Using Maize δ15N values to assess soil fertility in fifteenth- and sixteenth-century ad Iroquoian agricultural fields.

Native Americans developed agronomic practices throughout the Western Hemisphere adapted to regional climate, edaphic conditions, and the extent of dependence on agriculture for subsistence. These included the mounding or "corn hill" system in northeastern North America. Iroquoian language speakers of present-day New York, USA, and Ontario and Québec, Canada were among those who used this system. While well-known, there has been little archaeological documentation of the system. As a result, there is scant archaeological evidence on how Iroquoian farmers maintained soil fertility in their often-extensive agricultural fields. Using δ15N values obtained on fifteenth- and sixteenth-century AD maize kernels from archaeological sites in New York and Ontario, adjusted to take into account changes that result from charring as determined through experiments, we demonstrate that Iroquoian farmers were successful at maintaining nitrogen in their agricultural fields. These results add to our archaeological knowledge of Iroquoian agronomic practices. Our results also indicate the potential value of obtaining δ15N values on archaeological maize in the investigation of Native American agronomic practices.

Introduction

Native American farmers developed agronomic practices throughout the Western Hemisphere adapted to climatic and edaphic conditions and the degree of reliance on agricultural production for subsistence. Well known systems of groups who relied heavily on agricultural production include the terraced fields in the Andes of South America [1], the milpa systems of Central America [2], the irrigation systems of the American Southwest [3], and the ridge systems of the upper Mississippi drainage [4]. How these systems functioned is evinced by archaeological investigations of extant features, ethnohistorical documentation, and in some cases, ethnographic documentation. Well known, but less-well understood, are the mounding (“corn hill”) systems in temperate northeastern North America. While portions of several fields have been documented (e.g., [5]), and they are recorded in the seventeenth century ethnohistorical record (e.g., [6]), few have been subject to archaeological investigations [7,8]. As a result, there is little direct archaeological information on how these agronomic systems were managed.

Groups using the mounding system in northeastern North American included Iroquoian language family speaking peoples in present-day New York, USA, and Ontario and Québec, Canada. From the fourteenth century AD onwards, Iroquoians lived in villages and towns that were occupied for 20 to 40 years or more [9] and housed hundreds to well over 1,000 individuals [10-12]. A major source of calories for these communities derived from agricultural produce, primarily maize (Zea mays ssp. mays) [13], but also other crops, including common bean (Phaseolus vulgaris), squash (Cucurbita pepo), and sunflower (Helianthus annuus) [14]. Non-cultivated foods included a wide range of terrestrial animals and plants [14,15], but with freshwater fish being an important source of animal protein for at least some populations [16, 17]. The only domesticated animal present was dog (Canis lupus familiaris), which was consumed occasionally at feasts and ceremonies [18].

The typical seventeenth-century AD ethnohistorically documented Iroquoian agricultural field consisted of many small mounds measuring approximately 46–120 cm in diameter and spaced 76–180 cm apart, which were formed with wooden, bone, antler, or stone hoes ([18], p. 178). Each mound contained 3–4 maize plants [18]. Common bean vines often were grown in the same hills with maize whose stalks acted as climbing poles for the bean vines, while squash vines were planted at intervals and occupied spaces around the mounds [19,20]. These fields were highly productive with some estimates suggesting they surpassed that of contemporaneous European and Euro-American farms [21]. While the agronomic [22-24] and nutritional [25] benefits of such polycultures are well established, how earlier Iroquoian farmers maintained the productivity of their fields is not (but see [21]).

Archaeological estimates of the number of cultivated acres needed to feed individual Iroquoian settlement populations are in the hundreds to thousands of acres (e.g., [26], pp. 99–100). Extensive agricultural fields are attested by the seventeenth-century AD ethnohistorical record (e.g., [18]). Analyses of Iroquoian archaeological site locations demonstrate that settlements were sited in locations favorable to agricultural production. For example, Iroquoians in some areas located settlements and fields at elevations that took advantage of thermal belts, which extended growing seasons up to 30 days [27,28]. These analyses also showed that locations selected by some Iroquoian farmers correlated with heavy, moisture-retaining, upland soils with high lime content, which may have facilitated rhizobia bacteria symbionts that provide nitrogen to common bean-plants [28]. Other analyses have found that Iroquoian archaeological settlement sites in New York are located near loamy, well-drained soils [29,30]. Contrary to suggestions that well drained sandy soils were selected by seventeenth-century AD Wendat (Huron) farmers for agricultural fields south of Georgian Bay in Ontario [18], these were simply the most common soils in the area [31].

It is apparent, then, that at least some Iroquoian farmers selected their settlement locations in part with agricultural productivity in mind [26,32]. While the per-acre productivity of Iroquoian agriculture is debatable [18, 21, 33–36], production needed to be sustained throughout the occupational history of each settlement. Given the large number of acres under cultivation it seems unlikely that Iroquoian farmers practiced frequent shifting cultivation throughout the occupational spans of each settlement [37]. Ethnohistoric accounts indicate continuous cultivation of fields for 10–30 years; while 12 years may have been a more likely maximum for seventeenth-century AD Wendat (Huron) fields, where settlements were associated with sandy soils not well suited to maize production [18]. Seventeenth- and eighteenth-century Haudenosaunee (Iroquois) populations in New York, likely practiced continuous cropping over long periods of time, which was achieved by maintaining soil organic matter in naturally fertile Alfisols and Inceptisols [21,36].

Although many thousands of acres were under cultivation at any given time across the Iroquoian region, which had profound impacts on regional biota [32], only a portion of a single Iroquoian agricultural field located south of Georgian Bay in Ontario with extant mounds has been subjected to archaeological excavation [7]. The mounds at this site had a mean diameter of 102±11 cm and heights of 20 cm and were spaced an average of 140±23 cm apart. Unlike in a buried field remnant in southern New England [8, 38], no evidence for the use of fish as fertilizer in the form of bone and scales was found in the Ontario mounds. The most notable result of soil analysis of the mounds and interstices is that the mounds contained greater amounts of charcoal and at greater depths than did the interstices [8]. This led to the suggestion that the mounds were created from topsoil and wood ash from the initial clearing of the field [8]. It is also likely that charcoal and ash were incorporated into the mounds as a result of annual burning to rid the fields of unwanted vegetation and previous years’ crop detritus [8]. One of the primary limiting factors for maize production is nitrogen [39, 40]. Charcoal (biochar) can be effective at increasing or maintaining plant-available soil nitrogen [41-44]. The incorporation of ash may raise the pH of acidic soils [18].

