Domestication and lowland adaptation of coastal preceramic maize from Paredones, Peru
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eLife assessment
In this important article, the authors characterize ancient DNA from maize unearthed in archaeological contexts from Paredones and Huaca Prieta in the Chicama river valley of Peru, recovered by painstakingly controlled excavation. The genetic evidence is compelling, albeit from a small sample size, but the dating evidence, despite the excellent archaeological context, is inadequate. Since the age of the samples is so important for the inferences, the individual radiocarbon determinations should be subject to further scrutiny.
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Abstract
Archaeological cobs from Paredones and Huaca Prieta (Peru) represent some of the oldest maize known to date, yet they present relevant phenotypic traits corresponding to domesticated maize. This contrasts with the earliest Mexican macro-specimens from Guila Naquitz and San Marcos, which are phenotypically intermediate for these traits, even though they date more recently in time. To gain insights into the origins of ancient Peruvian maize, we sequenced DNA from three Paredones specimens dating ~6700–5000 calibrated years before present (BP), conducting comparative analyses with two teosinte subspecies ( Zea mays ssp. mexicana and parviglumis ) and extant maize, that include highland and lowland landraces from Mesoamerica and South America. We show that Paredones maize originated from the same domestication event as Mexican maize and was domesticated by ~6700 BP, implying rapid dispersal followed by improvement. Paredones maize shows no relevant gene flow from mexicana , smaller than that observed in teosinte parviglumis . Thus, Paredones samples represent the only maize without confounding mexicana variation found to date. It also harbors significantly fewer alleles previously found to be adaptive to highlands, but not of alleles adaptive to lowlands, supporting a lowland migration route. Our overall results imply that Paredones maize originated in Mesoamerica, arrived in Peru without mexicana introgression through a rapid lowland migration route, and underwent improvements in both Mesoamerica and South America.
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Author Response
Reviewer #1 (Public Review):
The authors sequence some of the oldest maize macroremains found to date, from lowland Peru. They find evidence that these specimens were already domesticated forms. They also find a lack of introgression from wild maize populations. Finally, they find evidence the Par_N16 sample already carried alleles for lowland adaptation.
Overall I think this is an interesting topic, the study is well-written and executed for the most part. I have a variety of comments, most important of which revolve around methodological clarity. I will give those comments first.
- The authors should say in the Results section how "alleles previously reported to be adaptive to highlands and lowlands, specifically in Mesoamerica or South America" were identified in Takuno et al. 2015. What method was used? I see …
Author Response
Reviewer #1 (Public Review):
The authors sequence some of the oldest maize macroremains found to date, from lowland Peru. They find evidence that these specimens were already domesticated forms. They also find a lack of introgression from wild maize populations. Finally, they find evidence the Par_N16 sample already carried alleles for lowland adaptation.
Overall I think this is an interesting topic, the study is well-written and executed for the most part. I have a variety of comments, most important of which revolve around methodological clarity. I will give those comments first.
- The authors should say in the Results section how "alleles previously reported to be adaptive to highlands and lowlands, specifically in Mesoamerica or South America" were identified in Takuno et al. 2015. What method was used? I see this partly comes in the Discussion eventually, but it would help to have it in the Results with more detail. The answer to this question would help a skeptical reader decide the appropriateness of the resource, given that many selection scans have been performed on maize genomes, the choice would ideally not be arbitrary.
This was explained in more detail in the Material and Methods section, to keep the Results and Discussion sections more concise. However, we agree that adding a brief explanation in the Results section would be useful and we have modified the revised version accordingly. Now the relevant part of the section Specific adaptation to lowlands in Mesoamerica and South America reads as follows: “To assess this, we identified in Par_N16 all covered SNPs with alleles previously reported to be adaptive to highlands and lowlands, specifically in Mesoamerica or South America by Takuno and coworkers (Takuno et al., 2015). These authors used genome-wide SNP data from 94 Mesoamerican and South American landraces and identified SNPs with significant FST values to infer which allele was likely adaptive. For example, those SNPs showing significant FST only in Mesoamerica, were characterized as adaptive for lowlands if they were at high frequency in the lowland population and at low frequency in the highland population, and vice versa. The same was applied for South America (Takuno et al., 2015). They identified 668 Mesoamerican and 390 South American previously reported adaptive SNPs, from which 32 and 20 were covered in Par_N16, respectively.”
- How were the covered putative adaptive SNPs distributed in the genome? Were any clustered and linked? The random sampled SNPs should be similarly distributed to give an appropriate null.
