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The role of genotypic diversity in determining grassland community structure under constant environmental conditions

2007; Wiley; Volume: 95; Issue: 5 Linguagem: Inglês

10.1111/j.1365-2745.2007.01275.x

1365-2745

Raj Whitlock, J. Phil Grime, Rosemary E. Booth, Terry Burke,

Botany and Plant Ecology Studies

Journal of EcologyVolume 95, Issue 5 p. 895-907 Free Access The role of genotypic diversity in determining grassland community structure under constant environmental conditions RAJ WHITLOCK, Corresponding Author RAJ WHITLOCK *Author to whom correspondence should be addressed: R. Whitlock. E-mail: r.whitlock@sheffield.ac.uk.Search for more papers by this authorJ. PHIL GRIME, J. PHIL GRIME Department of Animal and Plant Sciences, and Unit of Comparative Plant Ecology, Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UKSearch for more papers by this authorROSEMARY BOOTH, ROSEMARY BOOTH Department of Animal and Plant Sciences, and Unit of Comparative Plant Ecology, Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UKSearch for more papers by this authorTERRY BURKE, TERRY BURKE Department of Animal and Plant Sciences, and Unit of Comparative Plant Ecology, Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UKSearch for more papers by this author RAJ WHITLOCK, Corresponding Author RAJ WHITLOCK *Author to whom correspondence should be addressed: R. Whitlock. E-mail: r.whitlock@sheffield.ac.uk.Search for more papers by this authorJ. PHIL GRIME, J. PHIL GRIME Department of Animal and Plant Sciences, and Unit of Comparative Plant Ecology, Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UKSearch for more papers by this authorROSEMARY BOOTH, ROSEMARY BOOTH Department of Animal and Plant Sciences, and Unit of Comparative Plant Ecology, Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UKSearch for more papers by this authorTERRY BURKE, TERRY BURKE Department of Animal and Plant Sciences, and Unit of Comparative Plant Ecology, Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UKSearch for more papers by this author First published: 27 July 2007 https://doi.org/10.1111/j.1365-2745.2007.01275.xCitations: 75AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Summary 1 A recent experiment varied the genetic diversity of model grassland communities under standardized soil and management conditions and at constant initial species diversity. After 5 years' growth, genetically diverse communities retained more species diversity and became more similar in species composition than genetically impoverished communities. 2 Here we present the results of further investigation within this experimental system. We proposed that two mechanisms – the first invoking genetically determined and constant differences in plant phenotypes and the second invoking genotype–environment interactions – could each underpin these results. This mechanistic framework was used as a tool to interpret our findings. 3 We used inter-simple sequence repeat (ISSR) DNA markers to confirm which of the individuals of six study species initially included in the model communities were unique genotypes. We then used the molecular markers to assess the survival and abundance of each genotype at the end of the 5-year experimental period. 4 The DNA marker data were used to create, for the first time, a genotype abundance hierarchy describing the structure of a community at the level of genotypes. This abundance hierarchy revealed wide variation in the abundance of genotypes within species, and large overlaps in the performance of the genotypes of different species. 5 Each genotype achieved a consistent level of abundance within genetically diverse communities, which differed from that attained by other genotypes of the same species. The abundance hierarchy of genotypes within species also showed consistency across communities differing in their initial level of genetic diversity, such that species abundance in genetically impoverished communities could be predicted, in part, by genotypic identity. 6 Three species (including two canopy-dominants) experienced shifts in their community-level genotype abundance hierarchies that were consistent with an increased influence of genotype–environment interactions in genetically impoverished communities. 