Possible if the sequences designated for barcode is species-specific enough.

I think it depends on the level of divergence of those sub-species

DNA barcoding is addressed to identify species. As indicated by ANA AMARAL, only if divergence is high you can use barcoding to distinguish between sub-species. Barcoding in aninals uses COI mitochondrial gene, maybe you can use other mtDNA genes (cytb, control region,...) to analyse the divergence and to identify your sub-species.

That's depends entirely on the amount of variation in the taxa you work with and what the criteria for "sub-species" is!

Sub-species designations mean very different things in different taxonomic groups. Sometimes they are defined by geography (e.g. alopatric populations of the same species may), sometimes morphology (e.g. minor morpholgoical variation without fixed characters) etc. It is a much more difficult to define category than "species" (and even that is widely debated).

In many cases I have worked with sub-species were traditionally classified that way because they had minor-morphological differences (e.g overall body size) but they lacked obvious morphological characters that could be used to differentiate them in a museum. Once we re-investigate with DNA in many cases we have found that they are actually completely reproductively isolated groups and, when we look at DNA, morphology, behaviour etc. in combination, we realize they are really just unsubscribed species that could not easily be identified using traditional morphological approaches and we end up elevating the sub-species to species status.

Here's an example where we found some "sub-species" to really be species:

http://www.biomedcentral.com/1471-2148/13/26/abstract

In this case it is quite likely that they had not previously been considered species because the most obvious character which may differentiate them is the frequency of their echolocation. This is not preserved in a museum. So in this case, the COI DNA barcode differentiated sub-species and upon re-evaluation, we realized the sub-species actually meet the criteria for the biological species concept.

So the answer to your question will really depend on what your definition of a species is, what the definition of a sub-species is and how much variation exists within the region you use as a barcode (e.g. the "DNA barcode" based on COI). So the answer may be very taxon or geographically specific.

I hope that helps.

Best wishes

Elizabeth Clare

A subspecies is not a clear diagnostic entity. Often, it describes a phylogeographic group within a species, and if the split is deep enough, we tend to call it currently, based on DNA barcoding, a separate species. But essential is that there is no clear definition of what a subspecies really represents, and barcoding will not help there either. In some cases, barcoding will be able to distinghuish them, in others not. Subspecies are often defined based on regional and morpholigical coherence, but do not necessarily represent genetically or evolutionarily hierarchical units. Actually, the same applies to the generic level. In birds, anything that looks a bit different is placed in a different genus nowadays, especially as reciprocal monophyly is required in taxonomy. So we are getting an inflation of new genus names, all of which represent very young (molecular-clockwise) units. In reverse, go to invertebrates like water fleas. The genus Daphnia comprises species that have gone their own separate evolutionary ways some 150 million years ago, but still they are in the same genus. That's like putting all birds and mammals together in one genus. We tend to want to put organisms in taxonomic boxes and label them. The truth is, there aren't always clearcut distinctions. Everything is gradual in evolution. So there is no general answer to your question, as there is no general clear-cut treshold (in barcoding) of what represents a species, a subspecies, a genus or any other level in taxonomy and systematics. Hope this helps.

This strongly depends on the analysed taxa or species. For some species it may work, for others not. However, you need more than a few specimens to answer this question.

Ya sure

Since chloroplast sequences are very conserved and mutation rate is low it is very difficult to distinguish between varieties using standard barcoding regions.

Typically, when there is a "barcoding gap" of >5%, it gives more evidence for a species naming. Less than 5% can designate subspecies or subpopulations. I've only done barcoding for Crocodilia, so the 5% threshold is what I have used. I'm not sure if the same threshold value is true for other orders.

The subspecies should be a good "taxon". The barcoding locus is very important. If no hybridization, the nuclear locus is better than plastid locus.

Well Barcoding is not the tool to separate sub-species....as it is not the tool to separate close species. If you consider the classical Cox1, genetic entities had to be separated from around 3 millions of years to have a chance to see few significant mutations. It means that all the splits of populations/species (recent radiations) which occurred during pleistocene are not possible to being characterized.

I think that the discussion to determine what is a subspecies is interesting....as many people are mixing clines, local dominance of a form versus another etc...with: A population of a species which have no geographical contact with other populations of the same species and possess stable morphological or genetic différences.