Given the lack of reporting of Iroquoian agricultural fields that date prior to the adoption of European agronomic practices, it is likely that historical Euro-American and Euro-Canadian farming have obliterated those fields. This evident lack of Iroquoian agricultural field preservation precludes direct archaeological assessments of agronomic practices to maintain soil fertility. The seventeenth-century AD Ontario field cannot be taken as representative of agricultural practices across time and throughout the Iroquoian region [21, 36]. We can only surmise that Iroquoian farmers’ agronomic practices included efforts to maintain soil fertility adapted to local edaphic conditions based on indirect evidence. This includes site locations, actualistic experiments, ethnohistoric documentation, and general agronomic knowledge, including the evident need for soil amendments to maintain soil fertility over extended periods of time. It is likely that specific practices to maintain soil fertility varied across the Iroquoian region both spatially and chronologically. Intercropping maize with common bean can result in increased availability of nitrogen to maize [24,45-47]. The annual incorporation of unburned crop detritus into the mounds would have maintained soil organic matter, which in turn, provided needed mineralized nitrogen for maize production [21,24, 34, 45–47]; a critical aspect of agronomic systems in naturally fertile temperate soils [48,49]. To the east of the Iroquoian region, archaeologically excavated mounds in a seventeenth-century AD agricultural field on Cape Cod, Massachusetts evinced intensive use of organic inclusions, including fish [8,37]. However, there has been no archaeological evidence directly from Iroquoian mounds themselves to test this hypothesis in the Iroquoian region.

Soil organic matter tends to be depleted in plowed, continuously cropped fields (e.g., [36, 50–52]), thus necessitating the use of fertilizer, such as animal manure, to maintain productivity. Eastern Hemisphere grains (e.g., wheat, Triticum spp.) recovered from prehistoric archaeological sites in Europe often exbibit δ15N values higher than those of uncultivated plants. This evidently resulted from use of draft-animal manure as fertilizer, which increased plant-available nitrogen in plowed fields. Experiments have documented that manure results in high δ15N values in grains (e.g., [53-57]); high plant δ15N values are positively correlated globally with high nitrogen content in soil [58,59]. Ammonium (NH4+) and then nitrate (NO3˗) production by soil organisms increase with higher N availability. Soil 15N proportions increase from the loss of 14N through N mineralization, nitrification, leaching, denitrification, and ammonia volatilization, resulting in higher δ15N values in plants [58,60].

Farmers in eastern North America did not have draft animals—all cultivation was done by hand. There is no ethnohistorical documentation of the use of manure for fertilization by Native Americans prior to the widespread, often forced, adoption of Euro-American agronomic practices [61]. As a result, the use of δ15N values to assess Native American agronomic practices has not been pursued because it is generally thought that eastern Native American farmers did not practice any form of crop fertilization [18,26]. However, if Iroquoian farmers used varied soil amendments to increase and sustain plant-available nitrogen to maintain fertility of their agricultural fields including unburned crop detritus, then we would expect charred archaeological maize δ15N values to be consistently higher than values for uncultivated terrestrial plants. Here, we provide the results of δ15N analyses of charred maize remains, adjusted to take into account the effects of charring, from primarily fifteenth- and sixteenth-century AD Iroquoian sites in portions of New York and Ontario, prior to European ethnohistorical documentation and the consolidation of Wendat (Huron) settlements in an area with sandy soils deficient in natural fertility (Fig 1).

Fig 1

Locations of Iroquoian archaeological sites from which maize samples originate.

Yellow shading denotes distribution of Alfisols (New York) and Luviols (Ontario). This map was produced in ArcGIS v 10.6 at the New York State Museum in Albany by compiling GIS shape files obtained from publicly available sources including Statistics Canada, the United States Census, the United States Geological Survey, the United States Department of Agriculture, and the Canadian Soil Information Service.

Results

δ15N values were obtained on 81 maize kernels and 1 cob fragment from 36 Iroquoian archaeological sites dating primarily to the fifteenth and sixteenth centuries AD in northern New York, the Mohawk River valley of New York, and southern Ontario (Tables 1 and 2, Fig 1). Values ranged from +0.60 to +9.37‰ with a mean of +5.30±1.54‰ and a median of +5.25‰ (Table 2).

Table 1

Archaeological maize samples.

Site	Location	Age (century AD)	Samples (n)
Baker	Southern Ontario	15th	3
Coulter	Southern Ontario	16th	2
Damiani	Southern Ontario	16th	2
Dunsmore	Southern Ontario	15th	2
Grandview	Southern Ontario	15th	2
Hidden Spring	Southern Ontario	15th	4
Jarrett-Lahmer	Southern Ontario	16th	3
Jones	Southern Ontario	15th/16th	1
Mackenzie-Woodbridge	Southern Ontario	16th	2
Maynard-McKeown	Southern Ontario	16th	2
McNair	Southern Ontario	15th	2
New	Southern Ontario	15th	1
Parsons	Southern Ontario	16th	2
Sopher	Southern Ontario	16th	2
Spang	Southern Ontario	16th	4
Wapoos	Southern Ontario	16th	3
Wellington	Southern Ontario	14th	4
Carlos	Northern New York	15th/16th	1
Durfee	Northern New York	15th/16th	1
Durham	Northern New York	15th/16th	1
Morse	Northern New York	15th/16th	2
Pine Hills	Northern New York	15th/16th	1
Potocki	Northern New York	15th/16th	1
Sanford Corner	Northern New York	15th/16th	2
Talcott	Northern New York	15th/16th	2
Whitford	Northern New York	15th/16th	1
Cayadutta	Mohawk Valley	16th	4
Garoga	Mohawk Valley	16th	3
Getman#1	Mohawk Valley	15th	1
Klock	Mohawk Valley	16th	4
Otstungo	Mohawk Valley	16th	3
Pethick	Mohawk Valley	14th	3
Smith-Pagerie	Mohawk Valley	15th/16th	2
Snell	Mohawk Valley	13th	3
Roundtop	Susquehanna Valley	12th–16th	4
Kelso	Finger Lakes	14th	1

Table 2

δ15N and δ13C data for individual maize samples.

