The genus Crocuta (African spotted and Eurasian cave hyenas) includes several closely related extinct and extant lineages. The relationships among these lineages, however, are contentious. Through the generation of population-level paleogenomes from late Pleistocene Eurasian cave hyena and genomes from modern African spotted hyena, we reveal the cross-continental evolutionary relationships between these enigmatic hyena lineages. We find a deep divergence (~2.5 Ma) between African and Eurasian Crocuta populations, suggesting that ancestral Crocuta left Africa around the same time as early Homo. Moreover, we find discordance between nuclear and mitochondrial phylogenies and evidence for bidirectional gene flow between African and Eurasian Crocuta after the lineages split, which may have complicated prior taxonomic classifications. Last, we find a number of introgressed loci that attained high frequencies within the recipient lineage, suggesting some level of adaptive advantage from admixture.

The genus Crocuta (spotted and cave hyenas) includes several closely related extinct and extant lineages and is one of only two large-bodied African carnivores (the other being the saber-toothed cat genus Megantereon) whose migratory and evolutionary history has been compared to that of the genus Homo ( 3 ). Crocuta crocuta, the spotted hyena from sub-Saharan Africa, represents the only extant species. Spotted hyenas are the most common large carnivore in Africa today. They are a highly adaptable and opportunistic species, often living in large matriarchal clans and displaying complex social behaviors. Spotted hyenas are opportunistic feeders, and both hunting and scavenging play important roles in obtaining food ( 4 ). Females tend to stay within their natal clan, whereas males typically disperse to achieve reproductive success ( 5 ). Although now restricted to sub-Saharan Africa, the genus once had a much more extensive range, occupying most of Eurasia, from the British Isles to the far east of Asia ( 6 ). Initially, because of distinct morphologies, Eurasian cave and African spotted hyena lineages were considered distinct taxa. Cave hyenas had shorter distal limb elements than the extant species, indicating less cursorial ability. They also had a less trenchant check tooth morphology, indicating less active hunting and meat eating and a greater reliance on scavenging for their nutritional needs ( 7 ). Eurasian cave hyenas have further been split into European (C. crocuta spelaea) and Asian (C. crocuta ultima) subspecies ( 6 ). These classifications have, however, met with some resistance, with differences being attributed to phenotypic plasticity caused by different climates ( 8 ). This skepticism was further supported by a study using short fragments of mtDNA ( 9 ), which found African spotted hyenas to be intermingled within Eurasian cave hyenas’ mitochondrial haplogroups. This result indicated that, when only considering these mtDNA fragments, cave and spotted hyena appear to be inseparable taxa.

The late Quaternary was characterized by a substantial number of global extinction events ( 1 ). Despite this, many lineages still persist today, albeit with marked local extinctions, culminating in reductions in distribution and diversity. The study of ancient DNA and, more recently, paleogenomics are powerful approaches enabling direct comparisons between surviving and extinct lineages. The relationships between our own species (Homo sapiens) and extinct archaic hominins (Neanderthals and Denisovans) are arguably the best known example of this. The inclusion of paleogenomic data from nuclear genomes proved to be an invaluable tool for studying their evolution and most notably uncovered gene flow between archaic and modern humans that could not be detected using mitochondrial DNA (mtDNA) alone. The discovery of this gene flow markedly changed commonly accepted views of human evolution and highlighted the ability of paleogenomics to provide insights into the relationships between extinct and extant lineages ( 2 ).

