The mitochondrial genome as an evolutionary storyteller: a journey from dire wolf (Aenocyon dirus) to extant species of Canini (Carnivora: Canidae)

The mitochondrial genome as an evolutionary storyteller: a journey from dire wolf (Aenocyon dirus) to extant species of Canini (Carnivora: Canidae) | 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 Article The mitochondrial genome as an evolutionary storyteller: a journey from dire wolf ( Aenocyon dirus ) to extant species of Canini (Carnivora: Canidae) Iuri Batista da Silva, Igor Rodrigues-Oliveira, Renan Rocha, Priscila Assis, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8379789/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The dire wolf ( Aenocyon dirus ) was a large canid that roamed the Americas in the Late Pleistocene. The genome of A. dirus was only recently sequenced. This was a turning point in the studies on A. dirus , especially because it refuted the previous hypothesis of a close phylogenetic relationship between dire wolves and grey wolves, placing A. dirus as the sister group of the other canids. Despite this, population studies, usually based on mitochondrial genomes, were absent, and a proper description and annotation of the A. dirus mitochondrial genome are lacking. In our study, we aimed to address this issue. Using previously sequenced DNA from A. dirus , we de novo assembled the complete mitochondrial genome. We also assembled the mitochondrial genomes of several Canini species, many of which are the first descriptions so far, and conducted a phylogenetic analysis. We observed a population-level structure between the eastern and western USA samples of A. dirus . Our comparative analysis further reinforced the mitonuclear discordance by comparing our results with many mitochondrial and nuclear phylogenetic studies. This indicates a complex evolutionary history within different species, with a possible mitochondrial genome capture event happening within the group. Consequently, our work improved the current mitochondrial genome knowledge of the Canini tribe. Our findings will be valuable for future population genetics studies on A. dirus . They also conveyed insights into the mitonuclear discordance across Canini, which can help clarify introgression and mitochondrial genome capture events within the tribe. Biological sciences/Genetics/Genomics/Comparative genomics Biological sciences/Evolution/Phylogenetics Biological sciences/Biological techniques/Bioinformatics Biological sciences/Genetics/Evolutionary biology Biological sciences/Genetics/Genomics/Genome evolution ancient DNA Canidae Cerdocyonina Canina mitogenome Figures Figure 1 Figure 2 Figure 3 1. Introduction Aenocyon dirus (Leidy, 1858), often referred to as “dire wolf”, was a large canid species that roamed the Americas during the Late Pleistocene. Fossil records indicate that their presence spanned from South America, where they were scarce, to North America, where they were the most common large predator found in fossil sites (Dundas 1999; Anyonge and Roman 2006; Hodnett et al. 2009). More recently, fossil evidence of the A . dirus in Eurasia, a mandibular fragment, was found in Northeastern China (Lu et al. 2021), thus expanding their distribution in the Late Pleistocene. Unlike gray wolves and other canids, dire wolves were larger and preyed on large megafauna animals, possibly contributing to their downfall (Anyonge and Roman, 2006; DeSantis et al. 2019; Dundas, 1999). Until recently, dire wolves were thought to be closely related to the grey wolf ( Canis lupus lupus ) (Zrzavý et al. 2018). However, this changed with advancements in ancient DNA studies, which made it possible to extract high-quality DNA from fossil samples. Perri et al. (2021) obtained DNA from five different A . dirus fossil samples, with which they were able to reconstruct the phylogeny of the species alongside other canids. The results, obtained from numerous SNPs and incomplete mitochondrial sequences, refuted the previous hypothesis of a close phylogenetic relationship between dire wolves and grey wolves, placing A . dirus as the sister group of the other canids. Furthermore, the authors attempted to retrieve the mitochondrial genome using a reference-guided approach, which resulted in varying degrees of incomplete sequences (Perri et al. 2021). An unpublished study by Gedman et al. 2025 has significantly deepened the sequencing and assembly of the A . dirus genome. They also explored the mitochondrial genome using a similar approach to the one used by Perri et al. 2021, while also conducting a mitochondrial and a nuclear phylogeny. In both approaches, they find similar results to Perri et al. 2021. Despite these efforts, A . dirus is far behind many Pleistocene megafauna species that co-existed with them, which had their mitochondrial genome sequenced, assembled, annotated and used for populational studies, such as the saber-toothed cats (Paijmans et al. 2017; Rodrigues-Oliveira et al. 2023) and the mammoths (Chacón-Duque et al. 2025; Chang et al. 2017; Karpinski et al. 2020; Roca, 2008; van der Valk et al. 2021). Complete mitochondrial genome sequences are a reliable resource for phylogenetic and phylogeographic studies involving ancient DNA, as they enhance phylogenetic resolution when compared to the use of a few genes (Chacón-Duque et al. 2025; Karpinski et al. 2023, 2020; Paijmans et al. 2017; Zhang et al. 2021). The use of a mitochondrial dataset correctly partitioned by gene and codon position can further improve phylogenetic resolution due to the consideration of different evolutionary rates (Kainer and Lanfear, 2015; Miller et al. 2009; Ward et al. 2010). Moreover, this data must be generated and publicly available to be used in further studies. Therefore, we used publicly available ancient DNA libraries to de novo assemble and describe the complete mitochondrial genome of the A . dirus , as well as other species within the Canini tribe. We reconstructed the phylogenetic of these species to investigate their evolutionary history from a mitochondrial genome perspective. Our results reinforced that the A . dirus does not belong to the same lineage as the grey wolf and other Canis species and highlighted a significant mito-nuclear divergence spread across the tribe. Furthermore, our study presents the mitochondrial genome of all extant species of Canini and discusses the mito-nuclear discordance. This will be valuable for future studies aiming to understand introgression events in the unknown evolutionary history of this group. 2. Materials and Methods 2.1 Sampling of DNA-Seq libraries We sampled nine DNA-Seq libraries of A. dirus from the Sequence Read Archive (SRA) available under the BioProject PRJEB31639 (Perri et al. 2021). They successfully retrieved ancient DNA from five fossil samples, relying upon different sequencing strategies, as depicted in Table 1. DireSP fossil was located in Sheriden Pit, Ohio, and DireGWC in Guy Wilson Cave, Tennessee, both in the east of the USA, while DireGB and DireAFR were both collected near the American Falls Reservoir in Idaho, on the west of the USA (Figure 1). We also sampled DNA-Seq libraries corresponding to 11 species and two subspecies of Canina ( Canis aureus ; Canis latrans ; Canis lupaster ; Canis lupus familiaris ; Canis lupus lupus ; Canis lycaon ; Canis rufus ; Canis simensis ; Cuon alpinus ; Lupulella mesomelas ; Lupullella adusta and Lycaon pictus ); eight species of Cerdocyonina ( Atelocynus microtis , Chrysocyon brachyurus ; Lycalopex culpaeus ; Lycalopex fulvipes ; Lycalopex griseus ; Lycalopex gymnocercus ; Lycalopex sechurae and Speothos venaticus ) (Table 2), and one species of Vulpini ( Vulpes vulpes ). 2.2 Mitochondrial genomes assembly and annotation 2.2.1 De novo approach We used GetOrganelle 1.7.7 (Jin et al. 2020) to obtain the de novo mitochondrial genome sequences from the previously stated DNA-Seq libraries (Table 1 and Table 2). We applied the following parameters: five k-mers (-k 21,45,65,85,105), an auto-estimated wordsize (-w), 30 rounds (-R), and animal mitochondrial genome as the organelle type (-F animal_mt). When an assembly failed to recover the circularized mitochondrial genome, another assembly was executed, but the estimated wordsize was reduced by ten, and the number of rounds increased to 100. 2.2.2 Reference-guided approach for A. dirus Since the only successful attempt to de novo assemble the mitogenome of A . dirus was with library ERR3274215 (fossil DireGWC), we used the other libraries to recover the mitochondrial genome through a reference-based approach, except for ERR3274214, since it corresponds to the same BioSample as ERR3274215. To achieve this, we first trimmed the libraries with fastp v.0.24.0 (Chen et al. 2018), considering phred quality of >= 30 and removal of adapters. The trimmed libraries were aligned with Bowtie2 v.2.5.1 (Langmead and Salzberg 2012) under the preset ‘--very-sensitive-local’, against the de novo complete mitochondrial genome assembled in the previous step. A consensus sequence was constructed for each library using SAMtools mpileup v. 1.17 (Danecek et al. 2021) and iVar consensus tool v.1.4.2 (Grubaugh et al. 2019) with the following parameters: -q 20 -t 0.0 -c 0.8 -m 5 -n N. We aligned the resulting sequences against the de novo reference with MAFFT v.7.508 (Katoh and Standley 2013). We visualized and manually curated each reference-based mitochondrial genome sequence using AliView v.1.28 (Larsson 2014) by comparing it to the de novo sequence. For the libraries ERR3274210-ERR3274213, which correspond to the fossil sample DireAFR, an additional alignment and consensus construction were executed to reconstruct a more complete mitochondrial genome. We aligned the four sequences obtained from the iVar step using MAFFT. We then create another consensus with the tool ‘Consensus sequence from aligned FASTA’ (Keck 2020), available at Galaxy Webserver (Afgan et al. 2022), and then, as previously stated, we aligned the DireAFR consensus sequence against the reference. 2.2.3 Mitochondrial genomes annotation We annotated the mitochondrial genome sequences that we assembled using MitoZ v.3.6 (Meng et al. 2019), with the best-suited options for vertebrates (--genetic_code 2 --clade Chordata). The resulting annotation was reordered using a custom in-house Python script using trnF as the starting point for the coordinates and reannotated using the previously stated parameters. We took this approach to obtain a common starting point in the annotation files across all mitochondrial genomes. We also annotated the tRNA sequences with tRNAscan-SE v.2.0 (Chan et al. 2021) under mammalian mitochondrial as the sequence source and default parameters. 2.2.4 Relative synonymous codon usage (RSCU) analysis Using the protein-coding gene sequences, we conducted the RSCU analysis between species using the MitoRSCU tool, available at https://github.com/silvaib/MitoRSCU (Batista da Silva 2025). We run the analysis under the vertebrate mitochondrial genetic code. All sequences used in the phylogenetic inference were submitted to this step, except for A . dirus sequences, of which we only used the DireGWC reference sequence. Clustered heatmaps of RSCU’s values were generated with the same tool. 2.3 Phylogenetic analysis and divergence time estimation For the phylogenetic analysis, in addition to the mitochondrial genomes assembled and annotated in this study, we sampled the sequences of 13 protein-coding genes (PCGs) from species of Canini and Vulpini with available mitochondrial genomes at GenBank or from published papers, giving preference to RefSeq sequences when available. The complete list of species and accession numbers is available at Supplementary Table S01. Next, we aligned each protein-coding gene sequence using MAFFT under the L-INS-I approach. We visualized the resulting alignments in AliView and manually curated them. We then processed aligned gene sequences with Concatenator 0.3.1 (Vences et al. 2022), taking into account the codon position. We conduct a phylogenetic analysis using the Maximum Likelihood (ML) method with IQ-TREE v.2.3.6 (Minh et al. 2020), under 10,000 ultrafast bootstrap (Minh et al. 2013) replicates, and 10,000 bootstrap replicates for SH-aLRT (Guindon et al. 2010). All models and partitions for this analysis were estimated using the MFP+MERGE option. For the Bayesian Inference (BI) with MrBayes v.3.2.7 (Ronquist et al. 2012), we used PartitionFinder2 (Lanfear et al. 2017) under default parameters and with branch lengths = linked; models = mrbayes; model_selection = aicc, and the greedy algorithm to infer the best-fit partitioning schemes and models for the dataset. We ran the BI with 10,000,000 generations, four chains, and two runs, with sampling occurring every 100 generations and a burn-in set to 25% (sump relburnin=yes burninfrac=0.25 sumt relburnin=yes burninfrac=0.25). We used Tracer v.1.7.2 (Rambaut et al. 2018) to evaluate the Effective Sample Size (ESS) of the MCMC run. All generated trees were rooted at the Vulpini clade and visualized with iTOL v.7 (Letunic and Bork 2024). 