The efficacy of single mitochondrial genes at reconciling the complete mitogenome phylogeny—a case study on dwarf chameleons

Although genome-scale data generation is becoming more tractable for phylogenetics, there are large quantities of single gene fragment data in public repositories and such data are still being generated. We therefore investigated whether single mitochondrial genes are suitable proxies for phylogenetic reconstruction as compared to the application of full mitogenomes. With near complete taxon sampling for the southern African dwarf chameleons (Bradypodion), we estimated and compared phylogenies for the complete mitogenome with topologies generated from individual mitochondrial genes and various combinations of these genes. Our results show that the topologies produced by single genes (ND2, ND4, ND5, COI, and COIII) were analogous to the complete mitogenome, suggesting that these genes may be reliable markers for generating mitochondrial phylogenies in lieu of generating entire mitogenomes. In contrast, the short fragment of 16S commonly used in herpetological systematics, produced a topology quite dissimilar to the complete mitogenome and its concatenation with ND2 weakened the resolution of ND2. We therefore recommend the avoidance of this 16S fragment in future phylogenetic work.


INTRODUCTION
Mitochondria, the subcellular organelles that enable eukaryotes to respire aerobically, likely began their evolution as unicellular organisms that made their way into the ancestral progenitor of eukaryotic cells via a process of endosymbiosis approximately 2.5 billion years ago (Gray, 2017;Sagan, 1967).Mitochondria possess a distinct haploid genome, independent of the nuclear genome.Among vertebrates, mitochondria are, for the most part, uniparentally inherited along the maternal line and rarely, if ever, experience any recombination (Bágeľová Poláková et al., 2021;Shtolz & Mishmar, 2019).Compared to protein-coding genes in the nuclear genome, mitochondria experience fast, sometimes clock-like, substitution rates (Avise et al., 1987;Konrad et al., 2017).Furthermore, mitochondria are far more abundant in the cell than the nuclei within which nuclear genomic DNA is located (Bollmann et al., 2021;Filograna et al., 2021).The abundance, haploidy and fast mutation rate of mitochondria have made mitochondrial DNA an attractive target for phylogenetic reconstruction of eukaryotic life (Hajibabaei et al., 2007).
In contrast to the long-standing reliance on mitochondrial genes in phylogenetics, the rapid expansion of next-generation sequencing technologies has ushered in a new era characterised by mitogenome-scale data (Hayden, 2014;Lewin et al., 2022;Muir et al., 2016).Despite the pace with which these genome-scale approaches are being assimilated into phylogenetics, many researchers still do not have access to resources to produce and analyse mitogenome-scale data.This is particularly pertinent for researchers based in low-and middle-income countries where funding resources are scarce, but biodiversity is plentiful (Helmy, Awad & Mosa, 2016).Indeed, researchers globally continue to make use of single or multiple gene fragments for phylogenetics, typically sequenced via Sanger Sequencing technologies.The abundance of mitochondria in tissues results in high DNA yield, which can overcome instances where DNA has been degraded.The popularity of sequencing single mitochondrial gene fragments over the past two decades has meant that public sequence data repositories have amassed immense stockpiles of single gene fragment data (Joly et al., 2014).By 2016, GenBank (https://www.ncbi.nlm.nih.gov/genbank/)alone had accumulated sequence data from 1,125,514 accessioned individuals of tetrapods, and this number continues to grow (Gratton et al., 2017).
As the cost of sequencing drops and mitogenome-scale data become more widely available, some important and interesting questions emerge.In particular, can the stockpiles of single gene data generated over the past three decades still be utilised in phylogenetic analyses, or are these data inferior and obsolete?It is therefore necessary to assess whether single gene data can be used accurately to reflect the evolutionary history of the mitochondrion, and/or to what extent mitogenome-scale data are necessary.These questions are not simple to answer; some nuance is required when interrogating them.Of course, more data allows for greater confidence in the assessment of complex genealogies.This is not being disputed, but, if we triage funding resources, then mitogenome-scale data may not be cost effective in terms of financial trade-off, particularly when research funds are limited.Even if the cost of sequencing drops to a point where mitogenome-scale data are accessible to all researchers worldwide, if single gene data are reliable proxies for mitogenome-scale data, then the democratisation of mitogenome-scale data should not render previously generated single gene data obsolete.
