The complete mitochondrial genome of a threatened loach (<Emphasis Type="Italic">Sinibotia reevesae</Emphasis>) and its phylogeny

In present study, the complete mitochondrial genome of Sinibotia reevesae was first sequenced using the next-generation sequencing technology and annotated using bioinformatic tools. The circular mitochondrial genome was 16,572 bp in length, and contained 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 1 displacement loop locus. It presents a typical gene organization and order for completely sequenced cypriniformes mitogenomes. The control region could be divided into three parts included the extended termination associated sequence domain, the central conserved domain and the conserved sequence block. Interestingly, two stem-loop domains were found in control region and O_L region, respectively. Furthermore, phylogenetic analyses using concatenated amino acid and nucleotide sequences of the 13 protein-coding genes with two different methods (Maximum likelihood and Bayesian analysis) both highly supported the close relationship of S. reevesae and Sinibotia superciliaris, which was in line with the previous classifications based on morphological and molecular studies. These data provide useful information for a better understanding of the mitogenomic diversities and evolution in fish as well as novel genetic markers for studying population genetics and species identification.     

Complete mitochondrial genome of the miiuy croaker Miichthys miiuy (Perciformes, Sciaenidae) with phylogenetic consideration

The complete sequence of the 16,493 nucleotide mitochondrial genome from the single species of the family Sciaenidae, the miiuy croaker, Miichthys miiuy, was determined. The nucleotide sequences of M. miiuy mitochondrial DNA have been compared with those of three other Sciaenidae fishes. The contents of the M. miiuy mitochondrial genome are 13 protein-coding genes, two ribosomal RNA genes and 22 transfer RNA genes, and two non-coding regions (L-strand replication origin and control region), the gene order of which is identical to that observed in most vertebrates. The L-strand replication origin of M. miiuy is not pyrimidine-rich compared to those of most bony fishes. Within the control region, we identified the extended termination associated sequence domain, the central conserved sequence block domain and the conserved sequence block domain, while the typical central conserved blocks CSB-D, -E and -F could not be detected in the three other Sciaenidae species. In the ML phylogenetic analyses, the monophyly of Pseudosciaeniae was not supported, which is against with the morphological results. Collichthys niveatus is most closely related to Larimichthys polyactis, and Collichthys and Larimichthys may be merged into one genus, based on the current datasets.     

麦穗鱼线粒体基因组序列测定及分析

利用麦穗鱼Pseudorasbora parva和相关鱼类的部分线粒体基因序列,设计出2对长批引物和30对短批引物,采用基于长PCR的2次PCR扩增法测定并注释麦穗鱼线粒体基因组全序列。结果表明,麦穗鱼线粒体基因组长16600bp,A+T含量为58.9%,37个基因位置及组成与其它硬骨鱼一致,均由13个蛋白编码基因、22个tRNA、2个rRNA基因和1个控制区(D-loop)组成。其中L链仅含8个tRNA(Pro、T yr、Ser、Ala、Asn、Cys、Glu、Gln)及ND6基因,其余基因皆由H链编码。基因排列紧密,间隔序列共计13处64bp,长度从1～32bp不等;基因重叠区7处23bp,重叠碱基数在1～7bp之间。13个蛋白编码基因中,除COI起始密码子为GTG外,其余均以ATG为起始密码子;有8个基因(ND1、ND2、COI、ATP6、ATP8、ND4L、ND5、ND6)3’端有完全的TAA或TAG终止密码子,其它5个基因终止密码子为不完整的TA(ND3和ND4)或T(COⅡ,COⅢ,Cyt b)。除tRNASer(AGY)外,其余21个tRNA基因的二级结构均为典型的三叶草结构。预测的lrRNA二级结构共有6个结构域,53个茎环结构,srRNA二级结构包含43个茎环结构。控制区(D-loop)存在3个结构区:终止序列区(TAS)、中央保守区(CSB-F、CSB-D)和保守序列区(CSB-1、CSB-2、CSB-3),其中TAS与DNA复制终止相关,出现茎环结构。     

Complete mitochondrial genome of the rabbitfish Siganus fuscescens (Perciformes, Siganidae).

