Investigating the direct and indirect effects of forest fragmentation on plant functional diversity.

Ongoing habitat loss and fragmentation alter the functional diversity of forests. Generalising the magnitude of change in functional diversity of fragmented landscapes and its drivers is challenging because of the multiple scales at which landscape fragmentation takes place. Here we propose a multi-scale approach to determine whether fragmentation processes at the local and landscape scales are reducing functional diversity of trees in the East Usambara Mountains, Tanzania. We employ a structural equation modelling approach using five key plant traits (seed length, dispersal mode, shade tolerance, maximum tree height, and wood density) to better understand the functional responses of trees to fragmentation at multiple scales. Our results suggest both direct and indirect effects of forest fragmentation on tree functional richness, evenness and divergence. A reduction in fragment area appears to exacerbate the negative effects resulting from an increased amount of edge habitat and loss of shape complexity, further reducing richness and evenness of traits related to resource acquisition and favouring tree species with fast growth. As anthropogenic disturbances affect forests around the world, we advocate to include the direct and indirect effects of forest fragmentation processes to gain a better understanding of shifts in functional diversity that can inform future management efforts.

Introduction

Forest loss and fragmentation result in long-lasting and complex changes in biodiversity that may go beyond the loss of species to include the alteration of functional diversity of remaining communities. Forest fragmentation threatens the long-term persistence of species [1-3], as well as the goods and services provided by those ecosystems [4]. Fragmentation is a hierarchical process that involves breaking apart the habitat of a focal species into populations isolated from each other in a matrix of modified habitat [5-7]. Changes in the spatial configuration of the landscape alter the abiotic and biotic filters that govern community assembly, selecting individuals with suites of traits that enable them to survive, grow, reproduce, and colonize remaining fragments. Species traits relate to physiological, morphological, and phenological functions [8-10], and local functional diversity can influence ecosystem functioning [11,12]. If species’ traits in remaining fragments become more similar over time, a process known as functional homogenization, this could severely alter a variety of ecosystem functions performed by remaining communities and, by extension, the ecosystem services they provide. Previous studies provide evidence that forest fragmentation often favours plant species with traits within a specific range of values [e.g. 13], potentially leading to functional homogenization by reducing alpha diversity of functional traits [14]. By taking into account functional diversity within a community, we can better understand how species respond to fragmentation processes that alter the abiotic and biotic filters that govern community assembly.

Trait values that allow species to take advantage of recent disturbances are commonly hypothesised to determine species success in fragmented landscapes [15,16]. Recent studies have shown that reductions in fragment area and increases in the amount of edge habitat locally favour tree species with faster growth rates (e.g. pioneers), smaller seeds, shorter leaf life span and lower wood density [15-20]. Additionally, increased spatial isolation and an inhospitable matrix habitat are expected to select for abiotically-dispersed tree species and/or small-seeded, animal-dispersed tree species that have the potential for wide dissemination by attracting many seed-disperser species [16,18,20,21]. However, because of the variable results across studies and systems, there is limited consensus on the generality of the magnitude of these shifts and their drivers.

The process of landscape fragmentation can be considered at multiple, interacting scales. Fragmentation effects via fragment isolation or matrix quality that impact dispersal among fragments or meta-population dynamics may manifest most strongly at the landscape scale. In contrast, fragmentation effects via edge effects, fragment shape or size that impact fine-scale habitat quality and individual persistence may be best detected at the fragment-scale (as with our study with fragments ranging from 0.011–9.51 km2). Furthermore, these landscape- and fragment-scale changes typically occur concurrently which may lead to interactions among various fragmentation effects. For example, dispersal between fragments typically declines with isolation and an inhospitable matrix habitat may exacerbate the effects of fragment isolation on species diversity [7]. In forest fragments, altered abiotic conditions such as greater desiccation through increased wind and light, causing higher temperatures and lower humidity, are among the main edge effects as the shape of fragments becomes narrower and/or as the size of fragments decreases [6]. Decreasing fragment size could both directly impact species persistence by lowering local population sizes and increasing edge effects as the relative amount of edge habitat is greater in smaller fragments. Teasing apart these co-occurring changes across spatial scales has posed a major challenge in predicting the net response of functional diversity to forest fragmentation to date [6,22,23].

Previous investigations have yielded mixed results, with functional diversity responding either positively or negatively to forest fragmentation [14,24,25]. This lack of consensus could be the result of not accounting for the direct and indirect effects of both landscape- and fragment-level effects. While measuring the independent effects of individual landscape properties is useful to identify mechanisms behind fragmentation-driven biodiversity changes, such approaches may miss critical indirect effects between fragment-level and landscape-level fragmentation variables [26], and potentially leads to incorrect inferences and predictions regarding the impacts of forest fragmentation on communities. Structural Equation Models (SEM) have been proposed as an alternative tool to jointly study the direct and indirect effects of habitat amount and configuration because SEMs specify predictor variables that may not have been measured or that may be difficult to observe directly, and therefore measure the strength of causal relationships among predictors and provide rigorous estimates of direct and indirect effects [27].

Here, we use a SEM approach that permits the evaluation of direct and indirect effects of forest fragmentation on plant functional diversity in the East Usambara Mountains of Tanzania. This approach in particular allows us to tease apart the attributes of forest fragmentation that operate across different spatial scales and to compare the relative importance of local versus landscape-scale processes affecting different dimensions of functional diversity (e.g richness, evenness and divergence). In this study, we censused trees in plots across a fragmented rainforest in the East Usambara Mountains, an area in Africa known for its high levels of biodiversity and endemism [28] that is currently protected under the United Nations Educational, Scientific and Cultural Organization (UNESCO) Biosphere Reserve status. We hypothesize that:

The variation in functional diversity in response to fragmentation is mediated by both fragment- and landscape-scale factors (Fig 1). We expect that the impacts of fragment size, shape complexity, and edge effects on functional diversity are indirectly affected by landscape-level processes such as fragment isolation and matrix quality. For example, edge effects tend to be more severe in small and/or narrow or irregularly-shaped fragments, which would therefore affect functional diversity. Finally, we anticipate a greater negative effect of isolation on functional diversity for fragments surrounded by an inhospitable matrix habitat.

Fig 1

Conceptual model illustrating the directional relationships between fragmentation processes occurring at the landscape and fragment level affecting functional diversity.

Functional diversity was defined in terms of functional richness, evenness and divergence. Functional metrics were fitted in separate models. Arrows indicate the hypothesized causal relationships, with dashed arrows representing indirect effects and continuous lines representing direct effects.

The effects of fragmentation are expected to impact functional diversity in several ways: a) functional richness, evenness and divergence of resource use traits are expected to decline with reduced fragment area and shape complexity as the amount of forest edge increases; b) low quality of matrix surrounding the remaining fragments is expected to exacerbate the environmental stress in edge habitats, further reducing functional richness, evenness and divergence (Fig 1); c) trait distribution is expected to become more skewed towards species with trait values associated with fast resource use (e.g. short stature, light-demanding species with low wood density and small seeds) within edge habitats; and d) functional richness, evenness and divergence of dispersal traits are expected to decrease with increasing fragment isolation and decreasing matrix quality. Specifically, we expect abiotically-dispersed species and small-seeded, animal-dispersed species to dominate in more isolated fragments surrounded by a less hospitable matrix.

Materials and methods

Study area

The forest of the East Usambara Mountains stretches continuously from about 250 m to 1100 m asl in the southern part of this mountain range to form what is now protected as Amani Nature Reserve (8380 ha; -5°04'58.80" S 38°40'1.20" E). To the north of this reserve is Nilo Nature Reserve, and eastwards is the Derema corridor and several large fragments of lowland forest. Rainfall averages at 2000 mm per annum, falling largely from March to May and October to November; however, with the exception of January and February, precipitation is prevalent in most other months due to moisture carried across from the adjacent Indian Ocean [29]. The forest on the submontane plateau, in and around the primary study area of Amani Nature Reserve, is dominated by a suite of wet rainforest species. These include two emergent species Newtonia buchananii (Fabaceae) and Maranthes goetzeniana (Chrysobalanaceae), and several canopy and midstory/understorey species such as Allanblackia stuhlmannii (Clusiaceae), Cephalosphaera usambarensis (Myristicaceae), Sorindeia madagascariensis (Anacardiaceae), Parinari excelsa (Chrysobalanaceae), Isoberlinia schefflerii (Fabaceae), Greenwayodendron suaveolens (Annonaceae), Anisophyllea obtusifolia (Anisophylleaceae), Leptonychia usambarensis (Sterculiaceae), Myrianthus holstii (Urticaceae), Macaranga capensis (Euphorbiaceae), Trilepisium madagascariense (Moraceae) and Strombosia scheffleri (Olacaceae). The forest also contains Maesopsis eminii (Rhamnaceae), an exotic, invasive gap- and edge-specialist tree species [29,30].

