Linkage between exotic earthworms, understory vegetation and soil properties in sugar maple forests

Forest Ecology and Management 364 (2016) 113–121

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Linkage between exotic earthworms, understory vegetation and soil properties in sugar maple forests Mélanie Drouin a, Robert Bradley a, Line Lapointe b,⇑ a b

Département de biologie, Université de Sherbrooke, 2500 boul. de l’Université, Sherbrooke, Québec J1K 2R1, Canada Département de biologie, Université Laval, 1045 avenue de la Médecine, Québec, Québec G1V 0A6, Canada

a r t i c l e

i n f o

Article history: Received 24 July 2015 Received in revised form 10 January 2016 Accepted 11 January 2016 Available online 18 January 2016 Keywords: Arbuscular mycorrhizal fungi Forest floor thickness Hardwood forests PLFA NLFA Plant biodiversity

a b s t r a c t The comminuting and soil mixing activities of earthworms can affect soil physical, chemical and biological properties, which in turn can influence plant growth and survival. Accordingly, there is growing concern that the spread of exotic earthworms into northern temperate forests may compromise biodiversity and tree species recruitment. We report on a study where we sampled earthworms, soils, and understory plants in plots established in 40 mature sugar maple stands distributed over 3 areas in the Eastern Townships of Southern Québec (Canada). Earthworms were found in 19 of 40 plots, and earthworm frequency of occurrence (Efo) as well as the complexity of earthworm communities reflected human accessibility to the plots. Plant species richness decreased, and species evenness increased, with Efo. The Efo was related to a decrease in the cover of 5 plant species, and to an increase in the cover of 2 other plant species or plant functional groups. Increasing Efo also correlated with higher soil pH, lower forest floor thickness and lower soil C:N ratio. Among these 3 variables, redundancy analysis (RDA) revealed that soil pH and forest floor thickness correlated with plant community composition. Based on neutral lipid and phospholipid fatty acid profiles, we found that soil bacteria and fungi increased with a decrease in forest floor thickness, bacteria and arbuscular mycorrhizal fungi (AMF) increased with soil pH, whereas actinobacteria and AMF increased with Efo. We discuss the possible mechanisms by which earthworms might directly or indirectly alter understory plant community composition. By considering the location and land use management of each study site, our study provides further evidence that the spread of exotic earthworms in sugar maple stands of Southern Québec may be linked to human activities, with implications for further research and conservation issues. Ó 2016 Elsevier B.V. All rights reserved.

1. Introduction In southern Québec (Canada), it has been established that native earthworm species did not survive the Wisconsin glaciation, which ended over 11,000 years ago (Gates, 1970; James, 1995). Accordingly, 17 of the 19 known earthworm species in southern Québec were introduced in recent centuries by European settlers (Addison, 2009). Given that their natural rate of spread is no more than 5–10 m yr 1 (Marinissen and van den Bosch, 1992), and given that they are predominantly found in agricultural fields, along roads and near fishing lakes, exotic earthworm dispersal throughout the landscape is thought to mainly be mediated by human activities (Gundale et al., 2005; Keller et al., 2005; Tiunov et al., 2006; Holdsworth et al., 2007a; Cameron and Bayne, 2009;

⇑ Corresponding author. E-mail addresses: [email protected] (M. Drouin), Robert.Bradley@ USherbrooke.ca (R. Bradley), [email protected] (L. Lapointe). http://dx.doi.org/10.1016/j.foreco.2016.01.010 0378-1127/Ó 2016 Elsevier B.V. All rights reserved.

Sackett et al., 2012). In northern temperate forest ecosystems similar to those found in southern Québec, studies have revealed negative effects of exotic earthworms on the recruitment of certain plant species (e.g., Hale et al., 2006; Corio et al., 2009), which has prompted others to reflect on the need for land management policies that could mitigate earthworm dispersal (e.g., Callaham et al., 2006; Hale, 2008). In order for policy makers in southern Québec to engage in such a dialogue, empirical data is required to show a correlation between earthworm abundance, and earthwormmediated impacts on forest vegetation and soil properties in this region. The introduction of exotic earthworm species into previously earthworm-free northern temperate forest soils, such as in Minnesota and Wisconsin, was shown to either increase or decrease the abundance of different plant species, resulting in an overall net reduction of understory plant diversity (Gundale, 2002; Hale et al., 2006; Holdsworth et al., 2007b; Corio et al., 2009; Gibson et al., 2013). Various mechanisms may explain a negative effect

