Guadalupe Peralta1, Ian A Dickie2, Gregor W Yeates1, Duane A Peltzer1. 1. Manaaki Whenua Landcare Research, Lincoln, New Zealand. 2. Bio-Protection Research Centre, School of Biological Sciences, University of Canterbury, Christchurch, New Zealand.
Abstract
Success of invasive non-native plant species management is usually measured as changes in the abundance of the invasive plant species or native plant species following invader management, but more complex trophic responses to invader removal are often ignored or assumed. Moreover, the effects of invader removal at different stages of the invasion process is rarely evaluated, despite a growing recognition that invader impacts are density or stage-dependent. Therefore, the effectiveness of invasive species management for restoring community structure and function across trophic levels remains poorly understood. We determined how soil nematode diversity and community composition respond to removal of the globally invasive tree species Pinus contorta at different stages of invasion by reanalysing and expanding an earlier study including uninvaded vegetation (seedlings removed continuously), early invader removal (saplings removed), late removal (trees removed), and no removal (invaded). These treatments allowed us to evaluate the stage-dependent belowground trophic responses to biological invasion and removal. We found that invaded plots had half the nematode taxa richness compared to uninvaded plots, and that tree invasion altered the overall composition of the nematode community. Differences in nematode community composition between uninvaded nematode communities and those under the tree removal strategy tended to dilute higher up the food chain, whereas the composition of uninvaded vs. sapling removal strategies did not differ significantly. Conversely, the composition of invaded compared to uninvaded nematode communities differed across all trophic levels, altering the community structure and function. Specifically, invaded communities were structurally simplified compared to uninvaded communities, and had a higher proportion of short life cycle nematodes, characteristic of disturbed environments. We demonstrate that a shift in management strategies for a globally invasive tree species from removing trees to earlier removal of saplings is needed for maintaining the composition and structure of soil nematode communities to resemble uninvaded conditions.
Success of invasive non-native plant species management is usually measured as changes in the abundance of the invasive plant species or native plant species following invader management, but more complex trophic responses to invader removal are often ignored or assumed. Moreover, the effects of invader removal at different stages of the invasion process is rarely evaluated, despite a growing recognition that invader impacts are density or stage-dependent. Therefore, the effectiveness of invasive species management for restoring community structure and function across trophic levels remains poorly understood. We determined how soil nematode diversity and community composition respond to removal of the globally invasive tree species Pinus contorta at different stages of invasion by reanalysing and expanding an earlier study including uninvaded vegetation (seedlings removed continuously), early invader removal (saplings removed), late removal (trees removed), and no removal (invaded). These treatments allowed us to evaluate the stage-dependent belowground trophic responses to biological invasion and removal. We found that invaded plots had half the nematode taxa richness compared to uninvaded plots, and that tree invasion altered the overall composition of the nematode community. Differences in nematode community composition between uninvaded nematode communities and those under the tree removal strategy tended to dilute higher up the food chain, whereas the composition of uninvaded vs. sapling removal strategies did not differ significantly. Conversely, the composition of invaded compared to uninvaded nematode communities differed across all trophic levels, altering the community structure and function. Specifically, invaded communities were structurally simplified compared to uninvaded communities, and had a higher proportion of short life cycle nematodes, characteristic of disturbed environments. We demonstrate that a shift in management strategies for a globally invasive tree species from removing trees to earlier removal of saplings is needed for maintaining the composition and structure of soil nematode communities to resemble uninvaded conditions.
Invasive species management programs are widely pursued to prevent or mitigate the impacts that invaders have on native communities (e.g., [1,2]). Despite these efforts, many invasive plant eradication programs either fail to meet their stated management goals or do not rigorously evaluate success (e.g., [3-5]). Those plant invasive removal programs that do measure effectiveness typically focus on responses of the targeted invasive plant species to management per se [6], even though assessing the recovery of ecosystems should represent an important part of the evaluation of success, as it is usually an implicit goal of management (e.g., [7]). Some successful invasive eradication programs that have measured ecosystem responses have shown an increase in species richness within a trophic level (e.g., plants) following invasive plant removal [8], whereas others have found that the removal of plant invaders has no net effects, or even negative effects, on native species [9,10].Because the outcomes of invasive plant species removal, in terms of ecosystem impacts, are variable [11,12], it is essential to evaluate the performance of different management strategies within a system. Previous studies have largely assessed the effectiveness of physical and chemical removal techniques [8,9,12,13], but few have compared removal strategies at different stages of the invasion process, which is needed to determine the best timing for management. Furthermore, most studies that assess the effectiveness of invasive plant eradication programs have considered only the response of plant communities [13-15], with only a few examining changes across trophic levels [6,16,17], even though multitrophic level approaches are necessary to provide management guidance [18]. In addition, invader impacts are usually measured in terms of effects on species numbers [15,19], although more holistic approaches, like the use of ordination techniques, can help to capture community-level effects by quantifying shifts in the composition of species assemblages [20,21].Among the few studies assessing the effects of invasive plant species removal strategies at different stages of the invasion process, Dickie et al. [16] showed that removing invasive plants at later stages of the invasion process generates a plant community composition that does not resemble communities from either earlier invaded nor uninvaded areas. Conversely, removing saplings allows plant community composition to more closely resemble that of uninvaded areas. Furthermore, Dickie et al. demonstrated that different management strategies can cause changes in soil chemistry, soil microbial composition, and the abundance of different invertebrate feeding groups. Despite these significant findings, whether different stage-dependent invasive removal strategies also alter the diversity and composition of soil invertebrate communities remains unknown. Moreover, understanding whether removal strategies at later stages of the invasion process also have a stronger impact on the soil food-web structure and function could be useful for informing management decisions. We therefore, took advantage of the soil biota data collected by Dickie et al. [16] to perform an in-depth assessment of soil biota responses to the removal of an invasive plant at different invasion stages.Within the soil biota, nematodes dominate soil food webs and their functioning [22], providing important information on the structure and complexity of the soil food web. Nematodes are also useful bioindicators of ecosystem processes, resource availability and disturbance of the soil environment [23,24]. Furthermore, because nematodes are intrinsically affected by changes in plant communities [25], they can be greatly affected by plant invasions and plant removal strategies. For example, bacterial feeding nematodes are more abundant in both invaded areas and in areas where invasive trees have been removed, compared to uninvaded areas [16]. Moreover, nematode communities dominated by bacterial feeders are also associated with higher rates of litter decomposition, suggesting that changes in the composition of nematode communities are strongly linked to ecosystem processes such as nutrient cycling. Despite these changes in the abundance of particular nematode feeding groups [16], it remains unknown how invasive plants affect the diversity, composition, structure and function of nematode communities and whether invasive removal strategies can reverse such impacts. Furthermore, because nematodes are present in different trophic levels, they could be useful indicators of bottom-up cascading effects of invasive plants and invasive removal strategies.We evaluated the effectiveness of different management strategies of a globally invasive tree species, Pinus contorta [26], by using nematode abundance data previously reported in Dickie et al. [16], and additional data on taxa identity and life strategies. We determined whether the diversity and composition of soil nematodes differ amongst invaded (i.e., no removal), early invader removal (i.e., sapling removal), late removal (i.e., tree removal) or uninvaded (i.e., seedling removal) treatments to resolve three hypotheses. (1) Because plant invasions strongly reduce the abundance, and alter the composition of, native plant communities that nematodes largely rely on [14,16], nematode taxa richness will decrease in invaded compared to uninvaded sites, and the nematode community composition will strongly differ between these two treatments. In addition, we expect invaded plots to present a higher proportion of nematode taxa with short life cycles, because these taxa are better adapted to disturbances [27]. (2) After removal of the invasive plant and recolonization by grasses, which represent an important food resource and habitat for nematodes, the richness and composition of the nematode community will recover to resemble uninvaded communities. The removal of the invasive plant should also help to restore the nematode-based soil food web structure and function. Therefore, we expect uninvaded communities, as well as those communities where the invasive plant species was removed, to have a more complex structure, with higher abundance of long life cycle nematode taxa characteristic of undisturbed habitats [27] compared to invaded areas. Furthermore, (3) we expect changes in nematode composition should be observed across all trophic levels of the nematode-based food web due to bottom-up regulation by resource availability [28,29]. Nevertheless, if more advanced stages of the invasion process have stronger legacy effects, the removal of invasive trees might not allow nematode communities to recover and resemble those of uninvaded plots, whereas the removal of saplings would. Finally, we aim to identify which nematode taxa have the greatest contribution to the compositional changes observed, and which removal strategy would be the most appropriate to adopt to restore the soil nematode community after P. contorta invasion.