Amani Nature Reserve is surrounded by several forest fragments of varying sizes in the submontane plateau (S1 Fig) and is primarily separated by a homogenous matrix of tea plantations. Apart from subsistence cultivation, which has shaped the forested landscape in more recent decades, much of the extensive forest loss and fragmentation arose from initial human occupation in the early pre-colonial period [31], but more extensively from the historical expansion of tea plantations, starting in the late 1800s [32]. Loss of original forest cover is estimated to exceed 50% [33]. Ten forest fragments and a large portion of the continuous forest were used to sample tree communities in 67 vegetation plots between May and July 2000; all sites are at 900–1100 m asl (S1 Table). Each vegetation plot was 20 x 20m and the plots were randomly located at ~25, ~150, ~250 and ~ 400 m from the forest edge towards the interior; smaller fragments (i.e. < 20 ha) did not have plots sampled at >200 m from the forest edge. All trees ≥ 10 cm Diameter at Breast Height (DBH) within each plot were identified to species.

Functional trait data

We collated five traits (seed length, dispersal mode, shade tolerance, maximum tree height, and wood density) that correspond to key dimensions of species ecological strategies and have been previously used to explain competitive ability, growth, and reproduction in the context of forest fragmentation [14]. Traits for all the tree species were obtained through an exhaustive search of existing literature as well as online databases (S2 Table). Data on seed length and dispersal mode were obtained from Chapman et al. [34] and the African Tree Database (https://figshare.com/articles/Plant_animal_interactions_from_Africa/1526128). Seed length was based on the largest average length of the diaspore that is transported by the vector, and not necessarily the seed kernel size. Seed length reflects a seed number-seedling survival trade-off with small seeds being produced in large quantities and being better colonizers than larger seeds at the expense of withstanding lack of resources or different hazards thus reducing seedling survival and establishment [35]. Dispersal mode included zoochory (animal-dispersal), anemochory (wind-dispersal), and barachory (gravity or explosive dispersal). Dispersal mode influences the capacity of an individual to colonize newly formed or isolated fragments [36]. Maximum tree height was obtained from the literature [37] and an online database (http://www.prota.org). Maximum tree height is associated with competitive ability for light with taller trees displaying greater carbon assimilation potential than smaller trees [38-40]. Wood density for each species, or genus (when data for a species was not known), was derived from the global wood density database [41,42]. Wood density is a critical component for many essential functions, such as mechanical support and nutrient storage [43] and reflects a trade-off between radial growth to acquire physical stability at the expense of vertical growth [44,45]. Finally, for shade-tolerance guilds, we followed the classifications of Ouédraogo and collaborators [46,47], and where necessary, supplemented information from other sources [37,48]. Plant successional guilds included pioneer (species dependent on gaps or forest edge to establish), shade-tolerant (species dependent on shade across different ontogenetic levels), and light-demanding non-pioneer (species that establish in shade but initially require light to maximize growth) [46, sensu 49].

Fragmentation metrics

Fragments were mapped using the high-resolution satellite imagery from Google Earth Pro. After mapping, metrics were calculated for each plot and all forest fragments using the GRASS GIS software [50,51]. Edge effects were evaluated based on the calculated distance of the center of the sampled plot to the forest edge. We also calculated fragment area (km2), distance from the edge of a fragment to the closest edge of the continuous forest (m), matrix quality based on the surrounding cultivated land, and shape complexity.

For assessing matrix quality, we first characterized the matrix habitat of the study area into three land cover types: tea plantations (the primary matrix habitat), Eucalyptus woodlots, and subsistence cultivation of mixed crops such as bananas, beans, maize, cassava, cardamom, cloves and cinnamon. Tea and eucalyptus plantation represent a more hostile environment surrounding fragments, while subsistence cultivation is less hostile as it includes a mix of small forest patches and multi-crop species farms. To calculate matrix quality (MQ), we quantified the percentage of the forest edge in contact with each of the mentioned land covers as MQ = 100—(%tea + %eucalyptus). Thus, matrix quality is reduced as the percentage of forest edge in contact with the hostile matrix habitats comprising of tea or eucalyptus plantation increases.

To describe the shape complexity of the fragment, we calculated a fractal dimension index [52,53] where lower values correspond to regular shapes and higher ones to convoluted shapes, as follows: where P represents the perimeter and A the fragment area. FDI measures how much a shape deviates from a circumference thus excluding the effect of area on the edge complexity; if fragments are more complex, the perimeter increases and yields a higher fractal dimension.

Analysis

Functional diversity

We used functional indices that capture three major components of functional diversity: functional richness (FRic) or the amount of niche space occupied by the community [54], evenness (FEve) or regularity of the distribution of species abundances in the functional space [55] and divergence (FDiv) or variance of species trait distribution in the trait space [54]. We calculated multivariate FRic, FEve and FDiv using all five functional traits, as studies have demonstrated that considering a single trait can lead to an oversimplification of results [e.g. 56]. All indices were calculated within the FD package [57] in R (Version 3.6.1; R Core Team 2019) and Gower distances were used to calculate functional distance between species pairs as it allows for the inclusion of both continuous and categorical traits. We calculated a weighted FEve index using the abundance of species (defined using number of stems) with different trait values within a community and an unweighted FEve index independent of species abundances. We then compared both weighted and unweighted FEve indices to improve the interpretation of this index as suggested by Legras and Gaertner [56]. To determine the functional trait value of a species community and explore how shifts along individual trait axes underlie the observed FRic, FEve and FDiv patterns, we calculated community-weighted mean (CWM) values (i.e. mean plot-level species trait values weighted by their relative abundance). To explore how community shifts along single traits underlie the observed FRic, FEve and FDiv patterns, we also calculated community-weighted mean (CWM) trait values (i.e. species trait values weighted by their relative abundance in each plot).

To further examine the potential drivers of functional diversity from the forest edge to interior of forests, we examined patterns of recruitment between edge and interior plots. It is possible that edge habitats have a more even distribution in wood density due to a mix of pre-fragmentation trees with high wood density and new post-fragmentation trees characterized by mostly low wood density. Thus, to evaluate whether there was a difference in tree size, we compared size distributions between interior and edge habitats for each guild using a Kolgomorov-Smirnov test. Edge plots were defined as all plots within <100 m of the fragment/forest edge and interior plots included all plots >100 m from the edge following Laurance [58] as 100 m being the threshold for edge effects.

Statistical modelling

We implemented a structural equation modelling approach using the R package piecewiseSEM [59] to investigate direct and indirect relationships of fragment- and landscape-scale variables in predicting local functional diversity. The general model investigated in this study (Fig 1) hypothesizes that variation in functional diversity among plots can be explained by the interacting direct and indirect effects of processes occurring at the fragment-level and the landscape-level. These include distance of plot to the nearest forest edge, distance of fragment to continuous forest as a measure of isolation, fragment area, matrix quality, and shape complexity. We used linear functions for all relationships in the structural equation models and ran separate models for each functional metric. To derive comparable estimates, we standardized all quantitative predictors to a mean of zero and standard deviation of one. In some cases, variables were log-transformed to achieve a normal error distribution. To further explore potential shifts in trait distributions underlying variation in FRic FEve and FDiv, we used general linear models as a post-hoc test to determine changes in CWM traits values as a result of fragment- and landscape-scale variables. In these models, the responses are CWM values and the predictors are fragment- and landscape-level variables.

Results

Relationships among fragmentation variables

We found a strong positive relation between matrix quality and fragment isolation (coef = 0.704, se = 0.11, p < 0.001; Fig 2) and found a weak relationship between matrix quality and distance from plot to forest edge (coef = -0.595, se = 0.189, p = 0.568; Fig 2). The distance from plot to forest edge increased with fragment area (coef = 0.603, se = 0.156, p < 0.001; Fig 2) and tended to decrease as shape complexity of the fragment was reduced (coef = -0.423, se = 0.167, p = 0.015; Fig 2). Finally, shape complexity was positively associated with fragment area (coef = 0.541, se = 0.116, p < 0.001; Fig 2), with larger fragments characterized by more complex shapes than small fragments.

