Sunday, July 26, 2015

A Four-legged fossil snake discovered, and a growing controversy regarding its legal status in Europe

The following has been adapted from a press release and a blog post, with some light editing by me (JCM).
A new fossil, named Tetrapodophis amplectus, claims to be a four-legged, burrowing snake. (A) Counterpart, showing skull and skeleton impression. (B) Main slab, showing skeleton and skull impression. Figure 1 from Martill et al. 2015.  (C) The snake has small ‘hands’ that are approximately 1cm long. Credit: Image courtesy of University of Portsmouth.
The first known fossil of a four-legged snake, and the team -- led by Dr. Dave Martill from the University of Portsmouth -- say that this discovery could help scientists to understand how snakes lost their legs. The research was published in the journal Science.
Dr Martill said: "It is generally accepted that snakes evolved from lizards at some point in the distant past. What scientists don't know yet is when they evolved, why they evolved, and what type of lizard they evolved from. This fossil answers some very important questions, for example it now seems clear to us that snakes evolved from burrowing lizards, not from marine lizards."
The fossil, from Brazil, dates from the Cretaceous period and is 110 million years old, making it the oldest definitive snake. Martill discovered the fossil as part of a routine field trip with students to Museum Solnhofen, Germany, a museum that is well-known for its prestige with regard to fossils.
Dr Martill said: "The fossil was part of a larger exhibition of fossils from the Cretaceous period. It was clear that no-one had appreciated its importance, but when I saw it I knew it was an incredibly significant specimen."
Martill worked with expert German palaeontologist Helmut Tischlinger, who prepared and photographed the specimen, and Nick Longrich from the University of Bath's Milner Centre for Evolution, who studied the evolutionary relationships of the snake.
Dr Longrich, who had previously worked on snake origins, became intrigued when Martill told him the story over a pint at the local pub in Bath.
He said: "A four-legged snake seemed fantastic and as an evolutionary biologist, just too good to be true, it was especially interesting that it was put on display in a museum where anyone could see it."
He said he was initially skeptical, but when Dr Martill showed him Tischlinger's photographs, he knew immediately that it was a fossil snake.
The snake, named Tetrapodophis amplectus by the team, is a juvenile and very small, measuring just 20 cm from head to toe, although it may have grown much larger. The head is the size of an adult fingernail, and the smallest tail bone is only a quarter of a millimeter long. But the most remarkable thing about it is the presence of two sets of legs, or a pair of hands and a pair of feet.
The front legs are very small, about 1cm long, but have little elbows and wrists and hands that are just 5mm in length. The back legs are slightly longer and the feet are larger than the hands and could have been used to grasp its prey.
Dr Longrich said: "It is a perfect little snake, except it has these little arms and legs, and they have these strange long fingers and toes.
"The hands and feet are very specialized for grasping. So when snakes stopped walking and started slithering, the legs didn't just become useless little vestiges -- they started using them for something else. We're not entirely sure what that would be, but they may have been used for grasping prey, or perhaps mates."
Interestingly, the fossilized snake also has the remains of its last meal in its guts, including some fragments of bone. The prey was probably a salamander, showing that snakes were carnivorous much earlier in evolutionary history than previously believed.
Helmut Tischlinger said: "The preservation of the little snake is absolutely exquisite. The skeleton is fully articulated. Details of the bones are clearly visible and impressions of soft tissues such as scales and the trachea are preserved."
Tetraphodophis has been categorized as a snake, rather than a lizard, by the team due to a number of features:
· The skeleton has a lengthened body, not a long tail.
· The tooth implantation, the direction of the teeth, and the pattern of the teeth and the bones of the lower jaw are all snake-like.
· The fossil displays hints of a single row of belly scales, a sure fire way to differentiate a snake from a lizard.
Tetrapodophis would have lived on the bank of a salt lake, in an arid scrub environment, surrounded by succulent plants. It would probably have lived on a diet of small amphibians and lizards, trying to avoid the dinosaurs and pterosaurs that lived there.
At the time, South America was united with Africa as part of a supercontinent known as Gondwana. The presence of the oldest definitive snake fossil in Gondwana suggests that snakes may originally have evolved on the ancient supercontinent, and only became widespread much more recently.
However there is a controversy brewing around the specimen, and it is not the creationist controversy you might expect.
Discover Magazine’ blog has a piece by Christie Wilcox  titled “Four-Legged Snake Shakes Up Squamate Family Tree – Or Does It?” (posted July 24, 2015)
Ancestral state analyses, use math and science to estimate the biological and ecological traits of the most recent common ancestor of a group of species, suggested that early snakes were nocturnal hunters, preying upon the small vertebrates of their era through stealth, not constriction. Their analysis didn’t find that snakes were burrowers, however — there was no strong support of a fossorial lifestyle, just that the snakes lived on land.
According to Hsiang, morphological data “strongly influenced” the snake tree. “Our study helped to demonstrate how important and essential it is to include fossils when we are trying to understand how and when organisms evolved.” In the paper, the author’s note that the inclusion of fossil data resulted in relationships that would be “unexpected” given current snakes, and that the fossils’ influence was sustained “even when such data are vastly outnumbered by genetic sequence data,” thus including the new fossil in a similar analysis might be even more informative.
“Now that Martill et al.’s paper on Tetrapodophis has been published, the obvious next step is to include it in large-scale, comprehensive analytical studies looking at snake evolutionary history and phylogenies.”