Somewhat surprisingly, whether single mito-gene data can accurately reconcile a mitogenome inferred phylogeny has rarely been investigated, with examples limited to cetaceans, and metazoans more generally (Duchêne et al., 2011;Havird & Santos, 2014).In both cases, certain single mito-genes and gene combinations were able to reconcile the mitogenome phylogeny.The most reliable genes for cetaceans, however, were not the same as for metazoans.This suggests that the phylogenetic informativeness of mito-genes may be, at the least, broadly taxon-specific.It should be noted that even the complete mitogenome can only be used to infer the genealogy of the mitochondrion, but this genealogy may not necessarily reflect that of the taxon.For example, discordance between the mitochondrial and nuclear phylogenies has been documented for many taxa, and has been attributed to incomplete lineage sorting, assortative mating, asymmetric introgression and/or sex-linked selection (Després, 2019;Fossøy et al., 2016;Tóth et al., 2017;Wendt et al., 2022).Thus, keeping in mind the potential for discordance between genomes is key for studies that incorporate mitochondrial DNA.
Despite the increased availability of mitogenome-scale data, studies comparing single gene phylogenies to the mitogenome phylogeny are notably lacking for reptiles-a group for which there exists an abundance of single gene data in publicly available repositories.For example, southern African dwarf chameleons of genus Bradypodion have largely been the subject of single locus (concatenated mito-genes) phylogenetics (Tolley et al., 2004;Tolley, Chase & Forest, 2008;Tolley, Tilbury & Burger, 2022).As a result of this, single mito-gene data are widely available for the genus on public data repositories.Most relationships between Bradypodion and other chameleon genera and most deep nodes within Bradypodion have been resolved through multi-locus phylogenetics that include mito-genes and nuclear genes (Tolley, Townsend & Vences, 2013).However, there are some nodes within the genus that are not yet well-resolved.Given the abundance of publicly available single gene fragment sequence data for Bradypodion, we used Bradypodion as a case study to evaluate the efficacy of single mitochondrial gene fragments as proxies in reconstructing the evolutionary history of the complete mitochondrial genome.With near complete taxon sampling for the genus, we contrasted topologies generated from single and concatenated mito-gene datasets to the full mitogenome topology.In particular, we hypothesised that the individual mito-gene topologies might show differing topologies amongst them as compared to the mitogenome, and that the individual gene trees would have lower node support than the topology generated by the mitogenome.
Maximum likelihood phylogenetic analyses were run for the complete mitogenome in IQ-TREE v2.0.3 (Minh et al., 2020b) with the optimal model of molecular evolution  estimated for each gene separately using the built-in ModelFinder implemented in IQ-TREE (Chernomor, von Haeseler & Minh, 2016;Kalyaanamoorthy et al., 2017).To assess whether node support would be improved by merging models of evolution for similarly evolving genes into larger partitions, a merged partitioned analysis was also run.This analysis makes use of the PartitionFinder (Lanfear et al., 2017) algorithm to generate a partitioning scheme within IQ-TREE and merge genes for model estimation to assess whether this reduces over-parameterization.
For the Bayesian analyses, we estimated the best evolutionary models and partitions for the complete mitogenome in PartitionFinder v2.1.1 (Lanfear et al., 2017).The output suggested that the dataset be partitioned into 10 partitions with GTR + I + G the optimal model of evolution for each partition (Table 3).
Bayesian phylogenetic analyses were carried out on all datasets separately and the complete mitogenome in MrBayes v3.2.7 (Ronquist et al., 2012) with partitions and models included as per the PartitionFinder results (Table 3).For each of the 22 datasets (13 separate coding genes, two ribosomal genes, 16S fragment, COI fragment concatenated 16S and ND2 alignment, concatenated ND2 and ND5 alignment, concatenated protein-coding genes, complete mitogenome), two separate runs were made, each with four independent Table 3 The partitioning scheme and models of evolution used for each partition generated by Parti-tionFinder for the phylogenetic analyses of Bradypodion.

