Were domestic camelids present on the prehispanic South American agricultural frontier? An ancient DNA study.

The southern boundary of prehispanic farming in South America occurs in central Mendoza Province, Argentina at approximately 34 degrees south latitude. Archaeological evidence of farming includes the recovery of macrobotanical remains of cultigens and isotopic chemistry of human bone. Since the 1990s, archaeologists have also hypothesized that the llama (Lama glama), a domesticated South American camelid, was also herded near the southern boundary of prehispanic farming. The remains of a wild congeneric camelid, the guanaco (Lama guanicoe), however, are common in archaeological sites throughout Mendoza Province. It is difficult to distinguish bones of the domestic llama from wild guanaco in terms of osteological morphology, and therefore, claims that llama were in geographic areas where guanaco were also present based on osteometric analysis alone remain equivocal. A recent study, for example, claimed that twenty-five percent of the camelid remains from the high elevation Andes site of Laguna del Diamante S4 were identified based on osteometric evidence as domestic llama, but guanaco are also a likely candidate since the two species overlap in size. We test the hypothesis that domesticated camelids occurred in prehispanic, southern Mendoza through analysis of ancient DNA. We generated whole mitochondrial genome datasets from 41 samples from southern Mendoza late Holocene archaeological sites, located between 450 and 3400 meters above sea level (masl). All camelid samples from those sites were identified as guanaco; thus, we have no evidence to support the hypothesis that the domestic llama occurred in prehispanic southern Mendoza.

Introduction

Southern Mendoza is thought to be the southern limit of the dispersion of prehispanic (>500 years ago) agriculture in South America [1-7]. Evidence of agriculture includes macrobotanical remains of domestic plants, the presence of human osteopathies that relate to an agricultural diet, and δ13C and δ15N signatures measured in human remains [4, 8, 9]. While the timing of the first arrival of cultigens to southern Mendoza is debated, it is estimated to be approximately 2,200 radiocarbon years before present. However, the initial presence of cultigens may not represent the onset of agriculture, and apparently during this early stage their contribution to the diet was not important [1–4, 10]. Traditionally, in South American archaeology, agriculture was associated with the presence of pastoral methods, and therefore domestic animals, such as llama (Lama glama) or alpaca (Vicugna pacos) [11-14]. Although the prehispanic presence of cultigens in southern Mendoza is supported by multiple lines of evidence, pastoral practices using domestic camelids have only recently been proposed based on archaeological remains from this area [13, 15–17]. The unequivocal presence of llama and pastoralism at this latitude is only established for the neighboring central Chile during the Inka empire between AD 1470 and 1536 [18-Universidad de Tarapacá. Facultad de Ciencias Sociales y Jurídicas. 2009 ">21].

Southern Mendoza province represents the northern limit of Patagonia from an environmental perspective [10, 22]. In this region, zooarchaeologists have identified wild camelid faunal remains as guanaco (Lama guanicoe) [23-31]. During the early Holocene, however, faunal remains of another wild camelid the vicuña (Vicugna vicugna), have also been identified at the Agua de la Cave site (Fig 1A) located in the Andes at 200 km north to Southern Mendoza [15, 24, 32].

Fig 1

Camelid distribution, boundaries and archaeological sites from the study area.

A) The map shows the boundary for accepted archaeological evidence of prehispanic domestication and Inka expansion near the study area [14, 33, 34]. Archaeological sites where ancient DNA samples were recovered: 1) Volcán El Hoyo; 2) Agua de Pérez; 3) Los Leones-6; 4) Agua de los Caballos; 5) Cueva de la Luna; 6) Zanjón El Morado; 7) Ojo de Agua; 8) Cueva Salamanca; 9) Cueva Palulo; 10) Arroyo El Desecho-4; 11) Cueva Arroyo Colorado; 12) Los Peuquenes; 13) El Indígeno; 14) Risco de los Indios; 15) El Carrizalito; 16) Alero Montiel; 17) Fuerte San Rafael del Diamante. LD-S4 site (red star) where llama was identified using morphometric analyses. High elevation sites near to LD-S4 site: Los Peuquenes (12), El Indigeno (13), and Risco de los Indios (14). The map uses NASA open data (https://earthdata.nasa.gov). B) The historic and present distribution of guanaco, llama and vicuña from Franklin [35].

Today, the guanaco is the only wild camelid in Patagonia, because the distribution of vicuña extends only as far south as northern San Juan Province, which is 500 km to the north (Fig 1B) [35, 36]. South American domestic camelids include llama and alpaca, but only llama is considered in this paper due to minimal evidence for the presence of alpaca from precolumbian sites in Argentina [37] and Chile [38, 39].

In addition to the morphometric identification of llama remains in the northern Mendoza archaeological record [15, 40, 41] its presence was assumed due to the late but strong presence of the Inka empire in the region [14, 33, 34]. In contrast, in southern San Juan province (500 km to the North), there are clear archaeological indicators of the presence of llama, such as dung deposits, textiles, and a large number of depictions in rock art, pottery, and "furniture art" [42-45]. The southernmost evidence of Inka occupation on the eastern slope of the Andes has been proposed for the Agua Amarga site in Tunuyán, located 100 km to the north of the study area [46]. The distribution of the Inka empire was similar on the western side of the Andes (central Chile), with an absence of pre-Inka evidence of domestic camelids at this latitude in the archaeological record [47-49].

Despite the absence of archaeological and ethnohistorical evidence, some researchers argue that prehispanic farming groups in the northern limit of Patagonia were camelid herders [13, 16, 17, 50]. These arguments focus on the presence of remains of domestic plants and pottery at archaeological sites with camelid herding inferred by extrapolation. It is important to note, however, that plant domestication, pastoral practices, and the adoption and development of pottery technology do not necessarily occur contemporaneously in many areas of the world [51-54]. Other inferences of camelid herding in southern Mendoza is based on morphometric analysis of camelid osteological remains [15], leading to the claim that domesticated llama, specifically, were present in the region. This claim is based on analysis of zooarchaeological remains from Laguna del Diamante-S4 (LD-S4) site (Fig 1A) from archaeological contexts dated from 700 to 950 years BP [15]. The identification of the remains as llama as opposed to guanaco was based on osteometric analysis indicating large body size, as llamas were intentionally bred for increased body mass. At LD-S4, 25% of the total faunal assemblage (n = 12 samples) was identified as llama. Gasco [15] identified a similar proportion of llama from zooarchaeological assemblages from northern Mendoza and central and southern San Juan using the same morphometric criteria.

Claims that prehispanic llama and "herders" occurred south of 34° latitude have important implications for archaeological interpretations of past human lifeways in the region. The presence of such groups would imply changes in subsistence and settlement systems including reductions in residential mobility, technological and dietary changes, as well as a differentiated materiality from the previous occupations [40, 55–57]. If the presence of herder groups in the northern limit of Patagonia is confirmed, it would also require revision of many interpretations of how people used the landscape, interpretations of human demography, and conclusions about human impacts on the environment, all of which would be dramatically different if herding societies were present in those areas. The morphometric-based identification of camelid bones as llama [15] is the only empirical evidence used to support such claims.

The use of morphometry to study domestication has been widely used in South America [39, 40, 57–62]. Even though morphometry has many advantages, researchers recognize the limitations of the approach, especially for making inferences concerning past geographic range extension based on small sample sizes [38, 59–62]. In contrast, analysis of ancient mitochondrial DNA (aDNA) generated from zooarchaeological samples can provide a stronger approach for species identification including population-level comparisons in current and archaeological contexts when species identification is less conclusive based on morphology [63-67]. Analysis of DNA from domestic and wild species can also help delineate domestication events [68-70]. Using DNA to study South American camelids, Marin et al. [69, 71] determined that the llama was domesticated from guanaco and the alpaca from vicuña. Analysis of aDNA can also be used to assess whether llamas were present in southern Mendoza/northern Patagonia, which would indicate the presence of a pastoral society in the region and subsequently extend the southernmost limit of prehispanic agriculture in the Americas.

In this paper, we employ analysis of aDNA as a method for identifying species among camelid remains from archaeological sites in Mendoza. We analyze whole mitochondrial DNA genomic sequence data generated from 50 archaeological samples of camelid bone from central and southern Mendoza, 41 of which produced sufficient aDNA for analysis. Remains from LD-S4 were not analyzed in this study because we were not able to obtain permission. Diagnostic nucleotide positions within the mitochondrial genome were used to assign samples to species based on taxon-specific DNA sequence data publicly available on GenBank (www.ncbi.nlm.nih.gov/genbank). This approach made it possible to assess whether any of the archaeological samples support the hypothesis that domestic llamas were present in prehispanic southern Mendoza.

