Multiple nuclear insertions of mitochondrial cytochrome b sequences in callitrichine primates

We report the presence of four nuclear paralogs of a 380-bp segment of cytochrome b in callitrichine primates (marmosets and tamarins). The mitochondrial cytochrome b sequence and each nuclear paralog were obtained from several species, allowing multiple comparisons of rates and patterns of substitution both between mitochondrial and nuclear sequences and among nuclear sequences. The mitochondrial DNA had high overall rates of molecular evolution and a strong bias toward substitutions at third codon positions. Rates of molecular evolution among the nuclear sequences were low and constant, and there were small differences in substitution patterns among the nuclear clades which were probably attributable to the small number of sites involved. A novel method of phylogenetic reconstruction based on the large difference in rates of evolution at different codon positions among mitochondrial and nuclear clades was used to determine whether different nuclear paralogs represent independent transposition events or duplications following a single insertion. This method is generally applicable in cases where differences in pattern of molecular evolution are known, and it showed that at least three of the four nuclear clades represent independent transposition events. The insertion events giving rise to two of the nuclear clades predate the divergence of the callitrichines, whereas those leading to the other two nuclear clades may have occurred in the common ancestor of marmosets.     

Forty Million Years of Independent Evolution: A Mitochondrial Gene and Its Corresponding Nuclear Pseudogene in Primates

Sequences from nuclear mitochondrial pseudogenes (numts) that originated by transfer of genetic information from mitochondria to the nucleus offer a unique opportunity to compare different regimes of molecular evolution. Analyzing a 1621-nt-long numt of the rRNA specifying mitochondrial DNA residing on human chromosome 3 and its corresponding mitochondrial gene in 18 anthropoid primates, we were able to retrace about 40 MY of primate rDNA evolutionary history. The results illustrate strengths and weaknesses of mtDNA data sets in reconstructing and dating the phylogenetic history of primates. We were able to show the following. In contrast to numt-DNA, (1) the nucleotide composition of mtDNA changed dramatically in the different primate lineages. This is assumed to lead to significant misinterpretations of the mitochondrial evolutionary history. (2) Due to the nucleotide compositional plasticity of primate mtDNA, the phylogenetic reconstruction combining mitochondrial and nuclear sequences is unlikely to yield reliable information for either tree topologies or branch lengths. This is because a major part of the underlying sequence evolution model — the nucleotide composition — is undergoing dramatic change in different mitochondrial lineages. We propose that this problem is also expressed in the occasional unexpected long branches leading to the “common ancestor” of orthologous numt sequences of different primate taxa. (3) The heterogeneous and lineage-specific evolution of mitochondrial sequences in primates renders molecular dating based on primate mtDNA problematic, whereas the numt sequences provide a much more reliable base for dating.[Reviewing Editor: Dr. Rafael Zardoya]     

Analysis of nuclear copies of mitochondrial sequences in honeybee (Apis mellifera) genome

At least 0.08% of the Apis mellifera nuclear genome contains sequences that originated from mitochondria. These nuclear copies of mitochondrial sequences (numts) are scattered all over the honeybee chromosomes and have originated by multiple independent insertions of mitochondrial DNA (mtDNA) as evident by phylogenetic analysis. Apart from original insertions, moderate duplications of numts also contributed to the present pattern and distribution of mitochondrial sequences in honeybee chromosomes. Assimilation of mitochondrial genes in the nuclear genome is mediated by extensive fragmentations of the original inserts. Replication slippage seems to be a major mechanism by which small sequences are inserted or deleted from mtDNA destined to nucleus. Most of the honeybee numts (84%) are located in the nongenic regions. The majority (94%) of the numts that are located in predicted nuclear genes have originated from mitochondrial genes coding for cytochrome oxidase and NADH dehydrogenase subunits. On the other hand, the mitochondrial rRNA or tRNA gene sequences are predominantly (88%) located in nongenic regions of the genome. Evidences also support for exertion of purifying selection on numts located in specific genes. Comparative analysis of numts of European, African, and Africanized honeybees suggests that numt evolution in A. mellifera is probably not demarked by speciation time frame but may be a continuous and dynamic process.     

