Vegetation and vertebrate abundance as drivers of bioturbation patterns along a climate gradient.

Bioturbators shape their environment with considerable consequences for ecosystem processes. However, both the composition and the impact of bioturbator communities may change along climatic gradients. For burrowing animals, their abundance and composition depend on climatic and other abiotic components, with ants and mammals dominating in arid and semiarid areas, and earthworms in humid areas. Moreover, the activity of burrowing animals is often positively associated with vegetation cover (biotic component). These observations highlight the need to understand the relative contributions of abiotic and biotic components in bioturbation in order to predict soil-shaping processes along broad climatic gradients. In this study, we estimated the activity of animal bioturbation by counting the density of holes and the quantity of bioturbation based on the volume of soil excavated by bioturbators along a gradient ranging from arid to humid in Chile. We distinguished between invertebrates and vertebrates. Overall, hole density (no/ 100 m2) decreased from arid (raw mean and standard deviation for invertebrates: 14 ± 7.8, vertebrates: 2.8 ± 2.9) to humid (invertebrates: 2.8 ± 3.1, vertebrates: 2.2 ± 2.1) environments. However, excavated soil volume did not follow the same clear geographic trend and was 300-fold larger for vertebrates than for invertebrates. The relationship between bioturbating invertebrates and vegetation cover was consistently negative whereas for vertebrates both, positive and negative relationships were determined along the gradient. Our study demonstrates complex relationships between climate, vegetation and the contribution of bioturbating invertebrates and vertebrates, which will be reflected in their impact on ecosystem functions.

Introduction

Bioturbation, the biological reworking of soils and sediments [1, 2], shapes the environment and thus has considerable consequences for ecosystem processes [3] such as sediment transport, soil formation [4, 5], soil water cycles [6], litter decomposition [7], and nutrient availability [8, 9]. Soil excavating animals range from small invertebrates such as ants [10, 11] and earthworms [12, 13] to medium-sized vertebrates such as gophers [14, 15] and beavers [16, 17]. Generally, bioturbating animals have distinct adaptations to environmental conditions but recent studies reveal that bioturbating animals are intentionally able to modify their environment [18-20]. Thus, assessments of the relative contributions of bioturbators to soil-shaping processes across larger climate gradients, must consider both the composition of bioturbator communities and their relationships to the abiotic and biotic environment. Previous studies indicate that:

The abundance and composition of burrowing animal communities depend on climatic (= abiotic) factors such as temperature and humidity [21-26]. Ants and mammals are the most important bioturbators in semiarid and arid areas, and earthworms (Lumbricidae) dominate in humid areas [27]. Local soil characteristics affect bioturbating activity, which is highest after rainfall because the soil softens and the energy cost of digging is accordingly reduced [28] as shown for burrowing mammals [29] and in the nest site selection of ants [23].

Burrowing animals are closely associated with biotic components of the environment, especially vegetation which affects the abundance of bioturbators directly by providing food [30, 31] and indirectly by providing habitat [32]. In humid regions with dense vegetation cover, food resources are generally abundant and thus mammals have less need to dig for food. Vegetation also provides shelter further reducing the need to dig. In resource-limited environments, such as semi-arid and arid regions, the activity and quantity of bioturbating mammals correlate positively with vegetation cover, because of those animals’ need to seek subterranean food and shelter [33]. By contrast, invertebrates such as earthworms do not rely on surface resources offered by vegetation cover as they live entirely belowground, where they feed on dead roots in the soil [34].

Those studies demonstrate, that both, abiotic and biotic components influence bioturbation patterns, with the relationships between bioturbators and their environment varying between animal groups [35]. Detailed insights into the relative contributions of those groups can be obtained by associating them with their burrows, such as based on the diameter of the holes they create. A previous study has collected data on burrowing animals along a climate gradient and used a threshold of 2.5 cm to differentiate between vertebrates and invertebrates [36].

However, most studies have thus far focused either on the burrowing activity and quantity of single species (mostly vertebrates), or on individual climatic regions [36]. Studies on the overall patterns of bioturbation along broad climatic gradients are rare. To close this research gap, we examined the interaction of abiotic and biotic components along a broad climatic and vegetational gradient in Chile. For this purpose, we measured the abundance of burrow entrances (hole density) and the amount of soil excavated by burrowing animals (excavated soil volume) as parameters for bioturbation activity and quantity across seasons. Taking into account the available literature, we hypothesized that:

H1: Bioturbating activity decreases from arid to humid regions because climate drives the abundance of burrowing animals and the contribution of invertebrates and vertebrates to bioturbation patterns.