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of earthworms on the recruitment of certain plants. A few manipulative studies have shown, for example, that comminuting and soil mixing activities of earthworms might have a direct effect on seed viability (McCormick et al., 2013), seed germination (Milcu et al., 2006; Aira and Piearce, 2009; Eisenhauer et al., 2010; Drouin et al., 2014) or seedling survival (Eisenhauer et al., 2010; Drouin et al., 2014). Earthworms could also have indirect effects on the performance of understory plant species by modifying the physico-chemical and biological properties of surface soil in which plant roots thrive (Sackett et al., 2013). For example, earthworms may reduce or eliminate organic forest floors (Hale et al., 2006; Gundale et al., 2005), thereby increasing soil moisture loss resulting in a negative impact on plants with shallow root systems (Corio et al., 2009). A second mechanism by which earthworms might indirectly affect understory plant species is by modifying the structure of soil microbial communities. For example, Lawrence et al. (2003) reported a negative effect of earthworms on the colonization of sugar maple (Acer saccharum Marsh.) roots by beneficial arbuscular mycorrhizal fungi (AMF). A third indirect mechanism is that earthworms might alter the availability of soil nutrients such as N and P (Hale et al., 2005a; Groffman et al., 2015; Resner et al., 2015), as well as certain groups of soil fauna (Eisenhauer et al., 2007; Snyder et al., 2011), all of which could reduce the performance of certain understory plants. Sugar maple stands are found throughout the St. Lawrence Lowlands and in most of the geographic area between the St. Lawrence River and Vermont, which makes it one of the most abundant forest types in southern Québec. Moreover, sugar maple holds a high economic value because of its timber, its fibre for pulping, and for the production of maple syrup. Studies have reported negative effects of exotic earthworms on the recruitment of sugar maple in the understory (e.g. Hale et al., 2006; Holdsworth et al., 2007b; Corio et al., 2009), and these same studies also found a higher recruitment of various Fraxinus spp. and Carex spp., which are also abundant in sugar maple stands of southern Québec. Thus, mature sugar maple stands with similar understory species was a sensible choice of forest type for us to conduct the first regional survey on the ecological impacts of exotic earthworms in southern Québec. Our first objective was to test whether earthworm abundance correlated with changes in the relative abundance and diversity of various understory plant species. Our second objective was to explore possible relationships between soil properties and changes in understory plant communities potentially caused by the presence of earthworms. Our longer-term objective was to provide data that might foster dialogue on land management policies to mitigate earthworm dispersal in the province of Québec.

2. Materials and methods 2.1. Study area, experimental design and field sampling The Eastern Townships (10,508 km2) in southern Québec (Canada) lie within the sugar maple–basswood biogeoclimatic zone (Gosselin, 2007). Mean annual rainfall and temperature for the region, based on 30-year running averages for the City of Sherbrooke, are respectively 1144 mm and 4.1 °C (Environment Canada, 2013). In September 2010, a total of 40 square sampling plots (9 m2) were established across 5 sites located within a 25 km radius around Sherbrooke (Fig. 1). Two of these sites, each comprising 10 plots, were 11 km apart on land owned by Domtar Corporation (a pulp and paper company) and located northeast of the town of Windsor (Area #1). The area in which these sites are found is not freely accessible to the public but is traversed by secondary roads accessible only to logging trucks. Two other sites,

each comprising 5 plots, were located 8 km apart within the Parc National du Mont-Orford, which is 5 km north of the City of Magog (Area #2). One of these sites is a conservation area that is inaccessible to the public whereas the second site is visited by thousands of campers and fishermen each year. The fifth site, comprising 10 plots, was Mont-Bellevue, a municipal recreational forest in the City of Sherbrooke with heavy human traffic (Area #3). The 5 different sites are thus associated with different levels of human activities. The distance between neighbouring plots within each site varied between 100 and >1000 m. Each sampling plot was established in a mature sugar maple stand with common understory species. In September 2010, 3 parallel transects were established 1.5 m apart from one another. Fresh litter and woody debris were removed and 4 soil cores (7.5 cm dia., 30 cm depth) were sampled at 1 m intervals along each transect (n = 12 cores per plot). The thickness of the forest floor F horizon (Soil Classification Working Group, 1998) in each core was measured, and these values were used to calculate the average forest floor thickness in each plot. Each soil core was then placed in a plastic bucket and dissected by hand to ascertain the presence (i.e. at least 1 individual) of earthworms. The same earthworm sampling scheme was repeated in late June 2011, using 3 parallel transects that were established at 75 cm distance from the transects used in the previous year. The frequency of abundance of earthworms (Efo) in each plot was based on the total number of cores, with the 2 sampling dates combined (i.e. n = 24 cores per plot), in which at least one earthworm was detected. In October 2012, a final visit was made to each plot to collect earthworm specimens for identification purposes. We applied a mustard solution (4 g L 1) over the entire area of each plot to expel earthworms from the soil (Gunn, 1992; Lawrence and Bowers, 2002). The specimens were preserved in aqueous 90% ethanol and brought back to the laboratory. Sexually mature individuals were identified morphologically using Reynolds’ (1976) identification key. Following the first earthworm survey in September 2010, all soil cores within each plot were pooled, earthworms and other visible fauna were removed, the soil was gently mixed by hand, and a 500 g subsample was kept under ice packs in a cooler. These 40 bulk soil samples were transported to the University of Sherbrooke, where they were sieved to pass a 5 mm mesh and stored at 4 °C until their physicochemical properties could be analyzed. A soil subsample (ca. 20 g) from each plot was immediately frozen until analyzed for its lipid fatty acid profile. Other data that were gathered on this date included an estimate of tree basal area using a 2-factor wedge prism, and soil drainage class according to the criteria developed by the Expert Committee on Soil Survey (1982). In early June 2011, the understory vegetation in each plot was surveyed using the line-intercept method (Kent and Coker, 1992). Stakes were driven into the soil at the 4 corners of each plot and at 1 m intervals between the 4 corner stakes. Twine was strung across opposing stakes and all understory plants, including juvenile trees <5 cm stem dia., which intersected the twine along its length were noted. To account for plant species that might only appear later in the summer, an identical survey was conducted in late August 2011. Most species were recorded on both dates, and their abundance values were thus averaged over both sampling dates. For the few species that were recorded on a single date, the abundance value for that single date was used. 2.2. Soil physico-chemical properties For each of the 40 sampling plots, soil pH was measured electrometrically using 1:5 soil:water slurries. Total C and N were measured by high temperature combustion followed by thermoconductometric detection, using a Vario Macro elemental analyzer (Elementar Analysensysteme GMbH, Hanau, Germany). A 100 g

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Fig. 1. Five main sampling sites located in the Eastern Townships of Québec, Canada. Shaded areas represent the total areas of Domtar-owned land (Windsor 1 and 2), of Parc National du Mont-Orford (Orford 1 and 2) and of Mont Bellevue.

soil subsample was ashed at 550 °C for 16 h in a muffle furnace to determine its organic matter content (i.e. loss-on-ignition method), and the textural class of the remaining mineral soil was determined by particle-size analysis using the hydrometer method (Bouyoucos, 1962). Mehlich-extractable base cations (Na, Ca, Mg, K) were analyzed by atomic absorption spectroscopy (AAnalyst 100, Perkin–Elmer Corp., Waltham, MA), whereas Mehlich-P (Mehlich, 1984) was analyzed colorimetrically at 882 nm absorbance (Murphy and Riley, 1962) with a Spectro 1200 spectrophotometer (UNICO Corp., Princeton, NJ). Fresh soil subsamples (15– 20 g) were extracted with 100 mL of 1.0 N KCl, and the filtered extracts were analyzed colorimetrically for NH+4 and NO3 on a Technicon II autoanalyzer respectively using the Berthelot and Griess-Ilosvay methods (Mulvaney, 1996). The sum of cation charges (cmol + kg 1) that corresponded to Na+, Ca2+, Mg2+, K+, and NH+4 concentrations, together with exchangeable acidity, was used as a proxy for cation exchange capacity (CEC). 2.3. Soil microbial community structure Soil microbial community structure in each plot was determined from phospholipid and neutral lipid fatty acid (PLFA and NLFA) profiles, using the procedure described in detail by Hamel et al. (2006). PLFA and NLFA concentrations were estimated from the detection of methylated 19:0 FA (Sigma–Aldrich, St. Louis, MO), which was introduced as an internal standard. Concentrated PLFA and NLFA extracts were dissolved in hexane and injected into a HP 6890 gas chromatograph equipped with a flame ionization detector (300 °C), a 30-m Restek Rtx-1 column, and He as carrier gas. Microbial community structure was based on the FA nomen-

clature that was proposed by Ratledge and Wilkinson (1988). Peaks of interest were identified, based on the retention times of 41 FA standards (Supelco Bacterial Acid Methyl Ester Mix 47080-U, Sigma–Aldrich). The abundance of these FAs was estimated from the area under each peak relative to the area below the 19:0 peak, which was calibrated according to a standard curve made from 19:0 FA standards. FA 18:2x6c was ascribed to fungal biomass (Frostegård and Bååth, 1996). FAs i14:0, i15:0, a15:0, i16:0, i17:0, a17:0, 10me16:0, 10me18:0, br17:0, br18:0 were ascribed to Gram + bacteria (Ratledge and Wilkinson, 1988; Zelles, 1999), while FAs cy17:0, cy19:0, 16:1x7c, 16:1x7t, 16:1x9, 18:1x7 were ascribed to Gram– bacteria (Ratledge and Wilkinson, 1988; Zelles, 1999). Total bacterial biomass was calculated as the sum of Gram+ and Gram bacteria, plus FA 17:0 (Harwood and Russell, 1984). FAs 16:1w5 and 10Me18:0 were respectively ascribed to arbuscular mycorrhizal fungi (AMF) (Madan et al., 2002) and to actinobacteria (actinomycetes; Frostegård et al., 1993). Although membrane fatty acids are widely used as biomarkers of specific soil microbial groups (e.g. Contosta et al., 2015), caution is warranted when interpreting these data because specific fatty acids may be common to different bacterial groupings (Frostegård et al., 2011). Our prime reason for studying microbial community composition was to test the effects of earthworms on AMF abundance in sugar maple stands, as was reported by Lawrence et al. (2003). In their review paper, Allison and Miller (2004) gave a favourable appraisal of 16:1w5 as a biomarker, citing 10 papers that specifically correlated this fatty acid to AMF hyphal weight or AMF hyphal length. On the other hand, Ngosong et al. (2012) found that PLFA 16:1x5 as a biomarker could be hampered by background levels derived from bacteria whereas NLFA 16:1x5

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was a feasible indicator for AMF in complex field soils. Here, we analyzed both PLFA and NLFA fractions in order to increase confidence in the interpretation of our results.

2.4. Statistical analyses Redundancy analysis (RDA; Borcard et al., 2011) was used to explore inter-set correlations between soil properties (including Efo) and the abundance of 17 selected plant species (i.e. plant community response matrix), together with intra-set correlations within these 2 matrices. We first verified for collinearity among soil variables, which led us to exclude% silt (correlated with% sand) and total base cations (correlated with CEC). Total-N, NH+4–N and NO3 –N concentrations were replaced by the ratios of (NH+4–N: total-N) and (NO3 –N:total-N). We removed extractable-P since the measured concentrations of this soil nutrient were often near our instrument’s threshold detection limit (<5 mg kg 1). The soil data were normalized, given that different units and scales were employed for each measurement. Because of the frequent occurrence of zero values, we executed a Hellinger transformation on the plant data matrix in order to give proportionate weight to the rarer plant species (Legendre and Gallagher, 2001). A second RDA was performed after sorting the 17 selected plant species into 5 functional groups (tree seedlings, shrubs, ferns, graminoids, broadleaf herbs), to see if this would yield stronger relationships once the species with common traits were grouped together. Both RDAs were followed by Parsimonious RDA Analysis (Borcard et al., 2011), which extracts the% variance solely explained by the significant environmental variables. For each parsimonious RDA, we used a variance inflation factor (VIF < 5) threshold in order to control for multicollinearity among variables (Borcard et al., 2011). We used regression type II models (Legendre and Legendre, 2012) to test the effects of Efo on soil properties, understory plant species richness, evenness and Shannon diversity (Magurran, 1988). As our objective was to study the putative impacts of earthworms on common understory plant species, our analyses included only those plant species that occurred in all 5 sampling sites and which were present in at least 40% of plots per sampling site. Our analyses thus included 17 species or species groups: A. saccharum, Acer pensylvanicum L., Acer rubrum L., Arisaema triphyllum (L.) Schott, Athyrium fillix-femina (L.) Roth, Betula alleghaniensis

Britt., Cystopteris fragilis (L.) Bernh., Dryopteris intermedia (Mull. ex Willd.) A. Gray, Fagus grandifolia Ehrh., Fraxinus spp., Maianthemum canadense Desf., Prunus serotina Ehrh., Rubus idaeus L., Sambucus canadensis L., Thelypteris noveboracensis (L.) Nieuwl., Trillium erectum L., and graminoid species. Linear regressions were also used to test the effect of Efo on each individual plant species. The validity of these regressions depended on the respect of 2 conditions: (1) an adequate distribution of the Efo among all sampling sites, to separate the effect of sampling site location from that of earthworms on plant communities, and (2) that the variation in tree seedling abundance be independent of seed source and propagule pressure. To address the first of these conditions, we considered the fact that earthworms were absent in 15 of the 20 plots that were located in Windsor 1 and 2 sampling sites; hence, all significant regressions that were obtained using 40 plots were validated by repeating the regressions after excluding the 20 Windsor plots. To address the second condition, we performed correlation analysis between tree seedling recruitment and the abundance of mature individuals (i.e. those tallied with the wedge prism) in each plot. To explore possible correlations between soil physico-chemical properties and soil microbial community structure, we performed 2 more RDAs using PLFA and NLFA abundances as the response data matrices. Again, these 2 RDAs were followed by Parsimonious RDA analysis. Based on the results of RDA analyses (data not shown), we were led to further test the effects of forest floor thickness and soil pH on selected microbial groups using simple linear regressions. All statistical analyses were performed using the vegan package (Oksanen et al., 2012) in the R statistical environment (Version 2.14.0).

3. Results Values of Efo were highly variable across the 40 plots, with median = 0, mean = 7 and standard deviation = 10 (Table 1). Earthworms occurred in 19 of the 40 sampling plots, and collectively included a total of 7 exotic earthworm species: 1 epigeic species (Dendrobaena octaedra Savigny) 1 epi-endogeic species (Lumbricus rubellus Hoffmeister), 4 endogeic species (Allobophora chlorotica Savigny, Aporrectodea rosea Savigny, Aporrectodea turgida Eisen, Octolasion tyrtaneum Savigny) and 1 anecic species (Lumbricus ter-

Table 1 Means, variances, minimum and maximum values of Efo, of each soil physico-chemical property, as well as more general site properties (N = 40 plots). Results of linear regressions between the Efo and the different soil and site properties are also shown. NA = Not applicable, SD = Standard deviation.

a

Variable

Median

Mean

SD

Min–max

Slope

Adj. r2

P-value

Efo (# of cores/plot)

0

7

10

0–24

NA

NA

NA

Soil physico-chemical properties pH in water %C %N C:N ratio % Clay % Sand Na (mg (100 g) 1) Ca (mg (100 g) 1) Mg (mg (100 g) 1) K (mg (100 g) 1) CEC (meq (100 g) 1) NH+4–N: total-N ratio (%) NO+3–N: total-N ratio (%)

4.83 5.2 0.40 13.3 0.6 59.0 4.8 39.3 42.6 8.0 6.8 0.06 0.42

4.96 6.5 0.44 14.0 1.1 59.2 5.9 45.5 82.8 26.8 10.1 0.10 0.49

0.59 4.5 0.19 2.4 1.4 8.3 3.1 13.1 114.7 56.0 10.6 0.11 0.50

4.12– 6.35 3.2–28.1 0.28–1.24 10.7–22.7 0.00–5.00 34.0–77.5 2.6–17.1 33.5–73.5 13.4–610.8 1.9–295.4 3.2–55.4 0.02–0.55 0.00–2.25

0.020 0.106 0.002 0.120 0.017 0.237 0.020 0.143 0.017 1.106 0.021 <0.001 <0.001

0.087 0.030 0.010 0.227 0.011 0.057 0.026 0.014 0.026 0.013 0.026 0.009 0.015

0.036a 0.150 0.440 0.001 0.450 0.075 0.970 0.500 0.990 0.230 0.900 0.250 0.520

Site properties Drainage class (1–7) Forest floor thickness (cm) Tree basal area (m2 ha 1)

NA 3.5 26

NA 3.1 26

NA 2.0 6

Poor–rapid 0.0–6.0 10–42

0.012 0.172 0.049

0.000 0.720 0.020

0.320 <0.001 0.640

P value in bold are statistically significant (P 6 0.05).

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RDA1 -1.0

-0.5

0.0

Variable Adj. R 2 1.0 0.110 Efo 0.055 pH 0.026 NH4:N

0.5

NO3:N

P value 0.001 0.001 0.024

0.5 0.5 As Fspp

FF

0.0

Mg CEC

BA Ps Te BaSc Ap Fg CLAY Di C:NSAND Ar

E fo

At K Na

RDA2

RDA2

Cf

0.0

Aff RiCa

DRN

pH

Mc Gr

Tn

-0.5

-0.5

NH4:N

-0.5

0.0

0.5

RDA1 -1.0

-0.5

0.0

2

Variable 1.0 Adj. R FF 0.235 0.5 pH 0.017

0.5

P value 0.001 0.017

NH4:N

b

0.5

RDA2

pH DRN

0.0

E fo

Sh

0.0

Fe

FF

Fo

SAND

RDA2

Gr

CLAY

Tr

K Ca

Na BA

-0.5

C:N

NO3:N

Mg CEC

-0.5 -1.0

-0.5

0.0

0.5

1.0

RDA1 Fig. 2. Bi-plots generated from redundancy analysis using soil properties as the environmental data matrix and the abundance of (a) plants species or (b) plant functional groups, as response variables. Significant explanatory variables are framed in the upper right corner of each bi-plot. Symbols denoting plots are the same as for Fig. 1. The plant species are abbreviated as follows: Acer saccharum (As); Acer pensylvanicum (Ap); Acer rubrum (Ar); Arisaema triphyllum (At); Athyrium fillix-femina (Af); Betula alleghaniensis (Ba); Cystopteris fragilis (Cf.); Dryopteris intermedia (Di); Fagus grandifolia (Fg); Fraxinus spp. (Fspp); Maianthemum canadense (Mc); Prunus serotina (Ps); Rubus idaeus (Ri); Sambucus canadensis (Sc); Thelypteris noveboracensis (Tn); Trillium erectum (Te); graminoid species (Gr). The plant functional species are abbreviated as follows: broadleaf herbs (Fo); ferns (Fe); graminoids (Gr); shrubs (Sh); tree seedlings (Tr). The soil properties are abbreviated as follows: earthworm frequency of abundance (Efo), forest floor thickness (FF) and tree basal area (BA).

restris L.). No earthworms were observed in plots from the Orford 1 sampling site. A single species of epigeic earthworm, D. octaedra, was observed in Windsor 1 and 2 sampling sites. The Mont Bellevue and Orford 2 sampling sites had the most complex earthworm species assemblages, in which epigeic, endogeic and anecic earthworms were identified. Most plots in the Mont Bellevue site contained only 1 or 2 species, whereas plots in the Orford 2 site contained 3 or 4 species. Soil pH increased, whereas C:N ratio and forest floor thickness decreased, with increasing Efo (Table 1). According to RDA, soil properties (including Efo, forest floor thickness and physico-chemical characteristics) explained 54.8% of the variance in plant species composition (Fig. 2a). The first and second ordination axes collectively explained 32.2% of that variance, and 3 variables (Efo, pH and NH+4:total-N ratio) contributed significantly (VIF 6 1.2) to the variance. Of the plant species that were included in this first RDA, C. fragilis was the most negatively correlated to Efo and to soil pH, whereas Fraxinus spp.

and A. triphyllum were the most positively correlated to the same 2 soil variables. The parsimonious RDA (not shown) that used only these 3 explanatory variables showed that they collectively explained 29.1% of the variation in plant species abundances. In the second RDA that was performed after sorting plant species into 5 functional groups, soil properties explained 53.0% of the variance in plant community composition, with the first and second axes alone accounting for 45.8% of the total variance (Fig. 2b). In this second RDA, forest floor thickness and soil pH contributed significantly (VIF < 1.2) to plant community composition, and explained 32.1% of the variance in the parsimonious RDA. Both RDAs clearly indicated that the Efo and litter thickness were oppositely correlated to each other and that they significantly influenced plant community composition. Shannon diversity did not vary with Efo (r2 = 0.009, P = 0.25), however species richness decreased and species evenness increased with Efo (Fig. 3). The Efo had a significant effect on 7 of

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17 understory plant species or plant functional groups (Fig. 4). Of these plant species, A. pensylvanicum, A. rubrum, C. fragilis, D. intermedia, and F. grandifolia decreased as Efo increased. In contrast, the abundance of Fraxinus spp. and graminoids increased with increasing Efo. When we excluded the 20 Windsor plots from the analysis, the same significant relationships persisted except for F. grandifolia and graminoids, where the effect of Efo was no longer statistically significant (data not shown). Among the 4 tree species included in Fig. 4, only F. grandifolia showed a significant positive correlation (r2 = 0.224, P < 0.01) between seedling recruitment and the abundance of mature trees (data not shown). It was interesting for us to note that Acer saccharum seedling abundance did not correlate with Efo (r2 = 0.011, P = 0.39). We tested the effects of Efo, forest floor thickness and soil pH on microbial lipid fractions through linear regressions (Table 2). These last 2 variables were selected since they were both significantly related to Efo (Table 1) and to plant community composition (Fig. 4). As estimated from the 2 lipid fractions (NLFA and PLFA), bacterial groups and fungi (including AMF) increased with a decrease in forest floor thickness. Only bacterial groups and AMF increased with soil pH, and only with PLFA fractions in the case of gram bacteria and actinobacteria (Table 2). NLFAs of actinobacteria and PLFAs of AMF increased with Efo (data not shown).

fairly confident that differences in tree seedling recruitment was not the result of seed source and propagule pressure, as seedling abundances for most tree species were not related to the abundance of mature individuals. Thus, the evidence prompts us to explore the rest of our data set in order to speculate on the mechanisms by which exotic earthworms might affect the understory plant community. One mean by which earthworms could have altered the understory vegetation is by reducing seed germination and/or seedling survival rates of certain plant species. For example, Eisenhauer et al. (2010) observed anecic earthworms that actively buried and ostensibly consumed newly germinated seedlings, whereas Drouin et al. (2014) showed a similar effect of L. terrestris (an anecic species) on the survival of A. rubrum seedlings. This is consistent with our results that show A. rubrum to be the tree species the most negatively affected by earthworms. Seedling survival in the presence of earthworms can also decline if, for example, a plant’s rooting habit is adapted to a permanent forest floor. For example, our results suggest that species such as M. canadense, which produce shallow roots and rhizomes (Lapointe et al., 2010) that are likely confined to the forest floor, could be negatively affected by earthworms compared to more deeply rooted species such as graminoids. Finally, earthworms can also consume fine roots (Gilbert et al., 2014) which would increase the susceptibility of seedlings and shallow-rooted herbs to water stress. Efo increased the abundance of soil microbes as reported previously in similar habitats (Dempsey et al., 2013; Groffman et al., 2015), either directly or indirectly through a reduction of the forest floor thickness (Table 2). Increased biomass of different soil microbes could in turn influence plant growth and survival. Of particular interest to us was the positive effect of earthworms on the biomass of symbiotic AMF. Such a positive relationship had already been reported in northern hardwood forests dominated by sugar maple and white ash (Dempsey et al., 2013). It is generally accepted that ectomycorrhizal (ECM) fungi are more abundant in mor type forest floor humus, whereas AMF are more concentrated in mull type mineral soil horizons (Smith and Read, 2008). Hence, the comminuting and burrowing activities of earthworms could have favoured plant species strictly associated to AMF, such as Fraxinus spp. (Wang and Qiu, 2006), and reduced the competitive ability of those strictly associated to ECM, such as F. grandifolia or B. alleghaniensis (Wang and Qiu, 2006). This argument does not hold, however, for A. rubrum and A. pensylvanicum, that are also strictly associated to AMF (Wang and Qiu, 2006), but were negatively correlated to Efo. Similarly, Dobson and Blossey (2015) reported a decrease in survival of transplants of Fraxinus americana and of 11 other native understory species – mostly AMF dependant – in presence of earthworms. Therefore, the net effect of Efo on plant species abundance most likely depends on the resulting balance between its effects on plant germination and survival and its effects on the microbial communities, including changes in the abundance of mycorrhizal species.

4. Discussion

4.2. Impacts of exotic earthworms on soil properties

4.1. Impacts of exotic earthworms on understory plant communities

In our study, we report average soil physico-chemical properties pooled across a common depth of 30 cm rather than by taxonomic soil horizon, as this describes the different environments in which plant roots developed across the 40 plots. Thus, a thick forest floor in a given soil core resulted in proportionately less mineral soil in the sample, and vice versa. Of the 3 soil properties that were significantly related to Efo (i.e. soil pH, C:N ratio, forest floor thickness), soil pH is the most tricky to explain. Some studies have suggested that low soil pH will reduce earthworm abundance and survival (Haimi and Einbork, 1992), although it has long been known that many earthworm species may withstand the range of

Species richness

30

a

r 2 = 0.113 P = 0.019

25 20 15 10 5 0 0

Species evenness

0.25

6

b

12

18

24

18

24

r 2= 0.099 P = 0.027

0.20 0.15 0.10 0.05 0.00 0

6

12

Efo per plot Fig. 3. Relationship between earthworm frequency of occurrence (Efo) and (a) understory plant species richness and (b) evenness (N = 40 plots). The Efo is based on the number of cores per plot (maximum = 24) in which earthworms were observed. Symbols are the same as for Fig. 1.

Our study confirmed that Efo is statistically related to changes in understory plant communities, such as a decrease in species richness and an increase in species evenness. Furthermore, Efo was significantly related to the abundance of 7 plant species across the 40 sampling plots. Given that this relationship remained significant for 5 of the species after eliminating the Windsor plots from our regression analyses, we are fairly confident that the effects of earthworms on plant communities did not arise from zeroinflated distributions of the predictor variable. Likewise, we are

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20

20

a

Ap r2 = 0.081 P = 0.042

15 10 5

Ar r2 = 0.242r P = <0.001

10 5

0

0 0

250

6

12

18

24

0 40

c

Cf r2 = 0.101 P = 0.026

200 150

Number of individuals per plot

b

15

6

12

d

18

24

Di r2 = 0.292 P = <0.001

30 20

100 10

50

0

0 0 12

6

12

18

80

e

10

0

24

Fg r2 = 0.080 P = 0.043

8 6

6

f

12

18

24

18

24

Fspp r2 = 0.201 P = 0.002

60 40

4 20

2 0

0 0

160 140 120 100 80 60 40 20 0

6

g

0

12

18

24

0

6

12

Gr r2 = 0.124 P = 0.015

6

12

18

24

Efo per plot Fig. 4. Significant relationships between earthworm frequency of occurrence (Efo) and the number of individual plants per plot, for seven plant species (or groups of species). The species shown here were present on at least 40% of plots in all five sampling sites. The Efo is based on the number of cores per plot (maximum = 24) in which earthworms were observed. Symbols are the same as for Fig. 1. The plant species are abbreviated as follows: (a) Acer pensylvanicum (Ap), (b) Acer rubrum (Ar); (c) Cystopteris fragilis (Cf); (d) Dryopteris intermedia (Di); (e) Fagus grandifolia (Fg); (f) Fraxinus spp. (Fspp); (g) Graminoids (Gr).