Fig 2

Structural equation models examining the effects of forest fragmentation on functional diversity in the East Usambara Mountains, Tanzania.

A) functional richness, B) functional evenness and C) functional divergence. Grey lines represent indirect effects and dark lines representing direct effects. Values associated to lines represent standardized path coefficients. Significant results (p ≤ 0.05) are represented in dark blue.

Response of functional diversity to fragmentation

We found that direct and indirect effects between fragment-level fragmentation attributes appeared to be important drivers of local functional diversity within plots, while landscape-level attributes seemed to be less important in most cases. Functional richness tended to decrease with reduced shape complexity (coef = -0.936, se = 0.314, p = 0.004; Fig 2A). In addition, we found evidence that functional evenness tended to increase with fragment isolation (coef = 0.038, se = 0.017, p = 0.03; Fig 2B). No other significant effects at the landscape or fragment level were captured on functional evenness (Fig 2B). Finally, functional divergence tended to decrease with distance of a plot from the forest edge (coef = -0.028, se = 0.012, p = 0.031; Fig 2C), with no other significant effects on functional divergence (Fig 2C).

Shifts in community weighted mean traits with fragmentation

Values of community-weighted means were significantly associated with fragment isolation and distance from plot to forest edge (i.e. edge effects). CWM values for dispersal mode was significantly associated with fragment shape complexity, with anemochory increasing in more complex fragments (estimate = 0.182, se = 0.073). CWM values for wood density significantly declined with increasing fragment isolation (estimate = -0.015, se = 0.006). Furthermore, CWM values for successional guilds were significantly associated with fragment isolation and edge effects (distance from plot to forest edge). Specifically, CWM values for shade tolerance significantly declined with increasing fragment isolation (estimate = -0.071, se = 0.025) and increasing distance from plot to forest edge (estimate -0.388, se = 0.122). Distance from plot to forest edge was negatively associated with CMV values of light-demanding non-pioneer species (estimate = -0.171, se = 0.083), but, in contrast, positively associated with pioneer species (estimate = 0.097, se = 0.022). We also found distance of plot to forest edge was positively associated with maximum height (estimate = 1.305, se = 0.630) and wood density (estimate = 0.022, se = 0.005). See Table 1 for all results.

Table 1

Effects of isolation, shape complexity and distance to edge on community weighted means for plant functional traits in the East Usambara Mountains, Tanzania.

Trait	Metric	Estimate	SE	t	p-value
Anemochory	Isolation	-0.064	0.078	-0.812	0.424
Zoochory		-0.005	0.013	-0.410	0.683
Barochory		0.073	0.133	0.551	0.586
Height		-0.406	0.653	-0.621	0.537
Light-demanding Non-Pioneer		0.111	0.083	1.344	0.185
Pioneer		0.179	0.121	1.482	0.145
Shade-Tolerant		-0.071	0.025	-2.903	0.005
Seed length		-0.458	0.671	-0.068	0.946
Wood density		-0.015	0.006	0.006	0.012
Anemochory	Shape complexity	0.182	0.073	2.476	0.020
Zoochory		0.007	0.013	0.528	0.600
Barochory		0.059	0.113	0.526	0.603
Height		-0.009	0.655	-0.014	0.989
Light-demanding Non-Pioneer		0.119	0.081	1.473	0.147
Pioneer		0.038	0.122	0.309	0.759
Shade-Tolerant		0.013	0.026	0.505	0.616
Seed length		-0.478	0.668	-0.716	0.477
Wood density		0.001	0.006	0.210	0.834
Anemochory	Distance to edge	-0.006	0.104	-0.059	0.953
Zoochory		-0.015	0.013	-1.161	0.251
Barochory		0.104	0.106	0.980	0.334
Height		1.305	0.630	2.071	0.044
Light-demanding Non-Pioneer		-0.171	0.083	-2.070	0.044
Shade-Tolerant		-0.388	0.122	-3.176	0.003
Pioneer		0.097	0.022	4.326	< 0.001
Seed length		1.127	0.652	1.727	0.09
Wood density		0.022	0.005	4.249	< 0.001

Patterns of recruitment between edge and interior plots

We found significant differences in size distributions between the edges and the interior habitats for light-demanding non-pioneer species (D = 0.25, p = 0.03), with many small sized individuals found at the edges of the forest (Fig 3). We found a similar pattern for pioneer species (Fig 3), although this difference was not statistically significant (D = 0.18, p = 0.23). For shade-tolerant species, larger individuals were found at the interior of the forest (Fig 3), but, similar to pioneer species, this difference was not statistically significant (D = 0.07, p = 0.65).

Fig 3

Size distribution of each of the three successional guilds at edge versus interior vegetation plots in the East Usambara Mountains, Tanzania.

Discussion

Delineating the different processes that occur during landscape fragmentation and evaluating how they affect functional diversity is challenging. This is because landscape fragmentation leads to a series of changes in forest dynamics that occur at multiple spatial scales. Using a SEM approach with data from the rainforest of East Usambara Mountains, Tanzania, we detect several direct and indirect effects of forest fragmentation for different facets of functional diversity. We use this example to illustrate the great potential for significant advancements towards a more in depth understanding of the ecological consequences of forest fragmentation. At the landscape level, we find an indirect effect of matrix quality on functional evenness via its effect on increased fragment isolation. At the fragment-level, we find an indirect effect of fragment area on functional richness and functional divergence via its effects on shape complexity and edge effects (i.e. distance from plot center to forest edge), respectively. In this study, loss of shape complexity leads to significant changes in functional richness for traits related to dispersal mode. For resource use traits, we find that functional richness and divergence decline with decreasing shape complexity and distance from plot to forest edge, respectively, while functional evenness increased with isolation. Our results also suggested a negative relationship between fragment shape complexity and distance from plot center to forest edge, in line with previous work [7,60,61]. A reduction in fragment shape complexity might exacerbate edge effects on functional diversity, effects that might not be revealed when analysed using simple regression [7].

The relative importance of landscape and fragment-level factors vary considerably between traits, but fragment-level factors were generally more important than landscape characteristics in explaining variation in functional richness and divergence. By favouring tree species with fast growth, edge effects and shape complexity seem to be key drivers of changes in the competitive hierarchies of tree communities in the fragmented forests of the East Usambaras. The lesser importance of landscape-scale variables in the current study, only observed for functional evenness, may be driven in part by our focus on plot-scale functional diversity. While the plot-scale data are informative in assessing variation in diversity at small spatial scales, they might not capture changes in diversity at large scales (e.g., at landscape scales or patterns in beta-diversity). Future work examining patterns in functional diversity aggregated at larger scales and across landscapes will extend the work presented here and better inform models of the main factors driving tree communities in fragmented forests.

Functional diversity of resource use traits in response to fragmentation

A decrease in area of suitable habitat is expected to lead to the loss of species and a corresponding narrowing of trait value, resulting in lower alpha functional richness [62]. We found evidence of a decline in functional richness and divergence for traits related to resource use (e.g. wood density and regeneration strategy) due to reduced shape complexity and distance to edge respectively, both mediated by fragment area. Our results suggest the presence of strong post-fragmentation edge effects, leading to an increase in pioneer species while shade tolerant species are negatively impacted. In addition to area-based effects, fragmentation creates more edge habitat typified by elevated radiation, temperature and wind turbulence, and lower soil fertility and air moisture [63,64]. However, CWM trait values suggested that even if we found evidence of functional divergence decreasing with distance from plot to forest edge, species at the edges were more functionally diverse. This is likely the result of strong post-fragmentation edge effects leading to an increase of small stature, light-demanding species characterized by low wood density, combined with the older and taller, shade-tolerant and high wood-density species persisting from pre-fragmentation communities.

The studied area has experienced a long history of land use with varying levels of anthropogenic disturbance, resulting in significant forest loss and fragmentation [33]. In this highly fragmented landscape, edge effects on microclimate variables (air temperature, vapor pressure deficits and light intensity) are stronger within 60 to 94 m from the edge, as compared to the forest interior [65], explaining the observed changes in the competitive hierarchies of tree communities in the fragmented forest of the East Usambara Mountains. Functional divergence for resource use traits decreased with increasing distance of the plot from the forest edge, especially in large fragments as smaller fragments (< 20 ha) generally included far fewer interior plots (>200 m from the forest edge). The edge effects found here are in line with other studies [17,18,66-68], where species associated with slow growth rates are outcompeted in forest edges by light-demanding or pioneer species with fast growth. Old-growth species are particularly vulnerable to the detrimental effects of wind turbulence, desiccation, and liana dominance that characterise the edge of forest fragments [15], including those in the East Usambaras [65].