H2: Seasonal changes affect bioturbation with a higher activity of burrowing animals during rainy seasons, when the soil is softer, and the energetic cost of digging is therefore reduced.

H3: With increasing vegetation cover, the bioturbating activity of many invertebrates (including most earthworms) decreases, due to the subterranean food supply provided by fine roots in the soil independent of soil surface vegetation, while that of vertebrates increases, due to the increased availability of food and shelter.

Methods

Study area

Our study was conducted at four sites representing a climate gradient along the coastal range of Chile (26°S-38°S), extending from an arid desert with a mean annual temperature of 16.8°C and mean annual precipitation of 12 mm to a temperate humid rainforest with a mean annual temperature of 6.6°C and mean annual precipitation of 1469 mm [37]: arid Atacama Desert, located in Pan de Azúcar National Park, semi-arid shrubland in the private reserve Santa Gracia, a Mediterranean forest in La Campana National Park and a humid rainforest in Nahuelbuta National Park. All approvals from the relevant authorities, i.e. the Chilean National Forest Commission (CONAF), were obtained in advance to our study and granted access to the research sites. In 2019, the year of our field campaigns, the mean temperature in the arid desert was 14.6°C and the mean precipitation was 9.4 mm while in the humid rainforest, the mean temperature was 7.3°C and the mean precipitation was 1885 mm [38].

To sample each research site representatively, we established 12 10 m × 10 m plots with a distance of at least 30 m between them during the first field campaign, conducted in autumn of the southern hemisphere (March to April 2019). In a second field campaign conducted in spring of the southern hemisphere (September to November 2019) we established eight additional plots at each site to cover possible variation, resulting in a total of 20 plots per site. The 20 plots per research site were evenly distributed across two opposing hillsides, 10 on the north- and 10 on the south-facing hillslope.

Assessment of bioturbation activity and quantity

To evaluate bioturbation activity, we counted the number of all visually detectable burrow entrances on the soil surface (hole density) of each plot. We calculated the amount of soil excavated by burrowing animals (excavated soil volume) as an indicator of bioturbation quantity by using a caliper to measure the vertical (dv) and horizontal (dh) diameters. In addition, we defined the depth of each hole entrance (de) as the distance to the first barrier encountered by the caliper and measured this parameter. Raw data of burrow measurements can be obtained from S7 Table in S4 Appendix. Following [36, 39, 40], we calculated the (minimal) excavated soil volume assuming that the measured burrows were cone-shaped:

To distinguish between the burrows of invertebrates and vertebrates, burrows with a hole-entrance diameter < 2.5 cm were assumed to be created by invertebrates and burrows with a hole-entrance diameter ≥ 2.5 cm by vertebrates [36].

Assessment of vegetation data

Vegetation cover was estimated using unmanned aerial vehicle (UAV) red green blue (RGB) images and land cover classification [41]. For each plot, we calculated the ratio of pixels classified as any plant type (herbs, shrubs, cacti, trees) to the amount of all pixels. Following [42], the average elevation (hillside elevation) and the hillslope of each plot were estimated based on high resolution Lidar data [43].

Statistical analyses

For the burrows of invertebrates and vertebrates we analyzed the allometric relationship between their depth (de) and their diameter (mean of dv and dh). We regressed the mean diameter of the entrance versus the depth using the log10-transformed values of both variables and then determining the slope. In an isometric relationship, the log-transformed variables should be linearly related to a slope of one [44]. Since diameter and depth were measured with roughly equal error, in addition to an ordinary least squares (OLS) regression, we estimated the slope using a reduced major axis (RMA) regression [45]. To assess a deviation from a slope of one, we used the offset argument available in most regression functions. With the diameter serving as the independent variable and the offset, the estimate tests for deviations from one. For the slope of the RMA regression, we used the standard error and a t-test to test for deviations from one. The same approach was applied to the regression between excavated soil volume and hole density.

