Persistent Gene Flow Suggests an Absence of Reproductive Isolation in an African Antelope Speciation Model.

African antelope diversity is a globally unique vestige of a much richer world-wide Pleistocene megafauna. Despite this, the evolutionary processes leading to the prolific radiation of African antelopes are not well understood. Here, we sequenced 145 whole genomes from both subspecies of the waterbuck (Kobus ellipsiprymnus), an African antelope believed to be in the process of speciation. We investigated genetic structure and population divergence and found evidence of a mid-Pleistocene separation on either side of the eastern Great Rift Valley, consistent with vicariance caused by a rain shadow along the so-called 'Kingdon's Line'. However, we also found pervasive evidence of both recent and widespread historical gene flow across the Rift Valley barrier. By inferring the genome-wide landscape of variation among subspecies, we found 14 genomic regions of elevated differentiation, including a locus that may be related to each subspecies' distinctive coat pigmentation pattern. We investigated these regions as candidate speciation islands. However, we observed no significant reduction in gene flow in these regions, nor any indications of selection against hybrids. Altogether, these results suggest a pattern whereby climatically driven vicariance is the most important process driving the African antelope radiation, and suggest that reproductive isolation may not set in until very late in the divergence process. This has a significant impact on taxonomic inference, as many taxa will be in a gray area of ambiguous systematic status, possibly explaining why it has been hard to achieve consensus regarding the species status of many African antelopes. Our analyses demonstrate how population genetics based on low-depth whole genome sequencing can provide new insights that can help resolve how far lineages have gone along the path to speciation.

Extensive Population Structure Highlights an Apparent Paradox of Stasis in the Impala (Aepyceros melampus).

Impalas are unusual among bovids because they have remained morphologically similar over millions of years-a phenomenon referred to as evolutionary stasis. Here, we sequenced 119 whole genomes from the two extant subspecies of impala, the common (Aepyceros melampus melampus) and black-faced (A. m. petersi) impala. We investigated the evolutionary forces working within the species to explore how they might be associated with its evolutionary stasis as a taxon. Despite being one of the most abundant bovid species, we found low genetic diversity overall, and a phylogeographic signal of spatial expansion from southern to eastern Africa. Contrary to expectations under a scenario of evolutionary stasis, we found pronounced genetic structure between and within the two subspecies with indications of ancient, but not recent, gene flow. Black-faced impala and eastern African common impala populations had more runs of homozygosity than common impala in southern Africa, and, using a proxy for genetic load, we found that natural selection is working less efficiently in these populations compared to the southern African populations. Together with the fossil record, our results are consistent with a fixed-optimum model of evolutionary stasis, in which impalas in the southern African core of the range are able to stay near their evolutionary fitness optimum as a generalist ecotone species, whereas eastern African impalas may struggle to do so due to the effects of genetic drift and reduced adaptation to the local habitat, leading to recurrent local extinction in eastern Africa and re-colonisation from the South.

Population genomics of the muskox' resilience in the near absence of genetic variation.

Genomic studies of species threatened by extinction are providing crucial information about evolutionary mechanisms and genetic consequences of population declines and bottlenecks. However, to understand how species avoid the extinction vortex, insights can be drawn by studying species that thrive despite past declines. Here, we studied the population genomics of the muskox (Ovibos moschatus), an Ice Age relict that was at the brink of extinction for thousands of years at the end of the Pleistocene yet appears to be thriving today. We analysed 108 whole genomes, including present-day individuals representing the current native range of both muskox subspecies, the white-faced and the barren-ground muskox (O. moschatus wardi and O. moschatus moschatus) and a ~21,000-year-old ancient individual from Siberia. We found that the muskox' demographic history was profoundly shaped by past climate changes and post-glacial re-colonizations. In particular, the white-faced muskox has the lowest genome-wide heterozygosity recorded in an ungulate. Yet, there is no evidence of inbreeding depression in native muskox populations. We hypothesize that this can be explained by the effect of long-term gradual population declines that allowed for purging of strongly deleterious mutations. This study provides insights into how species with a history of population bottlenecks, small population sizes and low genetic diversity survive against all odds.

