In their heyday, woolly mammoths blanketed the Siberian steppe, Alaska and large parts of North America. But by 10,000 years ago, warming climates had turned many of the grasslands they grazed into forest. And humans, well they weren't so friendly either—one mammoth provides quite a feast. So the mammoths largely disappeared. Except there's this one holdout population. On an island in the Arctic Ocean, called Wrangel Island. Where the last mammoths hid out…for more than 6,000 years. 
 
 "So the pyramids have been built, they've started to grow tea in China, and civilization had formed, and here are these mammoths no one knew was there, for the longest period of time." Rebekah Rogers, an evolutionary geneticist at the University of North Carolina. "And then people finally found this island around 3,700 years ago, around the time they went extinct." 
 
 Rogers and her colleague Montgomery Slatkin analyzed the genome from the tooth of one of those island mammoths, which lived 4,300 years ago. They compared it to the genome of a mainland mammoth, from much farther back, 45,000 years. And they found that harmful mutations had polluted the island mammoth's genome in that time interval. Mutations that led to the loss of smell receptors, and urine proteins—compounds they probably needed for social signaling and mate choice. 
 
 The animals also developed this satiny coat, that shines in the light. A trait that's actually popular for pet breeders today, for rabbits and guinea pigs. And the reason for all these mutations? Rogers says there just weren't enough individuals on the island—1,000 at the most, 300 at their lowest—to allow natural selection to run its course. So it wasn't survival of the fittest. It was survival of… whoever randomly survived. Which meant they accumulated a lot of mutations. None of which made them drop dead—but they weren't all that fit, either. The study is in the journal PLoS Genetics. [Rebekah L. Rogers, Montgomery Slatkin: Excess of genomic defects in a woolly mammoth on Wrangel island] 
 
 Rogers says a similar process could happen for rare animals on Earth today, like cheetahs, pandas, gorillas. "If you have a very small population for a very long time you can get this accumulation of bad mutations in their genomes. And so we'd expect to see the same effect for them. It does take a long time period to get a signal as big as what we saw in the mammoths, so the earlier we can intervene for those species, the better off they'll be." 
 
 As for those humans who, long ago, found this last holdout of mammoths on Wrangel Island? "We don't have direct evidence they hunted them, but you kind of wish that they had taken them back and domesticated them." There's already one interested buyer. "Oh yes, if you can find one I would like to have one as a pet. Preferably a little smaller so it fits in my house." 
 
 —Christopher Intagliata 
 
 [The above text is a transcript of this podcast.] 

 Woolly mammoths (Mammuthus primigenius) populated Siberia, Beringia, and North America during the Pleistocene and early Holocene. Recent breakthroughs in ancient DNA sequencing have allowed for complete genome sequencing for two specimens of woolly mammoths (Palkopoulou et al. 2015). One mammoth specimen is from a mainland population 45,000 years ago when mammoths were plentiful. The second, a 4300 yr old specimen, is derived from an isolated population on Wrangel island where mammoths subsisted with small effective population size more than 43-fold lower than previous populations. These extreme differences in effective population size offer a rare opportunity to test nearly neutral models of genome architecture evolution within a single species. Using these previously published mammoth sequences, we identify deletions, retrogenes, and non-functionalizing point mutations. In the Wrangel island mammoth, we identify a greater number of deletions, a larger proportion of deletions affecting gene sequences, a greater number of candidate retrogenes, and an increased number of premature stop codons. This accumulation of detrimental mutations is consistent with genomic meltdown in response to low effective population sizes in the dwindling mammoth population on Wrangel island. In addition, we observe high rates of loss of olfactory receptors and urinary proteins, either because these loci are non-essential or because they were favored by divergent selective pressures in island environments. Finally, at the locus of FOXQ1 we observe two independent loss-of-function mutations, which would confer a satin coat phenotype in this island woolly mammoth. 
 
 We observe an excess of detrimental mutations, consistent with genomic meltdown in woolly mammoths on Wrangel Island just prior to extinction. We observe an excess of deletions, an increase in the proportion of deletions affecting gene sequences, and an excess of premature stop codons in response to evolution under low effective population sizes. Large numbers of olfactory receptors appear to have loss of function mutations in the island mammoth. These results offer genetic support within a single species for nearly-neutral theories of genome evolution. We also observe two independent loss of function mutations at the FOXQ1 locus, likely conferring a satin coat in this unusual woolly mammoth. 
 