Materials and methods

Fifty camelid bone samples (identified as Lama spp. based on osteological characters) from 25 cultural assemblages excavated from 17 archaeological sites located in central and southern Mendoza (Fig 1A) were selected for analysis. This represents 70% of the available archaeological sites with faunal remains from the region. Each sample was assigned to a temporal assemblage using associated radiocarbon dates and artifact types [28–30, 72]. Several types of sites are represented including base camps, kill sites, butchery stations, and small camps occupied for short periods. Contexts include open-air sites, rockshelters, and caves (see Table 1). Some of the sites have remains of domestic plants and/or association with human remains with values of δ13C that suggest corn consumption [73].

Table 1

Assemblage and site descriptions for the samples.

Site	Sample ID	Funcionality	Kind of site	Years BP	skeletal element
Agua de los Caballos	14	undetm	Cave	300	Metapodial
Agua de Perez	87	BC	Open air	685	Mandible
Agua de Perez	88*	BC	Open air	685	Ischium
Alero Montiel	17*	undetm	Rockshelter	1800	Phalanx
Alero Montiel	39	undetm	Rockshelter	2240	Tooth
Cave Arroyo Colorado	5*	SAS	Cave	770	Astragalus
Cave Arroyo Colorado	6	SAS	Cave	770	Carpal
Cave Arroyo Colorado	33	undetm	Cave	770	Metapodial
Cave de Luna	20	undetm	Cave	1400	Phalanx
Cave de Luna	38	undetm	Cave	500	Tooth
Cave Palulo	19	SAS	Cave	2050	Humerus
Cave Palulo	61	SAS	Cave	130	Scapula
Cave Palulo	63	SAS	Cave	130	Carpal
Cave Palulo	65	SAS	Cave	2030	Long bone shaft
Cave Salamanca	31*	undetm	Cave	1500	Metacarpal
Cave Salamanca	72	undetm	Cave	2200	Phalanx
Cave Salamanca	85*	undetm	Cave	2200	Metapodial
Cave Salamanca	86	undetm	Cave	2200	Magnum
Cave Salamanca	94	undetm	Cave	2200	Metapodial
Cave Salamanca	95	undetm	Cave	2200	Phalanx
Cave Salamanca	96	undetm	Cave	2200	Tibia
Cave Salamanca	97	undetm	Cave	1360	Metapodial
Cave Salamanca	102	undetm	Cave	7000	Phalanx
El Desecho 4	45	BC	Open air	5500	Phalanx
El Desecho 4	47*	BC	Open air	5500	Metapodial
El Desecho 4	49*	BC	Open air	5500	Long bone fragment
El Indigeno	106	BC	Structures	900	Phalanx
El Indigeno	107	BC	Structures	900	Metapodial
El Indigeno	108	BC	Structures	900	Metapodial
El Perdido 4	121	BC	Open air	2600	Third Phalanx
El Perdido 5	119	BC	Open air	2100	Cuneiform
Fuerte SRD	66	undetm	Historico	200	Metapodial epiphysis
Fuerte SRD	67	undetm	Historico	200	Proximal metacarpal
Fuerte SRD	71	undetm	Historico	200	Ulna
Fuerte SRD	110	undetm	Historico	200	Phalanx
Fuerte SRD	112	undetm	Historico	200	Metapodial
Fuerte SRD	113	undetm	Historico	200	Metapodial
Gruta Carrizalito	92	undetm	Cave	530	Cuboid
Los Leones 6	78	undetm	Open air	300	Calcaneus
Los Leones 6	79	undetm	Open air	300	Metatarsal distal
Los Peuquenes	25	BC	Structures	360	Phalanx
Los Peuquenes	80	BC	Structures	360	Carpal
Ojo de Agua	109	BC	Open air	200	Metapodial
Puesto Ortubia	59*	BC	Open air	900	Long bone shaft
Risco de los Indios	53	BC	Structures	500	Phalanx
Volcan El Hollo	91*	SAS	Cave	500	Second Phalanx
Volcan El Hollo	100	SAS	Cave	500	Metapodial
Zanjon Morado	81	SAS	Rockshelter	1200	Phalanx (juv)
Zanjon Morado	82	SAS	Rockshelter	1200	Phalanx
Zanjon Morado	83	SAS	Rockshelter	1200	Phalanx distal

Notes: Of the 50 samples, adequate sequence data coverage was obtained for further analysis for 41 samples. BC (base camp), SAS (specific activities site), undetm (undetermined). The date of each bone sample was assigned by direct association with radiocarbon dates from charcoal. The exceptions are the samples from Fuerte SRD (date is based on historical documents) and Ojo de Agua (date is based on cultural material association). The samples are available in the Museo de Historia Natural de San Rafael, Archaeology lab. No permits were required for the described study, which complied with all relevant regulations

(*) These samples were not used for analysis due to low coverage.

DNA extraction and library preparation were conducted in a dedicated aDNA laboratory at the University of North Texas using methods developed specifically to minimize contamination with contemporary DNA. It is also important to note that no camelid samples had been processed in the lab prior to this study. Genomic DNA extraction followed methods originally described elsewhere [74] with minor modifications as also described in [75, 76]. All tools and bench-top surfaces were cleaned with 10% bleach and 95% ethanol between each sample prep, and the surface of each bone sample was sterilized prior to tissue extraction using a UV crosslinker CL-1000 (UVP, Upland, CA, USA) for 10 minutes (5 minutes per side). All bone samples were also cleaned by removing approx 1mm of surface layer using a sterile drill and disc in an airflow hood in a separate room from aDNA extraction. Dense interior cortical bone was removed and pulvurized to a fine powder using a SPEX 6775 Freezer/Mill cryogenic grinder (SPEX SamplePrep, Metuchen, NJ, USA).

The aDNA laboratory is dedicated solely for work on DNA extraction from ancient samples. Extractions were conducted in groups of fifteen that included a negative control in each group to verify no cross-contamination or reagent contamination. Approximately 150 mg of bone powder from each sample was pre-digested for 1 h at 56°C in 1 mL of lysis buffer (Proteinase K 0.25 mg/ml, N-laurylsarcosyl 0.5%, EDTA 0.45 M, pH8) to limit contemporary bacterial contamination. After pre-digestion, samples were centrifuged at 500 xg for 5 minutes and the supernatant was removed. The undigested bone pellets were again incubated in 1mL of extraction buffer overnight at 37°C. After the second digestion, samples were centrifuged for 5 min at 500 xg and the supernatant was mixed with 3 ml Tris-EDTA (TE) buffer 1x, in an Amicon Ultra 4 30 kDa centrifugal filter (EMD Millipore) and centrifuged for 10 min at 4,000 xg. The filter was washed with 2 ml TE and centrifuged for 8 min at 4,000 xg, the remaining 50 ul of the sample in the filter was brought to a total volume of 100ul. The final purification step was carried out with a QIAquick column (Qiagen) and eluted in 45ul with elution buffer (EB, Qiagen).

Total DNA was treated with 1X USER enzyme mix (New England BioLabs) for 3 h at 37°C to reduce nucleotide substitution errors associated with cytosine deamination, which is common when working with aDNA [77-79]. DNA concentrations of all extracted samples were measured using a Qubit Fluorometer with the dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA, USA). Sequencing libraries were then generated with a starting concentration of approximately 1.95 ug/ml following a modified version of the Blunt-End Single-Tube method (BEST; [80]) to allow for double-indexing. Amplified libraries were validated using the Qubit Fluorometer and the Agilent 2100 Bioanalyzer (Agilent Biosystems, Santa Clara, CA, USA). The libraries were also enriched for mitochondrial fragments following the procedure described by Maricic et al. [81], with minor modifications using a predesigned Lama glama myBaits Mito panel (ArborBioSciences, Ann Arbor, MI, USA). All of the libraries were pooled and sequenced using a 150 cycles reagent cartridge (2x75) on an Illumina NextSeq 500 sequencing platform (Illumina, San Diego, CA, USA) at University of North Texas BioDiscovery Institute Genomics Center (Denton, TX, USA), targeting an initial 200,000 paired end reads (clusters) per library. The raw data are available in the National Center for Biotechnology Information Sequence Read Archive: SRA accession PRJNA603673.

