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Soil nematodes differ in association with native and non-native dune-building grass species
Applied Soil Ecology xxx (xxxx) xxx–xxx
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Soil nematodes differ in association with native and non-native dunebuilding grass species Sarah M. Emerya, , Matthew L. Reida,b, Sally D. Hackerc ⁎
a
Dept. of Biology, University of Louisville, Louisville, KY 40292, United States of America Dept. of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI 48824, United States of America c Dept. of Integrative Biology, Oregon State University, Corvallis, OR 97331, United States of America b
ARTICLE INFO
ABSTRACT
Keywords: Ammophila breviligulata (American beachgrass) Elymus (Leymus) mollis (American dune grass) Foredune Succession NINJA US Pacific Northwest dunes
Non-native plant species can alter soil communities, which can lead to changes in their ecosystem functioning and facilitate invasive plant spread. In this study, we asked whether abundance, composition, or function of freeliving soil nematode communities differed in association with two dune building grass species in US Pacific Northwest coastal dunes. In 2016, we surveyed soil nematode communities along foredune profiles at six sites in Oregon and Washington, USA, where the non-native beach grass Ammophila breviligulata co-occurred with the native dune grass Elymus mollis. We also measured plant-associated factors that might influence nematode communities, including plant tissue nitrogen, arbuscular mycorrhizal associations, litter production, and plant community diversity associated with the two grass species across the foredune profile. Elymus mollis plots had over twice as many nematodes on average as A. breviligulata plots and were also associated with a higher nematode enrichment index, especially on dune heels. This is possibly explained by higher percent leaf nitrogen content in E. mollis. Subtle differences in nematode community composition and plant tissue nitrogen between the two grass species indicate that A. breviligulata may be altering nutrient cycling across the dune profile, which could explain the arrested succession associated with this species on back dunes. However, overall, nematode communities shifted more in response to foredune cross-shore position and stabilization as opposed to changes in vegetation.
1. Introduction Invasive plant species can dramatically alter bacterial, fungal, and invertebrate communities in soils following non-native plant invasion (e.g., Batten et al., 2006; Chen et al., 2007; Lazzaro et al., 2018; Mummey and Rillig, 2006; Renco and Balezentiene, 2015; Tanner et al., 2013; van der Putten et al., 2007). Such changes in belowground communities can affect nutrient cycling and other belowground ecosystem functions (Nielsen et al., 2011; Wagg et al., 2014). In particular, changes to soil nematodes, which occupy multiple trophic levels in soil food webs (bacterivores, fungivores, plant-parasites, and predators), can reflect changes in ecosystem successional status, nutrient cycling, and overall soil ecosystem health (Yeates, 2003). For example, Heracleum sosnowsyi invasion in grasslands in Europe reduced nematode diversity and altered nematode feeding guild composition, reflecting a more disturbed (lower Maturity Index) soil condition with higher contribution of bacteria to decomposition processes, and faster turnover rates of organic matter (Renco and Balezentiene, 2015).
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Invasive plants in primary successional systems such as sand dunes may have particularly strong effects on nematode community structure and function. Nematode communities are very responsive to disturbance and are good indicators of successional state on dunes, with early successional communities often dominated by predators and bacterivores and later-successional communities dominated by fungivores, reflecting changes in energy transfer pathways belowground (Zhi et al., 2009). Shifts in nematode abundance and community composition following plant invasion may indicate changes in successional processes and thus dune functioning. Beach and dune habitat covers ~42% of the coastline in the states of Oregon and Washington USA (Wiedemann, 1984) and provides important ecosystem services such as coastal protection, carbon sequestration, and tourism (Barbier et al., 2011). These Pacific Northwest dunes were historically dominated by native forbs and the native dune grass Elymus mollis, which sparsely covered this ecosystem (Cooper, 1958; Copeland et al., 2002; Wiedemann and Pickart, 2004). Starting in the early 1900s, US Pacific coast dunes were systematically planted
Corresponding author at: Dept. of Biology, University of Louisville, 139 Life Sciences Bldg., Louisville, KY 40292, United States of America. E-mail address: [email protected] (S.M. Emery).
https://doi.org/10.1016/j.apsoil.2019.06.009 Received 31 January 2019; Received in revised form 10 June 2019; Accepted 14 June 2019 0929-1393/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Sarah M. Emery, Matthew L. Reid and Sally D. Hacker, Applied Soil Ecology, https://doi.org/10.1016/j.apsoil.2019.06.009
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with the non-native European beachgrass Ammophila arenaria to stabilize a shifting sand environment that naturally lacked vegetation. In the 1930s, a separate non-native congener, American beachgrass Ammophila breviligulata was planted in dunes near the Columbia River and, by the 1980s, had spread and dominated northward along the Washington coast (Hacker et al., 2012; Seabloom and Wiedemann, 1994), where it has transformed the dune landscape from small, sparsely vegetated hillocks of sand to tall and densely vegetated linear ridges. Ammophila breviligulata is a superior dune builder compared to the native E. mollis because, despite having thinner stems and blades, it grows more densely and more clumped than E. mollis (Hacker et al., 2012; Zarnetske et al., 2012). This shift in geomorphology was accompanied by a decline in plant species richness (Hacker et al., 2012; Zarnetske et al., 2013) and slowing of dune plant succession (David et al., 2015). Ammophila breviligulata invasion may alter nematode communities through multiple ways. Smaller, less productive plants can decrease soil nematode abundance and density due to decreased belowground biomass, while differences in plant nutritional status or nutrient content can alter belowground resources and shift soil nematode community composition (Cortois et al., 2017; Yeates, 1987). Non-native plant species like A. breviligulata may suppress plant-parasitic nematodes (PPN) via processes of “enemy release” (Heger and Jeschke, 2014) such as is seen for the European beachgrass Ammophila arenaria (van der Putten et al., 2005). While belowground differences in nematode communities are often most distinct when comparing plants from different functional groups, even plant species belonging to the same functional group can show differences in their associated nematode communities. For example, Viketoft et al. (2005) found that nematode community diversity and composition significantly differed between species within the same functional groups (e.g., grasses, legume, or forbs), possibly due to differences in plant metabolites. Reid and Emery (2017) found that the invasive blue lyme grass, Leymus arenarius, was associated with reductions in PPN abundance compared to native dune grasses in the Great Lakes region, possibly due to enhanced benefits from arbuscular mycorrhizal fungi (AMF). In this study, we ask whether abundance, composition, or function of free-living soil nematode communities differ between these two dune-building grasses, and if so, what plant-associated factors are correlated with these changes. We also test whether nematodes reflect changes in belowground succession associated with dune stabilization or the two grasses by comparing belowground nematode communities across the foredune profile (from foreshore to backshore). We expect that A. breviligulata will be associated with decreases in soil nematode abundance, as A. breviligulata above- and belowground biomass is lower than E. mollis (David et al., 2016; Hacker et al., 2012), and will have lower fractions of PPN than E. mollis, possibly due to enemy release. We also predict that the nematode community will reflect plant successional patterns, with more mature nematode communities associated with E. mollis compared to A. breviligulata in backshore dunes, possibly due to increased soil organic matter contributions associated with the larger E. mollis plants.
