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Title
Establishment success of trees planted in riparian buffer zones
along an agricultural intensification gradient
Authors
B. Bourgeois1,2, A. Vanasse1, D. Rivest3,4, M. Poulin1,2
1 Département de Phytologie, Faculté des Sciences de l’Agriculture
et de l’Alimentation, Université Laval, Québec, 2425 rue de
l’agriculture, Québec, G1V 0A6, Canada.
2 Québec Centre for Biodiversity Science, Department of Biology,
McGill University, Stewart Biology Building, 1205 Dr. Penfield
Avenue, Montréal, Québec, H3A 1B1, Canada.
3Département des sciences naturelles et Institut des sciences de la
forêt tempérée, Université du Québec en Outaouais, 58 rue
Principale, Ripon, Québec, J0V 1V0, Canada.
4Centre for Forest Research, Université du Québec à Montréal, PO
Box 8888, Centre-Ville Station, Montréal, Québec, H3C 3P8,
Canada.
Corresponding author: Bérenger Bourgeois, Monique Poulin.
Département de Phytologie, Faculté des Sciences de l'Agriculture
et de l'Alimentation, Université Laval, Pavillon Paul-Comtois,
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2425, rue de l'Agriculture, Québec, Québec, G1V 0A6, Canada;
[email protected]; [email protected]
Abstract
Although riparian zones provide numerous ecological services,
they have been widely degraded by agricultural intensification. To
recover water quality and restore other critical services, tree
planting has been implemented in agricultural riparian buffer zones
worldwide. However, intensive agricultural practices adjacent to
tree plantations are likely to impede their establishment. In this
study, we assessed the survival and size of trees planted in riparian
buffer zones along a gradient of agriculture intensification. We
studied 68 riparian buffer zones in two agricultural watersheds of
southeastern Québec (Canada) where trees had been planted 3 to
17 years prior to sampling. Tree survival and size (height, diameter
and crown width) were measured and related to agricultural
intensification, quantified as the frequency of annual crops in the
agricultural field adjacent to riparian zones during the seven years
prior to sampling. Tree survival decreased by 25% with increasing
frequency of annual crops (P < 0.0001; R2 = 35%), independently
of the planting year. Aside from the influence of tree age, tree size
varied with the frequency of annual crops but only for three of the
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six most frequently planted tree species (P = 0.0007; R2 = 46%).
These three species (Fraxinus pennsylvanica Marsh., Quercus
macrocarpa Michx. and Picea glauca (Moench) Voss) showed
reduced size with higher cultivation frequency of annual crops,
whereas the other three species (Acer saccharum Marsh., Larix
laricina (Du Roi) K. Koch and Quercus rubra L.) were more
tolerant to agricultural intensification. While tree planting is
carried out in riparian buffer zones to mitigate the environmental
impacts of agricultural practices, agricultural intensification in
turns impedes the establishment success of trees. To increase the
environmental benefits provided by agroforestry projects, tree
planting in riparian buffer zones should focus on species that
tolerate agricultural intensive practices. Additionally, more
frequent inclusion of hay meadows in the crop rotation of fields
adjacent to riparian buffer zones may be beneficial to the
establishement success of planted trees.
Keywords
Agroforestry; annual crop frequency; crop rotation; establishment
success; tree planting; tree survival
1. Introduction
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Riparian zones which correpond to the ecotones between upland
and aquatic ecosystems under the influence of flooding or shallow
groundwater, provide numerous ecological services (Neary et al.,
2010). Besides their high biodiversity, riparian zones reduce soil
erosion, increase water quality and regulate hydrological regimes,
especially when they include forested plant communities
(Lowrance et al., 1984; Marshall and Moonen, 2002; Boutin et al.,
2003; Lovell and Sullivan, 2006). Trees improve soil cohesion,
increase infiltration of runoff water and trap sediments, nitrogen
and phosphorus more effectively than herbaceous species (Osborne
and Kovacic, 1993; Schultz et al., 1995; Lee et al., 2000, 2003).
Despite these environmental benefits, riparian zones have been
degraded worldwide. Some authors have, for example, estimated
that up to 80% of pristine riparian zones have been lost over the
last 200 years in Europe and North America (Naiman et al., 1993).