Lab #a	Region	Site	Material	δ15N	δ15N adj	δ13C
UGAMS-37382	Northern New York	Carlos	kernel	+2.97	+2.43	−9.06
UCIAMS-205978	Northern New York	Durfee	kernel	+5.29	+4.75	−9.52
UCIAMS-205971	Northern New York	Durham	kernel	+4.74	+4.20	−9.21
UCIAMS-205977	Northern New York	Morse	kernel	+6.08	+5.54	−8.80
UGAMS-37383	Northern New York	Morse	kernel	+6.07	+5.53	−10.24
UGAMS-37380	Northern New York	Pine Hills	kernel	+2.67	+2.13	−9.17
UCIAMS-205969	Northern New York	Potocki	kernel	+5.44	+4.90	−11.13
UCIAMS-205974	Northern New York	Sanford Corner	kernel	+9.37	+8.83	−9.81
UCIAMS-205975	Northern New York	Sanford Corner	kernel	+5.46	+4.92	−9.21
UGAMS-34445	Northern New York	Talcott	kernel	+5.70	+5.16	−8.80
UGAMS-34446	Northern New York	Talcott	kernel	+4.87	+4.33	−8.80
UGAMS-205972	Northern New York	Whitford	kernel	+5.86	+5.32	−8.87
UCIAMS-205965	Mohawk Valley	Cayadutta	kernel	+4.85	+4.31	−8.56
UCIAMS-205966	Mohawk Valley	Cayadutta	kernel	+3.98	+3.44	−8.88
UCIAMS-205967	Mohawk Valley	Cayadutta	kernel	+3.06	+2.52	−8.88
UCIAMS-205968	Mohawk Valley	Cayadutta	kernel	+6.48	+5.94	−10.24
UCIAMS-218473	Mohawk Valley	Klock	kernel	+4.29	+3.75	−9.35
UCIAMS-218474	Mohawk Valley	Klock	kernel	+6.76	+6.22	−8.78
UCIAMS-218475	Mohawk Valley	Klock	kernel	+4.55	+4.01	−9.50
UCIAMS-218476	Mohawk Valley	Klock	kernel	+4.72	+4.18	−9.05
UCIAMS-218477	Mohawk Valley	Garoga	kernel	+5.54	+5.00	−8.84
UCIAMS-218478	Mohawk Valley	Garoga	kernel	+4.62	+4.08	−9.27
UCIAMS-218479	Mohawk Valley	Garoga	kernel	+5.80	+5.26	−8.55
UCIAMS-218480	Mohawk Valley	Getman #1	kernel	+4.33	+3.79	−8.07
UCIAMS-218483	Mohawk Valley	Otstungo	kernel	+5.02	+4.48	−9.00
UCIAMS-218487	Mohawk Valley	Otstungo	kernel	+4.27	+3.73	−9.98
UCIAMS-218489	Mohawk Valley	Otstungo	kernel	+6.71	+6.17	−9.11
UCIAMS-218494	Mohawk Valley	Pethick	kernel	+0.81	+0.27	−9.52
UCIAMS-218495	Mohawk Valley	Pethick	kernel	+2.50	+1.96	−8.96
UCIAMS-218496	Mohawk Valley	Pethick	kernel	+4.73	+4.19	−8.96
UCIAMS-218492	Mohawk Valley	Smith-Pagerie	kernel	+4.66	+4.12	−8.68
UCIAMS-218493	Mohawk Valley	Smith-Pagerie	kernel	+4.75	+4.21	−8.93
NYSM-A39855A	Mohawk Valley	Snell	kernel	+7.97	+7.43	−9.82
NYSM-A71102	Mohawk Valley	Snell	kernel	+5.08	+4.54	-10.08
NYSM-A71098	Mohawk Valley	Snell	kernel	+7.08	+6.54	−10.46
AA26541/114197	Susquehanna Valley	Roundtop, 12th/13th cen.	kernel	+0.60	+0.06	−8.70
AA21979/114195	Susquehanna Valley	Roundtop, 14th century	kernel	+2.80	+2.26	−8.70
AA26539/114196	Susquehanna Valley	Roundtop, 15th century	kernel	+2.40	+1.86	−8.70
AA21978/114194	Susquehanna Valley	Roundtop, 16th century	kernel	+4.30	+3.76	−8.80
UGAMS-35644	Finger Lakes	Kelso	kernel	+7.65	+7.11	−8.86
UGAMS-32991	Southern Ontario	Baker	kernel	+6.49	+5.95	−9.40
UGAMS-32992	Southern Ontario	Baker	kernel	+4.41	+3.87	−9.31
UGAMS-40364	Southern Ontario	Barrie	kernel	+5.06	+4.52	−9.71
UGAMS-32755	Southern Ontario	Coulter	kernel	+5.98	+5.44	−9.47
UGAMS-32756	Southern Ontario	Coulter	kernel	+3.84	+3.30	−9.31
UGAMS-33005	Southern Ontario	Damiani	kernel	+5.22	+4.68	−9.64
UGAMS-33006	Southern Ontario	Damiani	kernel	+4.48	+3.94	−8.89
UGAMS-40350	Southern Ontario	Dunsmore	kernel	+7.66	+7.12	−8.23
UGAMS-40351	Southern Ontario	Dunsmore	kernel	+4.37	+3.83	−9.34
UGAMS-40348	Southern Ontario	Grandview	kernel	+6.25	+5.71	−9.24
UGAMS-40347	Southern Ontario	Grandview	kernel	+3.94	+3.40	−8.87
UGAMS-40359	Southern Ontario	Hidden Spring	kernel	+5.89	+5.35	−10.34
UGAMS-40362	Southern Ontario	Hidden Spring	kernel	+5.83	+5.29	−8.79
UGAMS-40361	Southern Ontario	Hidden Spring	kernel	+5.28	+4.74	−9.84
UGAMS-40360	Southern Ontario	Hidden Spring	kernel	+5.01	+4.47	−9.80
UGAMS-40358	Southern Ontario	Jarrett-Lahmer	kernel	+6.43	+5.89	−7.95
UGAMS-40356	Southern Ontario	Jarrett-Lahmer	kernel	+5.37	+4.83	−9.86
UGAMS-40357	Southern Ontario	Jarrett-Lahmer	kernel	+4.96	+4.42	−9.25
UGAMS-40363	Southern Ontario	Jones	kernel	+7.52	+6.98	−9.31
UGAMS-40365	Southern Ontario	Mackenzie-Woodbridge	kernel	+6.86	+6.32	−9.00
UGAMS-40366	Southern Ontario	Mackenzie-Woodbridge	kernel	+4.20	+3.66	−9.66
UGAMS-41528	Southern Ontario	Maynard-McKeown	kernel	+5.75	+5.21	−9.50
UGAMS-41529	Southern Ontario	Maynard-McKeown	kernel	+6.23	+5.69	−9.08
UGAMS-32995	Southern Ontario	McNair	cob	+4.87	+4.33	−9.70
UGAMS-32994	Southern Ontario	McNair	kernel	+4.61	+4.07	−10.32
UGAMS-40353	Southern Ontario	New	kernel	+6.40	+5.86	−9.29
UGAMS-40352	Southern Ontario	New	kernel	+6.10	+5.56	−8.19
UGAMS-33009	Southern Ontario	Parsons	kernel	+6.54	+6.00	−9.81
UGAMS-33008	Southern Ontario	Parsons	kernel	+4.54	+4.00	−9.24
UGAMS-40154	Southern Ontario	Sopher	kernel	+8.83	+8.29	−9.10
UGAMS-40155	Southern Ontario	Sopher	kernel	+7.58	+7.04	−9.28
UGAMS-38398	Southern Ontario	Spang	kernel	+7.50	+6.96	−8.22
UGAMS-37834	Southern Ontario	Spang	kernel	+7.04	+6.50	−8.73
UGAMS-38397	Southern Ontario	Spang	kernel	+5.94	+5.40	−9.44
UGAMS-37833	Southern Ontario	Spang	kernel	+5.20	+4.66	−9.65
UGAMS-41530	Southern Ontario	Waupoos	kernel	+5.31	+4.77	−8.89
UGAMS-41531	Southern Ontario	Waupoos	kernel	+4.76	+4.22	−8.63
UGAMS-41532	Southern Ontario	Waupoos	kernel	+4.98	+4.44	−9.05
UGAMS-40346	Southern Ontario	Wellington	kernel	+6.98	+6.44	−8.99
UGAMS-40343	Southern Ontario	Wellington	kernel	+5.77	+5.23	−9.21
UGAMS-40345	Southern Ontario	Wellington	kernel	+5.38	+4.84	−8.92
UGAMS-40344	Southern Ontario	Wellington	kernel	+4.31	+3.77	−8.55