Fig. 1. Site locations from south to north along the Pacific coast of Oregon and Washington USA used for this study. Original ID names correspond to those in Hacker et al. (2012).
foredune at two cross-shore positions on the foredune (toe, which is the foreshore position closest to the beach, and heel, which is the backshore position furthest from the beach). Each plot had a monoculture of one or the other grass species and we paired the plots such that each E. mollis plot had a paired A. breviligulata plot that was roughly 5 m away. In each plot, plant species richness and litter cover were visually estimated. We found that the dune heel had higher plant species richness (2.38 ± 1.58 vs. 0.83 ± 0.76 species m−2) and increased litter cover (60.63 ± 31.15 vs. 4.92% ± 5.99) compared to the dune toe. While overall plant diversity was very low in general, some common species at the dune toe were Cakile edentula and C. maritima and at the dune heel were Achillea millefolium, Erechtites minima, Frageria chiloensis, Hypochaeris radicata, Lathyrus japonicus, Lupinus littoralis, and Rumex acetosella. We also collected soil cores (2 cm diameter × 15 cm deep; 5 per plot) from near the bases of haphazardly selected plants of A. breviligulata or E. mollis in the plots, combined the cores for each plot, and stored them at 4 °C until processing. Finally, leaf blades were clipped from three individual tillers of each grass species and air dried back in the laboratory. The three leaf samples from each site and for each species in each foredune profile position were combined (n = 24) and then ground to a powder using a Mixer Mill (SPEX SamplePrep; Metuchen, NJ). The samples were analyzed for % total N at the stable isotope lab in the College of Earth, Ocean, and Atmospheric Science at Oregon State University (Corvallis, OR). 2.2. Nematode sampling We extracted free-living nematodes from fresh 100 ml subsamples of soil from each plot using the centrifugal flotation method (Jenkins, 1964). Nematode samples were placed in 3% formalin solution for longterm storage and identification. Nematodes in each sample were counted under a dissecting microscope (50×) and identified to genus (Andrássy, 2005, 2007, 2009; Bongers, 1988) under a compound microscope (100–1000×) and classified into feeding group (bacterivore, fungivore, plant-parasite, omnivore, or predator) based on Yeates et al. (1993). Nematodes were also classified to one of five colonizer-persister groups (cp1-cp5) based on life history traits and sensitivity to disturbance (Bongers, 1990; Yeates et al., 1993). The genera richness, PPN abundance, Maturity Index (MI; a measure of environmental disturbance and successional status), Structure Index (SI; a measure of food web connectedness), Enrichment Index (EI; a measure of resource availability, especially nitrogen), and Channel Index (CI; a measure of relative flow of energy through fungal vs. bacterial decomposition
2. Methods 2.1. Vegetation and soil sampling In June 2016, we conducted field sampling at six sites along a 100 km section of US Pacific Northwest coast (northern Oregon and Washington) where A. breviligulata and E. mollis co-occur (Fig. 1; Hacker et al. 2012). These sites are a subset of a larger long-term study of dune dynamics, and site details can be found in Hacker et al. (2012). Foredunes (i.e., linear hills of sand parallel to the shoreline) at these sites are short (~4 m) and wide (110 m) compared to other regions of the coast, and vary in community structure along their cross-shore profiles (see below). At each site, we laid out two 1 m2 plots (at least 10 m apart) on the 2
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channels) were calculated for each plot based on the nematode data using the NINJA program (Sieriebriennikov et al., 2014). These indices provide insight into the successional status and functioning of the soil nematode community (Bongers, 1990; Ferris and Bongers, 2009; Ferris et al., 2001).
3. Results 3.1. Grass species differences in nematode abundance and community composition When examining the entire free-living soil nematode community, E. mollis plots had 80% more total nematodes than the A. breviligulata plots (Table 1, Fig. 2). The most abundant genera overall were Eudorylaimus (cp4 omnivore), Plectus (cp2 bacterivore), and Rhabditis (cp1 bacterivore) (Table S1), and the ISA showed that the predatory genus Nygolaimus (cp5) was associated with A. breviligulata plots, while Eudorylaimus (cp4 omnivore) and Clarkus (cp4 predator) were more abundant in E. mollis plots (Table 2). Free-living PPN had very low abundances in general, only making up 2–5% of nematode communities (Table S1), and did not vary across plant species (Table 1; average PPN abundance [mean ± SD] = 0.34 ± 0.76100 ml−1 soil). There were no significant differences in nematode richness at the genus level across dune grass species (Table 1; average genera richness = 11.3 ± 2.7100 ml−1 soil), and no significant differences in feeding group composition across grass species (MANOVA results [F5,35, p] using Pillai Trace Statistics: Grass Species 1.57, 0.19; Dune Position 4.17, 0.004; Grass × Position 0.92, 0.48; Site 1.31, 0.16; Fig. 3). Overall nematode community composition and community structure indices also did not significantly differ between the two plant species (Figs. 4, 5). AMF root colonization and ERH length were similar for both dune grass species (A. breviligulata AMF root colonization = 76.0 ± 11.7%, ERH = 87.7 ± 120.1 mm g−1 soil; E. mollis root colonization = 71.8 ± 15.5%, ERH = 62.1 ± 55.7 mm g−1 soil). (Table 3). Elymus mollis had higher plant tissue nitrogen than A. breviligulata (1.12% ± 0.30 vs. 0.83% ± 0.34) ((Table 3).
2.3. Plant-associated measures As AMF can influence belowground nematode communities (Schouteden et al., 2015), we characterized the abundance of AMF in plots by measuring both colonization in plant roots, and extraradical hyphae (ERH) growth. For plant root colonization measures, we extracted fine roots from 100 ml subsamples of composited soil cores for each plot using a wet-sieve process (500 μm sieve; Milchunas, 2012). Roots were cleared with 10% KOH and stained using a 5% vinegar-ink solution following methods in Vierheilig et al. (1998). Visual estimation of percent root colonization was made based on 100 fields of view per sample under 200× magnification. To estimate ERH, we extracted hyphae from 20 ml subsamples taken from the pooled cores for each plot. Soil subsamples were suspended in water, then stained and vacuum filtered through a 45 μm filter, following methods described in Staddon et al. (1999). Hyphal length was estimated using the gridlineintercept method (McGonigle et al., 1990) at 100× magnification. 2.4. Data analyses We compared nematode abundance, diversity, and functional indices associated with the two grass species using 2-factor mixed models with grass identity (A. breviligulata or E. mollis) and dune profile position (toe or heel) as fixed factors and site as a random block term. The specific response variables were: total nematode abundance (individuals 100 ml−1 soil), PPN abundance (individuals 100 ml−1 soil), nematode genera richness (100 ml−1 soil), nematode Maturity Index (MI), nematode Structure Index (SI), nematode Enrichment Index (EI), and nematode Channel Index (CI). Nematode abundance data were square-root transformed to better meet model assumptions for analyses. To compare feeding group composition of nematode communities, we used a 2-factor MANOVA with grass identity and dune profile position as fixed factors and site as a block term, with relative abundances of bacterivores, fungivores, predators, omnivores, and plant-parasites as response variables. Two-factor mixed models and MANOVAs were performed using Systat v.12 (SYSTAT Software Inc., 2007). We used a two-factor blocked PERMANOVA (Anderson, 2001) to examine overall differences in nematode community composition due to dune grass identity and dune profile position. To visualize any differences in nematode community structure, we performed Nonmetric Multidimensional Scaling (NMDS) ordinations (McCune et al., 2002) with Bray-Curtis dissimilarity measures based on square-root transformed nematode abundance data. We used a blocked indicator species analysis (ISA) (McCune et al., 2002) to evaluate which individual nematode taxa were associated with each grass species and dune profile position, with Monte-Carlo randomizations to test for indicator value significance. PERMANOVA and NMDS analyses were performed using Primer v.6 (Anderson et al., 2008), and ISAs were done using PC-ORD v.6.08 (McCune and Mefford, 1999). Finally, we examined how plant-associated variables differed in response to grass identity and dune profile position by using similar 2factor mixed models as described above. Specific response variables were Plant %N, AMF % root colonization, AMF-ERH (mm g−1 soil), % litter cover, and species richness of other plants (m−2). ERH data were square-root transformed to better meet model assumptions for analyses.