Warthog Genomes Resolve an Evolutionary Conundrum and Reveal Introgression of Disease Resistance Genes.

African wild pigs have a contentious evolutionary and biogeographic history. Until recently, desert warthog (Phacochoerus aethiopicus) and common warthog (P. africanus) were considered a single species. Molecular evidence surprisingly suggested they diverged at least 4.4 million years ago, and possibly outside of Africa. We sequenced the first whole-genomes of four desert warthogs and 35 common warthogs from throughout their range. We show that these two species diverged much later than previously estimated, 400,000-1,700,000 years ago depending on assumptions of gene flow. This brings it into agreement with the paleontological record. We found that the common warthog originated in western Africa and subsequently colonized eastern and southern Africa. During this range expansion, the common warthog interbred with the desert warthog, presumably in eastern Africa, underlining this region's importance in African biogeography. We found that immune system-related genes may have adaptively introgressed into common warthogs, indicating that resistance to novel diseases was one of the most potent drivers of evolution as common warthogs expanded their range. Hence, we solve some of the key controversies surrounding warthog evolution and reveal a complex evolutionary history involving range expansion, introgression, and adaptation to new diseases.

Colonialism in South Africa leaves a lasting legacy of reduced genetic diversity in Cape buffalo.

The iconic Cape buffalo has experienced several documented population declines in recent history. These declines have been largely attributed to the late 19th century rinderpest pandemic. However, the effect of the rinderpest pandemic on their genetic diversity remains contentious, and other factors that have potentially affected this diversity include environmental changes during the Pleistocene, range expansions and recent human activity. Motivated by this, we present analyses of whole genome sequencing data from 59 individuals from across the Cape buffalo range to assess present-day levels of genome-wide genetic diversity and what factors have influenced these levels. We found that the Cape buffalo has high average heterozygosity overall (0.40%), with the two southernmost populations having significantly lower heterozygosity levels (0.33% and 0.29%) on par with that of the domesticated water buffalo (0.29%). Interestingly, we found that these lower levels are probably due to recent inbreeding (average fraction of runs of homozygosity 23.7% and 19.9%) rather than factors further back in time during the Pleistocene. Moreover, detailed investigations of recent demographic history show that events across the past three centuries were the main drivers of the exceptional loss of genetic diversity in the southernmost populations, coincident with the onset of colonialism in the southern extreme of the Cape buffalo range. Hence, our results add to the growing body of studies suggesting that multiple recent human-mediated impacts during the colonial period caused massive losses of large mammal abundance in southern Africa.

African climate and geomorphology drive evolution and ghost introgression in sable antelope.

The evolutionary history of African ungulates has been explained largely in the light of Pleistocene climatic oscillations and the way these influenced the distribution of vegetation types, leading to range expansions and/or isolation in refugia. In contrast, comparatively fewer studies have addressed the continent's environmental heterogeneity and the role played by its geomorphological barriers. In this study, we performed a range-wide analysis of complete mitogenomes of sable antelope (Hippotragus niger) to explore how these different factors may have contributed as drivers of evolution in southcentral Africa. Our results supported two sympatric and deeply divergent mitochondrial lineages in west Tanzanian sables, which can be explained as the result of introgressive hybridization of a mitochondrial ghost lineage from an archaic, as-yet-undefined, congener. Phylogeographical subdivisions into three main lineages suggest that sable diversification may not have been driven solely by climatic events affecting populations differently across a continental scale. Often in interplay with climate, geomorphological features have also clearly shaped the species' patterns of vicariance, where the East Africa Rift System and the Eastern Arc Mountains acted as geological barriers. Subsequent splits among southern populations may be linked to rearrangements in the Zambezi system, possibly framing the most recent time when the river attained its current drainage profile. This work underlines how the use of comprehensive mitogenomic data sets on a model species with a wide geographical distribution can contribute to a much-enhanced understanding of environmental, geomorphological and evolutionary patterns in Africa throughout the Quaternary.

