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. Author manuscript; available in PMC: 2026 Jan 6.
Published in final edited form as: Curr Biol. 2024 Dec 6;35(1):208–216.e5. doi: 10.1016/j.cub.2024.10.079

Genomic reconstruction reveals impact of population management strategies on modern Galápagos dogs

Gabriella J Spatola 1,2, Tatiana R Feuerborn 1, Jennifer A Betz 3, Reuben M Buckley 1, Gary K Ostrander 4, Emily V Dutrow 1,5, Alberto Velez 6, C Miguel Pinto 7, Alex C Harris 1, Jessica M Hale 1, Bruce D Barnett 8, Timothy A Mousseau 2,3,+, Elaine A Ostrander 1,+,*
PMCID: PMC11706705  NIHMSID: NIHMS2039166  PMID: 39644893

Summary

Free-breeding dogs have occupied the Galápagos islands at least since the 1830s, however, it was not until the 1900s that dog populations grew substantially, endangering wildlife and spreading disease1–4. In 1981, efforts to control the population size of free-roaming dogs began1. Yet there exist large free-roaming dog populations on the islands of Isabela and Santa Cruz whose ancestry has never been assessed on a genome-wide scale. We thus performed a complete genomic analysis of the current Galápagos dog population, as well as historical Galápagos dogs sampled between 1969 and 2003, testing for population structure, admixture, and shared ancestry. Our dataset included samples from 187 modern and six historical Galápagos dogs, together with whole genome sequence from over 2,000 modern purebred and village dogs. Our results indicate that modern Galápagos dogs are recently admixed with purebred dogs but show no evidence of a population bottleneck related to the culling. Additionally, identity by descent (IBD) analyses reveals evidence of shared shepherd-dog ancestry in the historical dogs. Overall, our results demonstrate that the 1980s culling of dogs was ineffective in controlling population size and did little to reduce genetic diversity, instead producing a stable and expanding population with genomic signatures of modern purebred dogs. The insights from this study can be used to improve population control strategies for the Galápagos Islands and other endangered endemic communities.

Blurb:

Dogs pose a major threat to native wildlife on the Galápagos islands, resulting in major population control efforts beginning in the 1980s. Using modern and historical dog samples, Spatola et al., perform detailed genomic analyses describing contributions from non-native dogs and demonstrating the need for improved population control strategies.

Results

Alien invasives are among the top five threats to biodiversity, with challenges posed by invasive species acutely felt on the Galápagos islands where, to date, 1,579 alien terrestrial and marine species have been introduced5–8. Dogs pose a major threat to native wildlife, destroying insular fauna including turtles, giant iguanas, and sea lions, all of which represent endangered or vulnerable populations1,2,9. Dogs were first reported on Isabela island in 1868, prior to the island’s first permanent settlement10,11, but were most likely introduced ~1835 by hunters who later abandoned them1,12. Further accounts from the early 1900s report feral dogs of unknown origin on Santa Cruz Island1,13. Despite efforts made to control population size, which began in 1981, the Galápagos dog population remains large and problematic, suggesting that the historical populations have either been replaced or rebounded since the 1980s. In the present study, we perform a complete genomic analysis of the current Galápagos dog population, comparing them to six historical Galápagos dog samples collected between 1981 and 2003, and over 2000 modern purebred and village dogs, to determine the impacts of population control initiatives.

Modern Galápagos dog populations are not genetically differentiated based on location

DNA from dogs living on two of the three most populous islands were collected in 2021: Santa Cruz (n = 149) and Isabela (n = 38) (Figure 1A). Additionally, 18 hunting dogs from rural locations on Santa Cruz Island were sampled. These are believed to have descended from a free-breeding population selectively bred by Galápagos residents for their hunting skills. All samples were initially genotyped using the Illumina CanineHD BeadChip, yielding 150,119 SNPs after filtering for quality.

Figure 1. Modern Galápagos dog populations are not genetically differentiated based on location.

Figure 1.

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A) Map of modern Galápagos dog sampling locations. B) PCA of modern Galápagos dogs from Isabela (orange), Santa Cruz (green), and hunting dogs (red). C) Phenotypic diversity and family relationships. Connected circles represent parent-offspring relationships. Circles connected with a dotted line represent dam-sire-offspring trio relationships. Circle fill shows the coat type of individuals. The circle sizes are proportional to the weight of individuals >1 year old. Unfilled circles represent dogs <1 year old. See also Figure S6 and Table S1.

To analyze population structure, we first performed a principal component analysis (PCA) (Figure 1B). PC1 and PC2 axes explained 20% and 8.2% of the variance within the dataset, respectively. The dog populations on Santa Cruz and Isabela islands do not appear to be genetically distinct, nor do they cluster based on their location in either axis (Figure 1B). Further, Galápagos hunting dogs do not appear to be genetically differentiated from free-breeding dogs on Santa Cruz or Isabela. In addition, free-roaming dogs on Isabela and Santa Cruz islands are phenotypically diverse, representing a wide range of ages, sizes, and coat colors/textures, thus suggesting a lack of natural or human selection for such traits (Figure 1C).