After sequencing, paired-end reads were filtered based on quality and mapped to the guanaco mitochondrial genome (GenBank accession: NC011822). Prior to mapping, SeqPrep (https://github.com/jstjohn/SeqPrep) was used with default settings to trim adapters and merge overlapping paired-end reads. Merged reads were then aligned to the reference mitochondrial genome using Mitochondrial Iterative Assembler (MIA) [82], using a kmer filter of length 13 (-k 13). After mapping, the consensus mitochondrial (mtDNA) genome sequences were determined with a minimum of 3x coverage per base and 2/3 of those bases in agreement. Sites not meeting those criteria were identified as missing. The final mtDNA alignment (16,649 base-pairs (bp)) was created using default parameters of ClustalW Alignment [83], as implemented in Geneious v.7.1.9 [84]. The mtDNA sequence dataset was then reduced to two loci, specifically cytochrome b (cytB 1,140 bp) and the D-loop (1,215 bp), to allow the inclusion of additional samples available on GenBank to perform species identification of the aDNA samples. The two loci were chosen not only because the majority of mtDNA sequences available on GenBank for the three focal taxa were either cytB or D-loop, but also because the number of nucleotide substitutions for the two loci differ by at least 5% and 8%, respectively, between guanaco and domestic llama or vicuña based on all available mtDNA sequences for those two loci at the time of this study. The latter point is important when working with aDNA because their final consensus DNA sequences may include ambiguous bases (i.e., unknown nucleotide sites, or Ns) after sufficient quality filtering has been achieved depending on a nucleotide sites’ overall depth of coverage. Having reduncancy in the number of similar or differing bases between samples along a DNA alignment is important to ascertain species identification of unknown samples.

Phylogenetic relationships were reconstructed with mtDNA cytB and D-loop sequence data for a total of 71 individuals, including 41 ancient sequences analysed in this work and 30 previously sequenced ancient (n = 3) and contemporary (n = 27) samples of guanaco, llama and vicuña available on GenBank (S1 Table). Phylogenetic reconstruction was performed using a Bayesian method implemented in BEAST [85] and Maximum Likelihood (ML) using Garli 0.951 [86].

BEAUti was used to prepare alignments for phylogenetic tree reconstruction using BEAST. The model of DNA substitution that best fit the data was identified using a hierarchical likelihood ratio test and Akaike information criterion as implemented in the program MODELTEST 3.7 [87]. The model that best fit the cytB data was the HKY+G model, and the HKY+I+G model was identified for the D-loop sequences and a Speciation: Birth-Death process tree prior [88]. The Markov Chain Monte Carlo (MCMC) was run from a random starting tree for 20,000,000 iterations, sampling every 1,000th tree with a burn in of 200,000 states. The effective sample size for estimated parameters exceeded 200, which was verified using Tracer [89]. Posterior probabilities were annotated onto the BEAST output tree using TreeAnnotator. Maximum likelihood analyses were performed with Garli 0.951 [86] under the substitution model HKY+G+I. One hundred bootstrap (BS) replicates were performed.

Results

A total of 50 samples were sequenced, of which aDNA from the mitochondrial genome was recovered from 41 samples from 17 archaeological sites (Fig 1A). Illumina sequencing reads from each sequencing library were mapped to the guanaco, llama and vicuña mitochondrial reference genomes to assess potential ascertainment bias. In each case a higher depth of coverage was achieved when mapped with the guanaco mtDNA reference genome and used for subsequent analyses (average depth of coverage = 461, range = 9.4 to 2098.4, S2 Table). Moreover, the consensus sequences from different reference genomes were identical independent of whether the sequencing reads were mapped against guanaco, llama or vicuña. Phylogenetic analyses were conducted with the consensus sequences mapped to the guanaco due to the higher coverage mappings. The Maximum Likelihood (ML) and Bayesian phylogenetic reconstructions yielded a well-resolved tree topology, with all 41 archaeological samples placed unambiguously within the guanaco clade with high bootstrap support (>91) and posterior probabilities (>0.99) (Fig 2).

Fig 2

Phylogenetic relationship of South American camelids.

The 50% majority rule consensus tree results from the Bayesian analyses of the cytB and D-loop sequence dataset. Nodal support values represent the Bayesian posterior probabilities/maximum likelihood bootstrap values (1,000 replications). Clades with nodal support values below that 0.5 or 50% have been collapsed. As currently presented, the guanaco clade is one large collapsed polytomy. Sample names are color coded according to whether the ancient sample was sequenced for this study (blue and filled triangle) and originated near the high-altitude LD-S4 site (red and filled square), or were ancient (green and empty square) or contemporary (black and without symbol) mtDNA sequences obtained from GenBank.

The ancient Holocene samples were identified as Lama guanicoe based on their placement within the resulting concensus trees. As shown previously using mtDNA data [90], guanaco form a monophyletic clade separate from vicuña and domestic llama based on mtDNA cytB and D-loop loci, making species identification of the bone samples possible.

Discussion

This study of ancient camelid mtDNA identified the remains of prehispanic camelids from archaeological sites in central and southern Mendoza as guanaco. Multiple phylogenetic analyses using cytB and D-loop grouped the southern Mendoza camelid samples with contemporary guanaco, to the exclusion of domestic llama. These results are consistent with previous analyses based on morphological data, which also identified the southern Mendoza camelids from the archaeological sites as guanaco [10, 23–26, 28–31].

The results of this study do not confirm the presence of domestic camelids in southern Mendoza. Such is the case in a broad sense in that the 41 samples geographically represent ecosystems in the region, particularly for samples dating to the last 2,000 years BP when domestic plants arrived in southern Mendoza. Archaeologists have argued that prehispanic pastoralist societies occupied southern Mendoza by 2,000 years ago [13, 16, 17]. This argument is based on evidence of domestic plant remains and pottery technology from archaeological sites in the southern boundary of agricultural societies. However, such indicators do not necessarily provide support for prehispanic pastoralism. On the contrary, such evidence may be present without animal domestication as has been shown in different parts of the world [51-54].

Southern Mendoza has been characterized as the agricultural dispersion boundary in South America with human societies to the north developing agricultural methods for subsistence starting two thousand years ago and those to the south practicing hunting and gathering prior to Spanish colonization approximately 500 years ago [1–4, 6, 7, 73, 91]. This boundary extends from west to east along the Atuel and Diamante rivers at 34° 40’ south latitude near the Patagonia phytogeographic boundary. The specific location of the transitional zone between these two populations is unclear and has fluctuated over time depending on archaeological evidence [4, 5, 25, 32, 73]. The results from this study do not indicate that pastoralism was present south of the agricultural dispersion boundary.

The absence of herding, however, does not mean that people did not practice agriculture south of the dispersion boundary. The southernmost archaeological evidence of prehispanic farming in Argentina is the presence of remains of Zea mays, Phaseolus vulgaris, Cucurbita pepo, Lagenaria siceraria and Chenopodium quinoa [2, 3]. Corn (Zea mays) has been considered the most important crop for farming because of its ubiquity in the archaeological record. Dependence on crops, however, was minimal and variable with limited consumption that was restricted to specific biogeographic contexts, such as sites located in Monte desert area [73]. There is no evidence of irrigation or semi-permanent or perminent housing structures suggesting that crops served a minor role in prehispanic economies in the Southern Mendoza region [4, 32].

The archaeological site of Laguna del Diamante-4 (LD-S4)—where researchers claimed to have identified specimens of domesticated llama using morphometric analysis—is one of several high elevation camps located from 2,400 to 3,400 meters above sea level (masl) [92, 93]. LD-S4 is located near the sources of the Atuel and Diamante rivers, in Laguna El Diamante, near the boundary between Argentina and Chile. The most important features of these high elevations sites include: the presence of semicircular stone housing structures ("pircados"), high frequencies of fragmented pottery, strong evidence for the consumption of guanaco, with chronologies dating back to the last 2,000 years BP in Argentina and ca 3,000 years BP on the western slope of the Andes [26, 92–94]. Until recently, these sites were thought to represent hunter-gatherer occupations, but Gasco [15] argued that they could also have been occupied by herders based on the purported identification of camelid remains as llama. As with other biogeographic contexts south of the agricultural dispersion boundary, our results do not support that llama herding occurred in these high elevation contexts.

