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- Materials and Methods
- Supporting Information
Juvenile plants of Cakile edentula sharing a pot demonstrate an apparent kin recognition response, with lower root allocation in groups of siblings than in groups of strangers (Dudley & File, 2007). Because roots are entangled in the shared pots and large sample sizes are needed, the phenotype was measured for groups of plants instead of individuals, with the assumption that equal representation of families within each treatment provides sufficient control for genetic differences. The interpretation of plasticity to relatedness as kin recognition is supported by further studies that used a similar methodology to demonstrate kin recognition in shared pots in Impatiens pallida (Murphy & Dudley, 2009) and Chenopodium album (S. A. Dudley et al., unpublished data), and a study in Arabidopsis thaliana showing plasticity in individual seedlings to root exudates that depended on the relatedness of the exudate source (Biedrzycki et al., 2010). However, even at the juvenile stage, accumulated competitive interactions within the groups could change the group phenotype. Thus, a possible alternative explanation for the sibling and stranger differences in root allocation is that they result from competitive interactions that depend on differences in phenotypic variation between sibling and stranger groups (Klemens, 2008; Masclaux et al., 2010).
The kin recognition hypothesis draws from the growing body of research on plant identity recognition. In many species, roots respond differently to roots of the same physiological individual than to roots of other plants, even when the nonself plants are clones (Mahall & Callaway, 1991, 1992, 1996; Maina et al., 2002; Falik et al., 2003; Holzapfel & Alpert, 2003; Gruntman & Novoplansky, 2004). These responses are presumed to prevent competition between different parts of the same plant. Sagebrush (Artemisia tridentata) shows recognition of self/nonself that is not root-related: plants exposed to volatiles from wounded self plants experience less damage than those exposed to wounded non-self plants (Karban & Shiojiri, 2009). Species-specific responses to belowground neighbours have now been demonstrated in root growth and metabolites (Semchenko et al., 2007; Broz et al., 2010). Pollen characters respond to the genotype of belowground competitors (Lankinen, 2008).
Other research compares the performance of groups of siblings with that of groups of strangers to test contrasting hypotheses: kin selection predicts that siblings will have higher fitness than strangers because siblings cooperate with each other (Hamilton, 1964), while the niche partitioning hypothesis predicts that strangers will therefore have higher fitness than siblings because they differ more in niche use than siblings and so compete less (Young, 1981). The most notable feature of these performance comparisons in plants is the variation in results, with positive, negative, and no fitness benefits to growing with siblings compared with strangers (reviewed in Milla et al., 2009).
In A. thaliana, accessions vary in fitness, plant biomass and competitive ability, but do not show fitness benefits of growing with either siblings or strangers (Masclaux et al., 2010). Unlike most such studies, that by Masclaux et al. (2010) controlled genetic variation by creating stranger groups consisting of only two families. Consequently, the results imply an alternative hypothesis to kin selection or niche partitioning; the hypothesis that in larger groups a few families that are highly competitive could disproportionately dominate in competition. This competitive dominance would then depress the overall performance of stranger groups compared with sibling groups, in which weaker competitors contend among themselves (Masclaux et al., 2010). Among-family variation in competitive ability and in root allocation could also potentially bias measures of kin recognition. If the highly competitive families produce most of the biomass in the pot, and those highly competitive families differ in root allocation from less competitive families, then within-pot natural selection for greater competitiveness could affect root allocation in stranger pots (Fig. 1).
Figure 1. Alternative hypotheses for the increased root allocation in groups of strangers compared with siblings in Cakile edentula (Dudley & File, 2007). Top path: increased allocation results from kin recognition, that is, a competitive response to strangers but not siblings (Dudley & File, 2007). Middle path: increased root allocation results if stranger groups have greater size inequality, and if larger plants have more allocation to roots than smaller plants (Klemens, 2008). Bottom path: increased root allocation results if a few genotypes with higher competitive ability dominate stranger groups, and if the genotypes with higher competitive ability have greater root allocation (Masclaux et al., 2010).
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Measuring root allocation through an analysis of covariance approach (Dudley & File, 2007) removes any associations between size and allocation at the group level. However, size inequality within groups could potentially affect the root allocation results (Klemens, 2008). Groups with the same total plant mass could contain four plants of intermediate size (low size inequality), or one large plant and three small plants (high size inequality). The group phenotype for root allocation (Dudley & File, 2007) is weighted towards the largest plant in the pot (Klemens, 2008). Stranger groups, because of their greater genetic and therefore phenotypic variation, are expected to have greater size inequality than sibling groups. If root allocation is related to size or developmental stage such that larger plants differ in root allocation from smaller plants, differences in root allocation between sibling and stranger groups would result (Fig. 1).
These hypotheses provide plausible alternative explanations for sibling vs stranger differences in root allocation, and are consistent with the direction of the sibling vs stranger differences varying among species. As these mechanisms are somewhat related, rather than mutually exclusive, a combined mechanism is also plausible. Taken together, they provide a reasonable summary of competition-based mechanisms to explain away the plant kin recognition result. However, there has yet been no empirical test for them. Here, we used single seed descent lines of C. edentula descended from the field-collected seed families used by Dudley & File (2007) to determine whether kin recognition or competitive interactions best explain sibling vs stranger differences in root allocation. The pair-wise family design (Masclaux et al., 2010) has several advantages for testing the role of competitive interactions in root allocation. Growing pairs of seedlings together, in all possible combinations of families, provides a robust test for the prediction of kin recognition that root allocation will depend only on whether members of the pair are from the same family or from different families. Analysis of variance for the effects of target plant family and competitor family and their interactions on target plant biomass tests several hypotheses (Masclaux et al., 2010). Superior competitive ability is demonstrated by a significant main effect of competitor family, superior tolerance to competition is demonstrated by a significant main effect of target plant family, and kin vs stranger effects on biomass are demonstrated by significant interactions between target plant and competitor plant families. The absolute aboveground mass and height differences between the seedlings in a pair provide measures of size inequality to test for its relation with root allocation (Klemens, 2008).
