Next Article in Journal
A Missense Mutation c.1132G > A in Fumarate Hydratase (FH) Leads to Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) Syndrome and Insights into Clinical Management in Uterine Leiomyomata
Next Article in Special Issue
Terrain Ruggedness and Canopy Height Predict Short-Range Dispersal in the Critically Endangered Black-and-White Ruffed Lemur
Previous Article in Journal
Satellite DNAs—From Localized to Highly Dispersed Genome Components
Previous Article in Special Issue
Molecular Phylogenetic Relationships and Unveiling Novel Genetic Diversity among Slow and Pygmy Lorises, including Resurrection of Xanthonycticebus intermedius
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Genes
Volume 14
Issue 3
10.3390/genes14030743
genes-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Past Connectivity but Recent Inbreeding in Cross River Gorillas Determined Using Whole Genomes from Single Hairs

by
Marina Alvarez-Estape
1,
Harvinder Pawar
1,
Claudia Fontsere
1,2,
Amber E. Trujillo
3,4,
Jessica L. Gunson
3,4,
Richard A. Bergl
5,
Magdalena Bermejo
6,7,
Joshua M. Linder
8,
Kelley McFarland
9,
John F. Oates
10,
Jacqueline L. Sunderland-Groves
11,
Joseph Orkin
1,12,
James P. Higham
3,
Karine A. Viaud-Martinez
13,
Esther Lizano
1,14 and
Tomas Marques-Bonet
1,14,15,16,*
1
Institute of Evolutionary Biology (UPF-CSIC), PRBB, c/ del Dr. Aiguader 88, 08003 Barcelona, Spain
2
Center for Evolutionary Hologenomics, The Globe Institute, University of Copenhagen, Øster Farimagsgade 5A, 1352 Copenhagen, Denmark
3
Department of Anthropology, New York University, New York, NY 10003, USA
4
New York Consortium in Evolutionary Primatology, New York, NY 10065, USA
5
Conservation, Education and Science Department, North Carolina Zoo, Asheboro, NC 27205, USA
6
SPAC Scientific Field Station Network, Hasso Plattner Foundation (HPF), 14467 Potsdam, Germany
7
Department of Ecology and Environmental Sciences, University of Barcelona, 08028 Barcelona, Spain
8
Department of Anthropology, James Madison University, Harrisonburg, VA 22807, USA
9
Triton College, River Grove, IL 60171, USA
10
Department of Anthropology, Hunter College, City University of New York, New York, NY 10065, USA
11
Department of Forest Resources Management, University of British Columbia, Vancouver, BC V6T 1Z4, Canada
12
Department of Anthropology, Montreal University, Montreal, QC H3T 1N8, Canada
13
Illumina Laboratory Services, Illumina Inc., 5200 Illumina Way, San Diego, CA 92122, USA
14
Institut Català de Paleontologia Miquel Crusafont, Universitat Autònoma de Barcelona, Edifici ICTA-ICP, Cerdanyola del Vallès, 08193 Barcelona, Spain
15
Catalan Institution of Research and Advanced Studies (ICREA), Passeig de Lluís Companys, 23, 08010 Barcelona, Spain
16
CNAG-CRG, Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology, Baldiri i Reixac 4, 08028 Barcelona, Spain
 
add Show full affiliation list
 
remove Hide full affiliation list
*
Author to whom correspondence should be addressed.
Genes 2023, 14(3), 743; https://doi.org/10.3390/genes14030743
Submission received: 17 February 2023 / Revised: 14 March 2023 / Accepted: 15 March 2023 / Published: 18 March 2023
(This article belongs to the Special Issue Primate Phylogeny and Genetics)
Browse Figures
Review Reports Versions Notes

Abstract

:
The critically endangered western gorillas (Gorilla gorilla) are divided into two subspecies: the western lowland (G. g. gorilla) and the Cross River (G. g. diehli) gorilla. Given the difficulty in sampling wild great ape populations and the small estimated size of the Cross River gorilla population, only one whole genome of a Cross River gorilla has been sequenced to date, hindering the study of this subspecies at the population level. In this study, we expand the number of whole genomes available for wild western gorillas, generating 41 new genomes (25 belonging to Cross River gorillas) using single shed hairs collected from gorilla nests. By combining these genomes with publicly available wild gorilla genomes, we confirm that Cross River gorillas form three population clusters. We also found little variation in genome-wide heterozygosity among them. Our analyses reveal long runs of homozygosity (>10 Mb), indicating recent inbreeding in Cross River gorillas. This is similar to that seen in mountain gorillas but with a much more recent bottleneck. We also detect past gene flow between two Cross River sites, Afi Mountain Wildlife Sanctuary and the Mbe Mountains. Furthermore, we observe past allele sharing between Cross River gorillas and the northern western lowland gorilla sites, as well as with the eastern gorilla species. This is the first study using single shed hairs from a wild species for whole genome sequencing to date. Taken together, our results highlight the importance of implementing conservation measures to increase connectivity among Cross River gorilla sites.

Graphical Abstract

1. Introduction

Equatorial Africa is home to two species of gorilla, which are distributed across a distance of 900 km and are the subjects of major conservation concern. The vast majority of Western gorillas belong to the western lowland gorilla subspecies (WL, G. g. gorilla), which has the largest distribution range and census population size of all gorilla subspecies (362,000 individuals) [1]. In contrast, Cross River gorillas (CR, G. g. diehli), the other subspecies of Western gorilla, exist in geographically distinct subpopulations [2,3] across the border between Nigeria and Cameroon and have the smallest census population size (~250–300 gorillas) [4,5]. Eastern gorillas are split into Grauer’s gorillas (GR, Gorilla beringei graueri), which have the second largest distribution range and census population size (6800 individuals) [6], and mountain gorillas (MN, Gorilla beringei beringei), which live in two discrete regions, the Virunga Massif and Bwindi Impenetrable National Park. The MN census population size has increased in recent years (above 1000 individuals) [7] due to successful conservation efforts. With the exception of MN gorillas (considered endangered) [8], all other gorilla subspecies are listed as critically endangered in the IUCN Red List of Threatened Species [9,10,11].
Anthropogenic factors such as habitat loss and degradation and former hunting activities have impacted CR gorillas, confining them to refuge areas to avoid human pressure [12,13,14,15]. We expect this increase in habitat fragmentation to hinder connectivity among CR gorilla populations and lead to high inbreeding levels, which, together with drift, could lead to reduced genetic diversity.
Despite great effort, the present understanding of CR gorilla population genomics has been limited by the small number of high-quality samples available for whole-genome sequencing. Most studies have relied on microsatellites [14,16,17,18,19,20], and to date, only one individual CR genome has been sequenced [21]. These results indicated that the heterozygosity level of CR gorillas falls in between that of WL and GR gorillas [14,20,21]. The divergence between CR and WL from their common ancestor is estimated to have occurred 17–114 thousand years ago (kya), although gene flow continued until ~420 years ago (ya) [21,22,23]. Furthermore, it has been suggested that CR gorillas have suffered a recent reduction in population size with a strong bottleneck around 100–200 ya [14,22]. Fecal microsatellite studies have identified three primary subpopulations of CR gorillas that coincide with patterns of habitat fragmentation [19], with occasional migration between them [4,19]. Given the limitations of the available data, assessing present levels of genetic diversity, inbreeding, population substructure, and levels of gene flow among subpopulations will be important not only for the conservation of CR gorillas but also for understanding their evolutionary history.
In this study, we generated whole-genome data from 115 single hairs collected across the western gorilla range, comprising the largest dataset of CR gorilla whole genomes sequenced to date. We use this dataset to deepen our understanding of the demographic history and connectivity of CR gorillas. As suggested by previous studies, we uncover recent inbreeding among CR gorillas using runs of homozygosity. However, we also confirm the evidence of past gene flow both among CR subpopulations and between CR gorillas and the other gorilla subspecies. Taken together, these findings improve our understanding of the genetic diversity and evolutionary history of CR gorillas and highlight the urgent need to implement conservation measures for the Cross River gorillas.

2. Materials and Methods

2.1. Samples, DNA Extraction, and Library Preparation

A total of 115 single shed hairs from putative gorillas were used mainly from an archived collection in the Primatology Department at NYU. These were mostly collected between 1991 and the early 2000s from CR and WL gorilla sites and three unknown sites (Supplementary Table S1).
DNA was extracted from previously cleaned individual hairs using the QIAamp DNA Investigator Kit (QIAGEN, Cat. No./ID: 56504) following the manufacturer’s instructions. A total of 105 sequencing libraries were made in Claret Bioscience (Santa Cruz, CA, USA), with 18 µL of DNA extract following the Santa Cruz Reaction (SCR) protocol [24] modified for small fragment retention. The remaining libraries (n = 11) were prepared using 20 µL of DNA extract following the SCR protocol [24] at the Comparative Genomics Group (IBE-UPF, Barcelona, Spain). For one sample, two libraries were generated, resulting in a total of 116 libraries from 115 hairs. Libraries were sequenced on an Illumina NovaSeq 6000 with a 2 × 150 bp setup at the National Center for Genomic Analysis (CNAG, Barcelona, Spain).