We determined the complete nucleotide sequence of the mitochondrial genome for the rabbitfish Siganus fuscescens (Perciformes, Siganidae). This mitochondrial genome, consisting of 16,491 base pairs (bp), included 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a noncoding control region similar those found in other vertebrates; the gene order was identical to that of typical vertebrates. Most of the genes of S. fuscescens were encoded on the H-strand, while the ND6 and eight tRNA (Gln, Ala, Asn, Cys, Tyr, Ser [UCN], Glu, and Pro) genes were encoded on the L-strand. The reading frames of ATPase 8 and 6 and those of ND4L and ND4 overlapped by ten and seven nucleotides, respectively. All mitochondrial protein-coding genes began with an ATG start codon, except for CO1, which started with GTG. Open reading frames of S. fuscescens ended with TAA (ND1, CO1, ATPase 8, ND4L, ND5 and ND6), and the remainder had incomplete stop codons, either TA (ATPase 6 and CO3) or T (ND2, CO2, ND3, ND4, and Cytb). The origin of L-strand replication in S. fuscescens was located in a cluster of five tRNA genes (WANCY) and was 34 nucleotides in length. A major noncoding region between the tRNA-Pro and tRNA-Phe genes (828 bp) was considered to be the control region (D-loop). Within this sequence, we identified a conserved sequence block characteristic of this region. The rabbitfish was grouped with Siganus canaliculatus in most parsimony analyses, which showed 100% bootstrap support for their divergence. These findings are useful for inferring phylogenetic relationships and identification within the suborder Acanthuroidei.     

Complete mitochondrial genome of <Emphasis Type="Italic">Ampittia dioscorides</Emphasis> (Lepidoptera: Hesperiidae) and its phylogenetic analysis

The complete mitochondrial genome of Ampittia dioscorides (Lepidoptera: Hesperiidae) was determined. The sequenced genome is a circular molecule of 15313 bp, containing 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and an A + T-rich region. The gene arrangements and transcribing directions are identical to those in most of the reported lepidopteran mitogenomes. The base composition of the whole genome and genes or regions are also similar to those in other lepidopteran species. All the PCGs are initiated by typical ATN codons; the exception being COI, which begins with a CGA codon. Eight genes (ND2, ATPase8, ATPase6, COIII, ND5, ND4L, ND6, and Cytb) end with a TAA stop codon, and two genes (ND1 and ND3) end with TAG. The remaining three genes (COI and COII, which end with TA-, and ND4, which ends with T-) have incomplete stop codons. All tRNAs have the typical clover-leaf structure of mitochondrial tRNAs, with the exception of tRNA^Ser(AGY). On the basis of the concatenated nucleotide and amino acid sequences of the 13 PCGs and wingless gene of 22 butterfly species, maximum parsimony (MP) and Bayesian inference (BI) trees were constructed, respectively. Both MP and BI trees had the same topological structure: ((((Nymphalidae + Danaidae) + Lycaenidae) + Pieridae) + Papilionidae) + Hesperiidae). The results provide support for Hesperiidae as a superfamily-level taxon.     

A new primer set for amplification of the cytochrome <Emphasis Type="Italic">b</Emphasis> gene in lantern fishes (Myctophidae)

New universal primers are offered for amplification of complete sequence of mitochondrial cyt b gene in lantern fishes of the family Myctophidae. Analysis of cyt b variability in the species of seven genera (Bolinichthys, Ceratoscopelus, Diaphus, Diogenichthys, Lampanyctus, Lepidophanes, Nannobrachium) demonstrates considerable divergence between species: an average of 18.2% (p-distances). Diversity (nucleotide diversity, number of segregating and phylogenetically informative sites, average number of nucleotide differences) and divergence significantly exceed those of another widely used mitochondrial marker, a fragment of cytochrome c oxidase subunit 1 sequence (cox 1). The mitochondrial cyt b gene amplified with the developed primers can be recommended as an informative tool for phylogenetic and population studies of lantern fishes.     