Methods
Our research was covered under the global concession for research sampling and analysis issued by the New Zealand Department of Conservation to Manaaki Whenua Landcare Research (Permit Number CA-31615-OTH).
Study site
We studied tree invasions and their management at Craigieburn Forest Park (43°9’04”S, 171°43’52”E), Canterbury, New Zealand, as described previously in [16]. This area is dominated by southern beech (Nothofagus solandri var cliffortioides) and open native shrubland and tussock grassland. Parts of this site were previously used from the 1950s until 1970s as an experimental forest area in which several species of non-native trees were introduced including Pinus contorta Loudon (lodgepole pine) [30]. In recent years there have been several different management approaches applied to prevent further spread or local impacts of invasive trees. Within this site, twenty-four 0.04 ha (20 x 20 m) plots with different management histories of the non-native invasive tree species P. contorta were selected. From these plots, six had continuous removal of P. contorta seedlings (< 2 cm basal diameter) where invasion was prevented by removing new seedlings at least twice annually (‘seedling-removal’ plots, i.e., the closest approximation to uninvaded areas); seven plots had P. contorta sapling-removal (ca. 10 cm basal diameter and 3–5 m height), which represents the removal of the invader at an early stage (‘sapling-removal’); five plots where tree-removal (closed canopy ca. 10 m height stands) was carried out, i.e., invasion was allowed and then later removed (‘tree-removal’); and six plots where P. contorta trees (closed canopy ca. 10 m height stands) have not been removed (‘no-removal’, i.e., invaded plots). Removals were done as part of operational management by contractors rather than imposed as an experimental treatment to plot per se. Aboveground biomass was not removed, which has the advantages of avoiding any export of mineral nutrients as well as being a realistic scenario for either management or natural disturbance, but does result in a temporary increase in organic matter. Furthermore, any attempt at biomass removal would have been inherently incomplete as there is no practical method to remove root biomass. Treatments were spatially interspersed and sampling occurred ca. three years after sapling- and tree-removal treatments, with some reinvasion occurring at the time of sampling. Reinvasion was most extensive in the sapling removal treatment, with all new invasion < 1 m height and not forming a closed canopy. Additional information about the site and plot selection is provided in Dickie et al. [16].
Sampling
Soil samples were collected and homogenised from five locations within each plot (the centre and at four orthogonal points 7.07 m from the centre) using a 65 mm diameter metal coring device to sample the top 100 mm of mineral soil. Litter was not included in our samples because it was generally sparse, and even when present under the densest pine in the no-removal (invaded) treatment, there was an abrupt transition between litter and mineral soil (i.e., there was no appreciable development of an O soil horizon).We extracted soil nematodes using the tray method [31] from c. 80 g (dry mass) of each soil sample. We then identified approximately 100 individuals from each sample to nominal genus level and, when further identification was possible, we classified genera into morphospecies. Hereafter we use the term taxa to refer to nematodes ids (i.e., genus and morphospecies). We used the proportion of each taxa as an estimate of the taxa abundance of each sample. After taxonomic identification, we assigned individual nematode taxa to one of six feeding categories according to their feeding habits (plant feeders, plant associated, bacterial feeders, fungal feeders, predators or omnivores) [32]. We designated plant feeder and plant associated nematodes to the first trophic level of the nematode-based soil food web (TL 1), bacterial and fungal feeders to the second trophic level (TL 2), and predators and omnivores to the third trophic level (TL 3) [33]. Nematodes from TL 2 are microbial feeders and therefore indirectly affected by plant changes, as root exudates influence the microbial community [34]. Similarly, nematodes from TL 3 can also be indirectly affected by changes in plant composition, as well as directly by changes in nematodes from TL 1 and TL 2, from which they feed.
Nematode-based ecological indicators
To estimate changes in the structure and function of the nematode-based soil food web, we used three commonly used nematode community level indices. First, we calculated sigma maturity index (ΣMI) [27,35], which is based on the abundance of nematodes’ functional guilds [36]. To this end, nematodes were classified according to their life strategies along a coloniser-persister (cp) gradient, where colonisers and persisters are extremes of the cp scale from 1–5 respectively [27]. Briefly, coloniser nematodes are those typically having short life cycles, and under favourable conditions, can rapidly increase in abundance. In contrast, persister nematodes have long life cycles, low colonisation ability, low reproduction rate, and are more sensitive to habitat disturbances. The ecological indicator ΣMI reflects the proportion of the different cp groups in the community, with higher ΣMI values representing higher proportions of persister nematodes and hence indicating less disturbed environments.The second ecological indicator we used was the enrichment index (EI), which measures the resource status, i.e., soil fertility, of the ecosystem [23]. Finally, to assess the level of complexity of the community we used the structure index (SI). SI indicates the prevalence of trophic links in the soil food web, where higher SI values indicate higher number of trophic links, i.e., enhanced trophic structure and redundancy [23]. All indices were calculated using the Nematode Indicator Joint Analysis program [37].
Analyses
To determine whether nematode taxa richness differed across management strategies (seedling-removal, sapling-removal, tree-removal, or no-removal), we used the total number of taxa of the entire community as the response variable in a generalised linear model (GLM). We entered management strategies as a fixed factor (factor with four levels) in the model and used the Poisson error distribution. We also used Tukey’s tests to estimate differences between management strategies.In addition, we tested whether the taxa composition of the entire nematode community differed between management strategies using Permutational Analyses of Variance (PERMANOVA) [38]. To accomplish this, we used two dissimilarity metrics that differ in the emphasis they give to taxa composition vs. relative abundance of each taxon. We used the Jaccard dissimilarity metric, which only uses taxa presence-absence, and the Bray-Curtis dissimilarity metric which also incorporates differences in the relative abundances of taxa. We performed two PERMANOVAs, one with the dissimilarity among plots for the entire nematode community estimated with the Jaccard index as the response variable and management strategies as the predictor. The second PERMANOVA included the dissimilarity among plots for the entire nematode community estimated with the Bray-Curtis index as the response variable, and management strategies as the predictor. We also conducted pairwise multilevel comparisons (with both dissimilarity metrics) to assess differences between individual management strategies. Furthermore, as a way to assess functional changes related to nematodes across management strategies, we compared the nematode-based ecological indicators (ΣMI, EI, SI) across management strategies using ANOVAs and Tukey’s tests for comparisons across management strategies.To evaluate whether different invasive management strategies affected taxa richness and composition of each trophic level (TL), we used GLMs and PERMANOVAs, respectively. Specifically, we performed one GLM (with Poisson error distribution) for each TL, to assess differences in taxa richness across management strategies, and two PERMANOVAs for each TL (one with each dissimilarity metric) to test for differences in taxa composition across management strategies. Finally, we used indicator species analyses to determine which specific nematode taxa mostly drove community composition changes at each trophic level.All analyses were performed in the R environment [39]. We tested for the overdispersion of residuals assumption of all the Poisson models. We used the ‘adonis’ and ‘betadisper’ functions of the vegan package [40] for the PERMANOVAs (9999 permutations), and the ‘pairwise.adonis’ function [41] for the pairwise multilevel comparisons. We tested for the PERMANOVA homogeneity of multivariate dispersions assumption [42], and used Principal coordinate analysis (PCoA) to illustrate differences between management strategies. We also used the ‘multipatt’ function from the indicspecies package [43] to perform the indicator species analyses.
