Enhanced dynamicity: evolutionary insights into amphibian mitogenomes architecture

Enhanced dynamicity: evolutionary insights into amphibian mitogenomes architecture | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhanced dynamicity: evolutionary insights into amphibian mitogenomes architecture Yi Xiao, Gengyun Niu, Haihe Shi, Zhenyu Wang, Renzeng Du, Yankuo Li, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4830272/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Mar, 2025 Read the published version in BMC Genomics → Version 1 posted 4 You are reading this latest preprint version Abstract Mitogenomes are known for their structural dynamics and the complexity of their rearrangement patterns. However, their utility in metazoan comparative biology has not been fully exploited. Vertebrate mitogenomes are now sufficiently representative to allow the development of more advanced methods for comparing genome architecture. Furthermore, the relatively robust phylogeny of vertebrates at higher taxonomic ranks allows us to infer the patterns of genome evolution accordingly. In this study, using amphibians as an example, we performed data cleaning and manual annotation on 1777 samples from the NCBI and identified 88 rearrangement types, most of which were clade specific. In addition, we quantified genomic changes in an evolutionary framework and obtained stepwise growth curves of the architectural changes. This study provides new perspectives for understanding the evolution of the mitogenomes in amphibians and is expected to facilitate the qualitative and quantitative development of mitogenomes research. Mitogenome Comparative genomics Macroevolution Gene rearrangement Complexity Evolutionary transitions Figures Figure 1 Figure 2 Figure 3 1. Introduction The mitogenomes architecture, as the structural organization of genetic material, exhibits dynamic properties [ 1 ]. It displays remarkable plasticity in individuals [ 2 – 4 ], while taking radically various pathways in diverse lineages. The architectural diversity of the mitogenomes provides a noteworthy dimension for observing the complexity of mitochondria as an open system. This diversity encompasses a broad range of genomic rearrangements, including insertions, deletions, inversions, and transpositions, both within genes and in intergenic regions. These structural changes reshape the genome and influence its functionality, thereby affecting species adaptability and evolutionary trajectories. For instance, significant innovations in metazoans, such as the emergence of multicellularity and bilateral symmetry, are associated with specific changes in mtDNA organization [ 5 – 7 ]. Extensively reorganized mitogenomes in amphibians have attracted considerable interest since 1997 [ 8 ]. These intriguing features, such as exceptional enlargement (up to 28.8 kbp) [ 9 ], loss and gain of genes [ 10 – 12 ], and the stepwise nature of rearrangement [ 13 ], all suggest the hidden diversity of amphibian mitogenomes. Beyond the rearranged genes, the long noncoding region or control region (CR) is also the “hot spot”, not only because of its duplication or triplication [ 9 , 10 , 14 ] but also because the replication fork barrier lying in the CRs can often mediate recombination between duplicated CRs [ 13 ]. Across amphibian lineages, independent rearrangements have been observed, leading to the consensus that such reorganization patterns can serve as phylogenetic signals of amphibian evolution. For instance, the LTPF tRNA cluster is considered as a synapomorphic characteristic of Neobatrachia [ 15 ]. However, further shuffling within the cluster [ 11 ], or the involvement of nad5 gene adds complexity, especially when similar events are found in taxonomically distant groups, such as Afrobatrachia [ 9 ]. Moreover, the replication of trnM and the presence of IQMM gene clusters in multiple, distantly related taxa further complicate the picture, as in some cases, these clusters exist as two separate copies rather than tandem repeats [ 16 ]. Additionally, intraspecific rearrangement diversity cannot be ruled out, as evidenced in Dicroglossidae [ 17 , 18 ]. These known and unknown complexities urge us to expeditiously unravel the evolutionary landscape of mitogenomes variations in amphibians. In light of the decreasing costs of high-throughput sequencing technologies [ 19 ], advancements in long-read sequencing [ 9 , 20 – 22 ], along with optimized assembly and annotation techniques [ 23 – 25 ], we are now able to explore the mitogenomes architecture in greater depth, thereby examining its evolutionary patterns and trends [ 26 ]. In this study, we specifically aimed to investigate the genomic architectural diversity of amphibians. We used NCBI2GO (unpublished) to clean the available data from 1777 samples representing at least 710 species to generate a high-quality dataset. Employing qMGR [ 27 ] and qGO (unpublished), we conducted qualitative and quantitative comparative studies within a phylogenetic framework to elucidate the patterns of genome structural changes during amphibian evolution. Our findings revealed a phased growth trend in structural changes, providing valuable insights into the evolution of mitogenomes. These insights also have the potential for uncovering the origin and maintenance of animal biodiversity and broadening the scope of comparative genomics research. 2. Materials and methods 2.1. Cleaning and error checking Accurate annotation is often a challenge [ 29 ]. Despite considerable efforts[ 30 – 32 ], the errors in RefSeq annotations are still too severe for gene organization comparisons [ 33 , 34 ]. The problem is even worse at NCBI, where a large number of sequences are unannotated [ 35 ]. Cleaned datasets are also not always accurate [ 7 , 36 , 37 ]. Therefore, in this study, 2143 mitogenomes retrieved from NCBI Organelle Genome Resources using the search string "Amphibia[ORGN] AND (mitochondrion[TITL] OR mitochondrial[TITL]) AND 10000:50000[SLEN] NOT (RNA [TITL] OR gene[TITL] OR product[TITL] OR mRNA[TITL] OR rRNA[TITL] OR misc_RNA[TITL] OR nuclear[TITL])" on October 11, 2023, were cleaned using NCBI2GO (unpublished), a cleaning pipeline developed by the authors. In this pipeline, the gene order was extracted, and the intergenic length was calculated based on the location information to identify the control region (CR) by the threshold. A total of 2143 mitogenomes, including 366 from RefSeq, have been deposited in the GenBank mitochondrial database. 369 out of 1777 samples with missing annotations and those with unconventional gene order were entered into the re-annotation module. Manual verification was then performed to further ensure the reliability of the dataset. For the standardization, the gene nomenclature in [ 38 ] is used. The single-letter amino acid codes designate the corresponding transfer RNA genes (trns). L1, L2, S1, and S2 indicate trns for Leu (UUR), Leu (CUN), Ser (UCN), and Ser (AGY), respectively. 2.2. Taxonomic reconciliation Coherence between species names and their parent taxa is central to comparative analysis at higher taxonomic levels. The NCBI taxonomy was checked against an authoritative list of accepted species. We chose to initially base this comparison on AmphibiaWeb (AW), which contains 77 families with 569 genera and 8689 species downloaded on January 13, 2024 [ 39 ]. The 1777 samples were first de-duplicated based on NCBI taxonomy, yielding a list of 97 unidentified species samples (17 with “cf.”, 79 with “sp.”, and 1 unisexal lineage), 1 hybrid, and 40 with “aff.”, 695 species, and 13 subspecies. Matching of the last three of the above five cases returned the scientific names of 716 species, and the remaining 32 were manually matched to the GBIF Backbone Taxonomy [ 40 ]. After the final deduplication, our taxonomic matchup placed 1777 samples into 710 species of 244 genera and 65 families as showed in an additional Table 12. 2.3. Quantification of mtDNA rearrangements The mitogenomic structural changes can be analyzed quantitatively by calculating the pairwise distance between the rearrangements and the ancestral organization. We applied both the qMGR [ 28 ] and qGO algorithms separately to the above dataset. While both aim to quantify the rearrangement frequency (RF) of each individual gene and the rearrangement score (RS) of each mitogenome, qMGR is alignment free, which calculates the RF of a given gene by accumulating the changes in the two nearest neighboring genes. However, qGO, developed by the authors (unpublished), relies on homology alignment to calculate the RF of the target gene directly. It also supports weighting, so breakpoints need to be predefined to generate intervals that give weight to genes that translocate across the interval. Following the widely accepted processes of replication [ 41 ] and transcription [ 42 , 43 ], we set OriL and OriH as the two breakpoints that force the circular genome into two intervals. The genes within each interval are then manually aligned separately as the input for qGO. 2.4. Evolution of mitogenomic structures across amphibians The time-calibrated molecular phylogeny of Jetz and Pyron [ 44 ] was used for comparative analyses. Changes were made to update families (Hynobiidae, Ambystomatidae, Proteidae, Rhyacotritonidae, Amphiumidae, Plethodontidae, Ascaphidae, Alytidae, Rhinophrynidae, Scaphiopodidae, Megophryidae, Limnodynastidae) and prunes (Odontobatrachidae, Conrauidae, Nyctibatrachidae, Ceuthomantidae, Cycloramphidae, Batrachylidae, Allophrynidae) due to the updata and the lack of complete mitogenomic data. All types are first projected to the phylogeny, and the ancestral type for the diversity is inferred to be the relative outgroup. 