2.2. Sequencing Data Processing (FASTQ to Genotypes/Likelihoods)

Sequencing reads were trimmed, quality filtered, and merged with FASTP (version 0.23.1) [25]. Mapping to the hg38 reference genome (GenBank GCA_000001405.15) of merged and paired reads (not merged) was performed with the bwa mem algorithm (version 0.7.15) [26], and read groups were added with PICARD (version 1.95) [27]. To avoid spurious alignments, mapped reads with read lengths below or equal to 35 bp were removed (as suggested by Meyer et al. [28] and Kuhlwilm et al. [29]). All bam files corresponding to the same library were merged into one with samtools “merge” (version 0.7.15) [30]. Each bam file was further processed to remove duplicates with PICARD “markduplicates” (version 1.95) [27] and filtered with samtools (version 0.7.15) [30].
FASTQs from high-coverage sequencing of wild gorillas published in previous studies (HighCov samples from now on) [21,31] were mapped to the human reference (hg38) following the same pipeline as the hair samples. Several of these high-coverage published samples were downsampled to an average coverage of 0.75×, 1×, 3×, 7×, and 10× to be used in further analyses (Supplementary Table S1).

2.3. Quality Control (Depth of Coverage and Contamination)

The average coverage of each library was estimated with MOSDEPTH (version 0.2.6) [32]. Two different approaches were followed to check for potential contamination of the hair samples. The Human Contamination Test for Great Ape Samples (HuConTest) script [29] was used, first setting ‘gorilla’ and then ‘pan’ (chimpanzee or bonobo) as the species to test. This script allowed us to obtain an estimation of the percentage of putative human contamination of the samples, as well as to confirm the presence of several chimpanzee samples that were misidentified as gorilla hairs (Figures S1 and S2). The BBsplit software (BBMAP 38.57) [33] was used on those samples with HuConTest results above 5% (>5% human contaminated hairs) as a secondary approach to confirm HuConTest results. BBsplit consists of a competitive mapping strategy where each read is aligned to a range of different genome assemblies from other primates. Apart from the human and the gorilla reference genomes (GRCh38 and gorGor6, respectively), reference genomes corresponding to primate species with overlapping distribution ranges to gorillas were used: chimpanzee (panTro6), De Brazza’s monkey (CertNeg_v1_BIUU), Angolan colobus (Cang.pa_1.0), olive baboon (Panu_3.0), mandrill (mandrill_1.0), and drill (Mleu.le_1.0) (Figure S3). We considered a sample to pass the quality control (QC) if it had an estimation below 1% of putative human contamination from the HuConTest and coverage ≥0.5× (Figure S4).

2.4. Genotype Likelihoods and SNP Calling

Genotype likelihoods for the whole set of samples and several subsets were computed using ANGSD (version 0.931) [34]. We chose the SAMtools model as the method to estimate genotype likelihoods and restricted the computation to only autosomal chromosomes. In each set of samples, we specified that at least 80% of individuals in each variant site needed to be present. Furthermore, we applied several quality filters: “-uniqueOnly 1 -remove_bads 1 -only_proper_pairs 1 -trim 0 -C 50 -minMapQ 30 -minQ 20 -SNP_pval 1e-6”. We specified to output the genotype likelihoods in nonbinary beagle format. These genotype likelihoods were used for PCA and admixture analyses.
The whole set of samples (Set01) consisted of all hair samples before QC together with the HighCov samples (see Section 2.3); thus, it contains samples from all four gorilla subspecies. Set02 was generated from Set01, keeping those samples that passed QC (HuConTest <1% and average coverage ≥ 0.5×) and that were kept after relatedness analysis (see Relatedness section). Set03 and Set04 derive from Set02, keeping WL gorillas and CR gorillas, respectively. Finally, Set05, Set06, and Set07 derive from Set02, keeping western gorilla hair samples, WL hair samples, and CR hair samples, respectively (Supplementary Table S1).
Genotypes were called for those hair samples from Set02 with coverage above 7× together with the high-coverage reference samples [21,31] (HighCov dataset; Supplementary Table S1) using GATK Haplotype Caller (version 4.1.4.0) [35,36]. Genotyping was performed for these samples with GenotypeGVCFs from the GATK toolkit. The resulting VCF was filtered with GATK SelectVariants to keep only SNPs and further filtered following GATK hard-filtering best-practice guidelines. Furthermore, we used BCFTools Filter (version 1.9) [30] to keep only those positions with an average coverage between a third and three times the dataset’s average coverage. We filtered those heterozygous positions with an allele balance <0.15 or >0.85 with BCFTools (version 1.9) [30] to discard any potential residual contamination. We also generated a dataset of these high-coverage samples to an average coverage of 10x (Down10× dataset).

2.5. Genetic Sexing from Coverage

For each library, the coverage per chromosome was estimated with MOSDEPTH (version 0.2.6) [32], and from this, the average autosomal coverage was obtained. Then, we estimated the ratio between the X chromosome coverage and the average autosomal coverage. We assigned samples as belonging to a female, male or undetermined following Cabrera et al. [37] (Figure S5).

2.6. Relatedness Test

We ran NgsRelate (version 2) [38] per gorilla site (Figure S6) on samples passing the QC. Where first- or second-degree relatives were detected, one individual from each pair was excluded from population structure analyses. From each related pair, we chose to exclude the sample with the lowest coverage and the higher number of familial relationships in the dataset.

2.7. Population Structure Assessment

We obtained beagle files of the different sets of samples (described in Genotype likelihoods and SNP calling section; Supplementary Table S1) to be used as input for both PCAngsd (version 0.8) [39] and for NgsAdmix (ANGSD version 0.916) [40]. We ran NgsAdmix for multiple Ks and evaluated its results using EvalAdmix [41] (Figures S7–S12).
Pairwise Fst estimates between different sites were calculated with ANGSD [34]. We used three randomly selected samples per site when more were available (Afi Mountain, Mbe, Kagwene, Mbe, DengDeng, MonteAlen, and NgagaCamp) (Supplementary Table S1). Bai Hokou and Lobéké sites were not included in this analysis because we only had one sample for each after QC.
To calculate pairwise Fst, we first obtained site allele frequency likelihood (saf) files for each population as follows: “angsd -b $file -ref Hg38.fa -anc Hg38.fa -sites autosomes.file -dosaf 1 -gl 1 -uniqueOnly 1 -remove_bads 1 -only_proper_pairs 1 -trim 0 -C 50 -baq 1 -minMapQ 20 -minQ 20”. Then, we calculated the 2D site frequency spectrum (SFS) with realSFS. This output was used as priors together with the saf files from the first step, obtaining fst binary files with realSFS “fst index” setting “-whichFst 1”. This setting is preferable for small sample sizes such as our case. Finally, realSFS “fst stats” was used to extract the Fst values, which were plotted with R (version 4.1.2) [42].

2.8. Heterozygosity Estimates

We estimated genome-wide heterozygosity per autosomal chromosome for the hair samples that passed our QC (HuConTest < 1% and average coverage ≥0.5×) and for the HighCov samples (full coverage and downsampled several coverage ranges) using ANGSD (version 0.931) [34].
To account for differences in coverage in our dataset, we downsampled hair samples and HighCov samples with coverage >1.1× to 1× with Picard (version 2.20.0) [27] (Supplementary Table S1). Then, we estimated the genome-wide heterozygosity of the resulting dataset of coverage ≤1× (downsampled hairs and published data with hairs <1× coverage) (Supplementary Table S1).

2.9. Estimation of Runs of Homozygosity and Inbreeding Coefficients

To estimate runs of homozygosity (ROH) along the genome, we used BCFTools (version 1.14) [43] on the Down10x dataset (see Supplementary Table S1). We used this dataset to avoid biases in the estimation of runs of homozygosity from differences in coverage between samples. We used a recombination rate of 1 × 10−8 [31] and kept only RoH with size ≥50 kbp and quality ≥70. To estimate heterozygosity by windows on the same dataset (Down10x dataset), we used a 100 kbp window size with a 50 kbp of step size with custom scripts. We plotted these results together with the results from BCFTools roh on R (version 4.1.2) [42].
The age of coalescence of the ROH tracks was estimated using the rule of g = 100/(2 × RoHlength) [44], with g being the number of generations. For this, we assumed a constant recombination rate of 1 cM/1 Mbp following [45,46,47].

