Saturday, February 28, 2015

Anilius: The Pipesnake that Wasn't

This post will soon be available in Spanish

Anilius scytale, the only living member of the family Aniliidae,
one of only two snake families containing just a single species
Deep in the Amazon rain forest there lives a fairly small, fairly obscure, red and black snake called Anilius scytale. It is banded, like many red and black snakes, but it has no venom, so it may be a coralsnake mimic. It spends most of its time under ground or in the water. Morphologically, it has a mixture of characteristics that place it somewhere in the no-man's-land we call "henophidia"—it has pelvic vestiges like many boas and pythons, but it has a small gape and is not capable of eating large bulky prey. It mostly feeds on elongate vertebrates, including other snakes, amphisbaenians, caecilians, and eels, and like other snake-eating snakes individuals can eat prey approaching their own total length. Its ventral scales are only barely wider than its dorsal scales, and it has just a few enlarged head scales, including one large hexagonal scale covering the eye and the surrounding skin. Males are smaller than females, which are viviparous, capable of giving birth to as many as 24 live young at a time. In 1946, the great naturalist William Beebe wrote "This is a strange snake", meaning that it's not quite like any other snakes. It is alone in its family, Aniliidae.

Head of Anilius showing the large scale covering both
the eye and the surrounding skin, like blindsnakes but
unlike most heno- and caenophidians
Snake biologists have used the term "pipesnake" to refer to any of three different lineages of snakes: the cylindrophiids (10 species of "Asian pipesnakes"), anomochilids (3 species of "dwarf pipesnakes"), and aniliids (1 species of "red pipesnake"; i.e., Anilius scytale). I'd like to propose that we begin to think of Anilius as "the pipesnake that wasn't", because (as I alluded to last month), it is now thought to be most closely related to tropidophiids (aka "the boas that weren't), superficially boa-like snakes found mostly in the Caribbean. Molecular data and some morphological data, especially that of the soft anatomy of the lungs and reproductive system, suggests that these two groups are each others' closest relatives, and they are now placed together in the Amerophidia (aka Anilioidea), the basal-most lineage of alethinophidia, which was apparently isolated in South America during the split-up of west Gondwana. Details of the skull anatomy cast some doubt on this classification, suggesting a closer relationship between aniliids and other non-macrostomatan pipesnakes, although even if this is true there are undoubtedly deep splits between Anilius and any other living snakes. Like the tuatara and the coelacanth, Anilius has not had close living relatives for tens of millions of years. Only it knows if it's lonely out there on such a long branch of the snake family tree.

Top: The plate of Anilius and a caiman as it appeared in
the 1719 printing of Merian's Metamorphosis
Insectorum Surinamensium

Bottom: A later version of the plate,
recolored and with the eggs removed
If Anilius is lonely, it can take some solace from having been noticed and beautifully illustrated by one of the first ecologists, Maria Sibylla Merian. Merian was a remarkable artist and scientist who lived from 1647 to 1717. She was one of the first trained artists to conduct detailed, long-term studies of living organisms, and the first published female naturalist. Most of her drawings, which she sketched from life on vellum and later engraved herself on copper plates, depict the life cycles of insects and their plant hosts, which she raised in captivity. She was the first to document that caterpillars turned into butterflies, and she described the life cycles of hundreds of insects, amassing evidence that contradicted the then-widespread notion that insects were "born of mud" by spontaneous generation (although others were credited with this discovery for a long time because her work was largely ignored, because it was written in Dutch rather than Latin). In 1699, Merian and her fifteen year-old daughter traveled to Surinam, where they spent the next two years studying and drawing the indigenous animals and plants, including several snakes. Her most famous work, Metamorphosis Insectorum Surinamensium, contains plates of many of these snakes, including one of an Anilius eating the egg of a caiman and being simultaneously attacked by the adult crocodilian. Like most of her drawings, it shows aspects of the natural history and ecology of the organisms in it, and helped establish a style of scientific illustration that later inspired naturalists from Catesby to Audubon. She depicted most of her insects life-sized, from various angles, in all stages of their life cycles, and most importantly, interacting with their host plants and predators. Her observations of animal behavior and plant-animal interactions are so detailed that many consider her the first ecologist. Considering that she died when Linnaeus was only 10 years old, it is all the more remarkable that her writings and drawings emphasize where organisms live and what they do rather than how they should be classified. Her works became very popular among Europe's upper class, and Czar Peter the Great in particular purchased many of her original watercolors and recruited her daughter as an art advisor and teacher at the newly-founded Academy of Arts in St. Petersburg. The Argentine Black and White Tegu, Tupinambis merianae, is named after her. Merian's text has not been translated into English, but I have taken a stab at translating her paragraph about snakes here:

Like crocodiles, some snakes hatch from eggs. They lay many small ones. The head and the tail of this snake, the Amphisbona, are the same shape and size, but you can tell which is the head because it has a mouth and small eyes, whereas the tail does not. Of all snakes, this one is the cleanest in color, being black, red, and yellow; others are grayish white, yellow, and brown with bodies that are more flattened.