3. Results 3.1. Overview of mitogenomic organization in amphibians Numerous errors in the annotation were identified and reviewed. Error types include but are not limited to abnormal reading direction (strand), erroneous gene designations, missing gene or other feature annotations, mistaken identity of trnL1/trnL2 and trnS1/trnS2 tRNAs, and inconsistencies in gene names. The most misleading situation is when the errors were included in RefSeq [ 45 ]. After data cleaning, error checking and taxonomic reconciliation, we obtained a dataset containing 1777 samples representing 710 species in 244 genera under 65 families, yielding 88 gene rearrangement types (Table S1 ). Species coverage within each genus was variable, with a mean of 35% and a median of 25%. Among them, Caudata was the richest (mean 58%, median 50%), and Neobatrachia was the least abundant (mean 23%, median 13%) (Fig. 1 and Table S3 ). The unexpected complexity and variability among amphibian mitogenomes were revealed (e.g., Fig. 2 and Table S2 ). The major lineages of both Gymnophiona and Archaeobatrachia, as well as a few Caudata, were identical to the typical vertebrate gene order (orange circle in Fig. 1a), which was named the Amphibian ground pattern herein and labeled type 1 in additional tables. In addition, both Caudata and Neobatrachia have their own autapomorphies. The Caudata ground pattern (type 2, red circle) features one more CR derived between trnT and trnP. The Neobatrachian ground pattern (type 21, blue circle) has a strongly rearranged gene order, involving the long-term translocation of trnL1 to form the LTPF tRNA gene cluster located upstream of the 12S rRNA gene [ 46 ]. In contrast to all 10 Caudata families, which have consistently retained the ground pattern among their members, only 24 of 35 Neobatrachia families show the Neobatrachia ground pattern. Among these, 9 families have evolved new types. The remaining 11 families exhibited completely derived types, leading to a total of 69 rearranged types. This makes Neobatrachia the most variable group, followed by Archaeobatrachia with 10 types, Caudata with 8, and Gymnophiona with 5, as shown in Fig. 2 a. The most common rearrangement events observed in this study involved nad5, trnTP, trnL1 and CR [ 46 ]. Inversions are rare relative to translocations. Notably, consistent with findings from long-read sequencing, multiple rearrangement types involved duplication of genes or gene clusters, as well as repetition of CR or a combination of both [ 47 ]. Among them, trnM duplications were the most common, followed by trnP duplications, as detailed in Fig. 2 b and Table S2 . 3.2. Mitogenomic structural changes across four lineages Mosaic evolution and lineage specificity coexisted throughout mitogenome evolution in amphibians. In particular, the ground patterns of vertebrate (n = 162) and Caudata (n = 415) are shared among distantly related species of Gymnophiona, Caudata, and Archaeobatrachia. However, both patterns are notably absent in Neobatrachia. Moreover, the 67 derived types that evolved from the Neobatrachian ground pattern (n = 756) are unique to this lineage and are not observed in the other three lineages. These observations challenge our understanding of the organization of the ancestral amphibian mitogenome. Both Rhinatrematidae and Ichthyophiidae (Uraeotyphlus only), the two most basal families of Gymnophiona, possess the Caudata ground pattern [ 48 ]. Given the rearrangements found in Caudata and Archaeobatrachia, it is difficult to rule out the possibility that the ancestral state of the amphibians was a Caudata ground pattern. However, the majority of Gymnophiona, with the exception of the aforementioned families, exhibit this vertebrate pattern. Therefore, applying the principle of parsimony, the most plausible hypothesis is that the ground pattern of amphibian mitogenomes is a vertebrate pattern. Almost all mitogenomic structural changes in Gymnophiona are genus specific, except for Boulengerula, which has interspecific variation, with one of the two species having a duplicated trnP (Type 3). There are also two types (types 4 and 5) concentrated in the trnWANCY clusters that are restricted to two genera in Siphonopidae. The Caudata ground pattern is shared by all 10 Caudata families, yet two of them have derived taxa. Among the 168 Caudata species examined, there is intrageneric differentiation in the genus Tylototriton, and two of these species are type 11, involving the expansion of the CR and its flanking genes [ 14 ]. The seven Plethodontidae species from Tylototriton, Aneides, Hydromantes and Plethodon are all intragenerially diverse to types 6–9, and the single-sample Stereochilus is type 10. Archaeobatrachia comprises four successively evolved lineages, all of which can be traced back to the ancestral amphibian type. Two out of four genera of Pipidae, Pipa and Xenopus, possess both Type 1 and Type 2. Derived types 20 (swaps of nad6-trnE and cob-trnT) were found in both examined species of Leiopelmatida. The more diverse Megophryidae show an additional seven derived types occurring in Leptobrachium (Type 14), Leptobrachella (Type 13) [ 49 , 50 ], Scutiger (Type 17) [ 51 ] and Oreolalax [ 49 , 52 , 53 ], which intragenetically diversified into five derived types. These changes primarily involve the duplication of trnM [ 46 ] and/or the remote transposition of trnW. There is very high variation in the genomic structure of Neobatrachia. Whether derived in terms of majority rules or the status of the outgroups, the ancestral type should contain a unique rearrangement in which trnLTP forms a cluster of genes located between the CR and trnF. In addition to type 21, which is shared by 24 families and is therefore considered to be the ground pattern, there is another type shared by four distant families (type 48), which differs from the ground pattern only by an additional CR between nad5 and nad6. Another pair of sister groups shares type 27, which has an additional interchange of trnA and trnN on top of the ground pattern. The remaining 66 types other than these three are unique to a given family, meaning that they are no longer shared between families [ 46 ]. The clade comprising Dicroglossidae [ 17 , 46 , 54 – 56 ], Ranidae [ 55 , 57 – 63 ] and Rhacophoridae [ 64 – 66 ] exhibits the strongest changes, encompassing nearly half of the derived types (32 in total) with Breviceps, Hyperolius, Ptychadena, Cornufer, Limnonectes, Amolops, Nidirana, Odorrana, Rana, Polypedates, Nanorana, Quasipaa, and Nanorana presenting intrageneric changes and the latter two even presenting intraspecific changes [ 17 , 18 ]. Nested within this clade, Mantellidae and Ranixalidae each harbor family-specific types. Other intrageneric changes occur in Cornufer, Ptychadena [ 67 ], Hyperolius [ 9 ], Breviceps [ 9 , 68 ], Ischnocnema [ 69 ], Brachycephalus, Boana, and Bokermannohyla and reach an extreme of nine types in Pristimantis. The changes are varied and involve almost all regions of the mitochondria. The most notable variable regions involved the tRNA gene cluster WANCY [ 70 ]; in particular, multiple OLs were identified in the clusters [ 71 ]. 3.3. Quantitative analysis of mitogenomes changes across amphibians The hotspot is depicted in Fig. 1b on the heatmap plotted against the relative frequency (RF), detailed in additional table S5 . Each amphibian group is represented by a different color, with purple indicating the entire amphibian group. The gradient of color shading reflects the relative RF score. Among all the genes, trnL1 exhibited the highest score due to its long-range translocation across various amphibian taxa. However, when examined at a finer taxonomic level, trnL1 rarely undergoes changes within that specific taxon. It should be noted that hotspots differ between taxa. For instance, Gymnophiona primarily experiences changes in the trnWANCY gene cluster, while Archaeobatrachia scores highest for the flanking genes of nad2. Neobatrachia displayed significantly more variable regions than did the other taxa, with almost all the genes exhibiting some degree of rearrangement except for cox2, atp6, and nad4l, which had RF scores of 0. Notable hotspots common to all three taxa may be confined to the region between nad6 and trnF, including CR [ 16 ]. Further analysis of the RS distribution (Fig. 1c and Table S2 ) highlighted the significant variation in mitogenomes architecture among amphibians. Gymnophiona, the most primitive group, exhibited the lowest median RS intensity, suggesting a relatively low level of genomic change. Caudata has a greater median RS than Archaeobatrachian, possibly due to Pyxicephalus adspersus, which undergoes strong rearrangement with gene duplication, resulting in 49 genes [ 12 ]. This extreme value elevates the overall distribution profile with score peaks at 50 (qGO) and 46 (qMGR). However, disregarding this extreme value, both taxa exhibited relatively uniform distributions of RS that were generally greater than those observed in Gymnophiona. It is worth noting that although both Caudata and Archaeobatrachia had a minimum RS value of 0, considering that type 1 in Cadauta evolved from ancestral type 2, this score varied when type 2 was changed to the reference. The multimodal distribution observed in both Archaeobatrachia and Neobatrachia indicates heterogeneity, potentially suggesting divergent evolutionary directions. A more intriguing alternative inference concerns the emergence of Neobatrachia, which disrupts continuity and gives rise to fluctuations in Archaeobatrachia. Neobatrachia, as the most recently evolved lineage, stands out for its high RS values and several extreme values. There are ten values above 20, a magnitude that far exceeds that of any other taxon, suggesting that it may have evolved in an aggressive way, avoiding the fatal decrease in evolutionary potential. This is consistent with the evolutionary pattern observed in other vertebrates, where more recently evolved taxa tend to have greater complexity. The extremely high RS may also indicate that the taxa have undergone rapid adaptive radiation or evolutionary innovation, resulting in significant increases in complexity. 