2.10. Past and Present Demographic History

We performed pairwise sequential Markovian coalescent (PSMC) [48] on samples of comparably high coverage (Supplementary Table S1). This included hair samples with coverage >7× (three CR hair samples: Afi18, Ken12, Ken18; one WL hair sample: naA2), one CR blood sample (Ggd-Nyango), one GR blood sample (Gbg-Mkubwa), and one MN blood sample (Gbb-Maisha). We used samtools (version 1.11) [30,49] and BCFtools (version 1.6) [30] to call variants and filter for base and mapping qualities <30. We retained sites between a third of the average read depth (-d) and twice the average read depth (-D) for each sample. PSMC was applied using the standard parameters -N25 -t15 -r5 -p “4 + 25 × 2 + 4 + 6”; [48]. We scaled the output using a μ of 1.235 × 10−8 and a 19-year generation time, as previously inferred for gorillas by [50].
To study recent demographic changes, we used GONE [51] (Supplementary Table S1). GONE relies on linkage disequilibrium (LD), with high power between 0 and 200 generations ago [51]. We obtained PED and MAP files for each subspecies with PLINK [52,53,54] and ran 10 iterations of GONE for each subspecies, where each run had 40 internal replicates. For each run, we used standard input parameters except for the recombination rate of 1.193 cM/Mb [55] and 0.01 as the maximum recombination value. We reduced the maximum hc to avoid bias due to recent migrants and population structure, as suggested by [51].

2.11. Assessment of Gene Flow and Connectivity

Using Dsuite [56] on the Down10x dataset, we looked at possible gene flow across gorilla species and geographic areas. We ran Dtrios within the Dsuite package to obtain D statistic and f4-ratio values, which are input for F-branch. We ran the F-branch program and its accompanying Python plotting script to generate the matrix displaying excess allele sharing between each branch on the tree and/or its internal branches. First, we ran Dtrios on the species level using the Down10× dataset and one orangutan (Pab-Buschi) from Prado-Martinez et al. [21] as an outgroup. In another iteration, we divided the CR gorilla samples into four groups, each representing a site: Afi, Mbe, Nyango (unknown site), and Kagwene. Finally, to investigate if there are differences in allele sharing across CR sites and WL regions, we grouped several published wild WL gorilla samples into three regions based on their PCA clustering (Figure S13). The regions were the following: the Equatorial Guinea region, with one gorilla for Equatorial Guinea (Ggg-Coco) that clusters with our Monte Alen hair samples; the Naga region, with three samples that cluster around those from Ngaga Camp (Ggg-Carolyn, Ggg-Porta, Ggg-Tzambo), and the Unknown region, including the hair sample of unknown origin (coverage >7×) together with two other previously published samples (Ggg-Akiba-Beri and Ggg-Choomba).
We used ANGSD D-statistics [57] to further test for introgression at the site level. Since there is no site information for the published gorilla data, in this case, we restricted this analysis to western gorilla hair samples from Set02 (see Section 2.4, Supplementary Table S1). The option -doAbbababa2 [57] allows for multiple individuals per population and was run with the following settings: “angsd -doAbbababa2 1 -bam BAMsList.txt -sizeFile file.size -sites autosomes.file -doCounts 1 -sample 0 -blockSize 5,000,000 -maxDepth 50 -out -anc Hg38.fa -useLast 0 -minQ 20 -minMapQ 30 -p 1”. To estimate D-statistics at the species level, we used samples from Set02, in this case, keeping the previously published genomes, with a chimpanzee (Ptt-Clara) from Prado-Martinez et al. [21] as an outgroup. Finally, we also estimated D-statistics between WL gorilla regions and GR and between CR and WL gorilla sites.
To consider admixture at the CR region level (CR-west, CR-central, CR-east), we used an admixture graph approach. We considered five populations: CR-west, CR-central, CR-east, western lowlands, and easterns as an outgroup, using samples from the Down10x dataset. We calculated pairwise f2 statistics for these five populations using the function extract_f2 with blgsize = 500,000 using the R package ADMIXTOOLS 2 [58]. To assess the best-fit topology for a set of admixture events, we used the function find_graphs, with the easterns as the outgroup and varying the number of admixture events from 1 to 10. We assessed which graph had the lowest likelihood score using an out-of-sample score with the function qpgraph. We used the bootstrapping procedure of [58] to assess for significant differences between the graph with the best fitting number of admixture events and the graph with 0 admixture events, using the functions qpgraph_resample_multi with nboot = 100 and compare_fits. We also calculated confidence intervals for the drift lengths and admixture proportions inferred for the graph with 0 admixture events and the graph with the best-inferred number of admixture events using the function qpgraph_resample_snps with boot = 100.

3. Results

3.1. Single Hairs as a Source for Genomic DNA

We obtained whole-genome data from single hairs collected between 1991 and 2019 from 115 unique individuals, including samples from the poorly known CR subspecies, as well as WL and GR gorillas (Supplementary Table S1). The single-hair DNA extracts were sequenced to 30–60 gigabases (Gb) per library (n = 116; one sample had two replicate libraries). The average genome-wide coverage reached was 1.35× (min 0.001× and max 13.82×), and the median host DNA (hDNA) was 4.81% (min 0.02% and max 72%) (Supplementary Table S1). We identified and excluded five hair samples of chimpanzee origin from our dataset, three of which had less than 1% of human contamination (Figures S1–S3). After quality control (samples with >0.5× coverage and <1% putative human contamination; see Section 2.2), a total of 41 gorilla hair samples were kept (35.3% of the initial dataset), with a mean coverage of 3.23× (median 1.44×) and a mean hDNA of 20.64% (median 14.88%) (Figure S4). This final dataset consists of samples from three gorilla subspecies across eleven sites: four CR gorilla sites (Afi Mountain Wildlife Sanctuary, Afi; Mbe Mountains, Mbe; Cross River National Park Boshi Extension, Bos; and Kagwene Gorilla Sanctuary, Kag), six WL gorilla sites (Deng Deng, Den; Lobéké, Lob; Bai Hokou, Bai; Ngaga Camp, Nga; Monte Alen, MoA; and one unknown, Unk1), and one unknown GR site, Unk3 (Figure 1A, Supplementary Table S2). Most of the final 41 samples were identified as female (Figure S5). We identified several first-degree related pairs of gorillas in Afi and Kagwene CR sites that were excluded from population structure analyses (Supplementary Table S1, Figure S6).

3.2. Increased Resolution of Western Gorilla Population Structure

The four subspecies are clearly separated in the principal component analysis (PCA) (Figure 1B and Figure S14), with hair and blood samples from the same subspecies clustering together. PC1 separates the western and the eastern gorilla species, and PC2 separates the western species into WL and CR subspecies.
When looking specifically at WL gorillas, we observed a main cluster that includes samples from Deng Deng, Lobéké, Bai Hokou, and the unknown site, whereas samples from Monte Alen and Ngaga Camp separate from this cluster in PC1 and PC2, respectively (Figure 1C and Figure S15). This suggests that the sample of unknown geographic origin may belong to a northern WL site. The PCA for CR gorillas shows the Kagwene Gorilla Sanctuary site separated from the rest by PC1 while PC2 separates Afi Mountain Wildlife Sanctuary from CRNP Boshi Extension and Kagwene Gorilla Sanctuary D and Figure S16). As expected, FST analysis revealed that sampling sites from the same subspecies have higher genetic similarity than when comparing sites between the western gorilla subspecies (WL vs. CR) (Figure 1E, Supplementary Table S3). Among CR gorilla sites, Kag (Kagwene Gorilla Sanctuary) is the most genetically differentiated using FST (Figure 1E, Supplementary Table S3), supporting the PCA clustering and its separation along PC1 (Figure 1D).
Given that most hair samples included in the study have known GPS coordinates, we were able to better determine the putative geographic origin of the previously published high-coverage samples, for which only the area or country of origin are available as metadata (see the supplementary table in Prado-Martinez et al. [21] and Xue et al. [31]). For example, the only previously published whole genome sample of a CR gorilla, Nyango, clusters near the Mbe and Boshi sites (Figure S17). In the instance of WL, we were able to confirm that Ggg-Coco is from Equatorial Guinea, as observed from its clustering near the Monte Alen samples (Figure S13).

