In the majority of studies on S/NS recognition, an experimental design is used as depicted in Fig. 1: either a split-root plant had its own pot (this treatment is called ‘1 pot per plant no root competition’ and is also referred to as ‘Owners’ in the original studies), or its roots were spread over two pots together with another plant (this treatment will be called ‘2 pots per plant with root competition’ and is referred to as ‘Sharers’ treatment in the original studies). The rationale behind these treatments is that plants in both treatments have the same amount of nutrients available and the only factor that differs between them is the fact that sharing plants share the rooting space with another plant and plants that have the pot to themselves do not. However, a ‘1 ppp no rc’ plant and a ‘2 ppp with rc’ plant also differ in available rooting volume per plant, and here we explore its consequences following our Hypotheses I–III.
Our analysis starts with the results of Maina et al. (2002) with Phaseolus vulgaris L., who used two other treatments in addition to the ‘1 ppp no rc’ and ‘2 ppp with rc’ treatments, as illustrated in Fig. 1. The available rooting volume and the available nutrients per plant in each of these four treatments are as follows:
‘1 pot per plant no root competition’ (1 ppp no rc; ‘Owners’): each plant has one unit of rooting volume and one quantity of nutrients (1v, 1n);
‘2 pots per plant with root competition’ (2 ppp with rc; ‘Sharers’): each plant has access to two units of rooting volume and one quantity of nutrients (2v, 1n);
‘2 pots per plant no root competition’ (2 ppp no rc): the split root system of one plant is divided over two pots – each plant has two units of rooting volume and two quantities of nutrients (2v, 2n); and
‘1 pot per plant with root competition’ (1 ppp with rc): each plant has one unit of rooting volume and half a quantity of nutrients (1v, 1/2n).
The results of Maina et al. (2002) are consistent with our hypotheses I–III (Table 2). Plants that had access to two units of soil volume (two pots) produced a root mass per plant that was more than twice as large as the plants that had access to one unit of soil volume. Root mass can thus largely be explained by the available rooting volume per plant (Hypothesis I): the more soil volume is available, the more root mass a plant produced. This effect of volume on root biomass is independent of nutrient availability, as ‘2 ppp with rc’ and ‘1 ppp no rc’ plants have a different rooting volume but an equal amount of nutrients available. Shoot mass and total biomass were related to the available nutrients, and not to rooting volume or root mass (Table 2); the more nutrients available the more shoot and total mass was produced, in accordance with Hypothesis II: a neighbouring plant does not influence available soil volume, but does affect the availability of nutrients. Obviously, the results of Maina et al. (2002) are also in accordance with Hypothesis III, as ‘2 ppp with rc’ plants produce more roots than ‘1 ppp no rc’ plants without gaining more nutrients and allocate less biomass to reproduction.
Table 2. The effect of planting treatment on root, shoot and pod masses of 60-day-old Phaseolus vulgaris plants (from Maina et al. 2002). The weights are given on a per plant basis. The capital letters correspond to planting set-ups: A = ‘1 pot per plant no root competition’, B = ‘2 pots per plant with root competition’, C = ‘2 pots per plant no root competition’ and D = ‘1 pot per plant with root competition’ (see Fig. 1). Different superscripts indicate significant differences between treatments (P < 0.05). The analysis shows that root mass corresponds with the volume available per plant (v) following Hypothesis I, and that total mass (as well as shoot mass) corresponds with available nutrients per plant (n), following Hypothesis II. Hypothesis III states that increased allocation to root biomass because of more available rooting space, but equal biomass production because of nutrient limitation, will inevitably result in decreased allocation to reproductive biomass
|Hypothesis tested|| ||Corresponding volume (v) or nutrients (n) per plant||Biomass|
|H I||Root mass (g):||B (2v) = C (2v) > A (1v) = D (1v)||B (3.93)a= C (3.64)a > A (1.44)b ~ D (1.28)c|
|H II||Shoot mass (g):||C (2n) > B (1n) = A (1n) > D (1/2n)||C (4.93)a > B (2.08)b = A (1.99)b > D (0.76)c|
|H III||Pod mass (g):||A (1n & 1v) > B (1n & 2v)||C (7.03)a > A (4.82)b > B (2.29)c = D (1.80)c|
|H II||Total mass (g):||C (2n) > B (1n) = A (1n) > D (1/2n)||C (15.60) > B (8.30) = A (8.25) > D (3.84)|
The results of other studies using the same design as in Maina et al. (2002) also agree with our hypotheses I–III (Fig. 2). Doubling the rooting volume from ‘1 ppp no rc’ to ‘2 ppp with rc’ (i.e. from Owner to Sharer) consistently increased the root biomass 1.4–3-fold in seven out of eight cases. Only in the high-nutrient treatment of O’Brien et al. (2005) did doubling the rooting volume resulted in a marginal (but significant) increase in root mass. By contrast, and consistent with Hypothesis II, total biomass was similar in ‘1 ppp no rc’ and ‘2 ppp with rc’ plants in five out of eight cases. In the three exceptions, plants were larger in the ‘2 ppp with rc’ treatment than in the ‘1 ppp no rc’ treatment, and moreover in two of these cases the plants were harvested before seed-set (Falik et al. 2003; H.J. Schenk, unpublished data). Finally, in all six cases in which the plants flowered, ‘2 ppp with rc’ plants consistently produced a lower seed mass than ‘1 ppp no rc’ plants (cf. Hypothesis III).