3.4. Evolutionary dynamics underlying patterns of diversification By aligning the RS of each taxon with the time of origin of its corresponding branch and aggregating the RS values over time, a growth curve can be generated (Fig. 3 and Table S5 ). A comparison between the growth curves for all amphibians and those for each individual taxon revealed a consistent pattern of stepwise growth over time. This suggests that there were distinct periods of rapid expansion followed by relatively stable phases in amphibian evolution. Interestingly, while the absolute values of RS exhibited considerable variation across taxa, they demonstrated remarkably consistent patterns of increase, except for Gymnophonian, which consistently experienced a step change during the Cretaceous period. This indicates that Gymnophiona underwent unique evolutionary events or adaptations during this specific period. Furthermore, when examining the remaining three branches, it becomes evident that they all display a rapid increase with nearly comparable slopes soon after the beginning of the paleogene. This sudden surge in their RS values is preceded by an extended period of stagnation and a minor, limited magnitude increment occurring around or prior to the mid-Cretaceous epoch. The long periods of silencing in the early clade may indicate an underestimation of potential diversity for various reasons, such as extinctions associated with specific rearrangements. 4. Discussion 4.1. Unraveling the complexity: mitogenomes variability in amphibians The dogma that there is a frozen mitogenomes in vertebrates has long been broken, but vertebrates are still considered subphyla with probably the lowest variability in mt DNA gene content and gene order, with disparities thought to have a limited taxonomic distribution. This frosted glass exists mainly because of uneven sampling [ 72 ], a high percentage of erroneous annotations [ 33 ] and problematic mitogenomes [ 34 ], as well as the omission of duplicated regions [ 73 ]. During the last few years, the advent of high-throughput sequencing techniques has increased the number of sequenced mitogenomes. Vertebrates, in particular, account for more than half of all Metazoa [ 7 ]. Specifically, for amphibians, an adequate representation of 88% of the families is sufficient to infer consensus mitogenome characterizations of different taxa and to fully recognize exceptions that lie beyond this consensus. The present study revealed that 43% of the amphibian genera examined had at least one available mitogenome. However, the data still suffer from taxon bias. In the case of Gymnophiona, mitogenomic structural changes often occur at the genus level. The lack of novel rearrangements, despite a genus-level coverage of 82%, suggests data saturation. Conversely, although the number of Archaeobatrachia mitogenomes exceeds that of Gymnophiona mitogenomes, changes could occur within genera even within species, suggesting that as species diversity increases, the emergence of new types of mitogenomes is possible. In other words, the existing data for Archaeobatrachia may be insufficient [ 49 ]. The mitogenomes, traditionally considered highly conserved, exhibits unexpected dynamism, with 88 observed types in our study. This diversity, however, is likely an underestimate, suggesting that the complexity of the mitogenomes extends beyond current comprehension, especially when considering regions of high variability [ 13 , 47 ] that were excluded from this dataset due to stringent search criteria. In a phylogenetic framework, structural diversity is sensitive to the grain of phylogenetic resolution. Specifically, the differences between major types (type 1 and type 2) in amphibians, birds and reptiles [ 23 , 74 ] hinge on the presence of an additional CR between trnTPs, and Neobatrachia shows phylogenetic constraints from ancestral type 21. In contrast to the path-dependent evolutionary trajectories described above, fine-grained taxonomic diversifications appear to be stochastic and unpredictable, underpinning the individualized nature of mitochondrial evolution within specific families, [ 75 ] as depicted in Fig. 1a. These changes involve all kinds of components, including tRNAs, protein-coding genes, rRNAs, and the CR, occurring regardless of the gene position relative to the CR and affecting both strands. This observation challenges the prevailing notion of convergent evolution in vertebrate mitochondrial gene rearrangements, suggesting instead that the forces shaping mitochondrial genomic organization are diverse and influenced by unique evolutionary histories [ 36 , 76 ]. Based on such observations, it is reasonable to hypothesize that a variety of mechanisms can drive mitogenome structural changes, extending beyond the duplication-random loss (DRL) model traditionally invoked to explain genomic alterations [ 77 – 79 ]. The DRL model, while useful in explaining certain types of genomic changes at the individual level, may not fully account for the broad variability observed across different lineages [ 46 , 80 ] or proclivity, such as nested copies of duplicated segments [ 68 ]. This raises critical questions about the underlying mechanisms that confer flexibility and robustness to mitogenomes evolution, thereby enhancing its evolvability. 4.2. An asynchronous symphony of the episodic architecture variation Complexity often emerges from an intricate dance of genetic and environmental factors, resulting in the quantification of features that exhibit fluctuations across multiple dimensions. Through the use of RS and RF as proxies for measuring complexity, genetic innovations can be traced step by step, and amphibian evolution dynamics can be linked to genomic structure evolution. As shown in Fig. 1c, primitive taxa tended to exhibit simpler structures and lower levels of RS, while derived taxa, on the other hand, showed a significant increase in RS across several levels. This increase in complexity suggests that the genomic structure is able to evolve, enabling the exploration of new strategies and facilitating diversification [ 81 ]. In other words, evolvability evolved [ 82 ]. The case of Archaeobatrachia sheds further light on how this trend impacts specific groups within these taxa. The fluctuating RS suggests that Archaeobatrachia may have undergone a phase of complexity reduction or macroevolutionary freezing [ 83 ], which is precisely a consequence of the evolution of evolvability [ 84 ]. Rearrangement frequency (RF) helps us understand complexity by showing that genes or gene clusters within a genome may undergo independent evolution [ 85 , 86 ]. The trnW gene was used as an example. Although its overall RF appears normal across various amphibian groups, a zoomed-in examination revealed heterogeneity. Within Archaeobatrachia, all types involved trnW shifts, resulting in a remarkably high RF. In contrast, RF rapidly decreased in Gymnophiona but reached zero in Caudata. The heterogeneous behavior of gene rearrangement challenges the notion that the mitogenome evolves as a cohesive, inseparable evolutionary unit. It becomes evident that the way a genome evolves depends on the distinct patterns of independent evolution present among the different genes or gene blocks that a particular genome contains [ 87 ]. By examining changes in RS over time, it is possible to trace the increasing complexity of genomic structures and to infer the reasons for this complexity. As shown in Fig. 2 , across multiple time intervals, we observe a pattern where periods of stasis are punctuated by brief bursts of rapid evolution. The concept that the rate and pattern of evolutionary change are closely linked to selective pressures at the lineage level is a longstanding and vital part of macroevolutionary theory [ 88 ], and this concept also applies to mitogenomic evolution [ 89 ]. However, the episodic increase in the number of salientians (anurans and caudates) showed a similar pattern at significant timescales. The first increase, which was very small and occurred at the beginning of the Jurassic, was followed by an increase in limited growth near the Jurassic and Cretaceous boundaries, and a third significant increase occurred just after the K-Pg line, overlapping with the rapid diversification of species-rich clades [ 90 , 91 ]. Between these periods of growth are long periods of stagnation, which may reflect temporarily rising ‘challenging times’ for diversification of mitogenomes, possibly due to evolutionary constraints. The generalizability of this pattern across different amphibian clades highlights the dynamic nature of mitogenomes and the intermittent nature of the evolutionary events that shape their structure. Episodic mitogenomic evolution is also evident in other metazoans, such as fish and invertebrates [ 92 ] In these organisms, drastic changes in mitogenomic structure have been linked to episodes of adaptive radiation [ 93 ] or extreme environmental adaptation. The complexity and evolvability of amphibian mitogenomes structure could be shaped over evolutionary time by various external forces [ 94 ]. These forces include not only the deterministic effects of evolutionary events but also the stochastic influence of random changes. This interplay of factors has endowed mitogenomes with a capacity for adaptation and innovation that exceeds the predictions of traditional evolutionary models. The evolvability of mitogenomes through gene rearrangements highlights the dynamic role of genome structure in shaping the evolutionary trajectories of organisms. In addition to serving as repositories of genetic information, mitogenomes actively participate in evolutionary processes by responding to selective pressures and environmental challenges through structural changes. 