The Devil's Hole pupfish, as the name suggests, lives in a truly hellish environment.
263 of them are confined to a single deep limestone cave in Nevada's Mojave Desert, where the water temperature hovers around 93 degrees Fahrenheit year-round, food resources are scarce, and oxygen levels are so low that most other fish would die immediately. The pupfish, Cyprinodon diabolis, has the smallest habitat of any vertebrate known.
New research now shows the extreme impact that these harsh and isolated conditions have had on the genetic diversity of this fish.
Biologists report the first complete genome sequences of eight pupfish species from the American Southwest—30 individuals in total, including eight Devils Hole pupfish—in a paper published this week in the journal Proceedings of the Royal Society B, University of California, Berkeley. Surprisingly, the Devil's Hole pupfish is so inbred that, on average, 58% of the genomes of these eight individuals are identical.
"High levels of inbreeding are associated with a higher risk of extinction," said lead researcher Christopher Martin, UC Berkeley associate professor of integrative biology and curator of ichthyology in the campus's Museum of Vertebrate Zoology. "Although we were unable to directly measure fitness, we believe that increased inbreeding in these pupfish results in a significant reduction in fitness."
The researchers discovered that other pupfish species are also inbred, but only 10% to 30% of their genomes are identical.
According to the study's lead author, graduate student David Tian, the level of inbreeding in Devils Hole pupfish is equivalent to what would happen if four to five generations of siblings mated with one another. This tends to burn in or fix harmful mutations rather than weed them out, potentially dooming a population to extinction through mutational meltdown. The Devils Hole pupfish species is currently doing well in the wild and in captive or "refuge" populations, but the species' low genetic diversity may pose a problem as climate change and human impacts become more severe.
In the face of these potential threats, scientists and conservationists will be able to assess the health of native pupfish populations and potentially intervene in refuge populations to increase the genetic diversity of these species—particularly the Devils Hole pupfish.
"There's a lot of potential with this new genomic data to look not just at genetic diversity and how these species are related to each other phylogenetically, but also at inbreeding and mutation load to get an idea of what their current status is, how evolutionary history may have influenced their current genetic variation, and think about where the population is going and what we should do, if anything, to preserve these species," Tian said.
Population loss and rescue
Pupfish species are found all over the world and prefer isolated lakes and springs, often in harsh conditions that would kill most fish. In California and Nevada, about 30 species live in warm, salty desert springs and streams. Martin has studied various pupfish populations, including several on the Bahamas' San Salvador Island, to better understand the genetics of their adaptation to extreme conditions and unusual ecological niches.
The Devils Hole pupfish, on the other hand, is unique in its small range and perilous existence, according to Martin, making conservationists concerned about its fluctuating population in the wild.
"Part of the question about these declines is whether they are due to population genetic health," Martin said. "Perhaps the declines are due to harmful mutations that have become fixed due to the population's small size."
Martin believes that the small population is due in part to human incursions into their habitat. Local ranchers and developers pumped groundwater in the region in the 1960s and 1970s, drastically lowering the water level in Devils Hole and causing a population drop. Devils Hole and the resident population were saved by a 1976 Supreme Court ruling that allowed the federal government to limit groundwater pumping, while captive breeding at a nearby 100,000-gallon pool in the Ash Meadows National Wildlife Refuge saved the species. Nonetheless, a decline in the 1990s reduced the wild population to 35 individuals in 2013. Since then, the wild population has recovered, while the refuge population has grown to around 400, more than double the wild population.
However, humans are not entirely to blame for the Devils Hole pupfish's lack of genetic diversity. The UC Berkeley team also sequenced the genome of a pupfish that was collected in 1980 and is now housed at the University of Michigan. It exhibited inbreeding and a lack of genetic diversity similar to that found in recently collected individuals, the majority of whom died naturally. This implies that the pupfish has likely experienced frequent population bottlenecks over hundreds, if not thousands, of years.
Martin and Tian discovered that 15 genes have vanished entirely from the Devils Hole pupfish genome as a result of this. Five of them appear to be involved in the adaptation to low-oxygen or hypoxic environments.
"These deletions are perplexing because this is a habitat where you're most vulnerable to hypoxia," Martin explained. "It could have something to do with the habitat's long-term stability." However, it appears that the hypoxia pathway has been disrupted. It doesn't really matter if you break another gene in that regulatory pathway once you've broken one. Our next step will be to investigate what these deletions do. Do they improve hypoxia tolerance? Do they reduce hypoxia tolerance? At this point, I believe both scenarios are equally plausible."
He believes that selective breeding within a captive population of Devils Hole pupfish could help increase diversity and possibly save the species from extinction. CRISPR genome editing could also be used to restore genes that have already been lost.
The fact that the genome of the fish collected in 1980 was about as inbred as today's fish is "maybe good news," according to Martin, because the population has historically been highly inbred with very low genetic diversity, implying that the recent decline in the '90s, with population bottlenecks to only 35 fish in 2013 and 38 fish in 2007, didn't seem to have had much of an effect.
Tian is currently analyzing approximately 150 complete genome sequences of nine species of American pupfish to gain a better understanding of the deleterious mutations and gene deletions found in the various Southwestern populations. The study, he believes, exemplifies what conservation genomics can do for endangered and possibly inbred populations around the world.
"We're on a really cool cusp when it comes to using genomic data and applying it to conservation," he said, "especially at a time when it's a problem that's likely only going to get worse with climate change, increased habitat fragmentation, and just anthropogenic changes."
Tian, on the other hand, is wary of genetic interventions because little is known about how genes influence a species' physical and behavioral characteristics, and how this relates to fitness and adaptation to a specific environment. Conservation should remain a top priority.
"The answer remains increased funding for these populations, habitat protection, legal avenues for protecting these species, and figuring out how humans and these endangered species can coexist on this planet," he said.
Austin Patton of UC Berkeley and Bruce Turner of Virginia Tech in Blacksburg are co-authors with Martin and Tian.
Journal information: Proceedings of the Royal Society B