RESULTS AND DISCUSSION

Using paleogenomic data from several late Pleistocene cave hyenas from across Eurasia and population-level genomic data from sub-Saharan spotted hyenas, we investigated the evolutionary history of the genus Crocuta. We first assembled the mitochondrial genomes for 7 cave hyenas and 12 spotted hyenas. We then constructed a Bayesian phylogenetic tree using these assembled mitochondrial genomes, three previously published Crocuta mitochondrial genomes, and three outgroup mitochondrial genomes from Hyaena, Parahyaena, and Proteles (Fig. 1 and fig. S1). The tree shows the same major clades as previously found using short mitochondrial fragments and a similar topology, with the two taxa being polyphyletic with respect to each other, i.e., African spotted hyenas being intermingled within Eurasian cave hyenas’ mitochondrial haplogroups (9). However, the relationships between the major clades are slightly different. We find the East Asian clade D diverging first, followed by the purely European clade B, and a bifurcation of the African clade C and the African/Eurasian clade A. Moreover, the full mitochondrial genomes show the European and African sequences within clade A to form reciprocally monophyletic clades. In contrast to this picture for mtDNA, a principal components analysis (PCA) using nuclear genomic data from 7 cave hyenas and 10 spotted hyenas reveals a clear differentiation between cave and spotted hyenas along the PC1 axis (Fig. 2A). The PC2 axis then separates cave hyenas by geographical location, i.e., European versus East Asian individuals. As typical ancient DNA damage most commonly causes C-to-T transitions, this analysis only considered differences between individuals caused by transversions and should therefore not be biased by a mixture of modern and ancient samples.

Fig. 1 Sampling distribution and mitogenomic timetree of the hyaenidae family. (A) Map showing the geographic origins of our spotted and cave hyena samples. The color of the dot represents the age of the sample. (B) Dated Bayesian phylogenetic tree constructed using complete mitochondrial genomes and a strict molecular clock. Haplogroups are those previously defined in Rohland et al. (9). Red-colored labels show Pleistocene cave hyena and blue-colored labels show modern spotted hyena. The yellow star represents a putative mitochondrial introgression event of unknown direction. The Hyaena/Parahyaena node was fixed for fossil calibration (as indicated by the bone image), and Proteles was set as the outgroup. All major nodes had posterior probability values of 1 (fig. S1). Dark blue node bars show the 95% credibility interval of the divergence dating.

Fig. 2 Population structure analyses comparing nuclear genomic information from Pleistocene cave hyena and modern spotted hyena. (A) PCA based on genome-wide SNPs. Red shaded area encompasses cave hyenas and blue shaded area encompasses spotted hyenas. (B) Densitree constructed using 2 Mbp sliding windows and a maximum likelihood approach. Light gray lines represent single phylogenetic trees produced from each window. Dark black lines represent the root canal as defined by Densitree. Sample name colors represent the previously defined mitochondrial haplogroups.

To further investigate this distinct relationship, we constructed 467 maximum likelihood phylogenetic trees from 2–million base pair (Mbp) nonoverlapping windows along the nuclear genome and visualized the outputs simultaneously using Densitree (Fig. 2B) (10). We also combined each individual tree to produce a single consensus tree using PHYLogeny Inference Package (PHYLIP) (11) to investigate node support from the individual trees (fig. S2). Both the PHYLIP consensus tree and the Densitree root canal (consensus tree with the highest clade support) are consistent with the PCA results, again showing a separation between cave and spotted hyenas. Individual trees are highly concordant with respect to the major groupings, with 465 (99.6%) supporting the monophyly of cave hyenas and 462 (98.9%) supporting the monophyly of spotted hyenas. Tree topologies within each clade are, however, highly variable, likely reflecting the effects of both incomplete lineage sorting (ILS) and gene flow (fig. S2). A PCA of cave hyenas alone suggests three distinct clusters (fig. S3). The PC1 axis separates individuals by continent (European and East Asian), whereas PC2 separates the European cave hyenas into two groups, corresponding to their assignment to previously described mitochondrial haplogroups A and B and without any obvious correspondence with temporal or geographic proximity (9). This suggests an external barrier between geographically close populations, which may have hindered the successful dispersal of males to clans of different cave hyena matrilines. An alternative possible explanation for this finding has been suggested for European bison (12). In European bison, one mitochondrial haplogroup was dominant in individuals >50 thousand years old (ka). This haplogroup was then replaced by another between 50 and 32 ka, before returning to the original haplogroup. A similar phenomenon could explain our data with geographically close individuals displaying different mitochondrial haplogroups and clustering separately in the PCA. However, as Ccsp040 could not be dated owing to low collagen content, and the previously published Cc8 and Cc9 could not be reliably dated either, this hypothesis is purely speculative until more genetic information from reliably dated samples is available. A PCA of spotted hyenas alone did not show any clear clustering but indicates that there could be some level of isolation by distance (fig. S4). Demographic analyses provide an explanation for these contrasting patterns. Pairwise sequentially Markovian coalescent (PSMC) analyses indicate a large bottleneck in spotted hyenas over the late Pleistocene (Fig. 3). Furthermore, despite originating from markedly different areas on the African continent (Ghana, Namibia, and Somalia), all three spotted hyenas used within this analysis produced near-identical demographic histories, suggesting that these three individuals belonged to a single population or their demographics have been shaped by very similar drivers. While the exact causes behind this bottleneck remain unknown, population bottlenecks over a similar time period have also been reported in a large cohort of ruminant genomes (13). These bottlenecks coincided with increasing human effective population size, leading to the speculation that the decline might have somewhat been associated with human activities. An increasing human population size coupled with decreasing prey availability would have increased competition between spotted hyenas and other carnivorous species, potentially leading to the decrease in population size seen in the PSMC analysis. Furthermore, although our PSMC analyses only investigated African individuals, on the basis of the demographic findings by Chen et al. in European and Asian ruminants, Eurasian cave hyenas may have also faced a similar decline, ultimately leading to their demise at the end of the Pleistocene.