5. Conclusions We collected and curated all available mitogenomes for amphibians and discovered the unexpected dynamism of their architecture. The quantification of structural changes reveals the episodic pattern and highlights the diversity in terms of lineages and genes. This study sheds light on the mechanisms driving genome evolution and underscores the importance of genome structure in shaping the evolutionary fate of organisms. We challenge the simplistic view of genetic structural changes as discrete qualitative units and emphasize more specific quantitative descriptions of patterns of genome structural evolution. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials The data and materials presented in the study are included in the article/ Supplementary Material. Further inquiries can be directed to the corresponding author. Competing interests The authors declare no competing interests. Funding This research was supported by the National Natural Science Foundation of China (31970447; 32370500). Authors’ contributions Y.X.: Formal analysis, Writing, Original draft preparation, Visualization. G.N.: Conceptualization, Methodology, Validation, Writing, Reviewing and Editing. H.S.: Software, Data curation. Z.W.: Investigation. R.D.: Formal analysis. Y.L.: Resources. 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Supplementary Files Additionalfile1Typesforeachfamily.xlsx Additionalfile2mtATypes.xlsx Additionalfile3CoverageandTypesforeachgenus.xlsx Additionalfile4Typesofsamples.xlsx Additionalfile5RF.xlsx Additionalfile6RSacrosstime.xlsx Cite Share Download PDF Status: Published Journal Publication published 17 Mar, 2025 Read the published version in BMC Genomics → Version 1 posted Editorial decision: Revision requested 31 Jul, 2024 Editor assigned by journal 31 Jul, 2024 Submission checks completed at journal 31 Jul, 2024 First submitted to journal 30 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4830272","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":334527980,"identity":"6ffd1b66-5ebd-4f0c-81f1-90e46561eb40","order_by":0,"name":"Yi Xiao","email":"","orcid":"","institution":"Laboratory of Insect Systematics and Evolutionary Biology, College of Life Sciences, Jiangxi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Xiao","suffix":""},{"id":334527981,"identity":"31cfdd0f-d1d6-47e1-9a86-265f76e3a134","order_by":1,"name":"Gengyun Niu","email":"","orcid":"","institution":"Laboratory of Insect Systematics and Evolutionary Biology, College of Life Sciences, Jiangxi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Gengyun","middleName":"","lastName":"Niu","suffix":""},{"id":334527982,"identity":"4fd54c37-78c6-4a8e-b0c0-3396878cf337","order_by":2,"name":"Haihe Shi","email":"","orcid":"","institution":"School of Computer \u0026 Information Engineering, Jiangxi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Haihe","middleName":"","lastName":"Shi","suffix":""},{"id":334527983,"identity":"7a2db98a-6bbd-4a4f-aa9b-617d5fbe27d8","order_by":3,"name":"Zhenyu Wang","email":"","orcid":"","institution":"Nanchang Key Laboratory of Microbial Resources Exploitation \u0026 Utilization From Poyang Lake Wetland, College of Life Sciences, Jiangxi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Zhenyu","middleName":"","lastName":"Wang","suffix":""},{"id":334527984,"identity":"980f2910-906f-437e-a1b3-2df477ddf0de","order_by":4,"name":"Renzeng Du","email":"","orcid":"","institution":"School of Computer \u0026 Information Engineering, Jiangxi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Renzeng","middleName":"","lastName":"Du","suffix":""},{"id":334527985,"identity":"b2377520-bda1-4399-ad3d-3a6726822e94","order_by":5,"name":"Yankuo Li","email":"","orcid":"","institution":"Nanchang Key Laboratory of Microbial Resources Exploitation \u0026 Utilization From Poyang Lake Wetland, College of Life Sciences, Jiangxi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yankuo","middleName":"","lastName":"Li","suffix":""},{"id":334527986,"identity":"4a6eb6c2-450d-4897-b0fd-6a81d45ebebb","order_by":6,"name":"Meicai Wei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYFACxgaGDzwQpgTRWhhnkKiFgYEZqoNILQbHm1s328gczjM4wHzwNg+DXR5hLWcOtt3O4UkrNjjAlmzNw5BcTFCL2Y1EkBabxA0HeMykeRgOJDYQ1HL/YdttCx4JoBb+b0RqucHYdpsBYgsbcVrszyS23ezhSUuceZjN2HKOQTJhLZLtx5/d+NlzOLHvePPDG28q7AhrAQPGHiDBDGIZEKUeBH4QrXIUjIJRMApGIgAA0Q09F8E9bX0AAAAASUVORK5CYII=","orcid":"","institution":"Laboratory of Insect Systematics and Evolutionary Biology, College of Life Sciences, Jiangxi Normal University","correspondingAuthor":true,"prefix":"","firstName":"Meicai","middleName":"","lastName":"Wei","suffix":""}],"badges":[],"createdAt":"2024-07-30 16:41:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4830272/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4830272/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12864-025-11480-6","type":"published","date":"2025-03-17T15:56:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63336209,"identity":"dc50304d-041d-4791-8598-29000d7c24ea","added_by":"auto","created_at":"2024-08-27 05:38:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1621657,"visible":true,"origin":"","legend":"\u003cp\u003eBasic overview of the sampling of the amphibian mitogenome in this study, as visualized from additional table 3. \u003cstrong\u003ea.\u003c/strong\u003e The rising sun plot is based on the number of species in each genus, and the inner side shows the coverage statistics for each rank under the AW classification framework. \u003cstrong\u003eb.\u003c/strong\u003e The box plot shows the species coverage of each genus within each taxon.­\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4830272/v1/0d8a543e5d05be718cb36174.png"},{"id":63336207,"identity":"7fa4b3cf-ff88-41f7-b06c-e6521396c1e8","added_by":"auto","created_at":"2024-08-27 05:38:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1645329,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. The diagram illustrates 88 different types of structural variation in the amphibian mitogenome characterized by different color-coded blocks. Three ground patterns are represented by circular elements in orange, red and blue, corresponding to specific branches. In addition, the derived variations are represented by square elements with unique colors. \u003cstrong\u003eb. \u003c/strong\u003eHeatmap based on RF showing the activity of both genes and CR for each taxon.\u003cstrong\u003e c.\u003c/strong\u003e Cloud rain diagram and box plot comparing the RS distributions across four clades.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4830272/v1/c3d80f10b45fb025e0d5d0b8.png"},{"id":63335357,"identity":"dcaeb396-058d-410f-bb1b-af30d9170bc2","added_by":"auto","created_at":"2024-08-27 05:30:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":307729,"visible":true,"origin":"","legend":"\u003cp\u003eRS represents the change in the intensity of gene rearrangement over time, visualized from table S5.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4830272/v1/6dd431247d7bcba08904f135.png"},{"id":79120363,"identity":"1f3240d8-fcdf-4a68-a499-14e443876bf9","added_by":"auto","created_at":"2025-03-24 16:01:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3784990,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4830272/v1/8a93cd23-30fc-4fbe-b8ac-7b39aeda7744.pdf"},{"id":63335379,"identity":"4030621b-25ba-487d-a7c7-3a72cf633a98","added_by":"auto","created_at":"2024-08-27 05:30:05","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13248,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1Typesforeachfamily.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4830272/v1/5a4e3c5182f7995210943dd0.xlsx"},{"id":63335356,"identity":"26c0b02d-05d9-4c4a-b2db-d847d1386ba6","added_by":"auto","created_at":"2024-08-27 05:30:04","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15856,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2mtATypes.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4830272/v1/df7d9e6961a905ac2f31ff4c.xlsx"},{"id":63336897,"identity":"53945fef-86a6-403b-9359-4353537b2b36","added_by":"auto","created_at":"2024-08-27 05:46:04","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":30967,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile3CoverageandTypesforeachgenus.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4830272/v1/dcb92608eeededfb8ae40959.xlsx"},{"id":63335358,"identity":"b818a4da-e2fe-4e14-8b9b-6c487bb9573c","added_by":"auto","created_at":"2024-08-27 05:30:04","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":74173,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile4Typesofsamples.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4830272/v1/0383ef6780e79fd5ca433bb5.xlsx"},{"id":63335351,"identity":"97e79718-2d0a-4576-af76-bd86afdca4a6","added_by":"auto","created_at":"2024-08-27 05:30:04","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":15906,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile5RF.