I saw many recent papers in good journals where, checking well, only 4 or 5 nucléotides among 5000 sequenced are variables ......authors are building from such few differences great evolutionary theories......

I think that people have to stop with this tool (barcoding) and being more realistics.

If you want to work with populations use microsats or, if primers are not available, ISSR or AFLP (even if they are dominant markers).

Cheers from Luc

It quite depends on the barcode you use and the taxa you want to delimitate, because different barcodes(e.g. 16s, COI, ITS, matK, recL) have different resolution among groups.

Also, current popular barcode barely can separate sub-species, mostly species.

COI in many cases are effective in distinguishing populations or groups of populations. Also I'd ask a counter-question: what is a subspecies actually? After more of two centuries of scientific debate it is not completely clear what a species is, much less subspecies!

I think that Ferruccio is right....I've got to admit that 15 years ago I knew what was a species....actually.....not anymore lol !!!!

I proposed (see my last post) a definition for subspecies but which is based on a clear status for a species (which is not the case actually, unfortunately.....).

Coming back to barcode....of course how to say something on resolution if what we aim to characterize is not clear at all !!!! Anyway, I consider that building great evolutionary scenarii with less of 2 or 3% variation by gene is not valid, or only way, to sequence 15000 nucléotides (but then do we have to call such studies: barcoding?) and then these 2% may start to mean something.

It´s not clear sub-species concept, but you can use Barcode always had a good samples of "sub-species" (in the analyses must be "Genotypes" or OTU´s a operative concept) and compare with species evolutionary closed for obtained divergence or genetic distances and compare with all samples of that species (sub-species included), so with these methodology Barcode you definying MOTU´s and alternatives uses as Bayesian or ML you can allowed split lineages and maybe compare lineages with "sub-species" proposed with other researches. Is my opinion about how use DNA Barcode in your research problem, however, is clear that you use other molecular markers and the Sub-species problem is really an evolutionary problem. Greetins from Colombia

Can DNA barcoding apply to distinguish species level in all cases?

No, not working for some species at least for some vertebrates. I got 99 % similarity between two different species (different genus, same family)

The most importance thing about DNA barcoding: you should be sure about `did you work on enough samples and get high quality sequences or not`.

Arzu is right, for most of species barcoding is not able to separate them, with Arzu example with fishes, I've got A LOT of examples with insects !!!!

I worked recently with plants....and we compared barcoding with ISSR on a Mexican Fabaceae (in press in South African Journal of Botany, hopefully available on line in the next 3 weeks). Conclusion is mostly the same, barcode is not working for subspecies or very close species.

Cheers from Luc

@Arzu and Luc: have you done for all DNA plastid regions (atpF–atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–psbI spacer, and trnH–psbA

spacer etc) or only few?.

Some papers showed promising results on DNA barcode, particularly for plants.

Dear Tran

Of course if you combine all these genes you may have results...but barcoding in my mind is using 1 or 2 genes, you've got to measure the balance information/cost.

By sequencing to reach THE SAME number of variable sites (even if quality of information is different) you need 10 genes (around 7000 Euros = 9300 dollars for 96 samples one direction) versus 3000 Euros for microsats and 2000 Euros for ISSR.

Of course the best is to do SNP.......for 96 samples around 40000 dollars....

Is you lab is extremely rich.....???? If so I will visit you immediately !!! lol !!!

Cheers from Luc

Dear Luc,

cost depends on how you big is your samples and how long does sequence has. In my opinion, preliminary survey to find best combination of 2 or 3 genes does not much costly. Next step costly, of course, for the rest of samples. But sequencing companies in South Korea have lower price and good services are choices.

Yes, in brief, but depending on how you define subspecies and what gene you will use. Typically, CO1 is used, which may not provide the resolution to distinguish organisms at the intra-specific level. Some folks use the control region, which evolves more quickly.

Dear Michael

Yes we are finally restarting all the controversy around the use of Cox1 for everything....CoX is clearly the worst gene to separate close species (and of course subspecies) some are more variable like the control region and all the ND genes. But anyway, to reach a good level of resolution you need to combine minimum 3 or 4 of the most variable mitochondrial genes and then to balance results/costs/time when compared to use of microsats or ISSR.

Cheers from Luc