3. Results 3.1 A . dirus mitochondrial genome description 3.1.1 De novo assembled mitochondrial genome of A . dirus We have been successful in de novo assembling the complete mitochondrial genome of the A . dirus with the library ERR3274215, which corresponds to the fossil DireGWC, with a total length of 16,476 base pairs (bp) and a GC content of 39.35% (Table 3). We observed that the mitochondrial genome shares the same features and gene arrangement found in mammals and in most vertebrates, with 13 PCGs, 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNAs), and one control region (Figure 2A and Table 4). All PCGs presented ATG as the start codon, except for ND2 , ND3 , and ND5 , in which ATA was the start codon. The stop codon was more variable between genes, with TAA in ND1 , COX1 , ATP8 , ATP6 , ND4L , ND5 , and ND6 ; TAG in ND2 , COX2 , ND3 ; and AGA in Cytb . Incomplete stop codons were found in COX3 (TA-) and ND4 (T--). The de novo assembly significantly improved the completeness of the mitochondrial genome protein-coding genes compared to the DireGWC assembly of Perri et al (2021) (Figure 2A and Supplementary Table S02). While all genes were complete in our assembly, DireGWC had the following percentage of completeness: ATP6 (37%), ATP8 (58%), COX1 (35%), COX2 (96%), COX3 (17%), Cytb (40%), ND1 (29%), ND2 (37%), ND3 (25%), ND4 (51%), ND4L (63%), ND5 (40%), and ND6 (45%). Furthermore, we recovered the complete sequences of all 22 tRNAs, the two rRNAs (ribosomal 16S subunit and ribosomal 12S subunit), and the control region. The tRNAs showed the expected anticodons for mammalian mitochondrial genomes, with duplications of tRNA-Leu and tRNA-Ser , as well as a clover-leaf secondary structure, except for tRNA-Ser2 (Supplementary Table S03). The rRNAs ranged from 955 bp in the 12S rRNA to 1,582 bp in the 16S rRNA. Finally, we retrieved the control region sequences with a total length of 1,015 bp. 3.1.2 Mapped mitochondrial genomes While we failed to obtain mitochondrial genomes from the other libraries under the de novo method, we have successfully assembled three mitochondrial genomes from different fossil samples under the reference-guided approach: DireAFR (ERR3274210-13), DireSP (ERR3274216),and DireGB (ERR3274217) (Table 3). We recovered an almost complete mitochondrial genome of DireSP (99.48%), with only 86 gap sites. As for the DireGB and DireAFR, they were incomplete, comprising 78.32% completion and 64,04% completion, respectively. Due to incompletion, we were unable to 10 tRNAs and two rRNAs in DireAFR, and small gaps were found in ND1 , COX1 , COX2 , ND4L , and ND4 , while COX3 , ND5 , ND6 , and CYTB were incomplete, with only small fragments being recovered.The DireNTC (ERR3274218) sequence we obtained was severely gapped, with less than 10% completeness. Therefore, we discarded it from further analysis. Despite having incomplete sequences, when we compared the protein-coding sequences from Perri et al (2021) against our assemblies, we observed that we significantly improved the overall completeness, even when comparing the incomplete sequences of the DireGB and DireAFR (Figure 2B and Supplementary Table S02). Our sequences' completeness was only lower than the sequences of Perri et al (2021) on two genes and only in DireGB sequences: COX2 (47% vs 63%) and ND5 (25% vs 34%). 3.2 Other Canini mitochondrial genomes We successfully assembled all the target species’ mitochondrial genomes, exhibiting the same gene arrangement and organization pattern observed in the A . dirus mitochondrial genome (Supplementary Table S04). The mitochondrial genomes of A. microtis , L . griseus , L . gymnocercus , L . fulvipes and L . culpaeus are the first descriptions for these species. We have also improved the mitochondrial genome resources of L . adusta , L . mesomelas and L . sechurae , which have only a partial mitochondrial genome available at GenBank, by assembling a complete sequence. Regarding the length of the mitochondrial genomes, excluding A . dirus , the smallest was from C . lycaon , with 16,524 bp, and the longest was from C . lupaster, with 16,763 bp (Table 5). The GC ranged from 38.17 in C . brachyurus to 39.74 in C . lupaster . ATG was the start codon in the following genes in all species: ATP6 , ATP8 , COX1 , COX2 , COX3 , ND1 , ND4 , ND4L , ND6 , and Cytb . At the same time, ATA was the start codon for ND2 , ND3 , and ND5 . Only one exception was observed, in C . l . lupus , C . l . familiaris , C . simensis , L . culpaeus , L . fulvipes , L . griseus, and L . gymnocercus , ATA was used in place of ATG as the start codon of ND6 . Minimal differences were found in the length of the rRNAs, with the 12S ranging from 954 to 957 bp, and the 16S from 1,578 to 1,584 bp. Regarding the control region, C . lycaon had the shortest one with 1,065 bp, while C . lupaster had the longest one, with 1,303 bp. The annotated features for each mitochondrial genome are available at Supplementary Table S04. 3.3 RSCU Overall, the RSCU values were very similar across all species and codons, indicating conserved codon frequency and usage (Figure 2C). We observed some clusters between species, however, the patterns remain conserved across all species. Regarding the specific codons, Arg-CGA, Leu-CTA and Arg-CGC had the highest RSCU values, while codons with G at the third position had the lowest values (Figure 2C). 3.4 Phylogenetic relationships in Canini The estimated best-fit models and partitions for the ML and the BI analysis are available at Supplementary Tables S05 and S06, respectively. We successfully reconstructed the ML and BI trees, and they presented almost the same topology, except for two polytomies in the Canini clade on the BI tree. While a polytomy was not found in the ML tree, very low branch support values were found in the corresponding relationships in the BI tree. As such, we used the BI tree to be the representative tree, while showing the branch support values obtained from the ML approach (Figure 4). The individual figures for ML and BI are available as Supplementary Figures 01 and 02, respectively. We verified that the BI analysis reached convergence with ESS values highly superior to 200, with 3880.2 for the log likelihood (LnL) and 4805.9 for the log prior (LnPr). 3.4.1 The monophyly of the Aenocyon and non-monophyly of Lupulella We recovered the four dire wolves as a well-supported monophyletic group. Our results indicated that DireGWC and DireSP are more related to each other than the other dire wolves, with high branch support values supporting this relationship in both trees (100/100/100) (Figure 4). DireAFR and DireGB are more related to each other; however, this relationship is not well-supported, likely due to the incomplete sequences of DireAFR. In any case, there is an evident separation between the DireSP + DireGWC group and the DireGB + DireAFR group. The Aenocyon clade position was uncertain between the L . adusta clade and the Cuon alpinus + L . mesomelas + Canis group, indicated by a polytomy in the BI tree. In the ML tree, L . adusta was recovered as the sister group of the Aenocyon clade with very low branch support values (22.3/69). As such, in both trees, the exact relationship between those groups could not be properly inferred. Furthermore, we recovered Lupulella as a non-monophyletic genus, separated into two distinct groups. L . mesomelas was recovered as the sister group of the Canis clade, supported by a high branch support value in both BI (100) and ML trees (100/100), while L . adusta position, as stated before, was uncertain between Aenocyon and Cuon alpinus + L . mesomelas + Canis group. 3.4.2 The Canis Clade Canis group was recovered as monophyletic, therefore, forming a clade composed of all Canis species sampled in this study. The clade was divided into two well-supported sub-clades, one comprising C . rufus , C . lycaon and C . latrans (clade A) and the other C . simensis , C . aureus , C . lupaster , C . l . familiaris and C. l . lupus (clade B). We observed very short branch lengths between the three species of clade A, with C . rufus as the sister group of C . lycaon and C . latrans . Despite this, high support values were observed in the clade, except between C . lycaon and C . latrans taxa. In clade B, we recovered C . lupaster as the sister group of Canis lupus subspecies, with high branch support values. While C . simensis and C . aureus were reconstructed as a clade on their own, the relationship between the three clades couldn’t be solved, and a polytomy was observed in the BI tree and low branch support in the ML tree (22.3/69). 3.4.3 Cerdocyonina relationships We recovered Cerdocyonina as a non-monophyletic clade, exclusively due to S . venaticus being recovered as a sister group of Canina. The branch support for this was high in both BI (98) and ML (97.6/92) trees. All remaining species of Cerdocyonina were placed in the Cerdocyonina clade, which was recovered as the sister group of S . venaticus and Canina. We reconstructed C . brachyurus as the sister group of C . thous, A. microtis, and Lycalopex species with a high support in the BI tree (98) and not-so-high values (>= 95) in the ML tree (76.3/81). C . thous and A . microtis formed a well-supported monophyletic group (100/96.2/97) and were reconstructed as the sister group of the Lycalopex clade (100/100/100). Within Lycalopex , we observed a pectinate topology for the clade, with each species being the sister group of the remaining species. As such, we recovered L . vetulus as the sister group of all other Lycalopex species , L . sechurae as the sister group of L. gymnocercus , L. griseus , L. fulvipes , Lycalopex sp. CAN003, Lycalopex sp. CAN005 and L . culpaeus , and so forth . High support values were observed in all nodes. Discussion The absence of a complete and well-annotated mitochondrial genome for the A . dirus represents a significant gap in current genomic knowledge of the species. Previous attempts to reconstruct the mitochondrial genome failed to achieve a complete and annotated sequence. In this study, we were able to reconstruct the de novo complete mitochondrial genome of the A . dirus using publicly available libraries. We further reconstructed the mitochondrial genomes of other fossil samples using a reference-guided method, improving the previously assembled protein-coding sequences. Alongside the A . dirus , we also assembled multiple Canini mitochondrial genomes, including sequences corresponding to the species' first description. Differences in RSCU values were minimal. Furthermore, we reconstructed the phylogenetic relationship of Canini. We observed that the mitochondrial genome reinforced that the A . dirus belongs to a different lineage than that of the gray wolf and other Canis species. 4.1 The Aenocyon clade Within the Aenocyon clade, there are at least two main groups: DireSP + DireGWC, sampled from which are samples from the east side of the USA, and DireGB + DireAFR, which were sampled from the west side of the USA (Figure 1). This finding corroborates the separation between eastern and western populations and corroborates the subspecies definition of Aenocyon dirus dirus for eastern and Aenocyon dirus guildayi for western dire wolves based on morphological data (Anyonge and Roman 2006). Although the number of individuals and populations sampled is not enough to confirm this, with the recent advances in ancient DNA sequencing, it is expected that there will be more DNA samples of A . dirus in the future, thus allowing us to test this hypothesis with a more robust sampling. Nonetheless, despite the limitations, our findings are an important step towards a wide populational genetic study of A . dirus . 