ACKNOWLEDGMENTS

Thanks to Patrick Campbell and Andrew Snyder for allowing me to use their images.

REFERENCES

Anilius from d'Orbigny's 1849 Dictionnaire
Universel d'Histoire Naturelle
Beebe, W. 1946. Field notes on the snakes of Karatabo, British Guiana, and Caripito, Venezuela. Zoologica 31:11-52.

Duellman, W.E. 1978. The biology of an equatorial herpetofauna in Amazonian Ecuador. Miscellaneous Publications, Museum of Natural History, University of Kansas 65:1-352 <link>

Etheridge, K. 2011. Maria Sibylla Merian: The First Ecologist. in V. Molinari and D. Andreolle, editors. Women and Science: Figures and Representations – 17th century to present. Cambridge Scholars Publishing, Newcastle upon Tyne <link>

Marques, O. A. V. and I. Sazima. 2008. Winding to and fro: constriction in the snake Anilius scytale. Herpetological Bulletin 103:29-31 <link>

Martins, M. and E. M. Oliveira. 1998. Natural history of snakes in forests of the Manaus region, Central Amazonia, Brazil. Herpetological Natural History 6:78-150 <link>

Maschio, G. F., A. L. da Costa Prudente, A. C. de Lima, and D. T. Feitosa. 2007. Reproductive biology of Anilius scytale (Linnaeus, 1758) (Serpentes, Aniliidae) from eastern Amazonia, Brazil. South American Journal of Herpetology 2:179-183 <link>

Maschio, G. F., A. L. C. Prudente, F. S. Rodrigues, and M. S. Hoogmoed. 2010. Food habits of Anilius scytale (Serpentes: Aniliidae) in the Brazilian Amazonia. Zoologia (Curitiba, Impresso) 27:184-190 <link>

Merian, M.S. 1719. Metamorphosis Insectorum Surinamensium. Joannes Oosterwyk, Amsterdam <link>

Pieters, F. F. J. M. and D. Winthagen. 1999. Maria Sibylla Merian, naturalist and artist (1647-1717): a commemoration on the occasion of the 350th anniversary of her birth Archives of Natural History 26:1-18 <link>

Sawaya, R. J. 2010. The defensive tail display of Anilius scytale (Serpentes: Aniliidae). Herpetology Notes 3:249-250 <link>

Creative Commons License

Life is Short, but Snakes are Long by Andrew M. Durso is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.

Wednesday, January 28, 2015

Dwarf Boas

This post will soon become available in Spanish!
Este post pronto estará disponible en español!

Ambergris Cay Dwarf Boa (Tropidophis g. greenwayi)
Now that the USA and Cuba are finally warming up to one another after a chilly fifty years, we might be poised to learn a lot more about a really interesting group of snakes that reach their highest diversity on Cuba. These are the tropidophiids, or "dwarf boas". Their name is a little misleading—like the splitjaw snakes, they were once thought to be related to the true boas, and the name sticks even now that we now know better. At least the dwarf part is accurate: most tropidophiids are only 1–2 feet long. But this unassuming group of drab, nocturnal, live-bearing snakes holds more surprises and lessons about snake evolution that one would expect at first glance, with no shortage of interesting natural history to boot.

Top: Tropidophis melanurus constricts an anole
From Torres et al. 2014
Bottom: Madagascar Ground Boa
(Acrantophis madagascarensis)
constricts an oplurid lizard
Tropidophiids eat mostly frogs and lizards, and they constrict their prey in the same way as true boas: by winding the anterior part of their body neatly around their prey like a rope around a windlass, usually with an initial twist in the first loop, so that the snake's belly faces its head. This behavior, along with their relatively large gape size, seemed to suggest that they were related to the true boas (family Boidaesensu stricto), including well-known tree boas, boa constrictors, and anacondas. All true boas are neotropical and there are quite a few in the West Indies, so unlike many of the other boid "hangers-on" (such as the Malagasy Sanzinia & Acrantophis, African Calabaria, North American rosy and rubber boas, Pacific Candoia, and Old World sand boas), a close relationship between tropidophiids and boids was easy to accept in terms of the biogeography of the living species. A comparative analysis of constriction behavior in extant alethinophidian snakes done by Harry Greene and Gordon Burghardt showed that this pattern of constriction is shared by essentially all "henophidian" snakes, including booids, pythonoids, and some uropeltoids, notwithstanding a few fossorial species that have apparently secondarily lost constriction behavior alltogether, because it doesn't work in tight spaces.1