Functional diversity of dispersal traits in response to fragmentation

Fragments with more elongated shapes have higher proportion of total edge than interior habitat [60]. Reduced fragment shape complexity often results in low habitat heterogeneity, thus, communities in fragments with narrow and elongated shapes may exhibit reduced species richness and abundance [7]. Our results suggest that when fragments reach a certain reduced size, loss of shape complexity leads to significant changes in functional richness for traits related to dispersal mode. Increased complexity of fragment shape may limit impact of wind action to toppling large trees, which may explain why anemochorous species, like the emergent Newtonia buchananni (Fabaceae), remain in large and more complex East Usambara fragments. Small and less complex shaped fragments tend to be more vulnerable to edge-related wind damage increasing rates of windthrow and forest structural damage due to the higher ratio of perimeter to edge compared to larger and more complex shaped fragments [60]. Unfortunately, generalizing on the overall effects of fragment shape complexity on functional diversity is limited because it remains understudied compared to other fragmentation processes. It is important to highlight that our evidence of fragmentation effects on functional diversity comes from data of mature trees that represent the historical legacies of pre-fragmentation communities. Hence, without data on seedlings and saplings, it is difficult to ascertain whether the abundance of wind dispersed species is associated with reduced animal seed dispersers and therefore dispersal limitation which are negatively impacted by forest fragmentation in this study area [24,74,75]. Evidence from research in this study area has shown that several important frugivores are absent from or occur in lower abundance in forest fragments as compared to the continuous forest, threatening their persistence, as well as trees dependent on many these vectors [69-71].

Furthermore, we failed to uncover a relationship between traits related to dispersal (i.e. seed length, dispersal mode) and fragment isolation or matrix quality. Instead, our results suggest a less even distribution for traits related to resource use (e.g. wood density) as fragments became more isolated. Wood density is a strong indicator of successional dynamics with light wood often associated to early successional species (e.g. pioneer, light demanding species) that exhibit high fecundity and long-distance dispersal allowing them to colonize recently disturbed sites [41,72]. Therefore, light-wood species may be able to reach more isolated fragments perhaps due to better colonization abilities than hard-wood species. Fragmentation leads to increasing degree of isolation between fragments, hereby increasing the minimum dispersal distance for species from the regional pool to colonize fragments. However, it is important to highlight a potential correlation between dispersal and resource acquisition traits. Specifically, seed mass tends to define the mode of dispersal and is also related to the successional habit that determines resource acquisition strategies [40,73]. Light-demanding, early successional species often produce numerous small seeds and thus are considered better colonizers than shade-tolerant, late successional species [35], with potential to increase in abundance over time and negatively impacting the future establishment of late-successional trees.

The effects of isolation and matrix quality may require exploring functional diversity at a larger scale, beyond the local scale investigated in this study. Fragmentation leads to a high degree of isolation between the remaining fragments increasing the minimum dispersal distance for species from the regional pool to colonize fragments [61,74-76]. As species become more dispersal limited with decreasing fragment connectivity, we expect that fragments would become more similar in species composition, decreasing alpha functional richness and increasing evenness. Moreover, a more diverse matrix may promote habitat heterogeneity between fragments increasing the range of total available niches at the landscape scale [77-79], potentially increasing functional richness and evenness. However, with most studies conducted at a local scale, the effects of isolation and matrix type on functional diversity remains fairly unexplored.

Conclusion

By analysing trait variation due to processes occurring at the landscape scale and integrating this information with well-known fragment-scale processes using a structural equation approach, we were able to provide a more in-depth understanding of the different components of fragmentation and their impact on functional diversity. Specifically, the effects of fragment variables on functional diversity of trees were largely mediated by the indirect effect of fragment area on the amount of edge habitat and shape complexity. Our results demonstrate the power of this approach in detecting the effects of processes occurring at different spatial scales that may have been missed if only the direct impacts of landscape fragmentation would have been considered. This approach could greatly facilitate future empirical work in forest fragmentation and help advocate for management and restorations strategies that aim to achieve long-term persistence of remaining forests. Given that many fragmented forest systems will experience environmental conditions outside the range to which they are adapted, it is important to improve efforts to predict biodiversity responses to current human pressure to implement effective management and conservation strategies.

Map of study area in the East Usambara Mountains of Tanzania.

Map of the study area in the East Usambara Mountains of Tanzania. The protected area, Amani Nature Reserve, includes the continuous forest (dark green) and the largest forest fragment (largest fragment in light green). The landcover classification (i.e. tea plantation, Eucalyptus plantation, forest and subsistence farming) was based on a Landsat-8 images from 2016 (courtesy of the U.S. Geological Survey) and performed using the random forest classification extension (r.learn.lm) in GRASS GIS.

(TIFF)

Click here for additional data file.

Main fragment and continuous forest characteristics of study sites in the East Usambara Mountains, Tanzania.

(PDF)

Click here for additional data file.

Functional traits of tree species sampled in vegetation plots in the East Usambara Mountains, Tanzania.

(PDF)

Click here for additional data file.

20 Feb 2020

PONE-D-19-34303

DISENTANGLING THE DIRECT AND INDIRECT EFFECTS OF FOREST FRAGMENTATION ON PLANT FUNCTIONAL DIVERSITY

PLOS ONE

Dear Dr Zambrano,

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.

You will find further below the comments by three reviewers. They have in common that further work is required on the manuscript, though their recommendations are different from each other. Each of the issues they bring up has merits, so I think they all need attention. Forest fragmentation is a complex process, and providing clues about factors and their interactions needs careful attention. I find the hints by the reviewers all relevant.

We would appreciate receiving your revised manuscript by Apr 05 2020 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. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Berthold Heinze

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

After some struggle (19 potential reviewers were contacted), we finally obtained three high quality reviews that help a lot in shaping this manuscript further into a very interesting contribution. The three reviews are divided in their recommendation, maybe because they go to different levels of depth, but for me the long and short of it is that some aspects require additional thoughts (or analyses), some of the wording needs careful reconsideration, and some technical aspects need attention (e.g. in some figures). I would like to invite the authors to take care of all issues mentioned by the reviewers and provide comment/action for all of these.

Journal Requirements:

When submitting your revision, we need you to address these additional requirements:

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 http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In your Methods section, please provide additional location information of the study area, including geographic coordinates for the data set if available.

3. 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.

4. We note that Figure S1 in your submission contain map images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (a) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (b) remove the figures from your submission:

a.    You may seek permission from the original copyright holder of Figure S1 to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b.    If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

5.Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

[Note: HTML markup is below. Please do not edit.]

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: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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: Yes

Reviewer #2: No

Reviewer #3: 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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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: Zambrano et al. present an interesting work using a multi-scale SEM approach to tease apart the relative importance of suits of fragmentation effects on functional diversity in forest ecosystems. By exploring five key functional traits (i.e., seed length, dispersal mode, shade tolerance, maximum tree height and wood density), they found forest fragmentation directly and indirectly influences tree functional richness and evenness. Fragment area combing with edge habitat and shape complexity reduce functional richness and evenness for those traits related to resource acquisition as well as selecting fast grow tree species. Overall, I enjoy reading the work. The manuscript is well organized, and the framework, results and discussion are clear. This work is also timely important for forest management. I have only one major concern about quantifying functional diversity.

To account for unequal sampling effort, functional diversity indices were calculated through rarefied communities. It’s unclear for me about the rationale to use the mean 1000 random draws to represent the functional diversity indices. I am wondering whether the results for the expected functional indices are the same as the observed one. In addition, I cannot follow the rationale in Lines 230-234, too.

Minor concerns:

Line 112: What does UNESCO represent?

Line 248: Do you mean AICcs are less than 2?

Lines 299-307: This section can be moved to Methods section.

Line 476: Full journal name?

Reviewer #2: PONE_D_19_34303

GENERAL COMMENTS

A sound study that is well written with short-comings duly acknowledged by authors. My main comment focusses on the need for a greater consideration of animal vectors given that the majority of trees were animal dispersed (e.g. rather than wind dispersed). There was no mention on the habitat requirements/preferences on the animal vectors that would no doubt have an impact on plant colonisation (e.g. edge specialists vs core habitat requirements for fauna). More comments on this point provided below.

