The best tool for species delimitation is knowledge on its morphological variation, behavioiur, geographical distribution, cytogenetics, etc, supported by the molecular analysis. :)

I do not think there is a "global" tool for species delimitation that is 100% reliable and useful for all taxa. The combination of morphological characters, DNA sequences, morphometric tools are the best way to tell species apart. Of course, it depends on which taxa are you working with. You might be able to provide more information on which group of species you are trying to delimit in order to get more answers for our colleagues researchers.

I have just published a Review paper in Gondwana Research about the Problem of species recognition and delimitation. I think you can use them for the snakes.

The first thing would be to align sequences you have, I use Geneious for this, but there are lots of other software. Based on alignment you do all kind of manipulations like building haplotype networks (in TCS for example) or clustering (in BAPS) or visualising everything with some trees (BEAST). I work with species delimitation in lichenized fungi, don't know for sure how you "snake" people do this. Anyway, good luck!

To get more insight into this problem, apart the excellent article mentioned by Prof. Hohengger, I would recommend reading the book: Kunz, W. (2012). Do species exist? Principles of taxonomic classification. . Weinheim, Germany, Wiley-Blackwell.

According to the question if species exist and are real natural entities I also recommend my article on this problem published in Zootaxa 2012. You can find it in my list and download.

The best tool for species delimitation is knowledge on its morphological variation, behavioiur, geographical distribution, cytogenetics, etc, supported by the molecular analysis. :)

Hi Ashok:

I completely agree with Józef opinion and I think that the first step to understand the morphology of the group under study in depth.

You can try Bayes Factor Species delimitation on your dataset.

Dear Ashok

Don't search for any software which will give you an answer....you've got none (or they are gadgets).

First step is too have a look to all the articles of James Mallet who is the one (in my mind) who published the best papers on "real species".

Sequencing (genes of non coding DNA like your ITS) will provide you relationships and distances between your samples. Clearly (on reptiles) if you've more than 5% of differences between some individuals, they are surely belonging to different species. But I may recommend to have a look to all the recent papers on the Lacerta agilis complex of species. An author who was (a part from the very good papers of Johann, see above) is Zoltan Nagy (actually in Belgium). You may also check all the publications from the team of Darmstadt in Germany (many papers are posted by Michael Wink in research gate).

Cheers from Luc

iBPP which is a new program allows you to combine genetic data with phenotype data 

http://www.molecularecologist.com/2015/01/incorporating-phenotype-and-genotype-in-model-based-species-delimitation/