Anole Annals

Steven De Decker and Tess Driessens’ winning photo from the 2012 contest.

Thank you to everyone who has sent in photos for our 2016 calendar contest, we’ve received some great submissions! For those who haven’t yet gotten around to it, you’ve still got one week – the deadline is Saturday, November 21. If you have any great anole photos, we would love to have them in the contest! And just to remind you, the first and second place winners will receive a free Anole Annals 2016 calendar, woo! So send in your pics, let’s make the 2016 calendar great!

To remind you, here are the rules: submit your photos (as many as you’d like) as email attachments to anoleannals@gmail.com. To make sure that your submissions arrive, please send an accompanying email without any attachments to confirm that we’ve received them. Photos must be at least 150 dpi and print to a size of 11 x 17 inches. If you are unsure how to resize your images, the simplest thing to do is to submit the raw image files produced by your digital camera (or if you must, a high quality scan of a printed image).  If you elect to alter your own images, don’t forget that it’s always better to resize than to resample. Images with watermarks or other digital alterations that extend beyond color correction, sharpening and other basic editing will not be accepted. We are not going to deal with formal copyright law and ask only your permission to use your image for the calendar and related content on Anole Annals (more specifically, by submitting your photos, you are agreeing to allow us to use them in the calendar). We, in turn, agree that your images will never be used without attribution and that we will not profit financially from their use (nobody is going to make any money from the sale of these calendars because they’ll be available directly from the vendor).

Please provide a short description of the photo that includes: (1) the species name, (2) the location where the photo was taken, and (3) any other relevant information. Twelve winning photos will be selected by readers of Anole Annals from a set of 28 finalists chosen by the editors of Anole Annals.  The grand prize winning and runner-up photos will be chosen by a panel of anole photography experts. Deadline for submission is November 21, 2015.

Good luck, and we look forward to seeing your submissions!

Sarah Hykin and Jim McGuire

In our recently published paper in PLoS One, we provided ‘proof-of-concept’ that it is possible to obtain genome-scale data from formalin-fixed specimens. This study was proposed by one of us (SMH) about four years ago while contemplating how she might be able to undertake a phylogeographic study of a rare lizard species for which there was little hope of resampling the entire range.

Of course, people have been contemplating the challenge of obtaining DNA sequence data from museum specimens for decades, often with limited success. The typical approach involved targeting mitochondrial genes, developing sets of nested primers amplifying short fragments (often only 50-100 bp in length), and then a brute-force amplification and sequencing in an effort to score a few hundred usable base pairs.

However, our discussion was informed by the recent development of short-read Next-Generation Sequencing on the Illumina platform, which produces genomic-scale data 50-100 bp at a time. Surely, we thought, if any method could efficiently pull DNA sequence data from formalin- damaged DNA, this was it. Our timing was impeccable because our campus had just obtained a major foundation grant to support, among other things, the development of risky technology that could enhance the utility of historical museum specimens. We obtained a small subaward and Sarah went to work studying the literature on historical DNA sequencing, and figuring out how to perform NGS. Illumina sequencing and bioinformatic processing have become pretty routine now, but this was a very challenging undertaking at the time, and Sarah had to pursue this while focusing her energies on her unrelated dissertation research.

The first decision that we had to make was which species and specimens to select for sequencing. This turned out to be a ‘no-brainer’ because the only squamate genome available was Anolis carolinensis and we needed a genome to which we could map reads. Being a conservative museum curator, Jim suggested using no-data or limited-data specimens so that when the project inevitably failed we would not have cut up particularly important specimens. In retrospect, this was a mistake. For example, we used a limited-data specimen of Anolis carolinensis that was in the database as having been obtained in 1985. Further investigation of this specimen would have shown that this date was dubious, and it now appears the specimen was actually obtained and prepared between late 1986 and 1988 (and accessioned in 1990). We also have reason to believe the specimen was fixed in buffered formalin for one-week prior to rinsing and immersion in ethanol, though this is not known for sure since the specimen does not have associated field notes. If we had this to do over again, we would make sure that we knew as much as possible about the source specimens rather than taking a ‘let’s limit the damage if this experiment fails’ approach to specimen selection.

