Super variable California salamander is ‘an evolutionist’s dream’

The ensatina is a fairly common salamander. From southern British Columbia in Canada to northern Baja California in Mexico, it can be found lurking under logs in forests along the entire western coast of North America. But it’s in California where the little amphibian’s story takes an intriguing turn. Literally.

Depending on where you are, whether east of California’s Central Valley in the mountains of the Sierra Nevada, or west of the valley on the Coast Ranges, the ensatinas you encounter can look strikingly different. But they’re all thought to be the same species.

“As we like to say, the ensatina is a taxonomist’s nightmare, but an evolutionist’s dream,” said David Wake, a salamander expert and professor emeritus from the University of California, Berkeley, who has studied ensatinas for the last four decades.

Textbook case of speciation

This is because the ensatina demonstrates what some people refer to as a “textbook example” of speciation — it’s evolution in action. And it was Wake’s predecessor at U.C. Berkeley, Robert Stebbins, a herpetologist and illustrator, who first identified this in the late 1940s.

At the time, experts recognized four species of the ensatina based on their distinctive colors. But Stebbins, putting both his skills as an artist and a scientist to action, found an interesting pattern: he noticed that all the ensatinas could be arranged in the form of a ring encircling the Central Valley, a large flat valley that stretches for about 720 kilometers (450 miles) along the Pacific coast. On each side of the ring, neighboring ensatinas look similar to each other, but they differ considerably from the ensatina populations across the valley. In the Sierra Nevada, the salamanders have bright spots or blotches on their bodies. On the coast, they’re unblotched, with a more uniform brownish or dark reddish coloration.

The different ensatina populations could, in fact, be clubbed into just a single species, Ensatina eschscholtzii, Stebbins concluded, one that comprised seven subspecies. He named the four unblotched subspecies on the coast picta, oregonensis, xanthoptica and eschscholtzii, and the three blotched ones in the Sierra Nevada platensis, croceater and klauberi.

But these names are simply tags, Wake said. Researchers tend to identify the salamanders more based on the geographic regions and some general features of the salamanders.

“The fact that there are seven subspecies is kind of a historical mistake,” Wake said. “What happened is that Stebbins got tired of naming them. Six of them have distinctive features, the seventh, oregonensis, is sort of what’s left over. It has the greatest range and could be broken down more but nobody ever felt like adding.”

Names notwithstanding, Stebbins hypothesized that the ensatina represented a ring species, a concept first put forward by the famous evolutionary biologist Ernst Mayr.

A ring species, according to Mayr, was the “perfect demonstration of speciation”: it was a situation in which a chain of interconnected populations evolved around a geographic barrier, forming a loop, with older, foundational populations at one end and more recently emerged populations at the other. While the intermediate populations can mate and form hybrids, the two forms at the southern ends of the loop are so different that they can no longer interbreed, although they could eventually coexist in the same localities if geologic change brings their habitats together.

To Stebbins, the ensatina showed clear traits of a ring species. He thought that the various ensatina populations had originated from an ancestor living north of the Central Valley. This ancestor possibly had traits like E. e. picta (painted ensatina) now living in southwestern Oregon and extreme northwestern California. This salamander has sort of a mixed pattern — dark tan or brown interspersed with some fine yellow or orange spots — and Stebbins could imagine patterns on today’s ensatinas having emerged from a picta-like ancestor.

From this ancestor, ensatina populations slowly spread southward, expanding their ranges and avoiding the Central Valley as they moved.

This is because the ensatina is fully terrestrial, unlike most other salamanders, which means it spends all of its life stages on land, with its eggs hatching directly into miniature versions of the adults. On land, the ensatinas can tolerate a wide variety of habitats, from coniferous forest to scrub, as long as they find moist, but well-drained soil. What they don’t like, Wake said, is standing or flowing water, or swampy grounds.

Millions of years ago, when the ensatinas were migrating southward, the Central Valley was an area of swampland, creating conditions that would have been too wet and inhospitable for them, Wake added. Today the Central Valley is too hot and dry for them.

According to Stebbins, one group of populations went down the Sierra Nevada, becoming restricted to montane forests at higher elevations. As they evolved, they developed irregularly blotched, strongly contrasting color patterns, which researchers think offers them camouflage through disruptive coloration. This is akin to how military uniforms work: just like patterns of leaves and stems on military uniforms break up individuals’ outlines, hindering detection, the blotches on the salamanders make it hard for predators to spot their body shapes against the leaf litter on the forest floor.

Stebbins thought a second group of populations spread southward on the Coast Ranges. There they evolved to have more uniform body color. Again, researchers think such coloration helps them blend into the background, making it harder for predators to identify them. In one case, the ensatina seems to have developed a color pattern that’s very similar to that of another group of salamander: highly poisonous newts.