soil pH values observed in our study (Laverack, 1961). On the other hand, earthworms may increase soil pH through the secretion of calcium carbonate, as found in the oesophageal lumen of some Lumbricidae species (Piearce, 1972). A second explanation is that the mixing of forest floor material with surface mineral soil buffers the acidic litter via the exchange of basic cations that are held on negatively charged clay particles. As for the negative relationship between Efo and the soil C:N ratio, this is consistent with earthworms increasing litter decomposition rates (Lee, 1985; Groffman et al., 2015). The effects of earthworms on soil pH and C:N ratio are thus both consistent with the negative relationship between Efo and forest floor thickness. 4.3. Distribution of exotic earthworms The distribution and abundance of exotic earthworm species that we observed reflect human accessibility to the different sites, in line with current thinking that earthworm dispersal is mostly

mediated by humans (e.g. Callaham et al., 2006; Holdsworth et al., 2007a; Sackett et al., 2012). For instance, the absence of earthworms in the Orford 1 site coincides with plots that are located in a remote and protected area, far from human habitation or trails. On the other hand, Windsor 1 and 2 sites are located in a privately owned forest, with a few secondary roads used mainly by hunters and logging trucks. There, we found a single epigeic earthworm species, D. octaedra. Finally, the most complex earthworm species assemblages, comprising epigeic, endogeic and anecic species, were located in the Orford 2 and Mont Bellevue sites. These sites are the most accessible to humans, located near roads and public hiking trails. In the Orford 2 site in particular, located close to a lake used by anglers, we found 3–4 earthworm species within each plot. Our earthworm survey thus provides strong support that human-mediated dispersal can occur either by active transport of live earthworms into new habitats (e.g. dumping of fishing bait; Sackett et al., 2012) or by unintentional transport of cocoons or juveniles along roads (e.g. within tire treads).

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Table 2 Results of linear regressions testing effects of forest floor thickness (cm) and soil pH on neutral lipid and phospholipid fatty acids (NLFA and PLFA) of various soil microbial groups (ng g 1); NS = not significant. Forest floor thickness Microbial group Gram + Gram Total bacteria Total fungi Bacteria/fungi Actinobacteria AMF a

Lipid fraction NLFA PLFA NLFA PLFA NLFA PLFA NLFA PLFA NLFA PLFA NLFA PLFA NLFA PLFA

Slope 21.2 53.2 33.1 60.6 55.8 115.6 16.0 NS NS NS 2.5 5.2 81.1 10.1

Adj. r2 0.199 0.133 0.084 0.100 0.131 0.118 0.080 0.006 0.007 0.025 0.129 0.072 0.101 0.173

Soil pH P-value a

0.023 0.012 0.039 0.026 0.013 0.017 0.043 0.390 0.270 0.850 0.013 0.052 0.026 0.004

Slope

Adj. r2

P-value

81.0 222.5 NS 272.0 186.0 498.9 NS NS NS NS NS 18.2 263.3 37.2

0.260 0.220 0.060 0.196 0.130 0.208 0.020 0.020 0.020 0.003 0.060 0.080 0.090 0.211

<0.001 0.002 0.070 0.003 0.010 0.002 0.580 0.780 0.630 0.300 0.060 0.044 0.030 0.002

P value in bold are statistically significant (P 6 0.05).

It is interesting to note that Dendrobaena octaedra, found in the Windsor 1 and 2 sites (i.e. low human accessibility), is an epigeic species. There is no reason to believe that other earthworm species could not have been transported to these sites, but their absence supports the idea that endogeic and anecic life forms introduced into earthworm-free forests result in non-viable sink populations. This is consistent with suggestions that epigeic earthworms are at the leading edge of invasion fronts (Addison, 2009). Hale et al. (2005b) proposed that epigeic and epi-endogeic earthworms could facilitate the establishment of endogeic and anecic species, by altering litter properties and initiating its decomposition. Their activity ostensibly would make forest floor material more accessible to deeper dwelling earthworm species, although this remains to be verified. 4.4. Implications and future research Our study provides evidence that exotic earthworms compromise the biodiversity and tree species recruitment of sugar maple stands in southern Québec. This should be cause for concern for conservationists, as the introduction of exotic L. terrestris in similar forests in Northern Minnesota has been linked to the eradication of Botrychium mormo (Warner), a rare understory fern (Gundale, 2002). According to the Québec government, there currently are 43 endangered and 16 vulnerable plant species in the province many of which grow in forest habitats (Centre de données sur le patrimoine naturel du Québec, 2008). If some of these species are susceptible to exotic earthworms, it is important that we understand the mechanisms by which this occurs. For example, exotic earthworms may alter understory communities by imposing 3 successive ecological filters on certain plant species: (1) by altering relative seed germination rates through seed burial, (2) by diminishing seedling survival through direct physical interactions with seedlings, and (3) by altering the competitive abilities of surviving seedlings through indirect effects on soil properties. Such a model could be the basis for structuring further research aimed at understanding and abating the spread of exotic earthworm populations into new habitats. Acknowledgements Our study was funded by a ‘‘Team Grant” from the Fonds québécois de la recherche sur la nature et les technologies (FQRNT) and by a ‘‘Discovery Grant” awarded to R. Bradley by the Natural Sciences

and Engineering Research Council (NSERC) of Canada. We wish to thank Dr. Chantal Hamel (Agriculture Agri-Food Canada – Swift Current, Saskatchewan) for advice on fatty acid analyses. We also are grateful to Domtar Corporation and to SÉPAQ for giving us access to some of the research sites.

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