7 Our results indicate that under relatively constant environmental conditions the species abundance structure of plant communities can in part be predicted from the genotypic composition of their component populations. Genotype–environment interactions also appear to shape the structure of communities under such conditions, although further experiments are needed to clarify the magnitude and mechanism of these effects. Introduction It has long been held (Antonovics 1976) that the organization and structure of ecological communities is in part determined by the genetic diversity of their constituent populations. Genetic components to community structure are becoming evident in a variety of community processes such as succession (Proffitt et al. 2005), species coexistence (Booth & Grime 2003), and in the resistance and resilience of communities to environmental challenges (Hughes & Stachowicz 2004; Reusch et al. 2005). The possible role of intraspecific diversity in maintaining species diversity, although still poorly understood, has been the longest studied aspect of the genetics of plant communities (Antonovics 1976; Turkington & Harper 1979; Aarssen & Turkington 1985). In this sphere, genetic diversity potentially offers an attractive solution to the apparent paradox (Gause 1934) that mixtures of ecologically similar species are often observed in the natural world. Competitive hierarchies amongst coexisting species, often observed to be transitive at the species level (Goldberg & Landa 1991; Grace et al. 1993; Shipley 1993), may in fact be intransitive at the level of individual genotypes (Aarssen 1989; Taylor & Aarssen 1990), such that species identity is not always a reliable predictor of success through competition. Moreover, species that seem to overlap substantially in niche (Mahdi et al. 1989) may individually show adaptive differences at the scale of resolution of the genotype (Ennos 1985). Such adaptive diversity might allow a greater degree of species coexistence than is apparent from consideration of species-level niches alone (Vellend 2006). Adaptive genetic diversity is also thought to be critical in enabling species to persist when the prevailing environmental conditions change (Bradshaw 1952; Gregory & Bradshaw 1965; Kettlewell 1973; Snaydon & Davies 1982). Models of species coexistence that rely on intransitive competitive hierarchies amongst genotypes or on intraspecific adaptive diversity involve the interaction between individual genotypes and their environment, such that fitness is determined by a product of genotype and environment (a genotype by environment interaction, Haldane 1946). In the community context this leads to the expectation that no genotype of any species is maximally fit in all the environments contained within the community (e.g. Aarssen 1983, 1989). To date, experimental investigations of the significance of genetic diversity to plant communities have relied largely on extrapolation from much simpler experimental units, such as pairwise competition experiments between genotypes, to infer the community-level consequences of genetic diversity (e.g. Taylor & Aarssen 1990). Field-based studies that explore this area (Prentice et al. 1995, 2006; Odat et al. 2004) have encountered particular challenges because environmental and demographic complexities may affect both the species and the genetic structures within natural communities. An alternative strategy is to conduct replicated experiments using simplified model communities in which the genetic diversity of multiple component species is manipulated while other variables are held constant. In such an experiment, Booth & Grime (2003) demonstrated that genetically impoverished communities, each consisting of a unique set of genotypes, became more divergent in their species composition, and lost species diversity faster, than more genetically diverse communities of initially identical species composition. The divergence in composition observed between replicate communities was most conspicuous in the experimental treatment in which each species was represented by a single genotype. The effects of genetic impoverishment reported by Booth & Grime (2003) could have been caused by two mechanisms at the level of the genotype. These mechanisms are not mutually exclusive: 1 Under the deterministic model the fitness or abundance of each genetic individual in a community depends upon genetic characteristics that vary among individuals and that control the expression of traits, such as morphology or growth rate. The abundance of a species is determined additively by the total performance of all the genotypes that represent it within a population (i.e. by the genotypic composition and functional attributes of each genotype). In the experimental design applied by Booth & Grime (2003) reduction of genetic diversity acted to sample genotypes and their traits randomly from the total pool of genotypes. At low diversity, the abundance of species could change because the abundance characteristics of the genotypes representing them changed. Under such conditions a greater variance in species abundance is expected among genetically impoverished communities than among communities possessing genetic diversity. This is because particular traits are expressed consistently (fixed) in those populations comprising genetically impoverished communities. 