Partition
Genes Model MCMC chains of 20 million generations, sampling every 1,000 generations.For every run we assessed convergence using the R package RWTY v1.0.2 (Warren et al., 2017) with sampling considered adequate when all parameters have ESS values >200.We also made use of ASTRAL version 5.7.8 (Zhang et al., 2020) to reconcile the mitogenome tree from each of the previously estimated single mito gene trees.ASTRAL makes use of the multispecies coalescent and quartet scores to estimate the best-supported species tree assuming a model of gene tree discordance.This assumption is not necessarily relevant when estimating the mitogenome tree because the mitogenome is a single non-recombining locus and therefore each mitochondrial gene is expected to share a single coalescent history.Nonetheless, we performed this analysis as an additional measure of gene tree congruence.Finally, gene and site concordance factors were calculated by computing individual gene trees and comparing them to the best-supported merged-partitioned mitogenome tree in IQ-TREE v2.0.3 (Minh et al., 2020b).Concordance factors are typically used to measure topological discordance among independent loci but here all loci share a single history due to a lack of recombination (Minh et al., 2020a).Therefore, we used concordance factors to measure topological differences in resolution rather than independent history.

Topological congruency and phylogenetic resolution
To assess topological congruency between different gene trees and the complete mitogenome tree, Bayesian consensus trees for each of the 22 datasets were imported into R using the package Ape v5.6-2 (Paradis, Claude & Strimmer, 2004).Using the function tree.dist.matrix in the package RWTY, Robinson-Fould (RF) topological distances between trees inferred from different gene regions were calculated pairwise to produce a distance matrix of topological distances.We used the nj function in Ape to generate a neighbourjoining dendrogram from this topological distance matrix.To visualize if topological reliability relates to the length of the gene, we constructed a broken x-axis scatterplot in R, using the package ggbreak v0.1.1 (Xu et al., 2021) and the gap.plot function, of RF distance from the complete mitogenome against gene length (in 100 bp) for each gene as well as our different concatenated partitions.We also carried out Shimodaira-Hasegawa (SH) topology tests in IQ-TREE as an additional measure of topological congruency among gene trees (Shimodaira & Hasegawa, 1999).

Saturation
To ascertain whether topological discordance across different genes might be due to saturation, we estimated saturation for each gene separately and the complete mitogenome by plotting uncorrected genetic p-distances against model corrected p-distances for each gene (Everson, 2015;Philippe et al., 1994) using the package Ape and the dist and dist.correctfunctions.The difference between these estimates is an approximation of the unobserved mutations hidden by multiple mutations occurring at a site along a branch in our tree.Due to an abundance of gaps in the ribosomal RNAs, which would be ignored when calculating model-corrected p-distances and thereby inflate the slope of the linear regression, we did not estimate saturation plots for these genes.In these saturation plots, the slope of the linear regression is indicative of the degree of saturation, the lower the slope of the linear regression, the more saturated the gene is assumed to be.For this reason, we tabulated the slope of the saturation linear regression for each gene along with other metrics relevant to topology: number of polytomies, mean node posterior probability (calculated based only on the supported nodes within the phylogeny), number of fully bifurcating nodes, RF distance from mitogenome, number of parsimony informative sites, gene length, and standardized information content (SIC, see Macey et al., 2004;Table 4).

Explaining RF distance
In addition to comparing the topology of each gene tree to that of the complete mitogenome, we summed the number of polytomies, mean posterior probability support across the tree, and number of fully bifurcating nodes for each gene tree and partition in our dataset and tabulated these metrics together with gene/alignment length, number of parsimony informative sites, saturation and RF-distance.We sought to assess which of these metrics best explains RF distance.We therefore ran an analysis of variance (ANOVA) on the data tabulated above using the aov function in the stats package in R, omitting number of polytomies, number of fully bifurcating nodes, and mean posterior probability because these are all intrinsically linked to the topology of the tree and therefore to RF distance.