 We use these two ancient DNA sequences to identify retrogenes, deletions, premature stop codons, and point mutations found in the Wrangel Island and Oimyakon mammoths. We identify an excess of putatively detrimental mutations, with an excess of stop codons, an excess of deletions, an increase in the proportion of deletions affecting gene sequences, an increase in non-synonymous substitutions relative to synonymous substitutions, and an excess of retrogenes, reflecting increased transposable element activity. These data bear the signature of genomic meltdown in small populations, consistent with nearly-neutral genome evolution. They furthermore suggest large numbers of detrimental variants collecting in pre-extinction genomes, a warning for continued efforts to protect current endangered species with small population sizes. 
 
 Nearly neutral theories of genome evolution predict that small population sizes will lead to an accumulation of detrimental variation in the genome [ 5 ]. Such explanations have previously been invoked to explain genome content and genome size differences across multiple species [ 6 ]. Yet, within-species comparisons of how genomes are changed by small effective population sizes remain necessarily rare. These mammoth specimens offer the unique opportunity for within-species comparative genomics under a 43-fold reduction in population size. This comparison offers a major advantage as it will be free from confounding biological variables that are present in cross species comparisons. If nearly neutral dynamics lead to an excess of detrimental variation, we should observe an excess of harmful mutations in pre-extinction mammoths from Wrangel Island. 
 
 Woolly mammoths (Mammuthus primigenius) were among the most populous large herbivores in North America, Siberia, and Beringia during the Pleistocene and early Holocene [ 1 ]. However warming climates and human predation led to extinction on the mainland roughly 10,000 years ago [ 2 ]. Lone isolated island populations persisted out of human reach until roughly 3,700 years ago when the species finally went extinct [ 3 ]. Recently, two complete high-quality high-coverage genomes were produced for two woolly mammoths [ 4 ]. One specimen is derived from the Siberian mainland at Oimyakon, dated to 45,000 years ago [ 4 ]. This sample comes from a time when mammoth populations were plentiful, with estimated effective population size of N e = 13,000 individuals [ 4 ]. The second specimen is from Wrangel Island off the north Siberian coast [ 4 ]. This sample from 4,300 years ago represents one of the last known mammoth specimens. This individual comes from a small population estimated to contain roughly 300 individuals [ 4 ]. These two specimens offer the rare chance to explore the ways the genome responds to pre-extinction population dynamics. 
 
 Results 
 
 Excess of amino acid substitutions and stop codons We identified all SNPs in each mammoth genome as well as one Indian elephant specimen, Maya, using GATK [7]. We identified all non-synonymous and synonymous changes relative to the L. africana reference genome (https://www.broadinstitute.org/scientific-community/science/projects/mammals-models/elephant/elephant-genome-project) using r3.7 annotations lifted over to L. africana 4.0 genome sequences. We observe a significant increase in the number of heterozygous non-synonymous changes relative to synonymous changes in the Wrangel island genome compared with Oimyakon (χ2 = 68.799, df = 1, P < 2.2 × 10−16; S1 Table). There is also a significant increase in the number of homozygous mutations at non-synonymous sites relative to synonymous sites (χ2 = 9.96, df = 1, P < 0.0016; S1 Table). We further observe an excess of premature stop codons in the genome of the Wrangel Island mammoth, with 1.8X as many genes affected. There are 503 premature stop codons in the Oimyakon genome (adjusting for a 30% false negative rate at heterozygous sites) compared with 819 in the Wrangel island genome (Fig 1, Table 1). There are 318 genes that have premature stop codons that are shared across the two mammoths, and 357 genes that are truncated in both mammoths, including mutations that form at independent sites. A total of 120 of these genes have stop codons in the two mammoths as well as in Maya the Indian elephant, suggesting read through in the L. africana reference. Among truncated genes, there is a significant excess of olfactory genes and oderant binding receptors that appear to be pseudogenized with an EASE enrichment score of 9.1 (S2 Table) [8, 9]. We observe 85 truncated olfactory receptors and 3 vomeronasal receptors as well as multiple signal transduction peptides compared with 44 olfactory receptors and 2 vomeronasal receptors pseudogenized in the mainland mammoth. PPT PowerPoint slide 
 
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 larger image TIFF original image Download: Fig 1. Excess of putatively detrimental mutations in the Wrangel island genome. A) Deletions B)Genes deleted C) Retrogenes D) Premature stop codons. Numbers shown are corrected for false negative rates of 30% for heterozygous SNPs and 0.5% for deletions in the lower coverage Oimyakon mammoth. https://doi.org/10.1371/journal.pgen.1006601.g001 PPT PowerPoint slide 
 