2 The second proposed mechanism, the contingent model, implies an impact of genetic diversity on the community-level outcome of interactions between plants, or between plants and components of their biotic or abiotic environments (genotype by environment interactions). A key feature of this model is that genotypic fitness in genetically impoverished communities is contingent on elements of the environment (e.g. the identity of coexisting genotypes) that are heterogeneous within communities or whose prevalence varies with genetic impoverishment. As such, neither genotype abundance nor species abundance in these communities is expected to be predictable from the performance of each genotype in genetically diverse communities. All genotypes of a given species within impoverished communities may either 'win' or 'lose', resulting in the greatest variance in species abundance amongst these communities. Conversely, in more genetically diverse communities the expression of dominance by some species and the rate of exclusion of others may be moderated by intransitivity in the competitive relationships amongst genotypes (Aarssen 1983, 1989). There are many possible alternative environmental agents through which the contingent model may operate, in addition to the example given above involving competitive interactions with other genotypes. Specific examples are the reduced probability of pathogen epidemics developing in genetically diverse plant populations (Burdon 1993), the differential palatability of genotypes to herbivory (Graham et al. 2001), and the possibility that mutualist bacteria (Chanway et al. 1989; Turkington 1996) or mycorrhizal connections (Streitwolf-Engel et al. 2001) between specific genotypes can moderate plant growth and the outcome of competition between these genotypes. The two models represent extremes in a continuum where both mechanisms could contribute to community structure. Both models predict the same patterns of variance in abundance for species across communities with reduced genetic diversity. However, under the deterministic model, the abundance of genotypes is expected to be correlated between genetically diverse and impoverished communities. This pattern is not expected for differences in species abundance generated purely through the contingent model. Here, we use the model framework described above to explore and help interpret the genetic basis of experimentally generated differences in the species composition and structure of plant communities. Specifically, we used DNA markers to measure the survival and performance of genotypes of six plant species after they had grown for 5 years in experimental communities differing initially in their degree of genetic diversity. We describe, for the first time, the structure of a mature grassland community in terms of the success or fitness of genotypes composing its component species. We find that genetic determinism has a role to play in driving the species abundance structure of communities, but that such determinism cannot on its own account for the composition of genetically impoverished communities. This is the first study of the molecular ecology of multi-species, multi-genotype plant communities. We show that genetic markers can be used to help understand the role of genetic diversity in determining the composition and dynamics of an ecological community. Methods study system The long-term experiment exploited in this investigation is described in full elsewhere (Booth & Grime 2003). Here, we summarize particular aspects of the experimental design. In 1998, Booth & Grime (2003) assembled 36 model communities (0.6 m × 0.6 m in size) from experimental populations of 11 plant species, each population drawing upon a pool of 16 'biotypes'. We define a biotype as an individual plant and any of its clonally produced offspring that is presumed to be (but not necessarily) genetically distinct from other biotypes of the same species (i.e. a putative 'genotype'). We use the term genotype sensu stricto to denote an individual with demonstrable genetic differences from other genotypes. The experimental biotypes were originally derived from established plants sampled at random from within the same 10 m × 10 m area of limestone pasture at Cressbrookdale in Derbyshire, UK, and were assumed to represent unique genotypes. Some of the biotypes exhibited substantial differences in aspects of their morphology (e.g. Fig. 1), supporting the hypothesis that they were genetically differentiated. The plants were maintained in pots as an archive of clonal biotypes (hereafter designated the clonal archive) and vegetative propagation of this material was used to generate sufficient numbers of individuals to synthesize the communities. Figure 1Open in figure viewerPowerPoint Morphological differences between two genotypes of the sedge Carex caryophyllea that originated from the same 10 × 10 m area of calcareous grassland. The genotype shown in