In my experience studying the fish mitochondrial cytochrome B gene phylogenies, I get the impression that some sequences in GenBank are mislabeled or specimens were misidentified, or there is some other similar problem. One alternative explanation is that some researchers sequenced nuclear copies of mitochondrial genes (numnts). For many or most studies, the fish mitochondrial cytochrome oxidase gene phylogeny does "fit" with morphological estimates of evolution, and with phylogenetic analyses of many other genes. I am attaching a tree built from sequences of one study combined with BLAST hits from GenBank which illustrates the problem. The Astyanax sequences all seem to make sense, but there are some Alestes and Micralestes sequences in GenBank that seem to be mislabeled. I wrote to one fish sequence author about this, and they told me I just did not understand fish nomenclature, and it is not unexpected for Micralestes parensis (GenBank AY791387) to be within the Phenacagrammus clade. But I think sequence mislabeling or sample mix-up is a more likely explanation.

Thanks a lot for yr response... But in my case, I have not taken any sequence from database. I am working on some fishes... While doing the phylogeny, it didn't give desirable tree... I don't have any paper also that shows the limitation. I would be grateful to you if u could provide some reference in which cyt b phylogenetic tree didn't give desirable tree

For just one example, in the paper I am attaching here, figure 3 shows Rhabdalestes maurensis on a very long branch as if it evolved very rapidly compared to the other fish in this tree. But if you pull out the GenBank entries listed in table 2, and explore the data, it turns out that one of the sequences seems to be a typical Rhabdalestes (JF801095; 99 to 97% identical to other Rhabdaleses and Alestes) while the other is very odd (JF800946; 84% to 88% identical to other (JF800946). Sorry, these are mitochondrial COI and myh6 genes and not mitochondrial cytB, but it is the same issue. In my opinion, some problem with the sequence JF800946 makes more sense than very rapid evolution of this gene while other genes of the same mitochondria evolve normally).

I am attaching also a 1996 paper on the problem of nuclear copies of mitochondrial genes. It is possible that even if you did not have any sample mislabeling or mix-up (or PCR contamination etc) that you sequenced some mitochondrial genes and some nuclear copies of mitochondrial genes.

Thanks a lot sir....Is there any literature that explains that cyt b gene is not potent for some fishes for Phylogeny tree construction. CoI and 16s for the species I am working is fine-appropriate tree is coming in MEGA with appropriate intra and interspecies variation. But when I did cytb gene amplification from the same DNA which I used for COI and 16s, the results were erroneous. In BLAST, its showing here and there and Phylogenetic tree is also not proper.

The history of using cyt B for phylogenetic is tortured in my view. It was originally used as an arbitrary choice based exclusively on availability. It was the first bit of DNA sequence in any genome for which "universal" PCR primers were published. Axel Meyer used it for phylogenetic and others simply followed his lead. By the time it was possible to begin generating sequences from other homologous loci across many taxa, cyt B was the only locus with a published database to compare with. Axel Meyer even published a paper several years later regretting that cyt B arbitrarily became the standard in molecular phylogenetics, because it is largely a set of very slowly evolving sites and very fast (especially in homeotherms) evolving sites. This commonly results in either too much homoplasy or too little phylogenetic signal in many applications. It is also important to know that the 'true' tree topology can differ between any mtDNA gene and the consensus topology from nuclear DNA, so the notion of a "desirable" tree strikes me as odd. In addition to all of this, sex-biased dispersal (female philopatry) can increase differences between 'true' mtDNA trees and 'true' nDNA trees. Differences between observed and "desired" topologies can be informative.

Thanks a lot sir..... Do you have any reference for this kind of deviation...?

Here are some I found:

Farias, I.P., Ortî, G. Sampaio, I. Schneider, H. and Meyer, A. 2001. The cytochrome b gene as a phylogenetic marker: the limits of resolution for analyzing relationships among cichlid fishes. Journal of Molecular Evolution 53: 89-103.

Meyer, A. 1994. Shortcomings of the cytochrome b gene as a molecular marker. Trends in Ecology and Evolution 9: 278-280.

Melnick, D. J. and G. A. Hoelzer. 1992. Differences in male and female macaque dispersal lead to contrasting distributions of nuclear and mitochondrial DNA variation. International Journal of Primatology 13: 1-15.

Melnick, D. J., G. A. Hoelzer, R. Absher and M. V. Ashley. 1993. mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species. Molecular Biology and Evolution 10: 282-295.