Phylogeography of the Asian rice gall midge <Emphasis Type="Italic">Orseolia oryzae</Emphasis> (Wood Mason) (Diptera: Cecidomyiidae) in Thailand

The Asian rice gall midge (RGM) Orseolia oryzae (Wood Mason) (Diptera: Cecidomyiidae) is a major pest of rice, leading to yield losses in Thailand and many Asian countries. Despite an increasing number of reported midge outbreaks and the presence of many susceptible rice varieties, only a few studies have focused on the genetic variation of the midges. Therefore, we analyzed the phylogeography among Thai RGM populations covering north, northeast and central Thailand. Two mitochondrial DNA genes, cytochrome C oxidase I (COI) and 12S, and a non-coding repeat region (RR) situated just before COI were amplified. Overall, the haplotype diversity for COI and 12S genes of the Thai population was high, but the nucleotide diversity was quite low. Altogether, the phylogenetic tree and pairwise F _st values indicated that Thai RGM populations recently expanded and were homogeneously distributed throughout the country, except for some populations in the north, which most likely became recently isolated from the main population. Two non-coding repeat motifs, that were recently observed in the mitogenome of RGM in India, were absent in Thai populations and replaced by an 89 bp non-coding sequence. Tandem nucleotide repeats of the sequence TA were also observed. The repeat copy number varied from 2 to 11 and was not correlated with geographical repartition of the midge. Finally, COI barcoding divergence between Indian and Thai populations was high (6.3% in average), giving insights into the potential existence of an RGM species complex in Asia.     

Mitochondrial genome of <Emphasis Type="Italic">Pteronotus personatus</Emphasis> (Chiroptera: Mormoopidae): comparison with selected bats and phylogenetic considerations

We described the complete mitochondrial genome (mitogenome) of the Wagner’s mustached bat, Pteronotus personatus, a species belonging to the family Mormoopidae, and compared it with other published mitogenomes of bats (Chiroptera). The mitogenome of P. personatus was 16,570 bp long and contained a typically conserved structure including 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and one control region (D-loop). Most of the genes were encoded on the H-strand, except for eight tRNA and the ND6 genes. The order of protein-coding and rRNA genes was highly conserved in all mitogenomes. All protein-coding genes started with an ATG codon, except for ND2, ND3, and ND5, which initiated with ATA, and terminated with the typical stop codon TAA/TAG or the codon AGA. Phylogenetic trees constructed using Maximum Parsimony, Maximum Likelihood, and Bayesian inference methods showed an identical topology and indicated the monophyly of different families of bats (Mormoopidae, Phyllostomidae, Vespertilionidae, Rhinolophidae, and Pteropopidae) and the existence of two major clades corresponding to the suborders Yangochiroptera and Yinpterochiroptera. The mitogenome sequence provided here will be useful for further phylogenetic analyses and population genetic studies in mormoopid bats.     

Complete mitochondrial genome sequence of the Korean water deer Hydropotes inermis argyropus (Cervidae, Capreolinae)

The complete mitochondrial genome sequence of Hydropotes inermis argyropus consists of 13 protein-coding, 22 tRNA, and two rRNA genes, and 1 control region (CR). Three overlaps among the 13 protein-coding genes were found: ATP8/ATP6, ND4L/ND4, and ND5/ND6. The CR was located between the tRNA-Pro and tRNA-Phe genes and is 928 bp in length. The typical conserved domains, such as TAS and CSB, were identified in the CR.     

Complete mitochondrial genome sequence of Catla catla and its phylogenetic consideration

Complete nucleotide sequence of mitochondrial genome (mitogenome) of the Catla catla (Ostariophysi: Cypriniformes: Cyprinidae) was determined in the present study. Its length is 16,594 bp and contains 13 protein coding genes, 22 transfer RNAs, two ribosomal RNAs and one non-coding control region. Most of the genes were encoded on the H-strand, while the ND6 and eight tRNA (Gln, Ala, Asn, Cys, Tyr, Ser (UCN), Glu and Pro) genes were encoded on the L-strand. The reading frames of two pair of genes overlapped: ATPase 8 with 6 and ND4L with ND4 by seven nucleotides each. The main non-coding region was 929 bp, with three conserved sequence blocks (CSB-I, CSB-II, and CSB-III) and an unusual simple sequence repeat, (TA)₇. Phylogenetic analyses based on complete mitochondrial genome sequences were in favor of the traditional taxonomy of family Cyprinidae. In conclusion present mitogenome of Catla catla adds more information to our understanding of diversity and evolution of mitogenome in fishes.     