These losses are mainly attributed to agricultural intensification,
which has destroyed some riparian zone functions directly through
clearing or grazing, and indirectly disturbed others through
fertilizer inputs, pesticide use or soil tillage (Allan, 2004;
Tscharntke et al., 2005). Intensive cereal production has doubled
globally since 1960s (Tilman et al., 2002), and an 18% increase in
agricultural land is predicted by 2050, implying the conversion of
109 hectares of natural ecosystems to agriculture (Tilman et al.,
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2001). The maintenance of natural riparian zones and their
associated ecosystem services is thus increasingly critical
worldwide.
Important governmental measures, including legal protection and
restoration, have been implemented worldwide to recover the
ecological services provided by riparian zones among agricultural
landscapes. In the USA, guidelines for multispecies riparian buffer
installation have, for example, been developed by the USDA
Natural Resources Conservation Service to promote the reduction
of nonpoint source pollution (USDA, 1997). The Common
Agriculture Policy also fosters the establishment of environment-
friendly practices in agricultural landscapes throughout Europe
(Kleijn et al., 2006). In Québec (eastern Canada), agricultural
practices such as soil tillage, fertilization and pesticide applications
have been banned in a riparian zone of at least 3 m wide along
streams adjacent to agricultural fields since 1987, and financial
incentives encourage farmers to plant trees in riparian buffer zones
(Gouvernement du Québec, 1987). When planted with trees,
riparian buffer zones indeed offer multiple agronomic advantages
that result in increased crop productivity (e.g., through windbreak
effect and improved pollination and pest control; Brandle et al.,
2009) and stock safety and exclusion from rivers (when fenced).
Forested riparian zones also provide several external benefits to
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society, like increased landscape aesthetics, beyond their positive
impact on terrestrial and aquatic biodiversity and ecosystem
services (Jose, 2009; Kulshreshtha and Kort, 2009). Furthermore,
tree harvest can generate direct income for farmers (Lockaby et al.,
1997; Correll, 2005) without affecting water quality when best
management practices are used for forestry operations (such as
keeping machinery out of waterways, minimizing stream crossing
and establishing sediment control treatment; Naery et al., 2010;
Smethurst et al., 2012). Agricultural intensification has, however,
been shown to impact spontaneous plant communities on field
margins, reducing their species diversity and influencing their
composition in favour of nitrophilous and ruderal herbaceous
species (Boutin and Jobin, 1998; Mensing et al., 1998; Marshall
and Moonen, 2002). Similarly, trees planted in agricultural riparian
zones may also be negatively impacted by agricultural
intensification. Yet, to our knowledge, no previous study has
investigated the success of tree planting in riparian buffer zones
relative to agricultural practices on adjacent lands.
Intensive agricultural systems such as the cultivation of annual
crops require high chemical inputs of fertilizers and pesticides
(e.g., herbicides, fungicides, insecticides, plant growth regulators;
Tscharntke et al., 2005) to sustain productivity. In turn, drifts from
agricultural inputs represent strong environmental disturbances that
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can lead to biotic homogenization and biodiversity losses of
spontaneous plant communities in adjacent riparian zones
(Tscharntke et al., 2005). Similarly, for trees planted at field
margins, intensive agricultural practices have the potential to
decrease establishment success. While drifts of non-selective
herbicides (such as glyphosate) can reduce the survival of both
planted trees and spontaneous herbaceous plants, trees might be
more sensitive and regenerate slower especially when they are
small. Moreover, the leaching of fertilizers from intensive fields is
more likely to favour fast-growing herbaceous plants, and thereby
to decrease the growth of planted trees through competition. Since
hay meadows corresponds to low-intensity agricultural practices
with less chemical inputs than annual crops, they may attenuate the
detrimental environmental impact of annual crops when included
in crop rotation (Bignal and McCracken, 1996; Sutherland, 2002).
Understanding the response of planted trees to agricultural
intensification is needed to improve decision-making and
implement economically and environmentally productive tree
planted riparian buffer zones or other agroforestry systems in
agricultural landscapes (Jose et al., 2004; Smith et al., 2012).
The goal of this study was to assess the effect of agricultural
intensification on the survival and size of different trees species
planted in agricultural riparian zones. Agricultural intensification
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was quantified as the cultivation frequency of annual crops in
adjacent fields over the seven years preceding sampling. We
hypothesized that agricultural intensification decreases the survival
and size of planted trees. As growth rate is species-specific
(Cogliastro et al., 1990; Burton and Bazzaz, 1995), we also
hypothesized that tree species respond differently to agricultural
intensification. Our study fills a knowledge gap that has recently
been identified by different agricultural stakeholders in Canada
(Tartera et al., 2012; Masse et al., 2014).