4.1 The position of the A . dirus in the Canina tree The low support values on the relationships of A . dirus , L . pictus, and L . adusta indicate that the mitochondrial data alone cannot resolve their relationship. Interestingly, nuclear DNA was also not sufficient to determine these relationships (Perri et al. 2021), which indicates that the evolutionary history of these species may have been connected in the past. This also raises the possibility of an ancient introgression between these species or their ancestors, since introgression is recurrent among Canini, as evidenced by genetic studies revealing extensive admixture among canid species (Gopalakrishnan et al. 2018; Rutledge et al. 2012; Wang et al. 2019). For instance, the Tibetan and Himalayan wolves exhibit significant genetic contributions from an unknown lineage, complicating their classification within the Canini (Wang et al. 2020). Speciation, introgression, and incomplete lineage sorting (ILS) represent a challenge for species tree inference, often misleading traditional methods and hindering the accurate reconstruction of evolutionary histories (Hibbins and Hahn 2022). As such, ancient and present introgression must be investigated in future studies to comprehend the genetic dynamics and evolutionary story of Canina. 4.2 Relationships in the Canis clade In the Canis clade A, the close relationship between C . rufus , C . lycaon and C . latrans was expected due to current hybridisation between them (Fredrickson and Hedrick 2006; Hailer and Leonard 2008; Rutledge et al. 2012; Bohling and Waits 2015; vonHoldt et al. 2016; Hinton et al. 2017; Gopalakrishnan et al. 2018). It has been suggested that hybridization can occur as a result of anthropogenic factors (Galov et al. 2015; Barash et al. 2023), such as proximity to human cities and settlements and roaming dogs. Recurrent and intense hybridization can be harmful for species conservation from a genetic and ecological standpoint. The recovered polytomy in the Canis clade B also points to a rather complex relationship between the three sub-clades ( C . simensis , C . aureus, and C . lupaster + C . lupus ), and while their position in clade B couldn’t be asserted, each group was very well-supported. C . lupaster , previously known as Canis anthus , was recovered as the sister group of C. lupus through mitochondrial data in previous studies (Koepfli et al. 2015; Hassanin et al. 2021). In nuclear phylogenies, C . latrans is often recovered as the sister group of C . lupus (Lindblad-Toh et al. 2005; Perini et al. 2010; Perri et al. 2021; Chavez et al. 2022), with C . lupaster as their sister group when it was included in the study. 4.3 Cerdocyonina relationships The phylogeny we recovered was similar to previous studies on Cerdocyonina. The major divergence was the position of S . venaticus as the sister group of Canini, which is a finding of mitochondrial phylogenies. In nuclear phylogenies, S . venaticus is recurrently recovered as the sister group of C . brachyurus , forming the sister group of all other Cerdocyonina in nuclear phylogenies (Lindblad-Toh et al. 2005; Perini et al. 2010; Porto et al. 2019; Chavez et al. 2022). This is a major mitonuclear discordance in the Canini evolutionary history, and we discuss it in detail in the next section. Our findings on C . thous and A . microtis clade, which is the sister group of all Lycalopex species, are also recurrently, but no always, recovered in both nuclear and mitochondrial phylogenies (Perini et al. 2010; Koepfli et al. 2015; Porto et al. 2019; Chavez et al. 2022) . Within Lycalopex , the position of L . vetulus as the sister group of all other Lycalopex is recovered in both mitochondrial and nuclear phylogenies as we recovered. However, the divergence is centered around the position of L . sechurae , recovered here and in other mitochondrial phylogenies as the sister group of L . gymnocercus , L . griseus , L . fulvipes and L . culpaeus (Favarini et al. 2022), while recovered as the sister group of L . culpaeus in nuclear phylogenies (Lindblad-Toh et al. 2005; Chavez et al. 2022). Furthermore, we observed that Lycalopex sp. CAN003 and Lycalopex sp. CAN005 corresponds to mitochondrial genomes assembled from ancient DNA by Popović et al. (2020). In their study, they recovered these two samples as a sister group of L . griseus based on d-loop phylogeny, but with low branch support. The authors have also highlighted that pre-Columbian archaeological sites where CAN003 and CAN005 samples were found are located outside the currently known distribution of L . griseus . Furthermore, we recovered these two taxa as closely related to L . culpaeus , with CAN005 as the sister group to the group composed of CAN003 and L . culpaeus , with high support values. Considering our results, the unmatched distribution of L . griseus and the known distribution of L . culpaeus encompass the locations of the archaeological sites (Noguera-Urbano et al. 2016; Guntiñas et al. 2021), we found it more likely that the remains of CAN003 and CAN005 belonged to L . culpaeus specimens instead of L . griseus. 4.4 Mitonuclear discordance in Canini Comparing mitochondrial against the nuclear topologies, we observed a mitonuclear discordance in the Canini tribe, especially in Canina. We have recovered a tree topology incongruent with phylogenetic studies that used only or most of the data from a nuclear source: S . venaticus was recovered as the sister group of Canina and not grouped with other Cerdocyonina. Lupulella was retrieved as a non-monophyletic group. The relationships between L . pictus , A . dirus , L . adusta , C . alpinus, and L . mesomelas lineages also differed between our findings and those that used nuclear data. In nuclear data studies, S . venaticus has been recovered inside Cerdocyonina (Perini et al. 2010; Koepfli et al. 2015; Lamarca and Schrago 2020; Chavez et al. 2022). Our results, however, have been recovered in other studies, in which S . venaticus was recovered as the sister group of Canina and not Cerdocyonina (Hassanin et al. 2021; Lamarca and Schrago, 2020). One possible hypothesis is that ancient introgression may have happened between S. venaticus and Canina lineages, even extinct ones. Although generally rare, hybridization between subtribes was recently discovered in Canini, with a hybrid of C . l . familiaris (Canina) and L . gymnocercus (Cerdocyinina) (Szynwelski et al. 2023). These species belong to different subtribes and are estimated that their lineages diverged between ~6.8 Ma and ~3.5 Ma (Perini et al. 2010; Perri et al. 2021; Chavez et al. 2022). Despite that, they were able to generate a hybrid. Therefore, such hybridization is possible to have occurred in the evolutionary history of S . venaticus and some Canina species, which may have led to a mitochondrial genome capture event. However, further investigation is needed to clarify the events that resulted in this mitonuclear discordance and test this hypothesis. In the relationships between L . pictus , A . dirus , L . adusta , C . alpinus, and L . mesomelas , we also observed a mitonuclear discordance, especially in Lupulella . We identified evidence of this discordance by comparing our results with those of previously published phylogenetic studies. In the study of Lindblad-Toh et al. (2005), they used a dataset composed of only nuclear sequences, encompassing exonic and intronic sequences, and they not only recovered Lupulella as a monophyletic group but also inferred that Lupulella was the sister group of a clade composed of L . pictus , C . alpinus, and Canis species. The exact phylogenetic relationships were also reconstructed by Perini et al. (2010), who based their phylogenetic inference on 22 nuclear and three mitochondrial sequences; by Koepfli et al. (2015), who used sequences from segments of three exons and 17 introns; and by Lamarca and Schrago (2020), with the use of different orthologous segments and multiple nuclear genes based on the work of Lindblad-Toh et al. (2005). Similar results were also recovered by Zrzavý et al. (2018) with a diverse characters, including morphological, ontogenetic and cytogenetic, behaviour, ecology, and molecular data; by Perri et al. (2021), who used up to 28 mb of nuclear sequence alignments and by Chavez et al. (2022), that used a robust dataset composed of 6,716 regions of the nuclear genome, each of them with 25 kb, from 31 nuclear genomes of 22 canid species. In the work of Bardeleben et al. (2005), the dataset composed of only nuclear sequences resulted in an exception on the position of Lupulella on the Canina evolutionary tree, since they recovered the jackals as the sister group of Cerdocyonina, with the latter being a clade inside Canina. In the data set composed of nuclear and mitochondrial sequences, Lupulella was recovered as the sister group of Canis species. The non-monophyly of Lupulella was observed in their study in the phylogenetic tree based only on mtDNA sequences ( Cytb , COX1 and COX2 ), with L . adusta as the sister group of a group composed of C . alpinus , L . mesomelas , C . aureus , C . latrans , C . lupus lupus and C . lupus familiaris . This topology has also been recovered by Hassanin et al. (2021) through the use of mitochondrial genomes. While recovering the non-monophyletic nature of Lupulella with the same topology we recovered by using 13 PCGs + two rRNAs, Koepfli et al. (2015) observed a different tree topology based only on Cytb sequences, with a group composed of L . mesomelas , L . adusta , L . pictus, and C . alpinus . However, teir nuclear phylogenetic tree was similar to the nuclear topologies with Lupulella as a sister clade of L . pictus . Atickem et al. (2018) also recovered Lupulella as a non-monophyletic using Cytb sequences, but with a different topology, with a group composed of L . pictus , L . adusta, and C . alpinus , the sister group of L . mesomelas and Canis species. Furthermore, mitonuclear discordance is also found within the Lycalopex and Canis genera. When comparing our results with the multiple mitochondrial and nuclear phylogenetic studies, it is clear that mitonuclear discordance is spread across Canini. Mitonuclear discordance, which can occur without signals in the nuclear genome, has been proposed as the result of incomplete lineage sorting (McKay and Zink 2010; DeRaad et al. 2023), mitochondrial genome capture and introgression (Andersen et al. 2021; Bonnet et al. 2017; Mikkelsen and Weir, 2023). Introgression has been registered multiple times in Canini lineages. Interspecific hybridisation is common among canids, and numerous events have been documented in Canina and Cerdocyonina (Garcez et al. 2024). While the mitochondrial genome, as a phylogenetic marker, suffers from hybridization that occurred in the past or even in the present, it is a powerful tool to discover such events. Instead of viewing the mitochondrial genome as an insufficient source of phylogenetic signal in canids, we propose to see it as a fundamental tool for understanding admixture in the evolutionary history of the group, especially when only weak signs of hybridization are present in the nuclear genome. Therefore, efforts in describing and annotating mitochondrial genomes of the group must go on, strengthening the resources and enabling a more complete and in-depth understanding of hybridization in the group. Our findings reinforce the relevance of exploring mitochondrial genomes even in well-studied groups, such as Canini. Our findings reinforce the work of Perri et al. (2021), in which A. dirus was found not to be closely related to Canis lupus and belongs to a different lineage of Canina. Therefore, it is unlikely that they intimately resembled the Canis lupus lupus as dire wolves are usually reconstructed. Furthermore, we expanded the investigation on the mitonuclear discordance across Canini by presenting a more robust mitochondrial phylogenetic analysis. We expect that the description of the first complete mitochondrial genome of A. dirus and the advances in DNA sequencing from fossil samples will trigger complete mitochondrial genetic studies on their populations, such as phylogeographic studies, that have been done in other megafauna species using complete mitochondrial DNA. We expect that the description of almost the mitochondrial genome of Canini will improve genetic studies based on mitochondrial genomes, allowing investigations into introgression between species, leading to a better understanding of evolutionary events that have occurred in the evolutionary history of Canini. Declarations Acknowledgments Unpublished sequencing data for Lycaon pictus (SRR10331591) are used with permission from the DNA Zoo Consortium ( dnazoo.org ). Data Archiving All raw DNA-Seq data used in this work are publicly available at the Sequence Read Archive (Tables 1 and 2). 