Applying high-throughput rRNA gene sequencing to assess microbial contamination of a 40-year old exposed archaeological profile
Introduction
The analysis of ancient organic molecules such as nucleic acids, proteins, and lipids from the archaeological record has heralded a new field of research (Brown and Brown, 2011; Evershed, 2008) and led to revolutionary findings. Ancient DNA has been extracted from hominin bones, leading to the sequencing of the entire Neanderthal genome (Castellano et al., 2014; Green et al., 2010; Hajdinjak et al., 2018; Prüfer et al., 2014) and the discovery of new lineages such as the Denisovans (Krause et al., 2010; Meyer et al., 2012). Of equal significance, the technology to extract ancient hominin DNA from archaeological sediments has been developed (Slon et al., 2017). The extraction of microbial DNA from human remains, including dental calculus, has yielded key insights into past infectious diseases (e.g., Bos et al., 2011, 2014) and endogenous microbial communities, i.e. ‘oral’ and ‘gut’ microbiomes (Schnorr et al., 2016; Warinner et al., 2014, 2017).
In addition to nucleic acids, proteins are now also routinely recovered from archaeological materials. Proteinaceous binders in artworks can be characterized (Dallongeville et al., 2016; Vinciguerra et al., 2016), and dietary proteins can be extracted from dental calculus (Warinner et al., 2014; Hendy et al., 2018a, 2018b), which is also a source for proteins associated with ancient diseases (Warinner et al., 2014). Additionally, proteins are now routinely extracted from bones (Cappellini et al., 2012), leading to advances in phylogenetic reconstruction (Welker et al., 2016), and providing a valuable source of taxonomic information in archaeological contexts where morphological identifications are not possible due to fragmentation, thanks to the collagen peptide mass fingerprinting technique known as ZooMS, or Zooarchaeology by Mass Spectrometry (Buckley et al., 2009; Richter et al., 2011; Hofman et al., 2018). ZooMS has also led to the identification of hominin bones in archaeological sites, adding important samples to a rare taxonomic category (Brown et al., 2016; Welker et al., 2016; Devièse et al., 2017).
Lipids are also regularly extracted from archaeological contexts. Lipids derived from foods have been extracted from ceramic pottery, enabling the reconstruction of foodways (Reber and Evershed, 2004; Mukherjee et al., 2007; Evershed et al., 2008; Craig et al., 2011, 2013; Lucquin et al., 2018) and new cultural practices such as dairying (Copley et al., 2005a, 2005b, 2005c). A category of lipid from plants, the epicuticular waxes in leaves, can be recovered from sediments and used for paleoenvironmental reconstruction (Meyers, 2003; Gocke et al., 2013; Gamarra and Kahmen, 2015), even when charred (Jambrina-Enríquez et al., 2018, 2019).
As biomolecular techniques continue to be refined, the recovery of ancient biomolecules from archaeological contexts will become increasingly important. One key question in this regard relates to the preservation of ancient biomolecules. A better understanding of the circumstances favorable to the preservation of ancient biomolecules, or, conversely, destructive to them, will help us search for them more effectively. Therefore, it is imperative that we work to identify the factors that affect the preservation of ancient biomolecules, both in situ and during sampling for biomolecular analyses, as it is likely that some ancient biomolecules are at risk of being damaged by exposure to air, light, water, and microorganisms.
Microorganisms are well known to degrade diverse organic molecules in various sedimentary contexts (Meyers, 1997). As case in point, a recent study showed that the carbon and hydrogen isotopic ratios of n-alkanes in sediment samples were altered when microorganisms proliferated during storage (Brittingham et al., 2017). Such compounds come from the epicuticular leaf waxes of plants, and their δ13C as well as δD ratios have been shown to reflect the environmental conditions under which they form. Therefore, the extraction of these n-alkanes from archaeological sediments and the analysis of their carbon and hydrogen isotopic ratios can provide a valuable source of paleoenvironmental information. However, in a comparison of sediment samples from archaeological deposits dating to 45 ka at Lusakert Cave that were improperly stored at room temperature for three years versus sediment samples from identical locations in the site that were immediately frozen after collection, Brittingham et al. (2017) showed that the abundance of long-chain n-alkanes had dropped in the room temperature samples, while the abundance of medium-chain n-alkanes had increased. Furthermore, both the carbon and hydrogen isotopic values of the long-chain alkanes were altered in the improperly stored samples. Finally, DNA analysis revealed that bacterial genera, such as Rhodococcus and Aeromicrobium, which contain coding regions for n-alkane degrading enzymes, had increased in relative abundance in the room temperature samples versus the frozen samples. The authors conclude that during the period of storage, these microbes proliferated and resulted in the breakdown of longer-chain n-alkanes as well as the alteration of the isotopic ratios. The implications of these results are that the paleoclimatic inferences made from such altered data would be significantly skewed.