2. Materials and methods
2.1. Study area and sampling design
Riparian buffer zones planted with trees along relatively uniform
rivers (in terms of river width and flow) of two agricultural
watersheds in southeastern Québec, Canada were sampled during
the summer of 2012. These watersheds are characterized by
gleysolic and brunisolic soils. The region has a mean annual
temperature of 4 °C (19 °C in July and -12 °C in January) and
mean annual precipitation of 1300 mm, of which 24% falls as
snow (Environment Canada, 2015). In the Boyer watershed (216
km2 area; 46°41' N, 70°55' W), 66% of the land is used for
agriculture, of which 26% is farmed with annual crops (principally
wheat, corn and soybean). From 1984 to 1992, channelization of
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waterways was implemented to improve soil drainage for crop
cultivation along 73% of the 215 km of rivers (OBV Côte-du-
Sud/GIRB, 2011). In the Bélair watershed (43 km² area; 46°26' N,
70°56' W), agriculture covers 33% of the land, of which annual
crops account for about half, i.e. 56% (MAPAQ, unpublished
data).
In the study area, crop rotation of dairy farms generally consists in
four to five years of hay meadows (harvested each year; 2-3
cuts/year), followed by one or two years of genetically-modified
silage corn Roundup Ready (RR), one year of genetically-modified
soybean RR and one year of wheat. For cash crop farms, crop
rotation generally corresponds to two to three years of genetically-
modified grain corn RR, one year of genetically-modified soybean
RR and one year of wheat. Nitrogen fertilization is applied at about
150 kg N/ha in corn, 100 kg N/ha in wheat and 20 to 160 kg N/ha
in hay meadows according to year of production and cover of
grasses (CRAAQ, 2010). In corn and hay meadows, N fertilization
is fractioned in two or three applications (combination of liquid
manure and mineral fertilizers). In corn and soybean, herbicide is
sprayed in one or two applications of Roundup (glyphosate, a non-
selective herbicide). In wheat, an herbicide against broaded-leaf
weeds is applied at stage 3-5 leaves of cereals. In hay-meadow, no
herbicide is applied, or sometimes a broaded-leave herbicide in the
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first year. Minimum tillage is often used in cash crop (corn,
soybean, wheat) while conventionnal tillage (with plowing) is
generally conducted after hay meadows. Across the study area,
pastures were rare as livestock was generally kept indoor farming
facilities, and fenced when present to prevent livestock access to
riparian zones and rivers.
Between 1995 and 2009, after the provincial government banned
agricultural practices in buffer zones at least 3 m wide along
streams (Gouvernement du Québec, 1987), trees were planted
extensively on the flat edge of agricultural fields in the riparian
zones of these two watersheds, chiefly to reduce soil erosion and
improve water quality. Generally, tree plantations consist of a
single row of trees 30 cm tall planted every 3 to 5 m, on ca.
1.2 m-wide black polythene-film mulch.
To be sampled, a riparian zone had to meet four conditions: 1) be
adjacent to an agricultural field with a single crop, 2) have been
planted with trees within a single year, 3) measure at least 40 m
long, and 4) have a uniform vegetation structure. Depending on
site length, three to nine equidistant transects from field edge to
riverbank were staked out to account for intra-site variability, i.e.
three transects on sites less than 100 m long, five transects on sites
between 100 m and 150 m long, seven transects on sites between
150 to 200 m long and nine transects for sites longer than 200 m.
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At each transect, we measured the three planted trees closest to the
transect center. Overall, 923 trees representing 29 species were
sampled in 68 riparian zones that had been planted with trees 3 to
17 years prior to sampling.
2.2. Tree measurements and agricultural practices
Tree survival (%) was calculated from counts of the total number
of planted trees and the number of dead trees on each site. This
response variable thus corresponds to the survival of trees at the
site-scale, and not at the species level, as species could not be
identified for dead tree individuals. Among the three planted trees
measured at each transect, each living individual was identified to
the species. Size measurements were then taken for these trees,
namely height (m), calculated using a clinometer, diameter at
breast height (DBH in cm, at 1.3 m height), measured with a
metric diameter tape, and crown width (m), estimated visually by
two observers. This set of minimum variables was selected as it
accurately predict tree aboveground biomass (Lambert et al.,
2005).