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4830272/v1/5773b1ae74d20089097b56b3.xlsx"},{"id":63335353,"identity":"8782f3ff-f289-4129-a424-9a0f430d5d30","added_by":"auto","created_at":"2024-08-27 05:30:04","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":304908,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile6RSacrosstime.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4830272/v1/2e2343e4642ddc01f494b3b5.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhanced dynamicity: evolutionary insights into amphibian mitogenomes architecture","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe mitogenomes architecture, as the structural organization of genetic material, exhibits dynamic properties [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It displays remarkable plasticity in individuals [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], while taking radically various pathways in diverse lineages. The architectural diversity of the mitogenomes provides a noteworthy dimension for observing the complexity of mitochondria as an open system. This diversity encompasses a broad range of genomic rearrangements, including insertions, deletions, inversions, and transpositions, both within genes and in intergenic regions. These structural changes reshape the genome and influence its functionality, thereby affecting species adaptability and evolutionary trajectories. For instance, significant innovations in metazoans, such as the emergence of multicellularity and bilateral symmetry, are associated with specific changes in mtDNA organization [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExtensively reorganized mitogenomes in amphibians have attracted considerable interest since 1997 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These intriguing features, such as exceptional enlargement (up to 28.8 kbp) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], loss and gain of genes [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and the stepwise nature of rearrangement [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], all suggest the hidden diversity of amphibian mitogenomes. Beyond the rearranged genes, the long noncoding region or control region (CR) is also the \u0026ldquo;hot spot\u0026rdquo;, not only because of its duplication or triplication [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] but also because the replication fork barrier lying in the CRs can often mediate recombination between duplicated CRs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAcross amphibian lineages, independent rearrangements have been observed, leading to the consensus that such reorganization patterns can serve as phylogenetic signals of amphibian evolution. For instance, the LTPF tRNA cluster is considered as a synapomorphic characteristic of Neobatrachia [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, further shuffling within the cluster [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], or the involvement of \u003cem\u003enad5\u003c/em\u003e gene adds complexity, especially when similar events are found in taxonomically distant groups, such as Afrobatrachia [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, the replication of trnM and the presence of IQMM gene clusters in multiple, distantly related taxa further complicate the picture, as in some cases, these clusters exist as two separate copies rather than tandem repeats [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Additionally, intraspecific rearrangement diversity cannot be ruled out, as evidenced in Dicroglossidae [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These known and unknown complexities urge us to expeditiously unravel the evolutionary landscape of mitogenomes variations in amphibians.\u003c/p\u003e \u003cp\u003eIn light of the decreasing costs of high-throughput sequencing technologies [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], advancements in long-read sequencing [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], along with optimized assembly and annotation techniques [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], we are now able to explore the mitogenomes architecture in greater depth, thereby examining its evolutionary patterns and trends [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In this study, we specifically aimed to investigate the genomic architectural diversity of amphibians. We used NCBI2GO (unpublished) to clean the available data from 1777 samples representing at least 710 species to generate a high-quality dataset. Employing qMGR [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and qGO (unpublished), we conducted qualitative and quantitative comparative studies within a phylogenetic framework to elucidate the patterns of genome structural changes during amphibian evolution. Our findings revealed a phased growth trend in structural changes, providing valuable insights into the evolution of mitogenomes. These insights also have the potential for uncovering the origin and maintenance of animal biodiversity and broadening the scope of comparative genomics research.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Cleaning and error checking\u003c/h2\u003e\n \u003cp\u003eAccurate annotation is often a challenge [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. Despite considerable efforts[\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e], the errors in RefSeq annotations are still too severe for gene organization comparisons [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. The problem is even worse at NCBI, where a large number of sequences are unannotated [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. Cleaned datasets are also not always accurate [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. Therefore, in this study, 2143 mitogenomes retrieved from NCBI Organelle Genome Resources using the search string \u0026quot;Amphibia[ORGN] AND (mitochondrion[TITL] OR mitochondrial[TITL]) AND 10000:50000[SLEN] NOT (RNA [TITL] OR gene[TITL] OR product[TITL] OR mRNA[TITL] OR rRNA[TITL] OR misc_RNA[TITL] OR nuclear[TITL])\u0026quot; on October 11, 2023, were cleaned using NCBI2GO (unpublished), a cleaning pipeline developed by the authors. In this pipeline, the gene order was extracted, and the intergenic length was calculated based on the location information to identify the control region (CR) by the threshold. A total of 2143 mitogenomes, including 366 from RefSeq, have been deposited in the GenBank mitochondrial database. 369 out of 1777 samples with missing annotations and those with unconventional gene order were entered into the re-annotation module. Manual verification was then performed to further ensure the reliability of the dataset. For the standardization, the gene nomenclature in [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e] is used. The single-letter amino acid codes designate the corresponding transfer RNA genes (trns). L1, L2, S1, and S2 indicate trns for Leu (UUR), Leu (CUN), Ser (UCN), and Ser (AGY), respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Taxonomic reconciliation\u003c/h2\u003e\n \u003cp\u003eCoherence between species names and their parent taxa is central to comparative analysis at higher taxonomic levels. The NCBI taxonomy was checked against an authoritative list of accepted species. We chose to initially base this comparison on AmphibiaWeb (AW), which contains 77 families with 569 genera and 8689 species downloaded on January 13, 2024 [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eThe 1777 samples were first de-duplicated based on NCBI taxonomy, yielding a list of 97 unidentified species samples (17 with \u0026ldquo;cf.\u0026rdquo;, 79 with \u0026ldquo;sp.\u0026rdquo;, and 1 unisexal lineage), 1 hybrid, and 40 with \u0026ldquo;aff.\u0026rdquo;, 695 species, and 13 subspecies. Matching of the last three of the above five cases\u003c/p\u003e\n \u003cp\u003ereturned the scientific names of 716 species, and the remaining 32 were manually matched to the GBIF Backbone Taxonomy [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. After the final deduplication, our taxonomic matchup placed 1777 samples into 710 species of 244 genera and 65 families as showed in an additional Table\u0026nbsp;12.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. Quantification of mtDNA rearrangements\u003c/h2\u003e\n \u003cp\u003eThe mitogenomic structural changes can be analyzed quantitatively by calculating the pairwise distance between the rearrangements and the ancestral organization. We applied both the qMGR [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e] and qGO algorithms separately to the above dataset. While both aim to quantify the rearrangement frequency (RF) of each individual gene and the rearrangement score (RS) of each mitogenome, qMGR is alignment free, which calculates the RF of a given gene by accumulating the changes in the two nearest neighboring genes. However, qGO, developed by the authors (unpublished), relies on homology alignment to calculate the RF of the target gene directly. It also supports weighting, so breakpoints need to be predefined to generate intervals that give weight to genes that translocate across the interval. Following the widely accepted processes of replication [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e] and transcription [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e], we set OriL and OriH as the two breakpoints that force the circular genome into two intervals. The genes within each interval are then manually aligned separately as the input for qGO.