Top: Panamanian Dwarf Boa (Ungaliophis panamensis),
a member of the group to which tropidophiids were
once thought to be most closely related.
Bottom: Red Pipesnakes (Anilius scytale)
don't resemble tropidophiids very closely,
but we now think that they are each others'
closest living relatives.
In particular, what we now call tropidophiids were thought to be particularly closely related to two other small genera of neotropical boids, Exiliboa and Ungaliophis, which they superficially resemble both morphologically2 and ecologially. These still share their common name of "dwarf boa", but about 15 years ago a new picture began to emerge. While DNA from Exiliboa and Ungaliophis suggested that they were indeed related to true boas, evidence from both mitochondrial and nuclear DNA and immunological proteins of Tropidophis and Trachyboa, along with details of their muscular, circulatory, and reproductive anatomy, suggested that they were most closely related to the monotypic family Aniliidae, which contains a single South American species known as the Red Pipesnake (Anilius scytale). As far as we know, Anilius doesn't normally constrict its prey3, because it mostly forages underground on elongate vertebrates such as eels, caecilians, amphisbaenians, and other snakes, similar to various Asian pipesnakes to which it was once thought to be closely related. But, we are now fairly certain that these Asian pipesnakes are convergent with Anilius, that tropidophiids and aniliids are each others' closest relatives, and that the similarity between the gape size and constriction behavior of tropidophiids and that of boas and pythons probably still represents the shared retention of a paired morphology/action pattern used by their common ancestor, it's just a common ancestor that is much older than we originally thought. Estimates suggest that tropidophiids and aniliids diverged from one another 60-110 mya in South America4, after their common ancestors were isolated from those of all other modern alethinophidian snakes, which radiated in Africa following the mid-Cretaceous split-up of west Gondwana 70-120 mya. This was the split that formed South America and Africa, and we are now getting used to diving the alethinophidians into two major lineages, Amerophidia (tropidophiids and aniliids) and Afrophidia (everybody else), instead of into a monophyletic "crown-group" Macrostomata containing boas, pythons, and caenophidians, and a basal group of non-macrostomatan pipesnakes more similar in ecology to scolecophidians. My snake taxonomy article from 2013 is actually out-of-date with respect to this major shift in snake taxonomy, because at the time it was still unclear to me (and there are still some strong arguments from paleontologists that the molecular data may be misleading).

The Greater Antilles, Bahamas, and Turks & Caicos
The "new" family Tropidophiidae consists of two species of "eyelash dwarf boas" in the mainland genus Trachyboa (there we go with the boa thing again), and the diverse genus Tropidophis, which contains 32 species in total: 5 from mainland South America, and a West Indian radiation consisting of 17 Cuban species (one of which is shared with Jamaica and one with both Jamaica and Hispaniola), 1 on Hispaniola (shared with Cuba), 5 on Jamaica (two shared with Cuba), 2 in the Bahamas, one from the Turks & Caicos Islands, one each on the three Cayman Islands (Grand Cayman, Little Cayman, and Cayman Brac), and one endemic to Navassa Island, a small, uninhabited, disputed island in the Caribbean Sea between Cuba, Jamaica, and Hispaniola (which is known from four specimens and has not been seen in over 100 years). The West Indian species, particularly the Cuban ones, represent a radiation which rivals and parallels that of Darwin's finches. Morphological and molecular data suggest that the 17 species on Cuba are descended from a single colonization event, and that the island species appear to be more distantly related to the mainland ones than they are to Trachyboa, although four-fifths of the species of Tropidophis have no published sequence data yet so both of those conclusions could change.

Tropidophis xanthogaster bleeding from the mouth,
with blood behind the spectacle making the eyes appear red.
From Torres et al. 2013
As early explorers and biologists collected these snakes from bromeliads, within stone walls, and underneath rocks, they noted that species of Tropidophis made no effort to escape their collection, but rather coiled up into tight balls when captured. Another peculiar defensive behavior was soon noted—autohemorrhage of the nose and mouth. In other words, these snakes spontaneously bleed from these orifices and smear the blood all over themselves when handled. Creepily, the space between their spectacle and their eyes fills with blood momentarily beforehand, so that their eyes appear to flash red. Blood collected from their mouths doesn't clot for over half an hour, whereas blood collected simultaneously from their tails has clotted after 10 minutes, and the mouth blood is more acidic and has fewer red blood cells, presumably because it is mixed with saliva. However, it is not harmful to frogs or lizards, so it is not a substitute for venom. The exact function is unclear, but it appears to be to freak out would-be predators. Like many snakes, Tropidophis habituates to captivity and eventually does not exhibit this behavior.

