Plant Soil
https://doi.org/10.1007/s11104-017-3483-7
REGULAR ARTICLE
Biocrust tissue traits as potential indicators of global
change in the Mediterranean
Laura Concostrina-Zubiri & Paula Matos &
Paolo Giordani & Cristina Branquinho
Received: 8 April 2017 / Accepted: 31 October 2017
# Springer International Publishing AG, part of Springer Nature 2017
Abstract
Background and aims Functional traits are promising
indicators of global changes and ecosystem processes.
Trait responses to environmental conditions have been
examined widely in vascular plants. In contrast, few
studies have focused on soil lichens and mosses com-
posing biocrusts. We aimed to evaluate the potential of
biocrust tissue traits as indicators of changes in climate
and soil properties.
Methods Isotope ratios and nutrient content in biocrust
tissue were analyzed in 13 Mediterranean shrublands
along an aridity gradient. Differences in tissue traits
Responsible Editor: Matthew A. Bowker
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11104-017-3483-7) contains
supplementary material, which is available to authorized users.
L. Concostrina-Zubiri (*) : P. Matos : C. Branquinho
Centre for Ecology, Evolution and Environmental Changes
(cE3c), Faculdade de Ciências, Universidade de Lisboa, Campo
Grande, 1749-016 Lisbon, Portugal
e-mail: lczubiri@fc.ul.pt
P. Matos
e-mail: psmatos@fc.ul.pt
C. Branquinho
e-mail: cmbranquinho@fc.ul.pt
P. Giordani
Dipartimento di Farmacia, Università degli Studi di Genova, Viale
Cembrano 4, 16148 Genova, Italy
e-mail: giordani@difar.unige.it
between biocrust groups (lichens and mosses), and re-
lationships between tissue traits and climatic and soil
variables were examined.
Results Lichens and mosses differed in δ13C, δ15N and
N content, indicating distinct physical and physiological
attributes. Tissue traits correlated strongly with numer-
ous climatic variables, likely due to a modulator effect
on biocrust water relations and metabolism. We found
contrasting responses of lichen and moss traits to cli-
mate, although they responded similarly to soil proper-
ties. Overall, the most responsive trait was δ15N, sug-
gesting this trait is the best to reflect integrated processes
occurring in the atmosphere and soil.
Conclusions Biocrust tissue traits arise as cost-effective,
integrative ecological indicators of global change
drivers in Mediterranean ecosystems, with potential ap-
plications in response-effect trait frameworks.
Keywords Isotope ratios . Tissue nutrient content .
Biocrusts . Climate . Soil . Mediterranean
Introduction
Functional traits are measurable characteristics of an
organism with a demonstrable link to its function
(Lavorel et al. 1997). Recently, functional traits are
experiencing increased attention in ecological research
as indicators of global changes (response traits) and
because of their link to ecosystem processes (effect
traits). On one hand, traits respond to environmental
variables at multiple scales and contexts. Changes in