Figure 2. The effect of planting arrangement (‘1 pot per plant no root competition’ and ‘2 pots per plant with root competition’, i.e. Treatments A and B, respectively, in Fig. 1) and nutrient level on root, seed and total masses of plants in five different studies. Maina et al. (2002) grew Phaseolus vulgaris plants in either 0.1 or 0.5 Hoagland solution (a, b). Gersani et al. (2001) used two different cultivation treatments with Glycine max, either split-root plants in pots (c), or non-split root plants in boxes (d). To create two root systems for the split-root plants Gersani et al. (2001) cut the radicle of the seedlings; this was not done in the box treatments. O’Brien et al. (2005) grew Pisum sativum plants in either 0.1 or 0.6 Hoagland solution (e, f). Falik et al. (2003) grew seedlings of Pisum sativum in 0.1 Hoagland solution (g). Shoot biomass values were recalculated from the root : shoot ratios given in Falik et al. (2003). Schenk (2006, and unpublished data) grew Glycine max plants as in Maina et al. (2002) (h). In studies (g) and (h) the plants were harvested before seed set. Means and SE values were read from the graphs in the papers and re-plotted. Stacked bars indicate, from bottom to top, the mass of roots (dark shading), shoots (speckled) and reproductive structures (light shading). Statistical results were obtained from the original papers; a difference in letter indicates a significant difference in root, shoot or reproductive biomass between the treatments.
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Finally, we will discuss the study by O’Brien et al. (2005) in which not only was the effect of the presence of roots of another plant studied, but volume and nutrient availability per plant were also manipulated in an attempt to disentangle the effects of presence of non-self roots, the effects of rooting volume and the effects of nutrients on the root biomass of a plant. In a two-pot experiment, the roots of single Pisum sativum L. plants were distributed over two pots, and the volume or the nutrient availability of one of the two pots was manipulated. In the treatment with a half-sized pot, half the amount of root mass was produced in the half-sized pot compared with the pot of standard size (treatment M; Table 3a), following Hypothesis I. If the nutrient concentration was halved in one pot while the volume remained unaltered (treatment L), the plants selectively placed their roots in the control pot containing more nutrients (cf. Hodge 2004).
Figure 3. Volume and nutrient availability effects on root mass in Pisum sativum (from O’Brien et al. 2005). (a) Two-pot experiment with single split-root plants (cf. Treatment C in Fig. 1), where in one of the pots either the nutrient concentration was halved (treatment L) or the pot volume was halved (treatment M), in addition to the control treatment K in which the split roots had access to two similar pots. Statistically significant differences (manova as tested by O’Brien et al. 2005) are indicated by differences in superscript letters. (b) Three-pot experiment in which two split-root plants shared a single pot and each plant also had access to a control pot that they did not share with other plants. In treatment N, the roots in the common pot grew intermingled; in treatment O a divider was present in the shared pot so that the roots did not interact and the rooting volume was effectively halved. The root mass of a plant in treatments N and O in the pot with root competition (rc) was calculated by including half of the total root biomass in the pot that was shared by two plants, as given in O’Brien et al. (2005). Statistics on these plant values are not available. For both sets of treatments, the total nutrient availability per pot (n) is based on the assumption that when two plants have access to a pot, half of the nutrients were available to each plant. Means and SEs were read from the graphs in the original paper. Rc is an abbreviation for root competition
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O’Brien et al. (2005) further conducted a three-pot experiment in which two plants share one pot and each plant has access to an exclusive pot. In one treatment a barrier was placed in the shared pot, effectively creating a ‘1.5 ppp no rc’ treatment with access to 1.5 pot volume per plant: treatment O. Again, the rooting patterns followed Hypothesis I: the plants produced similar root biomass in the exclusive and the shared pots of similar volume (treatment N; Table 3b), while the plants produced only half of the root mass in the half-sized pot created by the barrier (treatment O).