Tropidophis melanurus, the largest species of Tropidophis
and the first described, from Cocteau & Bibron's 1843
volume on reptiles
in de la Sagra's Histoire physique, politique,
et naturelle de l’Ile de Cuba
Just when you thought things couldn't get any more interesting, brace yourself, because most Tropidophis can change color! They are light silver-white at night, when they are active, and dark grayish-brown during the day, when they are not. It takes a Tropidophis 1-2 hours to go from completely light to completely dark, which they accomplish via mobilization of melanosomes (organelles containing the light-absorbing pigment melanin) from the core of a melanophore cell deep within their skin into finger-like extensions of the melanophore that are closer to the surface of the skin, partially blocking stationary xanthophores and iridiophores, which contain yellow, blue, or green pigments. Both adults and juveniles undergo diel color change, and it does not seem to be affected by age, sex, pregnancy, or feeding, although prior to shedding snakes remain dark and inactive for several days. The change is probably predominantly triggered by photoperiod, but exposure to cool temperatures (<63°F) can elicit a partial change from dark to light even in the middle of the day. When captive snakes were transported from Cuba to Czechoslovakia, they became jet-lagged—it took them several days to synchronize their rhythm to the new photoperiod, and keeping them in complete darkness for several days desynchronized their rhythm from that of the sun. The proposed function of this color change is to help nocturnally-active snakes retain their body heat, as light-colored objects lose heat more slowly than dark-colored ones. This is probably similar to the reason that Round Island Splitjaw Snakes, Pacific Keel-scaled Boas (Candoia carinata), and the Hogg Island race of Boa constrictor also become lighter-colored at night.

Tropidophis pardalis on a Cuban stamp
There's much more to learn about tropidophiids, the Cuban radiation of Tropidophis in particular. To date, little ecological information has been collected on most species, owing in part to their rarity and in part to the difficulty of working in the region. How do five or six sympatric species partition resources and coexist in various parts of Cuba? What was the order of speciation and colonization of the islands, and when did it happen? Hopefully tropidophiids will be around long enough for us to find out. They are faced with numerous threats. As in many places, local people not especially fond of them, despite the fact that no Greater Antillean snakes are dangerous to people. Collection for the pet trade may also be a concern, particularly since one former government official in the Turks & Caicos Islands apparently granted a permit to reptile dealers to remove thousands of Tropidophis greenwayi from North Caicos for the pet trade, allegedly implying that it would be preferred if they removed all of the snakes! Throughout the West Indies, most native ecosystems have been absent for centuries, and increasingly rapid development, especially due to tourism, threatens what little remains. And introduction of non-native predators, particularly the Small Indian Mongoose (Herpestes javanicus), may be their biggest threat. As early as 1919, herpetologist Thomas Barbour wrote "In Jamaica [Tropidophis maculatus] is almost extinct owing to the appetite of the introduced mongoose". Ironically, Operation Mongoose was the codename for the Kennedy administration's attempt to create Cuban diplomatic, political, and economic isolation in hopes of weakening Castro’s regime. Cats, dogs, rats, goats, pigs, cane toads, and even other introduced snakes also threaten not just tropidophiids, but all 120+ snake species endemic to the West Indies as well as the rest of the native fauna. Improved PR and conservation programs have benefited several lizard species, and could help snakes too.

Tropidophis haetianus
I'm going to go ahead and wager that we'll discover a few new species of Tropidophis in the not-too-distant future, and that possibly the mainland species will get moved into a new genus. I also think that we need a more creative common name for them than "dwarf boa", preferably one that doesn't include the word "boa" at all. One existing option is "wood snakes", which is mediocre at best. They are also called "rock pythons" in the Caicos Islands, an equally misleading name as "dwarf boa", "culebras bobas" (dumb snakes) in Cuba, and "shame snakes" on Andros Island in the Bahamas, both of which may refer to their head-hiding defensive behavior. However, my favorite is the name they are known by in many parts of the West Indies: "thunder-snakes", because they are more frequently seen after severe rainstorms. Caribbean Thunder-snakes has a nice ring to it, and it could help improve their image.



1 1: Constriction behavior has become a lot more variable within the Colubroidea, where it has also been lost in several venomous lineages. Venom and constriction can be thought of as two different solutions to the same problem—how to kill large prey without exposing yourself to undue risk. Also, the contention that constriction and large gape size were lost in fossorial henophidians (aka "regressed" macrostomatans, including uropeltids, anomochilids, and aniliids) is seemingly contradicted by the complex multipinnate morphology of their jaw adductor muscles, which is sufficiently similar to that of their lizard ancestors that it is unlikely to have re-evolved in the exact same way multiple times. This problem might also be an issue for scolecophidians, given that they have similar jaw muscle morphology to pipesnakes but appear to be more closely related to other living snakes than they are to some basal fossil macrostomate snakes with limbs (symoliophiids). Stay tuned for more on the unresolved relationships at the base of the snake family tree, including a look at what fossil snakes can tell us.