Though there was some excitement when Hsiang and her colleagues published their analysis in May, a paper published a little over a month earlier in PLoS ONE slipped by the press unnoticed. The analysis, led by Tod Reeder from San Diego State University, looked beyond snakes to reconstruct the evolutionary relationships within the squamates, the group of reptiles that contains lizards and snakes. Using the largest dataset to date which, like Hsiang, included both genetic and morphological markers, Reeder and his colleagues affirmed one of the crucial pieces of evidence of a marine snake origin: the close relationship between mosasaurs and snakes.
“The most comprehensive analysis of the lizard evolutionary tree now reinstates these aquatic mosasaurs as the nearest relatives to snakes,” explains Michael Lee, associate professor at the University of Adelaide, who was one of the first scientists to suggest that snakes may have started in the water.
The lizard tree, which found the aquatic mosasaurs (Mosasauria) to be sister to modern snakes (Serpentes).
The lizard evolutionary tree, which places the aquatic mosasaurs (Mosasauria) sister to modern snakes (Serpentes) rather than the group used by Hsiang et al. (Anguimorpha).
Because of this, Reeder et al. calls into question the methods used by Hsiang et al., specifically one of the core assumptions in the paper: the closest relatives of snakes. When constructing evolutionary trees, assumptions have to be made to “root” the tree, or put the relationships into the context with regards to time. Scientists must compare their data to what is called an “outgroup”, which is ideally the closest relative or relatives to the group of interest. Hsiang and her colleagues used a subset of a group of lizards called anguimorphs, which includes land dwelling lizards like varanids that includes the Komodo dragon.
“The Hsiang paper was a terrific analysis of the evolution within snakes, but the fundamental core assumption they made in the paper was that terrestrial lizards were ancestral to snakes,” said Lee. “The direction of evolution was determined by that assumption. But if you assume, as the Reeder paper suggests, that mosasaurs are ancestral to snakes, then some of the inferences by Hsiang might not hold.”
Hsiang admits that there are differences between the phylogenies in her paper and Reeder’s, and that the choice of outgroup may have skewed their results. “There are differences between the Reeder et al. phylogeny and our phylogeny — it would be interesting to conduct an in-depth analysis to try and determine why the differences in phylogeny exist,” she said. While her team’s tree was strongly influenced by morphology, Reeder’s team found that genetics most strongly predicted the results. “In fact, the morphological data are really ambiguous,” co-author John Wiens said in a press release. “Or in some cases, even worse than ambiguous.”
“There’s certainly a possibility that our results would have been different if we had used different outgroups, as phylogenetic and ancestral state reconstruction analyses use the outgroup to determine the direction and polarity of character state evolution,” said Hsiang. However, she doubts the impact would have been large, as other close relatives of mosasaurs are land-lubbers. “Though the inclusion of mosasaurs would likely have increased the probability of an aquatic lifestyle for early snakes somewhat, this would probably have been “balanced out” by the many anguimorph lizards that are not aquatic.”
“Of course, we’d have to actually run the analysis to know for sure.”
Meanwhile, Bruno Simões from the Natural History Museum, London, UK and his colleagues were taking a very different approach to understanding snake evolution. Instead of looking at bones and unrelated genes, they very specifically examined the genes encoding for visual pigments in lizards and snakes. These genes are well-studied, and in other groups like mammals, are correlated with behaviors like burrowing and nocturnal activity.
“Visual pigments, like opsin and rhodopsin, are basically the business front-ends of the visual pathway,” says Simões. “So basically if anything is happening in the visual system, the visual pigments will be the first to be impacted.” Burrowing mammals, for example, have lost some visual pigment genes, as they no longer need them underground. But even more impressively, scientists can connect genetic changes in these pigment genes to ecology and function. “By checking their amino acid composition, you can estimate what kind of wavelengths the animal can see,” says Simões.
When Simões et al. compared the visual pigment genes in snakes to other lizards, they found something exciting: snakes have lost two of the five pigments found in the rest of the squamates. They retain the same three that we have. Simões explained that this means snakes likely went through an “ancestral nocturnal bottleneck,” just like mammals did. “Snakes have this contrasting pattern from lizards that converges with mammals.”
Evolutionary tree for the Rhodopsin 1 gene, the only one left in the most underground snakes.
Interestingly, in fossorial lizards, all five pigments were still around, but in fossorial snakes like the termite-decapitating blindsnakes, only one pigment remained. “The fact that the visual system was not so reduced suggests that the ancestor for all snakes was nocturnal, not fossorial” — a finding which coincides with the ancestral state reconstructions found by Hsiang et al.
As for the question of marine origins, Simões says that he “didn’t find evidence that it was a marine animal.” Marine environments have very different light conditions than terrestrial ones, with a quick loss of red wavelengths with depth, followed by an eventual loss of all light in the deep sea. Marine animals eyes often show a “shift in spectral tuning to a marine environment,” says Simões, which includes a higher sensitivity for blue wavelengths. In sea snakes, for example, the shorter-length opsin 1 becomes blue sensitive instead of UV sensitive. But Simões found no such shift in all snakes.
“I think that it’s a really interesting paper, in that they’ve discovered that snakes have lost a whole bunch of visual genes that are found in other lizards, which does suggest they went through some kind of semi-blind phase in their evolution,” says Lee. But he still would like to see more research before discounting the aquatic hypothesis. “One thing I’d like to see done is what genes are lost in living marine reptiles like sea turtles,” said Lee, to see if there are any opsin genes lost in other marine reptiles like the ones lost in snakes.