climate, land-use, pollution and disturbance regimes or
nutrient availability can reshape functional community
composition in terms of growth form, chemical compo-
sition, symbiotic relations and physiological attributes
among other traits (e.g., Concostrina-Zubiri et al. 2014b;
Cornwell and Ackerly 2009; van den Driessche 1979;
Ellis and Coppins 2006; Furr et al. 1979; Huckabee and
Janzen 1975; Matos et al. 2015; Pinho et al. 2012;
Verheyen et al. 2003). In turn, shifts in these functional
attributes will have an effect on the ecosystems process-
es with which they are directly or indirectly related
(Dı́az and Cabido 2001; Tilman et al. 1997). Further-
more, there are traits that reflect environmental variabil-
ity and at the same time exert an effect on ecosystem
properties. This, evidences the complex feedbacks be-
tween the environment and ecosystem structure and
functioning (Suding et al. 2008). For instance, soil or-
ganic matter (environmental factor) determines leaf nu-
trient content (trait), which in turn determines decom-
position rates (ecosystem process).
For plants, leaf traits (e.g., stable isotope composition,
nutrient content) are a promising area of ecological re-
search due to their sensitivity to abiotic conditions such
as nutrient, water or light availability (as Bresponse
traits^), their link to ecosystem processes such as primary
productivity or nutrient cycling (as Beffect traits^) and
their relatively ease of measurement (Cornelissen et al.
2003). In particular, the C isotope ratio (δ13C) is strongly
related to climate, since it reflects the discrimination of
the heavier 13C in favor of 12C in diffusion and enzymatic
processes during photosynthesis, which are mainly con-
trolled by temperature and water availability (Marshall
et al. 2007). Hence, δ13C is typically higher in dry
environments while lower values are found in the tropics
(Kohn 2010). The N isotope ratio can be used as a tracer
of N sources and availability and mechanisms of uptake
(Dawson et al. 2002), reflecting ecological and anthro-
pogenic processes occurring in the atmosphere and soil.
For instance, foliar N isotope ratio close to zero indicates
atmospheric N2 fixation (i.e., N fixer plants), while more
positive and more negative values suggest NO3− (i.e.,
after nitrification) and NH3 (i.e., after volatilization) as N
sources, respectively, due to fractionation. Furthermore,
N isotope ratio reflects environmental conditions since
temperature and water availability greatly influences N
related processes (Amundson et al. 2003; Craine et al.
2009); e.g., increased nitrification under warmer condi-
tions. On the other hand, leaf nutrient content not only
reflects the physiological response of plants to the
Plant Soil
environment; e.g., nutrient uptake, but also determines
its effects on decomposition rates and nutrient recycling
(Aerts and Chapin 1999). However, most of the work on
leaf traits focuses on vascular plants and in a context of
land-use change (Cornelissen et al. 2007; Hevia et al.
2017). In contrast, Bleaf^ traits of biocrusts (i.e., soil
lichens and mosses) have received much less attention,
although they are also highly sensitive to environmental
changes (Bowker et al. 2006; Concostrina-Zubiri et al.
2014a, b; Ellis and Coppins 2010; Giordani et al. 2012,
2014; Matos et al. 2015; Nimis et al. 2002; Pinho et al.
2009; Pinho et al. 2011, 2014) and play an important role
in C and N cycles at the global scale (Elbert et al. 2012;
Porada et al. 2013).
In drylands, ecosystem structure is generally charac-
terized by scattered vascular plant cover due to water
limitation. However, less water demanding organisms
such as mosses and lichens, and other soil organisms,
collectively known as biological soil crusts or biocrusts,
find in open microsites a suitable habitat for establish-
ment and development. Biocrusts can almost complete-
ly cover vascular plant interspaces and play an important
role in soil-atmosphere dynamics at the small scale by
fixing carbon and nitrogen, protecting soil surface from
erosion, and modulating soil water and temperature
(Bowker et al. 2008; Chamizo et al. 2016;
Concostrina-Zubiri et al. 2016; Elbert et al. 2012).
In general, mosses and lichens are the most conspic-
uous components of biocrusts in the Mediterranean
(Concostrina-Zubiri et al. 2014a, b; Maestre et al.
2011), accounting also for most of above-ground
biocrust biomass. The poikilohydric nature of lichens
and mosses makes them suitable organisms to be used
as ecological indicators (Branquinho et al. 2015; Nimis
et al. 2002), especially in relation to climate (Bowker
et al. 2006; Concostrina-Zubiri et al. 2014b; Giordani
et al. 2012; Matos et al. 2015). Unlike vascular plants,
lichens and mosses lack leaf cuticle or stomata, so they
cannot actively regulate their water content. In dry-
lands, lichens and mosses do have certain structural
characteristics and mechanisms to deal with drying-
rehydration cycles (e.g., the capacity to curve-unfold,
altered metabolism, the presence of recurved margins
and hairs in mosses, porous surfaces in lichens) (Cruz
de Carvalho et al. 2014; Kappen and Valladares 2007)
but their water content is generally in equilibrium with