The SNPs in Takuno et al. (2015) are in general at a median distance of 353 bp from each other. The 20 adaptive sites covered in Par_N16 for South America (SA) are at a median distance of 8,301,843 bp (approximately 8.3 Mbp), while the 32 for Mesoamérica (MA) are at a median distance of 24,295,968 bp (approximately 24.3 Mbp). SNPs in five pairs from Mesoamerica are closer than 100 bp between them, but each pair is at a considerable distance (beyond 1 cM) from each other and from other SNPs covered in Par_N16. This same happens for only one SNP pair from South America. Then, in general, the covered adaptive SNPs are not clustered. For our random samples, the range of genomic distances between SNPs is similar to those of adaptive SNPs. This shows that our null distributions are adequate for our statistical purposes. The genomic positions of covered adaptive sites in Par_N16 are now included in a new Table in the revised version (Supplementary File 2). We have included these observations in the main text (section Specific adaptation to lowlands in Mesoamerica and South America), as follows: “In general, adaptive SNPs represented in Par_N16 were not clustered. The 20 South American adaptive SNPs are at a median distance of 8,301,843 bp, while the 32 Mesoamerican SNPs are at a median distance of 24,295,968 bp (Supplementary File 2). SNPs in five pairs from MA are closer than 100 bp between them, but each pair is at a considerable distance (beyond 1 cM) from each other and from other SNPs. This same happens for only one SNP pair from SA. Then, although at low proportions, the adaptive SNPs in Par_N16 are a bona fide representation of different genomic responses to selection pressures...” and “We analyzed some of these random samples and observed a similar behavior as the adaptive SNPs regarding the range of distances between SNPs (Fig, S18).”
- How is genetic similarity calculated? It should be briefly described in the Results.
This is formally explained in the Material and Methods section, but now we have included a brief description in the Results section (Specific adaptation to lowlands in Mesoamerica and South America) as follows: “The allelic similarity is the average of the frequencies of the Par_N16 alleles in the intersected sites with each test population (see Material and Methods).”
- It would help for the authors to state why they focus on Par_N16, I did not see this in my reading. Presumably, the analyses done are because of the higher quality data, but it would also help to mention why Par_N16 was sequenced in an additional run.
Indeed, Par_N16 has an endogenous DNA content of 1.1 %, while the other two samples presented a very low DNA content (0.2%). Therefore, we decided to invest more in the best sample, as a cost/benefit decision for additional sequencing. We have included brief explanations of this in the revised text. In the Results section Paleogenomic characterization of ancient maize samples, it reads as follows: “Due to its higher endogenous DNA content (one order of magnitude larger, we further sequenced the Par_N16 library, obtaining 459M additional reads, to generate a total of 851M for this sample (Table 2).” and “To determine if the specific elimination of C to T and G to A modifications could bias the results in favor of maize rather than teosinte alleles, an additional database was generated in which all transitions were eliminated (i.e., only transversions were included) in Par_N16 only, because it was the only sample with enough sequencing data to conduct this experiment.” While in the section Tests of gene flow from mexicana, is as follows: “Par_N16 was the only sample with enough DNA sequence data to perform this analysis. All the samples showed the same phylogenetic position; therefore, Par N 16 was considered to be representative of ancient Paredones maize.”
- In the sections on phylogenetic analysis, introgression, and D statistics, the authors could do a better job specifically indicating how the results support their conclusions.
Precise indications of how our results support our conclusions are given in the Discussion section. Nevertheless, we added relevant sentences in the specified sections. In the section Relationship between ancient maize, extant landraces, and Balsas teosinte, we added the following: “Thus, based on genome-wide relatedness, Paredones maize clusters with extant domesticated Andean landraces, supporting both, a single origin for maize and that these Peruvian samples were already domesticated.” In the section on introgression and D-statistics (Tests of gene flow from mexicana), we improved the last sentence as follows: “These results consistently show the absence of significant gene flow between Par_N16 and mexicana, implying that the lineage that gave rise to Paredones maize left Mesoamerica without relevant introgressions from this teosinte.”
Reviewer #2 (Public Review):
In this foundational article, the authors conduct an ancient DNA characterization of maize unearthed in archaeological contexts from Paredones and Huaca Prieta in the Chicama river valley of Peru. These maize specimens were recovered by painstakingly controlled excavation. Their context would appear to be beyond reproach though the individual radiocarbon determinations should be subject to further scrutiny.