A Four-Legged Snake?
Which brings us back to the most recent finding, what Martill and his colleagues claim is a four-legged snake ancestor from Brazil. Though there’s no concrete information about where this fossil originated, the color and texture of the limestone it is encased in suggests it’s from the Crato Formation, a fossil deposit which was laid down some 100 million years ago when the area was a shallow sea.
“The Crato formation is about 20 million years older than the oldest fossil snake,” Martill explained. Thus this ten centimeter-long fossil, which Martill and his colleagues named Tetrapodophis amplectus, may shed light on the earliest snakes.
 “The Martill paper is going to be one of the most controversial papers around for a long time,” said Lee. “I’ve already had about 50 emails from colleagues about it, all expressing really different views.”
“It is a very unusual specimen,” Lee said, “because if it is a snake, it’s a tremendous missing link between lizards and snakes.”
But there are several lineages of lizards with lost or reduced limbs and longer bodies, so the evidence to place it as a snake ancestor must be more than just that. Martill notes that the short length of the tail in relation to the body, structure of the pelvis, impressions of body scales, recurved teeth, high vertebral count and the shape of the vertebrae all make Tetrapodophis a snake. “This thing is much much more of a snake than it is of a lizard,” he concluded. But some scientists don’t buy it. “I think the specimen is important, but I do not know what it is,” University of Alberta paleontologist Michael Caldwell told Ed Yong from National Geographic. But Lee is willing to give Martill the benefit of the doubt. “I’m prepared to provisionally accept that it’s a very unusual small snake,” he said. “But the specimen is so small and the skull is so badly crushed that I think there is going to be a lot of debate until all interested researchers are able to look at it.”
“It does seem to have some pretty intriguing snake features,” Lee admits. “Snake teeth have a very distinct curvature to them… and this animal does seem to have that. So that’s one feature that really makes me think this is probably a snake.” He’s also impressed by the animal’s spine. “It’s got a very large number of vertebrae — 160 backbone elements — which is also a very snake-like feature,” he added. “None of the other features that they list do I find particularly compelling.”
Hsiang, on the other hand, is entirely convinced. “Tetrapodophis does seem to possess many anatomical features that are unique to snakes — the recurved teeth, intramandibular joint, vertebral characters, et cetera,” she said. “So, based on Martill et al.’s report of the anatomy, it seems likely that Tetrapodophis is indeed an early snake.” She’s especially intrigued by what else is visible in the new fossil: its last meal. Martill et al. report that inside the snake’s stomach are a collection of vertebral bones, likely from a small mammal or lizard that it ate just before it died — the same diet that Hsiang et al. predicted with their ancestral state analyses. “The new fossil provides empirical confirmation of some of our results,” she noted. “For instance, the discovery of vertebrate bones in the stomach contents of Tetrapodophis aligns with our inference that the earliest snakes likely ate small vertebrates.”
According to Martill et al., the short tail and reduced limbs are evidence that Tetrapodophis was a burrowing snake. “Although this thing has been found in sediments that were laid down in water,” Martill says, “the shortened limbs and the little scoop-like feet that it’s got on its hind limbs look much more like they’re for burrowing than they are for swimming.”
“Also, they wouldn’t really function for swimming,” Martill said. “This thing is almost certainly using lateral undulatory locomotion to burrow through soft sand and leaf litter.”
“I think that’s fairly weak evidence,” said Lee. “There’s no living burrowing lizard or snake with those type of body proportions,” he added. “We can’t really say what it did at the moment, because there are too many contradictory traits in this animal.”