The immediate implications of these results are that proper storage of sediment samples is essential. The wider implications, however, are more troubling. Microbial activity in buried sediments is normally low due to low oxygen availability (ibid.), and it is well known that microbial growth in soils is dependent upon the availability of air, water, and nutrients (Adl, 2003). Presumably, then, exposure of sediments to air, light, and water as a result of excavation allows microbes to proliferate on the exposed surfaces, but whether microbial communities deeper in the profile are altered over timescales relevant to archaeological excavations is unknown. We decided to explore this question in Paleolithic sediments at the site of Crvena Stijena in Montenegro.
The rock shelter of Crvena Stijena (‘Red Rock’) in Montenegro contains one of the longest and best-preserved Middle Paleolithic (MP) sequences in southeastern Europe. Crvena Stijena is situated in a limestone cliff that is part of the Dinaric Karst in the southwestern part of the country, at 700 m above sea level and 32 km from the present Adriatic Sea (Fig. 1). The shelter is large, approximately 26 m wide at the mouth, and 15 m deep from the dripline to the back of the shelter.
Excavations in the 1950s and 1960s uncovered a stratified sequence of archaeological layers over 20 m deep, spanning the Middle Paleolithic through the Bronze Age (Vušović-Lučić et al., 2017). These early excavations removed vast quantities of archaeological sediment, leaving the interior of the talus terraced to maintain an overall slope down towards the interior of the shelter. Excavations from 1960 to 64 concentrated on the innermost part of the shelter, further sinking a deep sounding 10 m vertically into Middle Paleolithic sediments without reaching bedrock. The stratigraphy developed by geologist Brunnacker (1975) on the basis of these excavations (Fig. 2) has been recognized as still valid today by subsequent field workers (Morley, 2007; Baković et al., 2009). The resulting lithic collections have been the basis for many analyses in which Crvena Stijena serves as a critical type-site for the southern Balkans (Mihailović, 2009, 2014; Dogandžić and Đuričić, 2017; Mihailović and Whallon, 2017).
Excavations from 2004 to 2015 explored the sediments above Basler's deep sounding, uncovering in situ remains in Mesolithic and late Middle Paleolithic sediments, as summarized in Baković et al. (2009) and Whallon (2017). This multidisciplinary research project also documented the excellent preservation of fauna and combustion features and yielded the first absolute chronology for the site, based upon an extensive radiometric dating program using TL, OSL, ESR, and AMS 14C methods (Mercier et al., 2013; see Fig. 3). In addition, these investigations showed that the Middle Paleolithic levels (XII through XXXI) are capped by a thick tephra layer (layer XI), which was geochemically identified as the Y5 tephra from the Campanian Ignimbrite (CI) eruption at 39.9 ka (Morley and Woodward, 2011).
Faunal and taphonomic analyses have shown that hominins were by far the dominant bone accumulator in all levels and that red deer dominates the species list in all but a few of the MP layers (Morin and Soulier, 2017). Anthracological and biomarker analyses conducted on Basler's profile have shown that charcoal is well-preserved (Shaw, 2017) and that organic molecules are present (March et al., 2017). Finally, analysis of the lithic collections has shown cultural continuity throughout the Middle Paleolithic sequence and the presence of Uluzzian (transitional Middle-Upper Paleolithic) elements in the uppermost MP levels, immediately below the Y-5 tephra (Mihailović and Whallon, 2017).