Identical-by-descent segment analyses reveal mixed ancestries and purebred admixture in modern Galápagos dog populations

To identify modern breed contributions to the current Galápagos populations we investigated breed-derived identical-by-descent (IBD) haplotype sharing between 187 modern Galápagos and 1,296 modern purebred dogs from 157 established breeds, genotyped at 150,119 genome-wide SNPs14. “Breed-derived haplotype regions” are defined as those with a frequency >25% within a given breed, and a frequency of <0.1% outside the breed’s assigned clade. Purebred dogs share extensive genetic variation within breeds and clades, but not across clades, allowing us to exclude regions shared outside of a breed’s clade in our definition of breed-derived haplotype regions14. Breed clades were assigned based on the placement of purebred individuals within a bootstrapped phylogeny where related breeds, comprised of 2–10 individuals each, were grouped into the same clade14,15. Breed-derived regions were identified and classified according to clade, allowing total breed-derived ancestry to be quantified in each individual Galápagos dog14,15 (Figure 2A).

Figure 2. Identical-by-descent segment analyses reveal mixed ancestries and purebred admixture in modern Galápagos dog populations.

Figure 2.

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A) Individual breed-derived ancestry of modern Galápagos dogs. Each vertical bar represents a single Galápagos dog. Each stacked color bar represents the length of breed-derived regions from corresponding clades. On the x-axis, modern Galápagos dogs are grouped by location and sorted from most to least breed-derived regions going left to right across each population. Black triangles mark dogs with predominant ancestry proportions. B) Box and whisker plot showing breed-derived sharing within clades compared to breed-derived sharing within the modern Galápagos dogs. Individual dogs highlighted in red are those with the predominant ancestry proportions in the Galápagos populations. C) Longest ten identical-by-descent segments shared with purebred dogs for each individual modern Galápagos dog. Individuals in red have predominant ancestry proportions. The dotted red line indicates the threshold for dogs with segments that are longer than one standard deviation above the mean population length. See also Figures S1, S2, S7, and Table S1.

All three Galápagos populations share the most breed-derived regions with the same three clades: European mastiffs and related breeds (Mastiff clade), Asian spitz and related breeds (Asian spitz clade), and retrievers, pointers, spaniels and related breeds (Retriever clade). The purebred clade contributing the most to the Isabela dog population was the Asian spitz, which shared, on average, 151 Mb of breed-derived haplotypes. By comparison, dogs from Santa Cruz, including hunting dogs, share the most breed-derived haplotypes with the European mastiff clade, averaging 142 Mb of breed-derived haplotypes per dog. Altogether, this suggests either that dogs from Santa Cruz and Isabela come from diverse purebred founding populations, or that recent admixture within the modern populations has occurred with different breed types.

To determine which individuals from the modern Galápagos populations have recent purebred ancestry, we identified those exhibiting above average sharing with dogs from a single clade. Z-scores were calculated based on the distribution of maximum breed-derived sharing values for each of 187 Galápagos dogs. Of those, 21 from Santa Cruz and seven from Isabela had maximum ancestry Z-scores that were at least one standard deviation above the population mean and were, thus, considered to have “predominant ancestry proportions” (Figure 2A–B). Galápagos dogs found to have predominant ancestry proportions also showed similar levels of breed-derived sharing as purebred dogs within that clade, suggesting that these individuals are likely purebred dogs themselves, have purebred ancestry, or were recently admixed with purebreds (Figure 2B). To test the latter possibility, we examined the distribution of the ten longest IBD segments shared between each modern Galápagos dog and purebred dogs (Figure 2C). Ten out of the 28 dogs with predominant ancestry proportions had long IBD segments that were at least one standard deviation above the population mean in this distribution, providing further evidence of recent purebred admixture within the population. It is likely that admixed dogs, or recent relatives of these dogs, were imported via tourism or trade from the South American mainland.

Modern Galápagos dog populations show no evidence of genetic bottlenecks

If the modern Galápagos dogs are, in fact, primarily related to the historical Galápagos dog population, we would expect to find signatures of a bottleneck related to the culling in the genomes of the modern individuals. To test this we used the software ASCEND, which estimates the age and intensity of founder events or bottlenecks in a population using genotype data and a recombination map16. Specifically, ASCEND calculates bottleneck intensities (If) where If = duration of bottleneck/2*population size and is proportional to the probability of coalescence during the bottleneck16. We observed insignificant bottleneck intensities (If < 5%) for all modern Galápagos populations, with If values of 0.5% for Santa Cruz, 4.6% for hunting dogs, and 2.3% for Isabela, revealing no evidence of a bottleneck following the culling (Figure S1-A).

We also examined genome-wide heterozygosity of the modern Galápagos dogs compared to other publicly available village dog populations, including 783 dogs from 51 populations worldwide17. Of the 51 village dog populations we compared, the lowest average genome wide heterozygosity was 0.294 and the highest was 0.367 (Figure S1-B). Surprisingly, despite the targeted culling operation, modern Galápagos populations have genome-wide heterozygosity estimates between 0.22 and 0.39, falling within the range of heterozygosity estimates for all tested village dog populations. The absence of a reduction in genome-wide heterozygosity also argues that a population bottleneck did not occur. Thus, regardless of how the population restructured itself following the culling, a large amount of genetic diversity remained.

Whole genome sequence-based analysis reveals differential heterozygosity in historical and modern Galápagos dog populations

Of the 187 Galápagos dogs genotyped on the Illumina CanineHD BeadChip, we randomly selected ten dogs each from Santa Cruz and Isabela, and ten Galápagos hunting dogs for whole genome sequencing (WGS) at 20x coverage. Additionally, four historical Galápagos dog samples, including one each from Santa Cruz (2003), Floreana (1983), Isabela (1981) and San Cristóbal (2003) underwent WGS (Figures 4C and S4).