The biogeography and population biology of contemporary camelids in southern Mendoza also does not support the presence of llama in the region. Today, South American camelids include the wild species guanaco and vicuña and the domestic species llama and alpaca. In Argentina, contemporary guanaco populations are spread throughout Patagonia as wild and semicaptive ranching populations. The four Lama and Vicugna camelid species each have 72 chromosomes [95] and possess very similar C and G banding patterns [96] so it is difficult their differentiation from cytogenetically. Sequencing methods are required for distinguishing members of Lama, which are difficult to distinguish based on morphology of skeletal remains; the same is also the case for separating Lama spp. and Vicugna spp. [69], which highlights the importance of our results for investigating the late Holocene biogeographic distribution of camelids near the southern agricultural dispersion boundary.

Indeed, the northern guanaco population, L. g. cacsilensis, represents the parental population of the lineage that led to llama [71]. Analysis of a male-specific Y-chromosome marker in the genus Lama supports that there were independent domestication events of llama from guanaco and of alpaca from vicuña. This evidence is based on the major DBY patriline haplotypes, which originated prior to domestication. The maternal lineage divergence among vicuña–alpaca is greater than between guanaco and llama based on mitochondrial DNA [71]. However, it has proven difficult to describe the phylogenetic relationship among wild and domestic camelids due to extensive hybridization between llamas and alpacas and their near extirpation during the Spanish conquest [69, 97, 98]. Thus, the results of this study help clarify the Holocene population biology history of camelids in the region.

While hybridization among all four species can occur, previous research suggests that it is less likely to occur in the wild because shared haplotypes are uncommon between guanacos and vicuñas [71], which may indicate the presence of a reproductive barrier [99]. Analysis of the partial or complete sequence of the cytB gene and the control region [69, 98, 100], as well as analysis of the mitochondrial genome [90] have successfully resolved the identification of South American camelids. Phylogenetic analysis of the aDNA sequences presented here grouped all the samples in the same clade as guanaco. The phylogeny also showed a monophyletic group between high elevation individuals and an archaeological individual from Mocha Island in Chile. The results of our study indicate not only that remains from archaeological sites represent guanaco, but that there is no evidence of interbreeding among various camelid species in the region.

A limitation of this study is that sampling may not be extensive enough to represent camelid diversity in the region. However, our study includes samples from a comprehensive variety of environments and periods, from both prehispanic and Hispanic sites (Table 1) that include periods when agriculture was present in the region, yet none of the camelid samples from these contexts is identifiable to domestic llama based on their aDNA.

Previous osteometric analyses of bone specimens from a single high elevation village from southern Mendoza (Site LD-S4; [15]) appear to support the presence of domestic camelids in this region. While we were unable to include these samples in our study, six DNA samples from camelids obtained from three other high elevation sites (El Indígeno, Los Peuquenes and Risco de los Indios) that are in close geographic proximity to LD-S4 and that date to approximately the same period [26, 72] were included in the analysis. The archaeological record of these sites suggests a similar livelihood among their occupants, particularly in terms of subsistence strategies indicating that occupants probably belonged to the same socio-environmental system as the occupants of LD-S4 [26, 72, 92, 93]. All high elevation camelid bone samples strongly grouped with the guanaco samples instead of domestic llama (Fig 2). In addition, camelid remains from the high elevation sites of Risco de los Indios and El Indígeno exhibited a very high similarity with an archaeological sample from Isla Mocha in Central Chile (Fig 2). There is high statistical support for the similarity despite the geographic distance between sites. As has been found in this study, camelid remains from Isla Mocha have also been identified as guanaco [90].

Our results weaken the claim that domestic camelids were present in prehispanic, high elevation contexts, further suggesting that size may not be a reliable indicator alone for distinguishing guanaco and domestic llama skeletal remains. The conclusion that the identified remains were from guanaco hunted in the proximity of the site is similar to results of other aDNA and zooarchaeological analyses focused on camelid remains located at different archaeological sites at similar latitude and elevation in central Chile and Argentina [4, 47, 48, 90, 93, 94, 101].

Whether pastoralists were present during prehispanic periods in the region as others have proposed has important implications for southern Mendoza archaeology. Such claims should be assessed using multiple lines of evidence. We argue that the presence of agricultural activities is not sufficient evidence to support that pastoralism was adopted. Morphometric identification of domestic camelids should be questioned, particularly related to studies of the archaeological record in boundary areas, such as central Chile and Argentina. In addition to the absence of domestic camelid DNA in our study, another weakness of the claim that domestic llama was present at LD-S4 is that the area would only have been habitable by people and camelids during a few summer months. Such environmental conditions would not have supported year-round pastoralism because of high snowfall during the winter. As a result, there should be evidence of pastoralist sites located below 2,000 meters, where pasture would have been available during the winter. However, no corral structures, dung deposits, or semi-permanent camps have been found. On the contrary, only hunter-gatherers activities have been documented [26, 44, 94, 102–104].

Conclusion

In this study, we analyzed aDNA from camelid faunal remains from numerous sites from throughout southern Mendoza that date to the late Holocene. Bone samples from 41 individuals were identified as guanaco, and none of the remains were identified as llama based on phylogenetic analyses of aDNA including a subset of samples located near the LD-4 site that date to the same period as those reported in Gasco [15]. The results of our analysis support previous aDNA results from the western Andes in Chile, which also did not detect the presence of llama among camelid samples that were subsequently identified as guanaco [90]. The results of our analysis also support previous archaeological conclusions that there was no herding livelihood during the prehispanic period near the southern limit of farming. There is no evidence of corrals, artifacts that indicate herding technology, dung deposits, and other changes in material culture that would support the hypothesis that pastoralism was adopted. The results of our study and of previous ones indicate that despite that people adopted farming in some areas, insufficient evidence exists to support the presence of a pastoral lifestyle during the prehispanic period in southern Mendoza.

mtDNA sequences obtained from GenBank.

(XLSX)

Click here for additional data file.

Sequence data.

Percentages of total reads, endogenous DNA, identity and coverage of 41 consensus sequences.

(XLSX)

Click here for additional data file.

19 May 2020

PONE-D-20-06090

Were domestic camelids present on the preHispanic South American agricultural frontier? An ancient DNA Study

PLOS ONE

Dear Abbona,

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Reviewers' comments:

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript presents an interesting contribution to understand the domestication patterns in South America. It is well written, I have to signal only a couple of typos: line 90 "presence" and S1 Table "Provenience" (I suppose).

The study is based on a very good sampling for number of specimens, site representation and type of sites.

The authors choose to prepare UDG-treated libraries to be more confident with SNP calling. Even if I would be interest in knowing more details about the degree of preservation of the DNA in correlation with the age of the samples and altitude of the sites (and it is not possible to see the misincorporation patterns with this kind of library), I recognise that for the context of the study it is not strictly necessary to this detail to authenticate the results. Anyway, I would like to see in S1 Table at least the median length of the sequenced molecules mapping on the target.

I also suggest to spend some words to briefly describe the DNA extraction method, since it is not clear only through the cited reference.

It is a pity that the authors could not analyse any sample from Site LD-S4 that they mentioned as the one with evidence of domestic camelids according to osteometric evidence. Were those samples not available for the analysis?

Reviewer #2: The manuscript entitled “Were domestic camelids present on the preHispanic South American agricultural frontier? An ancient DNA Study” describes the application of ancient DNA analyses to clarify the past presence of domestic camelid (Lama glama) at the southern boundary of prehispanic farming in South America. The topic and the results are very interesting in the perspective of spread of llama domestication in this peculiar area but also in the perspective to add NGS data of ancient samples to databases, that will help to better analyse llama domestication in the past with a deep resolution respect to modern data.

The study is well developed, using cutting-edge NGS technologies and approriated phylogenetic analyses. However, there are some issues that need to be addressed:

1) In the abstract it would be better to indicate the number of samples for which the authors obtained results (41?) and not the total samples analysed (52?), or at least to specify that.

2) In Table 1 I think that the authors should: (i) add a colum where specify what kind of skeletal element was analysed (e.g. petrous bone, long bone, tooth...); (ii) add a column where they indicate if the chronology is based on radiocarbon dating or material culture; (iii) check the number of samples, because in the abstract and results the total number of samples is 52 and the ones with sufficient endogenous is 41, but in the table 1 there are 53 samples, of which 10 with sufficient endogenous; (iv) explain why some samples have the same ID, are they referred to the same individual?