Results
Across all management strategies we identified 46 nematode taxa (S1 Table), with an average of 16 ± 4 [mean ± SD] taxa per plot. Across all taxa, 8 belonged to trophic level 1 (TL 1, i.e., plant feeder and plant associated nematodes), 22 to TL 2 (bacterial and fungal feeders) and 16 to TL 3 (predators and omnivores) (S1 Table).Taxa richness of the overall nematode community was higher in seedling-removal plots only compared to no-removal plots (Z = -2.441, P = 0.015) (Fig 1A; S2 Table). On the other hand, the overall nematode community composition of seedling-removal plots differed significantly from both no-removal (Pseudo-F = 9.098, P = 0.003) and tree-removal plots (Pseudo-F = 2.234, P = 0.009), but not from sapling-removal plots (Pseudo-F = 0.843, P = 0.688) (Fig 1B; S3 and S4 Tables) when taking into account the relative abundance of taxa (Bray-Curtis dissimilarity). However, when assessing community composition changes by only considering the presence-absence of nematode taxa (Jaccard dissimilarity), only differences between seedling-removal and no-removal plots were observed (Pseudo-F = 4.147, P = 0.002) (S1 Fig, S3 and S4 Tables).
Fig 1
Responses of total nematode richness and community composition across each of four management strategies for an invasive tree.
Management strategies representing different stages of invasion process: seedling removal, sapling removal, tree removal, no removal. (A) Taxa richness; (B) community composition. (A) Different letters represent significant differences obtained from Tukey’s test (P < 0.05). In each box plot the middle line indicates the median, bottom and top box limits are the first and third quartiles, respectively, whiskers indicate most extreme points 1.5 times the interquartile range, and circles indicate outliers. (B) Principal Coordinate analyses were based on the Bray-Curtis dissimilarity metric. Sites closer together in multivariate space have similar compositions. Dashed lines represent convex hulls in ordination space.
Responses of total nematode richness and community composition across each of four management strategies for an invasive tree.
Management strategies representing different stages of invasion process: seedling removal, sapling removal, tree removal, no removal. (A) Taxa richness; (B) community composition. (A) Different letters represent significant differences obtained from Tukey’s test (P < 0.05). In each box plot the middle line indicates the median, bottom and top box limits are the first and third quartiles, respectively, whiskers indicate most extreme points 1.5 times the interquartile range, and circles indicate outliers. (B) Principal Coordinate analyses were based on the Bray-Curtis dissimilarity metric. Sites closer together in multivariate space have similar compositions. Dashed lines represent convex hulls in ordination space.When testing the effects of invasive removal strategies on the structure and function of the nematode-based food web, we found that two of the three nematode-based ecological indicators assessed differed between the no-removal plots and all other management strategies. More specifically, ΣMI and structure index (SI) were lower in no-removal plots compared to seedling-, sapling- and tree-removal plots (Fig 2A and 2C; ΣMI: F = 22.359, P < 0.001; SI: F = 21.222, P < 0.001), but no differences were observed for enrichment index (EI) (F = 1.166, P = 0.347; Fig 2B).
Fig 2
Nematode community level indices across four management strategies for an invasive tree.
Nematode community indices: (A) ΣMI = sigma maturity index, (B) EI = enrichment index and (C) SI = structure index. Management strategies for an invasive tree representing different stages of the invasion process: seedling removal, sapling removal, tree removal, no removal. Different letters represent significant differences obtained from Tukey’s test (P < 0.05).
Nematode community level indices across four management strategies for an invasive tree.
Nematode community indices: (A) ΣMI = sigma maturity index, (B) EI = enrichment index and (C) SI = structure index. Management strategies for an invasive tree representing different stages of the invasion process: seedling removal, sapling removal, tree removal, no removal. Different letters represent significant differences obtained from Tukey’s test (P < 0.05).When considering different trophic levels (TLs) separately, we found that taxa richness was at least two-fold higher in seedling-removal and in sapling-removal plots compared to no-removal plots for TL 1 (Z = -2.404, P = 0.016) (Fig 3A), but did not vary among management strategies for the other trophic levels (Fig 3C and 3E; S2 Table). In addition, the nematode composition of TL 1 in the seedling-removal plots differed significantly from the composition in no-removal plots (Jaccard dissimilarity: Pseudo-F = 7.487, P = 0.006; Bray-Curtis: Pseudo-F = 10.123, P = 0.002), but not from the sapling-removal plots (Jaccard dissimilarity: Pseudo-F = 1.241, P = 0.334; Bray-Curtis: Pseudo-F = 1.310, P = 0.209) both when using only presence-absence data (Jaccard dissimilarity) as well as when incorporating nematode abundance (Bray-Curtis dissimilarity) (Fig 3B; S3 and S4 Tables). Differences in TL 1 taxa composition between seedling-removal and tree-removal plots were only detected when incorporating the abundance of nematode taxa (Bray-Curtis: Pseudo-F = 3.330, P = 0.006) (Fig 3B; S4 Table).
Fig 3
Nematode taxa richness and community composition of different trophic levels across management strategies.
Trophic levels: TL1-TL3. Management strategies: seedling removal, sapling removal, tree removal, no removal. Principal Coordinate analyses were based on the Bray-Curtis dissimilarity metric. Sites closer together in multivariate space have more similar compositions. Different letters in (A), (C), (E) represent significant differences obtained from Tukey’s test (P < 0.05).
Nematode taxa richness and community composition of different trophic levels across management strategies.
Trophic levels: TL1-TL3. Management strategies: seedling removal, sapling removal, tree removal, no removal. Principal Coordinate analyses were based on the Bray-Curtis dissimilarity metric. Sites closer together in multivariate space have more similar compositions. Different letters in (A), (C), (E) represent significant differences obtained from Tukey’s test (P < 0.05).For both TL 2 and TL 3, the nematode composition of the seedling-removal plots differed significantly only from that of the no-removal plots, both when considering the presence-absence of nematode taxa (TL 2: Pseudo-F = 3.006, P = 0.007; TL 3: Pseudo-F = 3.519, P = 0.013) as well as when incorporating their abundances (TL 2: Pseudo-F = 9.512, P = 0.007; TL 3: Pseudo-F = 3.866, P = 0.009) (Figs 3D, 3F, S2B and S2C; S4 Table). The composition of TL 2 from seedling-removal plots also differed from tree-removal plots only when considering the presence-absence of taxa (Pseudo-F = 2.110, P = 0.048) (S4 Table). Nevertheless, the composition of TL 2 did not differ between seedling-removal plots and sapling-removal plots, both when excluding (Pseudo-F = 0.980, P = 0.466) and including (Pseudo-F = 0.730, P = 0.726) taxa abundance (Figs 3D and S2B; S4 Table).The indicator species analyses allowed us to identify taxa that were most strongly associated with a management strategy or a group of management strategies (S5 Table). For instance, Doryllaimellus (TL 1), Tylencholaimus sp 2 (TL 2) and Aporcelaimidae (TL 3) were significantly associated with seedling-, sapling- and tree-removal management strategies (Fig 4). In addition, Doryllium (TL 2) was a good indicator of seedling-removal plots, whereas Plectus robus (TL 2) was strongly associated with sapling-, tree- and no-removal plots (Fig 4).
Fig 4
Relative abundance of nematode taxa (i.e., percentage composition of each nematode taxon within a sampling site) across management strategies and trophic levels.
Trophic levels: TL1-TL3. Management strategies: seedling removal, sapling removal, tree removal, no removal.
Relative abundance of nematode taxa (i.e., percentage composition of each nematode taxon within a sampling site) across management strategies and trophic levels.
Trophic levels: TL1-TL3. Management strategies: seedling removal, sapling removal, tree removal, no removal.
