A field study was conducted at ACRE (Agronomy Center for Research and Education, 40°28′07″N, 87°00′25″W), West Lafayette, IN, USA and PPAC (Pinney Purdue Agricultural Center, 41°26′41″N, 86°56′41″W), Wanatah, IN, USA in 2013 and 2014. The soil was Chalmers silty-clay loam (Fine-silty, mixed, superactive, mesic Typic Endoaquolls) in 2013 and Raub-Brenton complex (Fine-silty, mixed, superactive, mesic Aquic Argiudolls) in 2014 at ACRE. The soil at PPAC was Sebewa loam (Fine-loamy over sandy or sandy-skeletal, mixed, superactive, mesic Typic Argiaquolls) in both years. Average soil pH, organic matter, Mehlich-3 P, and Mehlich-3 K were 6.9, 3.7 g 100 g⁻¹, 22 mg kg⁻¹, 106 mg kg⁻¹ at ACRE in 2013; 6.7, 4.4 g 100 g⁻¹, 17 mg kg⁻¹, 92 mg kg⁻¹ at PPAC in 2013; and 6.2, 2.9 g 100 g⁻¹, 75 mg kg⁻¹, 236 mg kg⁻¹ at ACRE in 2014, 6.2, 4.8 g 100 g⁻¹, 27 mg kg⁻¹, 129 mg kg⁻¹ at PPAC in 2014. Soil N was not measured at sowing in this study; however, it was measured at V14 and R1 stages at both ACRE (2013 and 2014) and PPAC (2013 only) in immediately adjacent maize hybrid studies planted on the same day in the same field where no N fertilizer was added (de Oliveira Silva et al., 2017). Soil NH4+ ranged from 3.3 to 4.8 mg kg⁻¹ and NO3- ranged from 1.8 to 3.5 mg kg⁻¹ to a 30-cm depth at these locations in spring before N application (de Oliveira Silva et al., 2017). In both years, maize was planted after soybean at ACRE, and after maize at PPAC. The 2013 ACRE location was chisel plowed in the fall and field cultivated in the spring. The 2014 ACRE field site was strip-tilled in both fall and spring with a Soil Warrior® (Environmental Tillage Systems Inc.) using coulter-based soil engaging tools. At PPAC, the tillage system was chisel plow in the fall and field cultivation in the spring for PPAC in both years.

Treatments were arranged in a split-split plot design in both years at both locations. Nitrogen rate was the main plot - 55 kg N ha⁻¹ (55N) or 220 kg N ha⁻¹ (220N). Plant density was the sub-plot – 54,000, 79,000, and 104,000 plants ha⁻¹. Hybrid was the sub-sub-plot, including 8 commercial DeKalb hybrids released from 1967 to 2005. They were: 2005, DKC61-69 (VT3); 2005, DKC61-72 (RR2); 2003, RX752 (VT3); 2003, RX752RR2 (RR2); 1994, RX730 (Conventional); 1982, DK636 (Conventional); 1975, XL72AA (Conventional), and 1967, XL45 (Conventional). VT3 hybrids are resistant to European corn borer, corn rootworm, and glyphosate. RR2 hybrids are resistant to glyphosate. Conventional hybrids have no transgenic pest-resistant and glyphosate resistant traits. These eight commercial hybrids were selected in consultation with Dekalb maize breeders because they were widely grown in the US Corn Belt following their release and had similar relative maturity days ranging from 111 to 115 days.

Six blocks were planted at ACRE and three blocks were planted at PPAC. All plots were 10 m long and 3.04 m wide with 4 rows and 0.76 m row spacing. Planting dates were 14 May, 2013 and 25 April, 2014 at ACRE and 1 Jun, 2013 and 5 May, 2014 at PPAC. Nitrogen was side-dressed as urea-ammonium nitrate (UAN, 28% N) applied 30 days after planting (DAP) in 2013 and 33 DAP in 2014 at ACRE and 38 DAP in 2013 and 24 DAP in 2014 at PPAC. All UAN was injected in mid-row positions with a DMI Nutri-Placer 2800. All grass and broadleaf weeds in the plot areas were controlled with a combination of pre-emerge residual herbicides as well as a single post-emerge application at approximately the V5 stage. All maize seeds were treated in a similar manner with Acceleron™ (Difenoconazole, Fludioxonil, Mefenoxam, and Thiamethoxam). Force 3G (Tefluthrin) was soil-applied at planting with all maize hybrids to control corn rootworm.

