Dear Hong

All the genes you mentioned are poorly variable and will not help to discriminate close species. As commented by our friends above ITS was the best (in term of resolution) but if you've got a bad discrimination it means that you've got to search among NDx genes (ND5 is often easy to sequence and highly variable) or the control loop of the mitochondria. Most of time nuclear and chloroplastic genes are less variable than those of mitochondria.

Among those that you proposed from best to worse: ITS>>28S>Cox1>>Rbcl

With ITS you can separate species which diverged 1.5/2 millions years ago, with 28S and Cox 4 millions years ago, Rbcl will separate genera but not species.

Cheers from Luc

I'm not familiar with this group, but I think ITS (1 or 2, or 1+2) should be a good choice for their variability. I would add a more conserved locus to add some resolution and/or robustness in the deepest nodes of the tree (maybe rbcL and/or cox1, according the literature that should exist... ;-))

Of course, it depend of your time for experiments, money for sequencing, etc...

If they are closely related species, as you say, I would choose one or more markers that are variable enough to give you resolution to answer your questions.

I would use more than one chloroplast marker AND ITS (1 and 2).

Don't build your assumptions based only on one marker (or only on one of the Genomes).

When it comes to Chloroplast markers, have you read the following papers?

1 - Shaw, J. et al - American Journal of Botany - 94(3): 275-288. 2007 - Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies...

2 - Stech & Dietmar - PHYTOTAXA - 9: 196-228. 2010 - 20.000 species and five markers: The status of molecular bryophyte phylogenetics?

Hope this helps...

Yes Dr. Hinsinger and Musili, I agreed with both of you that ITS show a great degree of genetic divergence, but from our studies some of the cryptic species revealed low divergence. I have tried different alignment strategies for ITS1/5.8S/ITS2, ie. alignment with T-Coffee and concatenate the sequence with G-Block. Cryptic species tend to form polytomy in phylogenetic tree and long-branch effect is found in the tree for certain species. Same goes to LSU, perhaps the sequences are too conserved.

The tree was resolved by using only ITS2 with structural information, but the proposed secondary structure are too subjective, and may varies among the researchers. So I'm trying to look for other gene, which can better resolve the phylogeny, and with relatively easier steps in whole analysis. Currently working on cox1, about 600bp.

Hi Gama, I do compared the phylogeny with LSU, whole ITS as well as ITS2 (with structural information). I understand that they're ribosomal nuclear gene and now try to work on mitochondrial and plastid gene. Do you have any idea on these two gene? Thanks for the papers, very informative.

I think that the better marker s to study the structure of non coding RNAs located in the noncoding area of DNA. These molecules have a particulñar structure but also specific functions.

I'm not familiar with this group, but if you have whole genome, you may try to infer the phylogenetic tree using the whole genome.

As far as I know, for prokaryotes, the tree based on whole genome is a little "better" than 16s rRNAs, especially for genera or species ("better" just means in some controversial branches, the genome-tree seems to match the biologic knowledges better than rRNA tree. However, this is just an observation not a strict definition).

And also these whole-genome methods have been used to infer the phylogenetic tree for fungi and virues.

But I'm not sure if they're appropriate for your question.

Here is one whole genome method: http://tlife.fudan.edu.cn/cvtree/

I am not familiar with this group, but I have done some close related species in fungi. I try five markers. Among them, ITS, tef1-α, and rpb2 do work very well. Maybe you could try. Good luck.

Actually, these genes are all eligible for phylogenetic study in my opinion. Moreover, ITS regions are now considered to perform better in species-specific identification and used in barcoding research in more and more organisms. In our research group, one has found out the polymorphisms in several sites in ITS regions in one genus of dinoflagellates. Thus, it is possible to study the relationship under the level of species by ITS.

Besides, the variable regions in 18S rDNA are also widely used in molecular taxonomy and phylogenetics in mircoorganisms due to their high species-specific features.

I think Cox1 should be a good marker to solve cryptic species. However rbcL also will be suitable. LSU is not appropriate marker for closely related species.

Dear Hong

All the genes you mentioned are poorly variable and will not help to discriminate close species. As commented by our friends above ITS was the best (in term of resolution) but if you've got a bad discrimination it means that you've got to search among NDx genes (ND5 is often easy to sequence and highly variable) or the control loop of the mitochondria. Most of time nuclear and chloroplastic genes are less variable than those of mitochondria.

Among those that you proposed from best to worse: ITS>>28S>Cox1>>Rbcl

With ITS you can separate species which diverged 1.5/2 millions years ago, with 28S and Cox 4 millions years ago, Rbcl will separate genera but not species.

Cheers from Luc