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Zoologica Scripta 47:373–389. https://doi.org/10.1111/zsc.12293 Additional Declarations There is no duality of interest Supplementary Files SupplementaryFile1.pdf Supplementary File 01 SupplementaryTable01.xlsx Supplementary Table 01 SupplementaryFigures.pdf Supplementary Figures Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8379789","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":561385251,"identity":"e2b8f1fb-10f2-4f1d-8623-81c502205098","order_by":0,"name":"Iuri Batista da Silva","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYBACNiA+wNjAwMAvAeZLyBCvRXIGA4iS4CHOKpBagxtgioGwFj6J3IcHGHcw2Bvfbj7+6EaNBQ8D++GjG/A6TCLd4ADjGYbEbXeOJTbnHAM6jCct7QZ+LWlAv7QxJJjdyDFszmEDapHgMSNKi73xDJCWfyRoYdwgAdSS20aMFp5nDAcSz0gkzriRljg7t0+Ch42QX+Tb05g/fNxhY88/I/nA55xvdXL87IeP4dUCBgkMEkj2ElQ+CkbBKBgFo4AgAAAuxkEjmmZ3lAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-2788-5665","institution":"Federal University of Minas Gerais (UFMG)","correspondingAuthor":true,"prefix":"","firstName":"Iuri","middleName":"Batista da","lastName":"Silva","suffix":""},{"id":561385256,"identity":"ddd3c69d-57a5-4cd3-9d90-da1989b2d928","order_by":1,"name":"Igor Rodrigues-Oliveira","email":"","orcid":"","institution":"Federal University of Minas Gerais (UFMG)","correspondingAuthor":false,"prefix":"","firstName":"Igor","middleName":"","lastName":"Rodrigues-Oliveira","suffix":""},{"id":561385258,"identity":"c28f9222-05a1-4621-af5a-382f5fa25734","order_by":2,"name":"Renan Rocha","email":"","orcid":"","institution":"University of Mogi das Cruzes (UMC)","correspondingAuthor":false,"prefix":"","firstName":"Renan","middleName":"","lastName":"Rocha","suffix":""},{"id":561385259,"identity":"c900b8cc-e6d6-491b-8702-b5197ad32a54","order_by":3,"name":"Priscila Assis","email":"","orcid":"","institution":"Federal University of Viçosa (UFV)","correspondingAuthor":false,"prefix":"","firstName":"Priscila","middleName":"","lastName":"Assis","suffix":""},{"id":561385261,"identity":"90923663-7727-4d7f-b4c3-87727b10c5b7","order_by":4,"name":"Luiz Pimentel","email":"","orcid":"","institution":"Federal University of Minas Gerais (UFMG)","correspondingAuthor":false,"prefix":"","firstName":"Luiz","middleName":"","lastName":"Pimentel","suffix":""},{"id":561385265,"identity":"b8616182-88c3-4c61-a1eb-3f75adee9dd7","order_by":5,"name":"Letícia Simas","email":"","orcid":"","institution":"São Paulo State University (UNESP)","correspondingAuthor":false,"prefix":"","firstName":"Letícia","middleName":"","lastName":"Simas","suffix":""},{"id":561385266,"identity":"68b7bee3-bed2-4b44-a415-f41b1e262f52","order_by":6,"name":"Karine Frehner Kavalco","email":"","orcid":"https://orcid.org/0000-0002-4955-2792","institution":"Universidade Federal de Viçosa","correspondingAuthor":false,"prefix":"","firstName":"Karine","middleName":"Frehner","lastName":"Kavalco","suffix":""},{"id":561385267,"identity":"8125ed4c-ccf6-49f7-b834-38b5f3d0438c","order_by":7,"name":"Caroline Garcia","email":"","orcid":"","institution":"State University of Southwestern Bahia (UESB)","correspondingAuthor":false,"prefix":"","firstName":"Caroline","middleName":"","lastName":"Garcia","suffix":""},{"id":561385268,"identity":"35a74c9d-fa49-4874-a5d5-015a06621436","order_by":8,"name":"Fabiano Menegidio","email":"","orcid":"","institution":"University of Mogi das Cruzes (UMC)","correspondingAuthor":false,"prefix":"","firstName":"Fabiano","middleName":"","lastName":"Menegidio","suffix":""},{"id":561385270,"identity":"d1b08cd0-93ad-4c3e-a16c-e0760e37533a","order_by":9,"name":"Rubens Pasa","email":"","orcid":"","institution":"Federal University of Viçosa","correspondingAuthor":false,"prefix":"","firstName":"Rubens","middleName":"","lastName":"Pasa","suffix":""}],"badges":[],"createdAt":"2025-12-16 21:11:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8379789/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8379789/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100998116,"identity":"1ee6cad9-83b4-4910-9514-0be7ebf28af9","added_by":"auto","created_at":"2026-01-23 15:52:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":612695,"visible":true,"origin":"","legend":"\u003cp\u003eGeographic localization of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus \u003c/em\u003efossil samples sequenced by Perri et al. 2021, under the Quaternary deposits mapped in the USA. Geological layers extracted from the geodatabase Cooperative National Geologic Map — Quaternary geology (Barrette et al. 2025).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8379789/v1/097053ba05fef03252296a8e.png"},{"id":100998120,"identity":"6d1b5699-87e7-4045-b619-079ade7e9c46","added_by":"auto","created_at":"2026-01-23 15:53:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3965754,"visible":true,"origin":"","legend":"\u003cp\u003eA) The circular representation of the complete mitochondrial genome of the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e (DireGWC) obtained from the \u003cem\u003ede novo\u0026nbsp;\u003c/em\u003eapproach. \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e silhouette was obtained from \u003cu\u003ehttps://www.phylopic.org/\u003c/u\u003e. B) Percentage of completion of the protein-coding sequences compared to the sequences obtained by Perri et al. 2021. C) Clustered heatmap showing the RSCU values per codon and species. Colors close to yellow indicate a high RSCU value, while colors close to dark blue indicate a low RSCU value. The asterisk (*) indicates mitochondrial genomes assembled in this study.\u003c/p\u003e","description":"","filename":"Figure21.png","url":"https://assets-eu.researchsquare.com/files/rs-8379789/v1/673fac5968411084c8ca813d.png"},{"id":100998119,"identity":"d3014462-9471-4190-b6b0-c79619f57983","added_by":"auto","created_at":"2026-01-23 15:53:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2099297,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree from the BI approach. Colored lines indicate the two Canini subtribes: Cerdocyonina (lavender) and Canina (cyan). Node labels indicate posterior probability, SH-aLRT and ultrafast bootstrap values, respectively. Polytomies are indicated by the black dot in the respective nodes and with the bootstrap values indicated by P, followed by the SH-aLRT and ultrafast bootstrap values. Silhouettes were obtained from \u003cu\u003ehttps://www.phylopic.org/\u003c/u\u003e and are colour-coded according to the corresponding genus. The asterisk (*) indicates mitochondrial genomes assembled in this study.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8379789/v1/c5e6976d3bd7bd367ffd1547.png"},{"id":108804273,"identity":"14b9d582-d694-4adc-8d22-36815c268c1a","added_by":"auto","created_at":"2026-05-08 15:18:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7492140,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8379789/v1/56c0ebb6-89a3-4478-9369-b3d1bc9f8918.pdf"},{"id":100998117,"identity":"46852e58-c7d9-45d5-89b5-d4f1067e9e94","added_by":"auto","created_at":"2026-01-23 15:52:59","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":90518,"visible":true,"origin":"","legend":"Supplementary File 01","description":"","filename":"SupplementaryFile1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8379789/v1/feaf0fd714381432b140713a.pdf"},{"id":100998118,"identity":"6fe5a057-c956-464f-bd22-174c7218d76c","added_by":"auto","created_at":"2026-01-23 15:53:00","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":58557,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 01\u003c/p\u003e","description":"","filename":"SupplementaryTable01.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8379789/v1/0413b0714bc053962d7b5812.xlsx"},{"id":100998122,"identity":"738a0a29-edb9-4fa2-b621-dc65627fe299","added_by":"auto","created_at":"2026-01-23 15:53:00","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":559935,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figures\u003c/p\u003e","description":"","filename":"SupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8379789/v1/d7dd2292e85a8261bf0bf000.pdf"}],"financialInterests":"There is no duality of interest","formattedTitle":"The mitochondrial genome as an evolutionary storyteller: a journey from dire wolf (\u003ci\u003eAenocyon dirus\u003c/i\u003e) to extant species of Canini (Carnivora: Canidae)","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cem\u003eAenocyon dirus\u003c/em\u003e (Leidy, 1858), often referred to as \u0026ldquo;dire wolf\u0026rdquo;, was a large canid species that roamed the Americas during the Late Pleistocene. Fossil records indicate that their presence spanned from South America, where they were scarce, to North America, where they were the most common large predator found in fossil sites (Dundas 1999; Anyonge and Roman 2006; Hodnett et al. 2009). More recently, fossil evidence of the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e in Eurasia, a mandibular fragment, was found in Northeastern China (Lu et al. 2021), thus expanding their distribution in the Late Pleistocene. Unlike gray wolves and other canids, dire wolves were larger and preyed on large megafauna animals, possibly contributing to their downfall (Anyonge and Roman, 2006; DeSantis et al. 2019; Dundas, 1999).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUntil recently, dire wolves were thought to be closely related to the grey wolf (\u003cem\u003eCanis lupus lupus\u003c/em\u003e) (Zrzav\u0026yacute; et al. 2018). However, this changed with advancements in ancient DNA studies, which made it possible to extract high-quality DNA from fossil samples. Perri et al. (2021) obtained DNA from five different \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e fossil samples, with which they were able to reconstruct the phylogeny of the species alongside other canids. The results, obtained from numerous SNPs and incomplete mitochondrial sequences, refuted the previous hypothesis of a close phylogenetic relationship between dire wolves and grey wolves, placing \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e as the sister group of the other canids. Furthermore, the authors attempted to retrieve the mitochondrial genome using a reference-guided approach, which resulted in varying degrees of incomplete sequences (Perri et al. 2021). An unpublished study by Gedman et al. 2025 has significantly deepened the sequencing and assembly of the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e genome. They also explored the mitochondrial genome using a similar approach to the one used by Perri et al. 2021, while also conducting a mitochondrial and a nuclear phylogeny. In both approaches, they find similar results to Perri et al. 2021.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDespite these efforts, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e is far behind many Pleistocene megafauna species that co-existed with them, which had their mitochondrial genome sequenced, assembled, annotated and used for populational studies, such as the saber-toothed cats (Paijmans et al. 2017; Rodrigues-Oliveira et al. 2023) and the mammoths (Chac\u0026oacute;n-Duque et al. 2025; Chang et al. 2017; Karpinski et al. 2020; Roca, 2008; van der Valk et al. 2021). Complete mitochondrial genome sequences are a reliable resource for phylogenetic and phylogeographic studies involving ancient DNA, as they enhance phylogenetic resolution when compared to the use of a few genes (Chac\u0026oacute;n-Duque et al. 2025; Karpinski et al. 2023, 2020; Paijmans et al. 2017; Zhang et al. 2021). The use of a mitochondrial dataset correctly partitioned by gene and codon position can further improve phylogenetic resolution due to the consideration of different evolutionary rates (Kainer and Lanfear, 2015; Miller et al. 2009; Ward et al. 2010). Moreover, this data must be generated and publicly available to be used in further studies.\u003c/p\u003e\n\u003cp\u003eTherefore, we used publicly available ancient DNA libraries to \u003cem\u003ede novo\u003c/em\u003e assemble and describe the complete mitochondrial genome of the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e, as well as other species within the Canini tribe. We reconstructed the phylogenetic of these species to investigate their evolutionary history from a mitochondrial genome perspective. Our results reinforced that the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e does not belong to the same lineage as the grey wolf and other \u003cem\u003eCanis\u003c/em\u003e species and highlighted a significant mito-nuclear divergence spread across the tribe. Furthermore, our study presents the mitochondrial genome of all extant species of Canini and discusses the mito-nuclear discordance. This will be valuable for future studies aiming to understand introgression events in the unknown evolutionary history of this group.