the environment. Thus, the physiology of lichens and
mosses is greatly determined by water availability,
temperature and light conditions. These attributes
Plant Soil
allow lichens and mosses to trace changes in environ-
mental conditions with high and fast sensitivity; e.g.,
early warning indicators of changes in relative humid-
ity (Matos 2016). Also, lichens and mosses can be
either long-lived organisms or present short-term life
cycles, respectively, offering information on environ-
mental conditions at varying temporal scales depend-
ing on the research question. Indeed, they have been
used as predictors of global change drivers (e.g., pol-
lution) since the early 30s (e.g., Potzger 1939).
To date, biocrusts have shown to be good indicators
of soil type, land-use change and disturbance at the
landscape level as well (Bowker and Belnap 2008;
Löbel et al. 2006; Read et al. 2008, 2014). However,
this research has been mostly restricted to morphology-
related functional traits, which are easily measurable
and linked to ecological properties and processes; e.g.,
ecosystem health and resilience, erosion rates and water
relations (Eldridge and Rosentreter 1999). More recent
studies have measured directly and quantitatively mea-
sured other functional traits such as biomass, water
absorption and retention capacity, sediment capture, soil
stabilization and temperature buffering, and various
chemical traits; e.g., secondary metabolites or enzyme
activity (e.g., Asplund and Wardle 2013; Bohuslavová
et al. 2012; Concostrina-Zubiri et al. 2016; Delgado-
Baquerizo et al. 2015; Mallen-Cooper and Eldridge
2016; Michel et al. 2013; Ochoa-Hueso and Manrique
2011; Pietrasiak et al. 2013; Rosso et al. 2014; Xiao
et al. 2016). Still, biocrusts comprise a wider range of
functional traits largely unexplored (Cornelissen et al.
2007); e.g., acidifying potential, allelopathic relations or
tissue composition. In particular, biocrust tissue traits
arise as promising response-effect traits since they are
relatively easy to measure and provide useful informa-
tion related to environmental changes at multiple scales
(e.g., biocrusts respond to climate but also to soil prop-
erties) and the effects of biocrusts in ecosystem process-
es, such as their role in nutrient cycling (Asplund and
Wardle 2014, 2015; Cornelissen et al. 2007). Indeed,
recent research has shown promising results for isotope
and nutrient composition in biocrust-mosses as indica-
tors of changes in atmospheric pollution in drylands
(Delgado et al. 2013; Izquieta-Rojano et al. 2016).
In this study, we aim to assess the suitability of
biocrust tissue traits (as Bresponse traits^) as ecological
indicators of global change drivers as they can reflect
changes in environmental variables related to water
availability, temperature and soil biogeochemistry. In
particular, we propose the existence of a strong relation-
ship between climate and soil properties and biocrust
tissue traits, namely C and N isotope ratio and C and N
content. If biocrust tissue traits are related to climate and
soil properties, they could be used to predict changes in
global change drivers such as climate change and eco-
system processes occurring in the soil (e.g., N cycling).
Specifically, we focused on soil lichens and mosses due
to their interest as ecological indicators in drylands
because i) they are common and abundant ecosystem
components, even in the most extreme conditions where
vascular plants can be scarce or absent (e.g., deserts,
boreal ecosystems), ii) they are easy to collect and
process, iii) they are long-lived organisms; which allows
to record environmental changes from years to decades,
and iv) their perennial nature; most of them are present
and ready to be active all year round.
We hypothesized that biocrust tissue traits reflect
differences in climate and soil properties as a result of
physiological responses to water and nutrient sources
and availability (Craine et al. 2009; Farquhar et al. 1989;
Lakatos et al. 2007). We expected mosses and lichens to
show different responses, likely due to different mor-
phological and physiological characteristics (e.g., differ-
ent water relations; Concostrina-Zubiri et al. 2016;
Larson 1981; Mallen-Cooper and Eldridge 2016) and
differential carbon and nitrogen metabolism (Lakatos
et al. 2007; Teeri 1981).
To test our hypotheses, we examined biocrust tissue
traits along an environmental gradient defined by a set
of climatic variables based on temperature and precipi-
tation, and soil properties in Mediterranean shrublands
of Italy and Portugal. We used a space-for-time ap-
proach and assumed that if shifts in biocrust tissue traits
and environmental variables are coupled, tissue traits
could be used as indicators of environmental conditions
in the future.
Materials and methods
Study sites
We pre-selected 10 sites in Sardegna, Italy, and 10 sites
in Southern Portugal along aridity gradients ranging
from the semi-arid to the dry sub-humid. These two
regions, although geographically distant, allowed us to
build an aridity gradient without any major gap in typ-
ical Mediterranean shrublands (e.g., Cistus, Pistacea).
 Plant Soil

37°29′58.08″N, 8°14′15.69″W
38° 9′15.10″N, 7°17′30.04″W

39°40′27.24″N, 8°51′55.19″E
39°39′53.89″N, 9°20′27.61″E

39°56′48.30″N, 8°47′42.90″E
40°32′14.93″N, 8°19′29.10″E
39° 1′13.02″N, 8°26′47.65″E
We used aridity to define the gradient as this is an

39°10′51.70″N, 9°9′35.09″E

39°29′34.52″N, 9°11′7.59″E
38° 6′9.32″N, 7°2′29.23″W
39°25′17.46″N, 9°7′6.36″E

40°36′24.85″N, 8°9′5.13″E

39°47′4.26″N, 9°9′59.70″E
integrated measure of several climatic features; i.e.,
aridity index: the ratio of mean annual precipitation
and mean annual potential evapotranspiration. Aridity
index ranged from 0.40 to 0.67 in Italy and from 0.42 to

Coordinates
0.52 in Portugal (http://www.cgiar-csi.org). From this
pre-selection, sites where biocrusts were well developed
and light or no disturbance had occurred in the last two
decades were selected to build the final environmental

Pleurochaete squarrosa,

Ptychostomum capillare
Pleurochaete squarrosa

Pleurochaete squarrosa
Pleurochaete squarrosa

Pleurochaete squarrosa
Pleurochaete squarrosa
Pleurochaete squarrosa
gradient of interest. We kept a total of 13 sites out of 20;

Trichostomum spp.

Trichostomum spp.

Trichostomum spp.

Trichostomum spp.
Trichostomum
ten in Italy and three in Portugal, and the composite

Pottiaceae sp.
gradient comprised a wide range of climatic variables
and soil properties (Table 1). Sampling was conducted

Mosses
in Portugal and Italy in Spring and Autumn of 2014,
respectively.

Cladonia aff. rangiformis

Climate variables

Cladonia foliacea

Cladonia foliacea

Cladonia foliacea
Table 1 Summary of environmental characteristics of the study sites and lichen and moss species collected at each site
Each sampling site was characterized by set of 19 stan-

Cladonia sp.
Cladonia sp.
dardized bioclimatic variables obtained from
WorldClim global database (www.worldclim.org, for Lichens
more details on variables with codes Bio 1 to Bio 19;
Hijmans et al. 2005), representing the mean, range and

Calcaric Cambisols-Regosols-Leptosols
seasonal variation of temperature and precipitation.

Calcic Luvisols; Humic Regosols

These climate data correspond to average monthly
climate data from 1960 to 1990 interpolated on a 30
arc-second resolution grid. The global aridity index was
also extracted and included in the set (http://www.cgiar-
Leptic Chernozems

Leptic Chernozems

Leptic Chernozems

Calcaric Regosols
Luvic Phaeozems
csi.org/data/climate/item/51-global-aridity-and-pet-

Arenic Fluvisols
Luvic Litosols

database; Zomer et al. 2008). This data is available at the

Dolomitic
Soil type

same spatial resolution as precipitation and temperature

Arenitic

Litosols

Litosols

variables, and was modelled using the available data

from WorldClim for the same time interval.
8.21

6.33
5.84

6.99
6.32

6.52

7.55
7.34

8.05
6.48

6.69
7.26
6.72
pH

Soil sampling and analyses

K (mg/Kg)

