No temporal changes in genomic parameters were observed in the mountain gorilla

Many endangered species have experienced severe population declines within the last centuries []. However, despite concerns about negative fitness effects resulting from increased genetic drift and inbreeding, there is a lack of empirical data on genomic changes in conjunction with such declines []. Here, we use whole genomes recovered from century-old historical museum specimens to quantify the genomic consequences of small population size in the critically endangered Grauer’s and endangered mountain gorillas. We find a reduction of genetic diversity and increase in inbreeding and genetic load in the Grauer’s gorilla, which experienced severe population declines in recent decades. In contrast, the small but relatively stable mountain gorilla population has experienced little genomic change during the last century. These results suggest that species histories as well as the rate of demographic change may influence how population declines affect genome diversity.

The biodiversity of species and their rates of extinction, distribution, and protection.

Results and Discussion

1 Waters C.N.

Zalasiewicz J.

Summerhayes C.

Barnosky A.D.

Poirier C.

Gałuszka A.

Cearreta A.

Edgeworth M.

Ellis E.C.

Ellis M.

et al. The Anthropocene is functionally and stratigraphically distinct from the Holocene. 2 Pimm S.L.

Jenkins C.N.

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Sexton J.O. The biodiversity of species and their rates of extinction, distribution, and protection. 3 Lande R. Risks of Population Extinction from Demographic and Environmental Stochasticity and Random Catastrophes. 4 Reed D.H.

Frankham R. Correlation between fitness and genetic diversity. 8 Smith K.F.

Sax D.F.

Lafferty K.D. Evidence for the role of infectious disease in species extinction and endangerment. 5 Lande R.

Shannon S. The Role of Genetic Variation in Adaptation and Population Persistence in a Changing Environment. 6 Keller L.F.

Waller D.M. Inbreeding effects in wild populations. 7 Lande R. Genetics and demography in biological conservation. 9 Díez-del-Molino D.

Sánchez-Barreiro F.

Barnes I.

Gilbert M.T.P.

Dalén L. Quantifying Temporal Genomic Erosion in Endangered Species. In the last centuries, continuously increasing anthropogenic pressures have dramatically accelerated wild animal population losses, especially affecting large-bodied mammals []. These population declines can have severe consequences for long-term species survival due to environmental and demographic stochastic events [], the negative effects of reduced genetic diversity on fertility [], resistance to infectious diseases [], and adaptability to changing environments []. Moreover, natural selection is thought to be less efficient in small populations, which may increase the probability that deleterious alleles drift to fixation, further lowering the fitness of the population []. Maintaining genetic diversity is thus of considerable importance for species conservation []. However, the genomic consequences of rapid population decline, as faced by many species today, remain largely unexplored due to limited empirical data [].

10 IUCN (2017). The IUCN Red List of Threatened Species. Version 2017.3. 11 Prado-Martinez J.

Sudmant P.H.

Kidd J.M.

Li H.

Kelley J.L.

Lorente-Galdos B.

Veeramah K.R.

Woerner A.E.

O’Connor T.D.

Santpere G.

et al. Great ape genetic diversity and population history. 12 Xue Y.

Prado-Martinez J.

Sudmant P.H.

Narasimhan V.

Ayub Q.

Szpak M.

Frandsen P.

Chen Y.

Yngvadottir B.

Cooper D.N.

et al. Mountain gorilla genomes reveal the impact of long-term population decline and inbreeding. 13 Gordon D.

Huddleston J.

Chaisson M.J.P.

Hill C.M.

Kronenberg Z.N.

Munson K.M.

Malig M.

Raja A.

Fiddes I.

Hillier L.W.

et al. Long-read sequence assembly of the gorilla genome. 11 Prado-Martinez J.

Sudmant P.H.

Kidd J.M.

Li H.

Kelley J.L.

Lorente-Galdos B.

Veeramah K.R.

Woerner A.E.

O’Connor T.D.