3.3. Genetic Diversity among Gorilla Species

Genome-wide heterozygosity estimates on hair samples show that Cross River gorillas present lower heterozygosity levels than WL but higher levels than both eastern gorilla subspecies (Figures S18–S19). We observe little variation in heterozygosity levels among CR sites. Monte Alen presents the highest heterozygosity among WL sites, and there is a high variance in heterozygosity among WL sites, in contrast to the trend among CR sites. The overall fraction of the genome in runs of homozygosity (ROH) is higher in CR gorillas than in WL gorillas but lower compared to the eastern gorilla species. Nonetheless, the fraction of the genome in long ROH, which is a proxy of recent inbreeding levels, showed that CR gorillas have higher levels of recent inbreeding than Grauer’s gorillas but lower than mountain gorillas (Figure 2A, Figures S20 and S21).

3.4. Strong Bottleneck in Cross River Gorillas

PSMC results of the CR gorilla hair samples with higher coverage (Supplementary Table S1) recapitulate the trends for changes in effective population size (Ne) inferred using the single CR blood sample (Ggd-Nyango) (Figures S22 and S23). The trajectories in Ne for these four CR individuals are highly concordant, except in very recent times where the method has reduced power. This is in agreement with recent population split times of CR gorilla populations. To assess population demography in more recent times, we implemented GONE, which revealed a strong recent bottleneck in CR gorillas (Figure 2B). In the last 10 generations, we infer the Ne of CR gorillas was more similar to that of MN than to GR gorillas. Considering ROH of length 5–10 Mb to calculate the age of these ROH tracks, we likewise infer an onset of inbreeding 5–10 generations ago, which would correspond to around 100–200 years ago. These results indicate recent inbreeding, similar to what is known for MN.

3.5. Past Gene Flow among Gorilla Subspecies

On the sharing of alleles among western gorillas, between CR sites and WL sites, with D statistics, we see the highest allele sharing between the northern WL site cluster (Bai-Hokou, Deng Deng, and Lobéké) and all CR sites excluding the CRNP Boshi Extension site (Figure 3A, Figures S24 and S25). To assess this signal at a higher resolution, we implemented the F-branch method, which takes into account the correlation among different branches of the tree. As such, we were able to infer that the common ancestor of the northern WL cluster had gene flow with CR gorillas (Figures S26 and S27).
Within CR sites, we infer the highest levels of allele sharing between Afi Mountain and Mbe, which are geographically closely located (Figure 3B and Figure S28). Furthermore, using the F-branch method and Dstats, we uncover a signal of past gene flow between CR gorillas and the eastern GR gorilla subspecies (Figure 3B and Figures S28–S31). Taking our gene flow analyses together, the CR CRNP Boshi Extension and the WL Monte Alen sites show weaker allele-sharing signals compared to other sites (Figure 3A and Figures S29B–D).
To investigate admixture within CR regions (CR-west, Afi Mountain Wildlife Sanctuary site; CR-central, Mbe Mountains site and Nyango sample; and CR-eastern, Kagwene Gorilla Sanctuary site), we constructed admixture graphs of varying numbers of admixture events (see Section 2.11). The best-fitting admixture graph (likelihood = 0.001712409) infers high shared ancestry between western lowlands and CR-east (Kagwene Gorilla Sanctuary) and models CR-west (Afi Mountain Wildlife Sanctuary) and CR-central (Mbe Mountains site and Nyango) populations as admixed with a major CR-east (Kagwene Gorilla Sanctuary) component and a more basal, minor component (Figure S32A). Despite having a much lower likelihood than a model with zero admixture events (likelihood = 100.9402) (Figure S32B,C), when considering SNP variance by bootstrapping, these two models are not significantly different (p = 0.4970), likely because they share topological features. Nonetheless, we consider the best-fitting admixture graph to be a reasonable model for this group of populations since the CR-east (Kagwene Gorilla Sanctuary) population lives in closer proximity to western lowland gorilla populations than the two other regions, CR-west and CR-central. In principle, such allele sharing could be the consequence of admixture at some point in the past or due to ancient substructures within the CR gorillas. However, given the very similar population size trajectories of CR gorillas, likely reflecting recent split times, the time depth might not be sufficient for such an ancient substructure to occur.

4. Discussion

In this study, we have considerably expanded the number of whole genomes available for CR gorillas, from only 1 whole genome previously available [21] to 25 new samples that pass our stringent QC. Together with the new 15 WL gorilla genomes, we provide the most comprehensive whole-genome dataset of wild western gorillas to date, including publicly available genomes derived from invasive samples [21,31]. As such, we have been able to explore at greater resolution genome-wide heterozygosity, inbreeding, demography, and connectivity of CR gorillas, the subspecies for which population-level data had been missing.
Previous studies on great apes using non-invasive samples have been restricted to analyses of neutral markers, e.g., microsatellites [18,20,59,60,61,62], and more recently target enrichment of specific genomic regions, e.g., chromosome 21 [45]. To date, only one study has obtained WGS data using multiple hairs from a wild-threatened species [63]. In this study, we have used a single hair per extraction and proven the feasibility of obtaining reliable whole-genomic data for population genomic studies from this type of sample.
Using our new genomes with the publicly available wild gorilla genomes, we observe for the first time four differentiated clusters using the first and second principal components of the PCA, which represent the four gorilla species and subspecies. This indicates that although we included different sample types (blood and hair) from different studies, we do not observe major technical effects, and the clustering seen here reflects biological differences.
Our genome-wide heterozygosity estimates are in line with previous studies, in which CR gorillas showed lower heterozygosity levels than WL gorillas but higher heterozygosity than the two eastern subspecies, GR and MN gorillas [14,18,21,31,46,61,64]. However, we also uncovered for the first time long ROH (>10 Mb) in CR gorillas, which is indicative of recent inbreeding caused by the mating of related individuals in such small populations. In fact, we dated the age of ROH tracks >5–10 Mb to around 100–200 ya. This matches the timing of a strong recent bottleneck in CR gorillas inferred in our GONE analyses. In fact, CR gorillas appear to have suffered an even more recent bottleneck than both eastern gorilla subspecies. Hence, while MN gorillas have experienced long-term low effective population size [46], our findings indicate that CR gorillas are currently at greater risk of a higher realized genetic load [65,66,67]. This finding is in agreement with previous studies that observed evidence of a recent and abrupt reduction in CR gorilla population size [14,22], probably caused by hunting in the past two hundred years, as suggested by Bergl et al. [14].
Previous studies using microsatellites on CR gorilla fecal samples found a population structure in CR gorillas consistent with geography and habitat fragmentation [14,19]. As such, three main CR subpopulations were defined: western, comprising Afi Mountain Wildlife Sanctuary; eastern, comprising Kagwene Gorilla Sanctuary; and central, comprising the remaining study sites (Mbe Mountains, Mone, Takamanda south, and Boshi). In this study, we included samples from these three regions, and our results using WGS support those obtained with microsatellite data.
Our analyses revealed past gene flow among two nearby CR sites and suggested the presence of an isolated CR site. Specifically, the observed gene flow between Afi Mountain and Mbe, which are geographically close, suggests that these two communities were connected in the past but have been subjected to high levels of habitat fragmentation and human disturbance over time. This confirms previous findings by Imong et al. [12]. On the other hand, the CRNP Boshi Extension site showed the lowest gene flow signal, confirming the reduced functional connectivity between this and other CR sites previously observed [3]. Nonetheless, obtaining samples from closer sites, such as CRNP Okwa Hills, could help increase the resolution of these results.
Additionally, we observed past gene flow between CR sites and the northern WL sites. This agrees with findings from mitochondrial haplogroups observed by Clifford et al. [16] and Anthony et al. [17], where one haplogroup extended from the CR area in Nigeria to southeastern Cameroon (part of WL distribution). This signal of gene flow between the two western subspecies is consistent with microsatellite results using feces and 100-year-old teeth roots [22]. However, this signal was not observable using only one WGS CR gorilla sample, Nyango [23], demonstrating the importance of population-level WGS data for such analyses. Moreover, Fontsere et al. [45] recently observed gene flow between a chimpanzee site located nearby Lobéké (WL gorilla site) and chimpanzees located in the northern distribution of CR gorillas. Taken together, these results indicate the existence of a connectivity corridor in the past used by both chimpanzees and gorillas.
Furthermore, we detected a signal of past gene flow between CR gorillas and eastern gorillas, with a stronger signal with GR than MN gorillas, consistent with previous studies [20,68]. This finding was not detected in a previous analysis using Nyango, the single WGS CR individual to date [23]. The increased number of CR samples in the current study improves the resolution of inferences. Hence, all the evidence points to a complex demographic history of CR gorillas with several episodes of gene flow, which would require future in-depth modeling to better understand their demography.
We performed thorough quality control, resulting in a filtered dataset that represents 35% of the original set of samples, highlighting the low quality and amount of data retrieved from shed hair samples. This was to be expected, given that most of the single shed hairs we used were collected in the early 1990s from gorilla nests. As such, the samples from the CRNP Okwa Hill, Takamanda National Park and Mt. Doudou sites were not of sufficient quality for further analysis. We stress the importance of quality control when working with non-invasive samples because most of the samples from the Takamanda National Park site belonged to chimpanzees instead of gorillas.
In this study, we have focused on the two subspecies of western gorillas and complemented previously published blood samples with newly sequenced WG data from single hairs. Additional samples from geolocalized eastern gorilla sites would allow future studies to further investigate past connectivity in the gorilla species. Additionally, increasing the number of geolocalized samples for the CR sites would help gain resolution on differences among regions or sites. Furthermore, studying which CR introgressed tracks are derived from WL or GR gorillas and the time of these admixture events could help characterize some functional consequences of these introgressed fragments.
We demonstrate that Cross River gorillas display recent inbreeding and a population bottleneck despite signals of past connectivity with other gorillas and gene flow within the Cross River population. These findings are almost certainly associated with the gorillas’ current concentration in small, restricted, hilly areas, which hinders genetic connectivity. If current conditions persist, ongoing inbreeding and the associated loss of genetic diversity would very likely reduce the fitness of the population and increase their extinction risk. However, our data also reinforce previous findings [14] that since the bottleneck in the Cross River population is relatively recent, further loss of genetic diversity could be minimized if the population is allowed to expand. Since large areas of unoccupied apparent gorilla habitat remain in the Cross River landscape [3,12], and camera trap images suggest the population is reproducing (WCS unpublished data), it is possible that population growth could be achieved if hunting pressure were relaxed. Our study also illustrates that single hairs are a viable source of genomic information. Future genomic studies aiming to help the conservation of threatened species will undoubtedly benefit from this non-invasive sample type.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/genes14030743/s1, Figure S1: Great ape contamination test (HuConTest); Figure S2: PCA without QC; Figure S3: Contamination test; Figure S4: Quality control; Figure S5: Sex determination from coverage; Figure S6: Relatedness; Figure S7: Admixture western gorillas; Figure S8: Admixture WL gorilla hairs; Figure S9: Admixture CR gorilla hairs, Figure S10: Admixture WL hairs and blood; Figure S11: Admixture CR hairs and blood; Figure S12: Evaladmix; Figure S13: PCA of western lowland gorillas; Figure S14: PCA with the 4 gorilla subspecies; Figure S15: PCA western lowland gorilla hairs; Figure S16: PCA Cross River gorilla hairs; Figure S17: PCA of Cross River gorillas; Figure S18: Genome-wide heterozygosity; Figure S19: Genome-wide heterozygosity with downsampling; Figure S20: RoH sum and number; Figure S21: RoH along Chr2; Figure S22: PSMC gorillas; Figure S23: PSMC Cross River gorillas; Figure S24: D statistics test (ABBA-BABA) between western populations (CR sites) calculated with ANGSD; Figure S25: D statistics test (ABBA-BABA) between western populations (WL sites) calculated with ANGSD; Figure S26: F-branch CR sites, WL regions and eastern subspecies; Figure S27: Dstats CR sites, WL regions; Figure S28: Dstats CR sites; Figure S29: Dstats populations; Figure S30: F-branch species; Figure S31: Dstats gorilla subspecies; Figure S32: Admixturegraph; Table S1: Excel spreadsheet with metadata; Table S2: Final dataset; Table S3: Fst values.