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003ch2\u003e2.1 Sampling of DNA-Seq libraries\u003c/h2\u003e\n\u003cp\u003e We sampled nine DNA-Seq libraries of \u003cem\u003eA. dirus\u003c/em\u003efrom the Sequence Read Archive (SRA) available under the BioProject PRJEB31639 (Perri et al. 2021). They successfully retrieved ancient DNA from five fossil samples, relying upon different sequencing strategies, as depicted in Table 1. DireSP fossil was located in Sheriden Pit, Ohio, and DireGWC in Guy Wilson Cave, Tennessee, both in the east of the USA, while DireGB and DireAFR were both collected near the American Falls Reservoir in Idaho, on the west of the USA (Figure 1).\u003c/p\u003e\n\u003cp\u003eWe also sampled DNA-Seq libraries corresponding to 11 species and two subspecies of Canina (\u003cem\u003eCanis aureus\u003c/em\u003e; \u003cem\u003eCanis latrans\u003c/em\u003e; \u003cem\u003eCanis lupaster\u003c/em\u003e; \u003cem\u003eCanis lupus familiaris\u003c/em\u003e; \u003cem\u003eCanis lupus lupus\u003c/em\u003e; \u003cem\u003eCanis lycaon\u003c/em\u003e; \u003cem\u003eCanis rufus\u003c/em\u003e; \u003cem\u003eCanis simensis\u003c/em\u003e; \u003cem\u003eCuon alpinus\u003c/em\u003e; \u003cem\u003eLupulella mesomelas\u003c/em\u003e; \u003cem\u003eLupullella adusta\u003c/em\u003e and \u003cem\u003eLycaon pictus\u003c/em\u003e); eight species of Cerdocyonina (\u003cem\u003eAtelocynus microtis\u003c/em\u003e, \u003cem\u003eChrysocyon brachyurus\u003c/em\u003e; \u003cem\u003eLycalopex culpaeus\u003c/em\u003e; \u003cem\u003eLycalopex fulvipes\u003c/em\u003e; \u003cem\u003eLycalopex griseus\u003c/em\u003e; \u003cem\u003eLycalopex gymnocercus\u003c/em\u003e; \u003cem\u003eLycalopex sechurae\u003c/em\u003e and \u003cem\u003eSpeothos venaticus\u003c/em\u003e) (Table 2), and one species of Vulpini (\u003cem\u003eVulpes vulpes\u003c/em\u003e).\u003c/p\u003e\n\u003ch2\u003e2.2 Mitochondrial genomes assembly and annotation\u003c/h2\u003e\n\u003ch3\u003e2.2.1 \u003cem\u003eDe novo\u003c/em\u003e approach\u003c/h3\u003e\n\u003cp\u003eWe used GetOrganelle 1.7.7 (Jin et al. 2020) to obtain the de novo mitochondrial genome sequences from the previously stated DNA-Seq libraries (Table 1 and Table 2). We applied the following parameters: five k-mers (-k 21,45,65,85,105), an auto-estimated wordsize (-w), 30 rounds (-R), and animal mitochondrial genome as the organelle type (-F animal_mt). When an assembly failed to recover the circularized mitochondrial genome, another assembly was executed, but the estimated wordsize was reduced by ten, and the number of rounds increased to 100.\u003c/p\u003e\n\u003ch3\u003e2.2.2 Reference-guided approach for \u003cem\u003eA. dirus\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003eSince the only successful attempt to \u003cem\u003ede novo\u003c/em\u003e assemble the mitogenome of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e was with library ERR3274215 (fossil DireGWC), we used the other libraries to recover the mitochondrial genome through a reference-based approach, except for ERR3274214, since it corresponds to the same BioSample as ERR3274215. To achieve this, we first trimmed the libraries with fastp v.0.24.0 (Chen et al. 2018), considering phred quality of \u0026gt;= 30 and removal of adapters. The trimmed libraries were aligned with Bowtie2 v.2.5.1 (Langmead and Salzberg 2012) under the preset \u0026lsquo;--very-sensitive-local\u0026rsquo;, against the \u003cem\u003ede novo\u003c/em\u003e complete mitochondrial genome assembled in the previous step. A consensus sequence was constructed for each library using SAMtools mpileup v. 1.17 (Danecek et al. 2021) and iVar consensus tool v.1.4.2 (Grubaugh et al. 2019) with the following parameters: -q 20 -t 0.0 -c 0.8 -m 5 -n N. We aligned the resulting sequences against the de novo reference with MAFFT v.7.508 (Katoh and Standley 2013). We visualized and manually curated each reference-based mitochondrial genome sequence using AliView v.1.28 (Larsson 2014) by comparing it to the \u003cem\u003ede novo\u003c/em\u003e sequence.\u003c/p\u003e\n\u003cp\u003eFor the libraries ERR3274210-ERR3274213, which correspond to the fossil sample DireAFR, an additional alignment and consensus construction were executed to reconstruct a more complete mitochondrial genome. We aligned the four sequences obtained from the iVar step using MAFFT. We then create another consensus with the tool \u0026lsquo;Consensus sequence from aligned FASTA\u0026rsquo; (Keck 2020), available at Galaxy Webserver (Afgan et al. 2022), and then, as previously stated, we aligned the DireAFR consensus sequence against the reference.\u003c/p\u003e\n\u003ch3\u003e2.2.3 Mitochondrial genomes annotation\u003c/h3\u003e\n\u003cp\u003e\u003cstrong\u003e \u003c/strong\u003eWe annotated the mitochondrial genome sequences that we assembled using MitoZ v.3.6 (Meng et al. 2019), with the best-suited options for vertebrates (--genetic_code 2 --clade Chordata). The resulting annotation was reordered using a custom in-house Python script using trnF as the starting point for the coordinates and reannotated using the previously stated parameters. We took this approach to obtain a common starting point in the annotation files across all mitochondrial genomes. We also annotated the tRNA sequences with tRNAscan-SE v.2.0 (Chan et al. 2021) under mammalian mitochondrial as the sequence source and default parameters.\u003c/p\u003e\n\u003ch3\u003e2.2.4 Relative synonymous codon usage (RSCU) analysis\u003c/h3\u003e\n\u003cp\u003e Using the protein-coding gene sequences, we conducted the RSCU analysis between species using the MitoRSCU tool, available at https://github.com/silvaib/MitoRSCU (Batista da Silva 2025). We run the analysis under the vertebrate mitochondrial genetic code. All sequences used in the phylogenetic inference were submitted to this step, except for \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e sequences, of which we only used the DireGWC reference sequence. Clustered heatmaps of RSCU\u0026rsquo;s values were generated with the same tool.\u003c/p\u003e\n\u003ch2\u003e2.3 Phylogenetic analysis and divergence time estimation\u003c/h2\u003e\n\u003cp\u003eFor the phylogenetic analysis, in addition to the mitochondrial genomes assembled and annotated in this study, we sampled the sequences of 13 protein-coding genes (PCGs) from species of Canini and Vulpini with available mitochondrial genomes at GenBank or from published papers, giving preference to RefSeq sequences when available. The complete list of species and accession numbers is available at Supplementary Table S01. Next, we aligned each protein-coding gene sequence using MAFFT under the L-INS-I approach. We visualized the resulting alignments in AliView and manually curated them. We then processed aligned gene sequences with Concatenator 0.3.1 (Vences et al. 2022), taking into account the codon position.\u003c/p\u003e\n\u003cp\u003eWe conduct a phylogenetic analysis using the Maximum Likelihood (ML) method with IQ-TREE v.2.3.6 (Minh et al. 2020), under 10,000 ultrafast bootstrap (Minh et al. 2013) replicates, and 10,000 bootstrap replicates for SH-aLRT (Guindon et al. 2010). All models and partitions for this analysis were estimated using the MFP+MERGE option. For the Bayesian Inference (BI) with MrBayes v.3.2.7 (Ronquist et al. 2012), we used PartitionFinder2 (Lanfear et al. 2017) under default parameters and with branch lengths = linked; models = mrbayes; model_selection = aicc, and the greedy algorithm to infer the best-fit partitioning schemes and models for the dataset. We ran the BI with 10,000,000 generations, four chains, and two runs, with sampling occurring every 100 generations and a burn-in set to 25% (sump relburnin=yes burninfrac=0.25 sumt relburnin=yes burninfrac=0.25). We used Tracer v.1.7.2 (Rambaut et al. 2018) to evaluate the Effective Sample Size (ESS) of the MCMC run. All generated trees were rooted at the Vulpini clade and visualized with iTOL v.7 (Letunic and Bork 2024).\u003c/p\u003e"},{"header":"3. Results","content":"\u003ch2\u003e3.1 \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e mitochondrial genome description\u003c/h2\u003e\n\u003ch3\u003e3.1.1 De novo assembled mitochondrial genome of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003e We have been successful in \u003cem\u003ede novo\u003c/em\u003e assembling the complete mitochondrial genome of the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003ewith the library ERR3274215, which corresponds to the fossil DireGWC, with a total length of 16,476 base pairs (bp) and a GC content of 39.35% (Table 3). We observed that the mitochondrial genome shares the same features and gene arrangement found in mammals and in most vertebrates, with 13 PCGs, 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNAs), and one control region (Figure 2A and Table 4). All PCGs presented ATG as the start codon, except for \u003cem\u003eND2\u003c/em\u003e, \u003cem\u003eND3\u003c/em\u003e, and \u003cem\u003eND5\u003c/em\u003e, in which ATA was the start codon. The stop codon was more variable between genes, with TAA in \u003cem\u003eND1\u003c/em\u003e, \u003cem\u003eCOX1\u003c/em\u003e, \u003cem\u003eATP8\u003c/em\u003e, \u003cem\u003eATP6\u003c/em\u003e, \u003cem\u003eND4L\u003c/em\u003e, \u003cem\u003eND5\u003c/em\u003e, and \u003cem\u003eND6\u003c/em\u003e; TAG in \u003cem\u003eND2\u003c/em\u003e, \u003cem\u003eCOX2\u003c/em\u003e, \u003cem\u003eND3\u003c/em\u003e; and AGA in \u003cem\u003eCytb\u003c/em\u003e. Incomplete stop codons were found in \u003cem\u003eCOX3\u003c/em\u003e (TA-) and \u003cem\u003eND4\u003c/em\u003e (T--).\u003c/p\u003e\n\u003cp\u003eThe de novo assembly significantly improved the completeness of the mitochondrial genome protein-coding genes compared to the DireGWC assembly of Perri et al (2021) (Figure 2A and Supplementary Table S02). While all genes were complete in our assembly, DireGWC had the following percentage of completeness: \u003cem\u003eATP6\u003c/em\u003e (37%), \u003cem\u003eATP8\u003c/em\u003e (58%), \u003cem\u003eCOX1\u003c/em\u003e (35%), \u003cem\u003eCOX2\u003c/em\u003e (96%), \u003cem\u003eCOX3\u003c/em\u003e (17%), \u003cem\u003eCytb\u003c/em\u003e (40%), \u003cem\u003eND1\u003c/em\u003e (29%), \u003cem\u003eND2\u003c/em\u003e (37%), \u003cem\u003eND3\u003c/em\u003e (25%), \u003cem\u003eND4\u003c/em\u003e (51%), \u003cem\u003eND4L\u003c/em\u003e (63%), \u003cem\u003eND5\u003c/em\u003e (40%), and \u003cem\u003eND6\u003c/em\u003e (45%).\u003c/p\u003e\n\u003cp\u003eFurthermore, we recovered the complete sequences of all 22 tRNAs, the two rRNAs (ribosomal 16S subunit\u003cem\u003e\u003c/em\u003eand ribosomal 12S subunit), and the control region. The tRNAs showed the expected anticodons for mammalian mitochondrial genomes, with duplications of \u003cem\u003etRNA-Leu\u003c/em\u003e and \u003cem\u003etRNA-Ser\u003c/em\u003e, as well as a clover-leaf secondary structure, except for \u003cem\u003etRNA-Ser2\u003c/em\u003e (Supplementary Table S03). The rRNAs ranged from 955 bp in the \u003cem\u003e12S\u003c/em\u003e rRNA to 1,582 bp in the \u003cem\u003e16S\u003c/em\u003e rRNA. Finally, we retrieved the control region sequences with a total length of 1,015 bp.\u003c/p\u003e\n\u003ch3\u003e3.1.2 Mapped mitochondrial genomes\u003c/h3\u003e\n\u003cp\u003eWhile we failed to obtain mitochondrial genomes from the other libraries under the de novo method, we have successfully assembled three mitochondrial genomes from different fossil samples under the reference-guided approach: DireAFR (ERR3274210-13), DireSP (ERR3274216),and DireGB (ERR3274217) (Table 3). We recovered an almost complete mitochondrial genome of DireSP (99.48%), with only 86 gap sites. As for the DireGB and DireAFR, they were incomplete, comprising 78.32% completion and 64,04% completion, respectively. Due to incompletion, we were unable to 10 tRNAs and two rRNAs in DireAFR, and small gaps were found in \u003cem\u003eND1\u003c/em\u003e, \u003cem\u003eCOX1\u003c/em\u003e, \u003cem\u003eCOX2\u003c/em\u003e, \u003cem\u003eND4L\u003c/em\u003e, and \u003cem\u003eND4\u003c/em\u003e, while \u003cem\u003eCOX3\u003c/em\u003e, \u003cem\u003eND5\u003c/em\u003e, \u003cem\u003eND6\u003c/em\u003e, and \u003cem\u003eCYTB\u003c/em\u003e were incomplete, with only small fragments being recovered.The DireNTC (ERR3274218) sequence we obtained was severely gapped, with less than 10% completeness. Therefore, we discarded it from further analysis.