It works (sometimes)!

As indicated in our paper, it is indeed possible to obtain genome-scale data from formalin-fixed specimens housed for decades in a museum collection. However, our method is far from fool-proof and we strongly suspect that idiosyncratic features of individual specimens will determine success or failure in many instances. The age of the specimen is likely to be one of the most important variables as suggested by our published study – sequencing of our 100-year old sample failed, whereas sequencing for our ~25 year old sample was successful. However, other parameters are likely to prove important. For example, others have shown via direct experiments that DNA is better protected by buffered (versus unbuffered) formalin. We also suspect that other features of the specimen’s preparation such as the time spent soaking in formalin prior to immersion in ethanol, the concentration of the formalin used, the quantity of formalin injected into the specimen, and the time that passed between the death of the specimen and its preparation could all make a difference. These conditions are rarely recorded at the time of preparation, which means that for the vast majority of specimens that might be targeted for NGS, the researcher cannot know ahead of time whether the specimen is likely to be a good versus poor candidate for sequencing.

Our paper received some attention on social media with some calling our study a “game changer” and others arguing that such a statement is overblown. From our perspective, this is semantics. Have we completely solved the issue of obtaining genomic sequence data from formalin-fixed samples? Certainly not. Have we identified in a controlled way, the precise conditions underpinning success or failure of NGS from formalin-fixed samples? Again, not by a long-shot.

However, we have shown some things that are likely to be important in moving this technology forward (which could be interpreted as changing the game). Most importantly, we have shown definitively that it is possible to obtain genomic data from old museum specimens. This had not been shown previously and we believe that this will encourage many more people to give it a try than would be the case if our paper had appeared in the Journal of Negative Results. Further, we were able to shed some light on methodological issues that are actually quite important. First, we obtained a sufficient quantity and quality of DNA for sequencing from liver tissue and not from either leg muscle or, most importantly, bone. Many of you will be aware of a terrific study published by Maureen Kearney and Bryan Stuart in 2004, in which they provided a phylogeny for amphisbaenians that was based in large part on sequences obtained from old museum specimens. In their groundbreaking study, they obtained mitochondrial and nuclear sequence data using laborious traditional Sanger sequencing of short DNA fragments, along with non-traditional extraction of DNA from bone tissues using methods developed for human forensic DNA analysis. Their successful extractions required sampling bone from pickled specimens, which can be quite destructive. In contrast, pulling liver from a museum specimen is minimally invasive – especially when you consider that most newly collected specimens will have had their livers removed prior to preparation. Thus, our finding that liver is an optimal DNA source for NGS is important.

Further, second, we found that a modified phenol-chloroform extraction protocol outperformed Qiagen extraction for NGS purposes. Indeed, we now suspect that our failed attempt to perform NGS on our older sample could very well be the result of our effort to systematically compare extraction protocols. For both of our Anolis specimens, we subsampled the entire liver, which was divided into a small piece and a much larger piece. For the younger sample, the larger piece was extracted using phenol-chloroform, whereas for the older sample, the larger chunk was extracted using Qiagen. Importantly, from that older sample,we obtained more DNA from a sample ~20 times smaller in mass using phenol-chloroform versus Qiagen extraction. If we had dedicated the larger subsample from the 100-year old specimen to phenol-chloroform, we believe this might have resulted in successful NGS.

Figure: Sample placement in the phylogeny. As expected, our sequencing effort generated low-coverage (~0.5X) of the Anolis carolinensis genome. However, we did obtain ~60X coverage of the mitochondrial genome, providing a means of evaluating the quality of our sequence data after processing. We aligned the ND2 sequence from our sample with GenBank sequences representing A. carolinensis from Louisiana, the Anolis carolinensis complete genome, a variety of additional Anolis species, and a more distant outgroup. The phylogram shown here suggests that our mitochondrial sequence data obtained from a formalin-fixed specimen are reliable. If sequencing errors were evident, we would minimally expect the branch representing our formalin-fixed specimen to be relatively long compared with other Louisiana A. carolinensis, or perhaps even misplaced on the tree.