Author Contributions

M.A.-E., C.F., J.O., J.P.H., E.L. and T.M.-B. conceived the study; C.F. and T.M.-B. supervised the study; R.A.B., M.B., J.M.L., K.M., J.F.O., J.L.S.-G. and J.P.H. provided samples; M.A.-E., A.E.T. and J.L.G. performed laboratory work; K.A.V.-M. contributed to data generation; M.A.-E. performed data curation; M.A.-E. and H.P. performed formal bioinformatic analysis; M.A.-E. carried out the writing—original draft preparation; M.A.-E., H.P., C.F., J.O., E.L., J.P.H., R.A.B., M.B., J.M.L., K.M., J.F.O. and J.L.S.-G. carried out the writing—review and editing; M.A.-E. performed visualization. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Ministerio de Ciencia, Universidades e Investigación (FPI, PRE2018) (to M.A.-E.), Generalitat de Catalunya (FI_B100131) (to H.P.), European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 864203), PID2021-126004NB-100 (MICIIN/FEDER, UE), “Unidad de Excelencia María de Maeztu”, funded by the AEI (CEX2018-000792-M), NIH 1R01HG010898-01A1 and Secretaria d’Universitats i Recerca and CERCA Programme del Departament d’Economia i Coneixement de la Generalitat de Catalunya (GRC 2021 SGR 00177) (to T.M.B.). Illumina Inc. provided support for next-generation library sequencing, Beatriu de Pinós postdoctoral programme of the Government of Catalonia’s Secretariat for Universities and Research of the Ministry of Economy and Knowledge 2017 BP 00265 (to J.O.). The project that gave rise to these results received the support of a fellowship from “la Caixa” Foundation (ID 100010434) and from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement (No 847648. The fellowship code is LCF/BQ/PI20/11760004) (to J.O.).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Genome resequencing data are freely available in ENA under the project number (PRJEB60463).