The mitochondrial genome of spotted green pufferfish Tetraodon nigroviridis (Teleostei: Tetraodontiformes) and divergence time estimation among model organisms in fishes

We determined the whole mitochondrial genome sequence for spotted green pufferfish, Tetraodon nigroviridis (Teleostei: Tetraodontiformes). The genome (16,488 bp) contained 37 genes (two ribosomal RNA genes, 22 transfer RNA genes, and 13 protein-coding genes) plus control region as found in other vertebrates, with the gene order identical to that of typical vertebrates. The sequence was used to estimate phylogenetic relationships and divergence times among major lineages of fishes, including representative model organisms in fishes. We employed partitioned Bayesian approaches for these two analyses using two datasets that comprised concatenated amino acid sequences from 12 protein-coding genes (excluding the ND6 gene) and concatenated nucleotide sequences from the 12 protein-coding genes (without 3rd codon positions), 22 transfer RNA genes, and two ribosomal RNA genes. The resultant trees from the two datasets were well resolved and largely congruent with those from previous studies, with spotted green pufferfish being placed in a reasonable phylogenetic position. The approximate divergence times between spotted green pufferfish and model organisms in fishes were 85 million years ago (MYA) vs. torafugu, 183 MYA vs. three-spined stickleback, 191 MYA vs. medaka, and 324 MYA vs. zebrafish, all of which were about twice as old as the divergence times estimated by their earliest occurrences in fossil records.     

Mitochondrial genome of the eurasian otterLutra lutra (Mammalia, Carnivora, Mustelidae)

We determined the complete mitochondrial genome of the Eurasian otterLutra lutra, which is an endangered species in Korea. The circle genome (16,536 bp in size) consists of 13 protein-coding, 22 tRNA, and 2 rRNA genes, and a control region, as found in other metazoan animals. Out of the 37 genes, 28 are encoded on the H-strand, and the nine (ND6 and 8 tRNA genes) on the L-strand. Three overlaps among the 13 protein-coding genes were found: ATP8-ATP6, ND4L-ND4, and ND5-ND6. A control region (1090 bp) including the origin of H-strand replication (OH), TAS (a conserved motif TACAT-16bp-ATGTA) and CSB (CSB-1, CSB-2. and CSB-3) was observed between tRNA-Pro and tRNA-Phe genes, and O_L, with 36 highly conserved nucleotides between tRNA-Asn (N) and tRNA-Cys (C) within a cluster of five tRNA genes (WANCY), as typically found in vertebrates. The other important characteristics of theL. lutra mitochondrial genome were described in detail. In addition, a maximum likelihood and Bayesian trees of 9 mustelid species and 1 outgroup were reconstructed based on the nucleotide sequences of 11 protein-coding genes excluding ATP8 and ND6. It showed that Lutrinae formed a monophyletic group with Mustelinae that is not monophyletic. Within the subfamily Lutrinae,L. lutra andEnhydra lutris were grouped together and thenLontra canadentis placed as a sister of the clade. The present result is the first complete mitochondrial genome sequence reported from the genusLutra, and is applicable to molecular phylogenetic, phylogeographic, conservation biological studies for mustelid members. In particular, exploration of sequence variations of the control region may be helpful for analyzing inter-and intra-species variations in the genusLutra.     