Comparison with other published phylogenies
Our complete mitogenome topology and our ND2-16S are largely congruent with published dwarf chameleon phylogenies constructed using concatenated 16S and ND2 gene fragments (Tolley et al., 2004;Tolley, Chase & Forest, 2008;Tolley, Tilbury & Burger, 2022).However, we find that our phylogeny based on ND2 alone is more robust and has a topology more similar to our complete mitogenome phylogeny.Despite the similarities between the complete mitogenome phylogeny and the previously published phylogenies, a few differences are apparent.Our full mitogenome topology places B. setaroi and B. caffrum as sister taxa although the relationship lacks node support (PP = 0.71; BS = 47) and therefore cannot be considered a compelling difference.More notably is that the full mitogenome phylogeny supports B. ngomeense as being nested within the B. transvaalense clade (PP = 0.99; BS = 92) instead of sister to that species.This could be due to our better geographic coverage for B. transvaalense than in previous studies (Tolley et al., 2004;Tolley, Chase & Forest, 2008) allowing for a more detailed assessment of these relationships.However, none of our other (single gene or concatenated) analyses showed B. ngomeense to be nested within B. transvaalense, all of which are similar to previously published topologies, and also show node support for the monophyly of the two species.This poses the question as to whether the full mitochondrial dataset has allowed for better resolution within this particular lineage, or whether certain mito-genes have experienced directional selection, providing differing topological outcomes even within the mitochondrion.

Phylogeny
To assess if the mitogenome phylogeny can be reconciled from single mitochondrial genes, we estimated Bayesian and maximum likelihood phylogenies for the entire mitogenome as well for each single gene and various combinations of genes (all single gene trees have been archived on the Harvard Dataverse: 10.7910/DVN/RY1RQI).We found some variation in the mean node support and number of fully bifurcating nodes between the whole mitogenome tree and the single gene trees (Table 4).For example, the whole mitogenome inferred tree yielded 50 fully bifurcating nodes with a mean posterior probability support of 0.98 while a short fragment of 16S only yielded 13 fully bifurcating nodes with a mean posterior probability support of 0.86 (Table 4).
The complete mitogenome consensus phylogenies (Bayesian and maximum likelihood) recovered Bradypodion as a well-supported monophyletic clade with most internal nodes well-supported both in terms of bootstrap and posterior probability support (Fig. 1).The coalescent phylogeny estimated using ASTRAL (Fig. S1) was very similar to the Bayesian and maximum likelihood phylogenies except that the coalescent phylogeny does not recover B. caffrum and B. setaroi as monophyletic sister taxa, but this is not particularly surprising given that the sister-taxon relationship of B. caffrum and B. setaroi received very low support in the Bayesian and maximum likelihood phylogenies (PP = 0.71; BS = 47).Gene concordance factors reveal that while most nodes in the complete mitogenome consensus phylogeny are well-supported (PP ≥ 0.95; BS ≥ 75), some of these nodes do not show concordance across all gene trees (Fig. 1).For instance, the node separating the B. caffrum-B.setaroi clade from the B. caeruleogula-B.transvaalense clade is well supported by both Bayesian and maximum likelihood methods, but it has a gene concordance factor of only 33.3, meaning that only 33.3% of the gene trees in the mitogenome support that node (Fig. 1).The placement of B. caeruleogula, while well-supported in both bootstrap and posterior probability support, is only supported by 53.3% of gene trees.In both of these instances, the site concordance factors are also low (34.09% and 38.58% respectively (Fig. S2).These low concordance factors might reflect a lack of phylogenetically informative information in some of the genes, hindering phylogenetic estimation for these genes.Typically, when site and gene concordance factors agree (which they tend to in our analyses), this suggests genuine topological conflict between genes.However, given that the Notes.*In this context, number of polytomies refers to the number of branches directly emanating from polytomies within the phylogeny † Robinson-Fould distance from mitogenome phylogeny # SIC stands for standardized information content and is calculated as the ratio of parsimony informative sites to the total alignment length per kilobase (Macey et al., 2004).
mitochondrion is a single non-recombining locus, this conflict is more likely an artefact of certain genes lacking the phylogenetic information to converge on the true topology.