\u003c/p\u003e\n\u003cp\u003eDespite having incomplete sequences, when we compared the protein-coding sequences from Perri et al (2021) against our assemblies, we observed that we significantly improved the overall completeness, even when comparing the incomplete sequences of the DireGB and DireAFR (Figure 2B and Supplementary Table S02). Our sequences\u0026apos; completeness was only lower than the sequences of Perri et al (2021) on two genes and only in DireGB sequences: \u003cem\u003eCOX2\u003c/em\u003e (47% vs 63%) and \u003cem\u003eND5\u003c/em\u003e (25% vs 34%).\u003c/p\u003e\n\u003ch2\u003e3.2 Other Canini mitochondrial genomes\u003c/h2\u003e\n\u003cp\u003e We successfully assembled all the target species\u0026rsquo; mitochondrial genomes, exhibiting the same gene arrangement and organization pattern observed in the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e mitochondrial genome (Supplementary Table S04). The mitochondrial genomes of \u003cem\u003eA.\u003c/em\u003e \u003cem\u003emicrotis\u003c/em\u003e, \u003cem\u003eL\u003c/em\u003e. \u003cem\u003egriseus\u003c/em\u003e, \u003cem\u003eL\u003c/em\u003e. \u003cem\u003egymnocercus\u003c/em\u003e,\u003cem\u003eL\u003c/em\u003e. \u003cem\u003efulvipes\u003c/em\u003e and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eculpaeus\u003c/em\u003e are the first descriptions for these species. We have also improved the mitochondrial genome resources of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eadusta\u003c/em\u003e, \u003cem\u003eL\u003c/em\u003e. \u003cem\u003emesomelas\u003c/em\u003e and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003esechurae\u003c/em\u003e, which have only a partial mitochondrial genome available at GenBank, by assembling a complete sequence.\u003c/p\u003e\n\u003cp\u003eRegarding the length of the mitochondrial genomes, excluding \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e, the smallest was from \u003cem\u003eC\u003c/em\u003e.\u003cem\u003elycaon\u003c/em\u003e, with 16,524 bp, and the longest was from \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elupaster,\u003c/em\u003e with 16,763 bp (Table 5). The GC ranged from 38.17 in \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachyurus\u003c/em\u003e to 39.74 in \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elupaster\u003c/em\u003e. ATG was the start codon in the following genes in all species: \u003cem\u003eATP6\u003c/em\u003e, \u003cem\u003eATP8\u003c/em\u003e, \u003cem\u003eCOX1\u003c/em\u003e,\u003cem\u003eCOX2\u003c/em\u003e, \u003cem\u003eCOX3\u003c/em\u003e, \u003cem\u003eND1\u003c/em\u003e, \u003cem\u003eND4\u003c/em\u003e, \u003cem\u003eND4L\u003c/em\u003e, \u003cem\u003eND6\u003c/em\u003e, and \u003cem\u003eCytb\u003c/em\u003e. At the same time, ATA was the start codon for\u003cem\u003eND2\u003c/em\u003e, \u003cem\u003eND3\u003c/em\u003e, and \u003cem\u003eND5\u003c/em\u003e. Only one exception was observed, in \u003cem\u003eC\u003c/em\u003e. \u003cem\u003el\u003c/em\u003e. \u003cem\u003elupus\u003c/em\u003e, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003el\u003c/em\u003e. \u003cem\u003efamiliaris\u003c/em\u003e, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esimensis\u003c/em\u003e, \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eculpaeus\u003c/em\u003e, \u003cem\u003eL\u003c/em\u003e. \u003cem\u003efulvipes\u003c/em\u003e, \u003cem\u003eL\u003c/em\u003e. \u003cem\u003egriseus,\u003c/em\u003e and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003egymnocercus\u003c/em\u003e, ATA was used in place of ATG as the start codon of \u003cem\u003eND6\u003c/em\u003e. Minimal differences were found in the length of the rRNAs, with the \u003cem\u003e12S\u003c/em\u003e ranging from 954 to 957 bp, and the \u003cem\u003e16S\u003c/em\u003e from 1,578 to 1,584 bp. Regarding the control region, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elycaon\u003c/em\u003e had the shortest one with 1,065 bp, while \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elupaster\u003c/em\u003e had the longest one, with 1,303 bp. The annotated features for each mitochondrial genome are available at Supplementary Table S04.\u003c/p\u003e\n\u003ch2\u003e3.3 RSCU\u003c/h2\u003e\n\u003cp\u003e Overall, the RSCU values were very similar across all species and codons, indicating conserved codon frequency and usage (Figure 2C). We observed some clusters between species, however, the patterns remain conserved across all species. Regarding the specific codons, Arg-CGA, Leu-CTA and Arg-CGC had the highest RSCU values, while codons with G at the third position had the lowest values (Figure 2C).\u003c/p\u003e\n\u003ch2\u003e3.4 Phylogenetic relationships in Canini\u003c/h2\u003e\n\u003cp\u003e The estimated best-fit models and partitions for the ML and the BI analysis are available at Supplementary Tables S05 and S06, respectively. We successfully reconstructed the ML and BI trees, and they presented almost the same topology, except for two polytomies in the Canini clade on the BI tree. While a polytomy was not found in the ML tree, very low branch support values were found in the corresponding relationships in the BI tree. As such, we used the BI tree to be the representative tree, while showing the branch support values obtained from the ML approach (Figure 4). The individual figures for ML and BI are available as Supplementary Figures 01 and 02, respectively. We verified that the BI analysis reached convergence with ESS values highly superior to 200, with 3880.2 for the log likelihood (LnL) and 4805.9 for the log prior (LnPr).\u003c/p\u003e\n\u003ch3\u003e3.4.1 The monophyly of the\u003cem\u003eAenocyon\u003c/em\u003eand\u003cem\u003e\u003c/em\u003enon-monophyly of \u003cem\u003eLupulella\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003e \u003c/em\u003eWe recovered the four dire wolves as a well-supported monophyletic group. Our results indicated that DireGWC and DireSP are more related to each other than the other dire wolves, with high branch support values supporting this relationship in both trees (100/100/100) (Figure 4). DireAFR and DireGB are more related to each other; however, this relationship is not well-supported, likely due to the incomplete sequences of DireAFR. In any case, there is an evident separation between the DireSP + DireGWC group and the DireGB + DireAFR group.\u003c/p\u003e\n\u003cp\u003eThe\u003cem\u003eAenocyon\u003c/em\u003eclade position was uncertain between the \u003cem\u003eL\u003c/em\u003e.\u003cem\u003eadusta\u003c/em\u003e clade and the \u003cem\u003eCuon alpinus\u003c/em\u003e+ \u003cem\u003eL\u003c/em\u003e. \u003cem\u003emesomelas\u003c/em\u003e+ \u003cem\u003eCanis\u003c/em\u003e group, indicated by a polytomy in the BI tree. In the ML tree, \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eadusta\u003c/em\u003e was recovered as the sister group of the \u003cem\u003eAenocyon\u003c/em\u003e clade with very low branch support values (22.3/69). As such, in both trees, the exact relationship between those groups could not be properly inferred.\u003c/p\u003e\n\u003cp\u003e Furthermore, we recovered \u003cem\u003eLupulella\u003c/em\u003eas a non-monophyletic genus, separated into two distinct groups. \u003cem\u003eL\u003c/em\u003e. \u003cem\u003emesomelas\u003c/em\u003e was recovered as the sister group of the \u003cem\u003eCanis\u003c/em\u003e clade, supported by a high branch support value in both BI (100) and ML trees (100/100), while \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eadusta\u003c/em\u003e position, as stated before, was uncertain between\u003cem\u003eAenocyon\u003c/em\u003e and \u003cem\u003eCuon alpinus\u003c/em\u003e+ \u003cem\u003eL\u003c/em\u003e. \u003cem\u003emesomelas\u003c/em\u003e+ \u003cem\u003eCanis\u003c/em\u003e group.\u003c/p\u003e\n\u003ch3\u003e3.4.2 The \u003cem\u003eCanis\u003c/em\u003e Clade\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eCanis\u003c/em\u003e group was recovered as monophyletic, therefore, forming a clade composed of all \u003cem\u003eCanis\u003c/em\u003e species sampled in this study. The clade was divided into two well-supported sub-clades, one comprising \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erufus\u003c/em\u003e, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elycaon\u003c/em\u003e and \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elatrans\u003c/em\u003e(clade A)\u003cem\u003e\u003c/em\u003eand the other \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esimensis\u003c/em\u003e, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003eaureus\u003c/em\u003e, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elupaster\u003c/em\u003e, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003el\u003c/em\u003e. \u003cem\u003efamiliaris\u003c/em\u003e and C. \u003cem\u003el\u003c/em\u003e. \u003cem\u003elupus\u003c/em\u003e(clade B). We observed very short branch lengths between the three species of clade A, with \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erufus\u003c/em\u003e as the sister group of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elycaon\u003c/em\u003e and \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elatrans\u003c/em\u003e. Despite this, high support values were observed in the clade, except between \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elycaon\u003c/em\u003e and \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elatrans\u003c/em\u003etaxa. In clade B, we recovered \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elupaster\u003c/em\u003e as the sister group of \u003cem\u003eCanis lupus\u003c/em\u003esubspecies, with high branch support values. While \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esimensis\u003c/em\u003e and \u003cem\u003eC\u003c/em\u003e. \u003cem\u003eaureus\u003c/em\u003e were reconstructed as a clade on their own, the relationship between the three clades couldn\u0026rsquo;t be solved, and a polytomy was observed in the BI tree and low branch support in the ML tree (22.3/69).\u003c/p\u003e\n\u003ch3\u003e3.4.3 Cerdocyonina relationships\u003c/h3\u003e\n\u003cp\u003e We recovered Cerdocyonina as a non-monophyletic clade, exclusively due to \u003cem\u003eS\u003c/em\u003e. \u003cem\u003evenaticus\u003c/em\u003e being recovered as a sister group of Canina. The branch support for this was high in both BI (98) and ML (97.6/92) trees. All remaining species of Cerdocyonina were placed in the Cerdocyonina clade, which was recovered as the sister group of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003evenaticus\u003c/em\u003e and Canina. We reconstructed \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachyurus\u003c/em\u003e as the sister group of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ethous, A. microtis,\u003c/em\u003e and \u003cem\u003eLycalopex\u003c/em\u003e species with a high support in the BI tree (98) and not-so-high values (\u0026gt;= 95) in the ML tree (76.3/81). \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ethous\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emicrotis\u003c/em\u003eformed a well-supported monophyletic group (100/96.2/97) and were reconstructed as the sister group of the \u003cem\u003eLycalopex\u003c/em\u003e clade (100/100/100).\u003c/p\u003e\n\u003cp\u003eWithin \u003cem\u003eLycalopex\u003c/em\u003e, we observed a pectinate topology for the clade, with each species being the sister group of the remaining species. As such, we recovered \u003cem\u003eL\u003c/em\u003e. \u003cem\u003evetulus\u003c/em\u003e as the sister group of all other \u003cem\u003eLycalopex\u003c/em\u003e species\u003cem\u003e, L\u003c/em\u003e.\u003cem\u003esechurae as the sister group of L. gymnocercus\u003c/em\u003e,\u003cem\u003eL. griseus\u003c/em\u003e,\u003cem\u003eL. fulvipes\u003c/em\u003e,\u003cem\u003eLycalopex\u003c/em\u003esp. CAN003, \u003cem\u003eLycalopex\u003c/em\u003e sp. CAN005 and \u003cem\u003eL\u003c/em\u003e.\u003cem\u003eculpaeus\u003c/em\u003e, and so forth\u003cem\u003e.\u003c/em\u003eHigh support values were observed in all nodes.