Santpere G.

et al. Great ape genetic diversity and population history. Figure 1 Geography and Genetic Structure Show full caption (A) Sampling location of historical and previously published modern samples. Two modern Grauer’s gorilla individuals are not shown on the map due to uncertainty about their exact geographic origin. † depicts samples from extinct Grauer’s gorilla populations. (B) Principal-component analysis of eastern gorilla genomes. See also Figure S2 In this study, we directly quantify genomic changes within the last century in the two eastern gorillas: the critically endangered Grauer’s (Gorilla beringei graueri) and the endangered mountain gorillas (Gorilla beringei beringei) [] by sequencing whole genomes from historical specimens collected up to ca. 100 years ago and comparing them to present-day (modern) genomes [] ( Figure 1 A). After low-depth sequencing of 59 historical gorilla specimens (see Data S1 ), we selected samples from unrelated adult individuals displaying high endogenous DNA content and DNA quantity for uracil-DNA glycosylase (UDG) treatment and subsequent deep genome sequencing. Following whole-genome sequencing, we collapsed the obtained forward and reverse high-quality sequencing reads generated on the Illumina HiSeq X platform and aligned them to the gorilla reference genome []. For seven Grauer’s and four mountain gorilla samples collected between 1910 and 1962 (median: 1923), we obtained adequate coverage (3.1–10.8 X) to infer genomic changes through time (see Data S1 ). We also included published modern genomes from eight Grauer’s, seven mountain, and 17 western lowland gorillas (Gorilla gorilla gorilla) (see Data S1 ) [], the last to be used as a comparative dataset.

14 Sawyer S.

Krause J.

Guschanski K.

Savolainen V.

Pääbo S. Temporal patterns of nucleotide misincorporations and DNA fragmentation in ancient DNA. To study changes in genetic diversity over time, we called genotype likelihoods and variants across all historical and modern samples. We filtered low-quality sites, recalibrated quality scores accounting for post-mortem DNA damage, and removed cytosine-phosphate-guanine (CpG) sites to limit possible biases from remaining DNA damage ( Figures S1 A and S1B) [].

16 Kardos M.

Åkesson M.

Fountain T.

Flagstad Ø.

Liberg O.

Olason P.

Sand H.

Wabakken P.

Wikenros C.

Ellegren H. Genomic consequences of intensive inbreeding in an isolated wolf population. Next, we inferred the effects of the recent population decline on inbreeding levels by identifying tracts of within-individual chromosomal sequence sharing (runs of homozygosity [ROH]) using a Hidden Markov Model. Whereas an average of 35.1% and 33.9% of the historical Grauer’s and mountain gorilla genomes, respectively, consisted of ROH above 100 kb, this proportion increased to 39.2% in modern Grauer’s and to 36.3% in modern mountain gorillas ( Figures 2 B and S3 ). This change is driven by the proportion of the genome contained in very long ROH (2.5–10 Mb, Figures 2 B–2C and S3 ), which increased by 24% in modern Grauer’s gorilla genomes ( Figures 2 B and 2C). Such long tracts are likely to arise from mating between closely related individuals less than ten generations ago []. In regions of the genome outside of ROH, heterozygosity levels were similar between historical and modern Grauer’s gorillas ( Figure 2 A), indicating that recent inbreeding is the main cause of the observed reduction in genome-wide genetic diversity. The proportion of the genome contained in long ROH increased by 6.9% in modern mountain gorillas; however, the difference with the historical samples was not statistically significant ( Figures 2 B and S3 ).

17 Pollard K.S.

Hubisz M.J.

Rosenbloom K.R.

Siepel A. Detection of nonneutral substitution rates on mammalian phylogenies. 18 Adzhubei I.

Jordan D.M.