Analysis of topological congruence and phylogenetic resolution
We found varying degrees of topological inconsistency across phylogenies inferred by different genes within the mitogenome (Fig. 2).The dendrogram of RF topological distances shows that ND2, ND5, COIII, the concatenated PCG alignment, the concatenated ND2-16S and the concatenated ND2-ND5 all cluster with the tree inferred from the complete mitogenome, suggesting that the phylogenies inferred from these single genes or concatenations most closely resemble that of the complete mitogenome.However, the concatenated ND2-16S alignment is more distant from the complete mitogenome than the ND2 gene on its own, despite the concatenated alignment consisting of more characters.Sister to this cluster is a grouping of ND4 and COI, and just outside this cluster is ND6.More distant from the complete mitogenome are the ribosomal RNAs and ND3, Cyt-b, COII, ND4l, ATP6, ATP8, the shortened 16S fragment, and ND1.
It is apparent that RF distance from the complete mitogenome tree shows an inverse correspondence with gene length; however, there is considerable variation which indicates that some genes hold more information than others (Fig. 3).For instance, our longest alignment was for the combined protein-coding genes which also had the lowest RF distance from the mitogenome tree, while the shortest alignment was the ATP8 gene which had one of the highest RF distances from the mitogenome.ND2 had one of the lowest RF distances from the mitogenome, even lower than the concatenated ND2-16S alignment.Furthermore, the concatenated rRNA alignment was longer than all individual genes and yet still had a relatively high RF distance from the mitogenome, so phylogenetic informativeness does not correlate highly with gene fragment length.Nonetheless, concatenating two of the most reliable single genes, ND2 and ND5 produced a topology even closer to the complete mitogenome than either of these genes on their own.While the SH-tests also showed ND5 to be the best single gene proxy for the complete mitogenome, there were a few discrepancies between the RF-distances from the complete mitogenome and the results of the SH-tests (Table 4).The SH-tests supported COIII as the second-best proxy for the mitogenome, with no other single genes being congruent to the complete mitogenome.Furthermore, the SH-tests supported the concatenated alignment of ND2 and ND5 as a better proxy for the complete mitogenome than the PCG alignment.
As might be expected, the complete Bradypodion mitogenome phylogeny scores best with the fewest polytomies, the most fully bifurcating nodes, and the highest mean posterior probability support and an SH-test p-value of 1.Based on these same criteria, the short 16S fragment scored lowest with the most polytomies, lowest mean posterior probability support, and the fewest fully bifurcating nodes.It should be noted that mean posterior probability node support is calculated based only on the supported nodes within the phylogeny.If there is an unsupported polytomy, this is left out of the calculation, so these values should be consulted in conjunction with the number of polytomies and It is apparent from our ANOVA that saturation is not driving topological discordance within the mitogenome (p = 0.59; F = 0.30).This is probably due to strong structural constraints on the evolution of protein-coding genes within the mitochondrion, constricting the degree to which third position bases mutate.We did, however, find RF distance from the mitogenome to correlate significantly with gene/alignment length (p = 0.006; F = 11.36), and we found a significant interaction between the number of parsimony-informative sites and total gene alignment length (p = 0.02; F = 7.36).