the upper section of the figure was sampled five times from randomly selected locations in the same 10 × 10 m field site. Each experimental community was initially identical in species composition because the total number of individuals of each species planted in any community was held constant at 16. However, the number of biotypes representing each species in each experimental community was manipulated. This design resulted in the creation of three community types with contrasting levels of genetic diversity: 16, 4 and 1 biotypes per species per community, replicated 10, 10 and 16 times, respectively. Each replicate of the 4- and 1-biotype communities was created by making a random draw from the total pool of 16 biotypes of each species, with the constraint that every biotype was represented in at least one replicate of each genetic diversity treatment. In order to maintain the integrity of the genetic diversity treatments the communities were managed by cutting to simulate grazing and by removing all flowers by hand to prevent plants from dispersing seeds back into their communities (Booth & Grime 2003). This feature of the experimental design also permitted the re-identification of known individuals within genetically diverse communities using DNA markers (see below). We selected six plant species for study from those included in the experimental communities of Booth & Grime (2003): Festuca ovina L., Koeleria macrantha (Ledeb.) Schult., Carex caryophyllea Latourr., Succisa pratensis Moench, Leontodon hispidus L. and Campanula rotundifolia L. These species included the five most abundant species in the experimental communities, as well as the most subordinate species, C. rotundifolia. individual identification using molecular markers The biotypes used to set up the experimental communities were originally sampled at random from a natural grassland community. It was not initially known whether each of the sampled biotypes represented a unique genotype, and therefore our first objective was to test this assumption. For this purpose, we extracted DNA from all the plants in the clonal archive with which to develop molecular markers and test the genetic identity of biotypes. DNA was extracted using the CTAB protocol of Rogers & Bendich (1994), with some minor species-specific modifications as described by Whitlock (2004). Inter-simple sequence repeat (ISSR) DNA markers (Zietkiewicz et al. 1994) were developed for each plant species. The PCR primers used for ISSR analysis are shown in Table 1. PCR was carried out in a total volume of 10 µL containing PCR reaction buffer IV (ABgene), 1.5–2.5 mm MgCl2 (ABgene), 0.2 mm dNTPs, 0.5 µm of each of two different ISSR primers, 0.25 units of Thermoprime plus DNA polymerase (ABgene) and 10 ng template DNA. Thermocycling conditions for PCR consisted of a 4-min denaturation stage at 94 °C; 35 cycles of 94 °C for 30 s, 52 °C for 1 min and 72 °C for 1 min; and a final extension of 10 min at 72 °C. Diluted PCR products were separated and sized by polyacrylamide gel electrophoresis on an ABI 377 sequencer using a ROX-labelled size standard. Table 1. Sequences of ISSR primers used for the identification of genotypes within genetically diverse plant communities Primer Sequence 5′–3′ Reference RF01 AGAGAGAGAGAGAGAGT Huang & Sun (2000) RF04 CTCTCTCTCTCTCTCTG Whitlock (2004) RF05 GAGAGAGAGAGAGAGAT Whitlock (2004) RF06 VDHTCTCTCTCTCTCTC Whitlock (2004) RF09 RRRTTCTTCTTCTTCTTCT Whitlock (2004) RF10 RRGATCATCATCATCATCA Whitlock (2004) RF11 YYCTAGTAGTAGTAGTAGT Whitlock (2004) ISSR fingerprints obtained from samples of the biotypes in the clonal archive were examined in order to determine whether each represented a unique genotype. A fingerprint was considered genetically unique when it exhibited at least one band that distinguished it from fingerprints of other individuals and when this result was consistent over independent genotyping trials. The apparent genetic identity of different individuals can result either from clonal propagation or insufficient resolution of the genetic markers. Therefore, the power of the ISSR fingerprints to distinguish unique genotypes was tested by computing an overall probability of genetic identity (PID) for the primer combinations used to identify genotypes in each species. This value was calculated for each ISSR band as PID = (pl2)2 + (2plql)2 + (ql2)2 (modified after Waits et al. 2001; L. P. Waits, pers. comm.) where p and q are the frequencies of the two alleles at the locus underlying an ISSR band. The total PID for a primer combination was calculated by chain multiplication of the individual PIDs for each locus within the primer combination. Different bands within and among primer combinations were assumed to represent independently inherited genetic loci. Table 2 gives the PID values calculated using ISSR genotypes from unique individuals in each experimental