Marking Techniques for Population Studies

A grass bush anole, Anolis olssoni, from the Dominican Republic. Note the three colored beads sewed into the tail musculature for easy identification in the field. Photo by Michele Johnson.

Many studies of natural selection, behavioral ecology, and population biology in anoles focus on one to several populations over the course of days, weeks, or months. These studies require reliable identification of individual lizards over time. I describe several ID’ing methods in this post. Read on!

Anolis carolinensis shortly after release, marked S93 using a Sharpie. Photo by Yoel Stuart.

Felt-tip Permanent Markers (or paint pens) are great for marking lizards for fast identification in the field. A number or blotch on either side is quickly applied and easily visible from afar, especially with binoculars, so this method is great for behavioral observations. The major drawback of the permanent marker, however, is its impermanence (ironic, I know). A mark lasts only until the lizard sheds its skin. Thus, the ID could last a month, or it could be gone tomorrow.

Colored Beads, sewn into the tail musculature  in different combinations using a thin wire, also allow for long range identification and behavioral study (see photo above). Akin to bird banding, beading is more permanent than marker. This method is more stressful to the animal, and quick and effective application takes some practice. It has been successful in lizards down to about 40 mm snout-vent length (Michele Johnson, personal communication).

Visible Implant Elastomers, made by Northwest Marine Technologies, are biologically inert solids that can be injected just beneath the surface of translucent tissues where they remain externally visible. By injecting any of the 10 available colors (6 colors will fluoresce under UV light) into different positions on the body, researchers have an inexhaustible number of unique combinations available for marking animals. Properly injected, these tags are permanent as the skin heals over the entry site. Because the tags aren’t visible from afar, the animal has to be in hand to determine its identity. Thus, for behavioral studies, combining permanent marker or colored beads with the VI elastomer allows for long range identification with the assurance that the animal is also permanently marked.

Anolis sagrei female from the Bahamas marked with a yellow visible implant elastomer tag in the right forelimb and an orange elastomer tag in the left hindlimb. Photo by Melissa Losos.

Visible Implant Alpha Tags, also made by Northwest Marine Technologies, are perfect for those of us who struggle with combinatorics or just don’t want to deal with multiple marks and locations per animal. Like the elastomers, these are biologically inert, implantable tags with the difference that they have a black letter-number combo (i.e. A00, A01, … Z98, Z99) printed on one of four fluorescent background colors. These tags carry the same advantages and drawbacks of the elastomer tags. A unique drawback to this method is that a poorly implanted tag can be difficult to read, even under UV fluorescence.

Toe clipping is a classic method for marking small vertebrates. By clipping toes in different combinations on different limbs, a large number of lizards can be marked uniquely. Toe clipping, like the elastomer and alpha tags described above, is permanent, but the lizard has to be in hand for identification. For studies in the wild, clipping toes may negatively affect performance and survival. The evidence for this negative impact is mixed, however, and many wild lizards lose toes naturally so perhaps this isn’t a big issue. That said, the high prevalence of lost toes in the wild can introduce error to the marking system and lead to misidentification.

Anolis sagrei from the Bahamas, painted with three different colors (blue, yellow, and a dash of red) on three different days. Photo by Jonathan Losos.

Spray paint is a great tool for mark-recapture studies estimating population size. A researcher visits the population on three different days, each day painting the lizard with a different color using a pump sprayer. The non-toxic, water-based paint is applied as minimally as possible while still allowing for color detection from all angles. The researcher uses a statistical model that uses the number of lizards painted with one, two, or three colors at the end of the survey to estimate population size.

About Yoel Stuart

Yoel studies evolutionary ecology of Anolis lizards. He is a graduate student at Harvard University, in the Department of Organismic and Evolutionary Biology.
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18 Responses to Marking Techniques for Population Studies

  1. julienneng says:

    Interesting – I had not heard of the bead technique before. I’ve used visible implant elastomer tags before and we currently use a toe-clipping technique on lab-born hatchlings. Do you know whether studies have been done looking at whether there is an increase in predation rates with the beads or colored markings (paint pens or spray paint)? I’d also be curious to know how much the beads affect behavior as well…

  2. Rich Glor says:

    What’s been your failure rate with the alpha tags? If fairly reliable, it sounds like a nice approach for permanent identification of large numbers of animals without toe-clipping. What’s the smallest size animal that could be safely tagged in this manner? I looked at the newest PIT tags this summer and they’re definitely getting small enough that they could be inserted in adults of the smaller anole species. They’re also relatively expensive.