Figure 4. Identical-by-descent segment analyses reveal shepherd-dog ancestry in historical Galápagos dogs.

Figure 4.

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A) Galápagos islands where historical dog samples were collected. Map adapted from Gaba and Chadwick22. B) Breeds originally introduced to the Galápagos islands. C) Timeline of events on the Galápagos islands. Events in colored boxes correspond to colored locations in A. D) IBD haplotype sharing analysis including historical Galápagos dogs and modern purebred dogs that have the greatest sharing with historical dogs. Black bars represent the range of haplotype sharing between purebred individuals that belong to the corresponding breeds on the x-axis. The black dot represents the average sharing within each breed. Diamonds represent the average total length of haplotype sharing between each historical Galápagos individual and each breed. Historical dogs are colored according to their location within the Galápagos islands. See also Figures S4, S5, S8, and Table S1.

Individual genetic diversity was analyzed by calculating rates of heterozygosity and runs of homozygosity (ROH) for each of the four historical Galápagos dogs, 30 whole genome sequenced modern Galápagos dogs, and 2231 purebred, mixed breed, and village dogs (Figure 3A). The historical dog from Isabela had the lowest genome-wide rate of heterozygosity (0.17), and that from Santa Cruz had the highest (0.28) (Figure 3A). This pattern is also reflected in ROHs, where the historical dog from Isabela had the largest fraction of the genome in ROHs and the dog from Santa Cruz had the smallest (Figure S3). Compared to modern Galápagos dogs, historical dogs had larger fractions of the genome in long ROHs overall, except for the historical dog from Santa Cruz (Figure S3). That dog had the highest heterozygosity of any dog in the dataset, indicating that this individual likely descended from a large, outbred founding population. In aggregate, this argues that historical Galápagos dogs have distinct population histories.

Figure 3. Whole genome sequence-based analysis reveals differential heterozygosity and diverse mtDNA haplotypes in historical and modern Galápagos dog populations.

Figure 3.

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A) Rates of heterozygosity for whole genome sequenced modern and historic Galápagos dogs, mixed breed dogs, purebred dogs, and village dogs. B) Mitochondrial haplotype networks including modern and historical Galápagos dogs. Tick marks represent the number of mutations differentiating haplotypes. Circles are proportional to the number of individuals with that haplotype. Dotted line connections between circles indicate where mitochondrial haplotypes are shared between historical and modern Galápagos dogs. See also Figures S3, S4, S5, and Table S1.

Historical and modern Galápagos dogs carry diverse mtDNA haplotypes

A previous study of historic Galápagos dogs from Isabela culled in the 1980s reported finding the A22 mitochondrial haplotype in 50 of 54 dogs12. We analyzed mitochondrial haplotypes from 152 modern dogs from Santa Cruz and Isabela, four historical Galápagos dogs described above, and an additional historical Galápagos dog from Floreana (Figure 3B). We identified 18 mitochondrial haplotypes within the 157 modern and historical dogs in the dataset. The B1, C16, A19, and A17 haplotypes were predominant, accounting for approximately 25%, 19%, 15%, and 14% of the modern dataset, respectively. Two modern dogs from Isabela had the A27 haplotype and one historical sample from Isabela had the A22 haplotype. Twenty-one dogs with seven different haplotypes were only found in the modern Santa Cruz population. All five historical samples had distinct haplotypes, three of which matched several individuals in the modern dataset (Figure 3B). This argues, as above, that the historical samples are diverse and likely reflect different ancestral backgrounds.

Identical-by-descent segment analyses reveal shepherd-dog ancestry in historical Galápagos dogs

Dogs introduced to the Galápagos in 1835 were reportedly of European origin, including the Ibizan and Pharoah Hounds, Pointer, German Shepherd Dog, Great Dane, and Borzoi (Figure 4B–C)1. To test the breed ancestry of historical dogs, we performed an IBD haplotype sharing analysis, comparing four historical dogs to a dataset of 1,757 modern purebred dogs representing 252 breeds, including the six suggested to have been original contributors. We excluded wild canids, village dogs from outside the Galápagos, and breeds with <4 individuals, thus ensuring accurate representation from each breed. Initially, total pairwise identity-by-descent for each combination of historical and modern purebred dog was determined. Next, we calculated the average total sharing of each historical Galápagos dog to each modern breed and determined the upper threshold for outliers in the distribution (41 Mb), providing a cutoff for significant sharing relative to the overall sharing found within the historical dogs. Historical Galápagos dogs shared more than 41 Mb IBD, on average, with 23 of the 252 breeds analyzed (Figure 4D). All four historical samples shared significantly with white Swiss shepherd dogs and three of the four shared significantly with German shepherd dogs, Czechoslovakian wolfdogs, and Bohemian shepherds (Figure 4D). As expected, historical dogs’ IBD sharing with these breeds falls outside the range of within breed sharing. Therefore, it is unlikely that any of the historical Galápagos dogs were purebred or recently admixed with purebred dogs, and significant sharing in this case reflects ancestral relationships.

