UNIVERSITY PARK, Pa. — The genome of a once-endangered songbird shows extensive signs of inbreeding, according to a new study by Penn State researchers. Because inbreeding can negatively impact survival and reproduction, the results could guide continuing conservation efforts for Kirtland’s warblers, whose populations still rely on extensive management. The new study, published this week (Dec. 9) in the journal Evolutionary Applications, also suggests how genetic information about inbreeding could be used when considering the removal of species from the endangered species list.

Kirtland’s warblers have also been known as jack pine warblers, as their breeding habitat is limited to young jack-pine forests in Michigan. The species nearly went extinct in the 1970s due to threatened habitat and the population decline was exacerbated by brown-headed cowbirds, who lay their eggs in the nests of other bird species — including warblers — and divert resources away from their chicks. Kirtland’s warblers were classified as endangered under the Endangered Species Act of 1973. Thanks to intensive, targeted management over several decades, Kirtland’s warbler populations have recovered. The species was “delisted” — no longer considered endangered — in 2019, though management efforts remain critical to their survival, according to the researchers.

“When any population goes through a period when few breeding individuals are present, called a ‘bottleneck,’ there is always a chance of inbreeding, which can lead to reduced survival of future generations,” said Anna María Calderón, graduate student in biology in the Penn State Eberly College of Science and first author of the paper. “The 2019 decision to delist Kirtland’s warblers referenced a study that did not find strong evidence of inbreeding, but the tests available at the time were not very high resolution. We used updated sequencing technology to get a clearer picture of the genetic diversity and potential for inbreeding among these songbirds.”

Like other animals, birds receive one copy of their genome from each parent. Some genetic variants can have negative impacts on an animal’s survival or reproductive success. The likelihood of receiving the same potentially damaging variant from both parents increases with inbreeding, when genetically similar individuals mate.

Previous tests of genetic diversity — such as those referenced in the delisting decision — often focus on a small set of specific markers on the genome called microsatellites, which only provide part of the picture. However, advancements in sequencing technology now allow inexpensive analysis of the entire genome from multiple individuals. 

“If you think of the genome as a movie of the blueprint of life, then using microsatellites is like trying to get the plot from half a dozen snapshots, while sequencing the entire genome is like watching an entire 4K-resolution film,” said David Toews, Louis Martarano Career Development Professor of Biology at Penn State and co-leader of the research team. “There’s so much more information to be obtained from sequencing the entire genome, and modern technology has made that analysis possible.”

The researchers sequenced the whole genome of Kirtland’s warblers and, as a point of comparison, they also sequenced whole genomes of two closely related species whose populations have remained large and stable, Hooded warblers and American redstarts. The team specifically looked at a measure of inbreeding called “runs of homozygosity.” 

A given spot on the genome is called homozygous when the genetic information obtained from an animal’s maternal parent is the same as the information obtained from its paternal parent. Runs of homozygosity (ROH) are long, connected stretches of homozygosity, sometimes millions of letters of the genetic alphabet in length. Particularly long ROH indicate that the individuals who carry them are products of recent inbreeding, when genetically similar individuals mated and were thus more likely to pass on the same genetic information. Fewer and shorter stretches indicate that an individual’s parents are more distantly related.

“Runs of homozygosity give us a unique look into the past and can be an indicator of the genomic health of the population,” said Zachary Szpiech, assistant professor of biology at Penn State and the other co-leader of the research team. “For example, long ROH have been associated with how well other species survive in their first year. ROH can also flag potentially damaging genetic variants, which may be useful when considering the conservation of the bird.”

The researchers found exceptionally long ROH in Kirtland’s warblers, indicating very recent inbreeding that had not previously been identified in this species. Additionally, they found many small to medium-sized ROH in Kirtland’s warblers, while the two closely related species had almost none.

“The contrast between Kirtland’s warblers and their closest related species, which haven’t undergone any population bottlenecks, couldn’t be more stark,” Toews said. “We found no evidence of inbreeding whatsoever in Hooded warblers or American redstarts, and in comparison, the level of inbreeding for the Kirtland’s warblers is almost off the charts. We also found a high frequency of potentially damaging genetic variants. This gives us a clear picture of how the demographic history of songbirds can shape their genetic diversity.”

The researchers noted that the Kirtland’s warblers they sampled did not appear to have any physical deformities. However, they said that inbreeding could manifest during the stages of life they did not observe, for example during development or the energy-intensive task of migration, or impact aspects of their reproductive success, such as how many eggs they lay or how many hatch. According to Toews, continued monitoring of these birds to clarify the impacts of inbreeding will be critical to future conservation efforts.

“An open question in this species has been whether the population of Kirtland’s warblers has always been small, or if it was once large and crashed,” Calderón said. “We can actually use ROH to look into the past, using rates of recombination — one way genomes can swap information around — to estimate when certain combinations of genetic information appeared. From this, we can see that most of the DNA segments within the short ROH in these birds originated between 1874 and 1954, mostly before known bottlenecks. This suggests that Kirtland’s warblers may have always had small population sizes. This makes sense given the high specificity of these birds to young jack-pine forests.”

Comparing these results with information about the distribution and abundance of the species, which has been collected since the 1940s, can provide insight into the events that shaped the genomes of these birds. For example, the researchers dated the origin of many DNA segments within long ROH between 1940 to 1981. This is consistent with a sudden population collapse beginning in the early 1940s, as this bottleneck would result in more, longer ROH. The researchers also plan to sequence the genomes of museum samples, including birds collected as early as the late 1800s, to provide additional context around genetically important events. 

“Although Kirtland’s warblers are celebrated as a conservation success story, they are highly inbred and have a high frequency of potentially damaging genetic variants that could impact their ability to persist,” Calderón said. “Our work serves as an important example of how measures of genetic health may tell a different story than simple population numbers. Both need to be considered when assessing a population’s recovery and when making conservation decisions.”

In addition to Calderón, Toews and Szpiech, the research team also includes Andrew Wood, a research technologist in Toews’ lab at the time of the research and now a postdoctoral researcher at the University of Minnesota.

Funding from the U.S. National Science Foundation Division of Environmental Biology, the Penn State Huck Institutes of the Life Sciences and the Penn State Eberly College of Science supported this research.