In 2014, a large amount of sterile overburden was removed from levels immediately above the top of the MP sequence. This allowed access to the layers below the Y-5 tephra, and a new excavation project designed to investigate Neanderthal pyrotechnological behaviors in the Middle Paleolithic layers was initiated in 2017 (Tostevin, 2017). Horizontal excavations currently under way are designed to expose the combustion features and associated artifacts. Equally important are a set of ‘vertical excavations’ from the 10-m deep profile to obtain samples for dating, anthracological analyses, faunal analyses, micromorphological analyses, molecular analyses, archaeomagnetic analyses, pollen analyses, and phytolith analyses. The benefit of this vertical excavation strategy is that it allows us to obtain valuable data for reconstructing chronology, paleoenvironments, and site formation processes through many thousands of years while maximizing preservation of the site. A key component of our investigations includes biomolecular analyses such as the identification of sterol and lipid biomarkers (Jambrina-Enríquez et al., 2019; Rodríguez de Vera et al., in press) and the extraction of hominin DNA (sensu Slon et al., 2017). Analyses of ancient biomolecules from the hearths are integrated with the detailed micromorphological study of the site formation processes impacting the combustion features (sensu Mallol et al., 2013).
Vertical sampling for these analyses begins by cleaning the profile (i.e., the excavation wall), which has been exposed since the 1960s excavations. Often, samples for several analyses can be extracted at the same time, which minimizes disturbance to the deposits. However, we have observed that green biofilms sometime develop on the surface of certain portions of the profile over a matter of months (Fig. 4). Given the results of the Brittingham et al. (2017) study, which showed that changes to the microbial community in archaeological sediments altered the biomolecules contained within them, we worried that the presence of these biofilms could signify microbial impacts to sediment-hosted organics. Concerned about the depth to which microbes might penetrate into the sediments each time these were freshly cleaned and exposed to air during sampling, and with an eye towards guiding sampling for biomolecular analyses as described above, we decided to evaluate microbial communities on and within the archaeological profile and determine whether this new microbial growth was associated with microbial proliferation deeper into the sediments. We address this by systematically exploring the following questions: 1) are microbial communities on the surface similar to microbial communities deeper in the profile? 2) how deeply into the profile do the surface communities extend? To our knowledge, this is the first study of its kind.
Section snippets
Sample collection
In January of 2019, we sampled sediments from 8 different locations in the profile, across 4 different sedimentary layers. We followed a sampling protocol optimized for microbial sampling and designed to limit contamination. The two individuals doing the sampling each wore nitrile gloves and used stainless steel ‘scoopulas’ to remove sediments from the profile. The scoopulas were sterilized before each sample collection by an ethanol rinse and combustion of the ethanol on the implement.
Results
We generated 16S rRNA gene libraries from 31 samples that represented depth profiles from 5 horizons and 7 additional samples from other surfaces in the pit (Table 1). In order to assess contamination, we also generated libraries from DNA extraction kit blank controls, from positive controls and no template blanks used in the PCR reaction, and from blanks included during the sequencing process (Table 1). Following quality filtering, all libraries with fewer than 2000 sequences were excluded
Discussion
Excavation is a disturbance that exposes archaeological sediments to oxygen and other new biogeochemical gradients, and could therefore change the natural microbial communities in them. Recent work by Brittingham et al. (2017) showed that microbial activity can alter the isotopic ratios of n-alkanes during improper sediment storage, and authors have raised concerns over microbial degradation or alteration of organic compounds and biomolecular data during sample storage and in other
Conclusions
The analysis of ancient biomolecules from the archaeological record is yielding an unprecedented amount of data relevant to archaeological and paleoanthropological questions. However, much is still unknown about the circumstances favorable to the preservation of these ancient biomolecules and, conversely, the processes that destroy or, more insidiously, alter them. A recent study showed that plant n-alkanes extracted from archaeological sediments, which are used to reconstruct
Declaration of competing interest
None.
Acknowledgements
G. Monnier, G. Tostevin, M. Baković, G. Pajović, and N. Borovinić wish to thank Bob Whallon for his continued guidance and help; the villagers of Petrovići for their immense hospitality; Annie Melton, Samantha Porter, Vasilije Marojević, Đuro Pribilović, and the many other talented students and workers who assist with fieldwork and laboratory work; and the Montenegrin Ministry of Culture, Montenegro the Montenegrin Academy of Sciences, the United States National Science Foundation, United States
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