A closer look at a classic ring species: The work of Tom Devitt
by the Understanding Evolution team
If you've skimmed a high school
biology textbook, you've probably
seen the picture: multicolored
salamanders meander around
California, displaying subtle shifts in
appearance as they circle its Central
Valley. This is Ensatina
eschscholtzii, and it's so well known
because it is a living example of
speciation in action. Adjacent
populations of the salamander look
similar and mate with one another
— but where the two ends of the
loop overlap in Southern California,
the two populations look quite
different and behave as distinct
species. The idea is that this
continuum of salamanders — called
a ring species — represents the
evolutionary history of the lineage
as it split into two.
Ensatina has been recognized as a
ring species since the 1940s, when
biologist Robert C. Stebbins trooped
up and down California to
investigate its range. Since then,
several generations of scientists in Stebbins' institution, the Museum of Vertebrate Zoology at UC
Berkeley, have continued these studies, digging deeper into Ensatina's history and biology. At this point,
one might think we'd know it all. What more could there be to learn after 60 years of research on a common
salamander? "Lots!" says Tom Devitt, a graduate student at the museum. Tom studies Ensatina to flesh out
its evolutionary history — but not just for Ensatina's sake. This classic example sheds light on the basic
evolutionary processes that shape all life.
Tom Devitt and a map showing the range of Ensatina
eschscholtzii in California. The colors correspond to the different
subspecies.
In this research profile we will explore these key questions:
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What are ring species?
How are multiple lines of evidence used to evaluate a single hypothesis?
How can experiments be used to learn about evolutionary history?
What biological mechanisms contribute to reproductive isolation and
speciation?
Discovering a ring species
Ensatina's basic story was laid out by Robert Stebbins 30 years
before Tom was born in 1977. Based on the ring-like distribution
of the different forms, Robert had proposed that the species started
off in Northern California and Oregon and then spread south along
both sides of the Central Valley, which was too dry and hot for
salamanders.1
According to Robert's hypothesis, as the pioneering populations
moved south, they evolved into several subspecies with new color
patterns and adaptations for living in different environments. By
the time they met again in Southern California as the subspecies
eschscholtzii and klauberi, he argued, they had each evolved so
much that they no longer interbred — even though the subspecies
blended into one another around the rest of the ring. Since species
are often defined by their inability to interbreed with other species,
Ensatina seemed to represent the whole process of speciation —
all the gradual changes that accumulate in two lineages and that
wind up making them incompatible with one another.
Robert Stebbins examining an
Ensatina salamander in 1951.
Of course, since this all would have happened millions of years ago, Robert wasn't around to observe any
of it. He based his ideas on the morphology, or body form, of the subspecies — in this case, their color
patterns. First, neighboring subspecies were more similar to one another than to those across the ring and
seemed to blend into one another. From this, he hypothesized that Ensatina represented a ring species.
Robert also noticed that the northern coastal form, called picta, had a pattern of colors that seemed to
encompass the other subspecies. It was easy to imagine how the more specialized southern forms could
have evolved from picta. Based on this, Robert hypothesized that the two southward-moving Ensatina
lineages had both emerged from picta's immediate ancestors.
The subspecies Ensatina eschscholtzii picta.
The molecules support the morphology
By the 1980s, when Tom was splashing in a pond by his house catching the frogs, snakes, and salamanders
that he's always loved, biologists had moved beyond Ensatina's morphology. New tools allowed
researchers2 to study the salamanders' proteins, the chemical building blocks of life — and what they found
supported Robert Stebbins' ideas. Even though eschscholtzii and klauberi live in the same places in
Southern California, their proteins are quite different from one another and each is more biochemically
similar to its northern neighbor. This is exactly what we'd expect to observe if these two subspecies
represent the endpoints of the ring species. Furthermore, populations of picta had great variety in their
proteins — and that's typically what we observe in populations that have been established for a long time
and have had plenty of time to accumulate mutations. This observation supported the idea that picta's
immediate ancestors gave rise to the whole ring species.