Acknowledgments

Thanks to the many field assistants who made the sample collection possible and the communities and government agencies who facilitated access to field sites. We are also thankful to the Wildlife Conservation Society’s programs in Nigeria and Cameroon for allowing sample collection. We are thankful to Marta Gut and Ivo Gut for their involvement in data generation. We would like to thank Paula Esteller-Cucala, Nuria Hermosilla, Irune Ruiz Garzia, Martin Kuhlwilm, and Javier Prado-Martinez for their helpful discussions.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. 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. Sci. Adv. 2018, 4, eaar2964. [Google Scholar] [CrossRef] [Green Version]
  2. Dunn, A.; Bergl, R.; Byler, D.; Eben-Ebai, S.; Ndeloh Etiendem, D.; Fotso, R.; Ikfuingei, R.; Imong, I.; Jameson, C.; Macfie, L.; et al. Revised Regional Action Plan for the Conservation of the Cross River Gorilla (Gorilla gorilla diehli) 2014–2019; IUCN/SSC Primate Specialist Group and Wildlife Conservation Society: New York, NY, USA, 2014; ISBN 9780963206428. [Google Scholar]
  3. Imong, I.; Robbins, M.M.; Mundry, R.; Bergl, R.; Kühl, H.S. Informing Conservation Management about Structural versus Functional Connectivity: A Case-Study of Cross River Gorillas. Am. J. Primatol. 2014, 76, 978–988. [Google Scholar] [CrossRef] [PubMed]
  4. Bergl, R.A.; Warren, Y.; Nicholas, A.; Dunn, A.; Imong, I.; Sunderland-Groves, J.L.; Oates, J.F. Remote Sensing Analysis Reveals Habitat, Dispersal Corridors and Expanded Distribution for the Critically Endangered Cross River Gorilla Gorilla gorilla diehli. Oryx 2012, 46, 278–289. [Google Scholar] [CrossRef] [Green Version]
  5. Oates, J.; Sunderland-Groves, J.; Bergl, R.; Dunn, A.; Nicholas, A.; Takang, E.; Omeni, F.; Imong, I.; Fotso, R.; Nkembi, L.; et al. Regional Action Plan for the Conservation of the Cross River Gorilla (Gorilla gorilla diehli); IUCN/SSC Primate Specialist Group and Wildlife Conservation Society: Arlington, VA, USA, 2007; ISBN 9781934151020. [Google Scholar]
  6. Plumptre, A.J.; Kirkby, A.; Spira, C.; Kivono, J.; Mitamba, G.; Ngoy, E.; Nishuli, R.; Strindberg, S.; Maisels, F.; Buckland, S.; et al. Changes in Grauer’s Gorilla (Gorilla beringei graueri) and Other Primate Populations in the Kahuzi-Biega National Park and Oku Community Reserve, the Heart of Grauer’s Gorilla Global Range. Am. J. Primatol. 2021, 83, e23288. [Google Scholar] [CrossRef] [PubMed]
  7. Granjon, A.-C.; Robbins, M.M.; Arinaitwe, J.; Cranfield, M.R.; Eckardt, W.; Mburanumwe, I.; Musana, A.; Robbins, A.M.; Roy, J.; Sollmann, R.; et al. Estimating Abundance and Growth Rates in a Wild Mountain Gorilla Population. Anim. Conserv. 2020, 23, 455–465. [Google Scholar] [CrossRef]
  8. Hickey, J.R.; Basabose, A.; Gilardi, K.V.; Greer, D.; Nampindo, S.; Robbins, M.M.; Stoinski, T.S. IUCN Gorilla beringei ssp. beringei. IUCN Red List Threat. Species 2018. [Google Scholar] [CrossRef]
  9. Maisels, F.; Strindberg, S.; Breuer, T.; Greer, D.; Jeffery, K.; Stokes, E. IUCN Gorilla gorilla ssp. gorilla. IUCN Red List Threat. Species 2016. [Google Scholar] [CrossRef]
  10. Bergl, R.A.; Dunn, A.; Fowler, A.; Imong, I.; Ndeloh, D.; Nicholas, A.; Oates, J.F. IUCN Gorilla gorilla ssp. diehli. IUCN Red List Threat. Species 2016. [Google Scholar]
  11. Plumptre, A.; Nixon, S.; Caillaud, D.; Hall, J.S.; Hart, J.A.; Nishuli, R.; Williamson, E.A. IUCN Gorilla beringei ssp. graueri. IUCN Red List Threat. Species 2016. [Google Scholar]
  12. Imong, I.; Robbins, M.M.; Mundry, R.; Bergl, R.; Kühl, H.S. Distinguishing Ecological Constraints from Human Activity in Species Range Fragmentation: The Case of Cross River Gorillas. Anim. Conserv. 2014, 17, 323–331. [Google Scholar] [CrossRef]
  13. Etiendem, D.N.; Funwi-Gabga, N.; Tagg, N.; Hens, L.; Indah, E.K. The Cross River Gorillas (Gorilla gorilla diehli) at Mawambi Hills, South-West Cameroon: Habitat Suitability and Vulnerability to Anthropogenic Disturbance. Folia Primatol. 2013, 84, 18–31. [Google Scholar] [CrossRef] [PubMed]
  14. Bergl, R.A.; Bradley, B.J.; Nsubuga, A.; Vigilant, L. Effects of Habitat Fragmentation, Population Size and Demographic History on Genetic Diversity: The Cross River Gorilla in a Comparative Context. Am. J. Primatol. 2008, 70, 848–859. [Google Scholar] [CrossRef] [PubMed]
  15. McFarland, K.L. Ecology of Cross River Gorillas (Gorilla gorilla diehli) on Afi Mountain, Cross River State, Nigeria; City University of New York: Ann Arbor, MI, USA, 2007. [Google Scholar]
  16. Clifford, S.L.; Anthony, N.M.; Bawe-Johnson, M.; Abernethy, K.A.; Tutin, C.E.G.; White, L.J.T.; Bermejo, M.; Goldsmith, M.L.; McFarland, K.; Jeffery, K.J.; et al. Mitochondrial DNA Phylogeography of Western Lowland Gorillas (Gorilla gorilla gorilla). Mol. Ecol. 2004, 13, 1551–1565, 1567. [Google Scholar] [CrossRef]
  17. Anthony, N.M.; Johnson-Bawe, M.; Jeffery, K.; Clifford, S.L.; Abernethy, K.A.; Tutin, C.E.; Lahm, S.A.; White, L.J.T.; Utley, J.F.; Wickings, E.J.; et al. The Role of Pleistocene Refugia and Rivers in Shaping Gorilla Genetic Diversity in Central Africa. Proc. Natl. Acad. Sci. USA 2007, 104, 20432–20436. [Google Scholar] [CrossRef] [Green Version]
  18. Städele, V.; Arandjelovic, M.; Nixon, S.; Bergl, R.A.; Bradley, B.J.; Breuer, T.; Cameron, K.N.; Guschanski, K.; Head, J.; Kyungu, J.C.; et al. The Complex Y-Chromosomal History of Gorillas. Am. J. Primatol. 2022, 84, e23363. [Google Scholar] [CrossRef]
  19. Bergl, R.A.; Vigilant, L. Genetic Analysis Reveals Population Structure and Recent Migration within the Highly Fragmented Range of the Cross River Gorilla (Gorilla gorilla diehli). Mol. Ecol. 2007, 16, 501–516. [Google Scholar] [CrossRef] [PubMed]
  20. Thalmann, O.; Fischer, A.; Lankester, F.; Pääbo, S.; Vigilant, L. The Complex Evolutionary History of Gorillas: Insights from Genomic Data. Mol. Biol. Evol. 2007, 24, 146–158. [Google Scholar] [CrossRef] [Green Version]
  21. 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. Nature 2013, 499, 471–475. [Google Scholar] [CrossRef] [Green Version]
  22. Thalmann, O.; Wegmann, D.; Spitzner, M.; Arandjelovic, M.; Guschanski, K.; Leuenberger, C.; Bergl, R.A.; Vigilant, L. Historical Sampling Reveals Dramatic Demographic Changes in Western Gorilla Populations. BMC Evol. Biol. 2011, 11, 85. [Google Scholar] [CrossRef] [Green Version]
  23. McManus, K.F.; Kelley, J.L.; Song, S.; Veeramah, K.R.; Woerner, A.E.; Stevison, L.S.; Ryder, O.A.; Ape Genome Project, G.; Kidd, J.M.; Wall, J.D.; et al. Inference of Gorilla Demographic and Selective History from Whole-Genome Sequence Data. Mol. Biol. Evol. 2015, 32, 600–612. [Google Scholar] [CrossRef] [Green Version]
  24. Kapp, J.D.; Green, R.E.; Shapiro, B. A Fast and Efficient Single-Stranded Genomic Library Preparation Method Optimized for Ancient DNA. J. Hered. 2021, 112, 241–249. [Google Scholar] [CrossRef]
  25. Chen, S.; Zhou, Y.; Chen, Y.; Gu, J. Fastp: An Ultra-Fast All-in-One FASTQ Preprocessor. Bioinformatics 2018, 34, i884–i890. [Google Scholar] [CrossRef]
  26. Li, H.; Durbin, R. Fast and Accurate Short Read Alignment with Burrows-Wheeler Transform. Bioinformatics 2009, 25, 1754–1760. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. Picard Toolkit Picard Toolkit. Broad Inst. 2019.
  28. Meyer, M.; Arsuaga, J.-L.; de Filippo, C.; Nagel, S.; Aximu-Petri, A.; Nickel, B.; Martínez, I.; Gracia, A.; Bermúdez de Castro, J.M.; Carbonell, E.; et al. Nuclear DNA Sequences from the Middle Pleistocene Sima de Los Huesos Hominins. Nature 2016, 531, 504–507. [Google Scholar] [CrossRef] [PubMed]
  29. Kuhlwilm, M.; Fontsere, C.; Han, S.; Alvarez-Estape, M.; Marques-Bonet, T. HuConTest: Testing Human Contamination in Great Ape Samples. Genome Biol. Evol. 2021, 13, evab117. [Google Scholar] [CrossRef]
  30. Danecek, P.; Bonfield, J.K.; Liddle, J.; Marshall, J.; Ohan, V.; Pollard, M.O.; Whitwham, A.; Keane, T.; McCarthy, S.A.; Davies, R.M.; et al. Twelve Years of SAMtools and BCFtools. Gigascience 2021, 10, giab008. [Google Scholar] [CrossRef] [PubMed]
  31. 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. Science 2015, 348, 242–245. [Google Scholar] [CrossRef] [Green Version]