3.2. Belowground successional patterns Plots located at the toe of the foredune had 66% more total nematodes than plots at the foredune heel (Fig. 2), but nematode genus richness did not vary across the dune profile (Table 1). Feeding group composition varied across dune profile position, with increased percentages of omnivores and decreased percentage of bacterivores and predators on dune heels (Fig. 3). Specifically, four predator genera
Fig. 2. Total nematode abundance in 100 ml dry soil from the two dune grass species and two foredune profile positions. Asterisks indicate significant differences between foredune profile positions and grass species at p < 0.05. Error bars ± SE.
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Fig. 3. Feeding group composition of nematode communities associated with two dune grass species and two foredune profile positions. Asterisks indicate significant univariate differences between the two foredune positions at p < 0.05.
Fig. 5. Non-metric multidimensional scaling (NMDS) ordination of nematode communities showing significant divergence between dune grass species and foredune profile positions in nematode community composition (PERMANOVA [pseudo F, p]: Grass Species = 1.78, 0.056; Dune Position = 8.04, 0.001; Grass × Position = 1.61, 0.103; Site = 2.54, 0.001).
Fig. 4. A) Nematode Maturity Index, B) Structure Index, C) Enrichment Index, and D) Channel Index in plots associated with two dune grass species and two foredune profile positions. Letters indicate significant differences between grass species and foredune positions at p < 0.05 (brackets indicate differences at p = 0.06). Error bars ± SE.
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Table 1 Results of mixed models examining the effects of dune grass species (A. breviligulata or E. mollis) and dune profile position (toe or heel) on soil nematode abundance, diversity, and functional indices. df
Grass Species Dune Position Grass × Position Site
1, 1, 1, 5,
39 39 39 39
Nematode abundance
Plant-parasite abundance
Nematode genera richness
Maturity Index (MI)
Index (SI)
Enrichment Index (EI)
Channel Index (CI)
F†
p
F
p
F
p
F
p⁎
F
p
F
p
F
p
0.003 0.009 0.65 0.29
0.03 0.61 0.003 0.99
0.87 0.44 0.96 0.32
0.06 0.25 0.57 1.26
0.80 0.62 0.46 0.21
1.65 9.27⁎⁎ 3.07 1.10
0.21 0.004 0.09 0.27
0.08 26.4⁎⁎⁎ 0.01 0.28
0.78 < 0.001 0.99 0.78
0.36 7.92⁎⁎ 11.51⁎⁎ 1.16
0.56 0.008 0.002 0.25
0.85 3.87 0.08 1.12
0.37 0.06 0.78 0.26
9.78 7.51⁎⁎ 0.22 1.06 ⁎⁎
†
Site effects were assessed using a Z-test which is similar to the F-test, but for random effects. Shows statistical significance at the p = 0.05 level. ⁎⁎ Shows statistical significance at the p = 0.01 level. ⁎⁎⁎ Shows statistical significance at the p = 0.001 level. ⁎
(Clarkus (cp4), Nygolaimus (cp5), Tobrilus (cp3), and Tripyla (cp3)) and two bacterivore genera (Anaplectus (cp2), Plectus (cp2)) had significant associations with dune toes while two genera, Alaimus (cp4 bacterivore) and Aporcelaimus (cp5 omnivore), were associated with dune heels (Table 2). This resulted in a higher Maturity Index and Structure Index in dune heels (Table 1, Fig. 4). While the Channel Index was not statistically significantly different at p < 0.05, there was a trend for dune heels to have a higher CI value than dune toes (Fig. 4). Overall, there was clear separation of nematode communities from toe and heel dune positions (Fig. 5). The nematode community Enrichment Index is the only measure that indicated that grass species were differentially influencing belowground succession: the nematode communities associated with E. mollis at the dune toe had the lowest enrichment index values, while E. mollis on the dune heel had the highest enrichment index values (Fig. 4).
Table 2 Results from blocked Indicator Species Analysis for nematode genera associated with dune grass species and dune profile position. P-values are calculated based on 1000 randomizations in Monte Carlo simulations. Feeding group and c-p/p-p assignments are from Bongers (1990) and Bongers et al. (1995). Nematode genus
Feeding group
c-p/pp value
Total counts
Acrobeles Acrobeloides Alaimus Amphidelus Anaplectus Aphelenchoides Aphelenchus Aporcelaimus Cephalobus Chiloplacus Choanolaimus Clarkus
Bacterivore Bacterivore Bacterivore Bacterivore Bacterivore Fungivore Fungivore Omnivore Bacterivore Bacterivore Predator Predator
2 2 4 4 2 2 2 5 2 2 4 4
44 1 50 2 93 4 3 35 1 10 28 59
Criconematidae Desmolaimus Diphtherophora Dorylaimoides Ecumenicus Eucephalobus Eudorylaimus Eumonhystera Filenchus Heterocephalobus Iotonchus Leptonchus Mesodorylaimus Miconchus Mylonchulus Nygolaimus
Plant-parasite Bacterivore Fungivore Fungivore Omnivore Bacterivore Omnivore Bacterivore Fungivore Bacterivore Predator Fungivore Omnivore Predator Predator Predator
3 3 3 4 4 2 4 2 2 2 4 4 4 4 4 5
15 1 3 10 71 17 611 3 11 91 11 64 4 23 48 35
Odontopharynx Paratylenchus Plectus Pratylenchus Rhabditis Rotylenchus Sectonema Thornia Tobrilus Tripyla Tylencholaimellus Tylencholaimus Tylenchorhynchus Tylenchus Wilsonema
Predator Plant-parasite Bacterivore Plant-parasite Bacterivore Plant-parasite Omnivore Omnivore Predator Predator Fungivore Fungivore Plant-parasite Plant-parasite Bacterivore
1 2 2 3 1 3 5 4 3 3 4 4 3 2 2
39 1 278 8 170 1 6 3 11 12 14 4 53 5 2
Indicator group(s)
Indicator value
n/s n/s Dune heel n/s Dune toe n/s n/s Dune heel n/s n/s n/s Elymus Dune toe n/s n/s n/s n/s n/s n/s Elymus n/s n/s n/s n/s n/s n/s n/s n/s Ammophila Dune toe n/s n/s Dune toe n/s n/s n/s n/s n/s Dune toe Dune toe n/s n/s n/s n/s n/s
n/s n/s 46.1⁎⁎ n/s 50.2⁎ n/s n/s 41.0⁎ n/s n/s n/s 49.7⁎ 60.0⁎⁎ n/s n/s n/s n/s n/s n/s 60.3⁎ n/s n/s n/s n/s n/s n/s n/s n/s 37.3⁎ 39.4⁎ n/s n/s 77.9⁎⁎⁎ n/s n/s n/s n/s n/s 29.2⁎⁎ 24.3⁎ n/s n/s n/s n/s n/s
4. Discussion Our finding that soil nematodes were almost twice as abundant in Elymus mollis plots compared to A. breviligulata plots indicates that there were more belowground resources to support the entire nematode community in those plots occupied by the native dune grass species (Chen et al., 2007; Cortois et al., 2017). These overall changes in nematode abundance were driven by two cp4 genera in particular: Clarkus sp., a predator, and Eudorylaimus sp., an omnivore that is both a predator and algal feeder and common in wet coastal dunes (McSorley, 2012). On a per plant basis, Elymus mollis has greater aboveground and belowground biomass than A. breviligulata (David et al., 2016; Hacker et al., 2012), and thus likely able to support a denser nematode community. In the resource-poor primary successional dune system, reductions in nematode abundance associated with plant invasion may reflect reductions in plant contributions to soil organic matter, reductions in soil microbial biomass, reductions in nitrogen-mineralization (Landesman et al., 2011; Neher, 2001), and a slowing of dune succession (Wall et al., 2002). Nematode community composition and structure differences between the two dune grass species were subtler than the larger differences in nematode communities along the foredune profile. While total nematode abundance was lower overall in Ammophila breviligulata plots, one taxon increased in these plots: the cp5 predatory genus Nygolaimus, which has high sensitivity to disturbance (Zhao and Neher, 2013). Elymus mollis plots, especially on dune heels, had a higher enrichment index, dominated by more cp1 (opportunist-lifestyle) taxa. Such taxa often respond quickly to increased resources, especially soil nitrogen (Ferris et al., 2001), and E. mollis has higher %N in vegetative tissues compared to A. breviligulata. Elymus mollis may accumulate N in live tissues in the earliest stages of succession, resulting in a lower EI, while buildup of E. mollis litter on dune heels could contribute to the increase in EI of nematode communities (Chen et al., 2007; Ferris and Matute,
Shows statistical significance at the p = 0.05 level. Shows statistical significance at the p = 0.01 level. ⁎⁎⁎ Shows statistical significance at the p = 0.001 level. ⁎
⁎⁎
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Table 3 Results of mixed models examining the effects of dune grass species (A. breviligulata or E. mollis) and dune profile position (toe or heel) on plant-associated variables. Plant %N df Grass species Dune position Grass × position Site †
1, 1, 1, 5,
F 39 39 39 39
†
33.9 20.2⁎⁎⁎ 0.69 1.49 ⁎⁎⁎
Root %AMF colonization
AMF Extra-radical hyphal length
% Litter cover
Other plant species richness
p
F
p
F
p
F
p
F
p
< 0.001 < 0.001 0.41 0.14
1.43 3.10 1.55 0.98
0.24 0.09 0.22 0.33
0.93 0.16 0.09 0.62
0.34 0.69 0.77 0.54
0.07 89.11⁎⁎⁎ 0.43 1.08
0.80 < 0.001 0.52 0.28
1.17 19.72⁎⁎⁎ 0.36 0.64
0.29 < 0.001 0.55 0.52
Site effects were assessed using a Z-test which is similar to the F-test, but for random effects. Indicates statistical significance at the p = 0.001 level.