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Evolution of mitogenomic structures across amphibians\u003c/h2\u003e\n \u003cp\u003eThe time-calibrated molecular phylogeny of Jetz and Pyron [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e] was used for comparative analyses. Changes were made to update families (Hynobiidae, Ambystomatidae, Proteidae, Rhyacotritonidae, Amphiumidae, Plethodontidae, Ascaphidae, Alytidae, Rhinophrynidae, Scaphiopodidae, Megophryidae, Limnodynastidae) and prunes (Odontobatrachidae, Conrauidae, Nyctibatrachidae, Ceuthomantidae, Cycloramphidae, Batrachylidae, Allophrynidae) due to the updata and the lack of complete mitogenomic data. All types are first projected to the phylogeny, and the ancestral type for the diversity is inferred to be the relative outgroup.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Overview of mitogenomic organization in amphibians\u003c/h2\u003e\n \u003cp\u003eNumerous errors in the annotation were identified and reviewed. Error types include but are not limited to abnormal reading direction (strand), erroneous gene designations, missing gene or other feature annotations, mistaken identity of trnL1/trnL2 and trnS1/trnS2 tRNAs, and inconsistencies in gene names. The most misleading situation is when the errors were included in RefSeq [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]. After data cleaning, error checking and taxonomic reconciliation, we obtained a dataset containing 1777 samples representing 710 species in 244 genera under 65 families, yielding 88 gene rearrangement types (Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Species coverage within each genus was variable, with a mean of 35% and a median of 25%. Among them, Caudata was the richest (mean 58%, median 50%), and Neobatrachia was the least abundant (mean 23%, median 13%) (Fig. 1 and Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe unexpected complexity and variability among amphibian mitogenomes were revealed (e.g., Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). The major lineages of both Gymnophiona and Archaeobatrachia, as well as a few Caudata, were identical to the typical vertebrate gene order (orange circle in Fig.\u0026nbsp;1a), which was named the Amphibian ground pattern herein and labeled type 1 in additional tables. In addition, both Caudata and Neobatrachia have their own autapomorphies. The Caudata ground pattern (type 2, red circle) features one more CR derived between trnT and trnP. The Neobatrachian ground pattern (type 21, blue circle) has a strongly rearranged gene order, involving the long-term translocation of trnL1 to form the LTPF tRNA gene cluster located upstream of the 12S rRNA gene [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eIn contrast to all 10 Caudata families, which have consistently retained the ground pattern among their members, only 24 of 35 Neobatrachia families show the Neobatrachia ground pattern. Among these, 9 families have evolved new types. The remaining 11 families exhibited completely derived types, leading to a total of 69 rearranged types. This makes Neobatrachia the most variable group, followed by Archaeobatrachia with 10 types, Caudata with 8, and Gymnophiona with 5, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea.\u003c/p\u003e\n \u003cp\u003eThe most common rearrangement events observed in this study involved nad5, trnTP, trnL1 and CR [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]. Inversions are rare relative to translocations. Notably, consistent with findings from long-read sequencing, multiple rearrangement types involved duplication of genes or gene clusters, as well as repetition of CR or a combination of both [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e]. Among them, trnM duplications were the most common, followed by trnP duplications, as detailed in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb and Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Mitogenomic structural changes across four lineages\u003c/h2\u003e\n \u003cp\u003eMosaic evolution and lineage specificity coexisted throughout mitogenome evolution in amphibians. In particular, the ground patterns of vertebrate (n\u0026thinsp;=\u0026thinsp;162) and Caudata (n\u0026thinsp;=\u0026thinsp;415) are shared among distantly related species of Gymnophiona, Caudata, and Archaeobatrachia. However, both patterns are notably absent in Neobatrachia. Moreover, the 67 derived types that evolved from the Neobatrachian ground pattern (n\u0026thinsp;=\u0026thinsp;756) are unique to this lineage and are not observed in the other three lineages. These observations challenge our understanding of the organization of the ancestral amphibian mitogenome. Both Rhinatrematidae and Ichthyophiidae (Uraeotyphlus only), the two most basal families of Gymnophiona, possess the Caudata ground pattern [\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e]. Given the rearrangements found in Caudata and Archaeobatrachia, it is difficult to rule out the possibility that the ancestral state of the amphibians was a Caudata ground pattern. However, the majority of Gymnophiona, with the exception of the aforementioned families, exhibit this vertebrate pattern. Therefore, applying the principle of parsimony, the most plausible hypothesis is that the ground pattern of amphibian mitogenomes is a vertebrate pattern.\u003c/p\u003e\n \u003cp\u003eAlmost all mitogenomic structural changes in Gymnophiona are genus specific, except for Boulengerula, which has interspecific variation, with one of the two species having a duplicated trnP (Type 3). There are also two types (types 4 and 5) concentrated in the trnWANCY clusters that are restricted to two genera in Siphonopidae. The Caudata ground pattern is shared by all 10 Caudata families, yet two of them have derived taxa. Among the 168 Caudata species examined, there is intrageneric differentiation in the genus Tylototriton, and two of these species are type 11, involving the expansion of the CR and its flanking genes [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. The seven Plethodontidae species from Tylototriton, Aneides, Hydromantes and Plethodon are all intragenerially diverse to types 6\u0026ndash;9, and the single-sample Stereochilus is type 10. Archaeobatrachia comprises four successively evolved lineages, all of which can be traced back to the ancestral amphibian type. Two out of four genera of Pipidae, Pipa and Xenopus, possess both Type 1 and Type 2. Derived types 20 (swaps of nad6-trnE and cob-trnT) were found in both examined species of Leiopelmatida. The more diverse Megophryidae show an additional seven derived types occurring in Leptobrachium (Type 14), Leptobrachella (Type 13) [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e], Scutiger (Type 17) [\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e] and Oreolalax [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e], which intragenetically diversified into five derived types. These changes primarily involve the duplication of trnM [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e] and/or the remote transposition of trnW.\u003c/p\u003e\n \u003cp\u003eThere is very high variation in the genomic structure of Neobatrachia. Whether derived in terms of majority rules or the status of the outgroups, the ancestral type should contain a unique rearrangement in which trnLTP forms a cluster of genes located between the CR and trnF. In addition to type 21, which is shared by 24 families and is therefore considered to be the ground pattern, there is another type shared by four distant families (type 48), which differs from the ground pattern only by an additional CR between nad5 and nad6. Another pair of sister groups shares type 27, which has an additional interchange of trnA and trnN on top of the ground pattern. The remaining 66 types other than these three are unique to a given family, meaning that they are no longer shared between families [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]. The clade comprising Dicroglossidae [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e], Ranidae [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e] and Rhacophoridae [\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e] exhibits the strongest changes, encompassing nearly half of the derived types (32 in total) with Breviceps, Hyperolius, Ptychadena, Cornufer, Limnonectes, Amolops, Nidirana, Odorrana, Rana, Polypedates, Nanorana, Quasipaa, and Nanorana presenting intrageneric changes and the latter two even presenting intraspecific changes [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. Nested within this clade, Mantellidae and Ranixalidae each harbor family-specific types. Other intrageneric changes occur in Cornufer, Ptychadena [\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e], Hyperolius [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e], Breviceps [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e], Ischnocnema [\u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e], Brachycephalus, Boana, and Bokermannohyla and reach an extreme of nine types in Pristimantis. The changes are varied and involve almost all regions of the mitochondria. The most notable variable regions involved the tRNA gene cluster WANCY [\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e]; in particular, multiple OLs were identified in the clusters [\u003cspan class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Quantitative analysis of mitogenomes changes across amphibians\u003c/h2\u003e\n \u003cp\u003eThe hotspot is depicted in Fig. 1b on the heatmap plotted against the relative frequency (RF), detailed in additional table \u003cspan class=\"InternalRef\"\u003eS5\u003c/span\u003e. Each amphibian group is represented by a different color, with purple indicating the entire amphibian group. The gradient of color shading reflects the relative RF score. Among all the genes, trnL1 