Such mimicry can be best seen in E. e. xanthoptica, or yellow-eyed ensatina, a species found on the coastal ranges east of San Francisco Bay. The yellow-eyed ensatina shares its habitat with two species of newts, Taricha granulosa and T. torosa, both known to be highly poisonous. All three have a brown back, a striking orange underside and a bright yellow patch in the eyes. Seeing their similarity, Stebbins thought that the ensatina had likely developed its color pattern to mimic the poisonous newts and avoid being eaten by predators. In 2008, herpetologist Shawn Kuchta, who was then Wake’s student, found experimental evidence to support this hypothesis.

When Kuchta presented some California newts (T. torosa) to western scrub jays, one of many predators of salamanders, the jays never attempted to eat one. Then, when he offered both the yellow-eyed ensatina and the Oregon ensatina to the jays, the birds were quicker and more likely to eat the Oregon ensatina, suggesting that the yellow-eyed ensatina resembled the newts.

A key feature of the ring species hypothesis is that all interconnected populations throughout the ring, except at the ends of the loop, can form hybrids wherever they meet.

The yellow-eyed ensatina demonstrates this midway down the ring. There, the unblotched salamander from the Coast Ranges has made its way to the foothills of the Sierra Nevada and made contact with the blotched Sierran subspecies E. e. platensis (Sierra Nevada ensatina). Wherever they’ve met, the two have hybridized extensively; Wake and his colleagues have confirmed this through genetic studies.

At the end of the loop, though, the two end products of these populations — the unblotched E. e. eschscholtzii (Monterey ensatina) from the Coast Ranges, and the blotched E. e. klauberi (large-blotched ensatina) from the Sierra Nevada — have diverged so much that they no longer interbreed everywhere they meet.

“There are four contact zones we know of where the two subspecies occur together and I believe hybridization occurs in three out of the four,” said Thomas Devitt, currently a research fellow at the University of Texas, Austin, who’s studied hybridization between the two end subspecies.

On Palomar Mountain, the two subspecies do hybridize sometimes. When Devitt looked deeper into the hybrids that form there — he could identify them from their very unusual color patterns that are unlike either parent subspecies — he saw something peculiar.

He found that nearly all the eschscholtzii-klauberi hybrids he studied possessed klauberi mitochondrial DNA. Since mitochondria is usually inherited from the mother in sexually reproducing animals, this suggested that most hybrids had resulted from female klauberi mating with either male eschscholtzii or male hybrids, but not vice versa.

Devitt conducted some courtship experiments that hinted at this pattern as well. He found that getting the salamanders to mate was generally incredibly difficult, and the results weren’t statistically conclusive. But in the few instances when the salamanders did mate, klauberi females mated with eschscholtzii males, while eschscholtzii females always rejected klauberi males.

Why this might be happening isn’t clear, Devitt said. “The main thing that I can actually speak to based on the data I collected is that there’s relatively strong selection against hybridization or hybrids in that hybrid zone although it does occur,” he said. “And we don’t exactly know why. It may just be intrinsic incompatibility between different gene complexes.”

Despite the information gaps, the ensatina is one of only two known species that broadly live up to the ring species concept.

While Stebbins painted the initial, basic scenario, Wake and his colleagues have since added more detail and complexity to the ensatina’s evolutionary story by digging into the salamander’s genes.

For example, Wake’s team found that ensatina populations do not show continuous gene flow throughout the ring as one might expect with an ideal ring species. Instead, he found that the populations evolved in fits and bursts, with sharp genetic breaks within the populations. As Wake wrote in a study published in 1997, the “history of this complex has probably featured substantial isolation, differentiation, and multiple recontacts. In effect, there are rings within rings in this complex…”

Given the complexities, some researchers have argued that the ensatina is not a “classic” ring species. Some have even suggested splitting the ensatina into multiple species. In fact, when Wake first began to look into the genetics of ensatinas, he expected to uncover several ensatina species. “Turns out, I was wrong,” Wake said. “It took me 40 years to understand what is going on in the ring species.”

Devitt agreed that while the ensatina may not meet the “classic” definition of a ring species, it comes “pretty close.” More importantly, it makes for a “fascinating study system,” he said.

“A lot of times with species, you end up with two end products of population divergence or speciation and you don’t have those intermediate forms that link those populations in the past,” Devitt said. “But in this case with ensatina you have both the end products as well as the intermediate populations that kind of link those populations.”

In fact, the ensatina shows how species are not “fixed entities,” Wake said. “I don’t think a species is very real. I think they’re an entity in space and time that’s ever changing and so for me it’s a matter of what criteria you want to apply.”

An important role

The ensatina has another claim to fame: wherever this salamander lives, there are usually lots and lots of them.

Michael Best, currently an associate faculty member at the College of the Redwoods, California, figured this out early while pursuing his master’s degree at Humboldt State University, Arcata, California.

“Just being here in the west in California, walking around and flipping cover objects, the ensatina would be the most encountered salamander,” Best said. “So I quickly learned it was a common species to encounter.”