⁎⁎⁎
2003). In other systems, increased EI of nematode communities is associated with increased nitrogen availability, faster nutrient cycling, and increased nitrogen mineralization (DuPont et al., 2009; Lei et al., 2015; Song et al., 2016). Thus, shifts in these nematode communities may also indicate that belowground ecosystem functions related to succession are slowing in response to A. breviligulata invasion (e.g., slower nutrient cycling) compared to dunes where native E. mollis continues to dominate. While our data indicate that A. breviligulata is slowing belowground succession, we found no direct evidence for enemy release from belowground PPN for A. breviligulata in our study. Indeed, free-living PPN were only a very small component (< 5%) of the nematode communities associated with both A. breviligulata and E. mollis. This is consistent with other research along Pacific coast dunes, where PPN were not very abundant (Beckstead and Parker, 2003; David et al., 2016). However, the lack of PPN in these foredune habitats may be one factor responsible for the net negative effect of A. breviligulata on E. mollis measured under high sand burial seen in this system (Zarnetske et al. 2013), especially if the presence of belowground herbivory had the potential to mediate their interaction (van der Putten and Peters, 1997). As a caveat, we only quantified free-living PPN as part of this study. While free-living PPN tend to be more abundant than sedentary taxa in coastal dunes (Brinkman et al., 2015), a global survey of dune habitats showed that escape from specialist, sedentary PPN taxa was associated with Ammophila arenaria invasion (van der Putten et al., 2005), and it is possible that specialist taxa are important for A. breviligulata invasion as well. For example, the sedentary endoparasite Meloidogyne has been implicated in A. breviligulata die-off in the Great Lakes region (Little and Maun, 1997), though ectoparasites such as Helicotylenchus and Longidorus are associated with A. breviligulata die-off along the U.S. Atlantic Coast (Seliskar and Huettel, 1993). Finally, this survey was limited to foredunes, which are not as stable as back-dune habitats where die-off is most prevalent in these other regions. We also found no evidence that AMF growth, either within or external to plants, influences nematode communities, despite evidence that this can happen in other dune systems (Reid and Emery, 2017). The two plant species did not vary in their rates of root colonization, which contrasts with previous experimental work in Oregon coast dunes that reported E. mollis having consistently higher root colonization than A. breviligulata (74.5% vs. 41.5%; David et al., 2016). Instead, the results fit the conclusions of a meta-analysis of 67 studies showing no differences in AMF associations between native and invasive plants (Bunn et al., 2015). While we did find increases in the Channel Index (CI, Ferris et al., 2001), which is sensitive to fungivore taxa, across the dune profile, this was not related to AMF hyphal abundance, indicating that saprophytic or other fungi may be contributing to this shift. Overall, dune profile position was more important that grass identity in influencing nematode community composition, and patterns matched expectations in terms of belowground succession. Foredune heels had increased Maturity and Structure Indices, indicating more trophic links and a general association with lower disturbance and later successional stage (De Deyn et al., 2003; Lei et al., 2015; Wall et al., 2002). This was also clear in the indicator species analysis, with cp4
and cp5 genera associated with dune heels, while cp2–3 genera were associated with dune toes. There was also a trend for dune heels sites to have a higher Channel Index, indicating a belowground shift from bacterial to fungal decomposition pathways. While this shift is common across successional gradients (Ferris and Matute, 2003), it is notable that such a shift is present even within short distances (~50–125 m) across a single foredune habitat. In general, bacterivores were the most common feeding group across all plots, consistent with work in other dune systems, and indicating that these are still early-successional communities (Ferris and Matute, 2003; Fitoussi et al., 2016; Hanel, 2010). It was somewhat surprising to find high abundances of predators in the toe positions of these dunes, though at least two other studies had similar findings, with predatory nematodes associated with the earliest successional unstable dunes in Poland and Scotland due to their dependence on high soil water content found in the beach transition zone (Goralczyk, 1998; Wall et al., 2002). Nematodes in the upper beach zone can feed on other nematodes as well as aquatic microorganisms and can connect aquatic and terrestrial food webs (Gheskiere et al., 2005). This overlap between aquatic and terrestrial communities may explain the higher nematode abundance we found in dune toe plots as opposed to the more stable and inland-facing heel plots. These patterns support other work showing that aquatic subsidies can be important to soil faunal communities in general (Korobushkin et al., 2016). Nematode communities in heel plots were also associated with higher cover of plant litter, consistent with these as more stable, later successional dune environments. In conclusion, this study provides evidence that A. breviligulata reduces total nematode abundance and may be altering nitrogen cycling across the dune profile, despite a lack of clear differences in nematode community composition associated with these two dune-building grass species. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.apsoil.2019.06.009. Acknowledgments Funding for this project was provided to SME by the University of Louisville EVPRI Internal Grant program and to SDH by Oregon Sea Grant (NA14OAR4170064). We thank Vanessa Constant, Erin Kinnetz, Sarah Benton, and Brad Kimbrough for field and lab assistance. Thanks also to Steve Yanoviak and two anonymous reviewers for helpful feedback on this manuscript. References Anderson, M.J., 2001. A new method for non-parametric multivariate analysis of variance. Austral Ecol. 26, 32–46. Anderson, M.L., Gorley, R.N., Clarke, K.R., 2008. PERMANOVA+ for PRIMER. Plymouth, UK. Andrássy, I., 2005. Free-living Nematodes of Hungary: Nematoda errantia, Vol. 1. Hungarian Natural History Museum, Budapest, Hungary. Andrássy, I., 2007. Free-living Nematodes of Hungary: Nematoda errantia, Vol. 2. Hungarian Natural History Museum, Budapest, Hungary. Andrássy, I., 2009. Free-living Nematodes of Hungary: Nematoda errantia, Vol. 3. Hungarian Natural History Museum, Budapest, Hungary.