Lee similarly points to the shape and size of the limbs and feet, but says they provide evidence of an aquatic lifestyle rather than a fossorial one. Species known for their burrowing habits, like moles, have short, squat, strong limb bones, but Tetrapodophis has “long, delicate fingers and toes.” There are also questions about the composition of the bones themselves; bones can vary in the amount of calcium they contain, with more calcium or “more ossified” bones resisting breakage better than less ossified ones. Lee notes that the limbs of Tetrapodophis seem to be “fairly poorly ossified.” “That’s not what you find in burrowers because you want your hands and feet to be as robust as possible to push through the soil.” Reduced ossification of limb bones is, however, a trait shared by other aquatic organisms. Furthermore, in the hind feet, two ankle bones that are fused in most lizards are separate. The only other group of lizards where these bones are apart? The aquatic mosasaurs. Rather than seeing the feet as scoop-like, Lee sees them as “paddle-like.” He also noted that the bones of the fingers and toes are perfectly aligned in parallel with one another. “That leads me to think that they were held together in something, like a flipper or sheath.”
All of that and the fact that the animal was found in what, at the time, was a shallow sea, does give credence to the idea that it could be an aquatic snake. “I wouldn’t come out and say that it’s aquatic, because I don’t think we can say that either,” Lee said, “but I don’t think that we can conclude that it’s burrowing.”
The aquatic idea of snake origins might be the minority view, but there’s enough accumulating evidence now that it needs to be reexamined rather than dismissed out of hand.”
So how die Brazil’s Big Discovery end up in Germany?
Though Tetrapodophis is perhaps the most scientifically-intriguing snake fossil to date, questions about how it arrived in Germany are already beginning to overshadow its scientific importance. Even before the paper officially published, rumors swirled about whether the remarkable specimen was illegally poached from Brazil. When I asked Martill about the specimen’s discovery, he was disturbingly cavalier about the fossil’s origins. “More or less, I discovered it,” he said, “I actually found it in a museum collection.”
“It was one of those serendipitous things,” he continued.  “I actually worked on fossils from this location in Brazil for many many years.” But Martill didn’t find Tetrapodophis on an excursion to the jungle; he found it labeled as an “Unknown Fossil” in the Bürgermeister-Müller Museum in Solnhofen, Germany on a routine class trip for his students. It just so happened that when he took his students to see the museum, on display was an exhibit on Brazilian fossils, which Martill — having written a book about the Crato formation — was excited to see. “All of a sudden, my jaw just dropped to the floor,” he recounts. “This looks like a snake!”
When pressed, he admitted that there was no information about the fossil’s origins — when it was found, who pulled it from the earth, and how or why it made its way across the ocean to a small museum in Germany. He was blunter when he spoke to Herton Escobar (quoted by Herton Escobar for Science). Martill told him that questions about legality are ‘irrelevant to the fossil’s scientific significance’ and said: “Personally I don’t care a damn how the fossil came from Brazil or when.”
As Shaena Montanari explains for Forbes, given the laws in Brazil since 1942, it’s likely that Tetrapodophis found its way to Europe illegally. Brazilian officials have gone as far as to say they’re certain the specimen illegally left the country. Many scientists are expressing their outrage that a prestigious journal like Science would even publish a paper based upon what is likely a black market specimen.
Thus, the specimen is controversial by its very nature but the issue of it being in Germany illegally also raises questions about the fossil trade.