3) The authors followed proper ancient DNA guidelines, but I suggest to add the results from MapDamage software to analyse damage patterns with the aim to authenticate the data here obtained.

4) Given that the identification of domestic from wild species through DNA analysis is often complicated and confused because of recent times of divergence and/or hybridisation phenomena, the paper will benefit from some more sentences about the power of mitochondrial DNA analysis in the identification of Lama glama and Lama guanicoe. For example, the authors stated that "previous research suggests that it is less likely to occur in the wild because haplotypes are uncommon between guanacos and vicuñas", but are there shared or private mitochondrial haplotypes between Lama glama and Lama guanicoe?

5) add more keywords, for example the name of the species, domestication, mitochondrial DNA, the geographic area, etc,

6) in the caption of the Figure 3 add an explanation about the use of the colors blue, red, green and black for the samples'ID.

Minor edits

The paper will benefit from a revision of the English, I suggested here some words or sentences that need to be revised.

- the term "presense" need to be changed in "presence" (e.g. lines 59, 67, 90, 92, etc)

- line 61 " Traditionally, In South..." change in "Traditionally, in South...

- check "reducancy" at line 245

- check the concentration of the library (9 ng/ml) line 211

- line 252 check the sentence "41 sampled ancient Holocene samples sequenced" and specify that 41 are referred to the samples analysed in the paper.

- check "socieites" at line 351

- line 389 check "intead"

**********

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12 Jun 2020

REVIEWER: 1

I have to signal only a couple of typos: line 90 "presence" and S1 Table "Provenience"

The line 90 "presence" and S1 Table with "Provenience" was corrected the typos

I would like to see in S1 Table at least the median length of the sequenced molecules mapping on the target.

We have added in S2 Table (samples sequenced in this study, S1 Table belong to sequences used from GenBank) a column with number fragments mapped to reference, total length of mapped fragments and average fragment length

I also suggest to spend some words to briefly describe the DNA extraction method, since it is not clear only through the cited reference.

The reviewer must have missed the information in the original submission. We describe the DNA extraction method in detail from lines 190 to 217.

It is a pity that the authors could not analyse any sample from Site LD-S4 that they mentioned as the one with evidence of domestic camelids according to osteometric evidence. Were those samples not available for the analysis?

We agree with the reviewer; we could not obtain the permission.

REVIEWER: 2

1) In the abstract it would be better to indicate the number of samples for which the authors obtained results (41?) and not the total samples analysed (52?), or at least to specify that.

It was corrected

2) In Table 1 I think that the authors should:

(i) add a colum where specify what kind of skeletal element was analysed (e.g. petrous bone, long bone, tooth...); It was added

(ii) add a column where they indicate if the chronology is based on radiocarbon dating or material culture; It was described below Table 1

(iii) check the number of samples, because in the abstract and results the total number of samples is 53 and the ones with sufficient endogenous is 41, but in the table 1 there are 53 samples, of which 10 with sufficient endogenous;

(iv) explain why some samples have the same ID, are they referred to the same individual?

(iii and iv) 19, 78, 31 were done duplicated for NGS.

41 was used for phylogenetic analyses, using the sequence available for two loci, specifically cytb (1,140 bp) and D-loop (1,215 bp)

3) The authors followed proper ancient DNA guidelines, but I suggest to add the results from MapDamage software to analyzse damage patterns with the aim to authenticate the data here obtained.

While we agree with the reviewer that being able to visualize damage patterns along mapped reads using a program such as MapDamage is helpful, we are unable to employ due to the method used for mapping. We used the mapping iterative assembler (MIA, https://github.com/mpieva/mapping-iterative-assembler) for mapping merged reads to the mtDNA reference genome, which does not generate the necessary file type required for running MapDamage (SAM/BAM format). MIA was originally developed for working specifically with ancient DNA (i.e., Neanderthal mtDNA) and provides a number of additional steps that help minimize erroneous SNP calls when working with ancient DNA. For example, the method uses a position specific substitution matrix that has been altered to work with damaged/ancient DNA to help reduce potential calling errors. The method will iterate until convergence on a consensus sequence, which is one of the main selling points of this method when mapping ancient mtDNA.

To further reduce potential erroneous SNP calls associated with cytosine deamination, “total DNA was treated with 1X USER enzyme mix (New England BioLabs) for 3h at 37°C to reduce nucleotide substitution errors …., which is common when working with aDNA [74,75]” (line 211). This mix combines two enzymes, the Uracil-DNA glycosylase and DNA glycosylase-lyase endonuclease VIII and replaces with abasic sites the cytosines that have been deaminated post-mortem into uracils. So, the transitions C-T has been corrected, which is what MapDamage is used to show when not corrected.

4) Given that the identification of domestic from wild species through DNA analysis is often complicated and confused because of recent times of divergence and/or hybridisation phenomena, the paper will benefit from some more sentences about the power of mitochondrial DNA analysis in the identification of Lama glama and Lama guanicoe. For example, the authors stated that "previous research suggests that it is less likely to occur in the wild because haplotypes are uncommon between guanacos and vicuñas", but are there shared or private mitochondrial haplotypes between Lama glama and Lama guanicoe?

There are sufficient differences between llama and guanaco at the cytb and D-loop to result in reciprocal monophyly in the gene trees as shown on Fig 3. In contrast to previous studies, we have used both markers, the cytb and D-loop, together increasing the conserved sites and maintaining the polymorphisms.

“As shown previously using mtDNA data [84], guanaco form a monophyletic clade separate from vicuña and domestic llama...” (line 293)

5) add more keywords, for example the name of the species, domestication, mitochondrial DNA, the geographic area, etc,

The suggestions have been done

6) in the caption of the Figure 3 add an explanation about the use of the colors blue, red, green and black for the samples'ID.

“Sample names are color coded according to whether the ancient sample was sequenced for this study (blue) and originated near the high altitude LD-S4 site (red), or were contemporary (black) or ancient (green) mtDNA sequences obtained from GenBank.” (Fig 3)

Minor edits

The paper will benefit from a revision of the English, I suggested here some words or sentences that need to be revised.

- the term "presense" need to be changed in "presence" (e.g. lines 59, 67, 90, 92, etc)

- line 61 " Traditionally, In South..." change in "Traditionally, in South...

- check "reducancy" at line 245 the word is “reduced”

- check the concentration of the library (9 ng/ml) line 211

- line 252 check the sentence "41 sampled ancient Holocene samples sequenced" and specify that 41 are referred to the samples analysed in the paper.

- check "socieites" at line 351

- line 389 check "intead"

All of the above indicated edits have been implemented in the revised manuscript.

These changes have clearly improved our manuscript. Thank you again for consideration of our revised manuscript.

Submitted filename: Response to Reviewers.pdf

Click here for additional data file.

26 Aug 2020

PONE-D-20-06090R1

Were domestic camelids present on the prehispanic South American agricultural frontier? An ancient DNA study

PLOS ONE

Dear Dr. Abbona,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Reviewer 1 asks you to correct a few minor issues before the publication. These are listed below.

Reviewer 3 has some issues with your choice to map to the guanaco mtDNA reference genome for the phylogenetic reconstructions. Furthermore, the reviewer thinks that the discussion and conclusions sections should be edited to primarily emphasize the contributions of this study. Please, also check the reviewer's suggestions concerning the figures. There is also a list of minor issues that should be addressed.

Please submit your revised manuscript by October 10th 2020 If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #2: (No Response)

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The authors addressed all the issues raised, however there are some minor issues to be checked and fixed before the publication:

- l.46 the term "reamain"

- Table 1 the terms: "Izqenon", "Cuneiforme" and "Cuboide"

- l. 201 "Dremmel"

l. 202 "negative" hairflow hood. The fact that it has negative pressure is in contrast with the criteria of aDNA analysis.

