I was hoping to get suggestions from the readers of AA about methods of tissue collection for genetic work other than tail tips. I’ve been working with the agamid lizard Sitana ponticeriana, and my work is now taking decidedly genetic directions. It remains unclear whether or not these lizards regenerate their lost tails–while they seem to lose tails easily, I didn’t see any lizards with noticeably regenerated tails in the field. Given this, I am a little uncomfortable with the idea of taking tail tips as tissue for genetic work. Are there other common and easy options for sampling tissue from lizards? Many thanks in advance for your responses!
(Feel free also to weigh in with whether or not you think it acceptable to collect tail tips in a species that certainly autotomizes its tail but does not grow it back–it seems like a grey area to me).
A male Sitana ponticeriana near Pune, India.
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Tony Gamble
There are several options for sampling DNA in lizards without using tail clips. Buccal swabs are widely used to collect DNA from amphibians and reptiles (Miller, 2006; Pidancier et al., 2003; Poschadel and Möller, 2004; Beebee, 2008). You can also extract blood from the caudal vein in medium to large sized lizards (Gorzula et al., 1976). If you collect blood make sure you use citrate or EDTA as an anticoagulant since heparin is co-extracted with DNA and will inhibit subsequent PCR.
References:
Beebee, T. J. C. 2008. Buccal swabbing as a source of DNA from squamate reptiles. Conservation Genetics 9:1087–1088.
Gorzula, S. J., C. L. Arocha-Pinango, and C. Salazar. 1976. A method of obtaining blood by venipuncture from large reptiles. Copeia 1976:838–839.
Miller, H. C. 2006. Cloacal and buccal swabs are a reliable source of DNA for microsatellite genotyping of reptiles. Conservation Genetics 7:1001–1003.
Pidancier, N., C. Miquel, and C. Miaud. 2003. Buccal swabs as a non-destructive tissue sampling method for DNA analysis in amphibians. Herpetological Journal 13:175–178.
Poschadel, J. R., and D. Möller. 2004. A versatile field method for tissue sampling on small reptiles and amphibians, applied to pond turtles, newts, frogs and toads. Conservation Genetics 5:865–867.
Ambika Kamath
Many thanks for these sources! I’m excited to try out buccal swabs, but I think these lizards (SVL: 40-50mm) might be too small for caudal vein sampling).
Martha Munoz
Although I agree that removing tail tips in lizards that don’t regenerate is definitely a gray area, it’s important to note that you do not need a lot of tissue to get a good DNA sample. A few centimeters is really all you need, enough to fill a 2mL eppendorf tube, actually. If they lose their tails in the wild, it shouldn’t be too stressful to lose a small piece of the tip. Of course, if the lizard has already lost a ton of tail, then maybe not.
Yoel Stuart
You could probably get away with <1cm if you want to affect the lizard minimally and anticipate having to extract DNA only once or twice. Of course, the very distal tip of the tail is going to have less tissue than if you were to take from the base of the tail so you may have to adjust your take based on how much tail the lizard has.
As for the grey area, if lots of lizards have lost their tails and they don't regenerate them, then perhaps it's not that damning to have lost a tail. Too adaptationist?
Ambika Kamath
I was thinking along similarly adaptationist lines, especially given how easily these lizards lose their tails. It would be interesting to x-ray some tails to see if they perhaps can regenerate them, just with their new tails very closely matched to the originals.
Martin Whiting
I don’t know of any lizards that autotomize their tails but are unable to regenerate them. While a regenerated tail is obvious in many species, I have seen examples of regenerated tails that require very close scrutiny to detect. I agree with Yoel, if you lose your tail voluntarily, and don’t regenerate a new one, then it would suggest its not that costly. In any case, I would be interested to hear of any examples of lizards that actually autotomize their tails without regeneration (as opposed to a physical break that they can’t control).
Tony Gamble
The New Caledonian gecko (Correlophus ciliatus – formerly Rhacodactylus ciliatus) has a very specialized, semi-prehensile tail which will readily autotomize. The tail is always lost at the base and once lost, never grows back (except for a very small nub that covers the break site, see photo).
Bill Bateman
Agamas don’t seem to be too great at regenerating tails, at least not that look like the original; removing a tail tip won’t influence behaviour in the same way as removing the whole tail so is probably ok.
Martin Whiting
Thanks for that example and the photo, Tony. Someone should take a look at the costs of tail loss on their next trip to New Caledonia!
Lori
How about droppings or molts? Guess it would depend on how much DNA you would need.
Ambika Kamath
Do you know of anyone who has tried those sources for DNA for relatively small lizards, and/or how much DNA they might yield?
Martha Munoz
Yes – Shane Campbell-Staton has extracted DNA from Pogona shed skin. This is a much larger lizard and even still he had many difficulties. He has published a modified protocol of how to do this, but my first gander is that you might have trouble getting enough from such a small lizard.
cybokat
There is the possibility of getting dirty bands (contaminated sample), or sequencing another organism instead of the lizard altogether, depending on the specificity of your primers. Like lizard parasites, etc.
cybokat
We did buccal swabs of chameleons once – worked like a charm, however, if the first DNA extraction fails, you won’t have tissue to go back to.
Tail tips are the easiest, however, I am accustomed to taking a complete femoral muscle of the specimens that are sacrificed.
Ambika Kamath
Kat, any thoughts on how much DNA you can get out of the buccal swabs, if they work well?
cybokat
I don’t have an exact concentration but if you are thinking anything complicated or microsats, it’s probably not a good idea. We used them only for barcoding.
Ambika Kamath
Good to know. Thanks!