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Soil nematodes differ in association with native and non-native dunebuilding grass species Sarah M. Emerya, , Matthew L. Reida,b, Sally D. Hackerc ⁎
a
Dept. of Biology, University of Louisville, Louisville, KY 40292, United States of America Dept. of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI 48824, United States of America c Dept. of Integrative Biology, Oregon State University, Corvallis, OR 97331, United States of America b
ARTICLE INFO
ABSTRACT
Keywords: Ammophila breviligulata (American beachgrass) Elymus (Leymus) mollis (American dune grass) Foredune Succession NINJA US Pacific Northwest dunes
Non-native plant species can alter soil communities, which can lead to changes in their ecosystem functioning and facilitate invasive plant spread. In this study, we asked whether abundance, composition, or function of freeliving soil nematode communities differed in association with two dune building grass species in US Pacific Northwest coastal dunes. In 2016, we surveyed soil nematode communities along foredune profiles at six sites in Oregon and Washington, USA, where the non-native beach grass Ammophila breviligulata co-occurred with the native dune grass Elymus mollis. We also measured plant-associated factors that might influence nematode communities, including plant tissue nitrogen, arbuscular mycorrhizal associations, litter production, and plant community diversity associated with the two grass species across the foredune profile. Elymus mollis plots had over twice as many nematodes on average as A. breviligulata plots and were also associated with a higher nematode enrichment index, especially on dune heels. This is possibly explained by higher percent leaf nitrogen content in E. mollis. Subtle differences in nematode community composition and plant tissue nitrogen between the two grass species indicate that A. breviligulata may be altering nutrient cycling across the dune profile, which could explain the arrested succession associated with this species on back dunes. However, overall, nematode communities shifted more in response to foredune cross-shore position and stabilization as opposed to changes in vegetation.
1. Introduction Invasive plant species can dramatically alter bacterial, fungal, and invertebrate communities in soils following non-native plant invasion (e.g., Batten et al., 2006; Chen et al., 2007; Lazzaro et al., 2018; Mummey and Rillig, 2006; Renco and Balezentiene, 2015; Tanner et al., 2013; van der Putten et al., 2007). Such changes in belowground communities can affect nutrient cycling and other belowground ecosystem functions (Nielsen et al., 2011; Wagg et al., 2014). In particular, changes to soil nematodes, which occupy multiple trophic levels in soil food webs (bacterivores, fungivores, plant-parasites, and predators), can reflect changes in ecosystem successional status, nutrient cycling, and overall soil ecosystem health (Yeates, 2003). For example, Heracleum sosnowsyi invasion in grasslands in Europe reduced nematode diversity and altered nematode feeding guild composition, reflecting a more disturbed (lower Maturity Index) soil condition with higher contribution of bacteria to decomposition processes, and faster turnover rates of organic matter (Renco and Balezentiene, 2015).
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Invasive plants in primary successional systems such as sand dunes may have particularly strong effects on nematode community structure and function. Nematode communities are very responsive to disturbance and are good indicators of successional state on dunes, with early successional communities often dominated by predators and bacterivores and later-successional communities dominated by fungivores, reflecting changes in energy transfer pathways belowground (Zhi et al., 2009). Shifts in nematode abundance and community composition following plant invasion may indicate changes in successional processes and thus dune functioning. Beach and dune habitat covers ~42% of the coastline in the states of Oregon and Washington USA (Wiedemann, 1984) and provides important ecosystem services such as coastal protection, carbon sequestration, and tourism (Barbier et al., 2011). These Pacific Northwest dunes were historically dominated by native forbs and the native dune grass Elymus mollis, which sparsely covered this ecosystem (Cooper, 1958; Copeland et al., 2002; Wiedemann and Pickart, 2004). Starting in the early 1900s, US Pacific coast dunes were systematically planted
Corresponding author at: Dept. of Biology, University of Louisville, 139 Life Sciences Bldg., Louisville, KY 40292, United States of America. E-mail address: [email protected] (S.M. Emery).
https://doi.org/10.1016/j.apsoil.2019.06.009 Received 31 January 2019; Received in revised form 10 June 2019; Accepted 14 June 2019 0929-1393/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Sarah M. Emery, Matthew L. Reid and Sally D. Hacker, Applied Soil Ecology, https://doi.org/10.1016/j.apsoil.2019.06.009
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with the non-native European beachgrass Ammophila arenaria to stabilize a shifting sand environment that naturally lacked vegetation. In the 1930s, a separate non-native congener, American beachgrass Ammophila breviligulata was planted in dunes near the Columbia River and, by the 1980s, had spread and dominated northward along the Washington coast (Hacker et al., 2012; Seabloom and Wiedemann, 1994), where it has transformed the dune landscape from small, sparsely vegetated hillocks of sand to tall and densely vegetated linear ridges. Ammophila breviligulata is a superior dune builder compared to the native E. mollis because, despite having thinner stems and blades, it grows more densely and more clumped than E. mollis (Hacker et al., 2012; Zarnetske et al., 2012). This shift in geomorphology was accompanied by a decline in plant species richness (Hacker et al., 2012; Zarnetske et al., 2013) and slowing of dune plant succession (David et al., 2015). Ammophila breviligulata invasion may alter nematode communities through multiple ways. Smaller, less productive plants can decrease soil nematode abundance and density due to decreased belowground biomass, while differences in plant nutritional status or nutrient content can alter belowground resources and shift soil nematode community composition (Cortois et al., 2017; Yeates, 1987). Non-native plant species like A. breviligulata may suppress plant-parasitic nematodes (PPN) via processes of “enemy release” (Heger and Jeschke, 2014) such as is seen for the European beachgrass Ammophila arenaria (van der Putten et al., 2005). While belowground differences in nematode communities are often most distinct when comparing plants from different functional groups, even plant species belonging to the same functional group can show differences in their associated nematode communities. For example, Viketoft et al. (2005) found that nematode community diversity and composition significantly differed between species within the same functional groups (e.g., grasses, legume, or forbs), possibly due to differences in plant metabolites. Reid and Emery (2017) found that the invasive blue lyme grass, Leymus arenarius, was associated with reductions in PPN abundance compared to native dune grasses in the Great Lakes region, possibly due to enhanced benefits from arbuscular mycorrhizal fungi (AMF). In this study, we ask whether abundance, composition, or function of free-living soil nematode communities differ between these two dune-building grasses, and if so, what plant-associated factors are correlated with these changes. We also test whether nematodes reflect changes in belowground succession associated with dune stabilization or the two grasses by comparing belowground nematode communities across the foredune profile (from foreshore to backshore). We expect that A. breviligulata will be associated with decreases in soil nematode abundance, as A. breviligulata above- and belowground biomass is lower than E. mollis (David et al., 2016; Hacker et al., 2012), and will have lower fractions of PPN than E. mollis, possibly due to enemy release. We also predict that the nematode community will reflect plant successional patterns, with more mature nematode communities associated with E. mollis compared to A. breviligulata in backshore dunes, possibly due to increased soil organic matter contributions associated with the larger E. mollis plants.