Weather data for ACRE were collected from Purdue University-Indiana State Climate Office at station “ACRE-West Lafayette” (http://www.iclimate.org/), and for PPAC were collected from station “Wanatah 2 WNW, IN US” (http://www.ncdc.noaa.gov/cdo-web). Weather recording began with the planting dates at each site-year and continued until biomass harvest at maturity on 24 September, 2013 and 15 September, 2014 at ACRE, and on 22 October, 2013 and 29 September, 2014 at PPAC.

At ACRE, R1 biomass harvest was taken at 7 days (2013) and 0 days (2014) after 50% silking (average of all hybrids). At PPAC, R1 biomass harvest was taken at 2 days (2013) and 4 days (2014) after 50% silking (average of all hybrids). More precise timing of R1 harvest on the actual mean dates of 50% silk emergence (following daily flowering measurements in each plot) could not be achieved because of weather and labor resource constraints. R6 biomass harvest was completed after all treatments reached black layer (representative ears of each hybrid from multiple replications were sampled to insure all treatments had reached black layer). For all biomass harvests, the sampling area was 3.04 m² for each plot. All plants in the sampling area were cut at soil level and weighed to determine the total fresh weight. Five representative plants were then chosen as subsamples from each plot. For the R1 harvest, subsamples were separated into leaf, stem (with husk) and earshoot in both years. For the R6 harvest, subsamples were separated into leaf, stem (with husk), grain and cob for three blocks in ACRE and PPAC in both years. The other three blocks in ACRE were separated into stover (stems, leaves, and husks) and ears (grain and cob) at ACRE in both years. Fresh weights were taken for all samples. All subsamples were dried at 60 °C at ACRE for 5–7 days until they reached a stable dry weight. All subsamples from the first three blocks were weighed, ground and sent to A&L Great Lakes Lab (Fort Wayne, Indiana) for determination of plant N composition using combustion analysis (AOAC International 990.03, 1995).

Post-silking dry matter accumulation was calculated by the following formulas:

Dry matter remobilization from vegetative organs (leaf or stem) was determined as the dry matter lost between vegetative and reproductive stages by using the following formulas:

Post-silking N uptake (PostN), remobilized N (RemN), leaf remobilized N (RemN_leaf), stem remobilized N (RemN_stem) and cob remobilized N (RemN_cob) were determined as the N content lost between vegetative and reproductive stages utilizing the same equations that were employed in Chen et al. (2015).

Whole-plant N concentration at maturity was calculated as one of the explanatory variables for nitrogen internal efficiency:

Nitrogen use efficiency metrics included partial factor productivity, nitrogen internal efficiency, nitrogen conversion efficiency, and nitrogen nutrition index; these were determined by following formulae:

The formula for critical N concentration at silking can be found in Gastal et al. (2015), and the NNI parameters for maize were defined in Plénet and Lemaire (1999).

Statistical analysis was conducted in SAS 9.4 (SAS Institute Inc., 2013). Nitrogen, density and hybrids were fixed effects. Year and block (nested in each year) were treated as random effects. Only the first three blocks (1–3) were used in statistical analysis due to lack of N test data for the last three blocks (4–6). Locations were analyzed separately. Three error terms for this split-split-plot design was main plot error: E(a) – Block(year) × Nitrogen, sub-plot error: E(b) – Pooled Block(year) × Density and Block(year) × Density × Nitrogen and sub-sub-plot error: E(c) – Pooled Block(year) × Hybrid, Block(year) × Hybrid × Nitrogen, Block(year) × Hybrid × Density, and Block(year) × Hybrid × Nitrogen × Density. If the random terms had P(F > F0) less than 0.05 for most of the variables, then the random term(s) was pooled with the corresponding error terms. Hence, Year × Nitrogen was pooled with E(a), Year × Nitrogen × Density and Year × Density was pooled with E(b), Year × Hybrid, Year × Nitrogen × Hybrid, Year × Density × Hybrid, and Year × Nitrogen × Density × Hybrid was pooled with E(c). The final model was used in SAS:

where, μ is the grand mean, α is N rate effect (i = 1, 2), β is density effect (j = 1, 2, 3), γ is hybrids effect (k = 1, 2, …, 8), αβ is N rate and density interaction, αγ is N rate and hybrid interaction, βγ is density and hybrid interaction, αβγ is N rate, density and hybrid three-way interaction, τ is year effect (l = 1, 2), δ is block effect that nested in each year (m = 1, 2, 3), αδ is N rate and block interaction effect, αβδ is N rate, density and block interaction effect, αβγδ is N rate, density, hybrid and block interaction, and ε is the error term.