In this study, we grew pairs of seedlings in small pots. For eight families, 10 replicates were produced for each possible combination of families, including each family paired with itself. Because C. edentula has a seed dimorphism that affects dispersal and the expected competitive environment, the 10 replicates included both seed types. We measured the dry masses of cotyledons, leaves, and stems for each seedling and the combined root mass for the seedlings sharing a pot. We performed two harvests to study early and late effects of neighbour interaction in C. edentula seedlings. We asked the following questions: Did root allocation depend on relatedness, seed type, or time of harvest? Did aboveground size differences depend on relatedness? Was root allocation correlated with aboveground size difference? Were there among-family differences in competitiveness and root allocation, and was there evidence for family × family interactions?
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- Materials and Methods
- Supporting Information
In this study, we asked whether pairs of C. edentula seedlings would show the same kin recognition response observed in groups of four juvenile plants (Dudley & File, 2007). Again, we found kin recognition in competition, with lower root allocation in siblings than strangers (Dudley & File, 2007). Because we used a pair-wise experimental design (Masclaux et al., 2010), we could further test whether competition-based mechanisms explained the difference in root allocation between siblings and strangers. However, we found relatively little evidence for any competition-based mechanisms other than kin recognition, and no evidence that such competition-based mechanisms affected root allocation. The only significant predictor of root allocation was whether the individuals in the pot were kin or strangers.
In the present study there was no difference between the seed types in plasticity of root allocation to kin vs strangers. Though the proximal and distal seeds differed markedly in size and allocation, few differences between seed types remained at the seedling stage. The cotyledon mass was higher and the radicle to cotyledon mass ratio was lower in distal seeds, suggesting that distal seeds had increased maternal investment in food reserves. Increased provisions may be adaptive as they can increase successful germination of distal seeds under unfavourable conditions (Zhang, 1993, 1994). Cakile edentula seedlings from larger seeds produce leaves and branches more rapidly and reproduce earlier than seedlings from smaller seeds (Zhang, 1996). This gives larger distal seedlings a competitive advantage when growing amongst strangers. However, distal and proximal seedlings did not differ in root allocation or in response to relatedness. Thus, despite the expected differences in dispersal, distal and proximal seed types were not predisposed towards competing with strangers or cooperating with kin.
We tested two overlapping hypotheses for how sibling vs stranger differences in root allocation could arise without kin recognition. Both are two-part mechanisms, postulating first that there are more size differences among plants in stranger groups than those in sibling groups, and secondly that larger plants differ in root allocation from smaller plants (Fig. 1). In one mechanism (Masclaux et al., 2010), genetic variation in competitive ability creates the size difference, and genetic variation in root allocation, if correlated with size, creates the change in root allocation. In the present study, aboveground mass was independent of the family of the competitor, indicating that no one particular family competed more fiercely than others at this early life stage. Aboveground mass did differ among families, with that difference depending on seed type. There was a minor interaction for focal × competitor × seed type which implied more focal × competitor interactions in the smaller proximal seedlings. However, because of the small sample sizes, this needs further investigation. Root allocation did not differ among the kin pairs, indicating a lack of genetic variation for root allocation. Thus, there is no evidence that a competition mechanism involving genetic differences in competitiveness and root allocation could explain sibling vs stranger differences. Consequently, the hypothesis that plasticity to relatedness resulted from uncontrolled genetic variation was not supported in this study.
Another competitive interaction hypothesis for root allocation differences between sibling and stranger groups (Klemens, 2008) suggests that greater phenotypic variation in stranger groups, potentially enhanced by asymmetric competition for light, creates the size differences. Then, allometry results in differences in root allocation between larger and smaller plants. We tested the predictions from this mechanism that stranger pairs will have greater size inequality than kin pairs, and that the size inequality of a pair affects its root allocation. Stranger pairs showed a greater height difference than kin pairs, but kin and stranger pairs had similar aboveground mass differences. This pattern may simply reflect differences in underlying genetic variation for these traits, but could potentially indicate differences in competitive processes in sibling and stranger pairs. Because aboveground competition is asymmetric and driven by height differences (Schwinning & Weiner, 1998), height inequality provides a mechanism for developing greater size inequality in stranger groups than siblings over time. But neither height nor mass differences explained the observed root allocation differences in the kin and stranger pairs. Thus, despite some support for the hypothesis that size inequality is greater in stranger groups, these results do not support the hypothesis of Klemens (2008) that bias in root allocation created by size differences within a group explains differences between the kin and stranger treatments.
Competition-based mechanisms clearly did not explain root allocation differences between siblings and strangers in this study. However, in these relatively young seedlings, little resource limitation and competitive interaction would be expected. Thus, in larger plants these competitive processes may affect root allocation as resources become limiting and plants shade one another. In fact, the increased height inequality seen in stranger groups offers a potential mechanism for the continued generation of increased size inequality in stranger groups as the plants grow and interact to a greater extent. The impact of genetic diversity on competitive processes could modify initial responses to relatedness of competitors.
The results of the present study not only allow rejection of the alternative competition-based hypotheses but also support the predictions from the kin recognition hypothesis of consistent sibling vs stranger differences for seedling pairs. However, more work needs to be done to determine whether these changes do affect belowground competitive ability. Remarkably, the kin recognition response was present in very young seedlings and so, like the stem elongation response to density (Ballare et al., 1990), may allow a plant to anticipate competition and identify the nature of its competitors before any resource depletion occurs.