ABSTRACT

L25, ‘fragment … scale’, I assume you mean ‘local … scale’? (e.g. as referred to in L109)

INTRODUCTION

The introduction lacks any material on the role of animals in seed dispersal and plant colonisation (of both edges and forest interior). Given that there are very few wind-dispersed species in the forest and that nearly all are animal dispersed (including most of the pioneer species, from Table S1), there needs to be some consideration of fragmentation effects on colonisation by animal dispersed species – and I would also think some associated hypotheses?

L52, L58, missing space

L67, clarify what you mean by a ‘harsh matrix’

L78, still not clear what you mean by ‘fragment-scale’ (given fragment size can vary by orders of magnitude) – clarify so that this is clear in its subsequent use throughout

L93, passive sentence structure – revise

L114-125, there are many predictions included here – would it be possible to tighten up this paragraph? E.g. L115, 116, 123 each relate to pioneer species and their preference for edges.

L116, what about potential for long-distance dispersal via animal vectors?

L127, ‘fragment-scale fragmentation’ – meaning? (also L128)

MATERIALS AND METHODS

L144-148, for an international audience, it would be useful to include associated plant families (for those not familiar with the species)

L147, closing bracket here but there is no opening bracket

L157, 55 plots does not match the total number of plots shown in FigS1, nor the number of plots listed in Table S1.

L158, 20 × 20 m (not 20x20m)

L159, some fragments seem to have many more plots (Fig S1)?

L161, define DBH, measured for what? Presumable DBH and height only? How was height measured? Some indication of level of accuracy is needed.

L199-200, does this mean the metric considered the proportion of edge in contact with each of the different types of cultivated land? (e.g. given that each would potentially represent different habitat value for dispersal vectors, pollinators etc., differential effects on microclimate …)

RESULTS

Figure 3, from the scale shown on the map (Fig S1), I don’t know how distance to the continuous forest could be in the order of 1000’s of km (the scale shown in Fig S1 may also be incorrect).

DISCUSSION

L339-345, yes!

L367, avoid starting paragraph with ‘Furthermore’

L373, ‘compared’ not ‘compare’

L398, I don’t quite follow – wouldn’t it be easier to pick up a trend is you have a large range of seed dispersal modes? Moreover, the majority of the species were zoochorous – so perhaps it is the lack of diversity in dispersal modes that prevented detection of any trends? Zoochorous species may be ant dispersed, dispersed via ingestion or dispersed via adhesion – perhaps considering the finer levels of these dispersal modes would help given the flow on implications to dispersal distances and also habitat requirements of the animal vectors. [I see you make reference to this point in L405-407; however outside of gape-width, could you make finer groupings based on the type of animal vector – or are they all bird dispersed via ingestion?]

CONCLUSION

L421-428, this is introductory material.

SUPPLEMENTARY

Multiple font types used, inconsistent number of decimal places used -this needs tightening up.

Figure S1, Latitude and Longitude should be included along the edges of the map border. It looks like the scale legends are incorrect (possibly in both maps). What does the ‘white’ area represent? If the area between the forest fragments supports other land uses (e.g. tea plantations as indicated in text), then this should be shown.

Table S1, the number of plots listed in the table does not seem to match the plots shown in Figure S1? I wouldn’t describe this table as one of forest metrics as the only metric shown in Fragment Area. Do you need to differentiate the sizes according to small, medium, large (categorical attributes) given you have the actual area? Is the large green fragment considered one fragment? The numbers of fragments listed in the table don’t seem to match Figure S1.

Table S2, units are needed for Seed length, Height, Wood density.

Reviewer #3: In this manuscript Zambrano et al analyze the response of different functional diversity metrics to fragmentation variables separated into landscape and fragment-level using SEM. They report various responses of functional richness, evenness and divergence to fragmentation. The paper is interesting and generally well-written, my main suggestions for improvments are:

- check the consistency between the reported results and the statements in the discussion, for instance in the result a decrease in functional richness with distance to the edge is reported while in the discussion the opposite is stated

- the result section could be improved by separating the discussion of the inter-relation between the fragmentation variables from the discussion of the effect of fragmentation on functional diversity

Additionaly I have the following more detailed comments:

Line 37: this manuscript is about functional diversity, I recomend to change „community composition“ by „functional diversity“ or similar

Line 52: Replace „their local diversity“ by „local functional diversity“

Line 58: Unclear here what multivariate / univariate refer to, maybe drop it?

Line 74-89: Nice paragraph.

Line 95: One verb to much, choose one between identify and understanding.

Line 96-97: The interesting properties of SEM in this context could be made a bit clearer here. Something like: „may miss critical indirect effects between fragment-level and landscape-level fragmentation variables“

Line 107: I would be more specific here, the introduction focused on fragmentation only, „drivers“ is a bit vague here in that regard. Would suggest to replace with „fragmentation effects“.

Line 114-131: The hypothesis would need some greater consistency in their generality. For instance the first part „forest fragmentation is decreasing functional diversity“, is very vague given the objectives of the manuscript to disentagle direct and indirect relation across multiple scales. In this hypothesis, which aspect of forest fragmentation are you talking about? Which aspect of functional diversity? Similarly, the text under the (ii) subheaders is also very vague. I would recommend to only keep the three detailed points (matrix quality / isolation, fragment size and edge effects) separated into (i) to (iii).

Line 136: What range?

Line 138: Precipitation is usually given in mm

Line 214: remove „that describe“ and add a colon :

Line 222: How was abundance derived? Number of stems? DBH? Crown cover?

Line 226: This require clarification because above it is stated that all trees with >1m DBH were identified to species.

Line 244: Stepwise selection via AICc is an exploratory procedure that does not seem to fit to the confirmatory framework of the manuscript (clear hypothesis with predictions are set in the introduction). Why not just use the full model?

Line 260: I find the results as they stand now rather confusing because (at least) two different aspects are discussed at the same time: (i) the relation between the fragmentation variables (potential indirect effects) and (ii) the response of functional diversity to the fragmentation variables: I would recommend to separate the results into different subsections, in a first one the relations between the fragmentation variables would be discussed, and in subsequent ones the response of the different functional diversity metrics. Dropping the stepwise approach would also make the message clearer because now the inter-relation between the fragmentation variables is changing between the different functional diversity metrics which is a bit counter-intuitive. Working with a well thought-off full model would prevent this dispersion.

Line 285: „functional divergence tended to decrease“, with a p-value of 0.6, I find this a bit cheeky to give a direction to this effect that clearly could go in any (or no) direction.

Line 289: Please also report here the slopes and their p-value to be consistant with previous sections

Line 327: This is in contradiction with results reported line 266 for functional richness and in the result section I can see no reference to edge effects on functional eveness. Please check once again your result and derive the relevant conclusions from it!

Line 343-345: Good point! Maybe it is an option to already start looking at beta and gamma functional diversity in this manuscript?

Line 351-352: Again not supported by your results …

Table 1: Some of the coefficient estimates are very tiny (1e-20), why is that? What is the unit of your response variables? Did you consider re-scaling your response to improve model estimation (underflowing issue might arise with such tiny values)?

Figure 1: This figure is a bit confusing because in the SEM literature using a box (functional diversity) with arrows to different variables (functional richness …) is traditionally used to represent a latent variable. This is not the case here so to prevent confusion I would recommend to have one box with functional diversity and below it in bracket functional richness … Please also add in the legend that the different functional diversity metric were fitted in separate models.

Figure 2: Where is functional divergence? Also difference between significant / non-significant paths is not clear at all. Consider using a color scale. Also I am not sure that using line types (dashed, not dashed) to differentiate between direct and indirect effects is helpful here.

Figure S1: Some plots seem to be very close together, did you consider trying a Mantel test on the model residuals to check if there are any spatial autocorrelation present?

**********

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: No

Reviewer #2: No

Reviewer #3: Yes: Lionel Hertzog

[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.

5 Apr 2020

April 5, 2020

Dear Dr. Heinze,

We thank you for handling our original submission of our manuscript entitled “Investigating the direct and indirect effects of forest fragmentation on plant functional diversity” for consideration as a Research Paper in PlosOne as part of the Biodiversity Conservation Collection. Enclosed you will find a revised version of the manuscript that has been approved by my coauthors. We appreciate your consideration on the revised version.

In the following, we respond to each individual comment one-by-one. We hope you find the revisions satisfactory.