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 larger image TIFF original image Download: Table 1. Mutations identified in mammoth genomes. https://doi.org/10.1371/journal.pgen.1006601.t001 It is possible that DNA damage in the archaic specimens could contribute to a portion of the observed stop codons. When we exclude A/G and C/T mutations, there is still a gross excess of premature stop codons, with 645 genes truncated in the Wrangel Island mammoth compared with 377 in the Oimyakon mammoth. Hence, the patterns are not explained solely by differential DNA damage in the two mammoths. Maya, the Indian Elephant specimen shows 450 premature stop codons, but 401 when A/G and T/C mutations are excluded. When putative damage to ancient DNA is excluded, Maya appears to house an intermediate number of premature stop codons, with a 6% increase compared to the Oimyakon mammoth. 
 
 Deletions We identify 27228 deletions over 1 kb long in the Wrangel island genome, and 21346 (correcting for a 0.5% false negative rate at heterozygous sites) in the Oimyakon genome (Table 1). There are 6147 deletions (23%) identified in the Wrangel Island mammoth that are homozygous (≤ 10% coverage) compared with 5035 (24%) in the Oimyakon mammoth. (S3 Table). A total of 13,459 deletions are identified in both mammoth genomes (S4 Table). Some 4813 deletions in the Wrangel Island mammoth and 4598 in the Oimyakon mammoth appear hemizygous but have stretches of zero coverage for at least 50% of their length. These sites may represent multiple independent homozygous deletions that cannot be differentiated via change point statistics. Alternatively, they might indicate smaller secondary deletions that appear on hemizygous haplotypes. Such secondary deletions are common when large loop mismatch repair attacks unpaired, hemizygous stretches of DNA [10, 11]. The Wrangel Island Mammoth has sharply increased heterozygosity for deletions in comparison with the Oimyakon mammoth (S3 Table). Some portion of the inflated heterozygosity for deletions in the Wrangel Island mammoth could be due to this difficulty in inferring genotypes in a high throughput setting. Alternatively, the effective mutation rate may have increased as fewer deletions were removed from the population via purifying selection, inflating θ del . It is also possible that there was an increase in the rate of deletions in the Wrangel Island lineage due to defective DNA repair mechanisms. An increase in non-homologous end joining after DNA breaks rather than double stranded break repair could putatively induce such a change in the deletion rate. Maya the Indian elephant shows a larger number of deletions than the Oimyakon mammoth, but with different character from the Wrangel Island mammoth. The bulk of these are derived from 22,954 hemizygous deletions (S3 Table). Maya houses only 5141 homozygous deletions, similar to the mainland mammoth (S3 Table). There is an increase in the number of hemizygous deletions that affect gene sequences, but only a modest increase in the number of homozygous deletions that affect gene sequences (S3 Table). Competing pressures of higher N e , longer time frames to accumulate mutations toward equilibrium frequencies, differences in mutation rates between the mammoths and elephants, differences in selective pressures, differences in the distribution of selective coefficients for deletions, different effective mutation rates due to different selective constraints, or differences in dominance coefficients might all contribute to differences in the number of deletions observed in elephants and mammoths. Additional samples would be necessary to determine the extent to which genetic declines may be influencing the diversity of deletions in modern Indian elephants. We currently have no basis for conclusions given this single sample, with no prior comparison. There is a significant difference in the size distribution of deletions identified in the two mammoth samples, with a mean of 1707 bp in Oimyakon and 1606 bp in the Wrangel mammoth (Wilcox W = 304430000, P < 2.2e − 16; Fig 2). This difference could reflect either differences in DNA replication or repair mechanisms in the two mammoths, or altered selective constraints for different types of duplications. No significant difference is observed between the Wrangel island mammoth down sampled sequence data (W = 2004400, P = 0.3917) suggesting that the observed decrease in size is not due to differences in coverage. Some 1628 genes have deleted exons in the Wrangel Island mammoth compared to 1110 in Oimyakon (Table 1), a significant excess of genes deleted compared to expectations based on the number of deletions (χ2 = 12.717, df = 1,P = 0.0003623). Among these deleted genes, 112 in the mainland mammoth are homozygous compared to 133 homozygous exon deletions in the Wrangel Island Mammoth. Gene functions for affected genes in the Oimyakon mammoth include synapse functions, PHD domains, zinc fingers, aldo-keto metabolism, calcium dependent membrane targeting, DNA repair, transcription regulation, and development (S5 Table). Gene functions overrepresented among deletions in the Wrangel Island mammoth include major urinary proteins, lipocalins, and pheromones, pleckstrins, transcription regulation, cell transport, DNA repair, chromatin regulation, hox domains, and development (S5 Table). PPT PowerPoint slide 
 