    One quick remark on the spray painting technique, which I used a bunch a number of years ago. You really can’t use this approach anyplace that you don’t want to leave looking like a paintball arena. If anoles are common you’re going to end up coating trees and leaves as well as lizards. I’m sure the paint fades over time, but the habitat might look like a mess for a bit.

  3. Yoel Stuart says:

    I don’t know of any studies regarding an increase in predation rates associated with marker or bead use. Sounds like a good opportunity for another clay model experiment!

    Michele Johnson has studied anole behavior pretty intensively and has used beads to mark her lizards. She’d have a better intuition than me with respect to changing behavior. But you’re right, that is a potential drawback to using beads.

  4. Yoel Stuart says:

    It’s hard to estimate failure rate in the wild because the only way to know for sure if an animal was marked is to find the tag in the animal. An unmarked animal could result from tag failure or the fact that you never caught it before. We marked hundreds of animals in my populations with alpha tags, but because the population sizes were large, we continued to catch unmarked animals throughout the study. That said, only one of those unmarked animals had an insertion scar where it would have lost its tag and the telltale, newly-broken tail, the tip of which we took for genetic analysis. Also, you have a lot of control when inserting the tag so you can place it a tag-length or more away from the incision; I don’t think the tag would be able to work its way back out. Thus, I think the failure rate is pretty low, although a lab experiment to be certain would be worthwhile.

    As far as smallest markable lizard goes, we were marking carolinensis down to about 37mm SVL. We inserted the tags into the ventral side of the hindlimb, between the knee and the hip. Each tag is 1.2 mm wide. You probably wouldn’t want to mark a lizard whose leg width was smaller than 2mm as you’d be making a relatively large incision and the tag might interfere with the operation of the limb. We didn’t experiment with tagging other body regions however, which might allow you to go smaller.

    Alpha tags are $0.75 a piece. What do the PIT tags cost?

  5. geneva says:

    Maybe someone out there can help me with this… I’ve always been confused by one component of the spray paint version of estimating census population size. Wouldn’t you want the animals found each subsequent day to have the same criteria for counting? In other words, on day one you go out and spray the animals you can catch, and of course you don’t spray the animals that you see but can’t catch. Shouldn’t day two counts also only be those that you can catch, rather than only those you can see? I’m probably missing something but it seems like using different criteria on each census day will bias a population estimate.

  6. Rich Glor says:

    When you use the paint spraying approach you generally don’t catch any of the animals, you just spray them from a distance.

  7. geneva says:

    Gotcha, but I’m left with the same question. There are bound to be animals you can see but can’t hit with paint because they are too high, too skittish, etc. The goal is to estimate the number of animals at a site by incorporating your count based on spraying plus an estimate of the number of animals that were at the site but not sprayed. That number of not sprayed is estimated based on the ratio of sprayed versus non-sprayed animals you see on a second collection day. My question is: on the second day, do you count all of the animals you see or just the animals within paint spraying distance?

  8. Ambika Kamath says:

    Michele Johnson also described a method using little cardboard bee tags for temporary marking, which likely suffers from the same problems as sharpie marking in being temporary, but the tags might be easier to spot from far away for behavioural work. Also, it’s a matter of debate whether it’s worse for a lizard to eat sharpie-covered skin or a piece of cardboard when they shed and eat their skin.

  9. Rich Glor says:

    My lab should be posting something soon our efforts to permanently mark hatchlings in our colony. Toe-clipping is the only approach that we’ve found feasible.