Slow mitochondrial DNA sequence evolution in the Anthozoa (Cnidaria)

Mitochondrial genes have been used extensively in population genetic and phylogeographical analyses, in part due to a high rate of nucleotide substitution in animal mitochondrial DNA (mtDNA). Nucleotide sequences of anthozoan mitochondrial genes, however, are virtually invariant among conspecifics, even at third codon positions of protein-coding sequences. Hence, mtDNA markers are of limited use for population-level studies in these organisms. Mitochondrial gene sequence divergence among anthozoan species is also low relative to that exhibited in other animals, although higher level relationships can be resolved with these markers. Substitution rates in anthozoan nuclear genes are much higher than in mitochondrial genes, whereas nuclear genes in other metazoans usually evolve more slowly than, or similar to, mitochondrial genes. Although several mechanisms accounting for a slow rate of sequence evolution have been proposed, there is not yet a definitive explanation for this observation. Slow evolution and unique characteristics may be common in primitive metazoans, suggesting that patterns of mtDNA evolution in these organisms differ from that in other animal systems.     

The incomplete natural history of mitochondria

Mitochondrial DNA (mtDNA) has been used to study molecular ecology and phylogeography for 25 years. Much important information has been gained in this way, but it is time to reflect on the biology of the mitochondrion itself and consider opportunities for evolutionary studies of the organelle itself and its ecology, biochemistry and physiology. This review has four sections. First, we review aspects of the natural history of mitochondria and their DNA to show that it is a unique molecule with specific characteristics that differ from nuclear DNA. We do not attempt to cover the plethora of differences between mitochondrial and nuclear DNA; rather we spotlight differences that can cause significant bias when inferring demographic properties of populations and/or the evolutionary history of species. We focus on recombination, effective population size and mutation rate. Second, we explore some of the difficulties in interpreting phylogeographical data from mtDNA data alone and suggest a broader use of multiple nuclear markers. We argue that mtDNA is not a sufficient marker for phylogeographical studies if the focus of the investigation is the species and not the organelle. We focus on the potential bias caused by introgression. Third, we show that it is not safe to assume a priori that mtDNA evolves as a strictly neutral marker because both direct and indirect selection influence mitochondria. We outline some of the statistical tests of neutrality that can, and should, be applied to mtDNA sequence data prior to making any global statements concerning the history of the organism. We conclude with a critical examination of the neglected biology of mitochondria and point out several surprising gaps in the state of our knowledge about this important organelle. Here we limelight mitochondrial ecology, sexually antagonistic selection, life-history evolution including ageing and disease, and the evolution of mitochondrial inheritance.     

Mitochondrial pseudogenes are pervasive and often insidious in the snapping shrimp genus Alpheus

Here we show that multiple DNA sequences, similar to the mitochondrial cytochrome oxidase I (COI) gene, occur within single individuals in at least 10 species of the snapping shrimp genus Alpheus. Cloning of amplified products revealed the presence of copies that differed in length and (more frequently) in base substitutions. Although multiple copies were amplified in individual shrimp from total genomic DNA (gDNA), only one sequence was amplified from cDNA. These results are best explained by the presence of nonfunctional duplications of a portion of the mtDNA, probably located in the nuclear genome, since transfer into the nuclear gene would render the COI gene nonfunctional due to differences in the nuclear and mitochondrial genetic codes. Analysis of codon variation suggests that there have been 21 independent transfer events in the 10 species examined. Within a single animal, differences between the sequences of these pseudogenes ranged from 0.2% to 20.6%, and those between the real mtDNA and pseudogene sequences ranged from 0.2% to 18.8% (uncorrected). The large number of integration events and the large range of divergences between pseudogenes and mtDNA sequences suggest that genetic material has been repeatedly transferred from the mtDNA to the nuclear genome of snapping shrimp. Unrecognized pseudogenes in phylogenetic or population studies may result in spurious results, although previous estimates of rates of molecular evolution based on Alpheus sister taxa separated by the Isthmus of Panama appear to remain valid. Especially worrisome for researchers are those pseudogenes that are not obviously recognizable as such. An effective solution may be to amplify transcribed copies of protein-coding mitochondrial genes from cDNA rather than using genomic DNA.     