  32. Pedersen, B.S.; Quinlan, A.R. Mosdepth: Quick Coverage Calculation for Genomes and Exomes. Bioinformatics 2018, 34, 867–868. [Google Scholar] [CrossRef] [Green Version]
  33. Bushnell, B. BBMap. 2015. Available online: https://sourceforge.net/projects/bbmap/ (accessed on 15 June 2022).
  34. Korneliussen, T.S.; Albrechtsen, A.; Nielsen, R. ANGSD: Analysis of Next Generation Sequencing Data. BMC Bioinform. 2014, 15, 356. [Google Scholar] [CrossRef] [Green Version]
  35. DePristo, M.A.; Banks, E.; Poplin, R.; Garimella, K.V.; Maguire, J.R.; Hartl, C.; Philippakis, A.A.; del Angel, G.; Rivas, M.A.; Hanna, M.; et al. A Framework for Variation Discovery and Genotyping Using Next-Generation DNA Sequencing Data. Nat. Genet. 2011, 43, 491–498. [Google Scholar] [CrossRef]
  36. McKenna, A.; Hanna, M.; Banks, E.; Sivachenko, A.; Cibulskis, K.; Kernytsky, A.; Garimella, K.; Altshuler, D.; Gabriel, S.; Daly, M.; et al. The Genome Analysis Toolkit: A MapReduce Framework for Analyzing next-Generation DNA Sequencing Data. Genome Res. 2010, 20, 1297–1303. [Google Scholar] [CrossRef] [Green Version]
  37. Cabrera, A.A.; Rey-Iglesia, A.; Louis, M.; Skovrind, M.; Westbury, M.V.; Lorenzen, E.D. How Low Can You Go? Introducing SeXY: Sex Identification from Low-Quantity Sequencing Data despite Lacking Assembled Sex Chromosomes. Ecol. Evol. 2022, 12, e9185. [Google Scholar] [CrossRef]
  38. Hanghøj, K.; Moltke, I.; Andersen, P.A.; Manica, A.; Korneliussen, T.S. Fast and Accurate Relatedness Estimation from High-Throughput Sequencing Data in the Presence of Inbreeding. Gigascience 2019, 8, giz034. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  39. Meisner, J.; Albrechtsen, A. Inferring Population Structure and Admixture Proportions in Low-Depth NGS Data. Genetics 2018, 210, 719–731. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  40. Skotte, L.; Korneliussen, T.S.; Albrechtsen, A. Estimating Individual Admixture Proportions from next Generation Sequencing Data. Genetics 2013, 195, 693–702. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  41. Garcia-Erill, G.; Albrechtsen, A. Evaluation of Model Fit of Inferred Admixture Proportions. Mol. Ecol. Resour. 2020, 20, 936–949. [Google Scholar] [CrossRef]
  42. R Core Team. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2021; Available online: https://www.R-project.org/ (accessed on 1 February 2023).
  43. Narasimhan, V.; Danecek, P.; Scally, A.; Xue, Y.; Tyler-Smith, C.; Durbin, R. BCFtools/RoH: A Hidden Markov Model Approach for Detecting Autozygosity from next-Generation Sequencing Data. Bioinformatics 2016, 32, 1749–1751. [Google Scholar] [CrossRef] [Green Version]
  44. Thompson, E.A. Identity by Descent: Variation in Meiosis, across Genomes, and in Populations. Genetics 2013, 194, 301–326. [Google Scholar] [CrossRef] [Green Version]
  45. Fontsere, C.; Kuhlwilm, M.; Morcillo-Suarez, C.; Alvarez-Estape, M.; Lester, J.D.; Gratton, P.; Schmidt, J.M.; Dieguez, P.; Aebischer, T.; Álvarez-Varona, P.; et al. Population Dynamics and Genetic Connectivity in Recent Chimpanzee History. Cell Genom. 2022, 2, 100133. [Google Scholar] [CrossRef]
  46. Van der Valk, T.; Díez-Del-Molino, D.; Marques-Bonet, T.; Guschanski, K.; Dalén, L. Historical Genomes Reveal the Genomic Consequences of Recent Population Decline in Eastern Gorillas. Curr. Biol. 2019, 29, 165–170.e6. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  47. Sánchez-Barreiro, F.; Gopalakrishnan, S.; Ramos-Madrigal, J.; Westbury, M.V.; de Manuel, M.; Margaryan, A.; Ciucani, M.M.; Vieira, F.G.; Patramanis, Y.; Kalthoff, D.C.; et al. Historical Population Declines Prompted Significant Genomic Erosion in the Northern and Southern White Rhinoceros (Ceratotherium simum). Mol. Ecol. 2021, 30, 6355–6369. [Google Scholar] [CrossRef] [PubMed]
  48. Li, H.; Durbin, R. Inference of Human Population History from Individual Whole-Genome Sequences. Nature 2011, 475, 493–496. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  49. Li, H.; Handsaker, B.; Wysoker, A.; Fennell, T.; Ruan, J.; Homer, N.; Marth, G.; Abecasis, G.; Durbin, R. 1000 Genome Project Data Processing Subgroup The Sequence Alignment/Map Format and SAMtools. Bioinformatics 2009, 25, 2078–2079. [Google Scholar] [CrossRef] [Green Version]
  50. Besenbacher, S.; Hvilsom, C.; Marques-Bonet, T.; Mailund, T.; Schierup, M.H. Direct Estimation of Mutations in Great Apes Reconciles Phylogenetic Dating. Nat. Ecol. Evol. 2019, 3, 286–292. [Google Scholar] [CrossRef] [PubMed]
  51. Santiago, E.; Novo, I.; Pardiñas, A.F.; Saura, M.; Wang, J.; Caballero, A. Recent Demographic History Inferred by High-Resolution Analysis of Linkage Disequilibrium. Mol. Biol. Evol. 2020, 37, 3642–3653. [Google Scholar] [CrossRef] [PubMed]
  52. Purcell, S.; Chang, C. PLINK 1.9. Available online: www.cog-genomics.org/plink/1.9/ (accessed on 26 October 2022).
  53. Chang, C.C.; Chow, C.C.; Tellier, L.C.; Vattikuti, S.; Purcell, S.M.; Lee, J.J. Second-Generation PLINK: Rising to the Challenge of Larger and Richer Datasets. Gigascience 2015, 4, s13742-015-0047–0048. [Google Scholar] [CrossRef]
  54. Purcell, S.; Neale, B.; Todd-Brown, K.; Thomas, L.; Ferreira, M.A.R.; Bender, D.; Maller, J.; Sklar, P.; de Bakker, P.I.W.; Daly, M.J.; et al. PLINK: A Tool Set for Whole-Genome Association and Population-Based Linkage Analyses. Am. J. Hum. Genet. 2007, 81, 559–575. [Google Scholar] [CrossRef] [Green Version]
  55. Stevison, L.S.; Woerner, A.E.; Kidd, J.M.; Kelley, J.L.; Veeramah, K.R.; McManus, K.F.; Great Ape Genome Project; Bustamante, C.D.; Hammer, M.F.; Wall, J.D. The Time Scale of Recombination Rate Evolution in Great Apes. Mol. Biol. Evol. 2016, 33, 928–945. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  56. Malinsky, M.; Matschiner, M.; Svardal, H. Dsuite—Fast D-Statistics and Related Admixture Evidence from VCF Files. Mol. Ecol. Resour. 2021, 21, 584–595. [Google Scholar] [CrossRef]
  57. Soraggi, S.; Wiuf, C.; Albrechtsen, A. Powerful Inference with the D-Statistic on Low-Coverage Whole-Genome Data. G3 2018, 8, 551–566. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  58. Maier, R.; Flegontov, P.; Flegontova, O.; Changmai, P.; Reich, D. On the Limits of Fitting Complex Models of Population History to Genetic Data. bioRxiv 2022. [Google Scholar] [CrossRef]
  59. Gerloff, U.; Hartung, B.; Fruth, B.; Hohmann, G.; Tautz, D. Intracommunity Relationships, Dispersal Pattern and Paternity Success in a Wild Living Community of Bonobos (Pan Paniscus) Determined from DNA Analysis of Faecal Samples. Proc. Biol. Sci. 1999, 266, 1189–1195. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  60. Nater, A.; Arora, N.; Greminger, M.P.; van Schaik, C.P.; Singleton, I.; Wich, S.A.; Fredriksson, G.; Perwitasari-Farajallah, D.; Pamungkas, J.; Krützen, M. Marked Population Structure and Recent Migration in the Critically Endangered Sumatran Orangutan (Pongo abelii). J. Hered. 2013, 104, 2–13. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  61. Fünfstück, T.; Vigilant, L. The Geographic Distribution of Genetic Diversity within Gorillas. Am. J. Primatol. 2015, 77, 974–985. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  62. Lester, J.D.; Vigilant, L.; Gratton, P.; McCarthy, M.S.; Barratt, C.D.; Dieguez, P.; Agbor, A.; Álvarez-Varona, P.; Angedakin, S.; Ayimisin, E.A.; et al. Recent Genetic Connectivity and Clinal Variation in Chimpanzees. Commun. Biol. 2021, 4, 283. [Google Scholar] [CrossRef]
  63. Khan, A.; Patel, K.; Bhattacharjee, S.; Sharma, S.; Chugani, A.N.; Sivaraman, K.; Hosawad, V.; Sahu, Y.K.; Reddy, G.V.; Ramakrishnan, U. Are Shed Hair Genomes the Most Effective Noninvasive Resource for Estimating Relationships in the Wild? Ecol. Evol. 2020, 10, 4583–4594. [Google Scholar] [CrossRef]
  64. Baas, P.; van der Valk, T.; Vigilant, L.; Ngobobo, U.; Binyinyi, E.; Nishuli, R.; Caillaud, D.; Guschanski, K. Population-Level Assessment of Genetic Diversity and Habitat Fragmentation in Critically Endangered Grauer’s Gorillas. Am. J. Phys. Anthropol. 2018, 165, 565–575. [Google Scholar] [CrossRef]