Combining these two experiments of O’Brien et al. (2005), we can make an interesting comparison between the two-pot treatment in which one of the pots had half of the nutrient concentration of the other (treatment L; ‘2 ppp no rc’) and the three-pot treatment in which a plant was subjected to a competitor plant in the shared pot (treatment N; ‘2 ppp with rc’). If we assume that the competitor plant consumes half of the soil nutrients, we have exactly the same nutrient and volume availability for the roots of the plants in both of these treatments (Table 3) except for the cause of the lower nutrient availability in the second pot, i.e. a lower nutrient concentration in treatment L, and a competitor plant in treatment N. Interestingly, the root responses in these two treatments were quite different. In the two-pot situation of treatment L, the plants showed selective root placement, resulting in a root distribution over the pots that mimicked the nutrient distribution. By contrast, in treatment N, the root mass was almost the same in both pots, suggesting root overproduction in the presence of the roots of another plant.
Here we discuss studies that investigate S/NS recognition in clonal plants in which independent physiological individuals are created through severing clonal connections, and studies in which ramets or seedlings are split into two halves. The experimental set-ups can be compared with the ‘2 ppp with rc’ and ‘1 ppp no rc’ design (i.e. Sharers and Owners design), which was discussed in the previous section. In treatment E (Fig. 3), two connected ramets (or one single ramet) were used, similar to the ‘1 ppp no rc’ treatment. In the ‘1 ppp with rc’ treatment F (Fig. 3), the connection between the two ramets is severed (or a ramet is split into two halves), resulting in two physiologically separate plants each exploring the soil volume. The two separate plants in treatment F, initially half the size of the original plant, share a single pot. In some studies, there were two different treatments with severed plants, all referred to as ‘1 ppp with rc’: in treatment F, the two plant ‘halves’ originated from the same plant (Fig. 3), while in treatment H (Fig. 3), the two halves originated from different plants, either of the same or a different genotype. Gruntman & Novoplansky (2004)) and Falik et al. (2006) combined these treatments with the split-root design (G in Fig. 3) in which two two-ramet plants were growing in two pots, comparable with the ‘2 ppp with rc’ treatment B in Fig. 1B.
Figure 3. Experimental set-up used in self/non-self studies with severed clonal plants or severed ramets. In treatment E (‘1 pot per plant no root competition’), two interconnected ramets of a clonal plant, or a single split-root seedling, grow together in a single pot. In treatment F (‘2 pots per plant with root competition’, also called ‘Severed Self’ or ‘twins’ in the original papers), the clonal connection between both ramets is severed or a seedling is split into two halves. Severing in F thus results in two physiological separate plants, initially half the size of the original plant, growing together in a single pot. Treatments G and H combine the severing design with the split-root design of Fig. 1, resulting in the ‘2 pots per plant with root competition’ similar to treatment B. The two original plants in G and H can be of the same clone (genotype) or a different clone (the latter situation usually referred to as ‘Alien’). For each of the treatments, the available rooting volume (v) and nutrient quantities (n) are given per plant (i.e. per physiological separate individual).
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When analysing the results of these experiments in the same way as those of the non-severed split-root experiments from the previous section, we should consider the number of physiological individuals and the rooting volume and nutrients available to them. Analogous to Fig. 1, we can characterize the four treatments in Fig. 3 as:
‘1 ppp no rc’ (Intact = Owner): each plant has access to one unit of rooting volume and one quantity of nutrients (1v, 1n);
‘1 ppp with rc’ (Severed Self): each plant has one unit of rooting volume and half a quantity of nutrients (1v, 1/2n); the two plants originate from the same plant;
‘2 ppp with rc’ (Intact Non-self = Sharer): each plant has two units of rooting volume and one quantity of nutrients (2v, 1n);
‘1 ppp with rc’ (Severed Non-self): each plant has one unit of rooting volume and half a quantity of nutrients (1v, 1/2n). The two plants in each pot originate from different plants.
One important difference with the split-root studies of Fig. 1 is that the starting weights of the physiological individuals to which rooting volume and available nutrients were assigned differed two-fold between the severed and intact treatments. However, if the plants grow considerably beyond their initial weight, and if nutrients were limiting final biomass production, these differences in initial weight should play a minor role in comparisons of final biomasses and allocation.