Discussion

The Galápagos islands are well known for their preservation of unique plant and animal species, attracting naturalists and tourists alike. They are part of Ecuador’s National Park system and are a UNESCO World Heritage Site and, as such, they have been protected since 1959. In 1981 authorities initiated their first attempt at population control of free-roaming dogs1. In 2003, legislation was passed to regulate the importation of introduced species to the Galápagos islands, including dogs18. Although their importation remains prohibited today, a large population of free-roaming dogs still lives on several islands. In this study, we uncover the ancestry of modern Galápagos dogs and compare it to that of historical samples from free-breeding dogs that lived on the islands between 1981 and 2003.

Some modern Galápagos dogs are largely related to a single clade (predominant ancestry), suggesting that these individuals are likely purebred or recently admixed with purebred dogs (Figure 2A). Additionally, individuals with predominant ancestry have reduced heterozygosity compared to other free-breeding dog populations (Figure S1-B) and share long IBD segments with purebred dogs (Figure 2C), providing further evidence of recent purebred ancestry. Altogether, this suggests that modern Galápagos dog populations did not solely descend from historical free-breeding populations, and some purebred dogs have been recently imported, likely through immigration, tourism or commerce. These results suggest that importation policies have been ineffective in barring the introduction of pets.

Unexpectedly, modern populations lack any signatures of a population bottleneck (Figure S1-A). This argues that either the number culled was not sufficient, or remaining diversity together with admixture from illegal importation of dogs was sufficient to quickly reconstitute a diverse population. Similar results were found in Brazil, where dog-culling programs initiated to reduce the spread of canine Leishmania infection failed, partially due to rapid population replacement alongside culling efforts19. It is possible that some dogs have local ancestry related to the historic populations, but the overall signature within the population is lost because Galápagos dogs are not part of a closed breeding pool.

WGS of historical samples allowed us to complete an in-depth investigation of the dogs’ ancestry. IBD analyses showed that each of the four historic Galápagos dogs had elevated sharing with shepherd breeds, including the German shepherd dog (Figure 4D), suggesting that the historic population was markedly descended from a shepherd lineage. This is consistent with early reports claiming German shepherd dogs as one of the first breeds brought to Isabela (1835) (Figure 4B)1. Although the German shepherd dog breed was not recognized by the AKC until ~1908, the introduced shepherd dog could have been an early predecessor of the modern breed. Alternatively, it is possible that other dogs were introduced at multiple other timepoints throughout colonization of the islands, such as with the arrival of whalers and pirate vessels beginning in the late 1500s. Further, different rates of heterozygosity and mitochondrial haplotypes among historical dogs suggests that they likely have separate population histories, and do not descend from the same small founding population. Without representative genomes from pre-1900s Galápagos dogs, it is difficult to know for certain.

In effectively recreating the recent history of dogs on the Galápagos islands, we better understand the long-term impact of introducing non-native species to unique environments. In the past, it has been largely impossible to assess effectiveness of management and control strategies for alien invasives8, accounting in part for the poor performance of many conservation interventions. We provide evidence suggesting that neither large scale extermination nor extensive importation policy and policing have been effective at controlling dog populations. Realistic modeling of various free-roaming dog population control methods suggests that targeted fertility control could be more effective than culling at reducing population sizes20. In recent years, efforts have shifted toward sterilization of free-roaming dogs and pets on the Galápagos islands, although early evidence based on census data suggests the current sterilization efforts are not enough to keep the population stable21. Additionally, more studies are needed to understand how culling influenced the abundance of native prey species. We suggest that the information provided in this study can be used to better plan population control strategies across the Galápagos Islands and other threatened endemic communities across the globe.

Resource availability

Lead Contact

Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Elaine. A. Ostrander (eostrand@nih.gov).

Materials Availability

All data in this paper are publicly available as follows: Modern Galápagos dog SNP genotypes generated using the Illumina Chip can be found with GEO: GSE276576. Purebred dog SNP genotypes generated using the Illumina Chip can be found with GEO: GSE90441, GSE83160, GSE70454, and GSE9673614. Village dog SNP genotypes generated using arrays can be found on Dryad: https://doi.org/10.5061/dryad.v9t5h17. All metadata and accession numbers for whole genome sequenced samples are located in Table S1. Photographs of the modern Galapágos dogs are available upon request to T.A. Mousseau (mousseau@sc.edu).

Data and Code Availability

All data reported in this paper will be shared by the lead contact upon request. This paper does not report original code. Any additional information required to reanalyze the data reported in this study is available from the lead contact upon request.

STAR Methods

Experimental model and subject details

Modern Galápagos dogs

Blood samples from 38 dogs on Isabela, 131 on Santa Cruz, and 18 hunting dogs were collected alongside a spay, neuter, and vaccination clinic run by the nonprofit organizations, Visiting Veterinarians International and Animal Balance, in 2021. As dogs were brought in for wellness exams and sterilization surgeries, blood samples were collected. Data collected for this study were gathered opportunistically while animals were under the care of veterinary professionals and is hence exempt from Institutional Animal Care and Use Committee (IACUC) approval. Export permits were provided by ABG.

Historical Galápagos dogs

Samples from historical Galápagos dogs were obtained from the Charles Darwin Research Station (CDRS). Two historical samples from Floreana were both collected in 1983 from free-roaming farm dogs. One historical sample was collected in 2003 from a street dog in Puerto Ayora on Santa Cruz Island, while two others, one sampled from Santa Cruz in 1969 and one from San Cristóbal in 2003, were from feral dogs that died apparently of natural causes. Finally, one historical sample found in 1981 on Isabela Island was from a dog that was culled (Figure 4A–C). The six historical Galápagos samples include three tooth samples, one bone tissue sample, one paw tissue sample, and one brain tissue sample.

