How To Distinguish Two Species From Est Data?

Question

[Andyh]

I have EST (cDNA) data from tissues from two cattle ticks, one N. American and the other Australian. I'd like to know if they are the same species. They don't produce viable offspring when they mate, but that is not an indicator of species relatedness in many insects for reasons I don't want to go into here. SO, I'm left with the transcript data but I'm not sure what constitutes enough of a difference in the DNA to say they are different species. I'd appreciate any ideas...

[Andyh]

correction: ticks are not insects actually but they are arthropods

Answer 1

[ALchEmiXt]

I think the NCBI taxonomy pages will help you to determine where the classification of genus, species and sub-species relies on; At least some of its references.... [ http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/index.cgi?chapter=resources#Other%20arthropods ]

Answer 2

[Larry_Parnell]

BLAST is a start. I'd use BLAST to find ESTs representing the same gene, preferably one that is part of a one-gene family in your two EST collections and also in insect species such as mosquito and honeybee and others. Do this for several genes and trim those genes so that their alignments contain no overhanging, unaligned ends (such would occur if one tick had more 5' seq data than the other). Then, build phylogenetic trees with these sequences and use the relationships you see (branching patterns) with the various Drosophila and Anopheles species as a guide to determine speciation. In my mind, those branching patterns need to be consistent across several gene families.

Answer 3

[John St. John]

Are there any papers describing a similar distinction from sequence data alone in arthropods? Seems like a very subjective question since some species could have quite a bit of population variation, and others could have recently gone through some kind of event to reduce sequence variability. I don't work with organisms where the lack of viable offspring isn't enough of a determinate to draw a species line. You could always pull out 16s sequences and look at divergence times, comparing those to similar closely related arthropod species. That doesn't solve the problem of having a population with an exceptionally high level of variability. It seems like you will have to come up with some kind of definition of a species for your organisms (or better find someone else's) and then go on from there.

Answer 4

[Malachi Griffith]

If your two ticks are indeed different species and EST or genome sequencing of these species has been completed in the past, you may be able to identify the species by BLAST alignment of your ESTs to existing databases of sequences. This assumes that someone else has already correctly distinguished the two species, produced sequences from them, and deposited them in GenBank with the correct species designations...

There will not be a simple rule for determining "what constitutes enough of a difference in the DNA to say they are different species." You could start by enumerating what the single nucleotide differences are by aligning the ESTs to each other and identifying discrepancies. However, unless the sequencing is flawless you will not know for sure which differences are true and which are sequencing errors. Also if the two ticks are the same species they may still have polymorphisms. In other words, you may need some reference points. Perhaps you could treat this as a classic (phylogenetic) or computational phylogenetic problem. If you go to the NCBI taxonomy browser and search for the order 'Ixodida' (ticks). You will get a summary of all the ESTs, protein sequences, etc. currently deposited in NCBI. Using this info you could identify a set of sequences for previously sequenced ticks and use these to build a phylogenetic tree. This should give you an idea how closely related your two species are relative to other species from the same order. If your two species have been sequenced before you may even be able to infer what they actually are, or at least what they are most closely related to of all previously sequenced species...

"molecular phylogenetics uses nucleotide sequences encoding genes or amino acid sequences encoding proteins as the basis for classification. Many forms of molecular phylogenetics are closely related to and make extensive use of sequence alignment in constructing and refining phylogenetic trees, which are used to classify the evolutionary relationships between homologous genes represented in the genomes of divergent species." [wikipedia page on computational phylogenetics]