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe absence of a complete and well-annotated mitochondrial genome for the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e represents a significant gap in current genomic knowledge of the species. Previous attempts to reconstruct the mitochondrial genome failed to achieve a complete and annotated sequence. In this study, we were able to reconstruct the \u003cem\u003ede novo\u003c/em\u003e complete mitochondrial genome of the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e using publicly available libraries. We further reconstructed the mitochondrial genomes of other fossil samples using a reference-guided method, improving the previously assembled protein-coding sequences. Alongside the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e, we also assembled multiple Canini mitochondrial genomes, including sequences corresponding to the species\u0026apos; first description. Differences in RSCU values were minimal. Furthermore, we reconstructed the phylogenetic relationship of Canini. We observed that the mitochondrial genome reinforced that the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003ebelongs to a different lineage than that of the gray wolf and other \u003cem\u003eCanis\u003c/em\u003e species.\u003c/p\u003e\n\u003ch2\u003e4.1 The \u003cem\u003eAenocyon\u003c/em\u003e clade\u003c/h2\u003e\n\u003cp\u003e Within the \u003cem\u003eAenocyon\u003c/em\u003e clade, there are at least two main groups: DireSP + DireGWC, sampled from which are samples from the east side of the USA, and DireGB + DireAFR, which were sampled from the west side of the USA (Figure 1). This finding corroborates the separation between eastern and western populations and corroborates the subspecies definition of \u003cem\u003eAenocyon dirus dirus\u003c/em\u003e for eastern and \u003cem\u003eAenocyon dirus guildayi\u003c/em\u003efor western dire wolves based on morphological data (Anyonge and Roman 2006). Although the number of individuals and populations sampled is not enough to confirm this, with the recent advances in ancient DNA sequencing, it is expected that there will be more DNA samples of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003ein the future, thus allowing us to test this hypothesis with a more robust sampling. Nonetheless, despite the limitations, our findings are an important step towards a wide populational genetic study of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e.\u003c/p\u003e\n\u003ch2\u003e4.1 The position of the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e in the Canina tree\u003c/h2\u003e\n\u003cp\u003eThe low support values on the relationships of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e, \u003cem\u003eL\u003c/em\u003e. \u003cem\u003epictus,\u003c/em\u003e and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eadusta\u003c/em\u003e indicate that the mitochondrial data alone cannot resolve their relationship. Interestingly, nuclear DNA was also not sufficient to determine these relationships (Perri et al. 2021), which indicates that the evolutionary history of these species may have been connected in the past. This also raises the possibility of an ancient introgression between these species or their ancestors, since introgression is recurrent among Canini, as evidenced by genetic studies revealing extensive admixture among canid species (Gopalakrishnan et al. 2018; Rutledge et al. 2012; Wang et al. 2019).\u003c/p\u003e\n\u003cp\u003eFor instance, the Tibetan and Himalayan wolves exhibit significant genetic contributions from an unknown lineage, complicating their classification within the Canini (Wang et al. 2020). Speciation, introgression, and incomplete lineage sorting (ILS) represent a challenge for species tree inference, often misleading traditional methods and hindering the accurate reconstruction of evolutionary histories (Hibbins and Hahn 2022). As such, ancient and present introgression must be investigated in future studies to comprehend the genetic dynamics and evolutionary story of Canina.\u003c/p\u003e\n\u003ch2\u003e4.2 Relationships in the \u003cem\u003eCanis\u003c/em\u003e clade\u003c/h2\u003e\n\u003cp\u003e In the \u003cem\u003eCanis\u003c/em\u003e clade A, the close relationship between \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erufus\u003c/em\u003e, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elycaon\u003c/em\u003e and \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elatrans\u003c/em\u003e was expected due to current hybridisation between them (Fredrickson and Hedrick 2006; Hailer and Leonard 2008; Rutledge et al. 2012; Bohling and Waits 2015; vonHoldt et al. 2016; Hinton et al. 2017; Gopalakrishnan et al. 2018). It has been suggested that hybridization can occur as a result of anthropogenic factors (Galov et al. 2015; Barash et al. 2023), such as proximity to human cities and settlements and roaming dogs. Recurrent and intense hybridization can be harmful for species conservation from a genetic and ecological standpoint. The recovered polytomy in the \u003cem\u003eCanis\u003c/em\u003e clade B also points to a rather complex relationship between the three sub-clades (\u003cem\u003eC\u003c/em\u003e. \u003cem\u003esimensis\u003c/em\u003e, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003eaureus,\u003c/em\u003e and \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elupaster\u003c/em\u003e + \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elupus\u003c/em\u003e), and while their position in clade B couldn\u0026rsquo;t be asserted, each group was very well-supported. \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elupaster\u003c/em\u003e, previously known as \u003cem\u003eCanis anthus\u003c/em\u003e, was recovered as the sister group of C. \u003cem\u003elupus\u003c/em\u003ethrough mitochondrial data in previous studies (Koepfli et al. 2015; Hassanin et al. 2021). In nuclear phylogenies,\u003cem\u003eC\u003c/em\u003e. \u003cem\u003elatrans\u003c/em\u003e is often recovered as the sister group of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elupus\u003c/em\u003e(Lindblad-Toh et al. 2005; Perini et al. 2010; Perri et al. 2021; Chavez et al. 2022), with \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elupaster\u003c/em\u003e as their sister group when it was included in the study.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003ch2\u003e4.3 Cerdocyonina relationships\u003c/h2\u003e\n\u003cp\u003e The phylogeny we recovered was similar to previous studies on Cerdocyonina. The major divergence was the position of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003evenaticus\u003c/em\u003e as the sister group of Canini, which is a finding of mitochondrial phylogenies. In nuclear phylogenies, \u003cem\u003eS\u003c/em\u003e. \u003cem\u003evenaticus\u003c/em\u003eis recurrently recovered as the sister group of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachyurus\u003c/em\u003e, forming the sister group of all other Cerdocyonina in nuclear phylogenies (Lindblad-Toh et al. 2005; Perini et al. 2010; Porto et al. 2019; Chavez et al. 2022). This is a major mitonuclear discordance in the Canini evolutionary history, and we discuss it in detail in the next section. Our findings on \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ethous\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emicrotis\u003c/em\u003e clade, which is the sister group of all \u003cem\u003eLycalopex\u003c/em\u003e species, are also recurrently, but no always, recovered in both nuclear and mitochondrial phylogenies (Perini et al. 2010; Koepfli et al. 2015; Porto et al. 2019; Chavez et al. 2022) . Within \u003cem\u003eLycalopex\u003c/em\u003e, the position of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003evetulus\u003c/em\u003e as the sister group of all other Lycalopex is recovered in both mitochondrial and nuclear phylogenies as we recovered. However, the divergence is centered around the position of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003esechurae\u003c/em\u003e, recovered here and in other mitochondrial phylogenies as the sister group of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003egymnocercus\u003c/em\u003e, \u003cem\u003eL\u003c/em\u003e. \u003cem\u003egriseus\u003c/em\u003e, \u003cem\u003eL\u003c/em\u003e. \u003cem\u003efulvipes\u003c/em\u003e and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eculpaeus\u003c/em\u003e (Favarini et al. 2022), while recovered as the sister group of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eculpaeus\u003c/em\u003e in nuclear phylogenies (Lindblad-Toh et al. 2005; Chavez et al. 2022).\u003c/p\u003e\n\u003cp\u003eFurthermore, we observed that \u003cem\u003eLycalopex\u003c/em\u003esp. CAN003\u003cem\u003e\u003c/em\u003eand \u003cem\u003eLycalopex\u003c/em\u003esp. CAN005 corresponds to mitochondrial genomes assembled from ancient DNA by Popović et al. (2020). In their study, they recovered these two samples as a sister group of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003egriseus\u003c/em\u003ebased on d-loop phylogeny, but with low branch support. The authors have also highlighted that pre-Columbian archaeological sites where CAN003 and CAN005 samples were found are located outside the currently known distribution of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003egriseus\u003c/em\u003e. Furthermore, we recovered these two taxa as closely related to \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eculpaeus\u003c/em\u003e, with CAN005 as the sister group to the group composed of CAN003 and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eculpaeus\u003c/em\u003e, with high support values. Considering our results, the unmatched distribution of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003egriseus\u003c/em\u003e and the known distribution of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eculpaeus\u003c/em\u003e encompass the locations of the archaeological sites (Noguera-Urbano et al. 2016; Gunti\u0026ntilde;as et al. 2021), we found it more likely that the remains of CAN003 and CAN005 belonged to \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eculpaeus\u003c/em\u003especimens instead of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003egriseus.\u003c/em\u003e\u003c/p\u003e\n\u003ch2\u003e4.4 Mitonuclear discordance in Canini\u003c/h2\u003e\n\u003cp\u003eComparing mitochondrial against the nuclear topologies, we observed a mitonuclear discordance in the Canini tribe, especially in Canina. We have recovered a tree topology incongruent with phylogenetic studies that used only or most of the data from a nuclear source: \u003cem\u003eS\u003c/em\u003e. \u003cem\u003evenaticus\u003c/em\u003e was recovered as the sister group of Canina and not grouped with other Cerdocyonina. \u003cem\u003eLupulella\u003c/em\u003e was retrieved as a non-monophyletic group. The relationships between \u003cem\u003eL\u003c/em\u003e. \u003cem\u003epictus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e, \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eadusta\u003c/em\u003e, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ealpinus,\u003c/em\u003e and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003emesomelas\u003c/em\u003e lineages also differed between our findings and those that used nuclear data.\u003c/p\u003e\n\u003cp\u003eIn nuclear data studies, \u003cem\u003eS\u003c/em\u003e. \u003cem\u003evenaticus\u003c/em\u003e has been recovered inside Cerdocyonina (Perini et al. 2010; Koepfli et al. 2015; Lamarca and Schrago 2020; Chavez et al. 2022). Our results, however, have been recovered in other studies, in which \u003cem\u003eS\u003c/em\u003e. \u003cem\u003evenaticus\u003c/em\u003e was recovered as the sister group of Canina and not Cerdocyonina (Hassanin et al. 2021; Lamarca and Schrago, 2020). One possible hypothesis is that ancient introgression may have happened between \u003cem\u003eS. venaticus\u003c/em\u003e and Canina lineages, even extinct ones. Although generally rare, hybridization between subtribes was recently discovered in Canini, with a hybrid of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003el\u003c/em\u003e.\u003cem\u003efamiliaris\u003c/em\u003e(Canina) and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003egymnocercus\u003c/em\u003e(Cerdocyinina) (Szynwelski et al. 2023). These species belong to different subtribes and are estimated that their lineages diverged between ~6.8 Ma and ~3.5 Ma (Perini et al. 2010; Perri et al. 2021; Chavez et al. 2022). Despite that, they were able to generate a hybrid. Therefore, such hybridization is possible to have occurred in the evolutionary history of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003evenaticus\u003c/em\u003eand some Canina species, which may have led to a mitochondrial genome capture event. However, further investigation is needed to clarify the events that resulted in this mitonuclear discordance and test this hypothesis.