How to handle the burden of deleterious mutations

Alpine ibex.

With the increasingly pressing matter of populations being threatened by fragmentation and isolation, and with progressively more efficient sequencing technologies and analytical tools at hand, conservation genetics is starting to turn the spotlight on the topic of genetic load. It has become clear that population survival is not only about population sizes and estimates of genetic diversity. Some populations thrive despite extremely low genome-wide diversity, while others go extinct despite seemingly much better prospects.

The buzzword that keeps coming up in recent publications, is purging. Purging is defined as the “increased purifying selection facilitated by inbreeding as it increases the homozygosity of partially recessive deleterious variants” (Hedrick and Garcia-Dorado 2016). In other words, in small populations, where the inbreeding usually increases homozygosity, more of the really nasty stuff [aka highly deleterious mutations] is revealed in homozygous state, which makes it easier for purifying selection to act upon.

The problem is that studies examining bottlenecked natural populations are not conclusive on how much mutational load accumulates in small populations and how much is purged by selection. Remember that in small populations, selection is assumed to be overshadowed by the effects of genetic drift, therefore it is not clear how much power it has for eliminating deleterious mutations.

While some studies found empirical evidence of accumulation of genetic load in a population with low effective population size, for instance in the woolly mammoth (Rogers and Slatkin 2017) and the crested ibis (Feng et al. 2019), others showed that purging of highly deleterious mutations does take place in populations of extremely bottlenecked species, for example in the Channel Island foxes (Robinson et al. 2018) and mountain gorillas (Xue et al. 2015).

The new study by Christine Grossen and colleagues (Grossen et al. 2020) looked at the problem in a beautiful model system of the once near-extinct Alpine ibex. Back at the beginning of the 19th century, there were less than 100 individuals left in a single population in Gran Paradiso, Italy. After recolonizations, the Alpine ibex is now at a census size of 50,000 individuals. Interestingly, the successful recolonized populations were used to found other populations, and thus, the extant populations experienced two to four bottlenecks, which are also well documented in the records.

Grossen et al. analysed 60 high-coverage genomes of seven species, the domestic goat and six wild goat species. The wild species cover a range of population sizes (from 200,000 in the Siberian ibex to 2,500 in the Nubian ibex), red-list categories (from Least Concern after near extinction, to Vulnerable), and also a range of demographic histories. Put together, the Alpine ibex wins the overall competition for being the most bottlenecked (down to 100 individuals), having the least nucleotide diversity and having a considerable part of the genome in runs of homozygosity (ROH).

The six species of wild ibex analyzed in the study. (Grossen et al. 2020, Fig. 1a)
Phylogenetic tree (b), nucleotide diversity (c), ROH (d), mutational load (e). (Grossen et al. 2020, Fig1b-e)

So let’s focus on the Alpine ibex from now on. Grossen et al. used demographic records to estimate long-term effective population sizes and compared these to the estimates of nucleotide diversity, concluding that nucleotide diversity decreased with smaller long-term population size. The same goes for heterozygosity, while the inbreeding (from ROH) showed inverse pattern.

Next, the authors estimated high-confidence deleterious mutations based on a) GERP analysis of conserved regions, b) transciptomic analysis and genes missing evidence of transcription, and for some analyses also c) functional annotation of each variant in snpEff. Grossen et al. used a whole battery of genetic load tests, starting with the proportion of segregating, highly deleterious mutations. This was inversely correlated with nucleotide diversity, and thus, most pronounced in the most bottlenecked populations – the Alpine ibex, Iberian ibex, and Markhor.

Site frequency spectra (SFS) analyses were used to look for evidence of purging selection.

“Both Alpine and Iberian ibex experienced severe bottlenecks due to overhunting and habitat fragmentation. We first analyzed evidence for purifying selection using allele frequency spectra. We focused only on derived sites that were polymorphic in at least one of the two sister species. … We found that frequency distributions of high and moderate impact mutations in Alpine ibex were downwards shifted compared to modifier (i.e. neutral) mutations, which strongly suggests purifying selection against highly deleterious mutations. … We found no comparable frequency shifts in Iberian ibex (Fig. 2b). This is consistent with purifying selection acting more efficiently against highly deleterious mutations in Alpine ibex compared to Iberian ibex.” (Grossen et al. 2020)

They also calculated the relative number of derived alleles Rxy, comparing the Alpine ibex to the Iberian ibex across the spectrum of different mutation impact categories, using a set of intergenic SNPs for standardization. It turns out that the Alpine ibex, compared to the Iberian ibex has a minor excess of low to moderate impact mutations; however, it has a strong downward allele frequency shift in the highly deleterious mutation category. Together with the observation of a lower individual allele count at highly deleterious sites and lower number of homozygous sites with highly deleterious mutations, this provides a good evidence of purging in the Alpine ibex.