To analyze the interaction of abiotic and biotic components in bioturbation activity and quantity, we applied generalized linear mixed effect models (GLMMs). We used hole density or excavated soil volume as response variables, site, season, hillside elevation and hillslope as abiotic fixed predictors and vegetation cover and animal group as biotic fixed predictors. The study plots were used as a random factor (Table 1). All data of the GLMM parameters can be obtained from S7 Table in S4 Appendix. We also included interaction terms between site and all other fixed predictor variables and between vegetation cover and taxon. We standardized the fixed predictor hillside elevation for each site because it varied and could not be assigned separately to each of the sites. We performed GLMMs for the 12 plots within each site (total of 48 plots) in the first field campaign, conducted in the southern-hemispheric autumn, and in the 20 plots within each site (total of 80 plots) during the second campaign conducted in the southern-hemispheric spring. Separation of the hole density of invertebrates and vertebrates resulted in 256 measurements (2 × (48 + 80)). For the GLMM of the excavated soil volume, we log10-transformed data for hole density and excavated soil volume to achieve normality of the residuals. For the log10-transformation, we only considered plots with a hole density > 0 [no/ 100 m2]. Thus, 46 plots without holes were not included in the GLMM for excavated soil volume, resulting in 210 valid measurements. Additionally, we integrated the interaction between hole density and taxon as another fixed predictor.

Table 1

Summary of all variables used in the GLMM.

Response variable	Abiotic fixed predictors	Biotic fixed predictors	Random factor
Hole density or excavated soil volume	Site	Vegetation cover	Plot number
	Season
	Hillside elevation	Animal group
	Hillslope

Depicted are the response variables, fixed predictors (abiotic and biotic) and the random factor.

All statistical analyses were performed using the R statistical environment (version 1.3.1093). We used the lmodel2 package [46] for OLS and RMA regression analysis. For the GLMM, we employed the buildmer function [47] of the lme4 package [48] to perform backward stepwise selection. To determine the proportion of variation explained by the model in total including fixed and random effects, we calculated R-squared for the fitted models using the rsq command from the rsq package [49]. We additionally performed an ANOVA between all possible combinations of fixed predictors retained within the fitted model to evaluate the significance of certain combinations between predictors using the anova command and performing a Chi-square test [50].

Results

First, we investigated the allometric relationships by examining the relationship between the mean diameter and mean depth of the burrows and between the hole density and excavated soil volume. While the respective estimates of the slope are presented herein, our focus is on the RMA slopes. For both, invertebrates and vertebrates, the slopes showed a positive allometric relationship (Fig 1A, Table 2) that was maintained also in the single-season analysis (S1 Fig, S2 Table in S2 Appendix). However, note that the statistical tests evaluating burrow characteristics and the excavated soil volume were not strictly independent, as the former parameter was used to calculate the latter.

Fig 1

Relationships between burrow parameters and bioturbation parameters created by burrowing invertebrates (yellow) and vertebrates (blue).

(A) Relationship between the depth and mean diameter of the holes, (B) relationship between the excavated soil volume and hole density. The regression lines are derived from the reduced major axis analysis. Note that both axes in (A) and (B) were log10-scaled. Data from both field campaigns and all sites were used.

Table 2

Ordinary least squares (OLS) and reduced major axis (RMA) regression analyses of the relationships between the depth and mean diameter of the holes and between the excavated soil volume and hole density for invertebrates and vertebrates (all variables log10-transformed).

		invertebrate				vertebrate
Relation	method	r	slope	SE	p	r	slope	SE	p
Depth and diameter	OLS	0.31	0.729	0.053	<0.001***	0.44	0.996	0.045	0.93
	(mixed model)
	OLS	0.32	0.628	0.057	<0.001***	0.66	1.04	0.043	0.3
	RMA	0.61	1.97	0.057	<0.001***	0.53	1.57	0.43	<0.001***
Excavated soil volume and hole density	OLS	0.55	0.933	0.10	0.5	0.66	1.76	0.13	<0.001***
	(mixed model)
	OLS	0.69	0.955	0.10	0.64	0.81	1.77	0.13	<0.001***
	RMA	0.48	1.38	0.10	<0.001***	0.66	2.17	0.13	<0.001***

A slope of one represents an isometric relationship. Depicted are statistical method, correlation coefficient, slope, standard error (SE) and p-value (p) of the offset. Significant effects are labelled with asterisks: *:<0.1, **:<0.01

***:<0.001. Data from both field campaigns and all sites were used. Further information on the statistical analysis is provided in the Methods section.