a Isotopic measures were obtained on maize samples submitted for previously published AMS radiocarbon dating as indicated in the methods section except those identified by NYSM catalog numbers, which were assayed for this project at the University of Florida Light Stable Isotope Mass Spectrometry Lab. No permits or new permissions were required. AA = University of Arizona AMS Laboratory, UCIAMS = University of California-Irvine Keck Carbon Cycle AMS Laboratory, UGAMS = University of Georgia Center for Applied Isotope Studies.

Experiments on Eastern Hemisphere grain kernels, including wheat indicate that charring results in average Δ15N values of 0.31‰ to 1.00‰ [62,63]. Similar experiments have not been performed on maize. For the present project, samples of contemporary dried Tuscarora White Flour and Dent maize kernels from collections used in previous experiments [64,65] and freeze-dried commercial canned hominy kernels were halved. Following established protocols for experimental charring of maize kernels in non-oxidizing conditions [66,67], one half of each kernel was placed in a loose foil packet, buried in sand within a ceramic crucible, and heated in a muffle furnace at 180°, 220°, or 260°C for 2 h. δ15N and δ13C measurements were obtained on fractions of the carbonized and uncarbonized halves. Whole kernels were also carbonized to assess heating effects on kernel integrity [66-68].

Results are presented in Table 3 and S1 Table. Kernels heated for 2 h at 180°C did not fully carbonize and those heated at 260°C for 2 h did not maintain their structural integrity, consistent with outcomes obtained by others [66,67]. As a result, it is unlikely that either would have survived in the archaeological record. The kernels heated at 220°C fully carbonized and maintained their structural integrity appearing much like charred kernels recovered from archaeological sites; the mean Δ15N for these kernels is 0.51 ‰. The experiments were repeated with different kernels for 24 h at 180° and 220°C, which duplicated the results for kernels heated for 2 hr. The 24 h Δ15N values are statistically the same as those kernels heated for 2 h (df = 26, t = 0.413, p = 0.6831). Combining the 220°C 2 and 24 h experiments results in a mean Δ15N of 0.54±0.53. This value was subtracted from the archaeological maize δ15N values, and the standard deviation for the archaeological maize mean value was adjusted with the standard deviation of the Δ15N mean through error propagation calculation. This resulted in a range in δ15N values for the archaeological kernels of +0.06 to +8.83 ‰, a mean of +4.76±1.63 ‰, and a median of 4.71 ‰.

Table 3

Results of experimental maize charring on δ15N and δ13C values.

°C	Time (h)	n	Mean Δ15N	Std. Dev.	Median Δ15N	n	Mean Δ13C	Std. Dev.	Median Δ13C
180	2	20	0.06	0.57	0.19	23	0.00	0.29	0.00
180	24	9	0.39	0.66	0.58	9	0.07	0.04	0.01
220	2	20	0.51	0.59	0.43	15	0.02	0.36	0.12
220	24	9	0.60	0.40	0.74	9	0.16	0.24	0.10
260	2	6	0.96	0.2	0.91	6	0.56	0.38	0.61

Terrestrial plants should have δ15N values 3–4 ‰ lower than terrestrial herbivore bone collagen ([42], p. 3). Archaeologists exploring Neolithic and later agronomic practices in Europe have used this as one means to establish baselines to identify the use of animal manure for crop fertilization. That is, δ15N values of crop seeds higher than estimated plant browse are interpreted as evidence for manure fertilization [53-55]. Following this line of reasoning we calculated an estimated mean for plant browse from collagen of bone recovered form archaeological sites in the study region. δ15N values from 227 white tailed deer (Odocoileus virginianus) bone collagen samples obtained from Iroquoian archaeological sites in the three areas with sampled maize kernels have a range of +2.8 to +8.6 ‰, a mean of +5.6±1.0 ‰, and a median of +5.5 ‰ (S2 Table). Captive, control-fed, white-tailed deer had a mean δ15N for antler collagen of +4.29±0.42 ‰ [69]; for a pure C3 diet, the mean was +3.73±0.43‰. Subtracting 25 red deer (Cervus elaphus) antler collagen values from same-individual bone collagen values [70] resulted in a mean difference of +0.38±0.37 ‰, suggesting that there is essentially no difference between bone collagen and antler collagen δ15N values. As a result, we subtracted 4.0 ‰ from individual archaeological deer bone collagen values to estimate average consumed plant δ15N values, resulting in a mean of +1.6±1.0 ‰ and a median of +1.5 ‰ (Fig 2). This range of results is consistent with similar estimates in Europe based on large herbivore collagen values, ranging from +0.9 to +3.1 ‰ [54].

Fig 2

Box plots of adjusted δ15N values of archaeological maize, archaeological white-tailed deer bone collagen, and estimated deer forage.

The horizontal lines within the boxes are medians, boxes represent the 25th to 75th percentile and whiskers indicate 10th and 90th percentiles.