Sincerely,

Jenny Zambrano (on behalf of my co-authors)

---------------------------------------------------------------------------------------------------------------------

Additional Editor Comments (if provided):

After some struggle (19 potential reviewers were contacted), we finally obtained three high quality reviews that help a lot in shaping this manuscript further into a very interesting contribution. The three reviews are divided in their recommendation, maybe because they go to different levels of depth, but for me the long and short of it is that some aspects require additional thoughts (or analyses), some of the wording needs careful reconsideration, and some technical aspects need attention (e.g. in some figures). I would like to invite the authors to take care of all issues mentioned by the reviewers and provide comment/action for all of these.

Journal Requirements:

When submitting your revision, we need you to address these additional requirements:

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 http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Authors: Done

2. In your Methods section, please provide additional location information of the study area, including geographic coordinates for the data set if available.

Authors: We have included the main latitude and longitude coordinate in the main text and all GIS coordinates in the online database available at https://knb.ecoinformatics.org/view/doi:10.5063/F1KS6PX9

3. 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.

Authors: The data is available at: https://knb.ecoinformatics.org/view/doi:10.5063/F1KS6PX9

4. We note that Figure S1 in your submission contain map images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

Authors: We have now use Landsat-8 images from 2016, courtesy of the U.S. Geological Survey.

We require you to either (a) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (b) remove the figures from your submission:

a. You may seek permission from the original copyright holder of Figure S1 to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

5.Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information:http://journals.plos.org/plosone/s/supporting-information

¬ Authors: Done ________________________________________________________________________________________________________

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1:

Zambrano et al. present an interesting work using a multi-scale SEM approach to tease apart the relative importance of suits of fragmentation effects on functional diversity in forest ecosystems. By exploring five key functional traits (i.e., seed length, dispersal mode, shade tolerance, maximum tree height and wood density), they found forest fragmentation directly and indirectly influences tree functional richness and evenness. Fragment area combing with edge habitat and shape complexity reduce functional richness and evenness for those traits related to resource acquisition as well as selecting fast grow tree species. Overall, I enjoy reading the work. The manuscript is well organized, and the framework, results and discussion are clear. This work is also timely important for forest management. I have only one major concern about quantifying functional diversity.

Authors: Thank you for your support.

To account for unequal sampling effort, functional diversity indices were calculated through rarefied communities. It’s unclear for me about the rationale to use the mean 1000 random draws to represent the functional diversity indices. I am wondering whether the results for the expected functional indices are the same as the observed one. In addition, I cannot follow the rationale in Lines 230-234, too.

Authors: We removed the analysis using 1000 random draws of the rarefied community because all plants were exhaustively sampled in each plot. We used Community Weighted Means as a complement to functional diversity indices to better understand the functional shift observed in the studied area.

Minor concerns:

Line 112: What does UNESCO represent?

Authors: We have written out UNESCO so all readers are aware of what it stands for.

Line 248: Do you mean AICcs are less than 2?

Authors: We have deleted this information since we used a full model and no model selection was required.

Lines 299-307: This section can be moved to Methods section.

Authors: Thank you for the suggestion; we moved part of this section to metods and left the results of this analysis to clarify our observations about distance to edge and, that complements the previous section.

Line 476: Full journal name?

Authors: Done

Reviewer #2:

GENERAL COMMENTS

A sound study that is well written with short-comings duly acknowledged by authors. My main comment focusses on the need for a greater consideration of animal vectors given that the majority of trees were animal dispersed (e.g. rather than wind dispersed). There was no mention on the habitat requirements/preferences on the animal vectors that would no doubt have an impact on plant colonisation (e.g. edge specialists vs core habitat requirements for fauna). More comments on this point provided below.

Authors: Thank you for your suggestions. The concerns raised about animal seed dispersal are valid given that we did not clarify fully that we are evaluating functional diversity of adult tree communities post-fragmentation. We censused all trees > 10 cm DBH. The mature trees sampled in our census are largely remnants from pre-fragmentation, and for the most part, pioneer trees sampled in our census would have colonized post-fragmentation. Had we analysed functional diversity of early life history stages (i.e., seed, seedling, and saplings/recruits), we would have been able to make inferences about seed dispersal.

ABSTRACT

L25, ‘fragment … scale’, I assume you mean ‘local … scale’? (e.g. as referred to in L109)

Authors: We replaced “fragment” by “local”

INTRODUCTION

The introduction lacks any material on the role of animals in seed dispersal and plant colonisation (of both edges and forest interior). Given that there are very few wind-dispersed species in the forest and that nearly all are animal dispersed (including most of the pioneer species, from Table S1), there needs to be some consideration of fragmentation effects on colonisation by animal dispersed species – and I would also think some associated hypotheses?

Authors: Thank you for your suggestions. However, as we previously mentioned we have analyzed mostly pre-fragmentation tree communities. Pioneers would be the main guild that have colonized post-fragmentation, and we have made this clearer throughout the Mss.

L52, L58, missing space

Authors: Corrected

L67, clarify what you mean by a ‘harsh matrix’

Authors: We mean a low quality matrix. We have included this clarification line 63.

L78, still not clear what you mean by ‘fragment-scale’ (given fragment size can vary by orders of magnitude) – clarify so that this is clear in its subsequent use throughout

Authors: We have clarified this in line 74.

L93, passive sentence structure – revise

Authors: Done

L114-125, there are many predictions included here – would it be possible to tighten up this paragraph? E.g. L115, 116, 123 each relate to pioneer species and their preference for edges.

Authors: We have completely rewritten the hypotheses to distinguish fragmentation effects on traits related to acquisition and traits associated with dispersal. Thank you for your suggestion.

L116, what about potential for long-distance dispersal via animal vectors?

Authors: As we previously mentioned we have analyzed mostly pre-fragmentation tree communities. In order, to determine the effects of long-distance dispersal we need to study seedling communities which was not the scope in this study.

L127, ‘fragment-scale fragmentation’ – meaning? (also L128)

Authors: We have removed “fragmentation”

MATERIALS AND METHODS

L144-148, for an international audience, it would be useful to include associated plant families (for those not familiar with the species)

Authors: Thank you for the suggestion. We have included the associated plant families.

L147, closing bracket here but there is no opening bracket

Authors: We have removed the closing bracket

L157, 55 plots does not match the total number of plots shown in FigS1, nor the number of plots listed in Table S1.

Authors: Corrected

L158, 20 × 20 m (not 20x20m)

Authors: Corrected

L159, some fragments seem to have many more plots (Fig S1)?

Authors: That’s correct. We already made this apparent in the text.

L161, define DBH, measured for what? Presumable DBH and height only? How was height measured? Some indication of level of accuracy is needed.

Authors: Corrected. In addition, we have removed “height” since it was not included in any of the analyses.

L199-200, does this mean the metric considered the proportion of edge in contact with each of the different types of cultivated land? (e.g. given that each would potentially represent different habitat value for dispersal vectors, pollinators etc., differential effects on microclimate …)

Authors: Here we mean matrix quality, and we have clarified in the methods (lines 209-217) how we define and calculate matrix quality based on the different types of cultivated land surrounding fragments.

RESULTS

Figure 3, from the scale shown on the map (Fig S1), I don’t know how distance to the continuous forest could be in the order of 1000’s of km (the scale shown in Fig S1 may also be incorrect).

Authors: Corrected

DISCUSSION

L339-345, yes!

Authors: Thank you.

L367, avoid starting paragraph with ‘Furthermore’

Authors: Revised.

L373, ‘compared’ not ‘compare’

Authors: Corrected.

L398, I don’t quite follow – wouldn’t it be easier to pick up a trend is you have a large range of seed dispersal modes? Moreover, the majority of the species were zoochorous – so perhaps it is the lack of diversity in dispersal modes that prevented detection of any trends? Zoochorous species may be ant dispersed, dispersed via ingestion or dispersed via adhesion – perhaps considering the finer levels of these dispersal modes would help given the flow on implications to dispersal distances and also habitat requirements of the animal vectors. [I see you make reference to this point in L405-407; however outside of gape-width, could you make finer groupings based on the type of animal vector – or are they all bird dispersed via ingestion?]

Authors: Thank you. This is a good point. Unfortunately, we do not have data on the finer-scale categories of dispersal mode. We have altered this section as the new results include an effect of fragmentation on anemochory discussed in lines 401-406, and in addition, we clarify earlier that we are analyzing post-fragmentation communities of mature trees, much of which are remnants of pre-fragmentation, and hence changes in the diversity of dispersal modes are driven by pioneer species.

CONCLUSION

L421-428, this is introductory material.

Authors: We have deleted part of this section and move part of it to the end of the conclusion to reflect the important of using an approach such as the one we use in this study to better understand fragmentation effects and apply it to management efforts.