Hole density was always greater for invertebrates than for vertebrates (Fig 2A). For invertebrates, hole density decreased continuously from the arid site Pan de Azúcar (raw mean and standard deviation: 14 ± 7.8 no/ 100 m-2) to the humid site Nahuelbuta (2.8 ± 3.1 no/ 100 m-2) while hole density for vertebrates was highest in the semi-arid site Santa Gracia (9.1 ± 9.7 no/ 100 m-2) and remained similar in the other three sites (Pan de Azúcar: 2.8 ± 2.9 no/ 100 m-2, La Campana: 5.6 ± 8.7 no/ 100 m-2, Nahuelbuta: 2.2 ±2.1 no/ 100 m-2, S1 Fig in S1 Appendix).

Fig 2

Bioturbation patterns of invertebrates (yellow) and vertebrates (blue) in each site (Pan de Azúcar, Santa Gracia, La Campana, Nahuelbuta).

(A) Median hole density based on the raw data, (B) median excavated soil volume of holes, (C) the residuals of the excavated soil volume (log10-transformed) after correcting for hole density (log10-transformed) using separate regressions for the two animal groups. Note that the x-axis in (B) and (C) was log10-scaled. Data from the field campaign from September to November were used.

Overall, the pattern of excavated soil volume from arid to humid was hump-shaped for vertebrates (largest in La Campana), whereas for invertebrates we could not determine a clear geographic pattern along the gradient (Fig 2B). In each site, the soil volume excavated by vertebrates was larger. This difference between the two groups of bioturbators was especially clear in the Mediterranean site La Campana (raw mean and standard deviation for invertebrates: 0.00019 ± 0.00016 m3 ha-1, for vertebrates: 0.06 ± 0.18 m3 ha-1) and the humid site Nahuelbuta (invertebrates: 0.00015 ± 0.00022 m3 ha-1, vertebrates: 0.012 ± 0.02 m3 ha-1, S1 Fig in S1 Appendix). Correcting the amount of excavated soil volume for the number of holes, the geographic pattern revealed by the residuals was similar to that obtained based on the analysis of the raw data (Fig 2C); thus, the excavated soil volume was larger for vertebrates than for invertebrates, especially large at the two southern sites.

All predictors for the response variable hole density were significant in the GLMM, with the fixed predictors explaining 48% and the random predictor plot number explaining 39% of the variation (AIC = 2030.7, p < 0.001, S3 and S5 Tables in S3 Appendix). The overall hole density was higher in Santa Gracia and Nahuelbuta during the field campaign from March to April than during the field campaign from September to November while in Pan de Azúcar there was no difference between the two seasons (Fig 3A). For invertebrates, hole density decreased at all sites with increasing vegetation cover. The hole density of vertebrates was positively associated with increasing vegetation cover in Santa Gracia and La Campana (Fig 3B). Overall, there was no clear trend in the relationship between the hole density of invertebrates and increasing vegetation cover whereas vertebrates’ hole density increased with increasing vegetation cover (S2A Fig in S3 Appendix).

Fig 3

Fitted relationship between the hole density and fixed effects for invertebrates (yellow) and vertebrates (blue) at each site (Pan de Azúcar, Santa Gracia, La Campana, Nahuelbuta).

(A) Season (autumn: March-April/ spring: September-November), (B) vegetation cover [%]. Data from both field campaigns were used.

After the exclusion of non-significant independent variables, the fixed predictors season, vegetation cover, hole density and hillside elevation within the fitted GLMM for excavated soil volume explained 85% of the model variation (AIC = 296.67, p < 0.001, S4 and S6 Tables in S3 Appendix). The patterns of excavated soil volume varied for invertebrates and vertebrates with increasing vegetation cover along the climate gradient (Fig 4A). The raw data revealed another trend, as the excavated soil volume increased with increasing vegetation cover for both, invertebrates and vertebrates (S2B Fig in S3 Appendix). In addition, the excavated soil volume increased disproportionally with increasing hole density, with a larger increase for vertebrates than for invertebrates (Fig 4B).