Subtracting 4.0 ‰ from rabbit/hare (Leporidae) collagen values from southern Ontario Iroquoian archaeological sites (n = 6) resulted in a mean of −0.39±0.73 ‰ and a median of −0.04 ‰. Subtracting the value from woodchuck (Marmota monax) collagen values from southern Ontario Iroquoian sites (n = 18) resulted in a mean of −0.93±0.70 ‰ and a median of −0.97 ‰ (S2 Table). These results suggest that the plants consumed by these herbivores had lower δ15N values than the plants consumed by deer. To be conservative, we used the values for deer to calculate the value for non-cultivated plants in the data evaluation that follows.

Approximately 80% of land plant species are mycorrhizal including maize [71]. Like the majority of these plants, maize is associated with arbuscular mycorrhizae [42,58]. One study found that plants associated with arbuscular mycorrhizae have mean δ15N values ~2‰ lower than nonmycorrhizal plants, with a mean value of −1.1±0.1 ‰ [58]. Because of the high percentage of arbuscular mycorrhizal plants globally, the estimated browse values should provide a reasonable baseline for interpretation of the archaeological maize and are consistent with the lowest δ15N values obtained on archaeological maize kernels. While maize plants may obtain some nitrogen from these fungal symbionts, they primarily obtain mineralized nitrogen from the soil with enhanced phosphorus uptake being the primary benefit to arbuscular mycorrhizal plants [72]. Given the global positive correlation between high soil N and plant δ15N values [58] these values and the overall range in values of +8.77 ‰ suggest that they resulted from varied N pools, with the higher values reflecting high plant-available nitrogen pools. The 72 values greater than two standard deviations above the estimated mean of plant values (>3.6 ‰) is +5.14 ± 1.16 ‰. Two lowest values, +0.06‰ and +0.26‰, are greater than two standard deviations below the mean for estimated plant values, suggesting low soil nitrogen. A t-test indicates that the maize values as a whole are statistically different from the estimated terrestrial plant values based on deer collagen values (p = 0.0000); the maize δ15N values are higher than would be expected for terrestrial plants (difference in means = 3.20‰; Fig 2). Similar results are obtained for the three subregions (Table 4).

Table 4

T-test results of maize and estimated plant δ15N values.

Region	maize (n)	planta (n)	t	p	Difference in means	Confidence interval (95%)
All	82	227	17.445	0.0000	3.2009	2.9002–3.5007
Southern Ontario	42	191	19.965	0.0000	3.4983	3.1531–3.8436
Northern New Yorkb	12	9	4.870	0.0000	3.0811	1.9681–4.1864
Mohawk Valleyb	23	27	10.388	0.0000	3.6347	2.9312–4.3382

aEstimates (see text)

bExact permutation p, bootstrap confidence interval,.

The large area encompassed in our study has varied edaphic and climatic conditions, which likely resulted in differing agronomic practices. However, one goal of the varied practices was to maintain productivity to support settlement populations. This included maintaining plant-available nitrogen levels. T-tests of adjusted maize δ15N values between subregions indicate no significant differences between Northern New York and the Mohawk Valley and southern Ontario. There is a significant difference, however, between the Mohawk Valley and southern Ontario; the southern Ontario mean is higher than the Mohawk Valley mean, suggesting the possibility of regional variation (Table 5; Fig 3).

Table 5

T-test results of sub-regional δ15N values.

(Monte Carlo permutation p-values, bootstrap confidence intervals).

Regions	t	p	Difference in means	Confidence Interval (95%)
Northern NY-Ontario	0.7852	0.4372	0.3298	-0.5900–1.3649
Northern NY-Mohawk	0.8447	0.4084	0.4797	-0.6906–1.5610
Mohawk-Ontario	2.3899	0.0204	0.8094	0.0903–1.5151

Fig 3

Box plots of δ15N values of archaeological maize from southern Ontario, northern New York, and the Mohawk Valley.

The solid horizontal lines within the boxes are medians, boxes represent the 25th to 75th percentile and whiskers indicate 10th and 90th percentiles.

Discussion and conclusion

There is little doubt that fifteenth‒sixteenth century AD Iroquoian farmers in present-day New York and Ontario needed to maintain the fertility of their extensive, hand-cultivated maize fields over extended periods of time lasting up to several decades. While it is possible that some of the high δ15N values we obtained are the result of initial field clearance involving cutting down and burning trees and other vegetation ([42], p.7), given the probable lengths of time Iroquoian agricultural fields were in continuous cultivation, the effects of these activities on the isotopic compositions of the plants are insufficient to explain the range of δ15N values observed. For example, one study found an initial increase in foliar δ15N values after wildfires, followed by sharp drop offs in the first post-fire decade [73]. A meta-analysis of fire effects on nitrogen pools found that NH4+ increased immediately following fire and then declined synoptically to pre-fire levels within 3 yr, while NO3‒ increased following the fire peaking at 1 yr and then decreasing to pre-fire levels within 5 yr [74]. An analysis of clearcutting forests also indicated short-lived increase in foliar δ15N values, reaching their peak in 2 yr and falling thereafter [75] (see [42] for an overview). Therefore, clearance involving burning may have resulted in a very short-term pulse of mineralized nitrogen into the soil, possibly elevating plant δ15N values, but this effect would have dissipated well before these fields ceased to be cultivated.

While we do not have direct evidence of soil amendments to increase and maintain plant available nitrogen, Iroquoian agronomic systems were evidently well adapted to local edaphic conditions. All but six (7.3%) of the 82 δ15N maize values used in this analysis exceed the minimal threshold of ~+2.5‰ suggested for identifying fertilization with manure in Europe, and 14 (17.07%) exceed ~+6.0‰ for identifying heavy use of manure [55] (Fig 4). The maintenance of soil organic matter in naturally fertile soils of New York through the incorporation of crop detritus into “corn hills” allowed continuous cropping systems over extended periods of time [21,34]. Long-term incorporation of organic matter with high nitrogen content (>1%), such as common bean and squash vine detritus, can promote the accumulation of nitrogen in soil organic matter [76]. The addition of organic matter to contemporary no-till systems results in high levels of microbial respiration and nitrogen mineralization [77]. Naturally fertile soils containing 4% organic matter can annually produce 90 lbs per acre (102 kg/ha) of plant-available nitrogen [21] in excess of amounts provided under some systems of manure fertilization [78]. Agronomic systems that included annual burning of fields may have increased plant-available nitrogen [44,79] while the incorporation of charcoal from maize detritus into “corn hills” may have helped maintain plant-available nitrogen [80] and in some cases increased plant nitrogen uptake [81]. Intercropping maize with common bean may also have enhanced the availability of nitrogen to maize [45-47].