The frequency (%) of annual crops (mainly corn, soybean and
wheat) and hay meadows in the field adjacent to the riparian zones
sampled was documented for the seven years (longest period
available) preceding sampling (Appendix 1). This information,
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obtained from a governmental georeferenced database (developed
by la Financière agricole du Québec), represented a gradient of
agricultural intensification from 0% (only hay meadows) to 100%
(only annual crops).
2.3. Statistical analysis
The effect of agricultural intensification on trees planted in riparian
zones was assessed based on two components of establishment
success. First, the effects of annual crop frequency on tree survival
was evaluated using a generalized linear model with a binomial
distribution accounting for overdispersion and including the year
of planting as a covariate (to account for any factor associated to
the specific year of planting, such as climatic variation). The six
most frequently planted tree species (776 individuals among the
923 measured), i.e. Fraxinus pennsylvanica Marsh., Acer
saccharum Marsh., Picea glauca (Moench) Voss, Larix laricina
(Du Roi) K. Koch, Quercus rubra L. and Quercus macrocarpa
Michx. (Appendix 2), were secondly selected to assess the effect of
annual crop frequency on tree size. A Principal Component
Analysis (PCA) was performed on the three size measurements
(height, DBH, crown width) whose tree scores along the first PCA
axis corresponded to a tree size index.This size index was then
used as a response variable in a linear mixed model including
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annual crop frequency and tree species as fixed explanatory
variables, age of tree planting as covariate, and site as a random
variable. Significance of slopes was assessed using contrasts with a
Bonferroni correction (α = 0.05/6 = 0.0083). The marginal R2
(variance explained by fixed factors) of these models were then
calculated. All analyses were conducted on R version 3.1.0 (R
Core Team, 2014) using the nlme (Pinheiro et al., 2014) and vegan
(Oksanen et al., 2013) packages.
3. Results
3.1. Tree survival along the gradient of agricultural intensification
Tree survival was largely influenced by agricultural intensification
(deviance = 3.30; P < 0.0001), which accounted for 35% of tree
survival (explained deviance; Fig.1). As the frequency of annual
crop increased, tree survival decreased from 85% on average in
riparian zones adjacent to fields only cultivated with hay meadows
to 60% in riparian zones adjacent to fields only cultivated with
annual crops. The year of tree planting used as a covariate did not
significantly influence tree survival (deviance = 1.96; P = 0.2945),
showing that tree survival was independent of between-year
variation related to uncontrolled variables such as climate.
3.2. Representativeness of the tree size index
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The three size parameters were well represented by the first axis of
the PCA which accounted for 88% of the variation in tree size. All
tree size measurements were highly positively correlated with tree
scores along the first PCA axis, as correlation coefficients
amounted to 92% for DBH, 89% for crown width and 84% for
height. Trees with high scores thus corresponded to tall, large trees
with wide stems. These scores (ranking from -0.30 to 1.06) were
then used as a size index representative of tree size.
3.3. Tree size index along the gradient of agricultural
intensification
Age of tree planting, tree species and frequency of annual crops
explained 46% of the tree size index (marginal R2). Unsurprisingly,
age of planting positively influenced tree size (Table 1): older trees
were taller and larger than younger trees. However, after taking
age into account as a covariate, frequency of annual crops affected
tree size, but differently for each species (Table 1). While the size
of Acer saccharum, Larix laricina and Quercus rubra remained
constant with the frequency of annual crops (α = 0.0083), Fraxinus
pennsylvanica, Quercus macrocarpa and Picea glauca showed
reduced size in riparian zones adjacent to fields more frequently
cultivated with annual crops (Fig. 2).
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4. Discussion
While tree planting is an efficient approach to restore both
ecological services and species diversity in agricultural riparian
zones (Lee et al., 2000, 2003; Marshall and Moonen, 2002), our
study showed that the establishment success of planted trees was
reduced by agricultural intensification. Tree survival decreased by
25% along the intensification gradient. Some species were also
significantly smaller when growing along fields cultivated with
annual crops, after accounting for the effect of tree age, indicating
that tree tolerance to agricultural intensification is species-specific.