Evaluating the roles of energetic functional constraints on teleost mitochondrial-encoded protein evolution

Mitochondria are the power plant of cells, which play critical roles not only in energy metabolism but also in thermoregulation. These two roles have been individually suggested to influence mitochondrial DNA (mtDNA) evolution, however their relative importance is still rarely considered. Here, we conduct a comparative genomic analysis of 401 teleost complete mitochondrial genomes and test the roles of these dual functional constraints on mitochondria to provide a more complete view of mtDNA evolution. We found that mitochondrial protein-coding genes of migratory fishes have significantly smaller Ka/Ks than nonmigratory fishes. The same data set showed that the genes of fishes living in cold climates have significantly smaller Ka/Ks than tropical fishes. In contrast, these trends were not observed for two nuclear genes that are not involved in energy metabolism. The differences in selection patterns observed between mitochondrial and nuclear genes suggest that the functional constraints acting on mitochondria, due to energy metabolism and/or thermoregulation, influence the evolution of mitochondrial-encoded proteins in teleosts.     

Utility of nuclear DNA intron markers at lower taxonomic levels: phylogenetic resolution among nine Tragelaphus spp

Phylogenetic relationships among the nine spiral-horn antelope species of the African bovid tribe Tragelaphini are controversial. In particular, mitochondrial DNA sequencing studies are not congruent with previous morphological investigations. To test the utility of nuclear DNA intron markers at lower taxonomic levels and to provide additional data pertinent to tragelaphid evolution, we sequenced four nuclear DNA segments (MGF, PRKCI, SPTBN, and THY) and combined these data with mitochondrial DNA sequences from three genes (cytochrome b, 12S rRNA, and 16S rRNA). Our molecular supermatrix comprised 4682 characters which were analyzed independently and in combination. Parsimony and model based phylogenetic analyses of the combined nuclear DNA data are congruent with those derived from the analysis of mitochondrial gene sequences. The corroboration between nuclear and mtDNA gene trees reject the possibility that genetic processes such as lineage sorting, gene duplication/deletion and hybrid speciation account for the conflict evident in the previously published phylogenies. It suggests rather that the morphological characters used to delimit the Tragelaphid species are subject to convergent evolution. Divergence times among species, calculated using a relaxed Bayesian molecular clock, are consistent with hypotheses proposing that climatic oscillations and their impact on habitats were the major forces driving speciation in the tribe Tragelaphini.     

Mitochondrial DNA-like sequences in the nuclear genome of the opossum genus Didelphis (Marsupialia: Didelphidae).

This study reports the occurrence of mtDNA-like sequences in the nuclear genome of the opossum genus Didelphis (Didelphidae, Marsupialia). A specific primer pair designed to amplify a region encompassing a 3' terminal 118 bp region of the cytochrome b gene, the Thr and Pro tRNA genes, and a 489 bp region of the D-loop of the D. virginiana mtDNA, was used in highly stringent PCR reactions. These PCR reactions resulted in several fragments per individual varying in size from 259 bp to 1 kb. The sequencing of some of these fragments showed the occurrence of paralogous mtDNA-like sequences among the PCR amplified fragments. Analyses of qualitative aspects of these sequences, their transition/transversion ratios, and phylogenetic relationships were conclusive in showing the occurrence of mtDNA-like sequences in the nuclear genome of the genus Didelphis. Comparisons and phylogenetic analysis of orthologous mtDNA from the four Didelphis species and paralogous nuclear sequences suggested that mtDNA migration to the nuclear genome occurred more than once in Didelphis evolution.     

Nuclear mitochondrial pseudogenes

As has been demonstrated recently, the transfer of genetic material from mitochondria to the nucleus and its integration into the nuclear genome is a continuous and dynamic process. Fragments of mitochondrial DNA (mtDNA) are incorporated in the nuclear genome as noncoding sequences, which are called nuclear mitochondrial pseudogenes (NUMT pseudogenes or NUMT inserts). In various eukaryotes, NUMT pseudogenes are distributed through different chromosomes to form a “library” of mtDNA fragments, providing important information on genome evolution. The escape of mtDNA from mitochondria is mostly associated with mitochondrial damage and mitophagy. Fragments of mtDNA may be integrated into nuclear DNA (nDNA) during repair of double-strand breaks (DSBs), which are caused by endogenous or exogenous agents. DSB repair of nDNA with a capture of mtDNA fragments may occur via nonhomologous end joining or a similar mechanism that involves microhomologous terminal sequences. An analysis of the available data makes it possible to suppose that the NUMT pseudogene formation rate depends on the DSB rate in nDNA, the activity of the repair systems, and the number of mtDNA fragments leaving organelles and migrating into the nucleus. Such situations are likely after exposure to damaging agents, first and foremost, ionizing radiation. Not only do new NUMT pseudogenes change the genome structure in the regions of their integration, but they may also have a significant impact on the actualization of genetic information. The de novo integration of NUMT pseudogenes in the nuclear genome may play a role in various pathologies and aging. NUMT pseudogenes may cause errors in PCR-based analyses of free mtDNA as a component of total cell DNA because of their coamplification.     