Discussion
Multiple management strategies are often deployed in an attempt to reduce the abundance or distribution of invasive species, but with an ultimate goal of avoiding or mitigating negative impacts on native ecosystems (e.g., [44]). Thus, selection of different management strategies should consider both reduction of the invader abundance and the response of native communities [45]. Despite the high relevance of assessing the effects of invasive species removal at different stages in the invasion process, most studies on invasive species impacts consider only the effects of the invader as invasion progresses, but not the effects of invader removal as invasion proceeds [46]. As a contribution to fill this gap in our knowledge, our study demonstrates that both invasion and removal strategies alter the taxonomic composition of the soil nematode community and that invasion also alters community structure and function.The decrease in the number of nematode taxa with invasion, with lower richness in invaded (no-removal treatment) compared to uninvaded (seedling-removal treatment) plots, is consistent with the decrease in diversity of nematodes and other invertebrates caused by other invasive species [47,48], including other pine species [24], although the opposite pattern has also been observed [49]. In addition, nematode communities from invaded plots differed in taxa composition, suggesting that P. contorta exerts a selective pressure over the soil nematode taxa that can inhabit the soil. Furthermore, nematode taxa that were most abundant in invaded plots were those that proliferate under disturbances or stressful conditions, as identified by the decrease in ΣMI. Similar proliferation of nematodes having short life cycles, as well as similar reductions in complexity and redundancy of nematode communities in invaded plots (SI decrease), have also been observed in areas invaded by other tree species [50,51].An unresolved issue is when invasive species management should be deployed to avoid or reverse negative impacts on communities or ecosystems [20,46]. Our findings demonstrate that relatively early management is needed to avoid impacts of an invasive tree species on the composition of belowground nematode communities. For example, when considering the entire nematode community composition we found that only the sapling-removal strategy resembled uninvaded (seedling removal) plots. Even though sapling removal proved to be a better management strategy for the preservation of the nematode community composition, both sapling- and tree-removal management strategies seem to result in similar community structure. In particular, both removal strategies had similar levels of the structure index (SI) and sigma maturity index (ΣMI) to uninvaded areas, and both indices were higher compared to invaded (no-removal treatment) areas. This suggests that invader removal allows nematode communities to increase their complexity and redundancy to a considerable extent, and also favours nematodes having longer life cycles, which tend to survive only in more stable, less disturbed, environments [23]. Nevertheless, to help restore and preserve the nematode diversity, especially under the tree-removal strategy, further management interventions might be required. To this end, restoration using planting seedlings or increasing the seedbank of native species should be considered [52,53]. Furthermore, inoculation with taxa that were completely eliminated could also be considered [54] to promote restoration of disturbed ecosystems [55]. These interventions could help to restore relatively slow nutrient cycling processes characteristic of uninvaded areas [16,56], and even steer the development of plant communities [55,57].Changes in the number of taxa is a widely used measure of the impacts and the effectiveness of invasive species removal [9,10,19]. However, we found that the number of taxa (richness) of nematodes failed to capture changes across the upper trophic levels of nematode communities caused by invasion and different removal strategies. Nevertheless, these differences were detected when using multivariate analyses that incorporated only the identity or the identity and abundance of species that are present at a specific place. Differences in nematode community composition among management strategies were strongly influenced by a few taxa. For instance, Doryllaimellus, Tylencholaimus sp 2 and Aporcelaimidae were indicator taxa from seedling- (uninvaded), sapling- and tree-removal management strategies. These three taxa belong to cp group 4 and 5, suggesting that their absence or scarcity in invaded plots is driving the low values of ΣMI in invaded plots. Furthermore, the plant feeder Doryllaimellus has also been recorded as absent in areas heavily invaded by Pinus nigra [51], suggesting that this taxa could be highly vulnerable to pine invasions in general. In addition, another indicator taxa from uninvaded plots was the fungal feeder Doryllium. Uninvaded plots, compared to invaded and afforested areas, tend to have a higher proportion of fungal feeders [24,50,58], which leads to slower decomposition rates compared to bacterial dominated systems [59,60].Overall, the composition of invaded and uninvaded soil nematode communities differed across all trophic levels (including the highest trophic level, TL 3), suggesting that the impacts of invasive species can cascade up the nematode-based food web. Because our study focuses on a subset of soil organisms, future research should assess the extent to which these findings apply to the entire soil food web, and in turn, feedback to aboveground communities [56].
Conclusions
Controlling invasive species is complex because it involves a de facto manipulation of complex systems [61,62]. However, both the effectiveness of invasive species management and the longer-term recovery of more complex community structure and ecological processes are rarely measured [9]. We demonstrate that a shift in management strategies for a globally invasive tree species (Pinus contorta) from removing trees to earlier removal of saplings is needed for maintaining the taxonomic composition of soil nematode communities to resemble uninvaded conditions. In addition, the community-level responses of nematodes to management closely resemble those observed for shifts in plant composition found in the same experiment [16]. Our findings support the early management of invasive species to prevent impacts and potentially strong belowground legacies that can undermine our ability to restore community composition and ecosystem functions over the longer-term.
Nematode community composition across each of four management strategies for an invasive tree.
Management strategies representing different stages of invasion process: seedling removal, sapling removal, tree removal, no removal. Principal Coordinate analyses were based on the Jaccard dissimilarity metric. Sites closer together in multivariate space have similar compositions. Dashed lines represent convex hulls in ordination space.(PDF)Click here for additional data file.
Nematode community composition of different trophic levels across management strategies.
Trophic levels: (A) TL1, (B) TL2, (B) TL3. Management strategies: seedling removal, sapling removal, tree removal, no removal. Principal Coordinate analyses were based on the Jaccard dissimilarity metric. Sites closer together in multivariate space have more similar compositions.(PDF)Click here for additional data file.
Nematode classification.
Nematode taxon, feeding group (plant feeder, plant associated, bacterial feeder, fungal feeder, predator, omnivore), trophic level (1–3) to which each taxa was assigned and management strategy where each taxa was present (U = seedling removal, SR = sapling removal, TR = tree removal, NR = No removal).(DOCX)Click here for additional data file.
Results of generalised linear models with Poisson error distribution comparing taxa richness of seedling-removal management strategy with taxa richness of sapling-removal, tree-removal and no-removal management strategies.
TL = trophic level. Bold values indicate significant results (α = 0.05).(DOCX)Click here for additional data file.
Results of PERMANOVA analyses of Jaccard and Bray-Curtis dissimilarity in the entire nematode community and TL 1, TL 2 and TL 3 community composition across different management strategies.
Management strategies: seedling removal, sapling removal, no removal, tree removal. Bold values indicate significant results (α = 0.05).(DOCX)Click here for additional data file.
Pairwise multilevel comparisons of nematode community composition between different management strategies of the entire nematode community composition and across different trophic levels.
Management strategies: seedling removal, sapling removal, no removal, tree removal. Jaccard and Bray-Curtis dissimilarity metrics were used to assess differences in community composition. Bold values indicate significant differences in community composition (α = 0.05).(DOCX)Click here for additional data file.
Indicator nematode taxa within trophic level.