2 2: All four genera (Exiliboa, Ungaliophis, Tropidophis, and Trachyboa) either completely lack a left lung or have a greatly reduced one, a characteristic they share with anomochilids and some caenophidians, but not with most other henophidians, which have a somewhat reduced but functional left lung. In addition, all four genera also have a "lung" on the dorsal wall of the trachea: the tracheal cartilages do not form closed rings but remain open on the top, where a greatly expanded ligament forms the tracheal lung. It has alveoli just like a regular lung, which are especially deep near the head, and is contiguous with the true lung in the vicinity of the heart. But, although this might seem like very strong evidence that these four genera are closely related, tracheal lungs of diverse structure are widespread among snakes, being found in certain scolecophidians, xenophidiids, acrochordids, vipers, atractaspidids, sea snakes, and many colubroid snakes.



3 3: A tantalizing bit of evidence emerged in 2008—biologists in Brazil videotaped the prey subjugation behavior of a captive Anilius scytale, which essentially constricted an amphisbaenian that they tried to feed it. In general its constriction behavior agreed with that of other henophidia, although it was more variable in the particulars, which could have been due to the difficulty of holding onto the elongate, "vigorous and constantly twisting prey". But, data from a single observation do not a generalization make, and more studies are needed.



4 4: Fossils of t
en extinct species in five genera from the Paleocene, Eocene, and Oligocene of Europe, Africa, & North and South America have been assigned to the Tropidophiidae, although all of them are probably actually either ungaliophiines or stem afrophidians. Two genera, Falseryx and Rottophis, both from the Oligocene of western Europe, have some similarities with living tropidophiids as well as with ungaliophiines, but for the most part their skulls are poorly preserved, leaving paleontologists to work on just their vertebrae. Paleogene erycines dominated the snake fauna of North America prior to the Miocene explosion of colubroids, but as far as we know all of these species were much more closely related to modern rosy and rubber boas than they were to tropidophiids. The only unequivocal tropidophiid fossils are from the Pleistocene of Florida and the Bahamas.

ACKNOWLEDGMENTS

Thanks to Kenny Wray, Nick Garbutt, Alex Figueroa, Patrick Campbell, Pedro Bernardo, and Carlos De Soto Molinari for the use of their photographs.

REFERENCES


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Tropidophis paucisquamis from Brazil
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Cocteau, J. and G. Bibron. 1843. Reptiles. In: M. R. de la Sagra. Histoire physique, politique, et naturelle de l’Ile de Cuba. Vol. 4. Arthus Bertrand, Paris <link>

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Szyndlar, Z., R. Smith, and J.-C. Rage. 2008. A new dwarf boa (Serpentes, Booidea,‘Tropidophiidae’) from the Early Oligocene of Belgium: a case of the isolation of Western European snake faunas. Zoological Journal of the Linnean Society 152:393-406 <link>

Thomas, R. 1963. Cayman Islands Tropidophis (Reptilia, Serpentes). Breviora 195:1-8 <link>

Tolson, P. J. and R. W. Henderson. 2006. An overview of snake conservation in the West Indies. Applied Herpetology 3:345-356 <link>

Torres, J., C. Pérez-Penichet, and O. Torres. 2014. Predation attempt by Tropidophis melanurus (Serpentes, Tropidophiidae) on Anolis porcus (Sauria, Dactyloidae). Herpetology Notes 7:527-529 <link>

Torres, J., O. J. Torres, and R. Marrero. 2013. Autohemorrage in Tropidophis xanthogaster (Serpentes:Tropidophiidae) from Guanahacabibes, Cuba. Herpetology Notes 6:579-581 <link>

Vidal, N., A. S. Delmas, and S. B. Hedges. 2007. The higher-level relationships of alethinophidian snakes inferred from seven nuclear and mitochondrial genes. Pages 27-33 in R. W. Henderson and R. Powell, editors. Biology of the Boas and Pythons. Eagle Mountain Publishing, Eagle Mountain, Utah, USA <link>

Wilcox, T. P., D. J. Zwickl, T. A. Heath, and D. M. Hillis. 2002. Phylogenetic relationships of the dwarf boas and a comparison of Bayesian and bootstrap measures of phylogenetic support. Molecular Phylogenetics and Evolution 25:361-371 <link>

Zaher, H. 1994. Les Tropidopheoidea (Serpentes: Alethinophidea) sont-ils reellement monophyletiques? Arugments en faveur de leur polyphyletisme. Comptes Rendus de l'Académie des Sciences Paris 317:471–478 <link>



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Life is Short, but Snakes are Long by Andrew M. Durso is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.

Tuesday, December 16, 2014

What's the big deal about these snake genomes anyway?