\u003c/p\u003e\n\u003cp\u003eIn the relationships between \u003cem\u003eL\u003c/em\u003e. \u003cem\u003epictus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edirus\u003c/em\u003e, \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eadusta\u003c/em\u003e, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ealpinus,\u003c/em\u003e and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003emesomelas\u003c/em\u003e, we also observed a mitonuclear discordance, especially in \u003cem\u003eLupulella\u003c/em\u003e. We identified evidence of this discordance by comparing our results with those of previously published phylogenetic studies. In the study of Lindblad-Toh et al. (2005), they used a dataset composed of only nuclear sequences, encompassing exonic and intronic sequences, and they not only recovered\u003cem\u003eLupulella\u003c/em\u003e as a monophyletic group but also inferred that \u003cem\u003eLupulella\u003c/em\u003e was the sister group of a clade composed of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003epictus\u003c/em\u003e, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ealpinus,\u003c/em\u003e and \u003cem\u003eCanis\u003c/em\u003e species. The exact phylogenetic relationships were also reconstructed by Perini et al. (2010), who based their phylogenetic inference on 22 nuclear and three mitochondrial sequences; by Koepfli et al. (2015), who used sequences from segments of three exons and 17 introns; and by Lamarca and Schrago (2020), with the use of different orthologous segments and multiple nuclear genes based on the work of Lindblad-Toh et al. (2005). Similar results were also recovered by Zrzav\u0026yacute; et al. (2018) with a diverse characters, including morphological, ontogenetic and cytogenetic, behaviour, ecology, and molecular data; by Perri et al. (2021), who used up to 28 mb of nuclear sequence alignments and by Chavez et al. (2022), that used a robust dataset composed of 6,716 regions of the nuclear genome, each of them with 25 kb, from 31 nuclear genomes of 22 canid species.\u003c/p\u003e\n\u003cp\u003eIn the work of Bardeleben et al. (2005), the dataset composed of only nuclear sequences resulted in an exception on the position of \u003cem\u003eLupulella\u003c/em\u003e on the Canina evolutionary tree, since they recovered the jackals as the sister group of Cerdocyonina, with the latter being a clade inside Canina. In the data set composed of nuclear and mitochondrial sequences, \u003cem\u003eLupulella\u003c/em\u003ewas recovered as the sister group of \u003cem\u003eCanis\u003c/em\u003e species. The non-monophyly of \u003cem\u003eLupulella\u003c/em\u003e was observed in their study in the phylogenetic tree based only on mtDNA sequences (\u003cem\u003eCytb\u003c/em\u003e, \u003cem\u003eCOX1\u003c/em\u003e and\u003cem\u003eCOX2\u003c/em\u003e), with \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eadusta\u003c/em\u003e as the sister group of a group composed of \u003cem\u003eC\u003c/em\u003e.\u003cem\u003ealpinus\u003c/em\u003e, \u003cem\u003eL\u003c/em\u003e. \u003cem\u003emesomelas\u003c/em\u003e, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003eaureus\u003c/em\u003e, \u003cem\u003eC\u003c/em\u003e.\u003cem\u003elatrans\u003c/em\u003e, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elupus lupus\u003c/em\u003e and \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elupus familiaris\u003c/em\u003e. This topology has also been recovered by Hassanin et al. (2021) through the use of mitochondrial genomes. While recovering the non-monophyletic nature of \u003cem\u003eLupulella\u003c/em\u003ewith the same topology we recovered by using 13 PCGs + two rRNAs, Koepfli et al. (2015) observed a different tree topology based only on \u003cem\u003eCytb\u003c/em\u003e sequences, with a group composed of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003emesomelas\u003c/em\u003e, \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eadusta\u003c/em\u003e, \u003cem\u003eL\u003c/em\u003e. \u003cem\u003epictus,\u003c/em\u003e and \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ealpinus\u003c/em\u003e. However, teir nuclear phylogenetic tree was similar to the nuclear topologies with \u003cem\u003eLupulella\u003c/em\u003e as a sister clade of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003epictus\u003c/em\u003e. Atickem et al. (2018) also recovered \u003cem\u003eLupulella\u003c/em\u003e as a non-monophyletic using \u003cem\u003eCytb\u003c/em\u003e sequences, but with a different topology, with a group composed of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003epictus\u003c/em\u003e, \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eadusta,\u003c/em\u003e and \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ealpinus\u003c/em\u003e, the sister group of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003emesomelas\u003c/em\u003eand \u003cem\u003eCanis\u003c/em\u003e species. Furthermore, mitonuclear discordance is also found within the \u003cem\u003eLycalopex\u003c/em\u003e and \u003cem\u003eCanis\u003c/em\u003e genera.\u003c/p\u003e\n\u003cp\u003eWhen comparing our results with the multiple mitochondrial and nuclear phylogenetic studies, it is clear that mitonuclear discordance is spread across Canini. Mitonuclear discordance, which can occur without signals in the nuclear genome, has been proposed as the result of incomplete lineage sorting (McKay and Zink 2010; DeRaad et al. 2023), mitochondrial genome capture and introgression (Andersen et al. 2021; Bonnet et al. 2017; Mikkelsen and Weir, 2023). Introgression has been registered multiple times in Canini lineages. Interspecific hybridisation is common among canids, and numerous events have been documented in Canina and Cerdocyonina (Garcez et al. 2024).\u003c/p\u003e\n\u003cp\u003eWhile the mitochondrial genome, as a phylogenetic marker, suffers from hybridization that occurred in the past or even in the present, it is a powerful tool to discover such events. Instead of viewing the mitochondrial genome as an insufficient source of phylogenetic signal in canids, we propose to see it as a fundamental tool for understanding admixture in the evolutionary history of the group, especially when only weak signs of hybridization are present in the nuclear genome. Therefore, efforts in describing and annotating mitochondrial genomes of the group must go on, strengthening the resources and enabling a more complete and in-depth understanding of hybridization in the group.\u003c/p\u003e\n\u003cp\u003eOur findings reinforce the relevance of exploring mitochondrial genomes even in well-studied groups, such as Canini. Our findings reinforce the work of Perri et al. (2021), in which \u003cem\u003eA. dirus\u003c/em\u003e was found not to be closely related to \u003cem\u003eCanis lupus\u003c/em\u003e and belongs to a different lineage of Canina. Therefore, it is unlikely that they intimately resembled the \u003cem\u003eCanis lupus lupus\u003c/em\u003e as dire wolves are usually reconstructed. Furthermore, we expanded the investigation on the mitonuclear discordance across Canini by presenting a more robust mitochondrial phylogenetic analysis. We expect that the description of the first complete mitochondrial genome of \u003cem\u003eA. dirus\u003c/em\u003e and the advances in DNA sequencing from fossil samples will trigger complete mitochondrial genetic studies on their populations, such as phylogeographic studies, that have been done in other megafauna species using complete mitochondrial DNA. We expect that the description of almost the mitochondrial genome of Canini will improve genetic studies based on mitochondrial genomes, allowing investigations into introgression between species, leading to a better understanding of evolutionary events that have occurred in the evolutionary history of Canini.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eUnpublished sequencing data for \u003cem\u003eLycaon pictus\u003c/em\u003e (SRR10331591) are used with permission from the DNA Zoo Consortium (\u003cu\u003ednazoo.org\u003c/u\u003e).\u003c/p\u003e\n\u003ch2\u003eData Archiving\u003c/h2\u003e\n\u003cp\u003eAll raw DNA-Seq data used in this work are publicly available at the Sequence Read Archive (Tables 1 and 2). The complete mitochondrial genome sequences are available at GenBank (Table 5), except the sequences from \u003cem\u003eAenocyon dirus\u003c/em\u003e DireAFR and DireGB, which are available as Supplementary File 1.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAfgan E, Nekrutenko A, Gr\u0026uuml;ning BA, et al (2022) The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2022 update. Nucleic Acids Research 50. https://doi.org/10.1093/nar/gkac247\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAndersen MJ, McCullough JM, Gyllenhaal EF, et al (2021) Complex histories of gene flow and a mitochondrial capture event in a nonsister pair of birds. Mol Ecol 30:2087\u0026ndash;2103. https://doi.org/10.1111/mec.15856\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAnyonge W, Roman C (2006) New body mass estimates for \u003cem\u003eCanis dirus\u003c/em\u003e , the extinct Pleistocene dire wolf. 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Zoologica Scripta 47:373\u0026ndash;389. https://doi.org/10.1111/zsc.12293 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ancient DNA, Canidae, Cerdocyonina, Canina, mitogenome","lastPublishedDoi":"10.21203/rs.3.rs-8379789/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8379789/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The dire wolf (\u003ci\u003eAenocyon dirus\u003c/i\u003e) was a large canid that roamed the Americas in the Late Pleistocene. The genome of \u003ci\u003eA. dirus\u003c/i\u003e was only recently sequenced. This was a turning point in the studies on \u003ci\u003eA. dirus\u003c/i\u003e, especially because it refuted the previous hypothesis of a close phylogenetic relationship between dire wolves and grey wolves, placing \u003ci\u003eA. dirus\u003c/i\u003e as the sister group of the other canids. Despite this, population studies, usually based on mitochondrial genomes, were absent, and a proper description and annotation of the \u003ci\u003eA. dirus\u003c/i\u003e mitochondrial genome are lacking. In our study, we aimed to address this issue. Using previously sequenced DNA from \u003ci\u003eA. dirus\u003c/i\u003e, we de novo assembled the complete mitochondrial genome. We also assembled the mitochondrial genomes of several Canini species, many of which are the first descriptions so far, and conducted a phylogenetic analysis. We observed a population-level structure between the eastern and western USA samples of \u003ci\u003eA. dirus\u003c/i\u003e. Our comparative analysis further reinforced the mitonuclear discordance by comparing our results with many mitochondrial and nuclear phylogenetic studies. This indicates a complex evolutionary history within different species, with a possible mitochondrial genome capture event happening within the group. Consequently, our work improved the current mitochondrial genome knowledge of the Canini tribe. Our findings will be valuable for future population genetics studies on \u003ci\u003eA. dirus\u003c/i\u003e. They also conveyed insights into the mitonuclear discordance across Canini, which can help clarify introgression and mitochondrial genome capture events within the tribe.","manuscriptTitle":"The mitochondrial genome as an evolutionary storyteller: a journey from dire wolf (Aenocyon dirus) to extant species of Canini (Carnivora: Canidae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-23 15:52:55","doi":"10.21203/rs.3.rs-8379789/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0bc63bc5-8461-405f-9255-1449b10245cc","owner":[],"postedDate":"January 23rd, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Reject after peer review","date":"2026-05-05T17:37:24+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59775790,"name":"Biological sciences/Genetics/Genomics/Comparative genomics"},{"id":59775791,"name":"Biological sciences/Evolution/Phylogenetics"},{"id":59775792,"name":"Biological sciences/Biological techniques/Bioinformatics"},{"id":59775793,"name":"Biological sciences/Genetics/Evolutionary biology"},{"id":59775795,"name":"Biological sciences/Genetics/Genomics/Genome evolution"}],"tags":[],"updatedAt":"2026-05-05T17:40:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-23 15:52:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8379789","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8379789","identity":"rs-8379789","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}