Fig 4

Fitted relation between excavated soil volume (log10-transformed) and fixed effects for invertebrates (yellow) and vertebrates (blue) at each site (Pan de Azúcar, Santa Gracia, La Campana, Nahuelbuta).

(A) Vegetation cover [%], (B) hole density (log10-transformed). Data from both field campaigns were used.

Discussion

Our study showed that while hole density decreased from arid to humid environments, no clear pattern could be discerned for the excavated soil volume along the investigated environmental gradient. However, the contribution of vertebrates to excavated soil volume was larger than that of invertebrates. For the latter, the relationship to the vegetation cover along the climate gradient was consistently negative while for vertebrates it was partly positive.

Before discussing the general results, a few comments should be made on the allometric relationships of the burrow characteristics. Based on the RMA analysis regressions, these relationships were not isometric, as the relative depth of a burrow increased with the increasing diameter of the entrance. This finding suggests that, for bioturbators, larger animals dig deeper into the soil [51]. This relationship presumably reflects the anti-predator behavior of larger animals: with increasing body size animals want to keep their entrance as small as possible to exclude predators [52], but on the same time minimize burrowing cost [53] on one hand, but have a comfortable nest site [51] on the other hand. Our data do not allow a test of this hypothesis, but further analyses of these allometric relationships are likely to provide a rich source of biological and behavioral information, particularly in studies comparing a large number of animal groups.

Our first hypothesis, that bioturbating activity decreases from arid to humid regions [21-26], was supported by our results for invertebrates, as their hole density decreased from arid to humid climates. Vertebrates, however, created fewer holes in arid than in semi-arid regions. Burrowing vertebrates are, on average, larger than invertebrates [51] such that fewer holes are consistent with a decline in animal density with increasing body size [54]. Accordingly, vertebrates were presumably less frequent in the arid region of our study than in the other climatic zones, such that fewer vertebrate than invertebrate burrows were present over a given area. Similarly, the higher hole density of invertebrates all along the climate gradient can be attributed to the generally higher abundance of invertebrates [55]. However, it is also the case that most invertebrates create their own new burrows while some vertebrates use previously existing burrows as an energy-saving strategy [56, 57]. In particular, larger animals, in our case vertebrates, invest more energy in burrowing effort than smaller invertebrates. Previous investigations showed that the energy cost of burrowing is directly proportional to the amount of soil moved by the bioturbator. Consequently, larger vertebrates, which need to move larger soil amounts to create a burrow of adequate size, will burrow fewer holes [15].

The excavated soil volume did not follow a clear pattern across the climatic gradient and it differed between invertebrates and vertebrates in our study. Similar results were obtained in a recent study measuring the excavated soil volume of bioturbators along the same environmental gradient [36]. The authors found that the excavated soil volume was greater in the semi-arid (0.56 m3 ha-1 yr-1) and Mediterranean (0.93 m3 ha-1 yr-1) than in the arid (0.34 m3 ha-1 yr-1) and humid (0.09 m3 ha-1 yr-1) climate zones and that the excavation rates were higher for vertebrates (0.01–56 m3 ha-1 yr-1) than for invertebrates (0.01–37 m3 ha-1 yr-1). These findings are in line with several studies showing that, due to their larger body size, vertebrates excavate considerably larger volume of soil (1–5 m3 ha-1 yr-1) than invertebrates (<1 m3 ha-1 yr-1) [55, 58–64] as well as our findings. Those studies together with our own demonstrate the importance of vertebrates as bioturbators along a climate gradient.

Our second hypothesis, that bioturbation activity and quantity respond to seasonal changes [23, 29], was supported by the higher hole density during autumn than spring of the southern hemisphere, as observed at both the semi-arid and humid site. In the arid desert, with a consistent lack of rainfall events, there was no difference between seasons. This is in agreement with previous studies and with the observation that in the southern hemisphere the bioturbation season ends in autumn [65]. Moreover, the climate in Central Chile during the study period in 2019 was drier than usual [66], which may have lessened the differences in bioturbation activity and quantity between seasons. While the relationship between seasons and bioturbation patterns is no doubt, our study suggests that, at least in Chile, the impact of bioturbation is largest in semi-arid and humid climate zones after the autumn rainfall.