But pinpointing how many ensatinas live in a forest can be incredibly hard: these salamanders spend a lot of time underground, so researchers trying to estimate their numbers are able to access only a small proportion of the animals that happen to be on the forest floor at any given time. However, by using sampling methods that account for uncertainties, researchers have come up with some estimates over the years, ranging from over 60,000 to nearly 300,000 ensatinas per square kilometer.

“It’s hard to give a number because it depends on very local micro conditions,” Wake said.

But what we do know is that the ensatina can be present in huge numbers. Moreover, since the ensatina is completely terrestrial, the females lay large eggs in dark, moist places on the forest floor, such as in the soil or in the hearts of big round logs. The female then guards her eggs for the next three or four months until they hatch into tiny versions of adult ensatinas. Spending all their life stages on land means that the salamanders are really tied to forests throughout their lives. This caught Best’s attention.

Given the ensatinas’ abundance, Best was curious about the role these salamanders play in the forests. They are, after all, among the key predators on the forest floors they occupy. They eat a wide variety of insects, from beetles to ants and flies. And because they are often so numerous, Best hypothesized that by eating the insects, the salamanders could be reducing the amount of leaf litter that the insects break down, thereby increasing carbon storage.

Best tested this out in a mixed conifer forest of tanoak, Douglas-fir and madrone in Ettersberg in northwestern California. He built 12 experimental plots on the forest floor, each 25 square feet (2.3 square meters) in area, using long sections of steel mounted together with bolts. From these plots, he removed all the salamanders he could find. Then, to half the plots, Best introduced a single male salamander, while the remaining half remained salamander-free. He also introduced bags of fresh, dried leaf litter, each weighing 3 grams (0.1 ounce), to all the plots, and removed them after four months to see how much leaf litter had been broken down.

In the first year of his experiment, Best found that the plots that had salamanders had fewer fly larvae and small beetles. These insects are leaf shredders. By chomping leaves down to tiny bits, they increase the surface area of leaves available for bacteria and fungi to colonize and decompose, an act that releases carbon dioxide into the atmosphere, Best said.

“With salamanders consuming those organisms, it seems that what’s happening is that fewer of the leaves are actually being broken down,” he added.

When Best pulled out the leaf litter bags after four months and re-weighed them, he found that there was 13 percent more leaf litter remaining in the bags that had been placed on the salamander plots compared to the salamander-free ones. What this means is that by eating the leaf-shredding insects, the ensatina was helping store more leaf litter and other forms of carbon, such as sticks and branches, on the ground for longer period of time.

While decomposition doesn’t stop just because there are fewer insects to shred the leaves — microbes and other invertebrates still work their magic — it slows down the process considerably, Best said. But since the leaf litter now has more time to sit on the forest floor, more of it gets converted to rich, organic matter called humus, which gets incorporated into the forest soil instead of being released into the atmosphere as carbon dioxide.

Best estimated that a single ensatina was capturing around 200 kilograms of carbon per hectare. By extrapolating his results to the entire range of ensatina, he estimated that the salamanders could be helping sequester more than 70 metric tons of carbon in a single season. “It’s totally conservative and kind of rough math,” Best said, but it gives an idea of the impacts that the salamanders could be having in their ecosystems.

Best is continuing with the experiments. And he’s seeing the results vary depending on moisture levels of the leaf litter and the number of salamanders that are introduced into the plots. The picture, he said, will become clearer once he’s finished analyzing all the invertebrate data.

What is evident, though, is that the ensatina is a critical member of North American forests.

A threat on the horizon

So far, researchers haven’t seen signs of any imminent danger to these salamanders, but that doesn’t mean there aren’t any.

“One thing that’s very difficult with amphibians, at least in my experience, is that it’s really hard to know when there are die-offs just because they decompose so quickly,” said Obed Hernandez-Gomez, a postdoctoral research fellow at U.C. Berkeley. “They really don’t leave any trace behind.”

One threat that is looming upon North America’s salamanders is the fungus called Batrachochytrium salamandrivorans (Bsal). On infecting a salamander, the fungal pathogen eats away at its skin, creating lesions that make it hard for the salamanders to breathe, ultimately killing them. The fungus has decimated several fire salamander populations in Europe, and researchers think the pet trade in these animals could bring the fungus to North America at any moment.

Researchers like Hernandez-Gomez are trying to figure out if North America’s salamanders have any natural defenses against the fungus. His team has been swabbing the skin of five species of salamanders, including ensatinas, to build a picture of the vast army of bacteria that live on them. If there are certain bacteria that can either kill Bsal or prevent the fungus’ growth, that would be a triumph. These bacteria could be cultured and used to make probiotics, Hernandez-Gomez said. “We can also feel some comfort knowing that if Bsal were to be introduced tomorrow that at least our salamanders have some natural protections,” he added.

The fairly common ensatina could be an important piece in this jigsaw puzzle.

In fact, Wake, whose lab has driven a large chunk of ensatina research in the past decades, thinks that there’s much more to be discovered about the animal.

“The salamanders themselves are important as a demonstration of a species in action and they’re important as critical components of local ecosystem. And I think they could use more study,” he said.

 

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