Linear correlations were conducted by using “Proc Reg” in SAS. Log-transformation was used on the linear correlations between NIE and grain dry matter at maturity, NIE and total N content at maturity, NIE and whole-plant N concentration at maturity, NIE and HI, NIE and NHI, as well as between NIE and grain N concentration at maturity. The R² of fitted linear model was used as the percentage of NIE that was explained by each explanatory variable. Annual rates of increase were calculated using the slope of linear fit based on the mean values of each parameter of interest for each hybrid. Bilinear function was conducted in SPSS 23.0 (IBM Corp., Armonk, N.Y., USA). The equation used for bilinear function was Y = a₁ + b × X at X < x₀, and Y = a₂ at X ≥ x₀. Parameter estimation is based on the loss function, which is (y − ŷ)².

There was less accumulated precipitation at ACRE than PPAC in the pre-silking period as well as the post-silking period during both years (Figure 1). For 2013, the accumulated precipitation was 187 mm less at ACRE during the pre-silking period than PPAC, and 25 mm less during the post-silking period (Figures 1A,B). For 2014, PPAC had 200 mm higher accumulated precipitation than ACRE during the pre-silking period and 47 mm more during the post-silking period (Figures 1C,D). In general, 2014 had 164 mm higher accumulated precipitation than 2013 across locations (Figure 1). The cumulative pre-silking precipitation was similar in ACRE between 2013 and 2014 (only 9 mm higher in 2014), which was also the case for PPAC (only 22 mm higher in 2014). However, the larger cumulative precipitation difference between years for ACRE and PPAC was during the post-silking period, which was 137 mm higher in 2014 for ACRE (Figures 1A,C) and 159 mm higher in 2014 for PPAC (Figures 1B,D). In contrast, the temperature ranges were similar between the four environments, except for the occurrence of minimum temperatures below 0°C after planting and right before harvest at PPAC in 2013 (Figure 1).

Although the 1967 hybrid always had the lowest dry matter at silking, total dry matter at silking did not increase as era increased from 1975 to 2005 at either location (Tables 1, 2). An N rate and hybrid interaction was observed for total dry matter at silking (TDM_R1) at PPAC (Table 2). This interaction was due to a higher TDM_R1 response to increased N rate for 2005 hybrids, which increased 1.1 and 0.6 Mg ha⁻¹ for 2005VT3 and 2005RR2, respectively, compared to a relatively small TDM_R1 increase of −0.1 to 0.4 Mg ha⁻¹ for 1967 to 2003 hybrids (Table 2).

There were considerably more dry matter differences among hybrids at maturity than at silking. The total dry matter at maturity (TDM_R6) of 2005 hybrids were 6% higher than that of 2003 hybrids at ACRE (Table 1) but the same as 2003 hybrids at PPAC (Table 2), while the 2005 hybrids were 13 and 11% higher than the average of hybrids prior to 2000s at ACRE and PPAC, respectively. The yearly increasing rate for TDM_R6 was 92 kg ha⁻¹ year⁻¹ in ACRE and 67 kg ha⁻¹ year⁻¹ in PPAC. The higher TDM_R6 in 2000s hybrids was mostly due to higher grain dry matter at maturity (GDM) at both locations (Tables 1, 2). The N rate and hybrid interaction for GDM at PPAC (Table 2) was due to the greater gain in GDM of 2005VT3 at 220N, which increased 36% (2.9 Mg ha⁻¹) compared to an average gain of 28% (2.0 Mg ha⁻¹) for the remaining hybrids. There was no consistency of hybrid era effects on leaf or stem DM remobilization at either location (Tables S1, S2).