The absence of a clear trend between vegetation cover and either bioturbation activity or quantity along the climate gradient was consistent with previous studies examining the distribution of burrow entrances as a function of vegetation [67, 68]. However, we were able to show that the bioturbation patterns of invertebrates and vertebrates differed. The consistently negatively association of invertebrates with vegetation cover supported our hypothesis that some invertebrates are entirely independent of surface resources due to their permanently belowground lifestyle [34]. By contrast, because vertebrates rely on a resource supply from the surface [33], a positive association with vegetation cover occurred only in the middle of the geographic gradient, as in the arid region the vegetation cover is sparse. Vertebrates living in regions of extreme temperatures characterized by limited resource must invest their energy in digging for food as well as shelter from extreme temperatures in such resource-limited habitats [69]. Furthermore, there is often no vegetation near freshly created burrows, because burrowing typically destroys the vegetation at and possibly adjacent to the burrow [39, 70]. This may have introduced a biased estimate of vegetation cover within plots with fresh burrows and would explain the absence of either a positive or a negative association between burrowing vertebrates and vegetation cover in the humid region. Nonetheless, in general, vegetation cover was shown to be positively associated with vertebrates with a complex influence on bioturbation patterns along the climate gradient.

Conclusion

Our study showed that climatic conditions and vegetation cover drive the activity and quantity of bioturbation as well as the amount of burrowing by different animal groups. The contribution of vertebrates to bioturbation quantity is large and only bioturbating vertebrates had a positive association, albeit a partial one, with vegetation cover. In its examination of the interaction of abiotic and biotic components, our study demonstrated the intricate relationships between climate, vegetation and the contribution of bioturbating invertebrates and vertebrates. These results provide further insights into the patterns that occur along broad climatic gradients and therefore into the impact of ecosystem engineers on ecosystem processes such as sediment transport, soil water cycling and nutrient availability. In a further study, we will therefore compare physical and chemical soil properties in areas with soil affected and unaffected by bioturbation along the same climatic gradient. Additionally, our findings support the importance of examining impacts of bioturbation on ecosystem processes on a broader climatic scale and thereby encourage similar further studies like the assessment of sediment redistribution rates caused by bioturbation [71].

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24 Jan 2022

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Additional Editor Comments:

The authors have done an excellent job of examining the effects of meteorological conditions and vegetation cover on bioturbation activity and quantity, as well as the amount of borrowing by various animal groups. The study also highlighted the complex interactions and linkages between abiotic and biotic components, such as climate, vegetation, and the role of bioturbating invertebrates and vertebrates. However, there are several criticisms and suggestions made by reviewer number one that require clarity, and those remarks must be addressed. As a result, before the paper is accepted for publication, I strongly advise a thorough revision.

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Please 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 study is totally unexplored and uncovered. The hypothesis are excellent and executed very well. In short, impressed with the work done. The only lacking thing i found in MS is future perspective of the study. Therefore, authors can look into this.

Reviewer #2: Comments to the authors

The title is required to undergo some changes. The proposed title is “Study of relationships between bioturbators and their environment in Chile”.

Soil texture depends on the proportions of soil components. Sandy loams are easier to excavate. Hence the second hypothesis needs to be revised.

The research gap is not well understood. With a wide range of microhabitats as well as diversity of life forms on earth along with diversified evolutionary histories, modes of adaptation to microclimates, morphology and behaviour, it is assumed naturally that a pattern in bioturbation activities will be hardly noticed. A detailed review is already done by Kirstin et al. (https://bg.copernicus.org/preprints/bg-2021-75/bg-2021-75.pdf). Hence, it seems like a repetition of what is already done.

Line no. 112: Write the number of quadrats in words to avoid confusion. How was the study sites selected? What kinds of burrowing animals were present at the studied locations? How did you standardize the quadrat size and number?

The reasons behind the findings are very much obvious and predictable. But where is the significance or implication of this study?

**********

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Reviewer #1: No

Reviewer #2: No

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7 Feb 2022

Dear Ph.D. Tunira Bhadauria,

Please find enclosed our detailed responses to the reviewers’ and editor’s comments for the manuscript PONE-D-21-39015, “Vegetation and vertebrate abundance as drivers of bioturbation patterns along a climate gradient”. The reviewers’ comments were very constructive and mainly concerned (1) the description of the research gap, (2) differentiation of previous work in the study area and (3) future perspectives in the field of research. We have dealt with all of the critical comments in full, and thoroughly revised the manuscript. We hope that you agree that we have satisfactorily dealt with all of the reviewers’ comments in full and that the manuscript is now suitable for publication in PLOS ONE.