Fig. 1. Site locations from south to north along the Pacific coast of Oregon and Washington USA used for this study. Original ID names correspond to those in Hacker et al. (2012).
foredune at two cross-shore positions on the foredune (toe, which is the foreshore position closest to the beach, and heel, which is the backshore position furthest from the beach). Each plot had a monoculture of one or the other grass species and we paired the plots such that each E. mollis plot had a paired A. breviligulata plot that was roughly 5 m away. In each plot, plant species richness and litter cover were visually estimated. We found that the dune heel had higher plant species richness (2.38 ± 1.58 vs. 0.83 ± 0.76 species m−2) and increased litter cover (60.63 ± 31.15 vs. 4.92% ± 5.99) compared to the dune toe. While overall plant diversity was very low in general, some common species at the dune toe were Cakile edentula and C. maritima and at the dune heel were Achillea millefolium, Erechtites minima, Frageria chiloensis, Hypochaeris radicata, Lathyrus japonicus, Lupinus littoralis, and Rumex acetosella. We also collected soil cores (2 cm diameter × 15 cm deep; 5 per plot) from near the bases of haphazardly selected plants of A. breviligulata or E. mollis in the plots, combined the cores for each plot, and stored them at 4 °C until processing. Finally, leaf blades were clipped from three individual tillers of each grass species and air dried back in the laboratory. The three leaf samples from each site and for each species in each foredune profile position were combined (n = 24) and then ground to a powder using a Mixer Mill (SPEX SamplePrep; Metuchen, NJ). The samples were analyzed for % total N at the stable isotope lab in the College of Earth, Ocean, and Atmospheric Science at Oregon State University (Corvallis, OR). 2.2. Nematode sampling We extracted free-living nematodes from fresh 100 ml subsamples of soil from each plot using the centrifugal flotation method (Jenkins, 1964). Nematode samples were placed in 3% formalin solution for longterm storage and identification. Nematodes in each sample were counted under a dissecting microscope (50×) and identified to genus (Andrássy, 2005, 2007, 2009; Bongers, 1988) under a compound microscope (100–1000×) and classified into feeding group (bacterivore, fungivore, plant-parasite, omnivore, or predator) based on Yeates et al. (1993). Nematodes were also classified to one of five colonizer-persister groups (cp1-cp5) based on life history traits and sensitivity to disturbance (Bongers, 1990; Yeates et al., 1993). The genera richness, PPN abundance, Maturity Index (MI; a measure of environmental disturbance and successional status), Structure Index (SI; a measure of food web connectedness), Enrichment Index (EI; a measure of resource availability, especially nitrogen), and Channel Index (CI; a measure of relative flow of energy through fungal vs. bacterial decomposition
2. Methods 2.1. Vegetation and soil sampling In June 2016, we conducted field sampling at six sites along a 100 km section of US Pacific Northwest coast (northern Oregon and Washington) where A. breviligulata and E. mollis co-occur (Fig. 1; Hacker et al. 2012). These sites are a subset of a larger long-term study of dune dynamics, and site details can be found in Hacker et al. (2012). Foredunes (i.e., linear hills of sand parallel to the shoreline) at these sites are short (~4 m) and wide (110 m) compared to other regions of the coast, and vary in community structure along their cross-shore profiles (see below). At each site, we laid out two 1 m2 plots (at least 10 m apart) on the 2
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channels) were calculated for each plot based on the nematode data using the NINJA program (Sieriebriennikov et al., 2014). These indices provide insight into the successional status and functioning of the soil nematode community (Bongers, 1990; Ferris and Bongers, 2009; Ferris et al., 2001).
3. Results 3.1. Grass species differences in nematode abundance and community composition When examining the entire free-living soil nematode community, E. mollis plots had 80% more total nematodes than the A. breviligulata plots (Table 1, Fig. 2). The most abundant genera overall were Eudorylaimus (cp4 omnivore), Plectus (cp2 bacterivore), and Rhabditis (cp1 bacterivore) (Table S1), and the ISA showed that the predatory genus Nygolaimus (cp5) was associated with A. breviligulata plots, while Eudorylaimus (cp4 omnivore) and Clarkus (cp4 predator) were more abundant in E. mollis plots (Table 2). Free-living PPN had very low abundances in general, only making up 2–5% of nematode communities (Table S1), and did not vary across plant species (Table 1; average PPN abundance [mean ± SD] = 0.34 ± 0.76100 ml−1 soil). There were no significant differences in nematode richness at the genus level across dune grass species (Table 1; average genera richness = 11.3 ± 2.7100 ml−1 soil), and no significant differences in feeding group composition across grass species (MANOVA results [F5,35, p] using Pillai Trace Statistics: Grass Species 1.57, 0.19; Dune Position 4.17, 0.004; Grass × Position 0.92, 0.48; Site 1.31, 0.16; Fig. 3). Overall nematode community composition and community structure indices also did not significantly differ between the two plant species (Figs. 4, 5). AMF root colonization and ERH length were similar for both dune grass species (A. breviligulata AMF root colonization = 76.0 ± 11.7%, ERH = 87.7 ± 120.1 mm g−1 soil; E. mollis root colonization = 71.8 ± 15.5%, ERH = 62.1 ± 55.7 mm g−1 soil). (Table 3). Elymus mollis had higher plant tissue nitrogen than A. breviligulata (1.12% ± 0.30 vs. 0.83% ± 0.34) ((Table 3).
2.3. Plant-associated measures As AMF can influence belowground nematode communities (Schouteden et al., 2015), we characterized the abundance of AMF in plots by measuring both colonization in plant roots, and extraradical hyphae (ERH) growth. For plant root colonization measures, we extracted fine roots from 100 ml subsamples of composited soil cores for each plot using a wet-sieve process (500 μm sieve; Milchunas, 2012). Roots were cleared with 10% KOH and stained using a 5% vinegar-ink solution following methods in Vierheilig et al. (1998). Visual estimation of percent root colonization was made based on 100 fields of view per sample under 200× magnification. To estimate ERH, we extracted hyphae from 20 ml subsamples taken from the pooled cores for each plot. Soil subsamples were suspended in water, then stained and vacuum filtered through a 45 μm filter, following methods described in Staddon et al. (1999). Hyphal length was estimated using the gridlineintercept method (McGonigle et al., 1990) at 100× magnification. 2.4. Data analyses We compared nematode abundance, diversity, and functional indices associated with the two grass species using 2-factor mixed models with grass identity (A. breviligulata or E. mollis) and dune profile position (toe or heel) as fixed factors and site as a random block term. The specific response variables were: total nematode abundance (individuals 100 ml−1 soil), PPN abundance (individuals 100 ml−1 soil), nematode genera richness (100 ml−1 soil), nematode Maturity Index (MI), nematode Structure Index (SI), nematode Enrichment Index (EI), and nematode Channel Index (CI). Nematode abundance data were square-root transformed to better meet model assumptions for analyses. To compare feeding group composition of nematode communities, we used a 2-factor MANOVA with grass identity and dune profile position as fixed factors and site as a block term, with relative abundances of bacterivores, fungivores, predators, omnivores, and plant-parasites as response variables. Two-factor mixed models and MANOVAs were performed using Systat v.12 (SYSTAT Software Inc., 2007). We used a two-factor blocked PERMANOVA (Anderson, 2001) to examine overall differences in nematode community composition due to dune grass identity and dune profile position. To visualize any differences in nematode community structure, we performed Nonmetric Multidimensional Scaling (NMDS) ordinations (McCune et al., 2002) with Bray-Curtis dissimilarity measures based on square-root transformed nematode abundance data. We used a blocked indicator species analysis (ISA) (McCune et al., 2002) to evaluate which individual nematode taxa were associated with each grass species and dune profile position, with Monte-Carlo randomizations to test for indicator value significance. PERMANOVA and NMDS analyses were performed using Primer v.6 (Anderson et al., 2008), and ISAs were done using PC-ORD v.6.08 (McCune and Mefford, 1999). Finally, we examined how plant-associated variables differed in response to grass identity and dune profile position by using similar 2factor mixed models as described above. Specific response variables were Plant %N, AMF % root colonization, AMF-ERH (mm g−1 soil), % litter cover, and species richness of other plants (m−2). ERH data were square-root transformed to better meet model assumptions for analyses.
