufacturer's instructions. Electrophoresis was performed on ABI 3500 XL (Thermo Fisher). Negative controls were included in all PCR reactions to confirm the absence of contaminants.

Sequence assembly, annotation, and analysis

After sequencing the fragments, the Bowtie tool, implemented in Geneious 9, was used to map the generated sequences against the reference and generate the consensus sequence. For mapping the consensus sequence, the species B. boddarti was used as a reference. The complete mitogenome was annotated using the Mitos 2 (http://mitos.bioinf.uni-leipzig.de)69 and later confirmed in MitosZ 3.6 (https://github.com/linzhi2013/MitoZ) with reference to Metazoa 63. The analysis of the repetition identification regions was performed using the finder tinder repeat tool (https://academic.oup.com/nar/article/27/2/573/1061099?login=true). The base composition, amino acid calculation as well as relative synonymous codon usage (RSCU) of PCGs were estimated using MEGA 1170.

The base composition values (AT- and GC-skews) were calculated using the following formulas: AT-skew = (A−T)/(A + T) and GC-skew = (G−C)/(G + C). The genetic order was calculated using the Phylosuite71.

Phylogenetic analysis

To investigate the phylogenetic relationship among all mudskippers, a phylogenetic tree was constructed using a final dataset of 20 goby species. This dataset was based on the combinations of 13 PCG and two RNAs genes.

To accomplish that, we performed Maximum Likelihood (ML) analysis in the IQTree v 2.2.0 program. The best partitioning scheme for the database were adopted considering the position of the codons of each gene, as well as the evolutionary models for the respective partitioning scheme. Subsequently the ML analysis was run with 1000 bootstrap pseudo replicates.

Divergence time analysis

Divergence time analyses were conducted using BEAST v.1.1072. The uncorrelated relaxed clock73 was employed, and the Yule process served as the prior model for the tree74. Calibration points were incorporated to estimate divergence time, including the separation between B. boddarti and B. pectinirostris being calibrated to 3.8 Ma, based on Chen et al.63, which used references from the study conducted by Mukai et al.75, considering the mutation rate (1.95 ± 0.17) % per million years per lineage per site of the Rhinogobius ND5 gene, while the separation in the Periophthalmus genus was calibrated to 20.09 Ma based on the calibration proposed by76 that used approximate (2 μ) paired molecular clock of 3.8%/myr for the ND5 gene from related gobies (Rhinogobius).

The analysis was run for 100 million generations, with log parameters recorded every 100 registered at each 5000 generations. Trees were summarized in TreeAnnotator v.1.8.472. A burn-in of 20% was used and the run was considered satisfactory when all ESS values checked in Tracer v.1.677 were equal to or larger than 200.