Sampling locations for the Iberian and Alpine ibex (a), SFS of the Alpine ibex (b), SFS of the Iberian ibex (c), Alpine’s ibex SFS downsampled to the iberian ibex’ sample size (d). (Grossen et al. 2020, Fig2a-d)

Zooming in, the authors looked at the genetic load across populations, focusing on whether the genetic load increases with the number of bottlenecks that the population went through.

“Bottlenecks affect the landscape of deleterious mutations by randomly increasing or decreasing allele frequencies at individual loci. We find that individuals from populations that underwent stronger bottlenecks carry significantly more homozygotes for nearly neutral and mildly deleterious mutations (i.e. modifier, low and moderate impact mutations; Fig. 4a). In contrast, individuals showed no meaningful difference in the number of homozygotes for highly deleterious (i.e. high impact) mutations across populations. The stability in the number of homozygotes for highly deleterious mutations through successive bottlenecks despite a step-wise increase in the number of homozygotes for weaker impact mutations, supports that purging occurred over the course of the Alpine ibex reintroductions.” (Grossen et al. 2020)

The recolonization history of the Alpine ibex (a), nucleotide diversity (d). (Grossen et al. 2020, Fig.3)
Homozygote counts per individual for each population. (Grossen et al. 2020, Fig. 4a)

I could continue, but I guess that you get the picture. The take-home message is that mutational load accumulates in bottlenecked populations through mildly deleterious mutations, while highly deleterious mutations can be purged under extreme bottlenecks.

I really liked how the authors formulated the conclusions, so I will leave the last words to them:

“Our empirical results are also in line with predictions that populations with an effective size below 100 individuals can accumulate a substantial burden of mildly deleterious mutations. Such mutation load constitutes long-term extinction risks in contrast to short-term risks associated with highly deleterious mutations. The burden of deleterious mutations evident in Iberian ibex supports the notion that even population sizes of ~1000 still accumulate mildly deleterious mutations. High loads of deleterious mutations have been shown to increase the extinction risk of a species. Thus, conservation efforts aimed at keeping effective population sizes above a minimum of 1000 individuals are critical for the long-term survival of managed species.” (Grossen et al. 2020)

P.S.: This paper has truly beautiful figures. I had to restrain myself from using them all, so go check out the paper.


Grossen, Christine, Frédéric Guillaume, Lukas F. Keller, and Daniel Croll. 2020. “Purging of Highly Deleterious Mutations through Severe Bottlenecks in Alpine Ibex.” Nature Communications.

Feng, Shaohong, Qi Fang, Ross Barnett, Cai Li, Sojung Han, Martin Kuhlwilm, Long Zhou, et al. 2019. “The Genomic Footprints of the Fall and Recovery of the Crested Ibis.” Current Biology.

Hedrick, Philip W., and Aurora Garcia-Dorado. 2016. “Understanding Inbreeding Depression, Purging, and Genetic Rescue.” Trends in Ecology and Evolution.

Robinson, Jacqueline A., Caitlin Brown, Bernard Y. Kim, Kirk E. Lohmueller, and Robert K. Wayne. 2018. “Purging of Strongly Deleterious Mutations Explains Long-Term Persistence and Absence of Inbreeding Depression in Island Foxes.” Current Biology.

Rogers, Rebekah L., and Montgomery Slatkin. 2017. “Excess of Genomic Defects in a Woolly Mammoth on Wrangel Island.” PLoS Genetics.

Valk, Tom van der, David Díez-del-Molino, Tomas Marques-Bonet, Katerina Guschanski, and Love Dalén. 2019. “Historical Genomes Reveal the Genomic Consequences of Recent Population Decline in Eastern Gorillas.” Current Biology.

Xue, Yali, Javier Prado-Martinez, Peter H Sudmant, Vagheesh Narasimhan, Qasim Ayub, Michal Szpak, Peter Frandsen, Yuan Chen, Bryndis Yngvadottir, and David N Cooper. 2015. “Mountain Gorilla Genomes Reveal the Impact of Long-Term Population Decline and Inbreeding.” Science 348 (6231): 242–45.

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