Hybrid and management impacts on PostDM gains were readily apparent. The two 2005 hybrids had 10% higher PostDM compared to the average of 2003 hybrids at ACRE, but were the same as 2003 hybrids at PPAC (Tables 1, 2); they were also 19 and 20% higher than the average of hybrids prior to 2000s at ACRE and PPAC, respectively (Tables 1, 2). There was no era effect on the ratio of PostDM to TDM_R6 at either location. The higher N rate increased both PostDM (by 1.8 and 2.7 Mg ha⁻¹) and the proportion of PostDM in TDM_R6 (by 4 and 8% at ACRE and PPAC, respectively (Tables 1, 2). In contrast, increased plant density reduced the proportion of PostDM in TDM_R6.

Both vegetative organs and grain had large variations in N concentrations among hybrids at maturity, but there was no consistent era impact. The 1967 hybrid always had the highest grain N concentration (GNC) and whole-plant N concentration (PNC_R6) at both locations (Tables 3, 4). The 2003 hybrids had 10–12% lower GNC and PNC_R6 compared with the 1967 hybrid, but the 2005 hybrids' GNC and PNC_R6 was within a range of just 5–8% lower.

Nitrogen rate and hybrid interactions were observed for leaf N concentration at maturity (LNC_R6), stem N concentration at maturity (SNC_R6), cob N concentration at maturity (CNC_R6) and GNC at PPAC (Table 4). In general, the 1994 hybrid had the largest response to N rate with gains of 51% in LNC_R6, 23% in CNC_R6, and 28% in GNC. The 1982 hybrid had the largest response to N rate in SNC_R6 with mean gains from 0.42 to 0.58 g 100 g⁻¹. In contrast, 2005VT3 hybrid experienced little change with the higher N rate in leaf, cob and grain N concentrations at maturity.

In general, increasing density decreased organ N concentrations at both locations (Tables 3, 4). At maturity, a hybrid and density interaction was observed for stem N at ACRE (Table 3), where the two 2005 hybrids had much reduced stem N from low to medium density (from 0.46 to 0.38 g 100 g⁻¹), while the 1967 hybrid had almost zero reduction (from 0.57 to 0.56 g 100 g⁻¹).

Hybrid era influences on total N uptake at maturity (TNU_R6) were consistent at both locations (Tables 5, 6). The annual rate of increase in TNU_R6 was 0.70 and 0.66 kg ha⁻¹ year⁻¹ at ACRE and PPAC, respectively. The era effect on TNU_R6 was primarily due to the increase in grain N content (GNU) in more recent hybrids since leaf N content (LNU_R6), stem N content (SNU_R6), and cob N content (CNU_R6) at maturity did not show any consistent changes over the decades (Tables 5, 6). The annual rate of increase for GNU was 0.66 and 0.42 kg ha⁻¹ year⁻¹ at ACRE and PPAC, respectively. The N rate by hybrid interaction of GNU and TNU_R6 at PPAC were due to 9 kg ha⁻¹ higher GNU and 6 kg ha⁻¹ higher TNU_R6 responses of 2000s hybrids to higher N rate than was observed in earlier hybrids.

Increasing GNU over the hybrid eras was accomplished by gains in both PostN and RemN. The RemN was 10 and 9 kg ha⁻¹ higher in 2000s hybrids than hybrids prior to 2000 at ACRE and PPAC, respectively. Additionally, the PostN was 11 and 4 kg ha⁻¹ higher in 2000s hybrids than hybrids prior to 2000 at ACRE and PPAC, respectively (Tables 5, 6).

Leaves contributed most to RemN at both locations (Tables 5, 6). Leaves accounted for 57 and 58% of RemN, whereas stems contributed 39 and 40% of RemN at ACRE and PPAC, respectively. Increased N fertilizer rate increased RemN_stem but not RemN_leaf at both locations, whereas increasing density enhanced RemN_leaf but had no impact on RemN_stem.

Partial factor productivity increased dramatically over time. The annual rate of increase in PFP was 1.0 and 0.8 kg kg⁻¹ year⁻¹ at 55N, and 0.4 and 0.3 kg kg⁻¹ year⁻¹ at 220N, at ACRE and PPAC, respectively (Table 7). The interaction of N rate by hybrids for PFP was due to the lower PFP reduction for the 1967 hybrid at ACRE (112 vs. 142 kg kg⁻¹ for average of rest hybrids) and the lower reduction for 1967 and 1975 hybrids at PPAC (Table 7). There was no consistent era benefit to NIE; in fact, the 2003 and 1994 hybrids always tended to have from 3 to 6 kg kg⁻¹ higher NIE compared with other hybrids at both locations and N rates. Hybrid differences in NCE demonstrated no era pattern and seemed to be mostly due to genetic variation, and NCE was directly affected by their whole-plant N concentration—which lead to a lower NCE for the 1967 hybrid at both N rates and both locations (Table 7).

