Stone Age Yersinia pestis genomes shed light on the early evolution,diversity, and ecology of plague

The bacterial pathogen Yersinia pestis gave rise to devastating outbreaks throughout human history, and ancient DNA evidence has shown it afflicted human populations as far back as the Neolithic. Y. pestis genomes recovered from the Eurasian Late Neolithic/Early Bronze Age (LNBA) period have uncovered key evolutionary steps that led to its emergence from a Yersinia pseudotuberculosis-like progenitor; however, the number of reconstructed LNBA genomes are too few to explore its diversity during this critical period of development. Here, we present 17 Y. pestis genomes dating to 5,000 to 2,500 y BP from a wide geographic expanse across Eurasia. This increased dataset enabled us to explore correlations between temporal, geographical, and genetic distance. Our results suggest a nonflea-adapted and potentially extinct single lineage that persisted over millennia without significant parallel diversification, accompanied by rapid dispersal across continents throughout this period, a trend not observed in other pathogens for which ancient genomes are available. A stepwise pattern of gene loss provides further clues on its early evolution and potential adaptation. We also discover the presence of the flea-adapted form of Y. pestis in Bronze Age Iberia, previously only identified in in the Caucasus and the Volga regions, suggesting a much wider geographic spread of this form of Y. pestis. Together, these data reveal the dynamic nature of plague’s formative years in terms of its early evolution and ecology.

The earliest known cases of human infection with the plague pathogen, Yersinia pestis, date to around 5,000 y ago (1–4). Analyses of ancient Y. pestis genomes from this period suggest that the time window between 6,000 and 4,000 y ago was critical and formative for the evolution and ecology of Y. pestis as we know it today. Four ancient Y. pestis lineages have been identified so far, which can be genomically distinguished based on their adaptations to the flea, the main vector of modern plague. Today, fleas are known to play a central role in the transmission of plague within rodent populations, which can act as reservoirs from where spillovers to human populations typically occur (5, 6). The transmission of Y. pestis by the flea is either facilitated by a blockage of the foregut (proventriculus), where the bacterium produces a biofilm (7), or in a biofilm-independent manner, also known as early-phase transmission (8, 9). The oldest lineages of Y. pestis (2, 4) (hereafter referred to as preLNBA−), and the Late Neolithic and Early Bronze Age (LNBA−) lineage (1, 3) present a genetic background that has been interpreted as being incompatible with flea transmission via the blockage of the foregut (indicated in the naming by the minus sign). While a recently identified ancient lineage also dating to the Bronze Age presents all the genetic adaptations for this highly efficient form of flea transmission (10) (LNBA+; the plus sign indicates the adaptation to the flea vector). Intriguingly, both variants coexisted for millenia and they might have occupied different niches. However, it remains unclear how the different forms of Y. pestis infected humans during prehistory and how the resulting diseases manifested in the human population. Whether plague ecology and transmission as we know it today can serve as a model to understand its manifestation in the past remains also unknown.Elucidating the ecology and transmission will be crucial for understanding how the LNBA+/− lineages of plague, which were widespread across Eurasia for thousands of years (1, 3, 10, 11), have impacted human societies, and how changes in human subsistence and economy have shaped the early evolution of this pathogen. It is currently unknown whether and which types of animal populations served as potential reservoirs of the disease and their identification will be essential for characterizing past Y. pestis transmission dynamics. The absence of an adaptation to the flea vector in some plague lineages suggests that the transmission dynamics were complex. Today, the flea-mediated model is not the only documented form of plague transmission: pneumonic plague can be acquired via respiratory droplets from close human-to-human contact. However, only a few reported outbreaks have been attributed to this transmission mode and usually in contexts of poor ventilation and direct contact with infected individuals (12–17). Additionally, plague has been documented in humans who handled or ingested parts of infected animals (18–22).Changes in human behavior may also have contributed to a higher risk of plague infection. During the LNBA period, archeological evidence attests to technological advances, such as the spread of oxen-drawn carts and wagons (23) and horse domestication (24), which enabled increased human mobility and exploitation of new habitats, such as the Eurasian steppe belt. This ultimately led to the establishment of long-distance networks, in which raw materials such as copper were circulated (25, 26). However, periods of unrest and war could also have played a role in the extended human mobility during the LNBA period. While earlier studies hypothesized that increased mobility was the cause for an early spread of Y. pestis across Eurasia (1, 3), it could also have been its effect. It is also during this period that animal husbandry and mobile pastoralism intensified in the steppe (27), thus facilitating the overlap of ecological niches for zoonoses to occur. The aforementioned changes could have played a role in the likelihood of transmission to humans and the long-distance spread of plague during its early evolution.Here we expand the number of Y. pestis genomes from the LNBA period to offer a higher genomic resolution for important stages in the evolution of the bacterium, as well as its diversity and geographical distribution in the past. By linking the genomic evidence with the available archeological context, we discuss potential transmission mechanisms of plague during its early evolution.