A salamander’s dangerous liaisons

Amphibians aren’t doing well. Worldwide, almost two out of five of the species that scientists have been able to assess are struggling. In the US, the same is true for more than 50 of its 270-plus native species. But the causes are far from self-evident, as many amphibians are difficult to spot, let alone follow around and study. So how to figure out what ails them?

In the Annual Review of Animal Biosciences, conservation geneticists argue that new, affordable ways to read the creatures’ genomes might revolutionize our understanding. Knowable spoke with review coauthor Evan McCartney-Melstad, a postdoc at UCLA, who studies the vulnerable California tiger salamander. This conversation has been edited for length and clarity.

I can imagine it is quite a challenge to cheek-swab a salamander. How do you get their DNA?

We catch the larvae, which live in temporary ponds, and snip a small piece of their tail — we’ve done experiments to show that they survive really well after we do that. We bring those pieces to the lab and keep them in the freezer, stored in ethanol. Then, when we’re ready to use them, we break down the cells, separate the DNA from the other parts, and put it through a DNA sequencer.

Very few amphibian genomes have been completely sequenced so far. Why would that be?

They’re difficult and expensive, because they’re big. Many frog species’ genomes are about three times larger than a human genome, and salamanders’ genomes can be ten to forty times larger. They have a very similar number of genes, but amphibians have accumulated a lot of repetitive DNA that they don’t seem to be able to get rid of. Assembling the genome of the axolotl [a Mexican salamander] has cost millions of dollars.

But once we have such a reference genome — a detailed, representative example — we can use it to map other salamanders’ genomes for just a couple hundred dollars. In practice, though, most people aren’t sequencing entire salamander genomes, since we are most interested in the parts that are relatively unique in a population or species. We can compare those and use them to reconstruct a family tree: the relationships between different species or individuals.

What can we learn that may benefit amphibian conservation?

First of all, genome analysis can give us an idea of the size of the population. This is a very important value to predict the chance of survival of threatened species, and one that is essentially impossible to get directly for many amphibians. The adults of the California tiger salamander I study, for example, live underground in old ground squirrel burrows and are almost never seen.

Patterns of genetic variation look different in small populations than in large ones. That’s because small populations lose genetic variation faster than large ones — if a gene variant is only present in one or a few adults, which is more commonly the case in smaller populations, the chance is higher that none of the offspring in the next generation will inherit it. So if we can measure the genetic variation in a population, and divide this by the rate at which genetic mutations create new variants, we get an estimate of the population size, or at least of the amount of animals that are reproducing.

Comparing genomes can also help us infer how animals move across the landscape, and whether human barriers like roads or fields may lead to less exchange of migrants between areas. If you find that individuals are more genetically similar to each other on one side of a barrier than they are to individuals on the other side, you can infer that the barrier prevents them from moving across.

This has led to some unexpected insights. For example, though California tiger salamanders are usually found in grasslands, it turns out that the dry, shrubby chaparral vegetation where they’re rarely if ever seen does not form a barrier. In fact, it appears to be their preferred vegetation type for travel.

This is important, because one of the goals in conservation is to increase “gene flow” — the exchange of genes between different populations to maintain their genetic diversity and evolutionary potential — by removing barriers and protecting the corridors that allow animals to move around. This may be particularly important for animals like salamanders that aren’t able to travel large distances.

In eastern tiger salamanders that live on Long Island in New York, for example, we recently found that roads and traffic patterns have profoundly reduced the amount of movement between certain ponds. This makes the populations around these ponds much more sensitive to local extinction. In California, we’re similarly working with the state’s Department of Transportation to find out how roads are affecting the movement of both native and nonnative salamander genes on the landscape.

Is it a good idea to help species cross barriers that we cannot remove?

It depends. If, for instance, a species is extinct from part of its former range — the foothill yellow-legged frog in California is a good example of this — and they’re not going to get there by themselves, should we reintroduce the species? Genetics can really help with that decision. We’d like to introduce animals from other populations that are a lot like the ones that were there before. That is important for conservation, because these populations may have been in the same spot for millions of years and are adapted to the local conditions. So we should understand how populations are related to one another before we start moving species around.

Some animals that people have released outside their home range are causing a lot of trouble. The California tiger salamander, for example, is at risk due to the arrival of another species, the barred tiger salamander, from Texas.

How does a salamander from Texas end up in California?