By the 1990s, when Tom was in high school considering a career in biology, technology had progressed
even further. Scientists could now study the sequence of DNA inside mitochondria — the cellular
organelles that help power cells. Ensatina's mitochondrial DNA3 showed the same patterns as their
proteins: the northern salamander lineages had the greatest variety of sequences, suggesting that Ensatina
got its start in the north and moved south through California.
Biologists also used mitochondrial DNA sequences to try to figure out the family tree for all the salamander
subspecies. They found that the northern salamander lineages branched off near the base of that family tree
— suggesting that they are closely related to the ancestor of the ring. The forms near the eastern and
western endpoints of the ring formed distinct clans—just as we'd expect if they each evolved separately
from Ensatina's ancestor, as proposed by Robert.
Ensatina phylogeny based on mitochondrial DNA. Notice that oregonensis is composed of four separate evolutionary
lineages, which happen to be morphologically similar to one another. Similarly, platensis is made up of two distinct
lineages. In this case, Ensatina's DNA reveals distinct evolutionary histories that morphology alone did not.
Digging into DNA
When Tom became intrigued by Ensatina as a graduate student in 2004, he wasn't sure he'd be able to find
an unanswered question to work on: "I knew that people had worked on Ensatina for a long time and so I
wasn't sure that there was enough room or that there was much left to do with it — until I started talking to
people and reading more papers." He realized that there were still gaps in our knowledge and that
understanding Ensatina's basic story would allow him to ask deeper questions about their evolution.
First, though, Tom and his colleagues wanted to test Ensatina's basic evolutionary story against a new line
of evidence: the DNA inside the nucleus, which encodes most of an organism's traits. One might wonder,
why bother? After all, Robert Stebbins' original hypothesis had already been supported by studies of the
salamanders' morphology, proteins, and mitochondrial DNA. But scientists do their best to evaluate their
hypotheses against as many different lines of evidence as is possible and practical. These new lines of
evidence don't always overturn our ideas — but they do often help refine and add detail to existing
hypotheses.
Besides, in the late 1990s a graduate student at Berkeley had collected some additional mitochondrial DNA
sequences and had interpreted their history differently.4 Perhaps, he argued, the species got its start south
of San Francisco. Another line of evidence might help resolve the issue and clarify the relationships
between the subspecies.
Gene hunting
If Robert Stebbins' original hypothesis is right, sequence data from Ensatina's nuclear DNA should show
the same patterns as its mitochondrial DNA, with the most diversity in the north and big genetic differences
distinguishing the southernmost lineages. Furthermore, the evolutionary tree based on these DNA
sequences should be consistent with a northern origin and southward diversification. Tom followed in
Robert's footsteps, tromping up and down California to catch the salamanders, taking a tiny tissue sample
from the tip of the tail, bringing the samples back to his lab, and then extracting the DNA from them.
At left, Tom flips logs on Palomar Mountain, San Diego County, California, looking for Ensatina. At right, Tom working
in the lab.
But Ensatina's nuclear DNA was a hard nut to crack. No one had yet made a detailed study of it because of
its quirks. Ensatina has a huge genome — roughly six times bigger than ours! — and it's littered with
repetitive DNA — chunks of DNA that probably don't code for anything and that repeat the same, short
genetic sequence over and over again. All that makes their DNA extremely difficult to sequence.
Tom and his colleagues have been working on the project on and off for about four years so far. They have
sorted through hundreds and hundreds of short genetic sequences, hunting for ones with enough genetic
variation to figure out Ensatina's evolutionary history. Different parts of Ensatina's genome have different
levels of variation — some are remarkably uniform among all lineages, and some vary more from
population to population. This is because different parts of the genome evolve at different rates. Since
Ensatina has diversified into subspecies relatively recently, the parts of its genome that evolve more slowly
haven't had time to accumulate mutations that would carry information about how the lineages are related
to one another. On the other hand, the parts of its genome that evolve more quickly, have accumulated
more mutations and now vary among populations. These stretches of DNA can be used to figure out which
lineages are most and least closely related.