Fig. 3 PSMC demographic analyses of three modern spotted hyena nuclear genomes. The y axis represents effective population size (×10,000), and the x axis represents millions of years before present. Faded lines show bootstrap values.

The contrast between the nuclear and mtDNA results suggests either extensive ILS or gene flow between spotted and cave hyena. We therefore investigated for the presence and directionality of gene flow using a previously published five-taxon phylogenetic sliding window analysis (14). This analysis revealed several instances of gene flow between cave and spotted hyenas after the split of the two lineages (tables S1 to S3). In regard to gene flow from spotted hyena into cave hyena, the Russian individual (Ccsp041—Geographical Society Cave, Russia) had the least putatively introgressed genomic regions, whereas the German individuals (Ccsp040—Aufhausener Höhle and Ccsp042—Lindenthaler Höhle) shared a similar introgression signal. This pattern occurred irrespective of which spotted hyenas were included in the comparison. The similarity between the German samples was further confirmed by nonsignificant D statistics results (z score between −3 and 3; tables S4 and S5). This result suggests that gene flow into cave hyenas occurred after the split between European and Asian populations, but before the split between the two European lineages. Conversely, gene flow from cave hyenas into spotted hyenas appears more complicated, and levels of admixture did not appear to correlate with either mitochondrial haplogroup or geographical proximity to Eurasia. The Namibian individual (NamCrocuta) contained the fewest windows associated with gene flow from cave hyenas, whereas the Kenyan individual (Kenya_795) contained the most. All other spotted hyenas in the comparison had similar levels. There are two explanations for the differential admixture patterns across spotted hyenas: either there were multiple gene flow events into spotted hyenas or a single admixture event was followed by random assortment and differential diffusion of the cave hyena loci. To investigate these two possibilities and infer the relative timing of the admixture events, we computed the cumulative lengths of windows showing signs of admixture (14) (fig. S4). Ccsp042 and Ccsp040 share similar cumulative lengths, again suggesting gene flow before the split of the two European lineages. The cumulative length of the least admixed spotted hyena (NamCrocuta) was similar to that of Ccsp042 and Ccsp040, suggesting that bidirectional admixture occurred at a similar time. This then appears to have been followed by a secondary gene flow event into Northern Africa (Ghana, Kenya, and Somalia), which may have diffused into Zambia and, possibly, even Namibia. However, as this is a relative test, it is difficult to separate gene flow from ILS in the individuals showing the least admixture. To further investigate these signs of admixture, we ran a fastsimcoal analysis (15). Results were generally consistent with the previous analyses and suggested bidirectional gene flow between spotted hyena and European cave hyenas. Moreover, fastsimcoal suggested substantially more gene flow in the direction of Europe from Africa (table S6), with the presence of directional gene flow being stronger in recent times (between 120,000 years before present and the sampling time of the cave hyenas) than before 120,000 years before present. The five-taxon sliding window analysis, on the other hand, showed more admixed windows from Eurasia into Africa than vice versa, potentially indicating more gene flow in the opposite direction to what was uncovered using fastsimcoal. However, these seemingly contradictory results can be explained by some caveats in assessing levels of gene flow using the sliding window analysis. First, as it is a relative test, i.e., places the least admixed individual as a baseline “nonadmixed” individual, total levels of admixture could be underestimated if this baseline individual is highly admixed. For example, if the Asian individual, Ccsp041, contained a substantial amount of admixture from Africa, the total number of windows recovered showing gene flow in the other cave hyena individuals would be relatively reduced. Second, the timing of gene flow can influence the number of admixed windows recovered. The more recent the gene flow event, the less time for consecutive windows to be broken through recombination. More recent gene flow could therefore be misinterpreted as more total gene flow simply because the total number of windows showing gene flow would be higher. This seems a likely explanation for the discordance as we find longer consecutive admixture windows in spotted hyena compared to cave hyena, suggesting more recent gene flow into Africa than vice versa.