Toward the resolution of an explosive radiation--a multilocus phylogeny of oceanic dolphins (Delphinidae)

Oceanic dolphins (Delphinidae) are the product of a rapid radiation that yielded ～36 extant species of small to medium-sized cetaceans that first emerged in the Late Miocene. Although they are a charismatic group of organisms that have become poster children for marine conservation, many phylogenetic relationships within Delphinidae remain elusive due to the slow molecular evolution of the group and the difficulty of resolving short branches from successive cladogenic events. Here I combine existing and newly generated sequences from four mitochondrial (mt) genes and 20 nuclear (nu) genes to reconstruct a well-supported phylogenetic hypothesis for Delphinidae. This study compares maximum-likelihood and Bayesian inference methods of several data sets including mtDNA, combined nuDNA, gene trees of individual nuDNA loci, and concatenated mtDNA+nuDNA. In addition, I contrast these standard phylogenetic analyses with the species tree reconstruction method of Bayesian concordance analysis (BCA). Despite finding discordance between mtDNA and individual nuDNA loci, the concatenated matrix recovers a completely resolved and robustly supported phylogeny that is also broadly congruent with BCA trees. This study strongly supports groupings such as Delphininae, Lissodelphininae, Globicephalinae, Sotalia+Delphininae, Steno+Orcaella+Globicephalinae, and Leucopleurus acutus, Lagenorhynchus albirostris, and Orcinus orca as basal delphinid taxa.     

ALG11--a new variable DNA marker for sponge phylogeny: comparison of phylogenetic performances with the 18S rDNA and the COI gene

Phylogenetic relationships within sponge classes are highly debated. The low phylogenetic signal observed with some current molecular data can be attributed to the use of few markers, usually slowly-evolving, such as the nuclear rDNA genes and the mitochondrial COI gene. In this study, we conducted a bioinformatics search for a new molecular marker. We sought a marker that (1) is likely to have no paralogs; (2) evolves under a fast evolutionary rate; (3) is part of a continuous exonic region; and (4) is flanked by conserved regions. Our search suggested the nuclear ALG11 as a potential suitable marker. We next demonstrated that this marker can indeed be used for solving phylogenetic relationships within sponges. Specifically, we successfully amplified the ALG11 gene from DNA samples of representatives from all four sponge classes as well as from several cnidarian classes. We also amplified the 18S rDNA and the COI gene for these species. Finally, we analyzed the phylogenetic performance of ALG11 to solve sponge relationships compared to and in combination with the nuclear 18S rDNA and the COI mtDNA genes. Interestingly, the ALG11 marker seems to be superior to the widely-used COI marker. Our work thus indicates that the ALG11 marker is a relevant marker which can complement and corroborate the phylogenetic inferences observed with nuclear ribosomal genes. This marker is also expected to contribute to resolving evolutionary relationships of other apparently slow-evolving animal phyla, such as cnidarians.     

The mitochondrial genome of Arabidopsis is composed of both native and immigrant information

Plants contain large mitochondrial genomes, which are several times as complex as those in animals, fungi or algae. However, genome size is not correlated with information content. The mitochondrial genome (mtDNA) of Arabidopsis specifies only 58 genes in 367 kb, whereas the 184 kb mtDNA in the liverwort Marchantia polymorpha codes for 66 genes, and the 58 kb genome in the green alga Prototheca wickerhamii encodes 63 genes. In Arabidopsis’ mtDNA, genes for subunits of complex II, for several ribosomal proteins and for 16 tRNAs are missing, some of which have been transferred recently to the nuclear genome. Numerous integrated fragments originate from alien genomes, including 16 sequence stretches of plastid origin, 41 fragments of nuclear (retro)transposons and two fragments of fungal viruses. These immigrant sequences suggest that the large size of plant mitochondrial genomes is caused by secondary expansion as a result of integration and propagation, and is thus a derived trait established during the evolution of land plants.     