Methods Details

Modern sample DNA extraction and genotyping

Modern Galápagos dog blood samples were preserved in acid citrate dextrose anticoagulant tubes and sent to the National Human Genome Research Institute (NHGRI) for processing. DNA was isolated using a phenol chloroform extraction protocol23. Thirty-eight dogs from Isabela, 131 from Santa Cruz, and 18 hunting dogs were genotyped using the Illumina CanineHD Whole-Genome Genotyping BeadChip, providing data from 170,000 single-nucleotide polymorphism (SNP) markers per sample. In addition, 10 samples from Isabela and 20 from Santa Cruz, including 10 Galápagos hunting dogs, underwent WGS at 20x coverage by the National Institutes of Health Intramural Sequencing Center (NISC). Modern Galápagos dogs that underwent WGS were selected randomly, excluding samples with predominant ancestry proportions in an attempt to exclude dogs with obvious non-native ancestry.

Genotype calls for the 187 Galápagos dogs were made with GenomeStudio (v2011.1) using genotyping module v1.9.4 (Illumina). To compare the Galápagos to purebred dogs the dataset was merged with a publicly available dataset of 1,296 purebred dogs representing 157 breeds14 that were also genotyped using the same BeadChip. Datasets were merged using PLINK (v1.9)24. Sites missing more than 10% of data were excluded, yielding a final dataset of 1,532 individuals genotyped at 150,119 whole-genome SNP markers with an overall genotyping rate of 99.8%.

Historical sample DNA extraction and genotyping

Upon receipt by NHGRI, samples were sent to Daicel Arbor Biosciences (Washtenaw County, MI, https://arborbiosci.com/) for DNA extraction and library preparation using a degraded DNA extraction protocol and ancient DNA library preparation method. Libraries were then sequenced at the NISC, yielding depths that ranged from 0.10 – 4.57x coverage (Figure S4). We imputed genotypes for the historical Galápagos samples with GLIMPSE25 using a panel of 1,929 published sequences from the Dog10K consortium as a reference26. Imputed sites were then filtered to exclude sites with a reference allele frequency < 0.01 or INFO scores < 0.90. The final set of imputed sites were extracted from a merged dataset of sequences published by the NHGRI Dog Genome Project and Dog10K Consortium26. Additionally, we extracted the imputed sites from the dataset of 30 modern Galápagos dogs. Both modern WGS datasets were merged with the historical Galápagos dataset, yielding a final set of 2,293 modern dogs and wild canids, 30 modern Galápagos dogs, and six historical Galápagos dogs genotyped at 1,104,110 biallelic SNPs. Two of the historical Galápagos samples were excluded from our analyses, as one sample from Santa Cruz had very low coverage at 0.10x and one from Floreana had uneven coverage across chromosomes, and both samples displayed an excess of sites with 0x coverage (Figure S4). The lowest quality sequences were both from tooth samples. Therefore, our nuclear analyses included four historical samples, each from a different island. In our mitochondrial haplotype analyses we include five historical samples instead of four because one sample with uneven whole-genome sequence coverage produced quality genotypes when the mitochondrial hypervariable region was amplified.

Quantification and Statistical Analysis

Dimensionality reduction

We performed PCA using the modern dataset of 38 dogs from Isabela, 131 from Santa Cruz, and an additional 18 hunting dogs from Santa Cruz using PLINK with –pca (Figure 1B). Explained variance was calculated using prcomp (R v4.0.0 Stats package). PCAs including the historical Galápagos dogs were calculated with smartPCA27(Figure S5-A–B). Imputed genotypes from the historical Galápagos dogs were projected onto the variation in the modern dogs after filtering for sites in linkage disequilibrium, sites with a maximum of 50% missingness, maf 0.01, and removing individuals with greater than 90% missing sites (Figure S5-A). Figure S5-B included breed and village dogs worldwide representing the large amount of genomic variation within dogs. Figure S5-A included only modern dogs from the Galápagos that underwent WGS with the historical genomes projected onto the variation.

Family structure

Family relationships among the 38 dogs from Isabela, 131 dogs from Santa Cruz, and 18 hunting dogs were analyzed using the KING (v2.2.7) software toolset for family inference28. The --related flag was used to calculate pairwise kinship estimates and produce KING relationship inferences for each pair of dogs. A zero identity-by-state (IBS) cutoff value of 0.0004 was used to identify parent-offspring relationships. Parent-offspring pairs had kinship values between 0.2170 and 0.2781 (Figure S6). We identified 16 families consisting of at least one parent-offspring pair from Santa Cruz and four similar families from Isabela (Figure 1C), with 50 and 75% of families found on Santa Cruz and Isabela, respectively, consisting of a single parent-offspring pair. In total, 43 out of 149 dogs from Santa Cruz and 9 out of 38 dogs from Isabela had a first-degree family relationship with another dog in the population.

After parent offspring pairs were identified, we quantified Mendel errors for every possible sire-dam-offspring trio. Taking sex into account, a list of all possible trios was created using a custom R script and then Mendelian errors were calculated for each using PLINK --mendel. We then plotted a frequency distribution of Mendelian errors for all possible trios. True trios appeared as clear outliers in the distribution with less than 60 Mendelian errors. Possible trios that were not identified as true carried 5,631– 63,718 Mendel errors.