exhibited the highest score due to its long-range translocation across various amphibian taxa. However, when examined at a finer taxonomic level, trnL1 rarely undergoes changes within that specific taxon. It should be noted that hotspots differ between taxa. For instance, Gymnophiona primarily experiences changes in the trnWANCY gene cluster, while Archaeobatrachia scores highest for the flanking genes of nad2. Neobatrachia displayed significantly more variable regions than did the other taxa, with almost all the genes exhibiting some degree of rearrangement except for cox2, atp6, and nad4l, which had RF scores of 0. Notable hotspots common to all three taxa may be confined to the region between nad6 and trnF, including CR [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eFurther analysis of the RS distribution (Fig. 1c and Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e) highlighted the significant variation in mitogenomes architecture among amphibians. Gymnophiona, the most primitive group, exhibited the lowest median RS intensity, suggesting a relatively low level of genomic change. Caudata has a greater median RS than Archaeobatrachian, possibly due to Pyxicephalus adspersus, which undergoes strong rearrangement with gene duplication, resulting in 49 genes [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. This extreme value elevates the overall distribution profile with score peaks at 50 (qGO) and 46 (qMGR). However, disregarding this extreme value, both taxa exhibited relatively uniform distributions of RS that were generally greater than those observed in Gymnophiona. It is worth noting that although both Caudata and Archaeobatrachia had a minimum RS value of 0, considering that type 1 in Cadauta evolved from ancestral type 2, this score varied when type 2 was changed to the reference. The multimodal distribution observed in both Archaeobatrachia and Neobatrachia indicates heterogeneity, potentially suggesting divergent evolutionary directions. A more intriguing alternative inference concerns the emergence of Neobatrachia, which disrupts continuity and gives rise to fluctuations in Archaeobatrachia. Neobatrachia, as the most recently evolved lineage, stands out for its high RS values and several extreme values. There are ten values above 20, a magnitude that far exceeds that of any other taxon, suggesting that it may have evolved in an aggressive way, avoiding the fatal decrease in evolutionary potential. This is consistent with the evolutionary pattern observed in other vertebrates, where more recently evolved taxa tend to have greater complexity. The extremely high RS may also indicate that the taxa have undergone rapid adaptive radiation or evolutionary innovation, resulting in significant increases in complexity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. Evolutionary dynamics underlying patterns of diversification\u003c/h2\u003e\n \u003cp\u003eBy aligning the RS of each taxon with the time of origin of its corresponding branch and aggregating the RS values over time, a growth curve can be generated (Fig. 3 and Table \u003cspan class=\"InternalRef\"\u003eS5\u003c/span\u003e). A comparison between the growth curves for all amphibians and those for each individual taxon revealed a consistent pattern of stepwise growth over time. This suggests that there were distinct periods of rapid expansion followed by relatively stable phases in amphibian evolution.\u003c/p\u003e\n \u003cp\u003eInterestingly, while the absolute values of RS exhibited considerable variation across taxa, they demonstrated remarkably consistent patterns of increase, except for Gymnophonian, which consistently experienced a step change during the Cretaceous period. This indicates that Gymnophiona underwent unique evolutionary events or adaptations during this specific period. Furthermore, when examining the remaining three branches, it becomes evident that they all display a rapid increase with nearly comparable slopes soon after the beginning of the paleogene. This sudden surge in their RS values is preceded by an extended period of stagnation and a minor, limited magnitude increment occurring around or prior to the mid-Cretaceous epoch. The long periods of silencing in the early clade may indicate an underestimation of potential diversity for various reasons, such as extinctions associated with specific rearrangements.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Unraveling the complexity: mitogenomes variability in amphibians\u003c/h2\u003e \u003cp\u003eThe dogma that there is a frozen mitogenomes in vertebrates has long been broken, but vertebrates are still considered subphyla with probably the lowest variability in mt DNA gene content and gene order, with disparities thought to have a limited taxonomic distribution. This frosted glass exists mainly because of uneven sampling [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e], a high percentage of erroneous annotations [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and problematic mitogenomes [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], as well as the omission of duplicated regions [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. During the last few years, the advent of high-throughput sequencing techniques has increased the number of sequenced mitogenomes. Vertebrates, in particular, account for more than half of all Metazoa [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Specifically, for amphibians, an adequate representation of 88% of the families is sufficient to infer consensus mitogenome characterizations of different taxa and to fully recognize exceptions that lie beyond this consensus. The present study revealed that 43% of the amphibian genera examined had at least one available mitogenome. However, the data still suffer from taxon bias. In the case of Gymnophiona, mitogenomic structural changes often occur at the genus level. The lack of novel rearrangements, despite a genus-level coverage of 82%, suggests data saturation. Conversely, although the number of Archaeobatrachia mitogenomes exceeds that of Gymnophiona mitogenomes, changes could occur within genera even within species, suggesting that as species diversity increases, the emergence of new types of mitogenomes is possible. In other words, the existing data for Archaeobatrachia may be insufficient [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe mitogenomes, traditionally considered highly conserved, exhibits unexpected dynamism, with 88 observed types in our study. This diversity, however, is likely an underestimate, suggesting that the complexity of the mitogenomes extends beyond current comprehension, especially when considering regions of high variability [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] that were excluded from this dataset due to stringent search criteria. In a phylogenetic framework, structural diversity is sensitive to the grain of phylogenetic resolution. Specifically, the differences between major types (type 1 and type 2) in amphibians, birds and reptiles [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e] hinge on the presence of an additional CR between trnTPs, and Neobatrachia shows phylogenetic constraints from ancestral type 21. In contrast to the path-dependent evolutionary trajectories described above, fine-grained taxonomic diversifications appear to be stochastic and unpredictable, underpinning the individualized nature of mitochondrial evolution within specific families, [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e] as depicted in Fig.\u0026nbsp;1a. These changes involve all kinds of components, including tRNAs, protein-coding genes, rRNAs, and the CR, occurring regardless of the gene position relative to the CR and affecting both strands.\u003c/p\u003e \u003cp\u003eThis observation challenges the prevailing notion of convergent evolution in vertebrate mitochondrial gene rearrangements, suggesting instead that the forces shaping mitochondrial genomic organization are diverse and influenced by unique evolutionary histories [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Based on such observations, it is reasonable to hypothesize that a variety of mechanisms can drive mitogenome structural changes, extending beyond the duplication-random loss (DRL) model traditionally invoked to explain genomic alterations [\u003cspan additionalcitationids=\"CR78\" citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. The DRL model, while useful in explaining certain types of genomic changes at the individual level, may not fully account for the broad variability observed across different lineages [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e] or proclivity, such as nested copies of duplicated segments [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. This raises critical questions about the underlying mechanisms that confer flexibility and robustness to mitogenomes evolution, thereby enhancing its evolvability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.2. An asynchronous symphony of the episodic architecture variation\u003c/h2\u003e \u003cp\u003eComplexity often emerges from an intricate dance of genetic and environmental factors, resulting in the quantification of features that exhibit fluctuations across multiple dimensions. Through the use of RS and RF as proxies for measuring complexity, genetic innovations can be traced step by step, and amphibian evolution dynamics can be linked to genomic structure evolution. As shown in Fig.