Sunyaev S.R. Predicting functional effect of human missense mutations using PolyPhen-2. Figure 3 Functional Consequences of Population Decline in Eastern Gorillas Show full caption (A) Relative genetic load measured as the fraction of homozygous-derived sites in evolutionary-constrained regions of the genome. (B) Ratio of derived alleles between historical and modern genomes at LoF and missense sites. Error bars, ±1 SD. This ratio is significantly different from 1 in Grauer’s gorillas. (C) The likelihood of an amino acid substitution being damaging for homozygous variants within eastern gorilla populations. Solid lines show population averages for all alleles classified as possible and likely damaging (PolyPhen2 score > 0.45). ∗∗p < 0.01, ∗∗∗p < 0.001; NS, not significant. See also p < 0.01,p < 0.001; NS, not significant. See also Figures S2 and S3 To test whether population decline and small population size led to an accumulation of deleterious mutations in eastern gorillas, we used three independent and complementary approaches to characterize genetic load on genome-wide, gene, and protein level. First, we estimated genetic load for each individual by measuring the proportion of homozygous-derived mutations in regions of the genome that are conserved across a whole-genome alignment of 20 vertebrates and thus can be assumed to be mostly deleterious []. Although the differences in genetic load between modern and historical eastern gorilla individuals were not significant, a larger proportion of modern genomes of both Grauer’s and mountain gorillas fell into the upper range of the distribution ( Figure 3 A). Second, we assessed the effects of DNA sequence variants on protein-coding genes and found a significant increase in frequency of both missense and loss-of-function (LoF) variants in modern Grauer’s gorillas compared to the historical individuals, consistent with higher genetic load in the modern population ( Figure 3 B). In contrast, our results suggest that the frequency of missense and LoF variants has remained stable in the mountain gorillas during the last century ( Figure 3 B). Finally, we predicted the possible deleterious impact of each amino acid substitution on the protein function using physical considerations []. We observed a significant increase in the frequency of amino acid substitutions classified as “probably damaging” in the modern Grauer’s gorilla individuals, whereas no significant change was observed between historical and modern mountain gorillas ( Figure 3 C).

19 Mudakikwa A.

Cranfield M.R.

Sleeman J.M.

Eilenberger U. Clinical medicine, preventive health care and research on mountain gorillas in the Virunga Volcanoes region. Genes affected by LoF mutations with the highest frequency increase in the modern Grauer’s gorillas were associated with functions related to immunity and methylation (see Data S2 and S3 ), possibly indicating a decline in pathogen resistance during recent decades. LoF variants with the highest frequency increase in modern mountain gorillas were enriched for genes related to Sertoli cell differentiation, thus possibly affecting male fertility (see Data S2 and S3 ). In addition, both Grauer’s and mountain gorillas show several high-frequency LoF variants that are related to malformation of fingers and toes, corroborating the reports of syndactyly in eastern gorillas (see Data S3 ) [].

12 Xue Y.

Prado-Martinez J.

Sudmant P.H.

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Ayub Q.

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Frandsen P.

Chen Y.

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Cooper D.N.

et al. Mountain gorilla genomes reveal the impact of long-term population decline and inbreeding. 20 Strindberg S.

Maisels F.

Williamson E.A.

Blake S.

Stokes E.J.

Aba’a R.

Abitsi G.

Agbor A.

Ambahe R.D.

Bakabana P.C.

et al. Guns, germs, and trees determine density and distribution of gorillas and chimpanzees in Western Equatorial Africa. By analyzing complete genomes from museum-preserved historical specimens, we have identified and quantified the genomic consequences of population decline in eastern gorillas during the last century. Comparisons to the modern western lowland gorillas (estimated population size: 360,000 individuals), which diverged from the eastern gorillas around 150,000 years ago [], demonstrate that differences in genetic diversity between species are dominated by long-term demographic processes. The observed decrease in genome-wide diversity and increase in putatively deleterious variants in eastern gorillas ( Figures 2 and 3 ) is striking given the short time period spanned by our study, corresponding to 4–5 gorilla generations. Grauer’s gorillas have been more severely affected than mountain gorillas, which we hypothesize can be attributed to their contrasting demographic histories.

12 Xue Y.

Prado-Martinez J.

Sudmant P.H.

Narasimhan V.

Ayub Q.

Szpak M.

Frandsen P.

Chen Y.

Yngvadottir B.

Cooper D.N.

et al. Mountain gorilla genomes reveal the impact of long-term population decline and inbreeding. 21 Roy J.

Arandjelovic M.

Bradley B.J.

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Stephens C.R.

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Kusamba C.

Kyungu J.C.

Smith V.

et al. Recent divergences and size decreases of eastern gorilla populations. 22 Tocheri M.W.

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Troy Case D.

Orr C.M.

Roach N.T.