  65. Kardos, M.; Armstrong, E.E.; Fitzpatrick, S.W.; Hauser, S.; Hedrick, P.W.; Miller, J.M.; Tallmon, D.A.; Funk, W.C. The Crucial Role of Genome-Wide Genetic Variation in Conservation. Proc. Natl. Acad. Sci. USA 2021, 118, e2104642118. [Google Scholar] [CrossRef]
  66. Bertorelle, G.; Raffini, F.; Bosse, M.; Bortoluzzi, C.; Iannucci, A.; Trucchi, E.; Morales, H.E.; van Oosterhout, C. Genetic Load: Genomic Estimates and Applications in Non-Model Animals. Nat. Rev. Genet. 2022, 23, 492–503. [Google Scholar] [CrossRef]
  67. Robinson, J.; Kyriazis, C.C.; Yuan, S.C.; Lohmueller, K.E. Deleterious Variation in Natural Populations and Implications for Conservation Genetics. Annu. Rev. Anim. Biosci. 2022, 11, 93–114. [Google Scholar] [CrossRef] [PubMed]
  68. Ackermann, R.R.; Bishop, J.M. Morphological and Molecular Evidence Reveals Recent Hybridization between Gorilla Taxa. Evolution 2010, 64, 271–290. [Google Scholar] [CrossRef] [PubMed]
Genes 14 00743 g001 550
Figure 1. (A) Map showing the geographic distribution of the four gorilla subspecies. The Cross River and MN gorilla distribution ranges are from [2], and the other subspecies shape files were obtained from the IUCN. The shape files showing the specific Cross River gorilla populations (darker green) have been adapted with permission from [2] (Supplementary File). Inset at the top right of the map shows the Cross River distribution in more detail. Black dots indicate sites for which we have samples in the final dataset. Black crosses indicate sampled sites for which samples were discarded after quality control (Takamanda NP (National Park), CRNP Okwa Hills (Cross River National Park, Okwa Hills), and Mt. Doudou). Samples of unknown geographic origin are not represented in this map. (B) Principal component analysis (PCA) including hair samples (filled circles) and blood samples (empty circles) of the four subspecies. (C) PCA of western lowland gorilla hair samples with color indicating each site (Monte Alen, MoA; Ngaga Camp, Nga; Deng Deng, Den; Bai Hokou, Bai; unknown origin, Unk1). (D) PCA of Cross River gorilla hair samples with color indicating each site (Afi Mountain Wildlife Sanctuary, Afi; CRNP Boshi Extension, Bos; Mbe Mountains, Mbe; and Kagwene Gorilla Sanctuary, Kag). (E) Heatmap of pairwise FST of hair samples for both western gorilla species, CR and WL. Site labels are colored to indicate species, with red for WL gorilla sites and green for CR gorilla sites. Cell color indicates Fst value.
Figure 1. (A) Map showing the geographic distribution of the four gorilla subspecies. The Cross River and MN gorilla distribution ranges are from [2], and the other subspecies shape files were obtained from the IUCN. The shape files showing the specific Cross River gorilla populations (darker green) have been adapted with permission from [2] (Supplementary File). Inset at the top right of the map shows the Cross River distribution in more detail. Black dots indicate sites for which we have samples in the final dataset. Black crosses indicate sampled sites for which samples were discarded after quality control (Takamanda NP (National Park), CRNP Okwa Hills (Cross River National Park, Okwa Hills), and Mt. Doudou). Samples of unknown geographic origin are not represented in this map. (B) Principal component analysis (PCA) including hair samples (filled circles) and blood samples (empty circles) of the four subspecies. (C) PCA of western lowland gorilla hair samples with color indicating each site (Monte Alen, MoA; Ngaga Camp, Nga; Deng Deng, Den; Bai Hokou, Bai; unknown origin, Unk1). (D) PCA of Cross River gorilla hair samples with color indicating each site (Afi Mountain Wildlife Sanctuary, Afi; CRNP Boshi Extension, Bos; Mbe Mountains, Mbe; and Kagwene Gorilla Sanctuary, Kag). (E) Heatmap of pairwise FST of hair samples for both western gorilla species, CR and WL. Site labels are colored to indicate species, with red for WL gorilla sites and green for CR gorilla sites. Cell color indicates Fst value.
Genes 14 00743 g001
Genes 14 00743 g002 550
Figure 2. (A) FROH (percentage of the genome in runs of homozygosity (ROH) for different ROH size ranges (0.5–1.5 Mb, dark grey; 1.5–2.5 Mb, grey; 2.5–5 Mb, light grey; 5–10 Mb, yellow; and >10 Mb, blue). (B) Inference of recent effective population size from GONE for each subspecies (Cross River gorilla, CR; western lowland gorilla, WL; Grauer’s gorilla, GR and mountain gorilla, MN).
Figure 2. (A) FROH (percentage of the genome in runs of homozygosity (ROH) for different ROH size ranges (0.5–1.5 Mb, dark grey; 1.5–2.5 Mb, grey; 2.5–5 Mb, light grey; 5–10 Mb, yellow; and >10 Mb, blue). (B) Inference of recent effective population size from GONE for each subspecies (Cross River gorilla, CR; western lowland gorilla, WL; Grauer’s gorilla, GR and mountain gorilla, MN).
Genes 14 00743 g002
Genes 14 00743 g003 550
Figure 3. (A) Representation of the main results from D-statistics analysis between Cross River (CR) sites and western lowland (WL) gorilla regions. Arrows indicate which western lowland (WL) sites have higher allele sharing with Cross River (CR) gorillas. Arrow thickness indicates the level of gene flow, with the thicker arrow indicating higher gene flow between WL and CR sites. Empty dots indicate sites (CRNP Boshi Extension and Monte Alen) that have no positive signal of allele sharing with the other gorilla subspecies. (B) F-branch (fb(C)) analysis on excess allele sharing between species/populations/regions. The x-axis and y-axis trees are the species tree and the expanded species trees (with internal branches indicated with dotted lines), respectively. Grey-colored cells indicate comparisons for which gene flow could not be inferred based on the given tree topology. Red color indicates fb value; the darker color indicates greater allele sharing between the expanded tree branch (relative to its sister branch) (b) and the populations on the x-axis (C).
Figure 3. (A) Representation of the main results from D-statistics analysis between Cross River (CR) sites and western lowland (WL) gorilla regions. Arrows indicate which western lowland (WL) sites have higher allele sharing with Cross River (CR) gorillas. Arrow thickness indicates the level of gene flow, with the thicker arrow indicating higher gene flow between WL and CR sites. Empty dots indicate sites (CRNP Boshi Extension and Monte Alen) that have no positive signal of allele sharing with the other gorilla subspecies. (B) F-branch (fb(C)) analysis on excess allele sharing between species/populations/regions. The x-axis and y-axis trees are the species tree and the expanded species trees (with internal branches indicated with dotted lines), respectively. Grey-colored cells indicate comparisons for which gene flow could not be inferred based on the given tree topology. Red color indicates fb value; the darker color indicates greater allele sharing between the expanded tree branch (relative to its sister branch) (b) and the populations on the x-axis (C).
Genes 14 00743 g003
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Alvarez-Estape, M.; Pawar, H.; Fontsere, C.; Trujillo, A.E.; Gunson, J.L.; Bergl, R.A.; Bermejo, M.; Linder, J.M.; McFarland, K.; Oates, J.F.; et al. Past Connectivity but Recent Inbreeding in Cross River Gorillas Determined Using Whole Genomes from Single Hairs. Genes 2023, 14, 743. https://doi.org/10.3390/genes14030743

AMA Style

Alvarez-Estape M, Pawar H, Fontsere C, Trujillo AE, Gunson JL, Bergl RA, Bermejo M, Linder JM, McFarland K, Oates JF, et al. Past Connectivity but Recent Inbreeding in Cross River Gorillas Determined Using Whole Genomes from Single Hairs. Genes. 2023; 14(3):743. https://doi.org/10.3390/genes14030743

Chicago/Turabian Style

Alvarez-Estape, Marina, Harvinder Pawar, Claudia Fontsere, Amber E. Trujillo, Jessica L. Gunson, Richard A. Bergl, Magdalena Bermejo, Joshua M. Linder, Kelley McFarland, John F. Oates, and et al. 2023. "Past Connectivity but Recent Inbreeding in Cross River Gorillas Determined Using Whole Genomes from Single Hairs" Genes 14, no. 3: 743. https://doi.org/10.3390/genes14030743

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop