Plant invasions have resulted in observable shifts in the above ground plant communities, and there is evidence that soil fungi are also being significantly altered as the invasion process progresses (Hallett 2006, Mummey and Rillig 2006, Stinson et al. 2006, Zhang et al. 2010). Arbuscular mycorrhizal plant-fungal symbioses form highly specialized ‘nutrient-exchange’ structures (vesicles and arbuscules) at the plant-fungus interface and are recognized as being a particularly beneficial group of root endophytes. Plants associating with arbuscular mycorrhizal fungi are typically more competitive and better able to tolerate environmental stresses than are non-mycorrhizal or poorly colonized plants (Biermann and Linderman 1983, Daniell et al. 2001, Bianciotto and Bonfante 2002, Vandenkoornhuyse et al. 2002, Bonfante 2003, Brundrett 2004, DeBellis and Widden 2006, Greipsson and DiTommaso 2006, Parniske 2008, Shah et al. 2010). AMF are also known to provide protection from a variety of microbial pathogens to their host plants (Newsham et al. 1994, Borowicz 2001, Klironomos 2002, Pozo and Azćon-Aguilar 2007, Van der Putten et al. 2007, Appoloni et al. 2008, Mogg et al. 2008, Krüger et al. 2009, Wehner et al. 2009).

AMF species richness has been shown to affect plant productivity, and observed shifts in plant-AMF associations could impact plant community structure (Simberloff and Von Holle 1999, Maherali and Klironomos 2007). Given that AMF are functionally diverse and confer differential benefits to plants (Sanders and Fitter 1992, Bever et al. 1996, Bever 2002, Klironomos 2003), responses of AMF communities to invaders would be potentially highly disadvantageous to established native species (Helgason et al. 2002, Kourtev et al. 2002, Greipsson and DiTommaso 2006, Hawkes et al. 2006, Stinson et al. 2006), and would cause shifts in native plant communities (van der Heijden et al. 1998). Thus, disruptions to community plant-AMF associations in local ecosystems may support system dominance by invaders (van der Heijden et al. 1998, Daniell et al. 2001, Helgason et al. 2002, Kourtev et al. 2002, Greipsson and DiTommaso 2006, Hawkes et al. 2006, Bastias et al. 2007, Curlevski et al. 2010).

We compared both total fungal (TF) and AMF associations of the non-native vine Vincetoxicum rossicum or dog-strangling vine (DSV), several field and woodland native plants growing in dense patches of DSV, and the same natives growing separately from DSV. DSV is widely distributed in Eastern North America and has established virtual monocultures, consequently reducing diversity in local native ecosystems (Pimm et al. 1995, Ernst and Cappuccino 2005). DSV has been shown to be more densely colonized by arbuscular mycorrhizal fungi than co-occurring native plants in Henderson Harbour, New York (Greipsson and DiTommaso 2006, Smith et al. 2008), and extracts of DSV tissue demonstrated antifungal activity against plant pathogenic yeast-like and filamentous fungi as well as broad-host-range plant pathogens such as Fusarium spp. (Mogg et al. 2008).

In this study, we investigated the fungal communities associating with the roots of DSV and native plants in two proximal watersheds where dramatic invasions of DSV have occurred, taking advantage of the natural invasion of DSV into native field and woodland areas. We hypothesized that fungal root colonizers of native plants growing within dense patches of DSV would increase significantly in both richness and density relative to those growing separately from DSV. These findings are novel with respect to the use of molecular techniques to characterize the fungal community associating with DSV and natives persisting in DSV patches. The alternative, more traditional methods, including soil spore analysis or root-piece microscopy, are not able to produce similarly reliable and replicable results.

Peak height data for each individual TRF or phylotype were normalized to a percentage of total fluorescence per sample to account for the differences in the amount of amplicon sent for analysis. The normalized terminal restriction fragment data were then aligned using a macro created by Christopher Walsh (Rees et al. 2004) in Excel 2007 (Microsoft Corporation), and then subsequently used in downstream analyses. TRFs were also manually aligned such that single base pair differences were binned together when warranted by obvious patterns in the data. TRFs less than 50 base pairs (bp) were not analyzed. The normalized dataset was exported to R open source statistical analysis software, v 2.12.0 (R Development Core Team 2010) using the Adonis function in vegan (Oksanen et al. 2012) and NMDS in MASS (Venables and Ripley 2002) packages. Statistica (StatSoft Inc., v 7.1) was used for multivariate analyses.