SUPPLEMENTARY

Multiple font types used, inconsistent number of decimal places used -this needs tightening up.

Authors: Corrected.

Figure S1, Latitude and Longitude should be included along the edges of the map border. It looks like the scale legends are incorrect (possibly in both maps). What does the ‘white’ area represent? If the area between the forest fragments supports other land uses (e.g. tea plantations as indicated in text), then this should be shown.

Authors: Corrected.

Table S1, the number of plots listed in the table does not seem to match the plots shown in Figure S1? I wouldn’t describe this table as one of forest metrics as the only metric shown in Fragment Area.

Authors: Corrected.

Do you need to differentiate the sizes according to small, medium, large (categorical attributes) given you have the actual area?

Author: We have deleted this column.

Is the large green fragment considered one fragment? The numbers of fragments listed in the table don’t seem to match Figure S1.

Authors: Corrected.

Table S2, units are needed for Seed length, Height, Wood density.

Authors: Corrected.

Reviewer #3: In this manuscript Zambrano et al analyze the response of different functional diversity metrics to fragmentation variables separated into landscape and fragment-level using SEM. They report various responses of functional richness, evenness and divergence to fragmentation.

Authors: Thank you for your support.

The paper is interesting and generally well-written, my main suggestions for improvments are:

- check the consistency between the reported results and the statements in the discussion, for instance in the result a decrease in functional richness with distance to the edge is reported while in the discussion the opposite is stated

Authors: Done.

- the result section could be improved by separating the discussion of the inter-relation between the fragmentation variables from the discussion of the effect of fragmentation on functional diversity

Authors: Thank you for the suggestion. We have divided the results from the SEMs into two sections: 1) the inter-relation between fragment variables and 2) effects on functional diversity.

Additionaly I have the following more detailed comments:

Line 37: this manuscript is about functional diversity, I recomend to change „community composition“ by „functional diversity“ or similar

Authors: Corrected.

Line 52: Replace „their local diversity“ by „local functional diversity“

Authors: Done

Line 58: Unclear here what multivariate / univariate refer to, maybe drop it?

Authors: Done.

Line 74-89: Nice paragraph.

Authors: Thank you!

Line 95: One verb to much, choose one between identify and understanding.

Authors: Corrected.

Line 96-97: The interesting properties of SEM in this context could be made a bit clearer here. Something like: „may miss critical indirect effects between fragment-level and landscape-level fragmentation variables“

Authors: Corrected.

Line 107: I would be more specific here, the introduction focused on fragmentation only, „drivers“ is a bit vague here in that regard. Would suggest to replace with „fragmentation effects“.

Authors: Corrected.

Line 114-131: The hypothesis would need some greater consistency in their generality. For instance the first part „forest fragmentation is decreasing functional diversity“, is very vague given the objectives of the manuscript to disentagle direct and indirect relation across multiple scales. In this hypothesis, which aspect of forest fragmentation are you talking about? Which aspect of functional diversity? Similarly, the text under the (ii) subheaders is also very vague. I would recommend to only keep the three detailed points (matrix quality / isolation, fragment size and edge effects) separated into (i) to (iii).

Authors: We have included the information of how functional diversity was defined and the expected results on diversity of traits related to resource acquisition and dispersal, as well as, the expected interactions between fragment- and landscape-level processes.

Line 136: What range?

Authors: Clarified.

Line 138: Precipitation is usually given in mm

Authors: Corrected.

Line 214: remove „that describe“ and add a colon :

Authors: Done

Line 222: How was abundance derived? Number of stems? DBH? Crown cover?

Authors: Number of stems. We have included this information.

Line 226: This require clarification because above it is stated that all trees with >1m DBH were identified to species.

Authors: Corrected

Line 244: Stepwise selection via AICc is an exploratory procedure that does not seem to fit to the confirmatory framework of the manuscript (clear hypothesis with predictions are set in the introduction). Why not just use the full model?

Authors: Thank you for the recommendation. We now present and discuss the results from full models.

Line 260: I find the results as they stand now rather confusing because (at least) two different aspects are discussed at the same time: (i) the relation between the fragmentation variables (potential indirect effects) and (ii) the response of functional diversity to the fragmentation variables: I would recommend to separate the results into different subsections, in a first one the relations between the fragmentation variables would be discussed, and in subsequent ones the response of the different functional diversity metrics. Dropping the stepwise approach would also make the message clearer because now the inter-relation between the fragmentation variables is changing between the different functional diversity metrics which is a bit counter-intuitive. Working with a well thought-off full model would prevent this dispersion.

Authors: Thank you for the recommendations. We have now divided the results into two sections and drop the stepwise approach.

Line 285: „functional divergence tended to decrease“, with a p-value of 0.6, I find this a bit cheeky to give a direction to this effect that clearly could go in any (or no) direction.

Authors: Following reviewer suggestions on analysis, we now report new results pertaining to functional divergence that show a strong relationship with shape complexity.

Line 289: Please also report here the slopes and their p-value to be consistant with previous sections

Authors: Done.

Line 327: This is in contradiction with results reported line 266 for functional richness and in the result section I can see no reference to edge effects on functional eveness. Please check once again your result and derive the relevant conclusions from it!

Authors: We have corrected this.

Line 343-345: Good point! Maybe it is an option to already start looking at beta and gamma functional diversity in this manuscript?

Authors: Thank you for your recommendation. This is something we tried in a previous analysis; however, due to the low replication at the landscape level and variation in sampling effort we were not able to draw strong conclusions from this analysis.

Line 351-352: Again not supported by your results …

Authors: Corrected.

Table 1: Some of the coefficient estimates are very tiny (1e-20), why is that? What is the unit of your response variables? Did you consider re-scaling your response to improve model estimation (underflowing issue might arise with such tiny values)?

Authors: Thank you for your suggestion. We have rescaled the CWM values.

Figure 1: This figure is a bit confusing because in the SEM literature using a box (functional diversity) with arrows to different variables (functional richness …) is traditionally used to represent a latent variable. This is not the case here so to prevent confusion I would recommend to have one box with functional diversity and below it in bracket functional richness … Please also add in the legend that the different functional diversity metric were fitted in separate models.

Authors: Corrected.

Figure 2: Where is functional divergence? Also difference between significant / non-significant paths is not clear at all. Consider using a color scale. Also I am not sure that using line types (dashed, not dashed) to differentiate between direct and indirect effects is helpful here.

Authors: Corrected and we have included a figure including functional divergence.

Figure S1: Some plots seem to be very close together, did you consider trying a Mantel test on the model residuals to check if there are any spatial autocorrelation present?

Authors: Thank you for the suggestion; however, we believe that the measurements of fragment and landscape properties (i.e. distance of plot to edge of fragment, distance of fragment to continuous forest), included in this study, take into count the spatial context of the focal units. We do include information on distance of plot to fragment edge and distance of fragment to forest, so we would expect this would take into account some of the spatial signature. Of course, we are not accounting for variation in variables such soil and topography, to give just two examples, hence we cannot be completely certain of the spatial signature stems from the spatial fragmentation processes or the underlying topography/soil.

Submitted filename: Responses to reviewers.docx

Click here for additional data file.

5 May 2020

PONE-D-19-34303R1

Investigating the direct and indirect effects of forest fragmentation on plant functional diversity

PLOS ONE

Dear Dr Zambrano,

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.

A few remaining suggestions of the reviewers are listed below. I think they will improve the manuscript further, so it is worth the extra round.

We would appreciate receiving your revised manuscript by Jun 19 2020 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. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Berthold Heinze

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

All reviewers are impressed by the additional work done for improving the manuscript. It is considered (almost) ready for publication. I think the few remaining points can easily be addressed (some may just be a matter of re-wording in order to gain clarity in expression); e.g. the hypotheses are mentioned by two of the reviewers. Also carefully consider the other comments please.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. 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: All comments have been addressed

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

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: Yes

Reviewer #2: Yes

Reviewer #3: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. 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: I've read the revised manuscript. The authors have done an excellent work. All of my concerns have been addressed. Congratulations!

Reviewer #2: GENERAL

A much improved manuscript. The authors have diligently addressed the numerous reviewer comments. I only have a few minor comments.

My comments relate to the unannotated version.

INTRODUCTION

L65, Are you suggesting that animal-dispersed species are highly dispersed due to their smaller seed? Please clarify – many animal-dispersed species have large seed and are dispersed long distances by being carried by animals (either internally / externally). L120 mentions abiotically-dispersed species with smaller seeds – to I suspect the sentence in L65 just needs to be rephrased for clarity.