Fig 4

Scatter diagram of archaeological maize δ15N and δ13C values.

The blue horizontal lines are estimates for boundaries for medium (lower) and high (upper) manuring rates for European Neolithic crops, respectively [53–55].

Our results suggest that Iroquoian agronomic practices were as effective at providing nitrogen to crops. The contrary interpretations of the seventeenth-century AD Ontario ethnohistorical record [7], which emphasizes the exhaustion of soils after short periods of time, may have been a development of that century when the Wendat (Huron) Confederacy occupied an area with sandy soils having low natural fertility as opposed to areas to the south in Ontario where most settlements were located prior to the seventeenth-century consolidation [31]. Southern Ontario has dominantly Luviols, the equivalent of Alfisols in the U.S.D.A. system [82], the latter of which were exploited by Iroquoian farmers in New York [21,32]. In central New York, Iroquoian village sites, for example, are associated with high fertility Alfisols and areas that experienced high frequencies of evidently anthropogenic fire, presumably from agronomic practices [32,83]. At least some fifteenth-and sixteenth-century Iroquoian villages in southern Ontario were sited to take advantage of soils with high natural soil fertility [26]. The low fertility of the acidic soils exploited by Wendat (Huron) farmers in the seventeenth century after consolidation of the Confederacy [18] were not typical of soils exploited by Iroquoian farmers in other areas and times. Mineralization of nitrogen from the microbial breakdown of soil organic matter is negatively affected by lower temperatures in the presence of low soil pH, higher sand content, and lower clay content [84,85], all of which characterize the acidic, sandy soils of the seventeenth-century Wendat (Huron) region [18].

While Iroquoian farmers may not have used fertilizer in the Eastern Hemisphere sense, our results suggest that as with Native Americans in other regions [86], their agronomic practices maintained plant-available nitrogen, as evinced by high δ15N values. These relatively high δ15N values are suggestive of the addition of soil amendments with δ15N values higher than that of the original soil organic matter. At this early stage it is not possible to say what these soil amendments might have been, but human and/or dog excrement or fish are plausible. Additional studies are required to examine the effects of these practices on plant δ15N values. Our results indicate that reliance on ethnohistoric accounts of seventeenth-century AD agriculture in Ontario to model Iroquoian agriculture in general is unwarranted [21]. The results also demonstrate the utility of obtaining δ15N values on maize as a tool for increasing knowledge of pre-Contact Native American agronomic practices.

Methods and materials

All statistical analyses were performed in PAST v. 3.25 [87].

Isotopic measures were obtained on maize samples submitted for AMS radiocarbon dating as reported in [88-91] except those obtained on the three samples from the Snell site, which were obtained independently of AMS dating for this project. The values from the Roundtop site were obtained on samples originally reported in [92] on remaining portions of the samples archived at the University of Arizona AMS Laboratory.

The stable isotope results in this study are expressed in standard δ-notation. X = [(Rsample/Rstandard)– 1] * 1000, where X (in units permil, ‰) is δ13C or δ15N and R = 13C/12C or 15N/14N. The δ13C values are reported relative to the V-PDB standard, while the δ15N values are reported relative to atmospheric N2.

In this study we directly analyzed the δ13C and δ15N values from modern maize kernels. For analysis, dried maize kernels were crushed to a powder using a mortar and pestle then weighed (@ 3.5mg) into tin capsules. The samples were analyzed in the Light Stable Isotope Mass Spectrometry Lab in the Department of Geological Sciences at the University of Florida, Gainesville, FL, USA. Specifically, tin capsules were loaded into a 50-position automated Zero Blank sample carousel on a Carlo Erba NA1500 CNS elemental analyzer. Each sample was combusted at 1020°C in a quartz column in an oxygen-rich atmosphere. The sample gas was transported and passed through a hot reduction column (650°C) consisting of elemental copper to remove oxygen in a He carrier stream. The remaining sample gas then passed through a chemical (magnesium perchlorate) trap to remove water followed by a 0.7-meter GC column at 120°C to separate N2 from CO2. The sample gas next passed into a ConFlo II interface and into the inlet of a Thermo Electron Delta V Advantage isotope ratio mass spectrometer running in continuous flow mode where the sample gas was measured relative to laboratory reference N2 from CO2 gases. Precision for the analyses were <0.2‰ for δ15N and <0.1‰ for δ13C.

Isotopic analyses of charred archaeological maize were carried out at Keck Carbon Cycle Facility at the University of California Irvine (UCIAMS) or the Center for Applied Isotope Studies at the University of Georgia (UGAMS). Samples at both facilities were subjected to standard acid-base-acid pretreatment. UCIAMS δ15N was measured to a precision of <0.2 ‰ using a Fisons NA1500NC elemental analyzer/Finnigan Delta Plus isotope ratio mass spectrometer. At UGAMS, δ15N was measured using an elemental analyzer isotope ratio mass spectrometer to a precision of <0.1 ‰.

Stable isotope results of maize kernel charring experiments.

(DOCX)

Click here for additional data file.

Herbivore bone collagen δ15N values used to calculate forage values.

(DOCX)

Click here for additional data file.

26 Nov 2019

PONE-D-19-26845

Using Maize δ15N Values to Assess Soil Fertility in Fifteenth- and Sixteenth-Century AD Iroquoian Agricultural Fields

PLOS ONE

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One of the reviewers has indicated that the interpretation of N isotopic signatures should be improved before publication. Please be sure to address this aspect in the revised manuscript.

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Reviewer #1: Dear Authors

I appreciate your effort do use d15N values to assess the ancient agriculture practices. Many are the examples of ancient agriculture that was able to support dense populations (Asia) using a variety of soil amendments (in the obvious absence of fertilizers). However, authors might use a more precise interpretation of d15N values to draw their conclusions. Nitrogen isotope natural abundance (expressed here as d15N values) is not an indicator to “plant- available nitrogen levels”. Thus, the assumption of authors that the higher the d15N values (plant), the higher the plant-available nitrogen levels” is not correct. I fact, the N transformation processes lead to fractionation of nitrogen isotope and the N loss (in soli or manure storage) results in an increasing of d15N values. Manures have high d15N values manly due to N loss as ammonia (volatilization). Plants tend to mirror the d15N values of available N in soil which can reflect the N source, i.e. manure and plant residues and biological N fixing most of the time (I am excluding NH3 deposition for this case). Thus, high d15N indicates loss of N from the system (i.e. soil). However, soil amended with animal manure tends to show high d15N values. I recommend to the authors to bring to support their discussion two good reviews: CHOI, W. J. et al. Synthetic fertilizer and livestock manure differently affect δ15N in the agricultural landscape: A review. Agriculture, Ecosystems & Environment, v. 237, p. 1-15, 2017, and Martinelli LA, Piccolo MC, Townsend AR, Vitousek PM, Cuevas E, McDowell W et al., Nitrogen stable isotopic composition of leaves and soil: tropical versus temperate forests. Biogeochemistry 46:45–65 (1999). The last shows d15N values of soil and leaves (temperate vs. tropical forests). Tropical forests have higher d15N values than Temperate Forests.