Villmoare B.

Eriksen A.B.

Kalthoff D.C.

et al. The evolutionary origin and population history of the grauer gorilla. 23 van der Valk T.

Sandoval-Castellanos E.

Caillaud D.

Ngobobo U.

Binyinyi E.

Nishuli R.

Stoinski T.

Gilissen E.

Sonet G.

Semal P.

et al. Significant loss of mitochondrial diversity within the last century due to extinction of peripheral populations in eastern gorillas. 24 Gazave E.

Chang D.

Clark A.G.

Keinan A. Population growth inflates the per-individual number of deleterious mutations and reduces their mean effect. 25 Peischl S.

Excoffier L. Expansion load: recessive mutations and the role of standing genetic variation. 26 Plumptre A.J.

Nixon S.

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Vieilledent G.

Critchlow R.

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Nishuli R.

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Hall J.S. Catastrophic Decline of World’s Largest Primate: 80% Loss of Grauer’s Gorilla (Gorilla beringei graueri) Population Justifies Critically Endangered Status. 22 Tocheri M.W.

Dommain R.

McFarlin S.C.

Burnett S.E.

Troy Case D.

Orr C.M.

Roach N.T.

Villmoare B.

Eriksen A.B.

Kalthoff D.C.

et al. The evolutionary origin and population history of the grauer gorilla. 23 van der Valk T.

Sandoval-Castellanos E.

Caillaud D.

Ngobobo U.

Binyinyi E.

Nishuli R.

Stoinski T.

Gilissen E.

Sonet G.

Semal P.

et al. Significant loss of mitochondrial diversity within the last century due to extinction of peripheral populations in eastern gorillas. 27 Harcourt A.H.

Fossey D. The Virunga gorillas: decline of an ‘island’ population. 28 Kalpers J.

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Nzamurambaho A.

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Mugiri G. Gorillas in the crossfire: population dynamics of the Virunga mountain gorillas over the past three decades. 29 Gray M.

Roy J.

Vigilant L.

Fawcett K.

Basabose A.

Cranfield M.

Uwingeli P.

Mburanumwe I.

Kagoda E.

Robbins M.M. Genetic census reveals increased but uneven growth of a critically endangered mountain gorilla population. 28 Kalpers J.

Williamson E.A.

Robbins M.M.

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Nzamurambaho A.

Lola N.

Mugiri G. Gorillas in the crossfire: population dynamics of the Virunga mountain gorillas over the past three decades. 30 Robbins M.M.

Gray M.

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Mburanumwe I.

Kagoda E.

Basabose A.

Stoinski T.S.

Cranfield M.R.

et al. Extreme conservation leads to recovery of the Virunga mountain gorillas. Eastern gorillas experienced continuous population decline for the last 100,000 years [], before diverging into Grauer’s and mountain gorillas, ca. 10,000 years ago []. Subsequently, Grauer’s gorillas went through a period of population growth and range expansion 5,000–10,000 years ago []. This demographic expansion may have led not only to a historically higher genetic diversity in Grauer’s compared to mountain gorillas ( Figure 2 ) but also to a higher number of low-frequency deleterious mutations in the Grauer’s gorilla population []. As Grauer’s gorillas went through a severe population decline of 80% in the last 20 years to less than 4,000 individuals today [], these deleterious mutations appear to have increased in frequency, likely due to increased drift and inbreeding ( Figure 2 ), thus leading to a more pronounced change in genetic load in Grauer’s compared to mountain gorillas ( Figure 3 ). In contrast, the population size of mountain gorillas from the Virunga Massif population has likely remained small since their divergence from the Grauer’s gorillas []. Numbering fewer than 1,000 individuals at least since the late 1950s [], mountain gorillas experienced a population low of ∼250 individuals in the 1980s [] but recovered to ∼450 individuals in 2013 []. However, we do not detect significant differences in genetic diversity, inbreeding, and genetic load between historical and modern mountain gorilla samples. This might be due to both purging of genetic load as a result of the continuously small population size in mountain gorillas during the last 10,000 years as well as the rapid population recovery in recent decades thanks to implemented conservation measures and/or a less pronounced population decline than previously reported [].