Molecular Ecology Resources (2011) 11, 185–195 doi: 10.1111/j.1755-0998.2010.02893.x MOLECULAR DIAGNOSTICS AND DNA TAXONOMY Molecular identification of roots from a grassland community using size differences in fluorescently labelled PCR amplicons of three cpDNA regions JOHN M. TAGGART, JAMES F. CAHILL JR, GORDON G. MCNICKLE and JOCELYN C. HALL Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 Abstract Elucidating patterns of root growth is essential for a better understanding of the functioning of plant-dominated ecosys- tems. To this end, reliable and inexpensive methods are required to determine species compositions of root samples con- taining multiple species. Previous studies use a range of PCR-based approaches, but none have examined a species pool greater than 10 or 30 when evaluating mixed and single species samples, respectively. We present a method that evaluates size differences in fluorescently labelled PCR amplicons (fluorescent fragment length polymorphism) of the trnLintron and the trnT-trnLandtrnL-trnF intergenic spacers. Amplification success of the trnT-trnL spacer was limited, but variation in the trnLintronandthetrnL-trnF spacer was sufficient to distinguish over 80% of the 95 species (97% of the 77 genera) evaluated from a diverse fescue grassland community. Moreover, we identified species known to be present in mixed sam- ples of 4, 8, 12, and 16 species on average 82% of the time. However, this approach is sensitive to detecting species known to be absent (false positives) when using our key of 95 species. Comparing unknowns to a limited species pool ameliorates this problem, comparable to a researcher using prior knowledge of what species could be found in a sample to constrain the identification of species. Comparisons to other methods and future improvements are discussed. This method is efficient, cost- effective and broadly applicable to many ecosystems. Keywords: community composition, fluorescent fragment length polymorphism, mixed samples, roots, species identifica- tion, trnL-trnF Received 9 April 2010; revision received 7 May 2010; accepted 26 May 2010 history should lead to the conclusion that elucidating Introduction patterns of root growth in natural systems is of primary Individual plants live both aboveground and below- importance for understanding biodiversity and function- ground, with interactions among roots, soil, fungi, ing of plant-dominated systems. Of course, the tradition microbes, herbivores, shoots, and the atmosphere driving in plant ecology is opposite, and descriptions of plant patterns of plant growth (Wardle 2002). Although con- communities are typically based upon shoot, rather than nected at the soil surface, plants encounter different whole-plant, distributions (e.g., Tilman & Pacala 1993). ecological challenges aboveground and belowground. For The factors that typically limit the inclusion of below- example, most plants capture more than 20 different ground plant distributions in studies of plant ecology resources belowground compared to only two above- have been methodological, rather than conceptual. The ground. Doing so involves unique foraging strategies fine roots of many plant species are visually indistin- (Kembel & Cahill 2005) resulting in highly asymmetric guishable, and thus it has been historically difficult to root systems (e.g., Brisson & Reynolds 1994), and in a root- accurately measure belowground diversity without using ing breadth much greater than the corresponding breadth excavations of entire plots (Brisson & Reynolds 1994), of the canopy aboveground (Schenk & Jackson 2002). using tracer injections (Jackson et al. 1996), or dyeing Even at a more basic level, in most systems plants allocate individual root systems (Holzapfel & Alpert 2003). To more biomass belowground than aboveground (Jackson allow for more detailed understanding of belowground et al. 1996). This combination of observations of natural patterns of plant diversity, plant ecologists need reliable, high throughput, and inexpensive methods to determine Correspondence: James F. Cahill Jr, Fax: +1 780 492 9234; E-mail: species identity and distributions of plant roots in natural [email protected] systems. Ó 2010 Blackwell Publishing Ltd 186 MOLECULAR DIAGNOSTICS AND DNA TAXONOMY To this end, a range of techniques were developed for distinguish 82 out of 95 species in a diverse, fescue species identification of roots: PCR-RFLP (restriction grassland community. Fluorescently labelled primers are fragment length polymorphism) analysis of rbcL (Bobow- cost-effective, accurate and efficient because they permit ski et al. 1999), the trnL intron (Brunner et al. 2001; Ridg- precise sizing of three regions simultaneously on a way et al. 2003) and the internal transcribed