Harvest index (HI) and nitrogen harvest index (NHI) did not increase consistently over time. Harvest index plateaued after 1990s at both N rates and both locations, except for the 220N rate at ACRE, where HI plateaued with the 2003 hybrids followed by a slight decline with the 2005RR2 hybrid (Table 7). Harvest index was 6 and 8% higher at 55N when hybrids prior to the 1990s were compared with hybrids after the 1990s in ACRE and PPAC, respectively, whereas HI was 10 and 11% higher for this comparison at 220N. In contrast, NHI levels were stable in hybrids after 2000 at both N rates and locations, except that the 1967 hybrid had a high NHI at 55N at PPAC (Table 7). The N rate by hybrid interaction of NHI at PPAC was due to a greater response to higher N rate in the 1975 and the 2005s hybrids (increasing rate ranged from 0.03 to 0.04 kg kg⁻¹) compared with rest of hybrids (increasing rate ranged from 0.00 to 0.02 kg kg⁻¹).

As for plant N status, even though higher N rates increased NNI by 0.22 at both locations, PPAC still had an NNI below 1.0 even at the high N rate whereas maize plants at ACRE were only categorized as deficient at low N but not at the high N rate (Table 7). At low N, the NNI was not different among hybrids at either location. As N supply increased, all hybrids reached NNI levels above 1.0 with 220N at ACRE (indicating a possible luxury N uptake occurred in all hybrids, and especially in the 2005 hybrids).

In general, the higher N rate decreased PFP, NIE, and NCE at both locations, and increased HI, NNI at both locations, but increased NHI only at PPAC (Table 7). There were no density and hybrid interactions for PFP, NIE, NCE, HI, NHI, and NNI at either location (Table 7). Density increased PFP at ACRE when density increased from low to medium density, but PFP decreased when density increased from medium to high density. Density had no impact on PFP at PPAC. Higher densities increased NIE (ACRE) and NCE (both locations), but decreased NHI (both locations).

The proportion of NIE variation that was explained by other measurement factors depended on the different explanatory variables examined. To examine the underlying causes of NIE variation, log-transformation was used for both predictor and explanatory variables to obtain linear regression.

Total N content at maturity explained 57 and 20% of NIE variation at ACRE and PPAC, respectively, at 55N whereas GDM explained 8 and 1% of NIE variation at ACRE and PPAC at 55N, respectively (Figures 2A,B). In contrast, at 220N, GDM explained a higher proportion of NIE variation compared to TNU_R6 at both locations. The GDM accounted for 20% of NIE variation at ACRE and 12% at PPAC, while TNU_R6 accounted for 9% of NIE variation at ACRE and 2% at PPAC (Figures 2A,B). Grain dry matter was also highly correlated with TNU_R6 (Figures 3A,B).

However, when NIE was dissected into PNC_R6 and HI, NIE variations were dominantly explained by PNC_R6 at both N rates and both locations (Figures 2C,D). At 55N, 86% of total variance of NIE was explained by PNC_R6 and only 0.3% was explained by HI at ACRE, and the same metrics were 54 vs. 1% at PPAC. At 220N, PNC_R6 explained 73 and 54% of total variance of NIE, and HI explained 34 and 14% of variance of NIE at ACRE and PPAC, respectively. The GNC explained even more of variation in NIE compared to NHI when NIE was dissected into GNC and NHI. Grain N concentration at maturity accounted for 87 and 65% of the total variance of NIE at 55N, and 75 and 48% of total NIE variance at 220N, respectively, at ACRE and PPAC (Figures 2E,F). In contrast, NHI accounted for only 2% of total variance of NIE at 55N, and for 3 or 9% of total NIE variance at 220N, respectively, at the same two locations (Figures 2E,F). Increased NHI occurred simultaneously with higher GNC, as evident from the positive correlations between these two variables, even though the coefficient of determination decreased from 0.34 to 0.11 at ACRE and from 0.49 to 0.27 at PPAC when N rate increased from 55 to 220N (Figures 3E,F).