DISCUSSION
Our findings suggest that, if chosen carefully, single mitochondrial protein-coding gene trees can be nearly as informative as full mitogenome datasets that are produced by expensive new technologies.For Bradypodion, we find that certain mitochondrial genes produce topologies that are essentially identical to that of the entire mitogenome and have almost equally strong support at most nodes.Discrepancies are largely limited to nodes that lack support across all datasets including the full mitogenome dataset.The ND2 and ND5 genes produced topologies that were the most similar to the mitogenome, as well as to the concatenated protein coding genes phylogeny.These topologies were further improved (at least in terms of RF distance from the complete mitogenome) when ND2 and ND5 were concatenated.Overall, Bayesian and likelihood methods recovered the same topology and similar node support regardless of dataset.However, one node (B.caffrum-B.setaroi) in the complete mitogenome phylogeny lacked support regardless of dataset used or analytical approach, and that node has never been recovered in previous phylogenies (Tolley et al., 2004;Tolley, Chase & Forest, 2008;Tolley, Tilbury & Burger, 2022).
While our findings demonstrate that datasets with a larger number of base pairs provide improved mean node support, the differences appear to be fairly subtle.Most mitogenomeinferred, species-level relationships within Bradypodion are also well reconciled using single gene fragments.In particular, single genes ND5, ND2, COI, COII, and ND4 resolved all major relationships.The phylogenetic utility of these markers is supported by multiple different analyses (i.e., RF distances, number of polytomies, mean posterior probability support, and number of fully bifurcating nodes).The finding that certain single mito-genes reflect the entire mitogenome adequately is encouraging given that markers such as ND2, ND4, COI as well as ND2-16S have been widely used in reptile systematics over the past few decades (Bates et al., 2013;Laopichienpong et al., 2016;Macey, Schulte & Larson, 2000;Main et al., 2022;Nagy et al., 2012;Taft, Maritz & Tolley, 2022).
The topological similarities between our complete mitogenome phylogeny and previous phylogenies that included ND2 (Tolley, Tilbury & Burger, 2022) echo the sentiment that ND2 is a good proxy for the complete mitogenome.However, we find that the resolution of ND2 is weakened by its concatenation with a short fragment of 16S.This suggests that, at least for Bradypodion, short of using genomic data, it might be more appropriate to use only ND2 in small-scale phylogenetic analyses going forward, or to combine ND2 with another gene with a low RF score, such as ND5.Overall, the shortened fragment of 16S commonly used in reptile and amphibian phylogenetics is probably a poor marker for phylogenetic reconstruction, and this finding is not unique.A previous study similarly found that, for amphibians, short 16S yields poor and erratic phylogenetic estimations and that these can be improved by using longer fragments of 16S (Chan et al., 2022).Correspondingly, we also found that the complete 1,653 bp 16S gene performed notably better than the shortened fragment (ca.461 bp), in terms of RF distance from the mitogenome, number of polytomies and number of fully bifurcating nodes (Table 4).However, the full 16S gene still performed poorly in recovering the full mitogenome topology, as compared to coding genes, i.e., ND2, ND4, ND5, COI, and COII.Considering the popularity of 16S in herpetological systematics (Alrefaei, 2022;Bates et al., 2013;Hay et al., 1995;Hertwig, de Sá & Haas, 2004;Lamb & Bauer, 2002;Main, Van Vuuren & Tolley, 2018;Main et al., 2022;Vences et al., 2004;Vences et al., 2005;Welton et al., 2010), the inadequacy of 16S at reconciling the complete mitogenome tree is concerning, particularly when used as a single gene marker for phylogenetic estimation.While most of these studies make use of other makers as well, in most cases ND2 or ND4, our results suggest that the inclusion of both short and long fragment 16S for phylogenetic analysis confounds the signal of the coding genes, and thus might reduce the resolution of the topologies.The commonly used COI barcoding fragment (Hebert et al., 2003) did not recover as much resolution as the complete COI gene but it still outperformed the shortened 16S fragment.We advocate that both effort and expense could be saved by avoiding the use of 16S.We acknowledge that our study has used only one reptile genus as a model, but it is likely that these inferences apply more widely than for only Bradypodion.However, it would be useful to confirm these results by carrying out additional phylogenetic mitogenome analyses using other taxa and comparing topologies amongst full genomes and single genes.
Gene concordance factors revealed that certain clades are more robustly supported across the mitogenome than others.For instance, over 90% of the mitochondrial gene partitions supported the clade containing B. pumilum, B. barbatulum, B. venustum, and B. damaranum as monophyletic; however, fewer than 15% of gene partitions support B. setaroi and B. caffrum as sister taxa.At the species level, the sister relationship of B. setaroi and B. caffrum has not been recovered in previous analyses (Tolley et al., 2004;Tolley, Chase & Forest, 2008), although this node also lacks node support in the present analysis.The relationship between B. transvaalense and B. ngomeense, albeit with less extensive geographic coverage than we have in our analyses, has historically been considered closely related (Tilbury & Tolley, 2009).Our full mitogenome phylogeny recovers B. ngomeense as paraphyletic with B. transvaalense.Further, the placement of B. caeruleogula has been uncertain, as it has either been part of a deep polytomy or placed with weak node support as sister to the B. thamnobates clade (Tilbury & Tolley, 2009).Both the placements of B. caeruleogula and B. occidentale have now been resolved with good support (PP ≥ 0.95; BS ≥ 75) by the complete mitogenome phylogeny.
While our results demonstrate that certain single mitochondrial genes are effective proxies for reconciling the mitogenome phylogeny two caveats should be mentioned.Firstly, phylogenetic trees are merely informed estimates, trees that are well-supported and well-resolved are not necessarily reflecting the evolutionary history of the taxa, they are just our best estimates given the data.We used the full mitogenome as our gold standard with which to compare other genes because it holds the most data, and we therefore assume it to have the best priors to inform phylogenetic estimation.We did not, however, set out to test for phylogenetic accuracy, we only sought to test whether single or concatenated mito-genes could reconcile the same mitogenome phylogeny as the full mitogenome.Secondly, even if we assume the tree to be accurate, this does not mean that the mitogenome tree necessarily reflects the species tree.Mito-nuclear discordance, due to factors like assortative mating, sex-linked selection, asymmetric introgression, and incomplete lineage sorting, is well documented for other taxa (Després, 2019;Fossøy et al., 2016;Toews & Brelsford, 2012;Tóth et al., 2017;Wendt et al., 2022).To assess whether this is the case for Bradypodion, a whole genome phylogenetic analysis is required.Fortunately, the mitogenome phylogeny serves as an important starting resource for future mito-nuclear comparisons.While our results are applicable to Bradypodion and might be more generally applicable to other reptile taxa, we still emphasize care when interpreting evolutionarily relationships based on single/concatenated mito-genes only.That is, for a fuller assessment of the history of a taxonomic group, nuclear genes that mutate at different rates must be included.In addition, new methods have allowed for genome scanning and these have proven very useful in the discovery of mito-nuclear discordance, and even speciation in the presence of gene flow with introgression (see e.g., Kunerth et al., 2022;Mikkelsen & Weir, 2023;Perea et al., 2016;Wright et al., 2022).