Hoelzer, G. A. 1997. Inferring phylogenies from mtDNA variation: mitochondrial gene-trees versus nuclear gene-trees revisited. Evolution 51: 622-626.

There is a much larger literature on these issues. I just grabbed a couple of Axel's papers (to put things into perspective from the founder of using cytb for phylogenetics) and a few relevant papers from my own CV. I hope these are helpful.

Guy

Thank you sir...This would be helpful for me.

Sir, is there any possibility that intraspecies divergence (within group) will be more than interspecies divergence (between group) value ?

In biology, pretty much anything is "possible", but we can estimate how "probable" or likely something is to happen. It is possible that the mitochondrial cytochrome B gene could have a freakishly high evolution rate in one species of fish, but very highly unlikely that this would happen, especially without also showing some evidence of rapid evolution in other mitochondrial genes. What would be the mechanism for just one gene evolving rapidly? Also, you need to consider just exactly what distances or level of evolution are you talking about. It is much more likely that we would see 2 nucleotide changes in the gene within one species while there is only one change between the same gene in two other species, than it is that we would see 20 nucleotide changes within one species while there is only 4 or 5 mutations difference between other species in one genus.

The next thing to consider is whether these differences within your species are unique, or are they shared with another species. If your COI genes all look like Tuna COI from various species of Tuna and the CytB gene sequences are just unique, it is one thing. But if your CytB sequences are nearly identical to Salmon CytB gene sequences, that is very different. Is there any mechanism for Tuna and Salmon mitochondria to get together and exchange mitochondrial genes?

In general mitochondria in eukaryotes (not just vertebrates but most eukaryotes) do not have any opportunity to exchange genetic material across species boundaries. Nuclear genes or pseudogenes or transposons can be moved between species by viruses on rare occasions, but mitochondrial DNA is far less likely to be transferred.

On the other hand, if you look at large data sets of single mitochondrial gene data, such as COI or CytB, you can see that there are at least dozens of data sets in GenBank where the sequences have been misidentified due to sample mislabelling, PCR contamination, or other problems. Any lab that does PCR will have PCR contamination events, no matter how careful they are. They can be reduced and they can be detected so that the data does not get into GenBank, but they cannot be eliminated. If you question your data, send sample off to another lab that has never handled fish mitochondria for confirmation. Hundreds of labs study mitochondrial genes from other organisms (lizards, amphibians, mammals etc) and could amplify and sequence your fish genes for confirmation.

The references I gave you from my postdoctoral work with Don Melnick, you can see that there is greater divergence within some macaque monkey species than between. In fact, the best example among macaques is the crab eating macaque, which occupies many islands in southeast Asia. This species includes mtDNA genomes that coalesce into a most recent common ancestor much older than the origin of the entire macaque genus (about 23 species now). Intraspecific para/polyphyly of mtDNA among macaque species is common. Our research led to the conclusion that this resulted from extreme sex-biased dispersal with female philopatry in these species.

Interestingly, there is a situation between a couple of northern macaque species where we think the data indicate horizontal transfer of mtDNA genomes between species. Note that the mtDNA genome is so packed with essential genes compared with the nuclear genome one might expect selective sweeps to be more common with mitochondria. Here are some relevant references:

Melnick, D. J., G. A. Hoelzer, R. Absher and M. V. Ashley. 1993. mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species. Molecular Biology and Evolution 10: 282-295.

Hoelzer, G. A., M. A. Hoelzer and D. J. Melnick. 1993. The evolutionary history of the sinicagroup of macaque monkeys as revealed by mtDNA restriction site analysis. Molecular Phylogenetics and Evolution 1: 215-222.

Hoelzer, G. A., W. P. J. Dittus, M. V. Ashley and D. J. Melnick. 1994. The local distribution of highly divergent mitochondrial DNA haplotypes in toque macaques (Macaca sinica) at Polonnaruwa, Sri Lanka. Molecular Ecology 3: 451-458.

Melnick, D. J., G. A. Hoelzer, R. Absher and M. V. Ashley. 1993. mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species. Molecular Biology and Evolution 10: 282-295.

Hoelzer, G. A. and D. J. Melnick. 1994. Patterns of speciation and limits to phylogenetic resolution. Trends in Ecology and Evolution 9: 104-107.

Cheers,

Guy

Thanks a lot sir...