- Radiocarbon determination for at least one of the maize cobs analyzed for aDNA is not a direct date, but dates associated material. The authors should provide a table of the direct dates on the specimens that were analyzed for ancient DNA. They should also specify the type and quantity of material sent and whether the cob, glumes, pith, or husks were submitted for dates. Include δ13C determinations for each cob with laboratory analysis numbers because there is justifiable concern that at least one of these cob dates has a δ13C value suggesting the material dated is not maize. Generally, the δ13C for maize ranges from -14 to -7. One or more of the specimens subjected to ancient DNA analysis in this paper have δ13C values far outside of this confidence interval.
The indirect radiocarbon date on a maize cob was derived from a single piece of wood charcoal in a hearth directly associated with the analyzed cob, both embedded in a thin intact floor in Unit 20 at the Paredones site. The assay on the charcoal and the floor are in an undisturbed stratigraphic context and are in agreement with assays on other maize and charcoal remains in floors both above and below the hearth. We have included this information in Table 1 in the revised version. The information sought by Reviewer 2 on the studied cobs was published previously in Grobman et al. 2012 and in Dillehay 2017. Since details of the cobs were published, we decided to submit only what we thought were pertinent data for this manuscript.
As for the δ13C reading of one cob outside of the confidence interval for maize, the dated specimen with this value is a maize husk fragment. Both the macro- and micro-morphology and the ancient DNA analysis of the husk demonstrated it was maize. We do not understand what affected the δ13C value for this specimen. Similarly, three human skeletons from deeper site levels have δ13C values greater than the expected range for human remains.
- From the perspective of future scientists being able to repeat the analyses performed here, I would hope that all details of specimen treatment, extraction methods, read length and quality would need to be assiduously described. Routine analytical results should be reported so that comparisons with earlier and future results are facilitated, and not made difficult to decipher or search for.
The general procedures for accurate ancient DNA extraction were described in Vallebueno-Estrada et al. 2016 and we do not see the need to repeat this information in this article. Specific aspects of sample treatment and DNA extraction of the samples analyzed here are described in the Material and Methods, section on Extraction and sequencing of ancient samples. Results on quality (percentage of endogenous DNA, quality-filtered reads, mapped reads to either repetitive or unique regions, amount of sequence mapped, mapping Phred scores, estimated error rates, percentage of deamination, fragment median lengths, percentage of sites with signatures of molecular damage, number of unique genomic sites covered and their corresponding average sequencing depth) are described in the Results, section Paleogenomic characterization of ancient maize samples. This section also includes the number of SNPs in relation to the reference and the number of intersected SNPs between our samples and the HapMap3 database. In addition, complementary information to this section is included in Tables 2-4 and supplementary Figures S2-S6, as properly referenced in the last mentioned section.
- The aDNA analysis may or may not be affected by the anomalous δ13C values but one would anticipate that standard aDNA extraction and analysis protocols would provide a means by which the specimen's preservation of the specimens could be ascertained, for example, perhaps deamination and fragmentation rates could be compared or average read length evaluated with modern-contemporary materials so that preservation of the Paredones samples relative to that of maize in the CIMMYT germplasm bank and the San Marcos specimens investigated by the same researchers can be evaluated.
Average read length from contemporary material depends more on the sequencing platform than sample preservation. For example, Illumina can only read fragments of hundreds of base pairs, while MinIon or PacBio can read fragments in the order of kb. Also, deamination is not an issue in DNA extracted from modern material (unless bisulfite is used for methylation detection). Comparison with San Marcos samples indicates that Paredones samples are heavily degraded, although this is not a function of time only (humidity, temperature, and pH are among other relevant factors). Therefore, to avoid misleading interpretations, we are not including a comparison with San Marcos samples in the revised version.
- The size and shape of the cobs depicted are similar to specimens occurring much later in Mesoamerican assemblages. For example, the approximate rachis diameter of the San Marcos specimens depicted by Valle-Bueno et al. (2016: Fig.1) averages less than 0.5cm while the specimens depicted in Valle-Bueno et al. (this manuscript) average 1.0 cm. The former - San Marcos - specimens are dated at 5300-4970 BP cal while the larger - Paredones - specimens date roughly 6777 - 5324 BP cal. The considerable disparity among the smaller more recent specimens compared to the very much larger putatively older specimens suggests the Paredones specimen's radiocarbon determinations are equivocal. The authors point this out but repeatedly state these cobs are the most ancient; a conundrum that should be resolved.