Reviewer #3: I really enjoyed reading this manuscript. While I was previously unfamiliar with the specific question this study addresses—whether domestic camelids were present on the pre-Hispanic South American agricultural frontier—the authors provided ample context to explain the importance of this question to non-experts. Further they pose a question—whether camelid remains in their study area belong to domesticate or camelid species—that is well suited to ancient mitochondrial DNA analysis, and they provide strong support for why other methods (such as morphological assessment) cannot sufficiently address this question. In their analyses, the authors present compelling evidence that weakens previous claims that domestic camelids may have been present in their study area during the pre-Hispanic period. Overall, I think that this is a nice study that will be of interest to archaeologists and to those curious about camelid domestication in the Americas generally.

I do have several suggestions that I think would help strengthen the paper, which are listed below.

Broad comments-

Reference genome choice –

I am concerned by the choice to map to the guanaco mtDNA reference genome for the phylogenetic reconstructions. While the authors state that this choice was made because the maximum coverage was achieved when this reference genome was used (lines 290-296), I worry that this will introduce bias into the analysis, the aim of which is to distinguish between guanaco, llama and vicuña. Ideally, I think it would be best to map to a reference genome from an outgroup species (I will leave it to the authors to determine what the best outgroup species might be, perhaps the Arabian or Bactrian camel). If it isn’t possible to replicate this analysis using a reference genome from a true outgroup species, I think it would be valuable for the authors to demonstrate whether they see consistent results when the llama and vicuña mitochondrial reference genomes are used.

Structure –

While the authors do an excellent job placing their study within the context of existing literature, I think that the discussion and conclusions sections should be edited to primarily emphasize the contributions of this study. Of the 12 paragraphs included in the discussion, only 4 directly discuss findings from this study. Similarly, only about half of the conclusion section is directly focused on explaining this study’s significance. This may make it difficult for readers to understand what the specific contributions of this study are to the field. I would therefore recommend moving many of these background details to the introduction and reworking the discussion and conclusion sections to focus more specifically on interpreting the findings of this study. That is not to say that these findings should not be placed within the context of findings from previous studies, but I think it would be best if these studies were already introduced in the introduction so that references to them in the discussion section can be more focused.

Figures-

Figure 1a – This map conveys really important information, but I find it quite difficult to read. I think it would be best to remake this figure using an underlying map that is less detailed, so that the things the authors are trying to highlight stand out more clearly. I think it would be sufficient to outline the study area with an unfilled box, as the patterned shading used makes it difficult to see the text within it. Also, I think that the dashed lines and labels at the top of the figure showing the limits of southern domestication are easy to miss, because the dashed lines blend in with the map and the white text is difficult to see. I also think it could be useful for the entire map to be zoomed out slightly, so that these limits and the southern-most point in the study area are not all so close to the edges of the map. I do really appreciate the insert showing the location of this map within the larger map of South America, so please keep something like this.

Here are some suggestions of free mapping tools that include simple maps that you might find useful. I only mention them in case you don’t already have a method of map making that you are happy with: Tableau (Academic version), ggmaps (in R), basemaps (in python), Simplemappr.

Figure 1b – Would it be possible for the authors to recreate this map? While it conveys interesting information, it’s quite blurry and the image quality feels a bit out of place.

Figure 2 – I think that this map should be combined with figure 1a, with all the points highlighted in it just inserted into the same map in figure 1a. I think that if a less detailed base map is used, the sites highlighted in this plot will also be easier to see. Right now, the sites that fall on top of high elevation areas are difficult to see.

Figure 3 – The use of red and green colors to distinguish between samples is not accessible to readers with red/green color blindness. Additionally, all colors will be lost to any reader who prints out the study on a black and white printer. Perhaps you could add some sort of differentiating symbols next to the colored names to help distinguish them from one another.

Line by line comments-

Line 54 – masl is a non-standard abbreviation. I assume it stands for meters above sea level, but please write this out in full the first time you use it and then explicitly define the acronym (or since you don’t use the term that often, I think you could just write it out fully each time).

Line 61 – “and the isotopic signal of δ13C and δ15N from human remains” – this phrasing is a little odd. I think it sounds like humans don’t usually have any sort of 13C or 15N signals. I suggest “and δ13C and δ15N signatures measured in human remains”

Line 63 – RCYBP is a non-standard acronym, so please write it out in full the first time you use it (I don’t think it is referenced again, so possibly there is no need to include an acronym).

Line 122 – I think it would be beneficial to add a line explicitly stating that no remains from LD-S4 were analyzed in this study. Since the site is referenced heavily, I didn’t initially realize that it wasn’t the focus of this study.

Line 126 – “25% (n=3 samples) of the total faunal assemblage (n=12 samples)” I think this line can be simplified to read: “25% of the total faunal assemblage (n=12 samples)”

Lines 158-178 – I think it would be helpful to more explicitly state that you are analyzing ancient mitochondrial DNA and discussing its specific value in this section. With the increasing prevalence of genome-wide ancient DNA studies, I think that readers don’t know what kind of analysis to expect when just the term ancient DNA is used.

Line 173 – “ancient DNA” should be abbreviated to “aDNA” since this abbreviation has already been established

Line 180 – Should read “Materials and Methods”

Line 181-184- “Fifty-two camelid bone samples identified to Lama spp. based on osteological characters were processed from 25 cultural assemblages from 17 archaeological sites located in central and southern Mendoza (Fig 2), which represents 70% of the available archaeological sites with faunal remains from the region.” This phrasing is a little awkward, I suggest “Fifty-two camelid bone samples (identified as Lama spp. based on osteological characters) from 25 cultural assemblages excavated from 17 archaeological sites located in central and southern Mendoza were selected for analysis. This represents 70% of the available archaeological sites with faunal remains from the region”

Table 1 – I recommend changing the column heading “Functionality” to “Site function.” The following words are not in English: Izqueon (Ischium), Cuneiforme (Cuneiform), Cuboide (Cuboid). Line 192- I am not sure what the authors mean when they state that samples 19, 78 and 31 were duplicated, especially since I count 50 rows in the table. Please clarify this.

Line 204 – Please explicitly state what methods were used to specifically minimize contamination with contemporary DNA. There is a lot of variation between labs, so this is helpful to know.

Line 206-207 – The paper cited here is actually a comparison of multiple methods, so this is confusing. Also, since the next lines are about sampling methods and not DNA extraction, I think it may be appropriate to make a more general statement about the ancient DNA processing methods used, or to remove this sentence.

Line 209-210 – Please specify how long bones were UV crosslinked for.

Line 218 – I don’t think ref 74 is the correct citation. This paper lists multiple extraction methods, all of which are described in other papers, and I don’t think they involve a pre-treatment. Please list the original paper that this pretreatment was based on and list any modifications if necessary.

Line 227 – 229 – I think this sentence should be moved to the next paragraph, as UDG treatment is typically considered part of the library preparation. Also, while the citations provided do discuss ancient DNA damage, I don’t think that either are the original description of the UDG treatment protocol used, please add this citation.

Line 231-232 – What method was used to ensure that this starting concentration was used? If you just used a standard volume of extract, please specify that instead.

Line 242 – Please write out this abbreviation in full (I assume it stands for Paired End)

Line 244 – I don’t think it is necessary to provide the submission ID

Line 248-249 – Please state what criteria were used to merge paired end overlapping reads (i.e. how much overlap was required, was there any allowance for mismatch within this overlapping region made)

Line 253-254 – “The resulting mitochondrial genomes were assessed by visual inspection” – please specify what this process entailed

Line 261-262- Please provide a reference for this statement if possible.

Line 264-267 – Ancient DNA C-to-T damage is another source of error that could impact this (note that while UDG treatment was used to remove this damage, it cannot fix damage that occurs at CpG sites).

Line 275 – Change “previous” to “previously”

Line 277-279 – Please specify all default parameters were chose or if any modifications were made in this process

Line 281-283 - Please specify all default parameters were chose or if any modifications were made in this process

Line 290-291 – Please state what criteria were used to define successful recovery

Line 353 – change “chrososome” to “chromosomes”

Line 354 – I’m not sure if C and G banding patterns are very commonly discussed anymore. I would recommend either leaving this reference out or describe what they are and their significance for readers who might be unfamiliar with this term.

Line 354-355 – The phrasing “DNA nucleotide methods” is a bit odd. I recommend saying “Genetic methods”

Line 370 – I think this should this read “because shared haplotypes are uncommon” otherwise I am not sure what the meaning of this sentence is.