NMDS (based on Bray-Curtis distances) and Adonis (non-parametric permutation MANOVA or analysis of difference in R, based on 99 permutations; analogous to redundancy analysis, Legendre and Anderson 1999) analyses were used as a means of data reduction, and to investigate the underlying structure of the fungal communities in each sample. Samples were grouped as types ‘DSV’, ‘natives-in-dsv’, or ‘natives’ to represent DSV, native plants growing in dense DSV patches, and native plants growing separately from DSV, respectively. NMDS is a common ordination method for T-RFLP community data that preserves the rank ordering of original distances among observations, and uses these ranks to map the objects in two-dimensional ordination space (Ramette 2007). The stress value (Krustal’s) looks at the rank-order between distances and dissimilarities of the NMDS analysis: as the agreement between distances and dissimilarities improves, the stress is lower. Stress values of 0.20 or less indicate a biologically relevant result (Rees et al. 2004). NMDS and Adonis are free from the assumption of normality (Rees et al. 2004). The Adonis function (analysis of difference) in R was used to determine the significance of differences between samples or plant types (i.e. DSV, natives-in-dsv, or natives).

To determine significant TRFs contributing to the different analyses, principal component analysis (PCA) was performed in R to reduce the TRF data dimensionality prior to performing discriminant analysis (DA); principal components with eigenvalues > 1 were used in the DA. Significant loadings (TRFs) from the principal components were used in the DA to produce graphs of sample cluster patterns based on the variables that best discriminated among the different defined groups. Differences in fungal phylotype density and richness levels between samples were tested using Student’s t-tests or ANOVA.

TF amplicons of approximately 700–900 bp and AMF amplicons of 500–800 bp were successfully obtained from root tip DNA. TRFs were successfully obtained from the samples using both FAM- and HEX-labeled primers and both sets of restriction enzymes. Replicate T-RFLP analyses for both TF and AMF produced highly similar results with regard to both TRF phylotypes and peak heights. Based on sequencing results, all TF sequences were fungal, and all AMF sequences were Glomeromycota.

The community composition of the TF and AMF communities for DSV, natives-in-dsv, and natives was investigated using multivariate analyses of the normalized FAM and HEX TRF data. Given the tendency of the data to sort significantly by season in initial analyses (data not shown), all data were grouped by season (spring or fall) for subsequent fungal community comparisons.

TF phylotypes showed a tendency to sort by site initially and to a lesser extent environment when woodland and field samples were analyzed collectively. However, once the data were parsed into field and woodland sets, site was no longer a significant factor in either the spring or fall data analyses. Sample type remained significant throughout the analyses, with FAM and HEX data showing similar results, indicating where TF community patterns were strongly affected by the presence of DSV. Ordination results for spring and fall TF sample sets are shown in Table 4. The NMDS stress values for all analyses are < 0.20, indicating that the ordination results for the data sets are biologically relevant (Rees et al. 2004). The R² results were determined using data grouped by the terms type (DSV, natives-in-dsv, and natives), plant (different plant species), environment (woodland or field), and site (UTSC or Zoo), with F-tests used to report p-values showing the tendency of data to sort according to the terms defined in the model.

For the AMF data, site was not a significant factor affecting sample type group association, even prior to taking environment into consideration for either the spring or fall data. Sample type remained significant throughout the analyses, indicating where DSV, natives-in-dsv, and natives support different AMF phylotype patterns. Ordination results for fall and spring AMF sample sets are shown in Table 4.

For discriminant analysis, loadings (FAM-labeled TRFs) used in the analyses were derived from principal components determined using the Kaiser (1960) criterion (eigenvalues >1) for PCA in R. The plots for all woodland data showed separation of fungal community by sample ‘type’ in the normalized TRF patterns for DSV, natives-in-dsv, and natives for both the TF and AMF data (Figures 1 and 2 respectively), significant loadings are indicated where relevant.

Using the ratio of the average FAM peak height values to the extracted root DNA concentration per sample as a measure of fungal colonization density, we observed that the TF colonization of roots differed significantly between the natives and the natives-in-dsv, with the natives-in-dsv collectively averaging 1.9 times greater TF colonization densities (Student’s t-test, p= 0.00003; Appendix 1). As well, AMF colonization of roots differed significantly between the natives and the natives-in-dsv, with the natives-in-dsv showing a difference of 3.9 times greater average colonization densities (p = 0.002).

Collectively, there were significantly fewer FAM AMF phylotype variants in all samples (mean of 6.5±2.3) than TF variants (mean of 27.4±13.0), p = 0.0001. There was a significant difference in the number of TF phylotypes between DSV and natives, as well as between natives and natives-in-dsv (p = 0.0014 and p = 0.016, respectively using ANOVA, with Fisher’s LSD). The average number of TF phylotypes for DSV, natives-in-dsv, and natives were 31±11.6, 29±12.4, and 19±11.9 respectively (Table 5). There was no significant difference in the number of AMF phylotypes among the DSV, natives-in-dsv, and native TRFs (p = 0.95). However, there were observed differences in the phylotype occurrences between groups/plants (Appendices 2 and 3).

The dominant FAM TF and AMF phylotypes for individual plant species is shown in Appendices 2 and 3, respectively. For the TF data, the spring goldenrod-in-dsv had greater or equal phylotype representation for all TRFs considered relative to goldenrod growing separately from DSV, while the fall goldenrod-in-dsv had greater or equal phylotype representation in all twelve dominant TRFs relative to goldenrod. For woodland plants, results were mixed with wild ginger-in-dsv showing greater phylotype representation in seven out of ten TRFs, meadow rue-in-dsv showing greater or the same phylotype representation in five out of eleven TRFs, raspberry-in-dsv showing greater or the same phylotype representation in six out of eleven TRFs, and anemone growing in DSV showing greater phylotype representation in seven out of eleven TRFs. Anemone-in-dsv had considerably more rare phylotypes (average of 61) relative to anemone (average of 9), the greatest difference between any native-in-dsv/native plant sampled. All TRFs found collectively in anemone-in-dsv samples (136 in total) were also found in the DSV samples except TRFs 236, 285, and 253bp. Six out of eight of the most dominant phylotypes in DSV (67bp, 134bp, 150bp, 224bp, 321bp, and 324bp) were significantly greater in natives-in-dsv relative to their native counterparts (Student’s t-test p = 0.04, 0.03, 0.003, 0.045, 0.03, and 0.05, respectively).

TRF 67bp was present in 88% of the total DSV samples, and TRF 324bp was present in 96% of the DSV samples. There was an increase in TRF 67bp representation in all natives-in-dsv relative to natives except anemone, which remained unchanged, and TRF 324bp representation in all natives-in-dsv groups except meadow rue (that showed a decrease in 324bp), suggesting that these phylotypes may be universally affected by DSV invasion. As well, 72% of the TF phylotypes that were dominant in DSV showed increased representation in the natives-in-dsv relative to natives (by varying amounts), and 23% of TF phylotypes not observed in DSV showed reduced representation in natives-in-dsv relative to natives. TRF 321bp, which was present in > 50% of the DSV samples, was only present in natives-in-dsv, with the exception of goldenrod. BLAST sequences were obtained for FAM TRFs 67bp, 138bp, 222–224bp, and 324bp (Bongard et al. 2013), suggesting colonization by uncultured Ascomycota, Tetracladium spp., Glomus spp., and uncultured Ascomycota respectively. BLAST sequences for HEX TRFs 99bp and 142bp were uncultured Ascomycota and Fusarium spp. respectively.

For the AMF data, while significantly greater colonization densities were observed in the natives-in-dsv relative to the natives (as mentioned earlier), phylotype representation of the dominant TRFs in the invaded samples generally decreased relative to uninvaded samples (Appendix 3). The goldenrod-in-dsv had the same or reduced phylotype representation in six/seven out of nine TRFs (spring/fall respectively) relative to the goldenrod (growing separately from DSV). Woodland plants had mixed results with wild ginger-in-dsv showing reduced phylotype representation in seven out of nine TRFs, raspberry showing the same or reduced phylotype representation in five out of seven TRFs, anemone showing the same or reduced phylotype representation in six out of nine TRFs, and meadow rue showing the same or increased phylotype representation in five out of eight TRFs. TRF 54bp was present in only 29% of the total DSV samples, and TRF 81bp was present in 50% of the DSV samples. There was a reduced or maintained representation by TRF 54bp in all natives-in-dsv groups, suggesting that this phylotype is universally affected by DSV invasion. Similarly, reduced representation by TRF 81bp occurred in all natives-in-dsv relative to natives except fall goldenrod (increased levels). Only 22% of the TRFs that were dominant in DSV showed increased representation in the natives-in-dsv over natives for the AMF phylotypes, and 33% of TRFs not observed in DSV showed reduced representation in natives-in-dsv. BLAST sequences obtained for AMF were Glomus spp.

Disruption or alteration of soil fungi is one mechanism by which introduced plants are able to preferentially succeed in a novel environment (Hawkes et al. 2006, Stinson et al. 2006, Vogelsang and Bever 2009). AMF may either be disrupted or harnessed by aggressive invasive plant species, resulting in altered native fungal communities in the soil as the invader attains dominance in the system (van der Heijden et al. 1998, Daniell et al. 2001, Greipsson and DiTommaso 2006, Bastias et al. 2007, Curlevski et al. 2010). Mummey et al. (2005) used T-RFLP and multivariate analyses to show that AMF communities associating with a common forage grass species Dactylis glomerata, naturalized in mid-western US, shifted to reflect the community composition associated with a noxious weed native to eastern Europe, Centaurea maculosa, post invasion. Our research supports this mechanism as a possible contributor to DSV success by demonstrating that there are significant shifts in both the total and arbuscular mycorrhizal fungal communities associating with native plant roots in invaded relative to non-invaded sites.

We were able to show that native plants living in close association with DSV differ from those living distantly from DSV in that they have higher TF and AMF colonization densities, higher TF phylotype richness levels, and different TF and AMF community structures overall. We found that the AMF colonization density increased in native plants invaded by DSV collectively relative to uninvaded natives, an observation that has been supported by other researchers (Greipsson and DiTommaso 2006, Smith et al. 2008). There was also a shift in the AMF phylotypes themselves. We observed a decrease in the quantity of significant AMF phylotypes (determined by PCA) colonizing natives in the invaded sites. The explanation for this decrease in AMF phylotypes could be varied; however, fungal infection loci in roots, which are limited in number, may be dominated by relatively few AMF species such as Glomus intraradices which can propagate via fungal fragments in mechanically disrupted sites (unlike many AMF) and preferentially colonize invasives (Daniell et al. 2001, Greipsson and DiTommaso 2006).

The majority of dominant TF phylotypes in DSV showed significantly increased representation in the natives-in-dsv over native counterparts. While the fungal species have not been elucidated in this investigation, there is a reasonable likelihood that a component of the observed increases in the general fungal phylotypes would include pathogenic or harmful fungi. We compared significant FAM- and HEX-labeled TRFs to BLAST sequences (Bongard et al. 2013), and the TF species with BLAST matches > 90% similarity included predominantly Tetracladium spp., Fusarium spp., or uncultured Ascomycota. These fungi have been described as generalists, and also include important plant pathogens (Roldán et al. 1989, Priest et al. 2003). Though invaders can accumulate pathogens in their introduced range, the Enemy Release hypothesis suggests that they may avoid deleterious effects of novel pathogens (Callaway et al. 2008), and local generalist pathogens may have a more negative effect on the native plant species than on the invader (Eppinga et al. 2006, Mangla et al. 2008). Eppinga et al. (2006) hypothesized that while the accumulation of pathogens may initially limit invader abundance in the novel environment, it might feed back more negatively to the native plant community.

The use of relative fluorescence units:DNA concentration ratio as a proxy measurement of colonization density in this study is based on the assumption that DNA extraction efficiency across samples is comparable. Only the wild red raspberry may not be supported by this assumption as woody species are known to have tannins that can make DNA extraction more challenging. There is no assumption that this proxy for colonization density infers that the relative biomass of the various root samples is consistent. However, given that the same approximate amount of root tissue was used for the initial DNA extractions (i.e. 200 mg), and that there was high fidelity between replicates of both PCR amplification and T-RFLP runs, it is reasonable to conclude that normalized peak heights/DNA concentration provides an adequate proxy for colonization density. Though bias may result from differences among fungal types in the copy number of the targeted gene and differences in extraction efficiency (Nagashima et al. 2003), Burke et al. (2006) found T-RFLP to be a reliable method of microbial abundance analysis across natural communities relative to traditional methods. Peak height was chosen over integrated peak area for relative colonization density analysis in this study due to the overlap of some peaks (Stephauskas et al. 2003).

It would be expected that the TF community in natives would be less diverse/abundant relative to natives-in-dsv if diverse fungi associated with invaders (that are not present in natives) are being introduced subsequent to invasion (Brundrett 2004). Our ability to differentiate natives from natives-in-dsv based on phylotype colonization patterns suggests that there is a generalizable phenomenon at play. While in some cases the individual plant species accounted for more of the variance in the fungal communities than the plant ‘type’ in the present study, our investigation was focused on comparing the uninvaded and invaded natives species collectively as a first step. It would be important to examine each native species independently, with an increased sample size.

Soil fungi are an important consideration in plant fitness, plant community composition, and larger ecosystem processes, and as such, alterations to fungal communities is one likely means to successful invasion by introduced plant species. Elucidating invader fungal association dynamics could support strategies used by restoration ecologists in reclaimed sites where fungal inoculation is being considered. Given the findings of this and other related studies, further investigations into the general fungal and AMF association dynamics pre- and post-invasion are strongly warranted.