Sincerely,

Diana Kraus on behalf of all authors

Response to reviewers

Thanks to all the reviewers and the editor for your constructive suggestions. We addressed your comments and give a detailed list of changes below.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Apologies for not adequately meeting the PLOS ONE guidelines. We carefully checked all style requirements and now renamed our files according to PLOS ONE guidelines.

2. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why.

Many thanks for pointing us to this important issue. We now explicitly mention the permit in the lines 111 to 113.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"This study was funded by the German Science Foundation DFG Priority Program SPP 1803: EarthShape: Earth Surface Shaping by Biota, sub-project “Effects of bioturbation on rates of vertical and horizontal sediment and nutrient fluxes” [grant numbers BE1780/52-1, LA3521/1-1, FA 925/12-1, BR 1293-18-1]. We thank the Chilean National Forest Corporation (CONAF). Rafaella Canessa provided valuable comments on the statistical analyses and manuscript. We express our gratitude to Robin Fischer and Alexander Klug who participated during the field work."

We note that you have provided funding information. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"This study was funded by the German Science Foundation DFG Priority Program SPP 1803: EarthShape: Earth Surface Shaping by Biota, sub-project “Effects of bioturbation on rates of vertical and horizontal sediment and nutrient fluxes” [grant numbers BE1780/52-1, LA3521/1-1, FA 925/12-1, BR 1293-18-1]."

Apologies for the double mentioning the funding information in the manuscript. We now mention the funding information only in the correct position.

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Many thanks for pointing us to the copyright issue. The images used in the figures 2, 3, and 4 are photos that were taken by the first author of the submitted manuscript. Therefore, Diana Kraus provides the Content Permission Form as an “Other” file with the submission. The photo was uploaded to a server of the university to create the link for the Content Permission Form:

https://hessenbox.uni-marburg.de/getlink/fiSgU3uz9ud9a4qzPcRM7Dtt/Chile_photos.jpg.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. 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: Yes

Reviewer #2: Partly

Many thanks for this generally positive feedback on our manuscript.________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

We appreciate the general agreement with our statistical approach.

________________________________________

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: Yes

Reviewer #2: Yes

We are in favor of an open data policy and thus naturally make our data publicly available.

________________________________________

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: Yes

Reviewer #2: Yes

Many thanks for the positive feedback.

________________________________________

5. Review Comments to the Author

Please 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 study is totally unexplored and uncovered. The hypothesis are excellent and executed very well. In short, impressed with the work done. The only lacking thing i found in MS is future perspective of the study. Therefore, authors can look into this.

We appreciate the reviewer’s assessment and now included future perspective of the research field in the conclusion statement (lines 333 to 339).

Reviewer #2: Comments to the authors

The title is required to undergo some changes. The proposed title is “Study of relationships between bioturbators and their environment in Chile”.

We appreciate your alternative suggestion for the title of our manuscript. However, we have decided to stick to our original title as it already indicates our major finding: “Vegetation and vertebrate abundance as drivers of bioturbation patterns along a climate gradient”.

Soil texture depends on the proportions of soil components. Sandy loams are easier to excavate. Hence the second hypothesis needs to be revised.

The reviewer is correct in stating that sandy loams are easier to excavate than soil dominated by clay. We thus currently analyze the soil texture across the climate gradient to shed more light on the relationship between bioturbation and soil texture. In our current work we are focusing on the influence of rainfall events on bioturbation activity and thus excluded the misleading term “texture”. We apologize for this lack of clarity.

The research gap is not well understood. With a wide range of microhabitats as well as diversity of life forms on earth along with diversified evolutionary histories, modes of adaptation to microclimates, morphology and behaviour, it is assumed naturally that a pattern in bioturbation activities will be hardly noticed.

We appreciate the notion that bioturbation patterns could be relatively similar along the climate gradient due to adaptation of life forms on earth. However, previous studies, e.g., Wilkinson et al. (2009) highlighted already that patterns of bioturbation activity change along the climate gradient. To take the reviewer’s point into account, we now added further evidence such as a recent paper by Corenblit et al. (2021) showing that burrowing animals are not only adapting to the existing environment but also co-construct their physical environment (lines 55 to 57). After conducting our study and analyzing the data, we saw variation between research sites in different climate regions and presented it in our results even though it was not as severe.