Line 373 – “Crossbreeding among those species, however, produces fertile hybrids in captivity [70,95]. Although hybridization between domestic species is possible, it does not interfere with sample identification.” I’m not sure if these lines are necessary

Line 408-413—“Osteometric evidence supporting the presence of domestic camelids is from bone specimens from a single high elevation village from southern Mendoza (Site LD-S4; [15]. Six DNA samples from camelids obtained from three others high elevation sites (El Indígeno, Los Peuquenes and Risco de los Indios) that are in close geographic proximity to LD-S4 and that date to approximately the same period [26,97] were included in the analysis.” I am not sure that this statement is clear enough that no samples from LD-S4 were included in this study. I recommend rephrasing this paragraph to make this clearer. For example: “Previous osteometric analyses of bone specimens from a single high elevation village from southern Mendoza (Site LD-S4; [15]) support the presence of domestic camelids in this region. While we were unable to include these samples in our study, six DNA samples from camelids obtained from three other high elevation sites (El Indígeno, Los Peuquenes and Risco de los Indios) that are in close geographic proximity to LD-S4 and that date to approximately the same period [26,97] were included in the analysis.”

Line 424 -429 – I am curious what the alternative conclusions for how these remains arrived at these archaeological sites if they were not from domesticated llama. I assume that the alternative hypothesis is that local people hunted wild guanaco, but it might be nice to specifically state this (assuming that it is true).

Table 2- Please provide more information about the meaning of the abbreviations masl, SL and WL. Additionally, I’m confused by the blank columns labeled “guanaco alignment” and “llama alignment.” Are these headers for the columns to the right? If so, could these titles instead be placed above the corresponding columns.

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9 Sep 2020

REVIEWER: 2

� The authors addressed all the issues raised, however there are some minor issues to be checked and fixed before the publication

� l.46 the term "reamain"

� Table 1 the terms: "Izqenon", "Cuneiforme" and "Cuboide"

� l. 201 "Dremmel"

� l. 202 "negative" hairflow hood.

� We thank the reviewer for identifying the above errors. All of the above indicated edits have been implemented in the revised manuscript.

REVIEWER: 3

Reference genome choice –

� I am concerned by the choice to map to the guanaco mtDNA reference genome for the phylogenetic reconstructions. While the authors state that this choice was made because the maximum coverage was achieved when this reference genome was used (lines 290-296), I worry that this will introduce bias into the analysis, the aim of which is to distinguish between guanaco, llama and vicuña. Ideally, I think it would be best to map to a reference genome from an outgroup species (I will leave it to the authors to determine what the best outgroup species might be, perhaps the Arabian or Bactrian camel). If it isn’t possible to replicate this analysis using a reference genome from a true outgroup species, I think it would be valuable for the authors to demonstrate whether they see consistent results when the llama and vicuña mitochondrial reference genomes are used.

� We understand the reviewer’s concern with our use of the guanaco mtDNA reference genome for mapping since that could introduce ascertainment bias. However, the consensus sequences generated for each sample using the guanaco, llama, and vicuña mtDNA reference genomes separately were identical. We reported that we used the consensus sequences mapped to the guanaco mtDNA genome due to their higher coverage compared to the sequences generated from the other two reference genomes. We think that the above is sufficient to address concerns about possible ascertainment bias, and was one of the options suggested by the reviewer. (line 364)

� Structure –

While the authors do an excellent job placing their study within the context of existing literature, I think that the discussion and conclusions sections should be edited to primarily emphasize the contributions of this study. Of the 12 paragraphs included in the discussion, only 4 directly discuss findings from this study. Similarly, only about half of the conclusion section is directly focused on explaining this study’s significance. This may make it difficult for readers to understand what the specific contributions of this study are to the field. I would therefore recommend moving many of these background details to the introduction and reworking the discussion and conclusion sections to focus more specifically on interpreting the findings of this study. That is not to say that these findings should not be placed within the context of findings from previous studies, but I think it would be best if these studies were already introduced in the introduction so that references to them in the discussion section can be more focused.

� We did several things to respond to this comment; however, we did not move portions of the discussion to an earlier part of the paper. The introduction efficiently provides the reader with the context of the study and flows well into the methods. However, we do appreciate that the reader did not like the structure of the discussion and conclusion. Thus, 1) we moved the two final paragraphs of the discussion to the beginning of that section as they state the direct implications of the study (we edited those paragraphs to fit there); 2) we edited the other paragraphs and added better topic and transition sentences to refer more directly to the results section; thus, the regional implications are clearly linked to the broader implications of the study. This helps the reader follow why each part of the discussion matters in the paper. 3) We deleted the first paragraph of the conclusion section because it simply reiterated in summary form what was just stated in the discussion. This will reduce that form of distraction for the reader. The conclusion now reiterates the central claims of the paper. We feel the discussion and conclusion sections are dramatically improved from these revisions.

Figures-

� Figure 1a – This map conveys really important information, but I find it quite difficult to read. I think it would be best to remake this figure using an underlying map that is less detailed, so that the things the authors are trying to highlight stand out more clearly. I think it would be sufficient to outline the study area with an unfilled box, as the patterned shading used makes it difficult to see the text within it. Also, I think that the dashed lines and labels at the top of the figure showing the limits of southern domestication are easy to miss, because the dashed lines blend in with the map and the white text is difficult to see. I also think it could be useful for the entire map to be zoomed out slightly, so that these limits and the southern-most point in the study area are not all so close to the edges of the map. I do really appreciate the insert showing the location of this map within the larger map of South America, so please keep something like this.

� Here are some suggestions of free mapping tools that include simple maps that you might find useful. I only mention them in case you don’t already have a method of map making that you are happy with: Tableau (Academic version), ggmaps (in R), basemaps (in python), Simplemappr.

� We thank the reviewer for the suggested edits. We have modified the map to make it easier to read as requested by the reviewer.

� Figure 1b – Would it be possible for the authors to recreate this map? While it conveys interesting information, it’s quite blurry and the image quality feels a bit out of place.

� We have recreated this map to make it easier to read.

� Figure 2 – I think that this map should be combined with figure 1a, with all the points highlighted in it just inserted into the same map in figure 1a. I think that if a less detailed base map is used, the sites highlighted in this plot will also be easier to see. Right now, the sites that fall on top of high elevation areas are difficult to see.

� Figure 2 was combined with Figure 1A.

� Figure 3 – The use of red and green colors to distinguish between samples is not accessible to readers with red/green color blindness. Additionally, all colors will be lost to any reader who prints out the study on a black and white printer. Perhaps you could add some sort of differentiating symbols next to the colored names to help distinguish them from one another.

� We thank the reviewer for this request. The figure has been modified to address potential concerns with accessibility due to color choice.

Line by line comments –

� Line 54 – masl is a non-standard abbreviation. I assume it stands for meters above sea level, but please write this out in full the first time you use it and then explicitly define the acronym (or since you don’t use the term that often, I think you could just write it out fully each time).

� We thank the reviewer for catching this error. We have now written the abbreviation in full the first time it is used in the manuscript. (line 53 and 443)

� Line 61 – “and the isotopic signal of δ13C and δ15N from human remains” – this phrasing is a little odd. I think it sounds like humans don’t usually have any sort of 13C or 15N signals. I suggest “and δ13C and δ15N signatures measured in human remains”

� The suggested edit was used in the revised manuscript (line 61)

� Line 63 – RCYBP is a non-standard acronym, so please write it out in full the first time you use it (I don’t think it is referenced again, so possibly there is no need to include an acronym).

� Thank you again! We have made the suggested edit by specifying “radiocarbon years before present” (line 63)

� Line 122 – I think it would be beneficial to add a line explicitly stating that no remains from LD-S4 were analyzed in this study. Since the site is referenced heavily, I didn’t initially realize that it wasn’t the focus of this study.

� We have added a sentence at the end of the Introduction stating “Remains from LD-S4 were not analyzed in this study because we could not obtain the permission” (line 204)

� Line 126 – “25% (n=3 samples) of the total faunal assemblage (n=12 samples)” I think this line can be simplified to read: “25% of the total faunal assemblage (n=12 samples)”

� We have used the reviewer’s suggested edit in the revised manuscript (line 136)

� Lines 158-178 – I think it would be helpful to more explicitly state that you are analyzing ancient mitochondrial DNA and discussing its specific value in this section. With the increasing prevalence of genome-wide ancient DNA studies, I think that readers don’t know what kind of analysis to expect when just the term ancient DNA is used.