The larvae of the tiger salamander are sometimes called “water dogs,” and they are popular fishing bait. California tiger salamander larvae come out of the pond earlier in the year than those of the barred tiger salamander, which stay in the larval form longer, and grow slightly bigger. This apparently makes them better fishing bait.

Sometime around 1950, a California fishing-bait dealer drove to Texas and picked up a bunch of these salamanders to improve his business. Some of them were released; anglers will often throw their bait in the pond if they have not used it. This happened in sufficient numbers for the released salamanders to interbreed with the Californian species, which has resulted in a big population of hybrids that now appear to be outcompeting the native ones.

That’s quite surprising, as hybrids are often infertile or unhealthy.

It is indeed unusual that the hybrids of two species that have been evolving separately for millions of years will be so successful, because some of their genes may be incompatible. Our best explanation for this, currently, is that fatal incompatibilities are apparently rare in this case. Secondly, mixing these two different species means that the negative impacts of bad mutations within either parental lineage sometimes get canceled out, while some of the good, new functions that have evolved in those lineages are maintained. This may be because in the hybrids, the two different copies of each gene that all animals have are often more dissimilar to each other than those of salamanders with two parents of the same species. This way, the genes’ effects might add up, or one might compensate if the other doesn't work well.

In a way, it’s a match made in heaven, then — a struggling salamander species has apparently been saved by the accidental introduction of another one. So why…?

Why is that wrong? [Laughs.] Well, the California tiger salamander had been doing OK for millions of years, and we can’t know what the situation would be now if the hybridization had not happened. It could very well be that there would be just as many native individuals as there are of the hybrids now.

But what we do know is that the genes of the native ones are being displaced by the genes of the invasive ones.I think it’s a shame that a species that has been evolving in California for so long is changing because of something we know humans did.

Maybe sometimes hybridization can be a good thing, when the genetic health of a population is really poor. But before this happened, there were no indications that this was the case for the California tiger salamander. Scientists weren’t observing birth defects or other abnormalities.

There seems to be disagreement between some conservationists who would like to subdivide species as much as possible, and find interesting varieties everywhere, and others, often evolutionary biologists, who warn that if you isolate populations, you put them at risk. Your thoughts?

Right. I think we really need to look at this on a case-by-case basis. In this particular case, there is no question that the California species is very different from the Texan species and other salamanders. There is no debate about that, and the genetic evidence is reinforcing this view. This also has legal implications, by the way.  When a population is considered a separate species or subspecies, that affects its protection status.

Is there anything we can do to stop those hybrids from spreading?

California tiger salamander larvae live in vernal pools that fill up in winter with the rains and dry down to nothing in April. Once they are big enough, they metamorphose into their adult forms, then crawl out and go underground.

Some hybrids follow a different strategy, however. Cattle ranchers often keep ponds filled with water all year. In those ponds, some hybrids may stay in their larval form all year. Robert Cooper, a graduate student in our lab, is studying whether drying these ponds down at a certain time of year could tip the balance back in favor of the California tiger salamanders.

Is there a wider lesson to be learned from this?

An important lesson is that one should never release live bait or pets into the wild. You may think you’re doing a favor by releasing the animal alive, but you might be doing a great deal of harm, causing the death of thousands of wild animals. And not just through hybridization: In Europe, a deadly fungus, Batrachochytrium salamandrivorans, or Bsal, introduced through the release of Asian salamanders from the pet trade, has caused the death of entire salamander populations.

Think of it: People who like these animals so much they want to keep them as a pet could make them go extinct in the wild. Fortunately, it is now prohibited to import many salamanders into the US. If you ask me, responsible pet owners should really hold off on buying salamanders until we better understand the risks.

Can genome analysis help us with that as well?

Certainly. Understanding why some species are susceptible to diseases like Bsal and why others are resistant is a key research goal. Bsal was only discovered five years ago, and so intensive research efforts are very young for this disease. There's a lot more known about the closely related Batrachochytrium dendrobatidis, Bd, which has decimated many frog populations across the world.

Certain genes  — most famously those in the major histocompatibility complex, MHC — govern an organism's ability to discern invaders like pathogenic fungi. There is evidence from both the laboratory and wild populations that certain mutations in MHC genes can confer resistance to Bd, leading to survival in the wild for the populations that have these mutations.

This could very well be the case for salamanders and Bsal resistance as well, but as far as I know this remains to be seen.