大卫绢蛱蝶线粒体基因组全序列测定和分析

目前有关蝶类线粒体基因组全序列及其分子进化的研究报道还不多见。本文利用long PCR和引物步移法得到大卫绢蛱蝶Calinaga davidis的线粒体基因组全序列, 同时就其基因组成和结构特点作了初步分析。结果显示： 其基因组全长为15 267 bp (GenBank登录号为HQ658143), 包括13个蛋白质编码基因(ATP6, ATP8, COI-III, ND1-6, ND4L, Cytb)、22个tRNA基因、2个rRNA基因(16S和12S)以及非编码的控制区。与其他鳞翅目昆虫相一致, 其基因组未出现基因重排现象。基因组共包含11个基因间隔区,总长度为130 bp, 间隔长度1~46 bp, 最大间隔在tRNA^Gln与ND2基因之间; 基因间共存在13处重叠, 总长度为66 bp, 重叠碱基数1~35 bp, 最长的重叠区位于COII与tRNA^Lys基因。lrRNA和srRNA基因长度分别为1 337 bp和773 bp; 除tRNA^Ser(AGN)缺少二氢尿嘧啶臂(DHU stem), 在相应的位置上只形成一个简单环外, 其余的tRNA基因都能形成典型的三叶草结构。13个蛋白编码基因总长度为11 247 bp, 共有3 737个密码子, 它们的碱基组成和密码子的使用具有明显的偏倚性; 除COI外(起始密码子TTG), 其余的12个蛋白质编码基因都以标准的ATN作为起始密码子; COI基因终止密码子为不完全T, ND4基因终止密码子为不完全TA, 其余基因都以TAA为终止密码子。A+T丰富区全长为389 bp, A+T含量高达92.0%, 其中存在2段类似微卫星的重复序列(TA)₆和(AAT)₄。本文的研究结果为探讨绢蛱蝶亚科在蛱蝶科中的系统学地位及其与其他亚科间的系统发生关系等问题提供了重要的分子生物学数据。     

First reported chloroplast genome sequence of <Emphasis Type="Italic">Punica granatum</Emphasis> (cultivar Helow) from Jabal Al-Akhdar,Oman: phylogenetic comparative assortment with <Emphasis Type="Italic">Lagerstroemia</Emphasis>

Pomegranate (Punica granatum L.) is one of the oldest known edible fruits. It has grown in popularity and is a profitable fruit crop due to its attractive features including a bright red appearance and its biological activities. Scientific exploration of the genetics and evolution of these beneficial traits has been hampered by limited genomic information. In this study, we sequenced the complete chloroplast (cp) genome of the native P. granatum (cultivar Helow) cultivated in the mountains of Jabal Al-Akhdar, Oman. The results revealed a P. granatum cp genome length of 158,630 bp, characterized by a relatively conserved structure containing 2 inverted repeat regions of 25,466 bp, an 18,686 bp small single copy regions, and an 89,015 bp large single copy region. The 86 protein-coding genes included 37 transfer RNA genes and 8 ribosomal RNA genes. Comparison of the P. granatum whole cp genome with seven Lagerstroemia species revealed an overall high degree of sequence similarity with divergence among intergenic spacers. The location, distribution, and divergence of repeat sequences and shared genes of the Punica and Lagerstroemia species were highly similar. Analyses of nucleotide substitution, insertion/deletions, and highly variable regions in these cp genomes identified potential plastid markers for taxonomic and phylogenetic studies in Myrtales. A phylogenetic study of the cp genomes and 76 shared coding regions generated similar cladograms. The complete cp genome of P. granatum will aid in taxonomical studies of the family Lythraceae.     

Complete sequence of the mitochondrial genome of Odontamblyopus rubicundus (Perciformes: Gobiidae): genome characterization and phylogenetic analysis

Odontamblyopus rubicundus is a species of gobiid fishes, inhabits muddy-bottomed coastal waters. In this paper, the first complete mitochondrial genome sequence of O. rubicundus is reported. The complete mitochondrial genome sequence is 17119 bp in length and contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes, a control region and an L-strand origin as in other teleosts. Most mitochondrial genes are encoded on H-strand except for ND6 and seven tRNA genes. Some overlaps occur in protein-coding genes and tRNAs ranging from 1 to 7 bp. The possibly nonfunctional L-strand origin folded into a typical stem-loop secondary structure and a conserved motif (5^′-GCCGG-3^′) was found at the base of the stem within the tRNA ^Cys gene. The TAS, CSB-2 and CSB-3 could be detected in the control region. However, in contrast to most of other fishes, the central conserved sequence block domain and the CSB-1 could not be recognized in O. rubicundus, which is consistent with Acanthogobius hasta (Gobiidae). In addition, phylogenetic analyses based on different sequences of species of Gobiidae and different methods showed that the classification of O. rubicundus into Odontamblyopus due to morphology is debatable.     