General properties and phylogenetic utilities of nuclear ribosomal DNA and mitochondrial DNA commonly used in molecular systematics

To choose one or more appropriate molecular markers or gene regions for resolving a particular systematic question among the organisms at a certain categorical level is still a very difficult process. The primary goal of this review, therefore, is to provide a theoretical information in choosing one or more molecular markers or gene regions by illustrating general properties and phylogenetic utilities of nuclear ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) that have been most commonly used for phylogenetic researches. The highly conserved molecular markers and/or gene regions are useful for investigating phylogenetic relationships at higher categorical levels (deep branches of evolutionary history). On the other hand, the hypervariable molecular markers and/or gene regions are useful for elucidating phylogenetic relationships at lower categorical levels (recently diverged branches). In summary, different selective forces have led to the evolution of various molecular markers or gene regions with varying degrees of sequence conservation. Thus, appropriate molecular markers or gene regions should be chosen with even greater caution to deduce true phylogenetic relationships over a broad taxonomic spectrum.     

Molecular markers and modern phylogenetics of mammals

Application of modern molecular techniques such as molecular cloning, sequences and polymerase chain reaction of DNA resulted in the increasing of resolution of the phylogenetic analysis and enhanced the role of molecular markers in the evolutionary and taxonomic studies. However, certain properties of the molecular markers are to be taken into consideration when results of the molecular phylogenetic analyses are discussed. This survey reviews the advantages and shortages of different molecular markers (mtDNA and nDNA genes, satellite sequences, long and short retroposons) at the various taxonomic levels. The most part of new phylogenetic reconstructions are established on the results of mtDNA analysis and must be interpreted cautiously because of non-mendelian inheritance of mitochondrial genome. The extremely rapid rate of nucleotide change in mtDNA as compared with nDNA reinforces the saturation in nucleotide sequence and screens the phylogenetic signal. The analysis of nuclear genomes in constrained by that only truly orthologous genes are suitable for the phylogeny reconstruction. So there is a problem to distinguish genes from pseudogenes. Besides, there are some general problems of gene reconstruction such as nucleotide and amino acid compositional shift, long branch attraction and the choice of outgroup. Short interspersed nuclear elements (SINEs) may provide the most valuable phylogenetic information. The markers of multilocus DNA analysis (RAPD-PCR, IS-PCR, RELP, ISSR-PCR), their advantages and shortages are also discussed. A brief survey of the recent studies of molecular phylogeny of mammals for the period of about ten years is presented. The results based on the combined analysis of the mitochondrial and nuclear genes reject the reliability of some previously recognized supraordinal Eutherian taxa in favour of independent range of four main super-order clades: Afrotheria, Xenarthra, Euarchontoglires, and Laurasiatheria. Within these clades, monophyly of some of traditionally recognized orders was proved by molecular data. The recent advances of molecular phylogenetics are very encouraging. However, its future developments are full off serious difficulties. The problem of accumulation of the data turns into the problem of their correct analysis that is more difficult from the methodological point of view. The careful analysis of the conformities and contradictions between different data sets and looking for congruent conclusions deduced from different characters are the most fruitful way of further phylogenetic development.     

Using secondary structure to identify ribosomal numts: cautionary examples from the human genome.

The identification of inadvertently sequenced mitochondrial pseudogenes (numts) is critical to any study employing mitochondrial DNA sequence data. Failure to discriminate numts correctly can confound phylogenetic reconstruction and studies of molecular evolution. This is especially problematic for ribosomal mtDNA genes. Unlike protein-coding loci, whose pseudogenes tend to accumulate diagnostic frameshift or premature stop mutations, functional ribosomal genes are not constrained to maintain a reading frame and can accumulate insertion-deletion events of varying length, particularly in nonpairing regions. Several authors have advocated using structural features of the transcribed rRNA molecule to differentiate functional mitochondrial rRNA genes from their nuclear paralogs. We explored this approach using the mitochondrial 12S rRNA gene and three known 12S numts from the human genome in the context of anthropoid phylogeny and the inferred secondary structure of primate 12S rRNA. Contrary to expectation, each of the three human numts exhibits striking concordance with secondary structure models, with little, if any, indication of their pseudogene status, and would likely escape detection based on structural criteria alone. Furthermore, we show that the unwitting inclusion of a particularly ancient (18-25 Myr old) and surprisingly cryptic human numt in a phylogenetic analysis would yield a well-supported but dramatically incorrect conclusion regarding anthropoid relationships. Though we endorse the use of secondary structure models for inferring positional homology wholeheartedly, we caution against reliance on structural criteria for the discrimination of rRNA numts, given the potential fallibility of this approach.     