\u0026nbsp;1c, primitive taxa tended to exhibit simpler structures and lower levels of RS, while derived taxa, on the other hand, showed a significant increase in RS across several levels. This increase in complexity suggests that the genomic structure is able to evolve, enabling the exploration of new strategies and facilitating diversification [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. In other words, evolvability evolved [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. The case of Archaeobatrachia sheds further light on how this trend impacts specific groups within these taxa. The fluctuating RS suggests that Archaeobatrachia may have undergone a phase of complexity reduction or macroevolutionary freezing [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e], which is precisely a consequence of the evolution of evolvability [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRearrangement frequency (RF) helps us understand complexity by showing that genes or gene clusters within a genome may undergo independent evolution [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. The trnW gene was used as an example. Although its overall RF appears normal across various amphibian groups, a zoomed-in examination revealed heterogeneity. Within Archaeobatrachia, all types involved trnW shifts, resulting in a remarkably high RF. In contrast, RF rapidly decreased in Gymnophiona but reached zero in Caudata. The heterogeneous behavior of gene rearrangement challenges the notion that the mitogenome evolves as a cohesive, inseparable evolutionary unit. It becomes evident that the way a genome evolves depends on the distinct patterns of independent evolution present among the different genes or gene blocks that a particular genome contains [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBy examining changes in RS over time, it is possible to trace the increasing complexity of genomic structures and to infer the reasons for this complexity. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e, across multiple time intervals, we observe a pattern where periods of stasis are punctuated by brief bursts of rapid evolution. The concept that the rate and pattern of evolutionary change are closely linked to selective pressures at the lineage level is a longstanding and vital part of macroevolutionary theory [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e], and this concept also applies to mitogenomic evolution [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. However, the episodic increase in the number of salientians (anurans and caudates) showed a similar pattern at significant timescales. The first increase, which was very small and occurred at the beginning of the Jurassic, was followed by an increase in limited growth near the Jurassic and Cretaceous boundaries, and a third significant increase occurred just after the K-Pg line, overlapping with the rapid diversification of species-rich clades [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e, \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e]. Between these periods of growth are long periods of stagnation, which may reflect temporarily rising \u0026lsquo;challenging times\u0026rsquo; for diversification of mitogenomes, possibly due to evolutionary constraints. The generalizability of this pattern across different amphibian clades highlights the dynamic nature of mitogenomes and the intermittent nature of the evolutionary events that shape their structure. Episodic mitogenomic evolution is also evident in other metazoans, such as fish and invertebrates [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e] In these organisms, drastic changes in mitogenomic structure have been linked to episodes of adaptive radiation [\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e] or extreme environmental adaptation.\u003c/p\u003e \u003cp\u003eThe complexity and evolvability of amphibian mitogenomes structure could be shaped over evolutionary time by various external forces [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e]. These forces include not only the deterministic effects of evolutionary events but also the stochastic influence of random changes. This interplay of factors has endowed mitogenomes with a capacity for adaptation and innovation that exceeds the predictions of traditional evolutionary models. The evolvability of mitogenomes through gene rearrangements highlights the dynamic role of genome structure in shaping the evolutionary trajectories of organisms. In addition to serving as repositories of genetic information, mitogenomes actively participate in evolutionary processes by responding to selective pressures and environmental challenges through structural changes.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eWe collected and curated all available mitogenomes for amphibians and discovered the unexpected dynamism of their architecture. The quantification of structural changes reveals the episodic pattern and highlights the diversity in terms of lineages and genes. This study sheds light on the mechanisms driving genome evolution and underscores the importance of genome structure in shaping the evolutionary fate of organisms. We challenge the simplistic view of genetic structural changes as discrete qualitative units and emphasize more specific quantitative descriptions of patterns of genome structural evolution.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe data and materials presented in the study are included in the article/ Supplementary Material. Further inquiries can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Natural Science Foundation of China (31970447; 32370500).\u003c/p\u003e\n\u003cp\u003eAuthors\u0026rsquo; contributions\u003c/p\u003e\n\u003cp\u003eY.X.: Formal analysis, Writing, Original draft preparation, Visualization. G.N.: Conceptualization, Methodology, Validation, Writing, Reviewing and Editing. H.S.: Software, Data curation. Z.W.: Investigation. R.D.: Formal analysis. Y.L.: Resources. M.W.: Supervision, Project administration, Funding acquisition.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThe members of the Lab of Insect Systematics and Evolutionary Biology (LISEB) at Jiangxi Normal University are thanked for their contributions to laboratory work. The authors thank Jia-Yong Zhang and Jin Che for the assistance with revision. We thank the anonymous reviewers for their careful reading and many constructive comments.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSmith DR, Keeling PJ. Mitochondrial and plastid genome architecture: Reoccurring themes, but significant differences at the extremes. Proc Natl Acad Sci U S A. 2015;112:10177\u0026ndash;84.\u003c/li\u003e\n\u003cli\u003eCameron SL. Insect Mitochondrial Genomics: Implications for Evolution and Phylogeny. Annu Rev Entomol. 2014;59:95\u0026ndash;117.\u003c/li\u003e\n\u003cli\u003eFeulner PGD, De‐Kayne R. Genome evolution, structural rearrangements and speciation. J Evol Biol. 2017;30:1488\u0026ndash;90.\u003c/li\u003e\n\u003cli\u003eRavinet M, Faria R, Butlin RK, Galindo J, Bierne N, Rafajlović M, et al. Interpreting the genomic landscape of speciation: a road map for finding barriers to gene flow. 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Proc Natl Acad Sci. 2007;104:887\u0026ndash;92.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mitogenome, Comparative genomics, Macroevolution, Gene rearrangement, Complexity, Evolutionary transitions","lastPublishedDoi":"10.21203/rs.3.rs-4830272/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4830272/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMitogenomes are known for their structural dynamics and the complexity of their rearrangement patterns. However, their utility in metazoan comparative biology has not been fully exploited. Vertebrate mitogenomes are now sufficiently representative to allow the development of more advanced methods for comparing genome architecture. Furthermore, the relatively robust phylogeny of vertebrates at higher taxonomic ranks allows us to infer the patterns of genome evolution accordingly. In this study, using amphibians as an example, we performed data cleaning and manual annotation on 1777 samples from the NCBI and identified 88 rearrangement types, most of which were clade specific. In addition, we quantified genomic changes in an evolutionary framework and obtained stepwise growth curves of the architectural changes. This study provides new perspectives for understanding the evolution of the mitogenomes in amphibians and is expected to facilitate the qualitative and quantitative development of mitogenomes research.\u003c/p\u003e","manuscriptTitle":"Enhanced dynamicity: evolutionary insights into amphibian mitogenomes architecture","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-27 05:29:59","doi":"10.21203/rs.3.rs-4830272/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-31T09:55:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-31T09:02:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-31T07:58:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2024-07-30T16:39:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"00b625de-03d7-45a2-adf5-6b5f6e69ec66","owner":[],"postedDate":"August 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-24T15:58:43+00:00","versionOfRecord":{"articleIdentity":"rs-4830272","link":"https://doi.org/10.1186/s12864-025-11480-6","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2025-03-17 15:56:51","publishedOnDateReadable":"March 17th, 2025"},"versionCreatedAt":"2024-08-27 05:29:59","video":"","vorDoi":"10.1186/s12864-025-11480-6","vorDoiUrl":"https://doi.org/10.1186/s12864-025-11480-6","workflowStages":[]},"version":"v1","identity":"rs-4830272","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4830272","identity":"rs-4830272","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}