L109-113, the hypothesis is poorly expressed – please clarify. I assume you are expecting a decline in functional richness, evenness and divergence?

MATERIALS AND METHODS

L255, space needed (100 m, not 100m)

RESULTS

L303-317, given the results are provided in Table 1, I see no need to list estimate and se in the text (the table could also include t and p).

L324-332, I could not find Figure 3 in the revised version?

DISCUSSION

L383, long ‘history’

ACKNOWLEDGEMENTS

Manuscript also improved by suggestions by reviewers.

SUPPORTING INFORMATION

Much better.

Reviewer #3: First of all, I would like to congratulate the authors for their impresive work in taking into account the large number of comments that were made in the previous version of the manuscript.

I have still two major issues with the manuscript as it now stands:

- Hypothesis: Hypothesis 1 is very long and rather hard to follow. I would recommend to split it up, for instance into a) functional richness, evenness and divergence of resource use traits are expected to decline with …., b) low matrix quality is expected to lead to stronger decline in …., c) trait distribution is expected to become more skewed towards …, d) functional richness, evenness and divergence of dispersal trais are expected to … I would switch hypothesis 2 and 1, hypothesis 2 present how you expect fragmentation effects operating at different scale to interact with each other. Hypothesis 2 is therefore more general. In hypothesis 2 you write: „distance to fragment edge tend to increase in small … fragments“, how is that possible? In smaller fragments every single points should be closer to the edge than in larger fragments.

- Discussion: Fragmentation and especially reduction in habitat area is a process that is usually expected to lead to declining biodiversity, yet looking at Figure 2 it seems that this is not the case here. Based on this main results I am missing a more explicit discussion of the absence of negative direct and indirect effect of reduced fragment area on functional diversity. This sounds rather provocative but your results tend to show that smaller forest patches do not have lower functional richness and evenness compared to larger patches but they even have higher functional divergence. I think that such discussion would be particularly interesting around the lines 368-369, where the opposite is expected. I think that this manuscript would be greatly enhanced by further interpretation of these results and their potential implications.

Minor comments:

- line 230: replace „determine“, maybe use „measure“ instead

- line 259: which package was used to compute the SEMs?

- line 309-312: does the decrease of CWM for shade tolerance with distance to forest edge means that plants are more shade-tolerant closer to the edge? This sounds rather counter-intuitive, do you have some explanation for this?

**********

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: No

Reviewer #2: Yes: Sabine Kasel

Reviewer #3: Yes: Lionel Hertzog

[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.

14 May 2020

Dear Dr. Heinze,

We thank you for your handling of our previous submission to PlosONE entitled: Investigating the direct and indirect effects of forest fragmentation on plant functional diversity (ID: PONE-D-19-3430R1). The manuscript received two detailed expert reviews. There were a few remaining points that needed to be addressed leading the decision of minor revision.

Detailed responses can be found below. The suggestions made by the reviewers greatly helped to improve the work and the manuscript.

We thank you again for your consideration and we hope you will find this new version of the manuscript suitable for publication in PlosONE.

Sincerely,

Jenny Zambrano

Additional Editor Comments (if provided):

All reviewers are impressed by the additional work done for improving the manuscript. It is considered (almost) ready for publication. I think the few remaining points can easily be addressed (some may just be a matter of re-wording in order to gain clarity in expression); e.g. the hypotheses are mentioned by two of the reviewers. Also carefully consider the other comments please.

Authors: Thank you for handling the paper.

Reviewer #1: I've read the revised manuscript. The authors have done an excellent work. All of my concerns have been addressed. Congratulations!

Authors: Thank you for your support and for your previous suggestions.

Reviewer #2: GENERAL

A much improved manuscript. The authors have diligently addressed the numerous reviewer comments. I only have a few minor comments.

My comments relate to the unannotated version.

Authors: Thank you for your support and for your previous suggestions.

INTRODUCTION

L65, Are you suggesting that animal-dispersed species are highly dispersed due to their smaller seed? Please clarify – many animal-dispersed species have large seed and are dispersed long distances by being carried by animals (either internally / externally). L120 mentions abiotically-dispersed species with smaller seeds – to I suspect the sentence in L65 just needs to be rephrased for clarity.

Authors: Corrected

L109-113, the hypothesis is poorly expressed – please clarify. I assume you are expecting a decline in functional richness, evenness and divergence?

Authors: Corrected

MATERIALS AND METHODS

L255, space needed (100 m, not 100m)

Authors: Corrected

RESULTS

L303-317, given the results are provided in Table 1, I see no need to list estimate and se in the text (the table could also include t and p).

Authors: Corrected

L324-332, I could not find Figure 3 in the revised version?

Authors: We had included Figure 3 in the revised version.

DISCUSSION

L383, long ‘history’

Authors: Corrected

ACKNOWLEDGEMENTS

Manuscript also improved by suggestions by reviewers.

Authors: Done

SUPPORTING INFORMATION

Much better.

Authors: Thank you.

Reviewer #3: First of all, I would like to congratulate the authors for their impresive work in taking into account the large number of comments that were made in the previous version of the manuscript.

Authors: Thank you for your support and for your previous suggestions.

I have still two major issues with the manuscript as it now stands:

- Hypothesis: Hypothesis 1 is very long and rather hard to follow. I would recommend to split it up, for instance into a) functional richness, evenness and divergence of resource use traits are expected to decline with …., b) low matrix quality is expected to lead to stronger decline in …., c) trait distribution is expected to become more skewed towards …, d) functional richness, evenness and divergence of dispersal trais are expected to … I would switch hypothesis 2 and 1, hypothesis 2 present how you expect fragmentation effects operating at different scale to interact with each other. Hypothesis 2 is therefore more general. In hypothesis 2 you write: „distance to fragment edge tend to increase in small … fragments“, how is that possible? In smaller fragments every single points should be closer to the edge than in larger fragments.

Authors: Thank you for the suggestion. We have split the presentation of the hypothesis accordingly. We have also clarified and replaced “distance to fragment edge tend to decrease in small…fragments”.

- Discussion: Fragmentation and especially reduction in habitat area is a process that is usually expected to lead to declining biodiversity, yet looking at Figure 2 it seems that this is not the case here. Based on this main results I am missing a more explicit discussion of the absence of negative direct and indirect effect of reduced fragment area on functional diversity. This sounds rather provocative but your results tend to show that smaller forest patches do not have lower functional richness and evenness compared to larger patches but they even have higher functional divergence. I think that such discussion would be particularly interesting around the lines 368-369, where the opposite is expected. I think that this manuscript would be greatly enhanced by further interpretation of these results and their potential implications.

Authors: Larger fragments (area?) tended to include more interior plots (>200 m from the forest edge), whereas smaller fragments (i.e. < 20 ha) generally included far fewer interior plots due to their smaller size. The observed decrease in functional divergence may be explained by the fact that interior plots showed higher functional divergence than plots located closer to the edge. The decrease in functional divergence might have not been so significant in smaller fragments because most studied plots were found within 100m from the forest edges. We have clarified this in the Discussion (494-497).

Minor comments:

- line 230: replace „determine“, maybe use „measure“ instead

Authors: Corrected

- line 259: which package was used to compute the SEMs?

Authors: Corrected

- line 309-312: does the decrease of CWM for shade tolerance with distance to forest edge means that plants are more shade-tolerant closer to the edge? This sounds rather counter-intuitive, do you have some explanation for this?

Authors: Shade tolerance significantly declined with increasing distance from the plot to forest edge (see Table 1), while we found an increase in light-demanding species characteristic of edge habitats (i.e. pioneers and light-demanding non-pioneers).

Submitted filename: PlosONE_response_to_reviewers.docx

Click here for additional data file.

11 Jun 2020

Investigating the direct and indirect effects of forest fragmentation on plant functional diversity

PONE-D-19-34303R2

Dear Dr. Zambrano,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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,

Berthold Heinze

Section Editor

PLOS ONE

Additional Editor Comments (optional):

My apologies for the long time it took me to handle this manuscript. I have now gone through all the changes and answers, it all makes good sense to me and I am happy to accept this manuscript, which I am sure will make an outstanding contribution in this journal.

Reviewers' comments:

19 Jun 2020

PONE-D-19-34303R2

Investigating the direct and indirect effects of forest fragmentation on plant functional diversity

Dear Dr. Zambrano:

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. Berthold Heinze

Section Editor

PLOS ONE