Significant indicator taxa of individual or group of management strategies as identified by indicator species analyses for each trophic level. Analyses were performed including taxa abundance and using only presence-absence data. P values were obtained by permuting the data 9999 times. Bold values indicate significant results (α = 0.05).(DOCX)Click here for additional data file.20 Aug 2019PONE-D-19-17858Multi-trophic level responses of belowground nematode communities to stage dependent removal strategies of an invasive treePLOS ONEDear Miss Peralta,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.We would appreciate receiving your revised manuscript by Oct 04 2019 11:59PM. 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We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere: "Part of the data has been used in a previous publication, as explained in the cover letter and in the manuscript (introduction and material and methods)."Please clarify whether this [conference proceeding or publication] was peer-reviewed and formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript.Additional Editor Comments:Both reviewers are pretty positive regarding the study itself and the results but both recommend substantial text work in more or less all parts of the manuscript.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.Reviewer #1: PartlyReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously?Reviewer #1: NoReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.Reviewer #1: NoReviewer #2: No**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: No**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)Reviewer #1: The reviewed manuscript describes the results of an experiment examining the responses of soil nematode communities to an invasive plant (Pinus contorta) management intensity gradient. I am always excited to see new studies examining the responses of soil nematodes to plant invasion and management. The results presented here are interesting and highlight the importance of considering soil community responses to plant invasion and management. This manuscript presents data from an experiment originally described in Dickie et al. 2014 (AoB Plants). I remain at least marginally concerned about potential overlap between this MS and Dickie at al. 2014, which also presents some soil nematode data. Dickie et al. 2014 reports abundance data of nematode feeding guilds (bacterial-feeding, fungal-feeding, etc.), but community composition and diversity/richness data were not reported. Thus in my opinion, the data presented here are a substantial expansion of the Dickie et al. 2014 paper, though not entirely independent.That being said, I would recommend restructuring the Introduction to better frame this work as a direct expansion of Dickie et al. 2014. That paper is cited six times prior to the reveal that this MS is an in-depth assessment of nematodes initially reported in that paper. Over that same stretch of MS, no other paper is cited more than twice. So this MS relies heavily on Dickie et al. 2014. For the sake of transparency, I’d recommend acknowledging that upfront. Additionally, maintaining similar terminology between Dickie et al. 2014 and this MS would facilitate an easier comparison between the two. For example, Dickie et al. 2014 refers to the four treatments as 1) seedling-removal, 2) sapling-removal, 3) no-removal, and 4) tree-removal. In this MS, the four groups are 1) uninvaded, 2) sapling-removal, 3) tree-removal, and 4) invaded.Additional methodological details are needed to fully assess experimental rigor. I also recommend new analyses to provide insights into nematode community responses to invasion and management.Abstract:-No direction or magnitude of invasion or management effects are included in the abstract. I recommend adding statements along the lines of “Invaded plots had XX% lower richness than seedling removal plots.”Introduction:-The hypotheses focus primarily on nematode richness and community composition. Composition data typically includes taxon abundance, which may be encroaching on the abundance data presented in Dickie et al. 2014.Methods:-The phrasing ‘we selected‘ plots (line 118) sounds strange. Were these not the same 24 plots established in Dickie et al. 2014?-Is removal of seedlings twice a year often enough to be considered ‘uninvaded’? I’d like to see more data on the number of seedlings removed. Early removal of 3 vs. 3000 seedlings would be an important factor in determining if you are maintaining this area as uninvaded, or if it is well into the early stages of invasion.-Why no removal of invasive plant biomass from the site (line 127)? I need some justification for this. With herbaceous plant biomass, it is more typical to remove the biomass since its decomposition will alter soil nutrients and soil fauna.-Sampling occurred three years after removal treatments, but more details are needed. Was sampling three years after the initiation of removal treatments if removal treatments were a one-time event? Seedling removal seems like it would need to be recurring regularly, which it may have been (line 121), but I’m still not clear on this. If reinvasion as occurred (lines 128-129) has this shifted plots from one group to another?-I have some concerns about the designation of trophic groups T1, T2, and T3. As presented, it invokes a linear food chain with T1 being directly associated with the plant (by feeding on the plant) with T2 and T3 being sequentially further removed from direct plant interactions. In reality, these are two pathways of energy transfer from the plant, either to 1) plant-feeding nematodes directly, or to 2) microbial-feeding nematodes indirectly via root exudation and root turnover stimulating bacteria and fungi, which are then consumed by nematodes. Predatory nematodes may serve to bridge these two energy pathways by feeding on plant-feeding, bacterial-feeding, and fungal-feeding nematodes. I’d argue that the term ‘trophic level’ is not appropriate, and perhaps use ‘trophic group’.-Were nematode analyses run on genera or morpho-species data? Line 172 states that number of species were used, but nematodes were only identified to genus or morpho-species (lines 144-146).-It seems that relative abundance data was used for the ordinations and PERMANOVA (line 181). Is this correct? I highly recommend use of absolute abundance data, since differences can be caused by both changes in species identity but also abundance, though use of abundance data might overlap more with the Dickie et al. 2014 paper.-Following up on my earlier comment regarding the designated trophic levels, I think the SEM needs justification for including a direct effect of plants on TL3. In a soil food web, effects of plants on predaceous nematodes would likely be modulated by the lower trophic levels (TL1 and TL2). Direct effects of management could still exist on TL3, likely through habitat disruption, and could be included in the SEM.Results:-My biggest concern with the Results is that no data are presented to show which nematode species, or trophic groups, are driving the observed changes in composition. The PERMANOVA is great for testing for differences between groups, and the PCoA is one method for visualizing differences among groups, but for food webs it is important to know which genera/trophic groups are driving the observed changes. Without these data, we have an incomplete picture of the soil nematode responses to invasion and management. Ideally, inclusion of additional analyses, such as an indicator species analysis, or at minimum, a breakdown of feeding groups and/or trophic levels by treatment are needed.Discussion:-The Discussion generally lacks comparison of these results to previously published studies examining soil nematode responses to invasive plant management (lines 289-308).-Are there comparisons you can make with other published studies examining the timing of invasive plant management on other organisms? Comparisons to aboveground fauna may also be of interest to assess the generality of invasion and management effects in higher trophic levels.Data accessibility:-No database is listed where data will be deposited upon successful publication.Minor comments:-line 253: remove extra period after Invaded-line 304: change ‘same area’ to ‘same experiment’-line 330: change ‘than’ to ‘to’Reviewer #2: 1. The manuscript reports changes of soil nematode community structure in response to ontogenic removal of an invasive tree species. The study was well designed and the methodology was proper to address the concerns. However, this manuscript is overall poorly written and may need reorganization.2. The title is confusing (e.g. stage-dependent, what stages?) and not informative enough as the authors estimated the responses of soil nematodes both at community (composition, indices etc.) and trophic level to the management strategies. I suggest it could be changed like “Community- and trophic-level responses of soil nematodes to removal of an invasive tree species at different invasion stages”.3. Sap- and tree-removal are the two comparable management strategies (treatments) employed in this study. However, in the introduction I did not read any information about “why the two strategies were comparably tested” or “what are the potential difference in responses of soil food webs to sap- and tree-removal”.4. The hypotheses need to be adjusted: (1) Line 92-93: I could not read how the nematode species richness will increase if invasions reduce the abundance and alter the composition of native plant communities. I guess the authors attempted to highlight the invasions may reduce the abundance and diversity of native plant communities, right? (2) I understand that removal of invasive trees would results in the recolonization of herbs, which may help restore the nematode communities…However, I am confused by the following sentence “We expect uninvaded communities to have more complex structure, with higher abundance of long-life cycle nematode taxa…”. Does that mean uninvaded habitat will have more complex structure or higher abundance of nematodes than “invaded habitat”?5. The authors claimed that the removal management strategies influence the soil nematodes via effects on plant community, such as indicated in Fig. 4, but I could not find any data or related analysis on plant community. I strongly suggest them to include data on plant community composition they scored (percentage of coverage). Otherwise, I could not see the value of Fig. 4 in this study.6. I would change the structure of the manuscript, showing the community- and trophic- level responses separately. Fig. 1a, b and Fig. 3 showed tree species invasion indeed resulted in a different nematode community responses from uninvaded soil, and both sap- and tree-removal could reverse this disturbance of invasion to the level of uninvaded treatment. On the other hand, at the trophic level, it seems only sap-removal of the invasive tree species could reverse the disturbance (represented as nematode community responses) back to the level of the uninvaded treatment (Fig. 1c and Fig. 2), suggesting different trophic levels would respond to management strategies in different ways.7. I found the results were not fully discussed in the current version. I would like to read discussion on (1) invaded vs. uninvaded: tree invasion indeed alter soil nematode communities; (2) removal of these invasive trees could reverse these invasion influences; (3) the extent of removal of invasive trees recovering nematode communities depends on the invasion stage of removal.8. Specific comments:L62: what functional traits?L73: NOT a sentenceL83-86: not logicL90: “changes” should be “differs”L100-102: confusing. What is the comparison?L128-129: what do you want to say by mentioning “with some reinvasion…”?L143: where is the data of soil moisture?L167: more details are needed on SI to introduce this index.L172: “total number of species”? I thought authors mostly identify the nematodes at genus levelL319-321: not understandableL329: “have” should be “result in”L331: “both” should be “all three”L334: “perturbations” should be “perturbations, such as plant invasions”L340: should be “soil nematode communities” instead of “communities”**********6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: NoReviewer #2: No[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.10 Oct 2019>> Dear Associate Editor and reviewers: the line numbers in our responses refer to the line numbers of the manuscript with tracked changes.Journal Requirements:1. When submitting your revision, we need you to address these additional requirements.Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttp://www.journals.plos.org/ and http://www.journals.plos.org/>> Our manuscript meets the PLOS ONE's style requirements.2. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.>> Upon manuscript acceptance we will provide the data in an on-line repository (the datastore system hosted by Manaaki Whenua, https://datastore.landcareresearch.co.nz) to facilitate access and use of this information.3. We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere: "Part of the data has been used in a previous publication, as explained in the cover letter and in the manuscript (introduction and material and methods)."Please clarify whether this [conference proceeding or publication] was peer-reviewed and formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript.>> As we explain in the manuscript and in the first cover letter (i.e. when we submitted the manuscript for the first time), we use nematode data that was included in a very broadly focused paper (Dickie et al. 2014, AoB Plants). In this previous publication, only abundances of different nematode trophic groups were published; most results in that paper focused on shifts in plant community composition and soil nutrient availability. In our study we provide results for more detailed analyses that include additional information about nematode species identities and life strategies; this allows us, for the first time, to estimate the overall nematode-based food-web composition and structure. Thus, while some of the underlying data was included in a previous paper, this new manuscript presents (a) new data on species identity and life strategies, (b) new theoretical and analytical approaches to the data, and (c) an entirely new understanding of nematode community composition following invasion.Additional Editor Comments:Both reviewers are pretty positive regarding the study itself and the results but both recommend substantial text work in more or less all parts of the manuscript.>> We appreciate the positive feedback. We have incorporated the comments made by the reviewers and responded specifically to each of them below, marking our responses with ‘>>’.Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.Reviewer #1: PartlyReviewer #2: Yes>> We have clarified all the points made by reviewer #1 in the methods section.2. Has the statistical analysis been performed appropriately and rigorously?Reviewer #1: NoReviewer #2: Yes>> We have now incorporated the additional analyses suggested by reviewer #1.3. Have the authors made all data underlying the findings in their manuscript fully available?The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.Reviewer #1: NoReviewer #2: No>> We will provide the data in an on-line repository (the datastore system hosted by Manaaki Whenua, https://datastore.landcareresearch.co.nz) to facilitate access and use of this information upon manuscript acceptance.4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: No>> We have re-written several sections of the manuscript and re-structured the discussion, as suggested by reviewer #2.5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)Reviewer #1: The reviewed manuscript describes the results of an experiment examining the responses of soil nematode communities to an invasive plant (Pinus contorta) management intensity gradient. I am always excited to see new studies examining the responses of soil nematodes to plant invasion and management. The results presented here are interesting and highlight the importance of considering soil community responses to plant invasion and management. This manuscript presents data from an experiment originally described in Dickie et al. 2014 (AoB Plants). I remain at least marginally concerned about potential overlap between this MS and Dickie at al. 2014, which also presents some soil nematode data. Dickie et al. 2014 reports abundance data of nematode feeding guilds (bacterial-feeding, fungal-feeding, etc.), but community composition and diversity/richness data were not reported. Thus in my opinion, the data presented here are a substantial expansion of the Dickie et al. 2014 paper, though not entirely independent.That being said, I would recommend restructuring the Introduction to better frame this work as a direct expansion of Dickie et al. 2014. That paper is cited six times prior to the reveal that this MS is an in-depth assessment of nematodes initially reported in that paper. Over that same stretch of MS, no other paper is cited more than twice. So this MS relies heavily on Dickie et al. 2014. For the sake of transparency, I’d recommend acknowledging that upfront.Additionally, maintaining similar terminology between Dickie et al. 2014 and this MS would facilitate an easier comparison between the two. For example, Dickie et al. 2014 refers to the four treatments as 1) seedling-removal, 2) sapling-removal, 3) no-removal, and 4) tree-removal. In this MS, the four groups are 1) uninvaded, 2) sapling-removal, 3) tree-removal, and 4) invaded.>> As suggested by the reviewer, we now acknowledge upfront that this study represents an expansion of Dickie et al. 2014, in which we provide an in-depth assessment of soil biota responses to different removal strategies of a widespread invasive tree species (L28, 72-86). In addition, we have changed the terminology in our manuscript to make it consistent with that of Dickie et al. 2014, to facilitate comparison between these studies.Additional methodological details are needed to fully assess experimental rigor. I also recommend new analyses to provide insights into nematode community responses to invasion and management.>> We have modified the methods section according to the reviewer’s suggestions and also incorporated the new analysis suggested by the reviewer (also see below for additional details).Abstract:-No direction or magnitude of invasion or management effects are included in the abstract. I recommend adding statements along the lines of “Invaded plots had XX% lower richness than seedling removal plots.”>> We have incorporated more specific results in the abstract as suggested by the reviewer (L32-33, 36-37, 39-41).Introduction:-The hypotheses focus primarily on nematode richness and community composition. Composition data typically includes taxon abundance, which may be encroaching on the abundance data presented in Dickie et al. 2014.>> We have rephrased the hypotheses following the suggestions of reviewer #2. We have also incorporated the idea of finding out which nematode taxa are mostly driving the community composition changes, as suggested by reviewer #1. Furthermore, we have now incorporated a community composition analysis using the Jaccard dissimilarity index, which basically evaluates differences in the composition of communities by using only presence-absence data. With this new analysis we show that changes in nematode community composition between invaded and uninvaded plots are also evident when only taking into account the identity (i.e. presence-absence) of taxa (L291-294, 331-337, 340-343, 345-353).Although we acknowledge that we use abundance data that was also used in Dickie et al. 2014, we use abundance data at the taxa (genus or morpho-species) level, whereas Dickie et al. (2014) evaluated changes in the abundance of nematode feeding groups (i.e. not of each taxa independently, but at the feeding group level). Finally, to avoid any potential overlap with data reported by Dickie et al. we have now removed the trophic group composition analysis (previous Fig. 1c).Methods:-The phrasing ‘we selected‘ plots (line 118) sounds strange. Were these not the same 24 plots established in Dickie et al. 2014?>> We have rephrased this sentence.-Is removal of seedlings twice a year often enough to be considered ‘uninvaded’? I’d like to see more data on the number of seedlings removed. Early removal of 3 vs. 3000 seedlings would be an important factor in determining if you are maintaining this area as uninvaded, or if it is well into the early stages of invasion.>>This is a good point. We did not carry out the seedling removal treatment ourselves, but rather this has been carried out by volunteer groups who did not record data for number of seedlings removed during each management event. We have assessed seedling densities in the ‘uninvaded’ area, and typically observe 1-2 seedlings at a 20 x 20 m plot scale (or 25-50 seedlings per ha). We have clarified the ‘uninvaded’ treatment in the text to explain this is not zero or never-invaded, but is relatively uninvaded compared to the other management treatments we considered (160-161). Moreover, we also refer to Dickie et al. 2011 and Peralta et al. 2019 where such low densities of tree invasion have minimal or no detectable impacts on belowground communities.-Why no removal of invasive plant biomass from the site (line 127)? I need some justification for this. With herbaceous plant biomass, it is more typical to remove the biomass since its decomposition will alter soil nutrients and soil fauna.>> The reviewer raises another valid point. Removals were done as part of operational management at the site rather than imposed strictly as an experimental treatment. Because it is not practical or feasible for managers to remove (aboveground) tree biomass from a controlled site, the normal management practice is to leave biomass in situ. This practice has the advantages of avoiding any export of mineral nutrients as well as being a realistic scenario for either management or natural disturbance, although it does result in a temporary increase in organic matter. In addition, any attempt at biomass removal would have been inherently incomplete as there is no practical method to remove root biomass. We now state this clearly in the revised methods section (L167-173).-Sampling occurred three years after removal treatments, but more details are needed. Was sampling three years after the initiation of removal treatments if removal treatments were a one-time event? Seedling removal seems like it would need to be recurring regularly, which it may have been (line 121), but I’m still not clear on this. If reinvasion as occurred (lines 128-129) has this shifted plots from one group to another?