population. The probability of identical genotypes matching purely by chance was less than 10−6. Table 2. Summary of ISSR analysis for each study species. ISSR primer combinations used to identify genotypes are shown along with the power of these ISSR markers to discriminate individuals (PID) Species ISSR primer combinations Total number of ISSR bands Total PID Festuca ovina RF04-09 43 3.20 × 10−7 Koeleria macrantha RF04-10 58 6.81 × 10−9 Carex caryophyllea RF04-06, RF05-01 68 2.53 × 10−11 Succisa pratensis RF04-06, RF04-11 75 6.19 × 10−13 Leontodon hispidus RF04-06 95 2.39 × 10−15 Campanula rotundifolia RF04-06 78 4.58 × 10−13 genetic survey of community composition The survival and relative abundance of each of the 16 genotypes of the six study species was determined within every 4- and 16-genotype experimental community by sampling leaf tissue and then identifying the genotype of the resulting samples using the ISSR molecular markers. This survey was conducted once after 5 years' growth of the experimental communities. All results presented in this paper refer to the survival and abundance of genotypes and species at this stage of the experiment. We used a 100-position (10 × 10 square) point-quadrat frame (described in Booth 2001) to determine locations for leaf-tissue sampling within each community. This quadrat frame had an area equal to that of the plant communities being sampled (0.6 m × 0.6 m). A random sequence of numbers was used to determine a sequence of positions within the point-quadrat frame through which a metal pin was dropped vertically into the leaf canopy of each community. Leaf material was removed from a single individual of each target species that contacted this pin. This process was repeated until the full target sample size within a community had been collected for a given species. As the aim of sampling was to estimate the relative abundance of genotypes within communities, we used a lower target sampling intensity (~30) to survey 4-genotype communities than that employed for surveying 16-genotype communities (~50; Table 3). It was necessary to reduce the sampling intensity for 16-genotype communities of Leontodon hispidus because of the lower abundance of this species in experimental communities. Every living shoot of Campanula rotundifolia was sampled, as this species achieved such a low abundance in experimental communities that point-quadrat sampling would have been inefficient (Table 3). Leaf-tissue sampling was carried out during autumn 2002, after the last species-level point quadrat analysis for that year. Samples of C. rotundifolia were collected in May 2003 as senescence of the shoots of C. rotundifolia prevented collection during autumn 2002. The relative abundance of a genotype in any 1-genotype community was considered to be 100%. Table 3. Sampling strategy for a survey of the survival and relative abundance of plant genotypes within 20 communities initially possessing either 4 or 16 genotypes of each component species. The numbers of samples of leaf tissue recovered within each replicate community using point quadrat sampling are indicated for each study species. ALL indicates that every visible living shoot was sampled within each community surveyed. The mean species abundance (measured as point-quadrat pin contacts) across all experimental communities in the year 2002 (R. E. Booth, unpubl. data) is given as an indication of the ease with which samples can be recovered by point quadrat sampling Species 16-genotype 4-genotype Total Mean species abundance Festuca ovina 50 30 800 52.7 Koeleria macrantha 50 30 800 66.5 Carex caryophyllea 50 30 800 64.7 Succisa pratensis 50 30 800 34.0 Leontodon hispidus 30 30 600 31.6 Campanula rotundifolia ALL ALL 471 11.1 genetic identification of survey samples DNA was extracted from frozen leaf tissue samples of each species and genotyped, as described above. Each sample was identified by comparing its ISSR fingerprint to those generated from individuals of known identity in the clonal archive. Samples that could not be identified unambiguously were discounted from the analysis. A small subset of the genetic fingerprints was identified independently by a colleague, to verify objectivity and consistency of identification. Failure of DNA extraction, and rejection of samples that could not be identified, together accounted for a loss of between 1.4% (C. caryophyllea) and 3.2% (K. macrantha) of samples. analysis of genetic survey data The ISSR survey data yielded counted observations of the number of quadrat pins at which a genotype i of species j was detected in a given community k (Cijk). These raw data were used to calculate the relative abundance (RAijk) for each of the i genotypes within species j for community k by division with Njk, the number of successfully genotyped leaf specimens of species j in community k. An absolute measure of