The genetic impact of an Ebola outbreak on a wild gorilla population.

BACKGROUND: Numerous Ebola virus outbreaks have occurred in Equatorial Africa over the past decades. Besides human fatalities, gorillas and chimpanzees have also succumbed to the fatal virus. The 2004 outbreak at the Odzala-Kokoua National Park (Republic of Congo) alone caused a severe decline in the resident western lowland gorilla (Gorilla gorilla gorilla) population, with a 95% mortality rate. Here, we explore the immediate genetic impact of the Ebola outbreak in the western lowland gorilla population. RESULTS: Associations with survivorship were evaluated by utilizing DNA obtained from fecal samples from 16 gorilla individuals declared missing after the outbreak (non-survivors) and 15 individuals observed before and after the epidemic (survivors). We used a target enrichment approach to capture the sequences of 123 genes previously associated with immunology and Ebola virus resistance and additionally analyzed the gut microbiome which could influence the survival after an infection. Our results indicate no changes in the population genetic diversity before and after the Ebola outbreak, and no significant differences in microbial community composition between survivors and non-survivors. However, and despite the low power for an association analysis, we do detect six nominally significant missense mutations in four genes that might be candidate variants associated with an increased chance of survival. CONCLUSION: This study offers the first insight to the genetics of a wild great ape population before and after an Ebola outbreak using target capture experiments from fecal samples, and presents a list of candidate loci that may have facilitated their survival.

Vicariance followed by secondary gene flow in a young gazelle species complex.

Grant's gazelles have recently been proposed to be a species complex comprising three highly divergent mtDNA lineages (Nanger granti, N. notata and N. petersii). The three lineages have nonoverlapping distributions in East Africa, but without any obvious geographical divisions, making them an interesting model for studying the early-stage evolutionary dynamics of allopatric speciation in detail. Here, we use genomic data obtained by restriction site-associated (RAD) sequencing of 106 gazelle individuals to shed light on the evolutionary processes underlying Grant's gazelle divergence, to characterize their genetic structure and to assess the presence of gene flow between the main lineages in the species complex. We date the species divergence to 134,000 years ago, which is recent in evolutionary terms. We find population subdivision within N. granti, which coincides with the previously suggested two subspecies, N. g. granti and N. g. robertsii. Moreover, these two lineages seem to have hybridized in Masai Mara. Perhaps more surprisingly given their extreme genetic differentiation, N. granti and N. petersii also show signs of prolonged admixture in Mkomazi, which we identified as a hybrid population most likely founded by allopatric lineages coming into secondary contact. Despite the admixed composition of this population, elevated X chromosomal differentiation suggests that selection may be shaping the outcome of hybridization in this population. Our results therefore provide detailed insights into the processes of allopatric speciation and secondary contact in a recently radiated species complex.

Targeted conservation genetics of the endangered chimpanzee.

Populations of the common chimpanzee (Pan troglodytes) are in an impending risk of going extinct in the wild as a consequence of damaging anthropogenic impact on their natural habitat and illegal pet and bushmeat trade. Conservation management programmes for the chimpanzee have been established outside their natural range (ex situ), and chimpanzees from these programmes could potentially be used to supplement future conservation initiatives in the wild (in situ). However, these programmes have often suffered from inadequate information about the geographical origin and subspecies ancestry of the founders. Here, we present a newly designed capture array with ~60,000 ancestry informative markers used to infer ancestry of individual chimpanzees in ex situ populations and determine geographical origin of confiscated sanctuary individuals. From a test panel of 167 chimpanzees with unknown origins or subspecies labels, we identify 90 suitable non-admixed individuals in the European Association of Zoos and Aquaria (EAZA) Ex situ Programme (EEP). Equally important, another 46 individuals have been identified with admixed subspecies ancestries, which therefore over time, should be naturally phased out of the breeding populations. With potential for future re-introduction to the wild, we determine the geographical origin of 31 individuals that were confiscated from the illegal trade and demonstrate the promises of using non-invasive sampling in future conservation action plans. Collectively, our genomic approach provides an exemplar for ex situ management of endangered species and offers an efficient tool in future in situ efforts to combat the illegal wildlife trade.