A detailed review is already done by Kirstin et al. (https://bg.copernicus.org/preprints/bg-2021-75/bg-2021-75.pdf). Hence, it seems like a repetition of what is already done.

Of course, we are aware of the review article by Übernickel et al. who exemplary collected data on bioturbation along the climate gradient of the EarthShape consortium which is part of a larger summary of literature research of burrowing vertebrates and invertebrates. These data were collected on a limited number of plots in each research site and thus gave a first insight into bioturbation patterns. We now collected independent data across the climate gradient that comprised 1) a larger spatial scale with an adequate number of plots per research site and 2) a temporal/seasonal component. The larger sample size enabled us 1) to compare our data with the pilot study of Übernickel et al. and thus to generalize bioturbation activity patterns along the climate gradient and 2) to depict these activity patterns across time and thus grain insights into temporal significance of bioturbation.

Line no. 112: Write the number of quadrats in words to avoid confusion.

We appreciate your comment, yet we decided to follow the guidelines in academic writing stating that numbers up to nine should always be written in words, anything higher than nine can be written in numerals.

How was the study sites selected?

Many thanks for mentioning this point. The study sites were preselected for the priority program EarthShape of the German Science Foundation (https://esdynamics.geo.uni-tuebingen.de/earthshape/index.php?id=129) which tackles the overarching research question how microorganisms, animals, and plants influence the shape and development of the Earth’s surface over time scales from the present-day to the distant geologic past. In our project we made use of the large climatic gradient by establishing 20 representative plots per study site to assess the activity of bioturbation. We now slightly reworded this part in the methods section to make the general context clearer. It now reads (lines 105 to 108): “Our study was conducted at four sites representing a climate gradient along the coastal range of Chile (26°S-38°S), extending from an arid desert with a mean annual temperature of 16.8 °C and mean annual precipitation of 12 mm to a temperate humid rainforest with a mean annual temperature of 6.6 °C and mean annual precipitation of 1469 mm.”

What kinds of burrowing animals were present at the studied locations?

We appreciate this important point. We had planned parallel to the assessment of activity patterns also to capture bioturbating animals in the study areas. Unfortunately, the trapping success was rather limited. This is the reason why we were only able to differentiate the bioturbation activities between vertebrates and invertebrates. To still enable the readers to learn which burrowing animals there are in Chile, we now refer to the study of Übernickel et al., who compiled a literature research on this topic (lines 83 to 84).

How did you standardize the quadrat size and number?

We apologize for the lack of clarity in the methods. We now reworded the explanation how we standardized the quadrat size and number to make this clearer (lines 122 and 123): “The 20 plots per research site were evenly distributed across two opposing hillsides, 10 on the north- and 10 on the south-facing hillslope. “

The reasons behind the findings are very much obvious and predictable. But where is the significance or implication of this study?

We appreciate your comment and now explain in more detail the significance and implication of our study (lines 329 and 334): “In its examination of the interaction of abiotic and biotic components, our study demonstrated the intricate relationships between climate, vegetation and the contribution of bioturbating invertebrates and vertebrates. These results provide further insights into the patterns that occur along broad climatic gradients and therefore into the impact of ecosystem engineers on ecosystem processes such as sediment transport, soil water cycling and nutrient availability.”________________________________________

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

10 Feb 2022

Vegetation and vertebrate abundance as drivers of bioturbation patterns along a climate gradient

PONE-D-21-39015R1

Dear Dr. Kraus

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. 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.

Kind regards,

Tunira Bhadauria, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

After reading the authors' amended article and their responses to the reviewers' comments/suggestions, I believe the work has sufficient scientific content to be approved for publication in its current form. As a result, I recommend that the paper be accepted in its current form for publication.

Reviewers' comments:

15 Feb 2022

PONE-D-21-39015R1

Vegetation and vertebrate abundance as drivers of bioturbation patterns along a climate gradient

Dear Dr. Kraus:

I'm 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 let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, 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.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Tunira Bhadauria

Academic Editor

PLOS ONE