To our knowledge, no previous study has determined the response
of planted trees to agricultural intensification in an agroforestry
context. We acknowledge that our study remains observational, but
the trends detected are among the first evidence of the detrimental
effect of agricultural intensification on the establishment of planted
trees in agricultural riparian buffer zones. These findings can be
related to similar results observed in spontaneous (non-planted)
plant communities (Tilman et al., 2001; Allan, 2004). After land
use intensification, generalist, ruderal and exotic species replace
specialist species, leading to the biotic homogenization of
spontaneous plant communities (Mensing et al., 1998; Vellend et
al., 2007). Consequently, plant communities of riparian forests and
field margins within intensive agricultural landscapes are generally
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characterized by reduced species richness and diversity (Mensing
et al., 1998; Marshall and Moonen, 2002; Boutin et al., 2003). In
our study, such an indirect causal pathway between intensive
agricultural practices and the survival and growth of trees
exemplify the complexity of interactions that occur in agroforestry
systems. Our findings imply that favouring low intensity farming
during the tree establishment phase may help to hasten the
recovery of ecological services in riparian buffer zones (Mize et
al., 2008).
The effect of agricultural intensification on adjacent riparian plant
communities can be related to several specific agricultural pratices
characterizing annual crop cultivation that are less essential to hay
meadow cultivation. Among them, high fertilizer and pesticide
inputs, deep and regular ploughing, heavy winter runoff and short
crop rotation cycles have previously been identified as factors
explaining the environmental degradations associated with
intensive agricultural systems (Tilman et al., 2001; Allan, 2004;
Tscharntke et al., 2005). First, foliage-active herbicides used on
annual crops can reduce the survival and growth of trees planted in
riparian buffer zones more than for herbaceous species generally
characterized by high regenerative ability. The drift of herbicides
from agricultural fields, especially glyphosate, has been proven to
lead to the mortality of trees in adjacent habitats (Radosevich et al.,
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1980; D’Anieri et al., 1990) and to reduce the diversity,
productivity and integrity of spontaneous plant communities within
agricultural landscapes (Marrs et al., 1989; Skinner et al., 1997).
Second, the high inputs of fertilizers associated with annual crops
are also likely to reduce the establishment of planted trees in
riparian buffer zones through weed competition. The leaching of
fertilizers might indeed intensify competition by favouring fast-
growing species such as weeds more than slow-growing trees,
especially if trees have already been weaken by herbicide drifts. As
soil fertility drive competition among plant communities (Fynn et
al., 2005; Niu et al., 2008), leaching agricultural fertilizers could
foster nitrophilous herbaceous species in riparian zones, thereby
outcompeting planted trees. The species-specific response of tree
growth to agricultural intensification observed in this study can
also be related to differing sensitivity among tree species to
pesticide drift or fertilizer leaching (Radosevich et al., 1980;
D’Anieri et al., 1990) as well as differing competitive ability in
regard to herbaceous species (Cogliastro et al. 1990, 1997; Burton
and Bazzaz, 1995). For example, Quercus rubra is more resistant
to glyphosate application than Quercus macrocarpa in terms of
survival and height (Cogliastro et al. 1990). Moreover, it has been
determined that Fraxinus pennsylvanica grows better on humid
soils, while Acer saccharum grows taller on well drained nutrient-
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rich soils, and Quercus rubra on nutrient-poor soils (Cogliastro et
al. 1997). Draining intensively-cultivated fields is a third factor
that could explain low tree survival, because it fosters summer
water deficits (Davis et al., 1999; Coll et al., 2003). As livestock
was kept in indoor farming facilities across the study area,
herbivory or soil trampling might not have impacted planted trees.
Future controlled experiments are therefore required to disentangle
between pesticide, fertilizer, competition or water deficit impacts
on trees. Finally, the fact that intensive and extensive agricultural
systems generally occupy different physical environments could
have affected tree establishment success. However, intensive
agriculture generally extends on the most suitable areas for crop
cultivation in terms of physical environment (Long et al., 2014),
which should also favor tree survival and growth. As the opposite
result was observed here, this rather supports that agricultural
practices of intensive systems are detrimental to the establishment
success of planted trees.