Unrecognized circulation of SAT 1 foot-and-mouth disease virus in cattle herds around Queen Elizabeth National Park in Uganda.

BACKGROUND: Foot-and-mouth disease (FMD) is endemic in Uganda in spite of the control measures used. Various aspects of the maintenance and circulation of FMD viruses (FMDV) in Uganda are not well understood; these include the role of the African buffalo (Syncerus caffer) as a reservoir for FMDV. To better understand the epidemiology of FMD at the livestock-wildlife-interface, samples were collected from young, unvaccinated cattle from 24 pastoral herds that closely interact with wildlife around Queen Elizabeth National Park in Uganda, and analysed for evidence of FMDV infection. RESULTS: In total, 37 (15%) of 247 serum samples had detectable antibodies against FMDV non-structural proteins (NSPs) using a pan-serotypic assay. Within these 37 sera, antibody titres ≥ 80 against the structural proteins of serotypes O, SAT 1, SAT 2 and SAT 3 were detected by ELISA in 5, 7, 4 and 3 samples, respectively, while neutralizing antibodies were only detected against serotype O in 3 samples. Two FMDV isolates, with identical VP1 coding sequences, were obtained from probang samples from clinically healthy calves from the same herd and are serotype SAT 1 (topotype IV (EA-I)). Based on the VP1 coding sequences, these viruses are distinct from previous cattle and buffalo SAT 1 FMDV isolates obtained from the same area (19-30% nucleotide difference) and from the vaccine strain (TAN/155/71) used within Uganda (26% nucleotide difference). Eight herds had only one or a few animals with antibodies against FMDV NSPs while six herds had more substantial evidence of prior infection with FMDV. There was no evidence for exposure to FMDV in the other ten herds. CONCLUSIONS: The two identical SAT 1 FMDV VP1 sequences are distinct from former buffalo and cattle isolates from the same area, thus, transmission between buffalo and cattle was not demonstrated. These new SAT 1 FMDV isolates differed significantly from the vaccine strain used to control Ugandan FMD outbreaks, indicating a need for vaccine matching studies. Only six herds had clear serological evidence for exposure to O and SAT 1 FMDV. Scattered presence of antibodies against FMDV in other herds may be due to the occasional introduction of animals to the area or maternal antibodies from past infection and/or vaccination. The evidence for asymptomatic FMDV infection has implications for disease control strategies in the area since this obstructs early disease detection that is based on clinical signs in FMDV infected animals.

Foot-and-mouth disease virus serotype SAT 3 in long-horned Ankole calf, Uganda.

After a 16-year interval, foot-and-mouth disease virus serotype SAT 3 was isolated in 2013 from an apparently healthy long-horned Ankole calf that grazed close to buffalo in Uganda. The emergent virus strain is ≈20% different in nucleotide sequence (encoding VP1 [viral protein 1]) from its closest relatives isolated previously from buffalo in Uganda.

Genetic consequences of population expansions and contractions in the common hippopotamus (Hippopotamus amphibius) since the Late Pleistocene.

Over the past two decades, an increasing amount of phylogeographic work has substantially improved our understanding of African biogeography, in particular the role played by Pleistocene pluvial-drought cycles on terrestrial vertebrates. However, still little is known on the evolutionary history of semi-aquatic animals, which faced tremendous challenges imposed by unpredictable availability of water resources. In this study, we investigate the Late Pleistocene history of the common hippopotamus (Hippopotamus amphibius), using mitochondrial and nuclear DNA sequence variation and range-wide sampling. We documented a global demographic and spatial expansion approximately 0.1-0.3 Myr ago, most likely associated with an episode of massive drainage overflow. These events presumably enabled a historical continent-wide gene flow among hippopotamus populations, and hence, no clear continental-scale genetic structuring remains. Nevertheless, present-day hippopotamus populations are genetically disconnected, probably as a result of the mid-Holocene aridification and contemporary anthropogenic pressures. This unique pattern contrasts with the biogeographic paradigms established for savannah-adapted ungulate mammals and should be further investigated in other water-associated taxa. Our study has important consequences for the conservation of the hippo, an emblematic but threatened species that requires specific protection to curtail its long-term decline.