Despite of my opinion that the authors might refine the discussion using the right interpretation of d15N values as above mentioned, they are right suggesting a more relevant role of N-fixing from common bean instead use of animal manures based on d15N values obtained. But at this point the paper also needs to give more information about the d15N of N-fixing plants (ranges, common values, etc). This is crucial to support their right conclusions. Thus, I recommend the publication of this manuscript after revision.

Best regards

Reviewer

Reviewer #2: The following text is the same as the attached file for the Editor and Authors.

Review of PONE-D-19-26845

Title: Using Maize δ15N Values to Assess Soil Fertility in Fifteenth- and Sixteenth-Century AD Iroquoian Agricultural Fields

Authors: John P. Hart and Robert S. Ferenec

General Comments to Editor and Authors:

This paper provides a compelling case for Iroquoian farmers in New York State, USA and Ontario and Québec in Canada having maintained the fertility of their agricultural fields during the 15th and 16th centuries AD. Specifically, the authors obtained δ15N and δ13C values on maize kernels from >60 Iroquoian archaeological sites, and performed statistical analysis on these isotopic data, to elucidate upon the fertility of the soils that once supported maize at these sites. The authors analyzed these isotopes in the absence of preserved Iroquoian fields, therefore, δ15N and δ13C values on maize kernels can be viewed as an indirect proxy for soil productivity (an idea not fully fleshed out in the manuscript, see below). To strengthen the hypothesis that Iroquoian farmers burned their maize fields after harvest to input plant-available nitrogen, suggested from analysis of the archaeological material, the authors conducted an experimental study. The authors charred and performed isotopic analysis upon modern maize kernels as well as grass seeds collected in the 1930s-1940s, which validated their interpretations of the isotopic data based on Iroquoian archaeological material. The authors’ results were also compared to the experimental data on wheat kernels derived from archaeological sites in the Eastern Hemisphere.

The key finding of this research, presented for the first time in this paper (to my knowledge), is that the amount of nitrogen input into the soils through burning by Iroquoian farmers during the 15th and 16th centuries AD was comparable to adding manure to agricultural fields by Neolithic farmers in Europe. This finding is highly significant and requires to be more strongly highlighted in the paper (below I indicate where in the manuscript). Additionally, I recommend that the authors expound upon other aspects of their findings and the broader significance of their research in their revision of this manuscript.

In my opinion, this manuscript is worthy of publication in PLOS ONE pending minor revisions. The paper describes original research not published elsewhere. The theoretical and methodological approaches and explanations are robust and the statistical analyses are excellent; for example, the reasoning expressed in the paragraph on P. 7, Lns 188-205, is exceptional. The conclusions are well supported by the data. The research adheres to integrity standards, and the data will be available upon publication. The paper is also well written. Below are more specific comments to aid the authors when revising this paper.

Specific Comments (my suggested textual additions are in bolded red font):

Page # Line # Comments:

* My textual additions are in red font.

1 6, 32 Abstract & Introduction: insert “State, USA” after New York, and “Canada” after “Québec” [use the letter e with an acute accent, which is the official spelling of this province’s name], to read “New York State, USA and Ontario and Québec, Canada,”.

1 9-12 The authors did not mention an experimental component of their research, where they charred modern maize kernels and 20th century grass seeds to valid their isotopic values obtained from maize kernels obtained from archaeological results. Therefore, a short sentence regarding this work should be added to the Abstract.

1 13 Abstract: add a phrase to the end of this sentence, or an additional sentence, that identifies the broader significance of this research beyond Iroquoian studies. Here is a suggestion: “These results…Iroquoian agronomic practices, and identified that Iroquoian farmers augmented nitrogen through burning, producing values comparable to European Neolithic farmers who utilized manure to upkeep soil productivity.”

1 46 Provide the source for this sentence about 3-4 maize plants per mound.

3 98-99 Write a sentence or two that explains why there is an “apparent current lack of Iroquoian agricultural field preservation.” I suggest that the authors move up the first sentence of the following paragraph (Ln 116-117) about depletion of soil organic matter to Ln 98. Also, add a statement that the areas where Iroquoian agriculture fields were once located have been heavily farmed by Euro-Americans and Euro-Canadians in subsequent decades, thereby obliterating the soil characteristics of the fields farmed centuries ago by indigenous populations.

3 101-105 Please divide this long sentence into two.

3 113 Insert “directly from the mounds themselves” between “evidence” and “to test,” given that the paper does provide archaeological evidence to test this hypothesis.

3 116 Insert an introductory sentence, before the one beginning with “Eastern Hemisphere grains…”, which explains that in the absence of preserved Iroquoian agriculture fields one can use δ15N values obtained from cultigens as an indirect proxy for past soil productivity.

6 152 Table 2: at the bottom of this table, provide footnotes with details, including the full names, pertaining to the two labs used, UGAMS and UCIAMS (which are identified on p. 12). Note: I approve of the authors using only two labs to obtain these δ15N and δ13C data, to minimize any possible data discrepancy between them. While looking at the data in this table, I did not see any indication of variation in the results produced between the two labs, as both labs gave a similar range of values for each isotope. Additionally, these AMS labs have reputations for producing outstanding (reliable) results.

6 155 The colored dots are not identified in Fig. 1. Therefore, the authors should insert a legend explaining the color scheme for these dots, which indicate site locations in southern Ontario, northern New York, and the Mohawk Valley. This information could be added to the figure caption, but a better option is to embed a legend in this figure.

6 161 Identify the grain as being wheat (Triticum spp.).

6 173-175 Move this sentence starting with “The kernels heated at 220°C fully carbonized…” to integrate with the sentence starting on Ln 177, “Those heated to 220°C…” to reduce redundancy.

7, 9 201, 250 Is “cal” necessary, given that BC/AD are used?

7 211 Mention the three subregions at the end of this caption for Fig. 2 (d).

8 213 Replace Quebec with Québec.

10 285 Delete “in” before “can.”

11 315 Insert “involving fire” after agronomic practices.