>> As mentioned above, removal treatments were imposed by weed managers at the site. The tree removals were done within a single season by contractors, whereas the seedling removal treatment for the ‘univaded’ treatment was carried out by a volunteer group twice per year and is still ongoing. Because we took advantage of these operational management practices that had already occurred, we could not control the fact that ca. three years have passed since the sapling- and tree-removal treatments. Although there was some reinvasion at the time of sampling (L173-177), all new invasion was < 1 m height and not forming a closed canopy.-I have some concerns about the designation of trophic groups T1, T2, and T3. As presented, it invokes a linear food chain with T1 being directly associated with the plant (by feeding on the plant) with T2 and T3 being sequentially further removed from direct plant interactions. In reality, these are two pathways of energy transfer from the plant, either to 1) plant-feeding nematodes directly, or to 2) microbial-feeding nematodes indirectly via root exudation and root turnover stimulating bacteria and fungi, which are then consumed by nematodes. Predatory nematodes may serve to bridge these two energy pathways by feeding on plant-feeding, bacterial-feeding, and fungal-feeding nematodes. I’d argue that the term ‘trophic level’ is not appropriate, and perhaps use ‘trophic group’.>> Although the definition of trophic group is ‘a group of organisms consuming resources from a similar level in the energy cycle’, and hence the reviewer is correct that could apply to our classification, in nematode studies the term trophic group is usually associated to feeding types (i.e. plant-feeders, bacterial-feeders, etc.). Because in our classification we are combining different feeding types, we prefer to keep the term trophic level, to avoid any potential confusion with specific feeding types. The assignment of different nematode feeding groups to different trophic levels is well spread, from institutions such as the United States Department of Agriculture (https://www.nrcs.usda.gov/wps/portal/nrcs/detailfull/soils/health/biology/?cid=nrcs142p2_053866), to scientific nematode studies (e.g. Laliberte et al. Ecology Letters 2017). Nevertheless, we acknowledge that nematodes from higher trophic levels also depend indirectly on plants and, therefore, we have clarified this point in our manuscript (L200-203).-Were nematode analyses run on genera or morpho-species data? Line 172 states that number of species were used, but nematodes were only identified to genus or morpho-species (lines 144-146).>> Nematode analyses were run using the maximum classification possible for each taxa, i.e. genus or morphospecies. We recognised the term species was not accurate in line 172 and have now replaced it by the term taxa. In addition, we have clarified in the methods (previous lines 144-146) that we used the term taxa to refer to the maximum classification possible (L193-194).-It seems that relative abundance data was used for the ordinations and PERMANOVA (line 181). Is this correct? I highly recommend use of absolute abundance data, since differences can be caused by both changes in species identity but also abundance, though use of abundance data might overlap more with the Dickie et al. 2014 paper.>> Because the Bray-Curtis dissimilarity index is calculated based on the relative abundance of the different taxa, the use of absolute abundance data would reflect the same differences among treatments. In addition, to assess whether the differences in nematode community composition across treatments were mostly driven by changes in the relative abundance of taxa or whether the presence-absence of taxa was also a significantly important factor, we have now incorporated additional analyses using the Jaccard dissimilarity index. This index estimates the dissimilarity between communities based on the presence-absence of taxa only, disregarding the taxa relative abundance which the Bray-Curtis index incorporates. By using both of these indices, we show that not only changes in the relative abundance of taxa, but also changes in the identities of the nematode taxa are driving differences in community composition between treatments.-Following up on my earlier comment regarding the designated trophic levels, I think the SEM needs justification for including a direct effect of plants on TL3. In a soil food web, effects of plants on predaceous nematodes would likely be modulated by the lower trophic levels (TL1 and TL2). Direct effects of management could still exist on TL3, likely through habitat disruption, and could be included in the SEM.>> Because additional analyses on plant species composition cannot be added to this manuscript, as suggested by reviewer #2, as these are already published (Dickie et al. 2014), we decided to remove this analysis. We believe removing this analysis does not affect our conclusions and it also allows us to incorporate the additional species indicator analysis suggested by the reviewer.Results:-My biggest concern with the Results is that no data are presented to show which nematode species, or trophic groups, are driving the observed changes in composition. The PERMANOVA is great for testing for differences between groups, and the PCoA is one method for visualizing differences among groups, but for food webs it is important to know which genera/trophic groups are driving the observed changes. Without these data, we have an incomplete picture of the soil nematode responses to invasion and management. Ideally, inclusion of additional analyses, such as an indicator species analysis, or at minimum, a breakdown of feeding groups and/or trophic levels by treatment are needed.>> We have now included indicator species analyses (L255-256), as suggested by the reviewer. We believe with these additional analyses and the new Figure 4, it becomes clear which are the species that have the highest influence in the differences between community compositions across treatments.Discussion:-The Discussion generally lacks comparison of these results to previously published studies examining soil nematode responses to invasive plant management (lines 289-308).>> We now compare our results with those of previous studies examining soil nematode responses to plant invasions and management (e.g. Meisner et al. 2014, Čerevková et al. 2019, Peralta et al. 2019).-Are there comparisons you can make with other published studies examining the timing of invasive plant management on other organisms? Comparisons to aboveground fauna may also be of interest to assess the generality of invasion and management effects in higher trophic levels.>> Despite the huge literature available on biological invasions and their ecology or management, there are very few that consider the effects of removal at different stages in the invasion process (see review by Simberloff et al. 2013). Rather, most studies on impacts consider only the effects of the invader as invasion progresses and the abundance or biomass of the invader increases, but not the effects of invader removal or management as invasion proceeds. We now mention this in the revised discussion (L406-409).Data accessibility:-No database is listed where data will be deposited upon successful publication.>> As we mentioned previously, we will provide the data in an on-line repository (the datastore system hosted by Manaaki Whenua, https://datastore.landcareresearch.co.nz) to facilitate access and use of this information.Minor comments:-line 253: remove extra period after Invaded>> Done.-line 304: change ‘same area’ to ‘same experiment’>> Done.-line 330: change ‘than’ to ‘to’>> Done.Reviewer #2: 1. The manuscript reports changes of soil nematode community structure in response to ontogenic removal of an invasive tree species. The study was well designed and the methodology was proper to address the concerns. However, this manuscript is overall poorly written and may need reorganization.>> We have re-written several parts of the manuscript, clarified our hypotheses and re-structured the discussion as suggested by the reviewers.2. The title is confusing (e.g. stage-dependent, what stages?) and not informative enough as the authors estimated the responses of soil nematodes both at community (composition, indices etc.) and trophic level to the management strategies. I suggest it could be changed like “Community- and trophic-level responses of soil nematodes to removal of an invasive tree species at different invasion stages”.>> We have changed the title as suggested by the reviewer.3. Sap- and tree-removal are the two comparable management strategies (treatments) employed in this study. However, in the introduction I did not read any information about “why the two strategies were comparably tested” or “what are the potential difference in responses of soil food webs to sap- and tree-removal”.>> We have now incorporated in the hypotheses the potential difference in responses of soil food webs to sap- and tree-removal treatments (L137-139).4. The hypotheses need to be adjusted: (1) Line 92-93: I could not read how the nematode species richness will increase if invasions reduce the abundance and alter the composition of native plant communities. I guess the authors attempted to highlight the invasions may reduce the abundance and diversity of native plant communities, right? (2) I understand that removal of invasive trees would results in the recolonization of herbs, which may help restore the nematode communities…However, I am confused by the following sentence “We expect uninvaded communities to have more complex structure, with higher abundance of long-life cycle nematode taxa…”. Does that mean uninvaded habitat will have more complex structure or higher abundance of nematodes than “invaded habitat”?>> We apologise for the lack of clarity of our hypotheses. We have now rephrased them to improve their comprehensibility (L116-143).5. The authors claimed that the removal management strategies influence the soil nematodes via effects on plant community, such as indicated in Fig. 4, but I could not find any data or related analysis on plant community. I strongly suggest them to include data on plant community composition they scored (percentage of coverage). Otherwise, I could not see the value of Fig. 4 in this study.>> We agree with the reviewer that the presence of the plant community variable in the previous Fig 4 does not make sense if we do not present plant community data in other forms in the manuscript. Given that the plant community data has already been published in a previous publication (Dickie et al. AoB Plants, 2014), we decided to remove this analysis and instead focus on the additional analyses suggested by reviewer #1.6. I would change the structure of the manuscript, showing the community- and trophic- level responses separately. Fig. 1a, b and Fig. 3 showed tree species invasion indeed resulted in a different nematode community responses from uninvaded soil, and both sap- and tree-removal could reverse this disturbance of invasion to the level of uninvaded treatment. On the other hand, at the trophic level, it seems only sap-removal of the invasive tree species could reverse the disturbance (represented as nematode community responses) back to the level of the uninvaded treatment (Fig. 1c and Fig. 2), suggesting different trophic levels would respond to management strategies in different ways.