Substitution saturation and nuclear paralogs of commonly employed phylogenetic markers in the Caryophyllidea, an unusual group of non-segmented tapeworms (Platyhelminthes)

Caryophyllidean cestodes (Platyhelminthes) represent an unusual group of tapeworms lacking serially repeated body parts that potentially diverged from the common ancestor of the Eucestoda prior to the evolution of segmentation. Here we evaluate the utility of two nuclear and two mitochondrial molecular markers (ssrDNA and lsrDNA, nad3 and cox1) for use in circumscribing generic boundaries and estimating interrelationships in the group. We show that these commonly employed markers do not contain sufficient signal to infer well-supported phylogenetic estimates due to substitution saturation. Moreover, we detected multiple trnK+nad3+trnS+trnW+cox1 haplotypes within individuals, indicating a history of gene exchange between the mitochondrial and nuclear genomes. The presence of such nuclear paralogs (i.e. numts), to our knowledge described here in cestodes for the first time, together with the results of phylogenetic, saturation and split-decomposition analyses all suggest that finding informative markers for estimating caryophyllidean evolution is unusually problematic in comparison to other major lineages of tapeworms.     

The influence of demography,population structure and selection on molecular diversity in the selfing freshwater snail Biomphalaria pfeifferi

Several forces may affect the distribution of genetic diversity in natural populations when compared to what is expected in a random-mating, constant size population of neutral genes. One solution for unravelling their respective influence is to study several genes at once in order to better reflect the true genealogy. Here we reconstruct the evolutionary history of the freshwater snail Biomphalaria pfeifferi over its entire distribution, using eight African populations, and three congeneric species as outgroups. A phylogenetic analysis was conducted using amplified fragment length polymorphism markers, and sequences at eight nuclear non-coding loci and one mitochondrial gene were used to analyse population structure. The geographic distribution of variation suggests greater affinities within than among regions. The pattern of variability at both the nuclear and mitochondrial DNA (mtDNA) loci is consistent with a bottleneck, although population structure may also partly explain our results. Our results are also indicative of the role of selection, whether positive or purifying, in the mtDNA. This highlights the fact that the interfering influences of population structure, demography and selection on molecular variation are not easily distinguished.     

Nucleotide sequence and evolution of the 18S ribosomal RNA gene in maize mitochondria.

The nucleotide sequence of the gene coding for the 18S ribosomal RNA of maize mitochondria has been determined and a model for the secondary structure is proposed. Dot matrix analysis has been used to compare the extent and distribution of sequence similarities of the entire maize mitochondrial 18S rRNA sequence with that of 15 other small subunit rRNA sequences. The mitochondrial gene shows great similarity to the eubacterial sequences and to the maize chloroplast, and less similarity to mitochondrial rRNA genes in animals and fungi. We propose that this similarity is due to a slow rate of nucleotide divergence in plant mtDNA compared to the mtDNA of animals. Sequence comparisons indicate that the evolution of the maize mitochondrial 18S, chloroplast 16S and nuclear 17S ribosomal genes have been essentially independent, in spite of evidence for DNA transfer between organelles and the nucleus.     

Complete mitochondrial DNA of the hagfish, Eptatretus burgeri: the comparative analysis of mitochondrial DNA sequences strongly supports the cyclostome monophyly

The phylogenetic position of cyclostomes, i.e., the relationships between hagfishes, lampreys, and jawed vertebrates is an unresolved problem. Anatomical data support the paraphyly of cyclostomes, whereas nuclear genes data support monophyly of cyclostomes. Previous results obtained using mitochondrial DNA are ambiguous, presumably due to a lack of informative sequences. By adding the complete mtDNA of a hagfish, Eptatretus burgeri, we have generated a novel data set for sequences of hagfishes and of lampreys. The addition of this mtDNA sequence to the 12 taxa we have already used becomes sufficient to obtain unambiguous results. This data set, which includes sequences of mtDNA of animals closely related to the lamprey/hagfish node, was used in a phylogenetic analysis with two independent statistical approaches and unequivocally supported the monophyly of cyclostomes. Thus molecular data, i.e., our results and those obtained using nuclear genes, conclude that hagfishes and lampreys form a clade.