Where to from here?

One critique that we received in review was that we had failed to perform rigorous controlled experiments testing the various conditions that could impact success with NGS from formalin-fixed samples. Though we would have loved to perform such experiments ourselves, even limited NGS sequencing is still sufficiently expensive (thousands of dollars, rather than hundreds) that we were not in a position to pursue this. However, we would love to see someone – perhaps reviewer number 2 – grab the bull by the horns and perform this experiment! Such a follow-up study, should it identify via controlled experimental procedures the key parameters for successful NGS of formalin-fixed samples, would come closer to meeting the criteria of being a ‘game-changer.’

The last week has seen a spirited discussion of the pros and cons of splitting recognized genera into multiple, smaller genera. We’ve had 34 comments already. Check it out! And if you’re an advocate of splitting genera, that viewpoint has been getting the short end of the stick and could use more support.

As a tangent, the topic of subspecies has come up, and David Hillis has strongly argued for reviving its use. Here’s what he has to say:

First, I don’t think either species or subspecies are “clades.” Species are lineages (the branches on the tree of life). Sexual recombination among individuals results in tokogenetic relationships within species. Clades, on the other hand, are monophyletic groups of lineages on the tree of life. Rather than being defined by tokogenetic relationships, they are defined by phylogenetic relationships.

Traditionally, subspecies are geographical races of species. In other words, they are geographically distinct populations that nonetheless meet and interbreed at contact zones. Sometimes, these contact zones are very broad, as with broad-banded versus southern copperheads. If the contact zones are very narrow, and there is strong evidence that the contact zone is a genetic sink (there is no gene flow across the zone, because of strong selection against hybrids), then I agree that the two entities can be considered separate lineages, and hence species. But in many recent cases, as with the copperhead example, there is abundant evidence that the contact zone is NOT a sink, and that there is NO selection against hybrids. In this case, I disagree strongly with the authors who proposed to split these subspecies into distinct species. That is inconsistent with any lineage species concept…there is a huge area where these two forms intergrade, with no evidence of any loss of fitness. Thus, the two forms are geographical, intergrading races, or subspecies.

I think we will soon see a backlash against the splitting off of geographic races as species as well. Frank Burbrink (who was an author on the copperhead example I mentioned above) and I plan to write a pro/con article about this together, each arguing our respective points of view. Hopefully, this will re-kindle the conversation about subspecies.

Subspecies are unpopular right now because they were long abused in several ways. Inappropriate uses include (1) to describe non-geographic “varieties”; (2) to arbitrarily break up clines; and (3) to describe distinct, isolated lineages that clearly are species. But used in proper context to designate a geographically distinct race, they are certainly reasonable and often useful. They are rarely used in some groups, for several reasons: Groups like freshwater fishes have discrete ranges, so taxa don’t interbreed over broad areas. And many groups are too poorly studied to understand geographic variation. But in well-studied terrestrial groups (like herps), subspecies are perfectly reasonable and useful taxa to designate intergrading geographic races.

Over the last several years, ever since Nicholson et al. proposed dividing Anolis into eight genera, the topic of taxonomic splitting has periodically been discussed in these pages (for example, this post, its comments, and links to other posts).

The general question of when to split taxa recently has been revisited in several comments in AA. A week ago, David Hillis wrote:

“Anolis is a valid name for a monophyletic group on the Tree of Life. It is “special” as a genus only in that the genus name is used as part of a binomial for particular species. It doesn’t make sense to change the scope and application of generic names unless the names are actually misleading about phylogeny (e.g., if Anolis were polyphyletic, then that problem should be fixed). But splitting a valid, monophyletic genus into a bunch of smaller genera, and thereby needlessly changing the names of many species, without fixing any phylogenetic problems with the existing taxon names, is not science. It is just playing around with names. If someone wants to name the groups within genera, then do so…but there is no reason to change the meaning of a existing name (or the names of the all the affected species) in doing so. That is the kind of silliness that gives taxonomists such a deservedly bad reputation among biologists.”