Radiocarbon determinations in Paredones are not equivocal, on the contrary, they are perfectly in agreement with and supported by the unimpeachable stratigraphy of the site and by more than 150 other radiocarbon and OSL dates from Paredones and nearby excavated contexts. The difference in morphology between the more recent samples from Tehuacan and the more ancient samples from Paredones is exactly the paradox we try to address. Our results indicate that the rapid migration and adaptation of maize to the coast of Peru in comparison with a slower migration and adaptation to Tehuacan lands explains this apparent conundrum. This rapid movement and migration allowed the presence of more “modern” maize in Peru than in Tehuacan on the respective dates. This more rapid maize development also coincides with more rapid and advanced socio-cultural transformations in Peru, including proto-urbanism (i.e, first cities), early religious symbolism, long-distance irrigation canals, and other major innovations that far exceed what was happening in Mesoamerica at the time.
- I would suggest the authors consider redating these three specimens and if they do, hope that they will prepare the laboratory personnel with depositional environment information. MacNeish was skeptical about late dates on maize at Tehuacan, at first. Adovasio was initially certain about maize's associated dates from Meadowcroft. One would prefer to be reasonably certain the foundation this article creates is solid; the author's repeated reference to these cobs as the most ancient in the Americas should be reaffirmed so retraction will not be necessary.
As discussed in Grobman et al. 2012 and in Dillehay 2017, we do not confide in C14 dating of unburned corn remains due to the possible intrusion of fungi in the soft cellular structure of cobs. The chrono-stratigraphically acceptable dates on cobs and other maize remains were taken on burned and hard tissue remains, such as husks. See detailed discussion in Supplementary Materials.
MacNeish and Adovasio were excavating cave and rock shelter sites, which are known to often have areas of stratigraphically disturbed deposits. Paredones, Huaca Prieta, SR-18 and other Preceramic sites excavated in the study area here contain late to early varieties of maize and radiocarbon assays that are in chrono-stratigraphic agreement. As noted in the main text and in prior publications, these sites are open air localities with clear stratigraphy defined by intact floor and fill sequences, with no tree root, animal burrowing, or other major taphonomic disturbances.There were occasional hearths and pits (i.e., human burials) that intruded into deeper floor-fill sequences but none of the assayed and studied maize samples were derived from these contexts. Once again, we encourage readers to examine the stratigraphy shown in the main text and in Grobman et al. (2012) and Dillehay (2017). Moreover, as noted in the text, there is a growing number of Preceramic sites in South America that date between 6800 and 6000 years ago and later that contain micro-maize remains (see Kistler et al., 2018). Not all of these sites are well-dated and present reliable contexts, but several have good chrono-stratigraphic settings and micro-evidence (e.g., phytoliths, starch grains) indicative of a maize presence at or prior to 6000 years ago.
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eLife assessment
In this important article, the authors characterize ancient DNA from maize unearthed in archaeological contexts from Paredones and Huaca Prieta in the Chicama river valley of Peru, recovered by painstakingly controlled excavation. The genetic evidence is compelling, albeit from a small sample size, but the dating evidence, despite the excellent archaeological context, is inadequate. Since the age of the samples is so important for the inferences, the individual radiocarbon determinations should be subject to further scrutiny.
-
Reviewer #1 (Public Review):
The authors sequence some of the oldest maize macroremains found to date, from lowland Peru. They find evidence that these specimens were already domesticated forms. They also find a lack of introgression from wild maize populations. Finally, they find evidence the Par_N16 sample already carried alleles for lowland adaptation.
Overall I think this is an interesting topic, the study is well-written and executed for the most part.I have a variety of comments, most important of which revolve around methodological clarity. I will give those comments first.
The authors should say in the Results section how "alleles previously reported to be adaptive to highlands and lowlands, specifically in Mesoamerica or South America" were identified in Takuno et al. 2015. What method was used? I see this partly comes in the …
Reviewer #1 (Public Review):
The authors sequence some of the oldest maize macroremains found to date, from lowland Peru. They find evidence that these specimens were already domesticated forms. They also find a lack of introgression from wild maize populations. Finally, they find evidence the Par_N16 sample already carried alleles for lowland adaptation.
Overall I think this is an interesting topic, the study is well-written and executed for the most part.I have a variety of comments, most important of which revolve around methodological clarity. I will give those comments first.
