Toward understanding the ecology of fine roots, we measured the standing number of fine roots under natural levels of soil moisture and also fine root production in response to experimental watering treatments. We were interested both in the quantity of water needed to induce fine roots and whether induction is controlled at the level of the whole plant, the entire lateral root or the specific site of a fine root cluster.
Standing fine root crop during the growing season
We collected lateral root segments throughout the growing season and compared the number of live fine roots to soil moisture content. We removed ~5 cm long segments of
lateral roots from eight arbitrarily chosen individuals on 13 different days at ~7 day intervals beginning the last week of May until the last week of August, both in 2007 and 2008. Roots were obtained at approximately 25 cm depth and 20-30 cm away from the caudex and preserved immediately in 50% ethanol. We never re-sampled individuals. We quantified the standing fine root crop as the total length of white, apparently live and functional, fine roots (Fig. II.1) on each lateral root segment, normalized by the actual length of the sampled lateral root (mm fine root . mm-1 lateral root). Each fine root was measured to the nearest 0.1 mm with a caliper under a dissecting microscope (Olympus MV Plapo 2XC). We also collected soil
surrounding the sampled lateral root segment to calculate the gravimetric soil water content (SWC hereafter; Pearcy et al., 1989). We counted the total number of leaf rosettes, which correlates strongly with aboveground biomass (Salguero-Gómez & Casper, 2011; chapter III), and classified individuals as juveniles (non-flowering, < 25 rosettes individuals without evidence of having shrunk from a larger size) or adults (≥ 25 rosettes) to test whether ontogenetic stage and SWC affected standing fine root crop using a 2-way ANOVA.
We tested whether precipitation affects fine root production by carrying out three analyses (ANCOVA) for each year with plant size as covariate, standing fine root crop as the response variable, and either (i) SWC, (ii) monthly precipitation registered the
calendar month of root collection, or (iii) monthly precipitation registered the calendar month immediately preceding root collection date as the explanatory variable. Data met normality assumptions. We then fitted linear and polynomial regressions to describe changes in standing fine root and SWC over the 2007 and 2008 growing seasons separately and used adjusted R2’s to determine the best model.
Moisture threshold for producing fine root growth
We examined fine root production in response to simulated pulses of precipitation in August, both in 2007 and 2008. We chose three locations, 100 m apart, and supplied no water (0 cm), or a one-time pulse of precipitation of 2, 4.5 or 7 cm evenly distributed over the 1- m radius of a circle center on each individual (homogeneous watering treatment, hereafter). Long-term data show these amounts occur naturally with probabilities of 0.7-0.015 in August (Fig. II.2). To ensure accurate deliveries of water pulses, we place small rain gauges within the watered area. Each arbitrarily selected plant (nTotal = 72) was watered once between 17:00 h and
18:00 h. All watering took place over three days during the second week of August, when natural monsoonal precipitation might occur. No natural precipitation occurred during or two weeks prior to water in 2008, but a light rain (< 0.3 cm) occurred during the first week of August 2007. Prior to watering, for each individual, we counted the number of rosettes and collected ~100 g of soil 1.5 m south at 25 cm depth between 16:00 h and 18:00 h in order to obtain pre-treatment gravimetric soil water content (SWCPre-watering).
Between 16:00 h and 18:00 h on the seventh day after watering an individual, we collected one 5-cm lateral root segment from its east side and one from its west side at a depth of ~ 25 cm and 20-30 cm from the caudex. We counted white fine roots and measured the standing fine root crop as previously described. At the time of root collection, we also collected ~100 g of soil neighboring the lateral root in order to measure post-treatment soil water content (SWCPost- watering). We calculated the net change in soil water content caused by our watering treatment as
We used linear regressions to examine the effect of pulse intensity on standing fine root crop and on ∆SWC. We tested significant differences among pulse intensities in standing fine root crop and in ∆SWC using the Tukey-Kramer HSD tests (Sokal & Rohlf, 1995). Since date of sample collection, plant size (regression analyses), and whether the root segment came from the east or west side of the plant (paired t-test) did not significantly affect either fine root crop or ∆SWC, we excluded them from posterior analyses.