Veterinarians from two non-profit animal welfare organizations (Visiting Veterinarians International; Animal Balance) estimated the ages of sampled dogs from Santa Cruz to be between three months and twelve years, while ages ranged from three months to five years among dogs from Isabela. Santa Cruz dog weights ranged from 8.6 to 95.1 lbs., while Isabela dogs were slightly larger, ranging from 11.7 to 65.1 lbs.

Bottleneck analyses

Heterozygosity estimates were calculated using the dataset of modern Galápagos dogs and a publicly available dataset with 783 village dogs representing 51 populations worldwide17 (Figure S1-B). After merging the modern Galápagos dataset with the worldwide village dog dataset, the final dataset included 123,722 SNPs with a genotyping rate of 0.99. Average rates of heterozygosity were calculated using PLINK v1.9 with --het, producing observed counts of autosomal homozygosity across all loci for each individual. This was then used to calculate rates of heterozygosity per individual using the formula ((total # of sites – observed count of hom. sites / total # of sites).

We used the software program ASCEND16 to test for evidence of a population bottleneck in the modern Galápagos dog populations, running ASCEND separately for each modern Galápagos population and setting the outgroup population size equal to that of the test population (Figure S1-A). We used a random outgroup for each run, thus the outgroup was randomly selected from the input file, which included 38 dogs from Isabela, 18 hunting dogs, 131 dogs from Santa Cruz, and 1,296 purebred dogs representing 157 breeds14, making sure the random outgroup did not overlap with any individuals from the test population. We used a bin size of 0.04, a minimum distance of 0.001, and maximum distance of 0.2 for each run.

Breed-derived ancestry

Individual ancestry analyses were performed according to methods described previously15 using the dataset of 187 modern Galápagos dogs and 1,296 purebred dogs (Figure 2A). IBD genomic segments were identified using BEAGLE (v4.1)29 and subset to only those showing IBD sharing between a Galápagos dog and a purebred dog. Genomic regions were assigned as breed-derived from a particular breed by the following criteria: 1) the IBD segment is shared between Galápagos dogs and purebred dogs and is present at a haplotype frequency of >25% within the relevant breed and 2) the IBD segment is present with a haplotype frequency of <0.1% in purebred dogs from outside of that breed’s clade. Each breed-derived genomic segment was assigned a clade label based on where that breed appears on the bootstrapped phylogenetic tree composed of only purebred dogs in this dataset15. Clade rather than breed was used to analyze breed-derived ancestry because there is extensive sharing between breeds within the same clade but not outside of the clade, making it impossible to assign the precise breed origin of a given haplotype14.

To compare sharing of purebred dogs within assigned clades to sharing of Galápagos dogs with dogs from each clade, we plotted the total breed-derived IBD sharing length of each Galápagos dog with each clade and the total breed-derived IBD sharing length of each purebred dog to the clade that it was assigned based on phylogenetic analysis (Fig. 2B). We tested to see if sharing of Galápagos dogs to each clade was significantly greater than sharing of purebred dogs within assigned clades using a one-sided Wilcoxon Rank-Sum test in R (v4.0.3).

Individual ancestry analyses were similarly performed for all purebred dogs, treating each clade of purebred dogs as a separate population (Figure S7). IBD genomic segments were identified using BEAGLE (v4.1)29 and subset to only those showing IBD sharing between purebred dogs. Genomic regions were assigned as breed-derived from a particular breed by the following criteria: 1) the IBD segment is shared between purebred dogs and is present at a haplotype frequency of >25% within the relevant breed, and 2) the IBD segment is present with a haplotype frequency of <0.1% in purebred dogs from outside of that breed’s clade. The breed-derived ancestry of each individual purebred dog is plotted to show the range of sharing within each clade (Figure S7).

Registering bodies such as the American Kennel Club (AKC) or Fédération Cynologique Internationale (FCI) require that breed dogs with membership have registered sires and dams with ancestry that can be traced back to the parent breed through several generations. Therefore, to determine which individuals from the Galápagos population have recent purebred ancestry we identified those that have above average sharing with a single purebred clade. Z-scores were calculated in R (v4.0.3) based on the distribution of maximum breed-derived sharing values for each dog. Z-scores >1 represent individuals with maximum breed-derived sharing values more than one standard deviation above the mean and were therefore labeled using the term “predominant ancestry” (Figures 2A–C, S2-B). Additionally, we examined the ten longest IBD segments shared between each modern Galápagos dog and purebred dogs to test for recent admixture (Figure 2C). IBD segments were included in this analysis if they were shared between Galápagos dogs and purebred dogs and if they were present at a haplotype frequency of >25% within the relevant breed. We calculated the average length of the ten longest IBD segments for each Galápagos dog and assigned them Z-scores using R (v4.0.3) to investigate the population distribution. Using the same methodology, we examined the ten longest IBD segments shared between purebred dogs, in order to contextualize long IBD sharing found within the Galápagos population (Figure S2).