Lemaire and Gastal (1997) introduced the use of NNI as a determination standard for plant N status. If NNI is larger than 1.0, the actual plant N concentration should be sufficient for achieving maximum biomass and vice versa (Lemaire and Gastal, 1997). In this study, the NNI responses at two locations suggested very different apparent plant N supplies, with silking-stage N deficiencies occurring at both N rates at PPAC but only at 55N at ACRE (Table 7). Increasing N supply had dramatically different impacts on these two locations. For example, mean GDM was increased by 29% at PPAC (2.1 Mg ha⁻¹) and by only 14% at ACRE (1.5 Mg ha⁻¹) at the higher N rate (Tables 1, 2). Mean PostN increased by 105% at PPAC (21 kg ha⁻¹) and 50% at ACRE (23 kg ha⁻¹) at the higher N rate (Tables 5, 6). Ciampitti and Vyn (2011) also reported large GDM and PostN gains with 165–330 kg N ha⁻¹ vs. 0 kg N ha⁻¹ for four 2,000-era hybrids (regardless of their transgenic insect resistance features) using the same 3 densities that we utilized. In our research, the lack of N rate by hybrid interaction in PostDM and PostN at both locations indicated that all era hybrids responded similarly to greater N supply (Tables 1, 2, 5, 6). However, we observed that the two most recent hybrids achieved higher PostN than all other hybrids at ACRE but not at PPAC (Tables 5, 6), which indicated that these recent hybrids recovered more N as availability increased.

Hybrid selection is usually conducted with N sufficient conditions to insure comparisons are near peak grain production levels (Bänziger et al., 1997; Lafitte et al., 1997). This could lead to limitations or large variation in the resulting hybrid performances when soil N conditions are poor (Lafitte et al., 1997; Worku et al., 2007). Several research groups have addressed the need of conducting selection under low N conditions. For example, Moll et al. (1982) showed that conducting hybrid selection under low N was more effective at identifying high NIE genotypes. Worku et al. (2007) reported on the importance of PostN and NIE selection factors in improving grain yield under poor tropical soil conditions.

In our research, beyond the impact of N rate on PostDM and PostN dynamics, the higher N rate decreased PFP by 115 kg kg⁻¹, NIE by 11 kg kg⁻¹ and NCE by 30 kg kg⁻¹ when averaged over the two locations (Table 7). Overall NIE was 69 kg kg⁻¹ at 55N across locations, and it decreased to 58 kg kg⁻¹ at 220N. This reduction was consistent across all hybrids as no N rate by hybrid interactions occurred at either location (Table 7). The reduction of NIE at higher N rates agreed with Anderson et al. (1985) and Ciampitti and Vyn (2011).

The N rate also affected the correlations between PNC_R6 and HI, and between GNC and NHI, which were both stronger at 55 than 220N (Figure 3). Ciampitti and Vyn (2013) documented poor correlations between GNC and NHI (R² = 0.14) and found that the slope for log (GNC) and log (NHI) did not differ between old era (1940–1990) and new era (1991–2011) hybrid groups regardless of N rates. In our study, the poor correlations at 220N were related to higher PostDM and higher TDM_R6, which negatively affected GNC and PNC_R6 and weakened the effects of NHI and HI. In this case, a high N rate input resulted in less dilution of both PNC_R6 and GNC even though TDM_R6 and GDM increased dramatically. This finding agreed with Echarte et al. (2013), who also found reduced protein concentrations in newer hybrids in a series of DeKalb hybrids (1965–1993) when comparisons were made under zero N fertilized treatments, but not with N fertilizer applications.

The results of this study demonstrate the tradeoffs of how adding fertilizer N leads to greater DM accumulation by GDM and TDM_R6 (as well as PostDM and PostN), but leads to reduced PFP, NIE, and NCE. However, these tradeoffs were smaller at the more N deficient location even when the same N fertilizer rate was added. Moreover, the results also indicated that variation of selected traits could be large under severe N deficiency conditions if hybrid selection for selected traits were only conducted in adequate soil N conditions.