  10. Yoel Stuart says:

    As Ambika noted above, people have used bee tags. Michele’s paper on the method is linked here: http://www.trinity.edu/mjohnso9/files/JohnsonHR05.pdf

    Have you guys thought about bee tags for the hatchlings? They’re probably small and light enough but Ambika’s point on a lizard accidentally eating the tag may be especially important here.

  11. Michele Johnson says:

    Julienne, I’ve compiled something like 1500 hours of observation on anoles, mostly with bead tags, and I’ve not seen any obvious effects of the tags on their behavior. I’ve not done the experiment to compare the behavior of marked vs. unmarked individuals in the same population, but I’ve seen high rates of copulation, eating, running around chasing other lizards, etc. from bead-tagged lizards. The lizards almost never lose their tags, and in well-tagged lizards, they don’t bite at the tags either (but if the tag is loose, I’ve seen them bite at it)

    In terms of predation, I’ve bead-tagged a population of lizards in Puerto Rico and come back a year later, and seen ~75% of the tagged animals still there. I’ve observed predation of a marked animal exactly three times – one at night by a snake (Jamaica), one by a fish when a clumsy anole fell in the water (Louisiana), and once by a bird (Bimini). So, from my anecdotal, but extensive, experience, the effect of the beads on predation seems minimal.

  12. Michele Johnson says:

    Thanks Ambika and Yoel for the reference to the bee tags. They are definitely very easy to see in the field, as they’re neon colored, and I’ve used combinations of colors and locations on the lizard to mark individuals. They seem to work well in some places, but not others – they were fantastic on Puerto Rican and Dominican Republic anoles, and on Sceloporus in MIssouri, but they came off too frequently in Jamaica and Texas to be of use. On rare occasion, a lizard would shed its skin immediately after bee tag application and eat the skin and tags, and a couple of times I’ve found bee tags in lizard feces (whole; not digested at all).

    I think bee tags are a great method if you want to mark lizards to know you’ve already captured them (for example, during intensive tissue sampling in a small area, over a few days), or if you’re working with very cryptic anoles that are hard to see – the bee tags can definitely be seen! Again, this raises the issue of predation, but I haven’t noticed it to be a factor in my ~3 week studies. At that point, the lizard will have shed anyway, so it’s not a long-term problem. Whenever possible, I prefer the bead tag method for behavioral studies, though – maybe surprisingly, I think it has less impact on lizard behavior.

  13. Talia says:

    What about something like bird-banding? Putting a colorful belt, or a series of colorful belts would be less invasive. If you have them properly sized, then they would hopefully not impede locomotion or get stuck on anything.

  14. Ambika Kamath says:

    I was wondering the same thing after coming across a study using coloured aluminium bands in Agamids. A quick search showed that bands have been used in Teiids and Lacertids as well. Are anoles too small? Does it interfere with shedding?

  15. geneva says:

    For our hatchling marking, we need a method that is permanent and can be applied to an animal that weighs much less than a gram. We need to keep track of each hatchling individually for the life of the animal. Our colony has multiple hatchlings per cage, often each with different parents so marking method that comes off with shed risks the loss of lots of data. Really small PIT tags are an attractive idea, but I worry that might increase hatchling mortality. Maybe a mixed approach might be the best course of action.

  16. Rich Glor says:

    Can you provide more information or references for the use of aluminum bands to mark lizards?

  17. geneva says:

    In 2007 SSAR published a second edition of John Ferner’s Review of Marking & Individual Recognition Techniques For Amphibians & Reptiles. Although, we’ve discussed at least one technique absent from that circular (bee tags) there is a wealth of techniques and citations worth looking at. For instance, there are discussions of the ethical considerations of techniques like toe clipping and branding. The circular is also a wonderful collection of citations on Herp marking. For instance, it points to a 1973 paper, where Stamps was able to use body color and tail regeneration patterns to uniquely identify Anolis aeneus individuals.

  18. Jonathan Losos says:

    Yes, and it is important to use the same criteria for which animals to include each day. One complication is that weather is different from one day to the next, so that different animals may be active. Males, for example, tend to be out more than females, and it’s always possible that animals specialize for different climatic conditions–we assume they’re active every day, but at least in some lizard species, that’s probably not true.

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