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 larger image TIFF original image Download: Fig 2. eCDF for the size distribution of deletions in the Oimyakon and Wrangel island genomes. There is a significant reduction in the size of deletions identified in the Wrangel Island Genome. https://doi.org/10.1371/journal.pgen.1006601.g002 Among the genes deleted in the Wrangel Island mammoth, several have phenotypes of interest in other organisms. We observe a hemizygous deletion in riboflavin kinase RFK in the Wrangel Island mammoth, but normal coverage in the Oimyakon mainland mammoth (S1 Fig). Homozygous knockouts of riboflavin kinase, essential for B2 utilization/FAD synthesis, are embryonic lethal in mice [12]. Finally, we identify a hemizygous deletion in the Wrangel island mammoth that would remove the entire gene sequence at the FOXQ1 locus (S2 Fig). The alternative haplotype carries a frameshift mutation that disrupts the FOXQ1 functional domain. FOXQ1 knock-outs in mice are associated with the satin coat phenotype, which results in translucent fur but normal pigmentation due to abnormal development of the inner medulla of hairs [13], with two independent mutations producing this phenotype [13]. FOXQ1 also regulates mucin secretion in the GI tract, a case of pleiotropic functions from a single gene [14]. If the phenotype in elephantids matches the phenotype exhibited in mice, this mammoth would have translucent hairs and a shiny satin coat, caused by two independently formed knock-out alleles at the same locus. These genes each have functions that are conserved across mammals, though there is no guarantee that they would produce identical phenotypes in other species. 
 
 Retrogene formation Retrogene formation can serve as a proxy for retrotransposon activity. We identify retrogenes that display exon-exon junction reads in genomic DNA. We observe 1.3X more retrogenes formed in the Wrangel island mammoth. The Wrangel Island mammoth has 2853 candidate retrogenes, in comparison with 2130 in the Oimyakon mammoth and 1575 in Maya (Table 1). There are 436 retrogenes that are shared between the two mammoths, though some of these could arise via independent mutations. This excess of retrogenes is consistent with increased retroelement activity in the Wrangel Island lineage. During retrogene formation, highly expressed genes, especially those expressed in the germline, are expected to contribute to new retrogenes. To determine the types of loci that had been copied by retrotransposons, we performed a gene ontology analysis using DAVID [8, 9]. Functional categories overrepresented among candidate retrogenes include genes involved in transcription, translation, cell division/cytoskeleton, post translational modification, ubiquitination, and chaperones for protein folding (S6 and S7 Tables). All of these are expected to be highly expressed during cell divisions or constitutively expressed, consistent with expectations that highly expressed genes will be overrepresented. Gene ontologies represented are similar for both mammoths (S6 and S7 Tables). Although these retrogenes are unlikely to be detrimental in and of themselves, they may point to a burst of transposable element activity in the lineage that led to the Wrangel island individual. Such a burst of TE activity would be expected to have detrimental consequences, additionally contributing to genomic decline. 

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 Isolated on an island in the Arctic Ocean, not only were woolly mammoths the last of a dying species but they were also swamped with ‘bad genes’ that are likely to have stripped their sense of smell and saddled them with translucent coats. 
 
 A study1 published 2 March in PLOS Genetics gives a rare insight into how genomes change as a species dies out. Towards the end of the last Ice Age, around 11,700 years ago, woolly mammoths ranged through Siberia and into the colder stretches of North America. But by about 4,000 years ago, mainland mammoths had died out and only 300 remained on Wrangel Island off the Siberian coast. 
 
 In order to examine this disappearance at the genetic level, biologists Rebekah Rogers and Montgomery Slatkin at the University of California, Berkeley, compared the complete genome of a mainland mammoth (Mammuthus primigenius) that lived about 45,000 years ago with that of a Wrangel Island mammoth from about 4,300 years ago. The sequences were made available by Love Dalén at the Swedish Museum of Natural History in Stockholm. 
 