There was almost no gain apparent in NIE with more recent hybrids in this study, and what NIE gain there was relative to the oldest 1967 hybrid became fairly stable by the 1994 hybrid (Table 7). The Ciampitti and Vyn (2012) review of historic experiments documented an increase in NIE (49.7–56.0 kg kg⁻¹) when older hybrids (1940–1990) were compared with newer hybrids (1991–2011) with the same average N rates in the context of an average increase in plant density from 5.6 to 7.0 plants m⁻¹. The gains in NIE observed with plant densities across hybrid eras in this study, as well as for post-2000 hybrids by Ciampitti and Vyn (2011) suggests that conclusions about mean hybrid era impacts on NIE aren't fully valid unless comparisons are made at the same densities. Until more research is conducted, it is also possible to speculate that the grain yield improvements achieved in the Dekalb hybrids for this maturity zone did not coincide with the accompanying NIE gains observed in the maize genetic improvements of other commercial or public breeding programs. Certainly, the morphological changes in relative kernel weight gains observed in these Dekalb hybrids over time was rather unique (Chen et al., 2017).

The reasons for the NIE plateau in more recent hybrids in our research were exploited by dissecting NIE into its components of PNC_R6 and HI, or GNC and NHI. Plant N concentration at maturity and GNC had a dominant impact on NIE across all treatments (Figure 2). Newer hybrids did not have the lowest PNC_R6 or GNC across all treatments (Tables 3, 4) despite the larger kernel weights achieved in these newer hybrids (Chen et al., 2017) and the observation by Ciampitti and Vyn (2012) of a mean 10% decrease in both PNC_R6 and GNC in experiments utilizing hybrids after 1990. In our direct comparison on hybrids side by side, there was no consistent era effect on the proportion of NIE variation explained by either PNC_R6 or GNC.

We observed a strong positive correlation between GNC and PostN, and all hybrids responded similarly (the largest difference among slopes was 0.0015; p-value = 0.08) (Figure 4). In contrast, RemN affected GNC in a bilinear way for all hybrids (Figure 5). The lowest plateau for GNC was 1.02 g 100 g⁻¹ for the 1994 hybrid and the highest plateau for GNC was 1.30 g 100 g⁻¹ for the 1967 hybrid. Other authors have also reported correlations between GNC and PostN or RemN. Coque and Gallais (2007) showed that GNC had higher correlation with PostN than RemN when inbred lines were tested with a common 150 kg N ha⁻¹ and 90,000 plants ha⁻¹. Anderson et al. (1985) reported that higher N remobilization during grain filling period benefited NIE in prolific hybrids when then-current hybrids were compared. However, in this study, both PostN and RemN had positive effects on GNC which directly resulted in a lower NIE. Moreover, the correlation coefficient with NIE was much higher for PostN (−0.58, p < 0.001) compared to RemN (−0.33, p < 0.001).

Apparently, in the DeKalb hybrids used here, enhanced NIE occurred inconsistently and only via lower GNC or PNC_R6, which becomes more and more difficult to achieve as there will be a minimum GNC or PNC_R6 to maintain seed nutrient levels or grain yields (Ladha et al., 2005; Ciampitti and Vyn, 2013; Gastal et al., 2015). Furthermore, lower GNC or PNC_R6 may be undesirable for another N efficiency parameter because lower N fertilizer recovery efficiencies (NRE) occur when plant N concentrations at maturity are lowered. Lower NRE following N fertilizer applications will enhance N losses to the atmosphere (Omonode et al., 2017). However, we could not measure NRE in this study because there was no zero N control treatment.

Only very small proportions of NIE variation were explained by either NHI or HI in this study (Figure 2), which appeared to be related to lack of improvement of NHI and HI per se across the 38-year period in this hybrid series. The NHI difference between hybrids prior to 2000 and after 2000 only averaged 4% across treatments and locations, and the HI difference between hybrids prior to and after 1994 was only 4% (Table 7). Ciampitti and Vyn (2013) also indicated the lack of incremental gains in NHI and HI as era increased.

In this research, the lack of improvement of HI and NHI was related to lack of apparent era benefits to both the proportion of PostDM to TDM_R6 and in the proportion of PostN to TNU_R6, even though both PostDM and PostN per se were increasing in more recent hybrids (Tables 1, 2, 5, 6). Gastal et al. (2015) indicated the difficulty of increasing NIE by dilution of grain N concentration because of the lack of variation in N dilution curves between genotypes; they considered that achieving more genotype variation in NHI and HI was a more likely pathway to enhanced NIE. Hence, although higher PostN and PostDM were found in this study, the lack of improvement in the proportions of PostDM to TDM_R6, and PostN to TNU_R6, in more recent hybrids accounts for the lack of hybrid era differences in NHI and HI.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.