� We have added ancient mitochondrial DNA (Line 187)

� Line 173 – “ancient DNA” should be abbreviated to “aDNA” since this abbreviation has already been established

� We have made the suggested change here and throughout the revised manuscript (line 203)

� Line 180 – Should read “Materials and Methods”

� We have made the suggested change (line 211)

� Line 181-184- “Fifty-two camelid bone samples identified to Lama spp. based on osteological characters were processed from 25 cultural assemblages from 17 archaeological sites located in central and southern Mendoza (Fig 2), which represents 70% of the available archaeological sites with faunal remains from the region.” This phrasing is a little awkward, I suggest “Fifty-two camelid bone samples (identified as Lama spp. based on osteological characters) from 25 cultural assemblages excavated from 17 archaeological sites located in central and southern Mendoza were selected for analysis. This represents 70% of the available archaeological sites with faunal remains from the region”

� We have used the reviewer’s suggested edit in the revised manuscript (lines 212-215)

� Table 1 – I recommend changing the column heading “Functionality” to “Site function.” The following words are not in English: Izqueon (Ischium), Cuneiforme (Cuneiform), Cuboide (Cuboid). Line 192- I am not sure what the authors mean when they state that samples 19, 78 and 31 were duplicated, especially since I count 50 rows in the table. Please clarify this.

� We have made the suggested changes and have deleted the statement concerning duplicated samples as that was an error.

� Line 204 – Please explicitly state what methods were used to specifically minimize contamination with contemporary DNA. There is a lot of variation between labs, so this is helpful to know.

� We described in the methods used to specifically minimize contamination with contemporary DNA. For example, each bone sample was ground into a powder using a freezer mill with a sterile grinding vial. The aDNA laboratory is dedicated solely for work on extracting DNA from ancient samples where no camelid samples had been processed prior to this study. Extractions was conducted in groups of fifteen that included a negative control in each group to verify no cross-contamination or reagent contamination. Strict protocols for working with aDNA were employed to eliminate possibilities for contamination with other DNAs and all equipment was sterilized between use with samples using multiple methods (e.g. 10% bleach, 95% ethanol, and UV crosslinker). The NGS library preparation was carry out in a separate lab which also had not been exposed to any camelid samples prior to this study.

� Line 206-207 – The paper cited here is actually a comparison of multiple methods, so this is confusing. Also, since the next lines are about sampling methods and not DNA extraction, I think it may be appropriate to make a more general statement about the ancient DNA processing methods used, or to remove this sentence.

� The reviewer is correct that the reference paper compares three different methods, and we have modified the manuscript by referencing the original paper describing the extraction method used in this study (Yang et al. 1998) with specific modifications as described in our methods section. Some of the modifications we also used in Gamba et al. 2014 and 2016, which is why we originally cited the latter reference since it is the most similar in terms of how we conducted our extractions. We agree with the reviewer, however, that it is appropriate to cite the original paper describing the method, which we have now done (line 256-257).

� Line 209-210 – Please specify how long bones were UV crosslinked for.

� We have added the requested information to the revised manuscript (line 260)

� Line 218 – I don’t think ref 74 is the correct citation. This paper lists multiple extraction methods, all of which are described in other papers, and I don’t think they involve a pre-treatment. Please list the original paper that this pretreatment was based on and list any modifications if necessary.

� The reviewer is correct. We have no referenced the original paper (Yang et al. 1998) and described the modifications where appropriate.

� Line 227 – 229 – I think this sentence should be moved to the next paragraph, as UDG treatment is typically considered part of the library preparation. Also, while the citations provided do discuss ancient DNA damage, I don’t think that either are the original description of the UDG treatment protocol used, please add this citation.

� The indicated citation was added (line 285)

� Line 231-232 – What method was used to ensure that this starting concentration was used? If you just used a standard volume of extract, please specify that instead.

� We apologize that the specified information was not included in the previous version of the manuscript. DNA concentrations for all extracted samples was measured with a Qubit Fluorometer using a dsDNA HS Assay Kit (line 285).

� Line 242 – Please write out this abbreviation in full (I assume it stands for Paired End)

� The reviewer was correct, and we have now added the requested information to the revised manuscript (line 298)

� Line 244 – I don’t think it is necessary to provide the submission ID

� We deleted the submission ID as requested (line 299)

� Line 248-249 – Please state what criteria were used to merge paired end overlapping reads (i.e. how much overlap was required, was there any allowance for mismatch within this overlapping region made)

� This section of the methods has been modified. Default settings in SeqPrep were used to merge PE overlapping reads (line 303)

� Line 253-254 – “The resulting mitochondrial genomes were assessed by visual inspection” – please specify what this process entailed

� We agree with the reviewer that this statement was ambiguous. We have now deleted this sentence from the revised manuscript.

� Line 261-262- Please provide a reference for this statement if possible.

� The indicated sentence was not included in the revised manuscript.

� Line 275 – Change “previous” to “previously”

� We have made the suggested change (line 331)

� Line 277-279 – Please specify all default parameters were chose or if any modifications were made in this process and Line 281-283 - Please specify all default parameters were chose or if any modifications were made in this process

� We have included additional information as requested (line 341-352)

� Line 290-291 – Please state what criteria were used to define successful recovery

� “Successfully” was deleted (line 349)

� Line 353 – change “chrososome” to “chromosomes”

� We have corrected the misspelling as identified (line 458)

� Line 354 – I’m not sure if C and G banding patterns are very commonly discussed anymore. I would recommend either leaving this reference out or describe what they are and their significance for readers who might be unfamiliar with this term.

� C and G banding patterns method is cheaper than NGS, so if it was possible to differentiate them through banding patterns it was convenient.

� Line 354-355 – The phrasing “DNA nucleotide methods” is a bit odd. I recommend saying “Genetic methods”

� We changed for Sequencing methods (because Cytogenetic is also a genetic method)

� Line 370 – I think this should this read “because shared haplotypes are uncommon” otherwise I am not sure what the meaning of this sentence is.

� We have made the suggested change as indicated (line 483)

� Line 373 – “Crossbreeding among those species, however, produces fertile hybrids in captivity [70,95]. Although hybridization between domestic species is possible, it does not interfere with sample identification.” I’m not sure if these lines are necessary

� The indicated sentences were not included in the revised manuscript.

� Line 408-413—“Osteometric evidence supporting the presence of domestic camelids is from bone specimens from a single high elevation village from southern Mendoza (Site LD-S4; [15]. Six DNA samples from camelids obtained from three others high elevation sites (El Indígeno, Los Peuquenes and Risco de los Indios) that are in close geographic proximity to LD-S4 and that date to approximately the same period [26,97] were included in the analysis.” I am not sure that this statement is clear enough that no samples from LD-S4 were included in this study. I recommend rephrasing this paragraph to make this clearer. For example: “Previous osteometric analyses of bone specimens from a single high elevation village from southern Mendoza (Site LD-S4; [15]) support the presence of domestic camelids in this region. While we were unable to include these samples in our study, six DNA samples from camelids obtained from three other high elevation sites (El Indígeno, Los Peuquenes and Risco de los Indios) that are in close geographic proximity to LD-S4 and that date to approximately the same period [26,97] were included in the analysis.”

� We have changed the sentence as recommended by the reviewer (line 538-543)

� Line 424 -429 – I am curious what the alternative conclusions for how these remains arrived at these archaeological sites if they were not from domesticated llama. I assume that the alternative hypothesis is that local people hunted wild guanaco, but it might be nice to specifically state this (assuming that it is true).

� We agree with the reviewer that the stated alternative conclusion is possible, and have included a statement suggesting that possibility in the revised manuscript (line 568)

� Table 2- Please provide more information about the meaning of the abbreviations masl, SL and WL. Additionally, I’m confused by the blank columns labeled “guanaco alignment” and “llama alignment.” Are these headers for the columns to the right? If so, could these titles instead be placed above the corresponding columns.

� We have now written the abbreviation in full (Table 2).

The authors all agree that the reviewers’ comments and subsequent changes have improved the quality of our manuscript. Thank you again for considering our revised manuscript for publication.

Best regards,

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

28 Sep 2020

Were domestic camelids present on the prehispanic South American agricultural frontier? An ancient DNA study

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Reviewer #3: Yes: Eadaoin Harney

20 Oct 2020

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Were domestic camelids present on the prehispanic South American agricultural frontier? An ancient DNA study

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