Martill, Tischlinger & Longrich. (2015). A four-legged snake from the Early Cretaceous of Gondwana. Science. doi: 10.1126/science.aaa9208
Hsiang et al. (2015). The origin of snakes: revealing the ecology, behavior, and evolutionary history of early snakes using genomics, phenomics, and the fossil record. BMC evolutionary biology, 15(1), 87. doi: 10.1186/s12862-015-0358-5
Reeder et al. (2015) Integrated Analyses Resolve Conflicts over Squamate Reptile Phylogeny and Reveal Unexpected Placements for Fossil Taxa.PLoSONE 10(3): e0118199. doi: 10.1371/journal.pone.0118199
Simões et al. (2015). Visual system evolution and the nature of the ancestral snake. Journal of evolutionary biology 28(7): 1309-1320. doi: 10.1111/jeb.12663
 Read the full interview between Martill and Escobar here

Sunday, July 5, 2015

More on Pseustes poecilonotus as a nest predator

Fragmented tropical forest landscapes are becoming more abundant, and loss of species following fragmentation are often predictable. Larger animals, tend to disappear first from fragments due to the bushmeat trade. However,  another vulnerable group includes understory, insectivorous birds, and ant-following birds. Nest predation is one mechanism that may limit bird populations and has long been suspected as a factor threatening bird populations in temperate  and tropical forest fragments.  A potential influence on nest predation that remains understudied in the tropics is density dependence. Dense territories can increase predators’ ability to find the closely-spaced nests. Yet bird density and nest predation are not always positively correlated, and multiple life-history traits and contexts are relevant.

In a forthcoming paper in Biological Conservation Visco and Sherry (2015) compared nest predation rates, bird density, and predator identities in three habitats of lowland Caribbean Costa Rica: two fragments, a peninsular reserve (La Selva Biological Station), and unfragmented rainforest. Their results suggest an inversely density-dependent nest predation pattern: In fragments, chestnut-backed antbirds reached their highest density and—contrary to predictions—experienced their lowest nest predation rates; La Selva, on the other hand, experienced the lowest density and highest predation rate. Because nest predation decreased with fragmentation, it appears not to explain declines of understory insectivores from forest fragments generally. 

Nest survival models indicated that habitat best-described nest predation likelihood. Video surveillance of nests documented the bird-eating snake (Pseustes poecilonotus) causing 80% of nest loss (37 of 46 nests) and a larger variety of predators in fragments; thus, landscape factors influenced an understory bird’s nest predation. Given the large effect on our focal species, Pseustes likely affects other understory nesters, a topic warranting further study. Tropical reserve conservation plans should consider potential impacts of specialized nest predators on vulnerable understory birds

Visco, D. M., & Sherry, T. W. (2015 in press). Increased abundance, but reduced nest predation in the chestnut-backed antbird in Costa Rican rainforest fragments: surprising impacts of a pervasive snake species. Biological Conservation.pseutes

Sex reversal triggers rapid transition from genetic to temperature-dependent sex.

Hatchling Bearded Dragon
A climate-induced change of male dragons into females occurring in the wild has been confirmed for the first time, according to University of Canberra research recently published on the cover of international journal Nature.

The researchers, who have long studied Australia's bearded dragon lizards, have been able to show that a reptile's sex determination process can switch rapidly from one determined by chromosomes to one determined by temperature.

Lead author Dr. Clare Holleley, a postdoctoral research fellow at the University of Canberra's Institute for Applied Ecology, explained: "We had previously been able to demonstrate in the lab that when exposed to extreme temperatures, genetically male dragons turned into females."

"Now we have shown that these sex reversed individuals are fertile and that this is a natural occurring phenomenon."

Using field data from 131 adult lizards and controlled breeding experiments, Dr Holleley and colleagues conducted molecular analyses which showed that some warmer lizards had male chromosomes but were actually female.

"By breeding the sex reversed females with normal males, we could establish new breeding lines in which temperature alone determined sex. In doing so, we discovered that these lizards could trigger a rapid transition from a genetically-dependent system to a temperature-dependent system," she said.

"We also found that sex-reversed mothers -- females who are genetic males -- laid more eggs than normal mothers," Dr Holleley said. "So in a way, one could actually argue that dad lizards make better mums."

University of Canberra Distinguished Professor Arthur Georges, senior author of the paper, also highlighted the importance that these discoveries have in the broader context of sex determination evolution.

"The mechanisms that determine sex have a profound impact on the evolution and persistence of all sexually reproducing species," Professor Georges said.

"The more we learn about them, the better-equipped we'll be to predict evolutionary responses to climate change and the impact this can have on biodiversity globally."

Holleley CE, O'Meally D, Sarre SD, Marshall Graves JA, Ezaz T, Matsubara K, Azad B, Zhang X, Georges A.
Sex reversal triggers the rapid transition from genetic to temperature-dependent sex. Nature, 2015; 523 (7558): 79 DOI: 10.1038/nature14574