Degree of independence of fine root growth
Because we were interested in a plant’s response to soil water heterogeneity, given the species fragmented architecture (Salguero-Gómez & Casper, 2011; chapter III), we supplied pulses of precipitation to different portions of the root system to determine the spatial scale at which water induces fine root production.
First, we tested whether fine roots are produced in response to water applied to a cluster of fine roots. We arbitrarily chose 16 individuals in late May 2007, carefully excavated one live lateral root, and recorded the number and measured the cumulative length of live fine roots within a single cluster using a caliper. For eight individuals, we carefully placed beneath the lateral root a 30 mL plastic cup filled with water and used a sponge to wick water to that single cluster of fine roots. One end of the sponge was placed in the water and the other wrapped around the lateral root and fine root cluster and covered with plastic wrap to prevent conduction of water to neighboring portions of the root. In the remaining eight individuals, we tagged a targeted cluster of fine roots with tape on the lateral root and measured the number and cumulative length of white fine roots. In both cases, watered and unwatered, we replaced the soil and re-measured the
number and cumulative length of white fine roots within the targeted fine root clusters seven days later. No natural precipitation occurred during this experiment. We used repeated measures ANOVA (Sokal & Rohlf, 1995), with watering treatment and time of measurement as
explanatory variables, to examine cumulative live fine root length at day 0 and 7. Because six of the 16 targeted fine root clusters had no live fine roots, data were not normally distributed. We excluded these from the analyses, but we report their responses here.
Failing to induce fine roots at the level of the fine root cluster, we then tested whether fine roots are induced along lateral roots when the soil around them is watered. We arbitrarily selected individuals in each location used for the homogeneous watering treatment and watered a 60o sector of a 1-m radius circle centered on each plant (heterogeneous watering treatment, hereafter). We simulated precipitation pulses of 2 cm, 4.5 cm and 7 cm on 15, 15 and 10 individuals per location and year, respectively (nTotal = 240 plants). Watering took place at the
same time as the homogeneous watering treatment, and seven days later we collected two lateral root segments per individual, one from the watered sector and the other 180 o away in the unwatered sector. We counted the number of live fine roots on each sample and measured their standing fine root crops. We collected soil 1.5 m south from each individual at the onset of the experiment and at the time of root collection immediately adjacent to each sampled lateral root in order to calculate ∆SWC. For each year, we carried out 2-way ANOVAs with pulse intensity and sector (watered or unwatered, paired by individual as a random variable) and as explanatory variables and standing fine root crop or ∆SWC as response variables. Location, plant size and date of water application were not significant effects.
To determine whether fine root growth on lateral roots in watered sectors is the same as when the whole root system is watered, we compared standing fine root crop and ∆SWC
between the heterogeneous and homogeneous watering treatments each year. First, we made comparisons between the 0 cm pulse of the homogeneous watering treatment and the unwatered sector of the heterogeneous treatment using t-tests. Next, we made comparisons between the homogeneous watering treatment and the watered sectors of the heterogeneous watering treatment using 2-way ANOVAs with treatment and pulse intensity (2, 4.5 and 7 cm) as explanatory variables.
Finally, we determined whether the standing fine root crop induced by each of our watering treatments at the end of the growing season differed from the standing fine root crop under a comparable level of SWC during the growing season. To do so, we tested whether the slope of the relationship between SWC and standing fine root crop differed for natural levels of precipitation and our experimentally manipulated pulses. We used a two-way ANOVA with standing fine root crop as the response variable and SWC and pulse intensity (natural levels or pulses intensities of 2, 4.5, or 7 cm) as explanatory variables. Because in both years, the SWC x pulse intensity interaction was not
significant, we carried out ANCOVAs with SWC as main effect and pulse intensity as the covariate to compare the relationship between SWC and fine root crop among pulse intensities. We used JMP 8.0 (SAS Institute) for all statistical analyses.