Thus, on the basis of both the five-taxon phylogenetic sliding window analysis, D statistics results, and fastsimcoal (tables S1 to S6), we suggest a bidirectional gene flow event between cave and spotted hyenas after the split of cave hyenas into the European and Asian lineages and a subsequent unidirectional gene flow event into northern spotted hyenas, followed by differential diffusion of the admixed loci within the other spotted hyena lineages (Fig. 4). Furthermore, on the basis of the divergence time between the cave and spotted hyena in mitochondrial haplogroup A (Fig. 1), we suggest that gene flow occurred sometime before ~475 ka (95% CI, 388 to 570 ka), either from spotted hyena into cave hyena or vice versa. However, the fact that the spotted hyena mitochondrial haplogroup reached such a high frequency in some cave hyena populations, but not in others, or that one of the cave hyena haplogroups reached such a high frequency in northern Africa was most likely by chance (i.e., ILS and genetic drift) as opposed to selection.

Fig. 4 D statistics results and a schematic overview of the divergence of and post-divergence gene flow between spotted and cave hyenas. (A) D statistics results using two cave hyenas, one spotted hyena, and the striped hyena as outgroup. (B) D statistics results using two spotted hyenas, one cave hyena, and the striped hyena as outgroup. Filled circles show significant D scores while nonfilled circles show nonsignificant D scores. (C) Phylogenetic tree presenting the admixture events found between African spotted and Eurasian cave hyena. Blue color represents African inhabitancy and red color represents Eurasian inhabitancy. Arrows show the direction of gene flow events. (Illustration credit: Binia De Cahsan, https://sites.google.com/view/decahsanillustrations.)

To investigate whether this admixture may have had adaptive consequences, we determined the frequency and distribution of the different tree topologies across the genome recovered from the 100-kb five-taxon sliding window analysis. The most common topology within these comparisons was the monophyly of spotted and cave hyenas as 63 to 73% of all windows represented this topology with the rest assumed to have arisen due to ILS or admixture. However, we noted that some windows consistently showed an aberrant topology regardless of which individuals were used in the comparison and therefore were unlikely to have arisen by a stochastic process such as neutral drift. We further investigated these regions for the presence of protein-coding genes, the protein classes these genes belong to, and the biological functions of the genes. When investigating regions of introgression into spotted hyenas, we found 75 putative genes (table S7). These genes corresponded to 54 unique protein classes and 135 biological processes (tables S8 and S9). We further investigated Gene Ontology (GO) term enrichment in these genes using GOrilla (16) and found a significant enrichment in the glutamate receptor signaling pathway (false discovery rate q = 0.05). This pathway is involved in a variety of biological processes and plays a key role in the central nervous system. In humans, abnormalities in this pathway have been linked to both chronic disabling brain disorders (e.g., schizophrenia) and neurodegenerative diseases (e.g., Alzheimer’s, Parkinson’s, and multiple sclerosis) (17). On the other hand, when investigating regions of introgression into cave hyenas, we found only 12 putative genes (table S10) corresponding to 11 unique protein classes and 28 biological processes (tables S11 and S12). GO term enrichment analysis did not find any significantly enriched terms.