Tree planting success in riparian buffer zones can be improved by
different strategies. Implementing extensive agricultural practices,
such as cultivating hay meadows, should be more widely
encouraged adjacent to riparian buffer zones, at least in the initial
years following planting. Implanting a zone of unmanaged native
trees nearest the stream followed by a zone of managed
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commercial trees and further accompanied by a zone of native
grasses and forbs or non-native cool-season grasses is also advised
to mitigate agriculture impacts on planted native trees and water
quality (Schultz et al. 2004). Identifying the tolerance of tree
species to agricultural intensification should also help to define the
tree species most suited for planting in agri-environmental
schemes. For example, Acer saccharum should ideally be planted
in riparian buffer zones of the studied area. Planting plurispecific
rather than monospecific stands remains preferable, however, for
ensuring sufficient resilience of plantations to environmental
changes and exotic pathogens (Paquette and Messier, 2010) such
as the emerald ash borer, Agrilus planipennis Fairmaire, which has
become the most destructive forest insect to ever invade North
America (Herms and McCullough, 2014). The ability of riparian
buffer zones to provide ecological services depends not only on the
presence of arborescent species, but also on the buffer zone’s
dimensions (Mander et al., 1997; Syversen, 2005). Enlarging tree-
planted riparian buffer zones to more than 3 m wide could help
accelerate the recovery of associated ecological services such as
water filtration. To improve the efficiency of current agri-
environmental schemes in mitigating environmental degradation
caused by modern agriculture, determining their response to
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agricultural intensification deserves further attention in future
research.
Acknowledgements
The authors would like to thank local stakeholders from the
Organisme de Bassin Versant de la Côte-du-Sud and Club conseil
Bélair-Morency for their help in sampling design (François Lajoie
and Lisette Beaulieu), field assistants for data collection
(Annabelle Rablat, Philippe Israël-Morin and Mathieu
Vaillancourt), Hélène Crépeau for statistical advice, Karen Grislis
for English revision, each of the farmers who allowed us to
conduct tree measurements on their land, and the two anonymous
reviewers. This project was funded by a research grant from the
Ministère de l’Agriculture, des Pêcheries et de l’Alimentation du
Québec received by A.V. and M.P. and by a NSERC discovery
grant to M.P. (RGPIN-2014-05663).
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Table captions
Table 1. Effect of age of tree planting and cultivation frequency of
annual crops on the size index of the six most frequently planted
tree species, obtained by a linear mixed model. The cultivation
frequency of annual crops was quantified in the agricultural field
adjacent to riparian buffer zones over the seven years prior to
sampling. The size index corresponded to the scores of tree
individuals along the first axis of a PCA based on the three size
measurements (diameter at breast height, crown width and height)
and accounted for 88% of the variation.
Figure captions
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Fig 1. Survival of trees planted in riparian buffer zones as a
function of agriculture intensification. Agriculture intensification
was quantified as the cultivation frequency of annual crops in the
adjacent field over the seven years prior to sampling. The
explained deviance of the generalized linear model was 35%. The
frequency of annual crops was used as an explanatory variable, and
the year of tree planting as a co-variable.
Fig 2. Evolution of the size index for the six most frequently
planted tree species in riparian buffer zones as a function of
cultivation frequency of annual crops, obtained by a linear mixed
model (dotted lines corresponds to 95% confidence interval). The
cultivation frequency of annual crops was quantified in the
agricultural field adjacent to riparian buffer zones over the seven
years prior to sampling. The size index corresponded to the score
of tree individuals along the first axis of a PCA based on the three
size measurements (diameter at breast height, crown width and
height), and accounted for 88% of the variation: high index
correspond to tall trees with large crown and high diameter at
breast height. Frequency of annual crops and tree species were
used as explanatory variables, and age of tree planting as a co-
variable. The R2 of the mixed linear model was 46%. Significant p-
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values are indicated in bold. This slope significance was assessed
using contrasts with a Bonferroni correction (α = 0.0083).
Appendix caption
Appendix 1. Type of crop rotation in the agricultural fields
adjacent to the sampled tree-planted riparian zones during the
seven years prior sampling. These data were used to calculate the
cultivation frequency of annual crops.
Appendix 2. Planting frequency of the 923 trees measured in the
68 riparian buffer zones sampled. Only species above 1%
frequency are shown.
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Table 1
Figure 1
Figure 2
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Appendix 1
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Appendix 2
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