spacer (ITS; capillary sequencer. Moreover, this method avoids the Moore & Field 2005); sequencing of ITS (Jackson et al. need for procedures downstream of PCR, such as restric- 1999; Linder et al. 2000); PCR assay with species-specific tion digests, and generates digital, easily processed primers (McNickle et al. 2008; Mommer et al. 2008); and results. We developed a relational key based on size fluorescent fragment length polymorphisms (FFLP) anal- variation in 95 species, representing a significant percent- ysis of the trnL intron (Frank et al. 2010; Ridgway et al. age of species diversity within the community. We then 2003). These studies highlight the power of using PCR- examined the efficacy of our key in correctly identifying driven strategies for species identification, but the utility species in mixed samples of 4, 8, 12, and 16 species. of these approaches has been limited to low numbers of Benefits and limitations of this approach are discussed species. To date, the highest number of species investi- with emphasis in comparison with other methods. gated was 30 tree species from the Alps using the PCR– RFLP method (Brunner et al. 2001). However, most plant Materials and methods communities have a species pool much larger than 30. As a result, to understand belowground species diversity Focal community patterns in natural systems, methods are needed that readily discriminate among a greater diversity of species. The methods we developed should apply to any focal The standard method for harvesting roots in an eco- community; however, we focus on a specific fescue grass- logical study is to take a series of root cores of a given land in western Canada. The field site was located at the depth and diameter. These cores are then washed free of Kinsella Research Ranch, a 6000-ha research facility run soil, leaving a mass of tangled and morphologically by the University of Alberta in central Alberta. The ranch indistinguishable roots of an unknown number of spe- is located in the Aspen Parkland ecoregion, a savanna cies. Thus, to use molecular methods for species determi- consisting of aspen (Populus tremuloides) stands in the nation following this standard ecological sampling lowlands, and rough fescue (Festuca hallii) grasslands in protocol, the technological hurdle of having DNA from the uplands. We concentrated on the undisturbed fescue multiple species in a single sample (mixed or pooled grassland regions of the ranch. This community has been samples) must be overcome. Existing methods for deter- studied for 10 years by the Cahill lab, and the species pool mining plant species in mixed samples often rely on spe- and basic ecology is well understood (e.g., Coupe et al. cies-specific markers (McNickle et al. 2008; Mommer 2009; Lamb & Cahill 2008). In general, this is a species- et al. 2008). While highly accurate, these approaches are rich assemblage, with typical plots of 20 · 50 cm contain- designed for pot ⁄ mesocosm experiment where species ing 3–12 vascular species (pers. obs.) aboveground. Over pools are small and tightly controlled by the researcher. 100 vascular species were found throughout the fescue To date, such studies have included a maximum of ten regions of the ranch, including many representatives of species (McNickle et al. 2008) but more commonly four the species-rich families Asteraceae, Fabaceae, and Poa- (Mommer et al. 2008; Moore & Field 2005). If species ceae. We believe this system is well suited for develop- information is not already available in public databases ment of a community-level molecular key, and the such as GenBank, then developing species-specific mark- species diversity allows for a good test of this methods ers for communities with large species richness is not fea- ability to discriminate within families and within genera. sible as it would be time-consuming and potentially require significant DNA sequencing. Moreover, the Sampling and DNA extractions expensive and labour-intensive approach of cloning is required for sequencing-based identification of mixed We sampled 215 individuals, comprising 105 species from samples. In order for belowground plant community 29 angiosperm families, collected from the University of ecology to move forward, alternative methods must be Alberta Research Ranch near Kinsella, Alberta, Canada able to discern species within a high species pool as well (53°5¢N, 111°33¢W). Plants were chosen through a series as identify species in mixed samples. of walks (totalling over 11 km) from 2005 to 2006, with Here, we describe a method for species identification the intention of collecting multiple individuals of all of roots that evaluates size differences in fluorescently species that were found. Of these samples, we collected labelled PCR