CONCLUSIONS
Our results provide compelling evidence for the efficacy of single mito-genes such as ND5, ND2, COI, COII, and ND4 (or concatenation of some) for reconciling the mitogenome phylogeny within the genus Bradypodion.Whether mitochondria themselves are adequate proxies for evolutionary history remains to be demonstrated.Nevertheless, these results are promising and suggest that apart from relying on 16S as a marker of choice, there is still utility in producing single/few gene sequence phylogenies.Furthermore, for researchers that are not yet able to take advantage of whole genome sequencing, readily sequenced genes like ND2, ND5 or ND4 appear to be reasonable proxies for phylogenetic estimation.
• Bettine Jansen van Vuuren conceived and designed the experiments, authored or reviewed drafts of the article, acquired funding, and approved the final draft.
• Krystal A. Tolley conceived and designed the experiments, authored or reviewed drafts of the article, acquired funding, and approved the final draft.

Figure 1 A
Figure 1 A best-supported Bayesian phylogeny for Bradypodion based on the complete mitogenome.Black circles above branches represent nodes with ≥ 0.95 posterior probability and ≥ 75% bootstrap support.Gene concordance factors are indicated below branches in proportional pie charts.These pie charts represent the proportion of genes (out of 15) that support a given node within the phylogeny.Full-size DOI: 10.7717/peerj.17076/fig-1

Table 1 List of species used to generate mitogenomes for the study, with sample number and collection locality. See
Table S1 for GenBank accession numbers.

Table 4 Measures of topological congruency between gene trees.
Number of polytomies, saturation linear regression slope, mean posterior probability support, number of fully bifurcating nodes, alignment length, number of parsimony informative sites, SIC, RF distance from the complete mitogenome for each gene and partition, and Shimodaira-Hasegawa (SH) topology test p-value from the phylogenetic analyses of Bradypodion.