Characterisation of recent foot-and-mouth disease viruses from African buffalo (Syncerus caffer) and cattle in Kenya is consistent with independent virus populations.

BACKGROUND: Understanding the epidemiology of foot-and-mouth disease (FMD), including roles played by different hosts, is essential for improving disease control. The African buffalo (Syncerus caffer) is a reservoir for the SAT serotypes of FMD virus (FMDV). Large buffalo populations commonly intermingle with livestock in Kenya, yet earlier studies have focused on FMD in the domestic livestock, hence the contribution of buffalo to disease in livestock is largely unknown. This study analysed 47 epithelia collected from FMD outbreaks in Kenyan cattle between 2008 and 2012, and 102 probang and serum samples collected from buffalo in three different Kenyan ecosystems; Maasai-Mara (MME) (n = 40), Tsavo (TSE) (n = 33), and Meru (ME) (n = 29). RESULTS: Antibodies against FMDV non-structural proteins were found in 65 of 102 (64%) sera from buffalo with 44/102 and 53/102 also having neutralising antibodies directed against FMDV SAT 1 and SAT 2, respectively. FMDV RNA was detected in 42% of the buffalo probang samples by RT-qPCR (Cycle Threshold (Ct) ≤32). Two buffalo probang samples were positive by VI and were identified as FMDV SAT 1 and SAT 2 by Ag-ELISA, while the latter assay detected serotypes O (1), A (20), SAT 1 (7) and SAT 2 (19) in the 47 cattle epithelia. VP1 coding sequences were generated for two buffalo and 21 cattle samples. Phylogenetic analyses revealed SAT 1 and SAT 2 virus lineages within buffalo that were distinct from those detected in cattle. CONCLUSIONS: We found that FMDV serotypes O, A, SAT 1 and SAT 2 were circulating among cattle in Kenya and cause disease, but only SAT 1 and SAT 2 viruses were successfully isolated from clinically normal buffalo. The buffalo isolates were genetically distinct from isolates obtained from cattle. Control efforts should focus primarily on reducing FMDV circulation among livestock and limiting interaction with buffalo. Comprehensive studies incorporating additional buffalo viruses are recommended.

Extensive X-linked adaptive evolution in central chimpanzees.

Surveying genome-wide coding variation within and among species gives unprecedented power to study the genetics of adaptation, in particular the proportion of amino acid substitutions fixed by positive selection. Additionally, contrasting the autosomes and the X chromosome holds information on the dominance of beneficial (adaptive) and deleterious mutations. Here we capture and sequence the complete exomes of 12 chimpanzees and present the largest set of protein-coding polymorphism to date. We report extensive adaptive evolution specifically targeting the X chromosome of chimpanzees with as much as 30% of all amino acid replacements being adaptive. Adaptive evolution is barely detectable on the autosomes except for a few striking cases of recent selective sweeps associated with immunity gene clusters. We also find much stronger purifying selection than observed in humans, and in contrast to humans, we find that purifying selection is stronger on the X chromosome than on the autosomes in chimpanzees. We therefore conclude that most adaptive mutations are recessive. We also document dramatically reduced synonymous diversity in the chimpanzee X chromosome relative to autosomes and stronger purifying selection than for the human X chromosome. If similar processes were operating in the human-chimpanzee ancestor as in central chimpanzees today, our results therefore provide an explanation for the much-discussed reduction in the human-chimpanzee divergence at the X chromosome.

Integrating genetic data and population viability analyses for the identification of harbour seal (Phoca vitulina) populations and management units.