Elswhere, David posted a flowchart on his recommended decision-making process about whether and how to divide recognized genera:

Ivan Prates, in line with comments he made in a recent paper on A. punctatus, then remarked:

“This seems more like a sociological matter.

During the ‘taxonomic revolution’ of the amphibians, about 10 years ago, the (perhaps?) most influential (or faster?) group was the splitter one, and their taxonomic scheme prevailed. Currently, nobody is upset about which species were once named as Bufo, Hyla or Rana. A few do care about Dendrobates – like Anolis, a sexy group with a body of dedicated investigators.

It seems that a single genus makes sense for the community that investigates dactyloid lizards more closely. On the other hand, those who deal with overwhelming levels of herpetological diversity in the tropics (waaaay beyond lizards) see benefit in more partitioned schemes, which correlate more closely to morphology and geography.

So, when we discuss names, it may be healthy not to forget about our diversity as investigators as well. About science, splitting Anolis is not science, but well, not splitting Anolis isn’t science either.”

Over the last decade the term “model species” has taken on new meaning. Species that were once the building blocks for distinct disciplines have taken on new importance in comparative evolutionary studies that integrate perspectives across biological disciplines. Nowhere is this better illustrated than with Anolis lizards. For decades anoles were a workhorse of ecologists and evolutionary biologists, but have, more recently, been embraced by developmental biologists, genomicists, physiologists, and neurobiologists among others. This disciplinary expansion is perhaps most evident with the rapid increase of penis/hemipenis research that has been published using anoles within the year.

For many herpetologists, including those focused on anoles, the hemipenis is ripe with taxonomic characters, easily allowing for the identification of new species. Julia Klaczko and colleagues recently demonstrated that features of the hemipenis are some of the most rapidly evolving characters among anoles, a group already well known for its rapid anatomical evolution. Independent from these taxon-specific interests, developmental biologists became interested in the anole hemipenis because of its unique anatomy compared to other amniotes. Marissa Gredler and members of the Cohn Lab used anoles as one of their reptilian models of external genital development in what is arguably the broadest embryological survey of reptilian phallus development to date. In parallel, Patrick Tschopp and colleagues probed the cellular and molecular regulation of early phallus development among anoles, snakes, chickens and mice, demonstrating that the hemiphalluses (hemipenes and hemiclitores) and hindlimbs of squamates utilize similar molecular networks at the earliest embryonic stages of morphogenesis. Now, just within the last month, two more papers have used anoles in studies of phallus evolution and development, one using cutting-edge molecular techniques to better understand the relationship between limbs and external genitalia and the other addressing the fundamental question of external genital homology using museum specimens that are more than 100 years old.

Before getting into the findings of this new research, lets lay out some of the dirty details of penis evolution. First and foremost, the penises of amniotes are extremely diverse. Squamates have paired lateral phalluses while other clades have a single midline phallus. Each of the amniote lineages uses hydrostatic pressure to achieve an erection, yet accomplish this using different bodily fluids (lymph or blood). In mammals sperm is transferred to the female through a closed urethral tube, but other groups utilize an open channel. Most birds (97%) and the tuatara, have absent or highly reduced phalluses and reproduce with the famed “cloacal kiss.” These large differences in anatomy should not overshadow the spines, bulges, corkscrews, and dramatic differences in size that give species their distinctive features. But with such striking variation, we are forced to wonder how many times the penis evolved. Perhaps the amniote ancestor possessed an intromittent phallus capable to transferring sperm to the female that later diversified in each lineage independently. Or, perhaps the last common amniote ancestor used cloacal apposition to foster internal fertilization and unique phallus morphologies evolved independently at the origin of each lineage. Because adult anatomy provides few clues to phallus homology, Thom Sanger (me), Marissa Gredler, and Marty Cohn looked towards the embryo for help.

Table 1 from Sanger et al. 2015 summarizing phallus variation in amniotes

The tuatara, a species lacking an adult phallus, has presented a problem in attempts to reconstruct the last common ancestor of amniotes because it raises the distinct possibility that reproduction through cloacal apposition was the ancestral condition.

Read More