We can now test the results of S/NS studies on clonal plants and severed ramets against the volume and nutrient hypotheses I–III we set out at the beginning. We first analyse the results of Falik et al. (2006), who conducted an experiment with Trifolium repens L. plants in which all four treatments of Fig. 2 were represented. A ‘2 ppp with rc’ plant (treatment G) with access to two pot volumes produced 51% more root biomass than a ‘1 ppp no rc’ plant (treatment E) that had access to only one pot volume (Table 4a). This is in accordance with Hypothesis I, which predicts a larger root biomass as a result of a larger rooting volume. Severed plants in the ‘1 ppp with rc’ treatments F and H produced somewhat smaller root mass than the (Intact) plants of the ‘1 ppp no rc’ treatment E, although they had access to the same unit of rooting volume (Table 4a), perhaps because they started growing as a single rooted ramet rather than two-rooted ramets. Plants in the ‘1 ppp no rc’ treatment E had access to the same quantity of nutrients as plants in the ‘2 ppp with rc’ treatment G and their shoot masses did not differ significantly (Table 4a). The severed plants in treatments F and H, growing with only half the nutrient availability per plant, also produced half the shoot biomass of the intact plants in treatments E and G, indicating that the shoot mass of a plant closely follows the total nutrients available per plant, as in other studies described above.
Figure 4. Volume and nutrient availability effects in two severing studies with clonal plants. (a) Falik et al. (2006) arranged intact or severed two-ramet Trifolium repens plants over one or two pots resulting in treatments E–H as depicted in Fig. 3. The two original plants in the non-self treatments (G and H) belonged to the same genotype. The plants were harvested before seed set. Different superscript letters indicate statistically significant differences from a one-way anova and Tukey HSD post-hoc test (from Falik et al. 2006). Because we halved the original values for treatments F and H as given in the original paper to obtain masses per physiological individual, statistical information for these groups is not given. (b) Gruntman & Novoplansky (2004) used symmetrical two-branched ramets of the stoloniferous Buchloe dactyloides that were either longitudinally severed into two genetically identical but physiologically separate halves (treatment F) or left intact (treatment E). Severed ramet halves of different origins were placed together: the two halves originating from the same ramet were physiologically separated immediately before the onset of the experiment (treatment F1). In the Severed-7 days (F2) and Severed-60 days (F3) treatments, the halves originating from plants of the same genotype were grown in separate pots for 7 and 60 days, respectively, before severing. Finally, in treatment H, the two ramet halves originated from two different clones (genotypes). ‘Plant’ refers to a single physiologically separate individual, i.e. either an intact ramet (treatment E) or a severed ramet half (treatments F and H). Mean values were read from the graphs in the original paper and re-plotted; we recalculated values per physiological individual by halving the masses per pot. Shoot masses were computed from the root mass and the root mass ratio, and therefore there is no statistical information on shoot mass. Different superscript letters indicate statistically significant differences from a one-way anova, followed by a Fisher's least significant difference comparison (from Gruntman & Novoplansky 2004). We only show statistical results for the severed F and H treatments, because we halved the original numbers to obtain root mass per plant, and could not compare them statistically with treatment E (not halved)
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Gruntman & Novoplansky (2004) observed root growth responses in Buchloe dactyloides Engelm. after the plants were grown in the presence of neighbours of variable physiological and genetic identities. In addition to treatment E (‘1 ppp no rc’), in treatment F1 ramets were split longitudinally and the two halves were grown together (severed-self). In two further treatments, the plant-halves were alienated from the each other by letting them grow separately from each other (‘alienated’) for a period of 7 or 60 days before planting them together again (treatments F2 and F3, respectively). In the final treatment, treatment H, the second half originated from a plant of a different genotype (‘alien’). All the treatments depicted in Fig. 3 were thus conducted by Gruntman & Novoplansky (2004) except treatment G. Hence, all plants in this study had the same rooting volume and we do not expect a difference in root biomass between the treatments if root mass is solely determined by available volume per plant (Hypothesis I). The individual plants of each of the (Severed) treatments F2, F3 and H did indeed have a root biomass (17–21 mg) comparable with the root mass of plants of (Intact) treatment E (20 mg; Table 4b). Treatment F3 did produce a significantly greater root mass than treatment F2, suggesting that the period of alienation prior to the start of the experiment had affected root biomass. Remarkably, the un-alienated severed plants of the F1 treatment had only half the root biomass of the other four treatments (11 g), despite the same rooting volume. Shoot mass again closely followed the available nutrients per plant in all treatments (Table 4b).
The study of Falik et al. (2003), who split Pisum sativum plants longitudinally creating three treatments, i.e. E, F and H of Fig. 3 (‘1 ppp no rc’, ‘1 ppp with rc’ and ‘1 ppp with rc’, respectively), bears some resemblance with that of Gruntman & Novoplansky (2004). With the same rooting volume per plant, plants in treatment E and H had a similar root biomass (6.0 and 5.5 g, respectively). Interestingly, the plants in treatment F (‘1 ppp with rc’ or Severed Self), where two halves of the same severed plant remained growing together, had a much smaller root biomass (3.5 g), reminiscent of the lower root mass in treatment F1 of Gruntman & Novoplansky 2004).