Identification of populations and management units is an essential step in the study of natural systems. Still, there is limited consensus regarding how to define populations and management units, and whether genetic methods allow for inference at the relevant spatial and temporal scale. Here, we present a novel approach, integrating genetic, life history and demographic data to identify populations and management units in southern Scandinavian harbour seals. First, 15 microsatellite markers and model- and distance-based genetic clustering methods were used to determine the population genetic structure in harbour seals. Second, we used harbour seal demographic and life history data to conduct population viability analyses (PVAs) in the vortex simulation model in order to determine whether the inferred genetic units could be classified as management units according to Lowe and Allendorf's (Molecular Ecology, 19, 2010, 3038) 'population viability criterion' for demographic independence. The genetic analyses revealed fine-scale population structuring in southern Scandinavian harbour seals and pointed to the existence of several genetic units. The PVAs indicated that the census population size of each of these genetic units was sufficiently large for long-term population viability, and hence that the units could be classified as demographically independent management units. Our study suggests that population genetic inference can offer the same degree of temporal and spatial resolution as 'nongenetic' methods and that the combined use of genetic data and PVAs constitutes a promising approach for delineating populations and management units.

Giraffe lineages are shaped by major ancient admixture events.

Strong genetic structure has prompted discussion regarding giraffe taxonomy,1,2,3 including a suggestion to split the giraffe into four species: Northern (Giraffa c. camelopardalis), Reticulated (G. c. reticulata), Masai (G. c. tippelskirchi), and Southern giraffes (G. c. giraffa).4,5,6 However, their evolutionary history is not yet fully resolved, as previous studies used a simple bifurcating model and did not explore the presence or extent of gene flow between lineages. We therefore inferred a model that incorporates various evolutionary processes to assess the drivers of contemporary giraffe diversity. We analyzed whole-genome sequencing data from 90 wild giraffes from 29 localities across their current distribution. The most basal divergence was dated to 280 kya. Genetic differentiation, FST, among major lineages ranged between 0.28 and 0.62, and we found significant levels of ancient gene flow between them. In particular, several analyses suggested that the Reticulated lineage evolved through admixture, with almost equal contribution from the Northern lineage and an ancestral lineage related to Masai and Southern giraffes. These new results highlight a scenario of strong differentiation despite gene flow, providing further context for the interpretation of giraffe diversity and the process of speciation in general. They also illustrate that conservation measures need to target various lineages and sublineages and that separate management strategies are needed to conserve giraffe diversity effectively. Given local extinctions and recent dramatic declines in many giraffe populations, this improved understanding of giraffe evolutionary history is relevant for conservation interventions, including reintroductions and reinforcements of existing populations.

African bushpigs exhibit porous species boundaries and appeared in Madagascar concurrently with human arrival.

Several African mammals exhibit a phylogeographic pattern where closely related taxa are split between West/Central and East/Southern Africa, but their evolutionary relationships and histories remain controversial. Bushpigs (Potamochoerus larvatus) and red river hogs (P. porcus) are recognised as separate species due to morphological distinctions, a perceived lack of interbreeding at contact, and putatively old divergence times, but historically, they were considered conspecific. Moreover, the presence of Malagasy bushpigs as the sole large terrestrial mammal shared with the African mainland raises intriguing questions about its origin and arrival in Madagascar. Analyses of 67 whole genomes revealed a genetic continuum between the two species, with putative signatures of historical gene flow, variable FST values, and a recent divergence time (<500,000 years). Thus, our study challenges key arguments for splitting Potamochoerus into two species and suggests their speciation might be incomplete. Our findings also indicate that Malagasy bushpigs diverged from southern African populations and underwent a limited bottleneck 1000-5000 years ago, concurrent with human arrival in Madagascar. These results shed light on the evolutionary history of an iconic and widespread African mammal and provide insight into the longstanding biogeographic puzzle surrounding the bushpig's presence in Madagascar.

Introgression and disruption of migration routes have shaped the genetic integrity of wildebeest populations.