genotype abundance (Aijk) was defined as RAijk × SAjk, where SAjk is the species abundance (data from Booth 2001; Booth & Grime 2003) of species j in community k. Units of Aijk are on the same scale as the species abundance data measured by Booth & Grime (2003). In fact, the measure is an estimate of the abundance scores that would have been presented by Booth & Grime (2003) had these experimenters also been able to identify species down to genotype. The data from the genetic survey of communities were used to assess the survival rate (presence or absence) of genotypes originally planted in 4- and 16-genotype communities at the end of the 5-year experimental period. Species were compared to investigate whether they exhibited any differences in the loss of genotypes from genetically diverse communities across the experimental period. We explored the genetic structure of the communities with particular reference to the 16-genotype experimental replicates because each of the latter initially contained all 87 of the unique experimental genotypes (see Results) and so comprised a truly replicated set with a presumed close resemblance to natural limestone grassland. We used a chi-squared goodness-of-fit test to test the null hypothesis that the genotypes within species performed equally well over the replicates of the 16-genotype communities. This test compared the total count of each genotype across the 16-genotype communities (Cij16tot) with the number expected if each genotype achieved equal abundance. Before the analysis, the observed counts were weighted to take account of situations where some genotypes had been planted more frequently than others in the 16-genotype communities. A higher representation of certain genotypes had occurred inadvertently because it was discovered (see Results) that some of the biotypes (presumed genotypes) sampled in the Cressbrookdale field site were represented by clones that had been sampled more than once. The weighting procedure involved division of the number of counts achieved by a genotype by the number of times it had originally been planted in the community. We derived an abundance hierarchy of mean genotype abundance (Âij16) to describe the overall genetic structure of the 10 true replicates of the 16-genotype communities in their fifth year of growth. This hierarchy included the 87 unique genotypes of the six study species, and enabled the comparison of the hierarchical distribution of genotypes of different species within an ecological community. The hierarchy also allowed an assessment of the extent to which competitive relationships amongst species might have the potential for intransitivity at the genetic level. The mean abundance of each genotype, Âij, was weighted, as above, to take account of situations where certain genotypes had initially been planted more frequently than others in the 16-genotype communities. We compared the abundance (Âij) of each genotype across sets of communities differing initially in genetic diversity, species by species, in order to assess whether the genotype abundance hierarchy in 16-genotype communities predicted genotype abundance in more genetically impoverished communities. The Âij values were rescaled within each level of community genetic diversity to account for situations where certain genotypes had been planted more frequently than others due to genetic identity of biotypes. Correlations were used for each species to test whether the mean abundance of genotypes estimated in 16-genotype communities could predict the abundance of genotypes in 4- or 1-genotype communities. The deterministic model of community structure predicts a correlation of genotype abundance across communities differing in genetic diversity because, in communities lacking genetic diversity, species abundance is equal to genotype abundance. Under the contingent model, environmental interactions are expected to influence genotypes to a greater extent in 1-genotype communities, resulting in a breakdown of correlations across communities. Therefore, under the contingent model, any correlation in genotype abundance between the 16- and 1-genotype communities is expected to be weaker than the respective correlation between 16- and 4-genotype communities. Results genetic uniqueness of experimental biotypes We concluded that biotypes whose genetic profiles matched those of other experimental individuals were clonally identical, given the extremely small probability (Table 2) that such individuals are in reality genetically different. Comparison of ISSR fingerprints revealed that most of the biotypes originally sampled from the field and then included in the experimental communities represented genetically unique individuals (genotypes). However, the experimental populations of Carex caryophyllea and Campanula rotundifolia contained only 10 and 13 (out of a possible maximum of 16)