To better understand the various events in the evolutionary history of the genus Crocuta, we estimated the divergence time between the cave and spotted hyena lineages using a genome-wide distance-based approach based on the method from Heintzman et al. (18) with some adjustments. To remove any biases that admixture or ILS may incur, we only used windows from the above admixture analysis that showed cave and spotted hyenas as monophyletic. This resulted in 3993 100-kb windows for our analysis. With this method, we estimated the time to the most recent common ancestor (tMRCA) of cave and spotted hyenas to be 2.52 million years (Ma) (CI 2.21 to 2.83 Ma). This deep divergence was further strengthened through a fastsimcoal analysis, which specifically models both lineage sorting and gene flow. This analysis indicated a tMRCA between cave and spotted hyenas of 2.71 Ma (CI 2.15 to 2.84 Ma; table S6). As the earliest presence of Crocuta outside of Africa is Crocuta honanensis in the Longdan basin of China from ~2 Ma (19) and the oldest Crocuta fossils in Africa (Crocuta dietrichi) are from the early Pliocene (3.63 to 3.85 Ma) (20), we propose a dispersal from Africa into Eurasia, most likely into Asia, shortly after the divergence of the two lineages. This timing closely coincides with the oldest fossil of Homo found outside of Africa (Homo georgicus), found in Georgia and estimated at ~1.8 Ma (21), and the oldest Homo artifacts outside of Africa dated to ~2.1 Ma (22). This was most likely followed by subsequent dispersal into Europe from Asia as the earliest confirmed European occurrences of Crocuta are from Sierra de Atapuerca (Spain) [~0.9 Ma (23)] and middle Pleistocene Italy (24). Several other mammalian species (e.g., Theropithecus, Pachycrocuta, Panthera, and Hippopotamus) have also shown an Africa-to-Eurasia dispersal in the late Pliocene/early Pleistocene (25), with an overrepresentation of carnivores, most likely owing to their good dispersal abilities and broad ecological tolerances. However, as most of these dispersals only roughly correlate with hominin dispersals, any connection between these mammals would be speculative based on current evidence.

Beyond the timing of dispersal into Eurasia, in contrast to our own genus, very little information is available with regard to the evolutionary history of spotted hyena. Our analyses combining both modern and Pleistocene nuclear genomes reveal some notable similarities and provide new evidence to the theory that the evolutionary and migratory history of Crocuta is similar to that of Homo, first proposed decades ago (26). In addition to the similar migratory pattern and timing, we see highly similar discordances between the mitochondrial and nuclear genome trees for modern and archaic humans on the one hand and spotted and cave hyena on the other. In both cases, the deepest divergence in the mitochondrial tree is formed by an East Asian lineage, while the nuclear genomes show a sister group relationship for East and West Eurasian populations (Neanderthals versus Denisovans and European versus East Asian cave hyena, respectively), with the African populations forming the basal diverging lineage. Although these topological similarities are remarkable, since the underlying causes are largely unknown for both humans and hyenas, it remains to be determined if the similarities are due to a common cause or simply coincidence. However, replacements of forested to savannah-like terrain ~2.6 to 1.8 Ma outside of Africa have been suggested as a catalyst to early hominin dispersal out of Africa and may have played a similar role in Crocuta dispersal (27).

Despite all of these similarities, it should be noted that there are also a number of differences. Although humans and hyenas may have left Africa around the same time, on a nuclear genomic level, this first emigration did not leave any descendants in humans, whereas it may have done so in hyenas. Also, the population trajectories in spotted hyenas and modern humans are notably different, when considering both the PSMC analyses and the demographic outcome, with modern humans populating the entire globe, whereas spotted hyenas are today restricted to sub-Saharan Africa. Thus, certain similarities in the evolutionary history of species groups should not distract from the fact that the overall population history of an individual species or species group is almost certainly unique in most aspects.