So far, the team has found ten stretches of DNA that contain useful clues to Ensatina's evolutionary history.
Tom says that what they are discovering supports the basic hypothesis Robert laid out: "The relationships,
for the most part, make sense in terms of being consistent with the morphology and the mitochondrial DNA
… but at this point, we need more data." The extra data will help them figure out exactly how all the
subspecies are related to one another and will hopefully paint a more detailed picture of how the
salamander populations colonized new areas in their move south.
Love in the hybrid zone
Though Tom continues hunting for telltale stretches of DNA that provide clues to Ensatina's evolutionary
history, he is most intrigued by a question about Ensatina's evolutionary future: Why doesn't Ensatina's
ring join up fully? In the 1960s, one of Robert Stebbins' graduate students, Charles W. Brown, discovered a
few locations in Southern California where the muted western form (eschscholtzii) and the blotchy eastern
form (klauberi) live together and actually do interbreed, producing blurrily blotched hybrids.5 It was this
observation that piqued Tom's interest. Why do the two forms interbreed in some places and not others, and
— since they do sometimes interbreed — what's keeping the two forms distinct? Why don't these two
subspecies blend into one another, as the forms around the rest of the ring do?
Tom considered many possible hypotheses for why eschscholtzii and klauberi don't interbreed more than
they do:
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3.
Perhaps they rarely recognize each other as potential mates. Many animals use particular clues to
help them determine who would make an appropriate mate. Those clues may come in the form of
a smell (e.g., a pheromone), a physical trait (e.g., a color pattern), or a behavior (e.g., a particular
mating call or dance). Maybe eschscholtzii and klauberi have evolved such that they are attracted
to different cues and so now avoid each other in the salamander singles scene.
Perhaps they are reproductively incompatible. The two subspecies might have no qualms about
mating with one another but rarely produce healthy offspring because of basic biological
differences that have evolved as the two lineages moved south.
Perhaps they rarely mate because they rarely meet. For example, the two might prefer different
habitats or have such different lifestyles that they rarely even run into one another — let alone get
together and mate.
And of course, some combination of these factors might come into play — but to figure out which, Tom
would need to collect some evidence …
Love in the lab
To examine his first hypothesis — the idea that eschscholtzii and klauberi have trouble recognizing each
other as potential mates — Tom performed an experiment. He brought wild salamanders into his field lab
and set up the equivalent of a salamander love nest: a damp, dark aquarium with places to hide — a set of
conditions designed to encourage the animals to mate. He tested all possible combinations of animals
(eschscholtzii couples, klauberi couples, eschscholtzii females with klauberi males, and klauberi females
with eschscholtzii males) to try to figure out if the separate subspecies were willing to mate with one
another — or more accurately, if the females had any qualms about the males.
For the mating experiment, Tom collected salamanders in the field and brought them back to the lab, where they were
housed in these plastic boxes (left). Tom then placed combinations of salamanders in a bank of aquaria, each separated by
black dividers to provide privacy (right). With lights off, infrared cameras filmed any mating activity.
In salamanders, the female gets the final say about whether or not a potential mate meets her standards, and
the male does his best to convince her that he's worthy. In Ensatina, this process is elaborate and lengthy.
An amorous male will approach the object of his affection and nudge her neck and head with his snout. If
she's interested, she will let him slide underneath her until she straddles his tail. Then the two slowly walk
together — often for hours! — until the male deposits a spermatophore (or sperm packet) on the ground. A
willing female will walk over the sperm packet and take it up into her body to fertilize her eggs.
Unfortunately, Tom's love nest may not have been quite romantic enough. Only a few of the salamanders
were willing to mate — even with another salamander of their own subspecies. Nevertheless, his
preliminary results are intriguing. Klauberi females weren't picky at all; they mated with males of their own
subspecies and eschscholtzii males. Eschscholtzii females, on the other hand, seemed to be choosier; they
rejected klauberi males. To be sure, Tom needs to work on his matchmaking skills and convince more
salamanders to mate in the lab. Nevertheless, his initial results suggest that eschscholtzii, at least, has
evolved such that the females no longer recognize klauberi as potential mates.
