Seed yield increases of soybeans in response to increases in CO2 concentration of 180 to 200 μmol×mol−1 above the current ambient concentration applied using free air carbon dioxide enrichment (FACE) systems have ranged from about 0% to 45% in different cultivars  -  . Reasons for the wide range of responses among cultivars at the same location remain unclear. Delay in the transition from vegetative to reproductive growth in response to elevated CO2 varied among cultivars and was highly correlated with the seed yield increase both in indoor chambers and in field FACE systems  . Delayed transition to reproductive growth increased main stem and axillary node number, providing more sites for pods, and increasing seed yield. However, the cultivars compared in that study varied in maturity group, and it is not known whether variation exists within a soybean maturity group in effects of elevated CO2 on the duration of vegetative growth, or whether any such variation would be correlated with yield increase. Soybean cultivars used in North America have been assigned to “maturity groups” in order to specify the latitudinal band best suited to that cultivar. Reproductive development in soybean is affected by both photoperiod and temperature.
2. Materials and Methods
Experiments were conducted in 2013, 2014, and 2015 at the South Farm of the Beltsville Agricultural Research Center, Beltsville, Maryland, using maturity group IV cultivars adapted to the local conditions. Among the five cultivars used, Clark, Corsica, Kent, Spencer and Stress land, two or three cultivars were grown in each year, with Spencer grown every year (Table 1). The field site (39˚02'N, 76˚94'W, elevation 30 m) is on a flood plain with a Codorus silt loam soil, a fine-loamy, mixed, mesic Fluvaquentic Dystrochrept.
There were three plots with elevated CO2 and three plots with no CO2 added each year. Each plot covered 12 m2 and was equally divided among the two or three cultivars. The row width was 30 cm, and seedlings were thinned to an overall density of 40 plants per m2. Plots were tilled just prior to planting, and CO2 addition began before seed emergence. The plots had been fertilized with N, P and K for the prior winter wheat crop at locally recommended rates, but no fertilizer was applied to the soybean crops. The plots were not irrigated, but no severe water stress occurred in any of these years, because precipitation was near normal.
Elevated CO2 was applied continuously from planting using area distributed FACE systems  . The control system was set for a daytime CO2 elevation of 190 μmol×mol−1 above the ambient concentration, and 220 μmol×mol−1 above ambient at night. Whole season mean concentrations were 455 μmol×mol−1 for the ambient plots and 663 μmol×mol−1 for the elevated plots, averaged over the three seasons. Midday ambient CO2 concentration averaged 384 μmol×mol−1. One minute averages of CO2 concentration in the ele-
Table 1. Planting times and cultivars grown in each year of the experiment.
vated plots were within 10% of the target concentration 86% of the time during the daytime, and 73% of the time at night. Ambient CO2 concentrations at night were higher at low wind speeds, and ranged from about 400 to over 600 μmol×mol−1.
Within each plot 3 to 5 representative individual plants of each cultivar were tagged before flowering and the day of year when they reached the R1 stage of flowering (first open flower anywhere on the plant,  ), the day of year when the first open flower occurred at the apex of the main stem, and the total number of main stem nodes at maturity were recorded for each tagged plant. Mean values for each plot were used to compare the ambient and elevated CO2 plots for each cultivar, using ANOVA, with n = 3 replicate plots per treatment. Interactions between CO2 treatment and cultivar were tested using split-plot ANOVA.
At crop maturity in 2013 and 2014, 4 m of an interior row was harvested for each cultivar and plot for determination of seed yield. In 2015, weed control was not adequate to prevent reductions in crop yield by weeds, so yields were not obtained for that season.
3. Results and Discussion
The effect of the elevated CO2 treatment was to delay R1, delay flowering at the apex, and to increase the number of main stem nodes, or to have no effect on these parameters, depending on the cultivar. Thus elevated CO2 either prolonged vegetative growth or had no effect on its duration. Elevated CO2 never accelerated either vegetative or reproductive development in these field experiments, as also found by Castro et al.  . No increase in the rate of main stem node production at elevated CO2 was also found in indoor chambers with a wide range of day lengths  . In each year of this field study there were CO2 effects on the date of flowering at the apex, and on the number of main stem nodes at maturity for at least one cultivar, and no effects in other cultivar (s). This led to significant cultivar × CO2 interactions for both of these parameters in each year (Table 2). CO2 effects on the date of R1 were less consistent, and did not always occur even in cases in which the date of flowering at the apex and main stem node number
Table 2. Day of year (DOY) for reaching the R1 stage of development and for the first open flower at the apex of the main stem, the number of main stem nodes at maturity, and the probability of a CO2 × cultivar interaction for each year of the experiment.
*indicates a significant effect of CO2 treatment for that cultivar in that year at P = 0.05.
were affected (Table 2). Prior work also indicated no fixed relationship between CO2 effects on the date of R1 and on the duration of vegetative growth among soybean cultivars of different maturity groups  . Delays in the date of flowering at the apex and increases in the number of main stem nodes caused by elevated CO2 occurred for the cultivar Spencer in each of the three years. In these studies all cultivars reached maturity at least a week before the first frost occurred in the fall, even at elevated CO2.