>> We have re-structured the manuscript as suggested by the reviewer. Specifically, we now present first the results on composition, structure and function of the entire nematode communities under different treatments, and lastly the changes observed in the composition of the different trophic levels.7. I found the results were not fully discussed in the current version. I would like to read discussion on (1) invaded vs. uninvaded: tree invasion indeed alter soil nematode communities; (2) removal of these invasive trees could reverse these invasion influences; (3) the extent of removal of invasive trees recovering nematode communities depends on the invasion stage of removal.>> We have re-structured the discussion as suggested by the reviewer.8. Specific comments:L62: what functional traits?>> We have removed this sentence from the introduction.L73: NOT a sentence>> We have rephrased this sentence.L83-86: not logic>> We have revised the text.L90: “changes” should be “differs”>> We have changed this as suggested by the reviewer.L100-102: confusing. What is the comparison?>> We have now rephrased this sentence.L128-129: what do you want to say by mentioning “with some reinvasion…”?>> A few (literally) newly emerged seedlings had established within the 20 x 20 m area sampled. We have now clarified this in the manuscript (L173-176).L143: where is the data of soil moisture?>> We do not have data on soil moisture.L167: more details are needed on SI to introduce this index.>> We have now incorporated more details about the SI index (L218-220).L172: “total number of species”? I thought authors mostly identify the nematodes at genus level>> The reviewer is correct, we identified most of our species to genus and morphospecies level. We have now replaced the word ‘species’ by ‘taxa’ throughout the manuscript.L319-321: not understandable>> We have deleted this sentence in the current version of our discussion.L329: “have” should be “result in”>> We have changed this as suggested by the reviewer.L331: “both” should be “all three”>> We have not changed this because the Enrichment Index (EI) was not significantly lower in invaded areas compared to managed areas, i.e. only SI and MI were significantly lower in invaded areas compared to managed areas.L334: “perturbations” should be “perturbations, such as plant invasions”>> We have changed this as suggested by the reviewer.L340: should be “soil nematode communities” instead of “communities”>> We have incorporated the reviewer’s suggestion.Submitted filename: Response to Reviewers.pdfClick here for additional data file.12 Nov 2019PONE-D-19-17858R1Community- and trophic-level responses of soil nematodes to removal of a non-native tree at different stages of invasionPLOS ONEDear Miss Peralta,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.We would appreciate receiving your revised manuscript by Dec 27 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. 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If eligible, we will contact you to opt in or out.We look forward to receiving your revised manuscript.Kind regards,Martin SchädlerAcademic EditorPLOS ONEAdditional Editor Comments (if provided):The manuscript is much improved. There are only few minor comments. However, I think some changes to the new Figure 4 are warranted prior to publication. In its current form, it is not immediately intuitive how the data are depicted.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: (No Response)Reviewer #2: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.Reviewer #1: YesReviewer #2: Yes**********3. Has the statistical analysis been performed appropriately and rigorously?Reviewer #1: YesReviewer #2: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.Reviewer #1: YesReviewer #2: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)Reviewer #1: I appreciate the efforts of the authors to revise this manuscript. The manuscript is much improved, particularly regarding the new analyses and restructuring of the Introduction and Discussion. The authors have highlighted the original Dickie et al. 2014 paper in AoB Plants, and how this manuscript builds upon that published work. Additionally, the authors provide greater methodological details and justification for the tree, sapling, and seedling removal management treatments. Specific plans for making the data publicly available have now been stated, complying with the PLOS data policy. I have only a few concerns to be addressed, outlined below.Results:S2 Table: Table caption isn’t overly informative. This table appears to summarize results comparing the seedling removal treatment to the other treatments, so should be stated in the table caption.Figure 4: I’m a bit confused by this figure. For relative abundance data, figures are normally shown in percentages. Since an unequal number of nematodes were identified per sample ('approximately 100'), standardizing those to values to percentages would be most appropriate. A quick glance at Figure 4 reveals that adding up the bars of relative abundance for T1, T2, and T3 do not appear to sum to 100. That makes it harder to attribute differences to treatments versus sampling effort. It may be worthwhile to offer a more thorough explanation in the figure legend as well.Minor comments:Line 78: change ‘removal or…’ to ‘removal of…’Line 329: I think you mean ‘were’ not ‘where’Line 350: change ‘completed’ to ‘completely’Reviewer #2: The authors have considered my and other reviewer comments and made satisfactory and considerable changes in the new version of the manuscript "Community- and trophic-level responses of soil nematodes to removal of a non-native tree at different stages of invasion". The text has been edited significantly and the readability and flow of the manuscript has greatly improved. The change of an informative title and the reanalysis of the data, the addition of associated visual elements have resulted in a more interesting manuscript. Overall, this manuscript has been improved significantly.**********7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: NoReviewer #2: Yes: Minggang Wang[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.20 Nov 2019Response to reviewersAdditional Editor Comments (if provided):The manuscript is much improved. There are only few minor comments. However, I think some changes to the new Figure 4 are warranted prior to publication. In its current form, it is not immediately intuitive how the data are depicted.>> We appreciate the Editor and reviewers’ positive feedback and comments. We have incorporated all the comments made by the reviewers (including a modified version of Figure 4) and responded specifically to each comment below, marking our responses with ‘>>’.Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: (No Response)Reviewer #2: All comments have been addressed>> We have modified Figure 4 and incorporated all the minor comments suggested by Reviewer #1.2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.Reviewer #1: YesReviewer #2: Yes3. Has the statistical analysis been performed appropriately and rigorously?Reviewer #1: YesReviewer #2: Yes4. Have the authors made all data underlying the findings in their manuscript fully available?The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.Reviewer #1: YesReviewer #2: Yes5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)Reviewer #1: I appreciate the efforts of the authors to revise this manuscript. The manuscript is much improved, particularly regarding the new analyses and restructuring of the Introduction and Discussion. The authors have highlighted the original Dickie et al. 2014 paper in AoB Plants, and how this manuscript builds upon that published work. Additionally, the authors provide greater methodological details and justification for the tree, sapling, and seedling removal management treatments. Specific plans for making the data publicly available have now been stated, complying with the PLOS data policy. I have only a few concerns to be addressed, outlined below.Results:S2 Table: Table caption isn’t overly informative. This table appears to summarize results comparing the seedling removal treatment to the other treatments, so should be stated in the table caption.>> We have changed the S2 Table caption as suggested by the reviewer. It now reads:“S2 Table. Results of generalised linear models with Poisson error distribution comparing taxa richness of the seedling removal management strategy with taxa richness of the sapling removal, no removal and tree removal management strategies. TL = trophic level. Bold values indicate significant results (α = 0.05).”Figure 4: I’m a bit confused by this figure. For relative abundance data, figures are normally shown in percentages. Since an unequal number of nematodes were identified per sample ('approximately 100'), standardizing those to values to percentages would be most appropriate. A quick glance at Figure 4 reveals that adding up the bars of relative abundance for T1, T2, and T3 do not appear to sum to 100. That makes it harder to attribute differences to treatments versus sampling effort. It may be worthwhile to offer a more thorough explanation in the figure legend as well.>> We appreciate the comment and apologise for the imprecision. We have now modified Figure 4 following the reviewer suggestion. Specifically, we replaced total abundances by percentages as suggested by the reviewer. We have also expanded the figure legend to clarify that the figure shows the percentage composition of each nematode taxon within a sampling site across management strategies and trophic levels.Minor comments:Line 78: change ‘removal or…’ to ‘removal of…’>> We have changed this as suggested.Line 329: I think you mean ‘were’ not ‘where’>> We have changed this as suggested.Line 350: change ‘completed’ to ‘completely’>> We have changed this as suggested.Reviewer #2: The authors have considered my and other reviewer comments and made satisfactory and considerable changes in the new version of the manuscript "Community- and trophic-level responses of soil nematodes to removal of a non-native tree at different stages of invasion". The text has been edited significantly and the readability and flow of the manuscript has greatly improved. The change of an informative title and the reanalysis of the data, the addition of associated visual elements have resulted in a more interesting manuscript. Overall, this manuscript has been improved significantly.>> We appreciate the positive appraisal of our manuscript.Submitted filename: Response to Reviewers.pdfClick here for additional data file.13 Dec 2019Community- and trophic-level responses of soil nematodes to removal of a non-native tree at different stages of invasionPONE-D-19-17858R2Dear Dr. Peralta,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.With kind regards,Martin SchädlerAcademic EditorPLOS ONEAdditional Editor Comments (optional):Reviewers' comments:23 Dec 2019PONE-D-19-17858R2Community- and trophic-level responses of soil nematodes to removal of a non-native tree at different stages of invasionDear Dr. Peralta:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.For any other questions or concerns, please email plosone@plos.org.Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. Martin SchädlerAcademic EditorPLOS ONE
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