The blue wildebeest (Connochaetes taurinus) is a keystone species in savanna ecosystems from southern to eastern Africa, and is well known for its spectacular migrations and locally extreme abundance. In contrast, the black wildebeest (C. gnou) is endemic to southern Africa, barely escaped extinction in the 1900s and is feared to be in danger of genetic swamping from the blue wildebeest. Despite the ecological importance of the wildebeest, there is a lack of understanding of how its unique migratory ecology has affected its gene flow, genetic structure and phylogeography. Here, we analyze whole genomes from 121 blue and 22 black wildebeest across the genus' range. We find discrete genetic structure consistent with the morphologically defined subspecies. Unexpectedly, our analyses reveal no signs of recent interspecific admixture, but rather a late Pleistocene introgression of black wildebeest into the southern blue wildebeest populations. Finally, we find that migratory blue wildebeest populations exhibit a combination of long-range panmixia, higher genetic diversity and lower inbreeding levels compared to neighboring populations whose migration has recently been disrupted. These findings provide crucial insights into the evolutionary history of the wildebeest, and tangible genetic evidence for the negative effects of anthropogenic activities on highly migratory ungulates.

Cape buffalo mitogenomics reveals a Holocene shift in the African human-megafauna dynamics.

Africa is unique among the continents in having maintained an extraordinarily diverse and prolific megafauna spanning the Pleistocene-Holocene epochs. Little is known about the historical dynamics of this community and even less about the reasons for its unique persistence to modern times. We sequenced complete mitochondrial genomes from 43 Cape buffalo (Syncerus caffer caffer) to infer the demographic history of this large mammal. A combination of Bayesian skyline plots, simulations and Approximate Bayesian Computation (ABC) were used to distinguish population size dynamics from the confounding effect of population structure and identify the most probable demographic scenario. Our analyses revealed a late Pleistocene expansion phase concurrent with the human expansion between 80 000 and 10 000 years ago, refuting an adverse ecological effect of Palaeolithic humans on this quarry species, but also showed that the buffalo subsequently declined during the Holocene. The distinct two-phased dynamic inferred here suggests that a major ecological transition occurred in the Holocene. The timing of this transition coincides with the onset of drier conditions throughout tropical Africa following the Holocene Optimum (∼9000-5000 years ago), but also with the explosive growth in human population size associated with the transition from the Palaeolithic to the Neolithic cultural stage. We evaluate each of these possible causal factors and their potential impact on the African megafauna, providing the first systematic assessment of megafauna dynamics on the only continent where large mammals remain abundant.

Combining methods for non-invasive fecal DNA enables whole genome and metagenomic analyses in wildlife biology.

Non-invasive biological samples benefit studies that investigate rare, elusive, endangered, or dangerous species. Integrating genomic techniques that use non-invasive biological sampling with advances in computational approaches can benefit and inform wildlife conservation and management. Here, we used non-invasive fecal DNA samples to generate low- to medium-coverage genomes (e.g., >90% of the complete nuclear genome at six X-fold coverage) and metagenomic sequences, combining widely available and accessible DNA collection cards with commonly used DNA extraction and library building approaches. DNA preservation cards are easy to transport and can be stored non-refrigerated, avoiding cumbersome or costly sample methods. The genomic library construction and shotgun sequencing approach did not require enrichment or targeted DNA amplification. The utility and potential of the data generated was demonstrated through genome scale and metagenomic analyses of zoo and free-ranging African savanna elephants (Loxodonta africana). Fecal samples collected from free-ranging individuals contained an average of 12.41% (5.54-21.65%) endogenous elephant DNA. Clustering of these elephants with others from the same geographic region was demonstrated by a principal component analysis of genetic variation using nuclear genome-wide SNPs. Metagenomic analyses identified taxa that included Loxodonta, green plants, fungi, arthropods, bacteria, viruses and archaea, showcasing the utility of this approach for addressing complementary questions based on host-associated DNA, e.g., pathogen and parasite identification. The molecular and bioinformatic analyses presented here contributes towards the expansion and application of genomic techniques to conservation science and practice.