There are at least four genes affecting the photoperiodic control of flowering in soybean  . Studies using near isogenic lines for three of these genes indicated that each of those three genes influenced how elevated CO2 affected flowering time at some photoperiod  . There is considerable variation in the timing of flowering stages within maturity groups under natural photoperiods, presumably related to differences in photoperiod sensitive genes, so it is not surprising that variation exists within a maturity group in CO2 effects on the duration of vegetative growth.
The ratio of seed yield at elevated to that at ambient CO2 for each cultivar increased approximately linearly with the increase in the main stem node number caused by growth at elevated CO2 (Figure 1). Also shown in Figure 1 are data for two cultivars not of maturity group IV previously described  . This correlation between the yield ratio and the increase in the number of main stem nodes is probably consistent with the observation of Bishop et al.  that the two soybean cultivars with the largest relative yield increase at elevated CO2 (Loda and Dwight) also had the largest delay in reaching maturity at elevated CO2, although main stem node numbers were not presented. Increases in main stem node number in response to elevated CO2 have also been found in other soybean studies at Soy FACE   . Rising temperatures may prevent delayed maturity induced by elevated CO2 in some cultivars from resulting in yield losses due to low temperatures occurring before crop maturation. For example, no yield losses due to delayed maturity at elevated CO2 occurred in this three-year study. CO2 effects on the
Figure 1. The ratio of yield at elevated to ambient CO2 in relation to the additional number of main stem nodes at maturity at elevated CO2. Open symbols are for maturity group IV soybean cultivars, years 2013 and 2014. Closed symbols are for two other cultivars reported in bunce (2015)  of different maturity groups. The overall regression had r2 = 0.948.
duration of vegetative growth may be important in adapting soybean to higher atmospheric CO2, and this study indicates that variation in this CO2 response exists within a maturity group.
Variable effects of elevated CO2 on flowering occurred within a single maturity group, as evidenced by the fact that for each year of this study, there were cultivars in which elevated CO2 did not affect the duration of vegetative growth or the main stem node number, and other cultivars in which elevated CO2 prolonged vegetative growth and increased the number of main stem nodes and seed yield at maturity. The stimulation in yield by elevated CO2 was highly correlated with the increase in the number of main stem nodes, indicating that CO2 effects on the duration of vegetative growth may be important in adapting soybean to higher atmospheric CO2.
 Bishop, K.A., Betzelberger, A.M., Long, S.P. and Ainsworth, E.A. (2015) Is There Potential to Adapt Soybean (Glycine Max Merr.) to Future [CO2]? An Analysis of the Yield Response of 18 Genotypes in Free-Air CO2 Enrichment. Plant, Cell & Environment, 38, 1765-1774.
 Bunce, J.A. (2014) Limitations to Soybean Photosynthesis at Elevated Carbon Dioxide in Free-Air Enrichment and Open Top Chamber Systems. Plant Science, 226, 131-135.
 Bunce, J.A. (2015) Elevated Carbon Dioxide Effects on Reproductive Phenology and Seed Yield among Soybean Cultivars. Crop Science, 55, 339-343.
 Bunce, J.A. (2016) Responses of Soybeans and Wheat to Elevated CO2 in Free-Air and Open Top Chamber Systems. Field Crops Research, 186, 78-85.
 Hao, X.Y., Han, X., Lam, S.K., Wheeler, T., Ju, H., Wang, H.R., Li, Y.C. and Lin, E.D. (2012) Effects of Fully Open-Air [CO2] Elevation on Leaf Ultrastructure, Photosynthesis, and Yield of Two Soybean Cultivars. Photosynthetica, 50, 362-370.
 Fehr, W.R., Caviness, C.E., Burmood, D.T. and Pennington, J.S. (1971) Stage of Development Descriptions for Soybeans, Glycine max (L.) Merr. Crop Science, 11, 929-931.
 Castro, J.C., Dohleman, F.G., Bernacchi, C.J. and Long, S.P. (2009) Elevated CO2 Significantly Delays Reproductive Development of Soybean under Free-Air Concentration Enrichment (FACE). Journal of Experimental Botany, 60, 2945-2951.
 Bunce, J.A. and Cruz Hilacondo, W. (2016) Responses of Flowering Time to Elevated Carbon Dioxide among Soybean Photoperiod Isolines. American Journal of Plant Sciences, 7, 773-779.
 Cober, E.R. and Morrison, M.J. (2010) Regulation of Seed Yield and Agronomic Characters by Photoperiod Sensitivity and Growth Habit Genes in Soybean. Theoretical and Applied Genetics, 120, 1005-1012.
 Morgan, P.B., Bollero, G.A., Nelson, R.L., Dohleman, F.G. and Long, S.P. (2005) Smaller than Predicted Increase in Aboveground Net Primary Production and Yield of Field-Grown Soybean under Fully Open-Air [CO2] Elevation. Global Change Biology, 11, 1856-1865.