 “As I looked at the sequence data,” says Rogers, “it became very clear that the Wrangel mammoth had an excess of what looked like bad mutations.” 
 
 
 
 Some of these changes are only visible to a geneticist’s eye. Compared with the mainland mammoth, the Wrangel Island specimen's genome was riddled with deletions and an abundance of sequences called stop codons — which tell a cell when to stop transcribing a section of DNA — among other changes to the DNA. But some of these changes would also have been visible in the mammoth’s behaviour and appearance. 
 
 
 
 Rogers and Slatkin found that genes related to smell and urinary proteins, which in modern elephants are important for eliciting mating behaviours or signalling social status were shut down by the mutations. These might be related, the researchers hypothesize, because a duller sense of smell may have been hitched in a feedback loop to the loss of urinary proteins, leading to the rapid loss of both. Changes to the Wrangel mammoths’ coats would have been even more obvious. Rogers and Slatkin propose that a mutation in a part of the genome called FOXQ1 would have given the mammoths a ‘satin’ coat, marked by fur that is the same colour as normal but is shiny and translucent. 
 
 Reduced competition 
 
 What happened on Wrangel wasn’t a matter of inbreeding, Rogers says — the genetic signal is different. 
 
 “What did happen was that the population was simply small,” she says, and “under these circumstances any mammoth was better than no mammoth at all”, so natural selection did not operate in the usual way. This allowed unhelpful mutations to rack up, following a previously identified phenomenon called nearly neutral genome evolution. “Bad mutations that would normally be weeded out weren’t removed from the population because of reduced competition,” says Rogers. 
 
 “Isolation and reducing population size have long been recognized as important factors causing endangerment,” says palaeontologist Ross MacPhee of the American Museum of Natural History in New York City, but the recognition of the mammoth’s “genetic meltdown” is a sign of how far studies of ancient DNA have come, and the work that still lies ahead. 
 
 The changes on Wrangel Island took place after mammoths had already been wiped out on the mainland. Tracking the downfall of the species is an ongoing effort, says MacPhee. “With additional specimens, drawn from other times and parts of the woolly mammoths’ enormous range, we may get a better picture of the genetic load that this species was labouring under at the end of its tenure.” 
 
 Still, MacPhee adds, the study “is maybe telling us something very important about what happens in populations already under severe threat because of diminished range and numbers”. 
 
 Endangered mammals 
 
 MacPhee cautions that no single animal or genome can tell the whole story for an entire species. But he notes that human hunting, climate change or any other external factor wasn't enough by itself to wipe out the woolly mammoth. There had to be some other element operating within the animals, driving them to extinction. 
 
 As dramatic as genetic meltdown sounds, Rogers says that it’s difficult to tell whether the increase in bad mutations contributed directly to the final extinction of the woolly mammoth. Yet the findings have implications for the survival of the mammoths’ elephant cousins and other endangered mammals. Rogers notes that it’s better to prevent a species from becoming endangered in the first place than to try to recover its genetic diversity after a sharp plummet. 
 
 “Even though we can improve the number of individuals in endangered populations,” she says, “their genomes may still bear the hallmarks of genomic meltdown, which will be difficult to undo.”
– On Wrangel Island off Siberia, a small group of woolly mammoths managed to survive for some 6,000 years after the mainland mammoths died off. Though humans were a little late in discovering that—by the time we made it to the Arctic island 3,700 years ago, the beasts were on their last legs, Scientific American reports—the genes of one of that lived there 4,300 years ago is giving new insights into what may have fueled the creatures' undoing. When compared to the genome of a mainland mammoth who lived more than 40,000 years prior, researchers found that a "mutational meltdown" had taken place among the island dwellers, one that left the beasts with a poor sense of smell and possibly diminished mating. They also weren't so woolly by the end, with gene mutations blamed for a shiny, translucent coat, per Nature. Reporting in the journal PLOS Genetics, researchers found that gene mutations caused the island mammoths to lose olfactory receptors and urinary proteins, which in modern elephants are important for mating, per Nature. Researcher Rebekah Rogers explains it wasn't inbreeding that did the Wrangel mammoths in, but a population so small that it messed with natural selection. "Any mammoth was better than no mammoth at all," so "bad mutations that would normally be weeded out weren’t removed from the population because of reduced competition." Paleontologist Ross MacPhee tells Nature the findings could inform modern conservation efforts: They're "maybe telling us something very important about what happens in populations already under severe threat because of diminished range and numbers." (Mammoths could be de-extinct in two years.)