These results are also supported by genetic data. Nearly all the wild hybrids that Tom has found so far have
mitochondrial genes suggesting that they are the offspring of a klauberi female and an eschscholtzii male.
Waiting for data
Tom needs to collect more evidence to evaluate his other hypotheses. He knows that his second hypothesis
— the idea that eschscholtzii and klauberi are reproductively incompatible — is, at the very least, not
completely accurate. After all, he's found hybrids living in the wild, so the two subspecies can definitely
produce offspring together. However, almost all the hybrids he's studied so far have been from klauberi
females and eschscholtzii males. Why does the reverse pairing — an eschscholtzii female and a klauberi
male — seem to be so much more rare? That question is still unanswered.
Tom is using satellite images to study his third hypothesis — the idea that the two subspecies don't
interbreed in some places because they never actually meet in those locations. Two salamanders may live in
the same general area, but if their preferred habitats aren't intermixed in that area, they may hardly ever run
into one another. If this idea is accurate, we'd expect to observe more intermixing of habitat types in the
areas where the subspecies interbreed compared to the areas where they don't. Tom hopes that satellite
images — which often show different habitat types with different colors of vegetation — will reveal these
differences if they exist.
Tom's studies of Ensatina's hybrid zones are aimed squarely at understanding the process of speciation:
What differences have eschscholtzii and klauberi evolved that keep them distinct? But this has been a tough
question to answer. Says Tom, "It would be much easier to just collect the genetic data from the hybrid
zone, infer what might be going on, and leave it at that. But to me, the more interesting question is: What
are the processes that have led to this pattern? … Getting at the processes is the hard part, but it's also the
most interesting."
More answers, more questions
Using tools as simple as rulers and as complex as DNA sequencers, Tom and his colleagues have learned a
remarkable amount about Ensatina. We now have a fairly detailed picture of how the species moved
throughout California and Oregon, backed up by evidence from morphology, proteins, and DNA. We know
more about why the different subspecies have evolved the color patterns they did. And, with Tom's most
recent work, we are beginning to understand how all this squares with the most intriguing characteristic of
a ring species: the distinct endpoints of the ring (eschscholtzii and klauberi), which don't blend together
even though the two groups are connected by continuous variation throughout the rest of the ring. Over the
past 60 years of investigations, Robert Stebbins' initial hypothesis about Ensatina's evolutionary history has
grown into a much more detailed understanding of the animal. But there's still much to learn.
At left, Tom does some night collecting. At right, Tom and colleagues in the Sierra San Pedro Martir, Baja California, in
2006 collecting Ensatina. From the left are Tom, Angelo Soto-Centeno, Brad Hollingsworth, Clark Mahrdt, and Jorge
Valdez-Villavicencio.
Five years ago, Tom wondered if there was anything we didn't know about Ensatina. Now he's impressed
by the magnitude of what we still don't understand about these creatures. How long did it take them to
diversify into the different subspecies? What other salamanders are they most closely related to? Have the
differently colored subspecies evolved different strategies to avoid predators? What makes eschscholtzii
females reject klauberi males? Why do they interbreed in some places but not others? All these questions,
and many more, remain to be answered. Says Tom, "As you get new tools and new techniques, you're
always going to be able to ask new questions and learn new things. I don't think that we're ever going to get
to a point where Ensatina is 'done.'"
Discussion and extension questions:
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In your own words, describe what a ring species is.
This article described the Ensatina ring species. Research another possible example of a ring
species and explain why you think it might constitute a ring species.
What different lines of evidence support the idea that Ensatina is a ring species? List at least three
and explain how each line of evidence supports the idea.
As Ensatina spread into new habitats it evolved. Explain how evolutionary changes in one of the
following aspects of salamander biology might contribute to speciation: mating rituals, preferred
habitat type, or reproductive physiology.
What question about Ensatina's evolution would you most like to have answered? What sort of
evidence might help you answer that question?