Historical dog top breed sharing

Ancestry related to modern breeds was calculated for each historical Galápagos dog using the merged dataset of 2291 modern purebred dogs, village dogs, and wild canids. BEAGLE (v4.1) was used to identify pairwise IBD segments between all individuals. We subset the IBD segments to only those shared between purebred dogs with at least four individuals per breed and historical Galápagos dogs. First, we took the sum of the length of IBD segments shared between each individual historical Galápagos dog and each purebred dog. The total length of shared IBD with each historical Galápagos dog was then averaged for each breed. The average total length of sharing between each Galápagos dog and individuals from each breed was used to calculate the threshold for outliers in the distribution using the formula (1.5 × IQR) + Q3. IQR indicates the interquartile range calculated with IQR() function in R (v4.0.3). Q3 indicates the third quartile value found using summary() in R. For historical Galápagos dogs, the threshold for outliers was equal to 41 Mb. Any breeds sharing >41 Mb, on average, with a historical Galápagos individual were considered significant relative to the distribution of sharing, and therefore included in our analysis of top breed sharing (Figure 4D). We also calculated the total length of sharing between dogs within each breed in order to ascertain the range of sharing within breeds. Maximum and minimum sharing between individuals within breeds were then calculated and plotted against the average total sharing of each historical Galápagos individual to each breed (Figure 4D).

Historical dog rates of heterozygosity

Heterozygosity estimates were calculated using the whole genome sequence dataset of four historic and 30 modern Galápagos dogs, in addition to 2231 purebred, mixed breed, and village dogs from a merged dataset of sequences published by the NHGRI Dog Genome Project and Dog10K Consortium26 (Figure 3A). The final dataset included 1,101,329 SNPs. As described above, average rates of heterozygosity were calculated using PLINK v1.9 with --het, and then output was used to calculate rates of heterozygosity per individual using the formula ((total # of sites – observed count of hom. sites / total # of sites).

Runs of homozygosity

ROH were identified using PLINK v1.9 with the following parameters: het, homozyg, homozyg-density 50, homozyg-gap 1000, homozyg-kb 200, homozyg-snp 50, homozyg-window-het 3, homozyg-window-missing 2, and homozyg-window-snp 100. ROH were separated into bins based on length. We used a minimum length of 5154.6Kb to reflect the expected length of long ROH when the underlying IBD haplotypes had a most recent common ancestor 10 generations ago, calculated using the formula (100/(2*g))cM/0.97 cM/Mb where g is 10 generations and 0.97 is the sex-averaged genome-wide recombination rate in dogs30,31. Similarly, we used a minimum length of 1718.2KB to reflect the expected length of medium ROH, where g = 30 generations. Any ROH less than 1718.2KB in length was considered small (Figure S3).

mtDNA analyses

Primers were used to amplify the hypervariable-1 region (HV1) within the D-loop of the modern Galápagos dog mitochondrial genomes, including forward primer H15422: 5′-CTCTTGCTCCACCATCAGC-3′ and reverse primer L15781: 5′-GTAAGAACCAGATGCCAGG-3′. The second primer set included forward primer H15693 5′-AATAAGGGCTTAATCACCATGC-3′ and reverse primer L16106: 5′-AAACTATATGTCCTGAAACC-3′32. PCR was carried out on a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA) using AmpliTaq Gold (Thermo Fisher, Waltham, MA). A step-down PCR protocol was used, proceeding as follows: 95°C for five minutes, 95°C for 45 seconds, 65°C-55°C for 45 seconds (reducing by −0.5°C per cycle), then 72°C for 45 seconds, for 20 cycles. This protocol was then repeated for an additional 20 cycles without reducing the annealing temperature (55°C) for 45s. Finally, we carried out an elongation step at 72°C for 10 minutes. After PCR amplification was complete, 0.5 μl Exonuclease I, one μl Shrimp Alkaline Phosphatase (SAP), and 0.5 μl of 10X SAP Buffer was added to each reaction and then placed onto the thermocycler for 15 minutes at 37°C, then 15 minutes at 80°C to inactivate the enzyme. Resulting samples were diluted two-fold. One μl of amplified DNA was then used in a reaction with 0.3 μl BigDye (v3.1, Applied Biosystems, Foster City, CA), one μl BigDye buffer, 0.1 μl of primer, and water, totaling five μl in volume. Samples were amplified at 96°C for one minute, then 25 cycles of 96°C for 10 seconds, 50°C for five seconds, and 60°C for four minutes with an indefinite holding temperature of 4°C. PCR amplified DNA was run on a 3730xl sequence analyzer (Applied Biosystems). Variants were genotyped using Sequencher 5.4.6 (GeneCodes Corporation, Ann Arbor, MI). All 187 modern Galápagos samples were Sanger sequenced.

The reads from the 30 modern and six historical Galápagos dogs that underwent WGS were aligned to the dog mitochondrial reference genome (NC002008.4) with bowtie233. Consensus sequence calling from the reads aligned to the mitochondrial reference was performed with htsbox (https://github.com/lh3/htsbox). Whole mitochondrial genome sequences were aligned to the Sanger sequencing dataset with MAFFT34. Following the multiple sequence alignment the overlapping HV1 region was extracted from the combined dataset, and all sequences with missing sites were removed from the dataset. For haplogroup assignment, identical sequences were identified with DNAcollapser35. The collapsed haplogroup sequences were input into NCBI BLAST to label the haplogroups.

D-Statistics

To test for gene flow between purebred dogs and the historical dogs of the Galápagos D-statistics were calculated using ADMIXTOOLS v7.0.236 with the WGS dataset as input. We called pseudo-haploid by randomly selecting one allele at each site for each individual. The dataset was then filtered to exclude sites with >50% missingness, a minor allele count of three, and filtered for linkage disequilibrium with PLINK1v1.9 (--indep 50 5 2). We fixed the outgroup as the Coyote01 sample for all tests in the following formula: D(Out, Source; Target, Target Sister). Z-score <− 3.3 indicates gene flow occurred between the source and the target and Z-score > 3.3 indicates gene flow occurred between the source and the target sister. Dogs from 174 dog breeds, spanning 21 breed groups, were tested as potential sources and all tests were set with modern Galápagos dogs as the sister (Figure S8).