This post will soon become available in Spanish

King Cobra (Ophiophagus hannah; top) and
Burmese Python (Python bivittatus; bottom),
the two snake species whose genomes
were fully sequenced in 2013
One year ago today, the first snake genomes ever sequenced hit the newsstands. OK, so two papers in Proceedings of the National Academy of Sciences isn't exactly the cover of Time magazine to most people, but it was big enough news that it was covered by The Huffington Post and the two most prominent interdisciplinary scientific journals, Science and Nature, the former devoting a special section to the event. One year later, dear reader, welcome to the Life is Short, but Snakes are Long coverage of the snake genome project. So just what is the big deal about these snake genomes anyway, and what's changed in snake biology in the year that they've been available?

In one way, sequencing a snake genome means that snakes finally join the illustrious ranks of lab animals like the mouse, rat, guinea pig, fruit fly, and amoeba, all of whom have already had their genomes sequenced. By now the genomes of several hundred species have been sequenced, starting with a virus in the 1970s, and the first archaeon, bacterium, and eukaryote within one year of one another in 1995-96. The first animal genome sequenced was that of the model nematode Caenorhabditis elegans in 1998, and the first vertebrate was a pufferfish, so chosen because its genome is so small, in 2002 (although an incomplete first draft of the human genome preceded that by a year). As of 2014, we're now up to just over 100 vertebrate species, about 60 of which have been annotated and formally published, as well as numerous other animals, plants, fungi, protists, and prokaryotes. Last week, Science highlighted drafts of 38 new bird and 3 new crocodilian genomes, the largest single release of vertebrate genomes to date. But we are still a long way from sequencing the genomes of all known species. Why have we chosen the species we have? What does it mean to sequence a genome, exactly, and why do we do it?

Breakdown of what the human genome
consists of. Exons are coding DNA.
From Reece et al. (2013)
We use the word genome to refer to all the DNA within a single organism. Confusingly, this is not quite the same thing as saying all the genes in an organism, because we usually only call sections of DNA "genes" if we know what they do. You've probably heard that 98% of the human genome is "junk", or non-coding, DNA, which is just another way of saying that we haven't figured out what it does yet. Actually, we now know lots of things that non-coding DNA is good for, but we still usually don't call most of that DNA "genes" because we use that word specifically to mean sections of DNA that are read out via RNA and translated (usually) into proteins, which then have obvious effects on cells and the body. Non-coding DNA can also have effects on the body, often by regulating other genes, but it works in a more complicated way that we don't yet fully understand, so we tend make over-generalizations about it or dismiss it as unimportant.

Avian tree of life based on whole-genome
sequences. We're still several years away from
a tree like this for squamate reptiles.
From Jarvis et al. 2014
When we say we have sequenced the genome of an organism, we mean that we have read the sequences of all of its DNA, every one of its genes and all of its non-coding DNA, even if we don't know what it all does. The -ome suffix is added to the word 'gene' to signify "all". Yogis will be familiar with the Sanskrit word Om, which means "the whole thing", something that encompasses the entire universe in its unlimitnedness. Other fields in biology that consider all constituents of something collectively have picked up on this neologism, so we have proteomics (the study of all the proteins in a particular organism), transcriptomics (the study of all the RNA), and so on. Genomes are huge1, and we've strategically chosen species to sequence that are scattered across the diversity of life so that we can construct a skeletal tree of life based on genomic data. We have high confidence in such a tree2 because whole genomes contain so much data that trees built from them are more likely to reflect true evolutionary relationships than trees built from just one or a few genes. So we've selected exemplars from each major group of organisms to start out with (e.g., one sea urchin, one sea squirt, one lamprey), and eventually we'll go back and fill in the gaps. By sequencing the King Cobra (Ophiophagus hannah) and Burmese Python (Python bivittatus) genomes first, we're setting these species up to become model organisms, exemplars, and in some ways stand-ins for all of snake diversity in many future studies.

Understanding the genes controlling variation among individuals
of the same species, like the color morphs of these Groundsnakes
(Sonora semiannulata), must await population genomics
and a better understanding of gene expression regulation
When we sequence a genome we read all the DNA from a single individual3. This is different from knowing all the possible variants (often called alleles) of those genes. It's often said that a person has "the genes for" something, when in reality all people have the same genes, with different alleles. For example, if the person whose genome was sequenced in the Human Genome Project had brown eyes, we'll just have the gene sequences for brown eyes, not for blue or green. In order to get an idea of all the possible variants of all the genes in a species, we'll need to sequence the genomes of many individuals. Some genes, such as those involved in the immune system, have over 1,500 alleles (the "gene pool"), no more than two of which occur within the genome of a single individual (one from the mother and one from the father). So understanding the entire gene pool of a species is a very daunting task, given that we only have whole genomes for a few hundred species (one individual each), with multiple individuals of a few species, including humans.4 Population genomics is an emerging field, yet to be applied to snakes in any form, although apparently a few projects are in the works.