Complete mitochondrial genome of <Emphasis Type="Italic">Taharana fasciana</Emphasis> (Insecta,Hemiptera: Cicadellidae) and comparison with other Cicadellidae insects

Here, we describe the first complete mitochondrial genome (mitogenome) sequence of the leafhopper Taharana fasciana (Coelidiinae). The mitogenome sequence contains 15,161 bp with an A?+?T content of 77.9%. It includes 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and one non-coding (A?+?T-rich) region; in addition, a repeat region is also present (GenBank accession no. KY886913). These genes/regions are in the same order as in the inferred insect ancestral mitogenome. All protein-coding genes have ATN as the start codon, and TAA or single T as the stop codons, except the gene ND3, which ends with TAG. Furthermore, we predicted the secondary structures of the rRNAs in T. fasciana. Six domains (domain III is absent in arthropods) and 41 helices were predicted for 16S rRNA, and 12S rRNA comprised three structural domains and 24 helices. Phylogenetic tree analysis confirmed that T. fasciana and other members of the Cicadellidae are clustered into a clade, and it identified the relationships among the subfamilies Deltocephalinae, Coelidiinae, Idiocerinae, Cicadellinae, and Typhlocybinae.     

The complete mitochondrial DNA sequence of the guanaco (<Emphasis Type="Italic">Lama guanicoe)</Emphasis>: comparative analysis with the vicuña (<Emphasis Type="Italic">Vicugna vicugna)</Emphasis> genome

South American camelids comprise the guanaco (Lama guanicoe) and the vicuña (Vicugna vicugna), which are wild species, and the domestic llama (Lama glama) and alpaca (Lama pacos). This paper presents the first complete mitochondrial (mt) genome of the guanaco and the mt coding sequence of the vicuña. The guanaco mtDNA is 16,649 nt long and its composition and organization are similar to the mitochondrial genome of other mammals. Excluding the control region, comparison of the complete guanaco and vicuña mtDNA showed 4.4% sequence divergence. Nucleotide differences in peptide coding genes varied from 1.9% in ATP6 to 6.4% in Cyt b. These values are compatible with the close relatedness of both species identified by other authors. Based on the differences between the control region sequence here reported and that previously described, we also discuss the occurrence of NUMTs in the genome of South American camelids.     

Structure and evolution of the avian mitochondrial control region

The structural and evolutionary characteristics of the mitochondrial control region were studied by using control region sequences of 68 avian species. The distribution of the variable nucleotide positions within the control region was found to be genus specific and not dependant on the level of divergence, as suggested before. Saturation was shown to occur at the level of divergence of 10% in pairwise comparisons of the control region sequences, as has also been reported for the third codon positions in ND2 and cytochrome b genes of mtDNA. The ratio of control region vs cytochrome b divergence in pairwise comparisons of the sequences was shown to vary from 0.13 to 21.65, indicating that the control region is not always the most variable region of the mtDNA, but also that there are differences in the rate of divergence among the lineages. Only two of the conserved sequence blocks localized earlier for other species, D box and CSB-1, were found to show a considerable amount of sequence conservation across the avian and mammalian sequences. Additionally, a novel avian-specific sequence block was found.     

The complete mitochondrial genome and phylogenetic analysis of Nyctereutes procyonoides

The complete mitochondrial genome sequence of the raccoon dog (Nyctereutes procyonoides) was determined by using the long and accurate polymerase chain reaction. The entire mitochondrial genome sequence is 16,713 bp in length contains two ribosomal RNA genes, 13 protein-coding genes, 22 transfer RNA genes and 1 control region. Most mitochondrial genes are encoded on the H strand, except for the ND6 gene and 8 tRNA genes. The base compositions of mitochondrial genomes present clearly A–T skew. All the transfer RNA genes can be folded into the typical cloverleaf-shaped structure except tRNA-Ser (AGY), which lacks the dihydrouridine arm. Protein-coding genes mainly initiate with ATG and terminate with TAA. Some reading frame intervals and overlaps are found in the mitochondrial genome. The control region can be divided into three domains: the extended termination associated sequences (ETASs) domain, the central conserved domain and the conserved sequence blocks (CSBs) domain. Three conserved sequence blocks (CSBs) and one extended termination associated sequences (ETAS-1) is found in the control region. The phylogenetic analysis based on the concatenated data set of 14 genes in the mitochondrial genome of Canidae shows that the raccoon dog has close phylogenetic position with the red fox (Vulpes vulpes) and they constitute a clade which has an equil evolutionary position with the clade formed by the genera Canis and Cuon.