3.2. Belowground successional patterns Plots located at the toe of the foredune had 66% more total nematodes than plots at the foredune heel (Fig. 2), but nematode genus richness did not vary across the dune profile (Table 1). Feeding group composition varied across dune profile position, with increased percentages of omnivores and decreased percentage of bacterivores and predators on dune heels (Fig. 3). Specifically, four predator genera
Fig. 2. Total nematode abundance in 100 ml dry soil from the two dune grass species and two foredune profile positions. Asterisks indicate significant differences between foredune profile positions and grass species at p < 0.05. Error bars ± SE.
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Fig. 3. Feeding group composition of nematode communities associated with two dune grass species and two foredune profile positions. Asterisks indicate significant univariate differences between the two foredune positions at p < 0.05.
Fig. 5. Non-metric multidimensional scaling (NMDS) ordination of nematode communities showing significant divergence between dune grass species and foredune profile positions in nematode community composition (PERMANOVA [pseudo F, p]: Grass Species = 1.78, 0.056; Dune Position = 8.04, 0.001; Grass × Position = 1.61, 0.103; Site = 2.54, 0.001).
Fig. 4. A) Nematode Maturity Index, B) Structure Index, C) Enrichment Index, and D) Channel Index in plots associated with two dune grass species and two foredune profile positions. Letters indicate significant differences between grass species and foredune positions at p < 0.05 (brackets indicate differences at p = 0.06). Error bars ± SE.
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Table 1 Results of mixed models examining the effects of dune grass species (A. breviligulata or E. mollis) and dune profile position (toe or heel) on soil nematode abundance, diversity, and functional indices. df
Grass Species Dune Position Grass × Position Site
1, 1, 1, 5,
39 39 39 39
Nematode abundance
Plant-parasite abundance
Nematode genera richness
Maturity Index (MI)
Index (SI)
Enrichment Index (EI)
Channel Index (CI)
F†
p
F
p
F
p
F
p⁎
F
p
F
p
F
p
0.003 0.009 0.65 0.29
0.03 0.61 0.003 0.99
0.87 0.44 0.96 0.32
0.06 0.25 0.57 1.26
0.80 0.62 0.46 0.21
1.65 9.27⁎⁎ 3.07 1.10
0.21 0.004 0.09 0.27
0.08 26.4⁎⁎⁎ 0.01 0.28
0.78 < 0.001 0.99 0.78
0.36 7.92⁎⁎ 11.51⁎⁎ 1.16
0.56 0.008 0.002 0.25
0.85 3.87 0.08 1.12
0.37 0.06 0.78 0.26
9.78 7.51⁎⁎ 0.22 1.06 ⁎⁎
†
Site effects were assessed using a Z-test which is similar to the F-test, but for random effects. Shows statistical significance at the p = 0.05 level. ⁎⁎ Shows statistical significance at the p = 0.01 level. ⁎⁎⁎ Shows statistical significance at the p = 0.001 level. ⁎
(Clarkus (cp4), Nygolaimus (cp5), Tobrilus (cp3), and Tripyla (cp3)) and two bacterivore genera (Anaplectus (cp2), Plectus (cp2)) had significant associations with dune toes while two genera, Alaimus (cp4 bacterivore) and Aporcelaimus (cp5 omnivore), were associated with dune heels (Table 2). This resulted in a higher Maturity Index and Structure Index in dune heels (Table 1, Fig. 4). While the Channel Index was not statistically significantly different at p < 0.05, there was a trend for dune heels to have a higher CI value than dune toes (Fig. 4). Overall, there was clear separation of nematode communities from toe and heel dune positions (Fig. 5). The nematode community Enrichment Index is the only measure that indicated that grass species were differentially influencing belowground succession: the nematode communities associated with E. mollis at the dune toe had the lowest enrichment index values, while E. mollis on the dune heel had the highest enrichment index values (Fig. 4).
Table 2 Results from blocked Indicator Species Analysis for nematode genera associated with dune grass species and dune profile position. P-values are calculated based on 1000 randomizations in Monte Carlo simulations. Feeding group and c-p/p-p assignments are from Bongers (1990) and Bongers et al. (1995). Nematode genus
Feeding group
c-p/pp value
Total counts
Acrobeles Acrobeloides Alaimus Amphidelus Anaplectus Aphelenchoides Aphelenchus Aporcelaimus Cephalobus Chiloplacus Choanolaimus Clarkus
Bacterivore Bacterivore Bacterivore Bacterivore Bacterivore Fungivore Fungivore Omnivore Bacterivore Bacterivore Predator Predator
2 2 4 4 2 2 2 5 2 2 4 4
44 1 50 2 93 4 3 35 1 10 28 59
Criconematidae Desmolaimus Diphtherophora Dorylaimoides Ecumenicus Eucephalobus Eudorylaimus Eumonhystera Filenchus Heterocephalobus Iotonchus Leptonchus Mesodorylaimus Miconchus Mylonchulus Nygolaimus
Plant-parasite Bacterivore Fungivore Fungivore Omnivore Bacterivore Omnivore Bacterivore Fungivore Bacterivore Predator Fungivore Omnivore Predator Predator Predator
3 3 3 4 4 2 4 2 2 2 4 4 4 4 4 5
15 1 3 10 71 17 611 3 11 91 11 64 4 23 48 35
Odontopharynx Paratylenchus Plectus Pratylenchus Rhabditis Rotylenchus Sectonema Thornia Tobrilus Tripyla Tylencholaimellus Tylencholaimus Tylenchorhynchus Tylenchus Wilsonema
Predator Plant-parasite Bacterivore Plant-parasite Bacterivore Plant-parasite Omnivore Omnivore Predator Predator Fungivore Fungivore Plant-parasite Plant-parasite Bacterivore
1 2 2 3 1 3 5 4 3 3 4 4 3 2 2
39 1 278 8 170 1 6 3 11 12 14 4 53 5 2
Indicator group(s)
Indicator value
n/s n/s Dune heel n/s Dune toe n/s n/s Dune heel n/s n/s n/s Elymus Dune toe n/s n/s n/s n/s n/s n/s Elymus n/s n/s n/s n/s n/s n/s n/s n/s Ammophila Dune toe n/s n/s Dune toe n/s n/s n/s n/s n/s Dune toe Dune toe n/s n/s n/s n/s n/s
n/s n/s 46.1⁎⁎ n/s 50.2⁎ n/s n/s 41.0⁎ n/s n/s n/s 49.7⁎ 60.0⁎⁎ n/s n/s n/s n/s n/s n/s 60.3⁎ n/s n/s n/s n/s n/s n/s n/s n/s 37.3⁎ 39.4⁎ n/s n/s 77.9⁎⁎⁎ n/s n/s n/s n/s n/s 29.2⁎⁎ 24.3⁎ n/s n/s n/s n/s n/s
4. Discussion Our finding that soil nematodes were almost twice as abundant in Elymus mollis plots compared to A. breviligulata plots indicates that there were more belowground resources to support the entire nematode community in those plots occupied by the native dune grass species (Chen et al., 2007; Cortois et al., 2017). These overall changes in nematode abundance were driven by two cp4 genera in particular: Clarkus sp., a predator, and Eudorylaimus sp., an omnivore that is both a predator and algal feeder and common in wet coastal dunes (McSorley, 2012). On a per plant basis, Elymus mollis has greater aboveground and belowground biomass than A. breviligulata (David et al., 2016; Hacker et al., 2012), and thus likely able to support a denser nematode community. In the resource-poor primary successional dune system, reductions in nematode abundance associated with plant invasion may reflect reductions in plant contributions to soil organic matter, reductions in soil microbial biomass, reductions in nitrogen-mineralization (Landesman et al., 2011; Neher, 2001), and a slowing of dune succession (Wall et al., 2002). Nematode community composition and structure differences between the two dune grass species were subtler than the larger differences in nematode communities along the foredune profile. While total nematode abundance was lower overall in Ammophila breviligulata plots, one taxon increased in these plots: the cp5 predatory genus Nygolaimus, which has high sensitivity to disturbance (Zhao and Neher, 2013). Elymus mollis plots, especially on dune heels, had a higher enrichment index, dominated by more cp1 (opportunist-lifestyle) taxa. Such taxa often respond quickly to increased resources, especially soil nitrogen (Ferris et al., 2001), and E. mollis has higher %N in vegetative tissues compared to A. breviligulata. Elymus mollis may accumulate N in live tissues in the earliest stages of succession, resulting in a lower EI, while buildup of E. mollis litter on dune heels could contribute to the increase in EI of nematode communities (Chen et al., 2007; Ferris and Matute,
Shows statistical significance at the p = 0.05 level. Shows statistical significance at the p = 0.01 level. ⁎⁎⁎ Shows statistical significance at the p = 0.001 level. ⁎
⁎⁎
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Table 3 Results of mixed models examining the effects of dune grass species (A. breviligulata or E. mollis) and dune profile position (toe or heel) on plant-associated variables. Plant %N df Grass species Dune position Grass × position Site †
1, 1, 1, 5,
F 39 39 39 39
†
33.9 20.2⁎⁎⁎ 0.69 1.49 ⁎⁎⁎
Root %AMF colonization
AMF Extra-radical hyphal length
% Litter cover
Other plant species richness
p
F
p
F
p
F
p
F
p
< 0.001 < 0.001 0.41 0.14
1.43 3.10 1.55 0.98
0.24 0.09 0.22 0.33
0.93 0.16 0.09 0.62
0.34 0.69 0.77 0.54
0.07 89.11⁎⁎⁎ 0.43 1.08
0.80 < 0.001 0.52 0.28
1.17 19.72⁎⁎⁎ 0.36 0.64
0.29 < 0.001 0.55 0.52
Site effects were assessed using a Z-test which is similar to the F-test, but for random effects. Indicates statistical significance at the p = 0.001 level.