So what have we learned from these snake genomes? Here are the basics:
  • Snake genomes are about half the size of the human genome (although an organism's complexity is not directly proportional to its genome size; for example, some salamander genomes are more than 60 times larger than the human genome).
  • The proportion of repetitive elements ("junk DNA") in snake genomes is about the same as that in humans (~60%).
  • Snakes have a faster baseline rate of evolution than other reptiles, birds, or mammals, as
    Red represents fast rates of neutral substitution
    From supplement to Castoe et al. 2013
    evidenced by their larger accumulation of neutral substitutions. and colubroid snakes have rates even faster than that of snakes at large.
  • Adaptive evolution (as evidenced by functional, non-neutral, changes to genes) in snakes has happened to over 500 genes, especially those involved in the development of the limbs, spine, skull, and eye, and those regulating the function of the cardiovascular system, lipid and protein metabolism, and cell birth and death. We already knew that all of these systems in snakes were highly modified relative to other vertebrates, and now we know that the genes that underlie them are too.
  • Some groups of genes have grown or shrank in snakes - for example, snakes have a lot more genes coding for vomeronasal receptors, and a lot fewer genes coding for opsins, which are light-sensitive proteins in the eye. This makes sense given what we know about snake sensory systems.
  • Changes to gene expression that happen after a snake feeds involve thousands of genes that control rapid changes in organ size - but genes that control cell division change in the kidney, liver, and spleen, organs that grow by cell division, but not in the heart, which grows when individual existing cells get larger.
  • Snake genomes contain endogenous viral elements from three families of viruses that have recurrently infiltrated their DNA over the past 50 million years. This is actually not rare, although it is bizarre and awesome that the 'fossils' of these ancient viral genomes can be identified in their host genomes even after tens of millions of years, and it can help us better understand both the biology of viruses and that of their snake hosts, including how viruses have contributed functions to the genetic repertoires of their hosts.
From the cobra genome in particular, we've learned or confirmed a great deal about the evolution of snake venoms. In particular, we now know that, unlike the venom of the platypus, the only other venomous vertebrate with a sequenced genome, snake venom has evolved primarily through gene duplication and restriction. Many venom proteins probably evolved like this:
  1. A snake has a gene that makes a protein somewhere in its body, including possibly in its salivary or venom gland5
  2. The gene for that protein is duplicated by accident during routine DNA replication or repair, resulting in a new, spare copy of the gene
  3. The effects of selection are relaxed on the duplicate gene, which gives it opportunities to mutate
  4. Mutations to transcription-factor binding sites change the signal for where the duplicate gene should be expressed, causing the new protein to be made only in the venom gland
  5. If the new protein helps the snake catch more prey, it improves fitness and causes natural selection
  6. Because the old protein is still being made, the new gene and protein are free to evolve to become more toxic or to take on some new function
  7. The new copy of the gene may become duplicated again, and subsequent new copies may mutate further, leading to diversification within a gene/toxin family6
The King Cobra venom gland, with
expression profiles of the venom (left) and
accessory gland (right). From Vonk et al. 2013
It's not yet clear to what extent the evolution of these novel toxic venom proteins corresponded with a shift to higher levels of their expression in the venom gland and lower levels of expression elsewhere. Although it seems obvious that their expression in non-venom-gland tissues would be harmful, their non-toxic orthologs are expressed in tissues as diverse as the kidney and brain in pythons, and no one has yet measured their expression outside of the venom gland in venomous snakes. Alternatively, gene duplication might have taken place after the change in function, if the genes in question were alternatively spliced to produce both toxic and non-toxic proteins from the same gene. Evolution  of siRNA and other regulatory elements (which is hard to detect because there's still a lot we don't understand about how it works) could then restrict expression of a particular splice variant to the venom gland, which could explain why we're seeing evidence that the venom protein genes themselves are often still expressed in other tissues even though they are capable of coding for highly toxic proteins that must be maintained in the venom gland in a competent but inactive state.

The cobra genome by itself does not answer these questions, even with help from that of the python. In order to fully understand the evolution of snake venoms (with major implications for public health, particularly in developing countries, not to mention the potential of venoms to be used as drugs), we'll need genomic, transcriptomic, and proteomic data from numerous snake species.