The authors should say in the Results section how "alleles previously reported to be adaptive to highlands and lowlands, specifically in Mesoamerica or South America" were identified in Takuno et al. 2015. What method was used? I see this partly comes in the Discussion eventually, but it would help to have it in the Results with more detail. The answer to this question would help a skeptical reader decide the appropriateness of the resource, given that many selection scans have been performed on maize genomes, the choice would ideally not be arbitrary.
How were the covered putative adaptive SNPs distributed in the genome? Were any clustered and linked? The random sampled SNPs should be similarly distributed to give an appropriate null.
How is genetic similarity calculated? It should be briefly described in the Results.
It would help for the authors to state why they focus on Par_N16, I did not see this in my reading. Presumably, the analyses done are because of the higher quality data, but it would also help to mention why Par_N16 was sequenced in an additional run.
In the sections on phylogenetic analysis, introgression, and D statistics, the authors could do a better job specifically indicating how the results support their conclusions.
-
Reviewer #2 (Public Review):
In this foundational article, the authors conduct an ancient DNA characterization of maize unearthed in archaeological contexts from Paredones and Huaca Prieta in the Chicama river valley of Peru. These maize specimens were recovered by painstakingly controlled excavation. Their context would appear to be beyond reproach though the individual radiocarbon determinations should be subject to further scrutiny.
Radiocarbon determination for at least one of the maize cobs analyzed for aDNA is not a direct date, but dates associated material. The authors should provide a table of the direct dates on the specimens that were analyzed for ancient DNA. They should also specify the type and quantity of material sent and whether the cob, glumes, pith, or husks were submitted for dates. Include δ13C determinations for …
Reviewer #2 (Public Review):
In this foundational article, the authors conduct an ancient DNA characterization of maize unearthed in archaeological contexts from Paredones and Huaca Prieta in the Chicama river valley of Peru. These maize specimens were recovered by painstakingly controlled excavation. Their context would appear to be beyond reproach though the individual radiocarbon determinations should be subject to further scrutiny.
Radiocarbon determination for at least one of the maize cobs analyzed for aDNA is not a direct date, but dates associated material. The authors should provide a table of the direct dates on the specimens that were analyzed for ancient DNA. They should also specify the type and quantity of material sent and whether the cob, glumes, pith, or husks were submitted for dates. Include δ13C determinations for each cob with laboratory analysis numbers because there is justifiable concern that at least one of these cob dates has a δ13C value suggesting the material dated is not maize. Generally, the δ13C for maize ranges from -14 to -7. One or more of the specimens subjected to ancient DNA analysis in this paper have δ13C values far outside of this confidence interval.
From the perspective of future scientists being able to repeat the analyses performed here, I would hope that all details of specimen treatment, extraction methods, read length and quality would need to be assiduously described. Routine analytical results should be reported so that comparisons with earlier and future results are facilitated, and not made difficult to decipher or search for.
The aDNA analysis may or may not be affected by the anomalous δ13C values but one would anticipate that standard aDNA extraction and analysis protocols would provide a means by which the specimen's preservation of the specimens could be ascertained, for example, perhaps deamination and fragmentation rates could be compared or average read length evaluated with modern-contemporary materials so that preservation of the Paredones samples relative to that of maize in the CIMMYT germplasm bank and the San Marcos specimens investigated by the same researchers can be evaluated.
The size and shape of the cobs depicted are similar to specimens occurring much later in Mesoamerican assemblages. For example, the approximate rachis diameter of the San Marcos specimens depicted by Valle-Bueno et al. (2016: Fig.1) averages less than 0.5cm while the specimens depicted in Valle-Bueno et al. (this manuscript) average 1.0 cm. The former - San Marcos - specimens are dated at 5300-4970 BP cal while the larger - Paredones - specimens date roughly 6777 - 5324 BP cal. The considerable disparity among the smaller more recent specimens compared to the very much larger putatively older specimens suggests the Paredones specimen's radiocarbon determinations are equivocal. The authors point this out but repeatedly state these cobs are the most ancient; a conundrum that should be resolved.
I would suggest the authors consider redating these three specimens and if they do, hope that they will prepare the laboratory personnel with depositional environment information. MacNeish was skeptical about late dates on maize at Tehuacan, at first. Adovasio was initially certain about maize's associated dates from Meadowcroft. One would prefer to be reasonably certain the foundation this article creates is solid; the author's repeated reference to these cobs as the most ancient in the Americas should be reaffirmed so retraction will not be necessary.
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