Supplementary Material

1

Table S1. Metadata and accession numbers for samples used in this study, related to Figures 1– 4.

2

Key resources table

REAGENT or RESOURCE SOURCE IDENTIFIER
Deposited data
Illumina Canine HD SNP genotypes of modern Galápagos dogs This study GEO: GSE276576
Illumina Canine HD SNP genotypes of published purebred dogs Parker et al.14 GEO: GSE90441, GSE83160, GSE70454, and GSE96736
Village dog SNP genotypes Shannon et al.17 Dryad: https://doi.org/10.5061/dryad.v9t5h
Published whole genome sequences See Table S1 See Table S1
Galápagos dog whole genome sequences This study See Table S1
Software and algorithms
GenomeStudio (v2011.1) Illumina https://support.illumina.com/array/array_software/genomestudio/downloads.html
PLINK (v1.9) Chang et al.24 https://www.coggenomics.org/plink2/
GLIMPSE Rubinacci et al.25 https://odelaneau.github.io/GLIMPSE/
R (v4.0.0, 4.0.3) R Core Team https://www.rproject.org/
smartPCA Patterson et al.27 https://github.com/ChristianHuber/smartsnp/blob/master/R/smart_pca.R
Bcftools Li et al.37 https://www.htslib.org/download/
Python (v3.7) Python Software Foundation https://docs.python.org/3.7/reference/
KING (v2.27) Manichaikul et al.28 https://www.kingrelatedness.com/
ASCEND Tournebize et al.16 https://github.com/sunyatin/ASCEND
BEAGLE (v4.1) Browning et al.29 https://faculty.washington.edu/browning/beagle/b4_1.html
Sequencher (v5.4.6) Gene Codes Corporation https://www.genecodes.com/
Bowtie2 Langmead et al.33 https://bowtiebio.sourceforge.net/bowtie2/index.shtml
htsbox Li et al. https://github.com/lh3/htsbox
MAFFT (v7) Katoh et al.34 https://mafft.cbrc.jp/alignment/software/
DNAcollapser Villesen et al.35 https://usersbirc.au.dk/~palle/php/fabox/dnacollapser.php
NCBI BLAST NCBI https://blast.ncbi.nlm.nih.gov/Blast.cgi
ADMIXTOOLS (v7.0.2) Patterson et al.36 https://github.com/DReichLab/AdmixTools
Oligonucleotides
H15422: 5′-CTCTTGCTCCACCATCAGC-3′ L15781: 5′-GTAAGAACCAGATGCCAGG-3′ H15693 5′-AATAAGGGCTTAATCACCATGC-3′ L16106: 5′-AAACTATATGTCCTGAAACC-3′ Boyko et al.32
Critical commercial assays
CanineHD Whole-Genome Genotyping BeadChip Illumina Cat# WG-440-1001
Other
AmpliTaq Gold Thermo Fisher
BigDye Terminator 3.1 Applied Biosystems
3730xl sequence analyzer Applied Biosystems
SimplyAmp thermocycler Applied Biosystems
GeneAmp PCR system 9700 Applied Biosystems
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Highlights:

  • Historical and modern Galápagos dogs have distinct ancestral backgrounds

  • Genomes of modern Galápagos dogs reveal evidence of recent purebred admixture

  • No evidence of genetic bottlenecks related to population control efforts was found

  • Management strategies require improvement for effective dog population control

Acknowledgements

We gratefully acknowledge Visiting Veterinarians International and Animal Balance for supporting field work, with special thanks to Emma Clifford, Mike Greenberg, Byron Maas, Elsa Kohlbus, Jessica Gonzalez, Pan Dickinson, and the many other volunteers who participated during the spay/neuter clinics on Santa Cruz and Isabela Islands. Financial support for field work was provided by the Samuel Freeman Charitable Trust and Visiting Veterinarians International. We are especially grateful for the support of the Agencia de Regulación y Control de la Bioseguridad y Cuarentena para Galápagos (ABG) who helped with all permits, and facilitated field collections related to the hunting dogs on Santa Cruz Island. We also thank the Charles Darwin Foundation for providing samples from historical dogs. Laboratory work was supported by and completed at the Intramural Program of the National Human Genome Research Institute (HG200377). Additionally, we extend gratitude to the NIH Intramural Sequencing Center for laboratory work and sequencing related to this project. We thank members of the Ostrander lab, especially Heidi Parker and Dayna Dreger for suggestions and careful reading of this manuscript.

Footnotes

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Declaration of interests

The authors declare no competing interests.

Supplemental information

Document S1. Figures S1–S8

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Table S1. Metadata and accession numbers for samples used in this study, related to Figures 1– 4.

NIHMS2039166-supplement-1.xlsx (71KB, xlsx)
2
NIHMS2039166-supplement-2.pdf (4.6MB, pdf)

Data Availability Statement

All data reported in this paper will be shared by the lead contact upon request. This paper does not report original code. Any additional information required to reanalyze the data reported in this study is available from the lead contact upon request.

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