Characterization of genomic biodiversity has the potential to change our understanding of evolution in fundamental ways. From explaining how snakes are capable of physiological feats to helping us understand how new genes appearwhat "junk DNA" does, and what the tree of life looks like, genome sequencing is one of the most exciting current frontiers in biology. As in many things, snakes are (one of) the last groups of vertebrates to the party (although it's worth noting that there aren't any fully annotated salamander or caecilian genomes yet). A snake genome doesn't add a whole lot to the picture of the vertebrate tree of life, because the Green Anole genome, sequenced in 2011, represents squamates on the tree, and no one is arguing that snakes aren't squamates. But, within squamates there are a number of puzzling unresolved relationships, including such fundamental questions as the origin of snakes and the placement of iguanians. In the interest of helping to shed light on these, and on the aforementioned complexity of snake venom evolution, another 10 or so snake genomes are likely to come out within the next couple of years, including those of the:
  • Texas Blindsnake (Rena dulcis)
  • Reticulate Wormsnake (Amerotyphlops reticulatus)
  • Red Pipesnake (Anilius scytale)
  • Mexican Burrowing Python (Loxocemus bicolor)
  • Round Island Splitjaw Snake (or "boa"; Casarea dussumieri)
  • Boa Constrictor (Boa constrictor)
  • Western Diamond-backed Rattlesnake (Crotalus atrox)
  • Speckled Rattlesnake (Crotalus mitchelli)
  • Copperhead (Agkistrodon contortrix)
  • Eastern Coralsnake (Micrurus fulvius)
  • Cloudy Snail-eating Snake (Sibon nebulatus)
  • Common Gartersnake (Thamnophis sirtalis)
As you can probably see if you know your snake taxonomy, these species represent a scattering of well-known snakes from each of the major branches of the snake tree. They have been strategically chosen to enable snake biologists to use them to put together a well-supported skeleton of the snake tree of life. However, several branches (such as the dwarf pipesnakes, acrochordids, and lamprophiids) are still missing.7 In particular, an atractaspidid genome would be useful in building a better understanding of the role of convergence in snake venom evolution - resolving the debate between proponents of a single ancient origin for venom and those of several more recent, independent origins. Genomes of scolecophidian blindsnakes and toxicoferan lizards such as Gila monsters will also help resolve this question. Hopefully, these genomes and others will continue to illuminate evolutionary biology for us in ways Darwin could have scarcely imagined.



1 Because genome sequences contain so much data, they are stored electronically and require a large amount of computing power and storage capacity. The computing power is actually more limiting than the biochemistry right now. A human genome contains about 6 billion base pairs (one for each person on Earth in 1999), which take up a couple of gigabytes. If that doesn't sound that impressive, imagine all that information stored 
in every one of your cells, then compare the size of a cell with that of a microchip here.



2 This is not to say that (as has been presumed by many) molecular data are inherently superior to morphological data, especially in the case of extinct fossil taxa, from which we cannot garner much molecular information (although that generalization too has been challenged).



3 How are the individuals whose genomes are sequenced chosen? The unsatisfying answer is that the scientists involved typically use whatever individuals are convenient. Specifically, the cobra and python genomes seem to have been taken from animals from the pet trade. We may not know the true geographic origin of these individuals, or even whether they might be the offspring of animals from two or more different parts of the species' range. Why is this important? If we sequence the genome of a cobra from Indonesia, but cobras in India have evolved different venom genes because of different evolutionary pressures, then we won't know that until we get some cobras from India. Taxonomic conclusions drawn from 
Boa constrictor gene sequences on GenBank are dubious because of the ambiguous origins of many of these specimensThe primary reasons to sequence a whole genome are subtly different from the reasons to sequence individual genes, and scientists doing these tasks have different questions. But, we should be cautious about inferring too much from the genome sequence of a single individual of any species.



4 Right now if you're a human you can actually get your whole genome sequenced for less than $5000, even though the first human genome cost over $3 billion, because we've optimized the process.



5 It's unclear how many venom proteins were originally made in the venom gland before they became toxic, and how many were recruited to this tissue following duplication. The original cobra genome paper by Vonk et al. implies that the latter is most common, whereas subsequent work by Hargreaves et al. uses gene expression data from Leopard Gecko salivary glands
 to suggest the former. Reyes-Velasco et al. used the python genome and transcriptome to suggest that venom genes are recruited preferentially from genes that are expressed at low levels in most tissues but at more variable levels than average across tissues.



6 Of the approximately 24 gene families that code for snake venom proteins, those that produce toxins that are known to be important in prey capture (e.g., the three-finger neurotoxins) have undergone repeated duplication and selection, whereas venom components that perform ancillary functions, such as helping the snake to relocate its bitten prey, do not show high rates of duplication or selection. These rates are probably further influenced by the need to target diverse receptors in different types of prey (in snakes with broad diets), and by predator-prey co-evolutionary arms races (in snakes with narrow diets).



7 A recent effort by a different research group generated a tree for Caenophidia using 333 loci totaling 225,140 base pairs for each of 31 snake species, almost 80,000 of which were informative. This is a drastic improvement on the 10 loci and maximum of 5,814 base pairs of the most comprehensive previous studies, but it is still a long way from the entire genome. Incredibly, they were still unable to resolve certain difficult parts of the snake family tree.


ACKNOWLEDGMENTS

Thanks to JD Willson, Baloch Imrankhan, and Alison Davis Rabosky for the use of their photographs, and to Alison Davis Rabosky and Todd Castoe for providing me with information regarding genomics.

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Creative Commons License

Life is Short, but Snakes are Long by Andrew M. Durso is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.