********************

**********

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Reviewer #1: Yes: Caio T Inácio

Reviewer #2: No

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Submitted filename: Review of PONE-D-19-26845.docx

Click here for additional data file.

13 Feb 2020

Responses to Reviewers

Reviewer 1

I appreciate your effort do use d15N values to assess the ancient agriculture practices. Many

are the examples of ancient agriculture that was able to support dense populations (Asia) using

a variety of soil amendments (in the obvious absence of fertilizers). However, authors might

use a more precise interpretation of d15N values to draw their conclusions. Nitrogen isotope

natural abundance (expressed here as d15N values) is not an indicator to “plant- available

nitrogen levels”. Thus, the assumption of authors that the higher the d15N values (plant), the

higher the plant-available nitrogen levels” is not correct. I fact, the N transformation processes

lead to fractionation of nitrogen isotope and the N loss (in soli or manure storage) results in an

increasing of d15N values. Manures have high d15N values manly due to N loss as ammonia

(volatilization). Plants tend to mirror the d15N values of available N in soil which can reflect

the N source, i.e. manure and plant residues and biological N fixing most of the time (I am

excluding NH3 deposition for this case). Thus, high d15N indicates loss of N from the system

(i.e. soil). However, soil amended with animal manure tends to show high d15N values. I

recommend to the authors to bring to support their discussion two good reviews: CHOI, W. J.

et al. Synthetic fertilizer and livestock manure differently affect δ15N in the agricultural

landscape: A review. Agriculture, Ecosystems & Environment, v. 237, p. 1-15, 2017, and

Martinelli LA, Piccolo MC, Townsend AR, Vitousek PM, Cuevas E, McDowell W et al.,

Nitrogen stable isotopic composition of leaves and soil: tropical versus temperate forests.

Biogeochemistry 46:45–65 (1999). The last shows d15N values of soil and leaves (temperate

vs. tropical forests). Tropical forests have higher d15N values than Temperate Forests.

Despite of my opinion that the authors might refine the discussion using the right

interpretation of d15N values as above mentioned, they are right suggesting a more relevant

role of N-fixing from common bean instead use of animal manures based on d15N values

obtained. But at this point the paper also needs to give more information about the d15N of N fixing plants (ranges, common values, etc). This is crucial to support their right conclusions.

Thus, I recommend the publication of this manuscript after revision.

Thank you for your comments. We have addressed the issue of nitrogen loss and δ15N in lines 125 through 129 and the last paragraph. We appreciate your suggestions for additional references. While we did not add these specific we have added citations to several additional sources throughout the document that we feel are more pertinent to the issue at hand and revised/added text that draws on information contained within those surces.

Reviewer 2

1 6, 32 Abstract & Introduction: insert “State, USA” after New York, and “Canada” after

“Québec” [use the letter e with an acute accent, which is the official spelling of this province’s

name], to read “New York State, USA and Ontario and Québec, Canada,”.

We have made these modifications except adding State after New York.

1 9-12 The authors did not mention an experimental component of their research, where they

charred modern maize kernels and 20th century grass seeds to valid their isotopic values

obtained from maize kernels obtained from archaeological results. Therefore, a short sentence

regarding this work should be added to the Abstract.

Done

1 13 Abstract: add a phrase to the end of this sentence, or an additional sentence, that

identifies the broader significance of this research beyond Iroquoian studies. Here is a

suggestion: “These results…Iroquoian agronomic practices, and identified that Iroquoian

farmers augmented nitrogen through burning, producing values comparable to European

Neolithic farmers who utilized manure to upkeep soil productivity.”

Done

1 46 Provide the source for this sentence about 3-4 maize plants per mound.

Done

3 98-99 Write a sentence or two that explains why there is an “apparent current lack of

Iroquoian agricultural field preservation.” I suggest that the authors move up the first sentence

of the following paragraph (Ln 116-117) about depletion of soil organic matter to Ln 98. Also,

add a statement that the areas where Iroquoian agriculture fields were once located have been

heavily farmed by Euro-Americans and Euro-Canadians in subsequent decades, thereby

obliterating the soil characteristics of the fields farmed centuries ago by indigenous

populations.

Done

3 101-105 Please divide this long sentence into two.

Done

3 113 Insert “directly from the mounds themselves” between “evidence” and “to test,” given

that the paper does provide archaeological evidence to test this hypothesis.

Done

3 116 Insert an introductory sentence, before the one beginning with “Eastern Hemisphere

grains…”, which explains that in the absence of preserved Iroquoian agriculture fields one can

use δ15N values obtained from cultigens as an indirect proxy for past soil productivity.

We have chosen to keep the beginning of this paragraph as in the original.

6 152 Table 2: at the bottom of this table, provide footnotes with details, including the full

names, pertaining to the two labs used, UGAMS and UCIAMS (which are identified on p. 12).

Done

6 155 The colored dots are not identified in Fig. 1. Therefore, the authors should insert a

legend explaining the color scheme for these dots, which indicate site locations in southern

Ontario, northern New York, and the Mohawk Valley. This information could be added to the

figure caption, but a better option is to embed a legend in this figure.

There is now a key to the colors in the figure.

6 161 Identify the grain as being wheat (Triticum spp.).

Done

6 173-175 Move this sentence starting with “The kernels heated at 220°C fully carbonized…”

to integrate with the sentence starting on Ln 177, “Those heated to 220°C…” to reduce

redundancy.

We have rewritten this paragraph to clarify and avoid redundancy.

7, 9 201, 250 Is “cal” necessary, given that BC/AD are used?

We have deleted the “cal”.

7 211 Mention the three subregions at the end of this caption for Fig. 2 (d).

We have added a key to the dot colors.

8 213 Replace Quebec with Québec.

Done

10 285 Delete “in” before “can.”

We eliminated this table after receiving additional low values on archeological maize, which serve as a better baseline for understanding the elevated values on archaeological maize.

11 315 Insert “involving fire” after agronomic practices.

Because this is only one possible aspect of the agronomic practices be have declided to modify this sentence.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

13 Mar 2020

Using Maize δ15N Values to Assess Soil Fertility in Fifteenth- and Sixteenth-Century AD Iroquoian Agricultural Fields

PONE-D-19-26845R1

Dear Dr. Hart,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Remigio Paradelo Núñez

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Dr. Hart,

Thank you for having considered the questions raised by the reviewers during the revision of your manuscript. Your paper is now acceptable for publication. Congratulations.

Reviewers' comments:

18 Mar 2020

PONE-D-19-26845R1

Using Maize δ15N Values to Assess Soil Fertility in Fifteenth- and Sixteenth-Century AD Iroquoian Agricultural Fields

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