⁎⁎⁎
2003). In other systems, increased EI of nematode communities is associated with increased nitrogen availability, faster nutrient cycling, and increased nitrogen mineralization (DuPont et al., 2009; Lei et al., 2015; Song et al., 2016). Thus, shifts in these nematode communities may also indicate that belowground ecosystem functions related to succession are slowing in response to A. breviligulata invasion (e.g., slower nutrient cycling) compared to dunes where native E. mollis continues to dominate. While our data indicate that A. breviligulata is slowing belowground succession, we found no direct evidence for enemy release from belowground PPN for A. breviligulata in our study. Indeed, free-living PPN were only a very small component (< 5%) of the nematode communities associated with both A. breviligulata and E. mollis. This is consistent with other research along Pacific coast dunes, where PPN were not very abundant (Beckstead and Parker, 2003; David et al., 2016). However, the lack of PPN in these foredune habitats may be one factor responsible for the net negative effect of A. breviligulata on E. mollis measured under high sand burial seen in this system (Zarnetske et al. 2013), especially if the presence of belowground herbivory had the potential to mediate their interaction (van der Putten and Peters, 1997). As a caveat, we only quantified free-living PPN as part of this study. While free-living PPN tend to be more abundant than sedentary taxa in coastal dunes (Brinkman et al., 2015), a global survey of dune habitats showed that escape from specialist, sedentary PPN taxa was associated with Ammophila arenaria invasion (van der Putten et al., 2005), and it is possible that specialist taxa are important for A. breviligulata invasion as well. For example, the sedentary endoparasite Meloidogyne has been implicated in A. breviligulata die-off in the Great Lakes region (Little and Maun, 1997), though ectoparasites such as Helicotylenchus and Longidorus are associated with A. breviligulata die-off along the U.S. Atlantic Coast (Seliskar and Huettel, 1993). Finally, this survey was limited to foredunes, which are not as stable as back-dune habitats where die-off is most prevalent in these other regions. We also found no evidence that AMF growth, either within or external to plants, influences nematode communities, despite evidence that this can happen in other dune systems (Reid and Emery, 2017). The two plant species did not vary in their rates of root colonization, which contrasts with previous experimental work in Oregon coast dunes that reported E. mollis having consistently higher root colonization than A. breviligulata (74.5% vs. 41.5%; David et al., 2016). Instead, the results fit the conclusions of a meta-analysis of 67 studies showing no differences in AMF associations between native and invasive plants (Bunn et al., 2015). While we did find increases in the Channel Index (CI, Ferris et al., 2001), which is sensitive to fungivore taxa, across the dune profile, this was not related to AMF hyphal abundance, indicating that saprophytic or other fungi may be contributing to this shift. Overall, dune profile position was more important that grass identity in influencing nematode community composition, and patterns matched expectations in terms of belowground succession. Foredune heels had increased Maturity and Structure Indices, indicating more trophic links and a general association with lower disturbance and later successional stage (De Deyn et al., 2003; Lei et al., 2015; Wall et al., 2002). This was also clear in the indicator species analysis, with cp4
and cp5 genera associated with dune heels, while cp2–3 genera were associated with dune toes. There was also a trend for dune heels sites to have a higher Channel Index, indicating a belowground shift from bacterial to fungal decomposition pathways. While this shift is common across successional gradients (Ferris and Matute, 2003), it is notable that such a shift is present even within short distances (~50–125 m) across a single foredune habitat. In general, bacterivores were the most common feeding group across all plots, consistent with work in other dune systems, and indicating that these are still early-successional communities (Ferris and Matute, 2003; Fitoussi et al., 2016; Hanel, 2010). It was somewhat surprising to find high abundances of predators in the toe positions of these dunes, though at least two other studies had similar findings, with predatory nematodes associated with the earliest successional unstable dunes in Poland and Scotland due to their dependence on high soil water content found in the beach transition zone (Goralczyk, 1998; Wall et al., 2002). Nematodes in the upper beach zone can feed on other nematodes as well as aquatic microorganisms and can connect aquatic and terrestrial food webs (Gheskiere et al., 2005). This overlap between aquatic and terrestrial communities may explain the higher nematode abundance we found in dune toe plots as opposed to the more stable and inland-facing heel plots. These patterns support other work showing that aquatic subsidies can be important to soil faunal communities in general (Korobushkin et al., 2016). Nematode communities in heel plots were also associated with higher cover of plant litter, consistent with these as more stable, later successional dune environments. In conclusion, this study provides evidence that A. breviligulata reduces total nematode abundance and may be altering nitrogen cycling across the dune profile, despite a lack of clear differences in nematode community composition associated with these two dune-building grass species. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.apsoil.2019.06.009. Acknowledgments Funding for this project was provided to SME by the University of Louisville EVPRI Internal Grant program and to SDH by Oregon Sea Grant (NA14OAR4170064). We thank Vanessa Constant, Erin Kinnetz, Sarah Benton, and Brad Kimbrough for field and lab assistance. Thanks also to Steve Yanoviak and two anonymous reviewers for helpful feedback on this manuscript. References Anderson, M.J., 2001. A new method for non-parametric multivariate analysis of variance. Austral Ecol. 26, 32–46. Anderson, M.L., Gorley, R.N., Clarke, K.R., 2008. PERMANOVA+ for PRIMER. Plymouth, UK. Andrássy, I., 2005. Free-living Nematodes of Hungary: Nematoda errantia, Vol. 1. Hungarian Natural History Museum, Budapest, Hungary. Andrássy, I., 2007. Free-living Nematodes of Hungary: Nematoda errantia, Vol. 2. Hungarian Natural History Museum, Budapest, Hungary. Andrássy, I., 2009. Free-living Nematodes of Hungary: Nematoda errantia, Vol. 3. Hungarian Natural History Museum, Budapest, Hungary.
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