We measured organic carbon (OC), available phospho-

427.20

125.20
135.00

454.20
191.40

239.60

341.80
532.60

318.80
108.20

242.20
281.80
225.40

rus and potassium, and pH to characterize soil properties

at each study site. To do so, we randomly collected 5 soil
P (mg/Kg)

samples of the top 1–5 cm (depending on soil depth) of

soil apparently devoid of biocrusts; i.e., no lichens or
65.32

10.73
35.99

16.67

9.02
9.99

4.04
3.41

7.57
3.16
6.88

9.70
1.36

mosses were present, although we cannot discard the

presence of soil cyanobacteria invisible to the human
OC (%)

eye. All soil samples were passed through a 2-mm mesh

2.79

1.31

2.17
2.51

2.75
2.67

1.76

2.13

5.68
1.15
1.27

1.93
1.66

sieve to remove roots and rocks. Soil OC was deter-

mined with the oxidation method (Combs and Nathan
0.40

0.44

0.51

0.62

0.67
0.56

0.64
0.42

0.45

0.58
0.60
0.52

0.61
AI

1998). Soil pH was determined by dissolving 10 g of

soil in 25 ml of deionized water. Available P and K were
IT10
PT1

PT3

PT9
Site

IT1

IT2

IT8
IT3

IT4

IT9
IT5
IT6
IT7

determined using an acetic acid and ammonium lactate

Plant Soil
solution of pH = 3.5. Total nitrogen content was deter-
mined with the Kjeldahl method (Bremner 1996). Soil
analyses were performed at Escola Superior Agrária de
Bragança, Bragança, Portugal.
Biocrust sampling and tissue analyses
To measure tissue traits at each site, we collected 5
samples of lichen and/or moss species meeting the fol-
lowing criteria: i) being common along the gradient and/
or most abundant at each site, and ii) with a minimum
surface of 3 cm × 3 cm. We found that biocrusts were
usually dominated by mosses, so we collected mosses in
every site of the gradient. In contrast, lichens were
important biocrust components only in certain Italian
sites (Table 1). For lichens, the genus Cladonia domi-
nated along the gradient, but we observed a replacement
in the dominant species as follows: C. foliacea
(N = 15) > C. aff. rangiformis (N = 5) > Cladonia sp.
(N = 10); a crustose Cladonia with only primary thallus
present. For mosses, Pleurochaete squarrosa (N = 30)
was a common and abundant species along the gradient.
We also found patches dominated by Trichostomum
spp. (i.e., T. brachydontium and T. crispulum) in both
regions (N = 15), Ptychostomum capillare (N = 5) in site
IT7 and one Pottiaceae species (N = 5), in site PT9. In
site PT3, we collected both P. squarrosa and
Trichostomum spp. dominated patches (N = 5, Table 1).
A total of 30 samples of lichens and 65 of mosses were
collected for the whole gradient. Samples were collected
within a 0.25 Ha area from site centroid (Table 1). The
biocrust layer was carefully removed from the soil beneath,
and any soil particles and external debris were eliminated
from lichen and moss samples. Afterwards, the clean tissue
was oven dried (48 h at 60°) and ground in an automatic
mill (Retsch 400). For lichens, the whole thallus was
ground. For mosses, we included the green part of the
stem only. In both cases, reproductive structures were
absent. The resulting powder was analyzed in an elemental
analyzer (EURO VECTOR) coupled with a continuous
flow stable isotope ratio mass spectrometer (SIRA II, VG
ISOGAS for C and ISOPRIME, MICROMASS for N).
Isotope ratios are given in the notation δ calculated as δ =
(Rsample/Rstandard – 1) × 1000 (‰). International standards
IAEA CH6 (sucrose) and IAEA CH7 (polyethylene) and
IAEA N1 (ammonium sulphate) were used to calibrate C
and N isotope ratios. We report δ13C values standardized
against Vienna Pee Dee Belemnite and δ15N relative to
δ15N of atmospheric air. All tissue analyses were
conducted at the Stable Isotopes and Instrumental Analysis
Facility – Universidade de Lisboa, Lisboa, Portugal.
Data analysis
To examine if environmental gradients (i.e., climate
and soil properties) and a particular combination of
biocrust traits (i.e., δ13C, δ15N, and C and N content)
were related, and visually detect differences between
groups, we conducted a non-metric multidimensional
scaling (NMDS) ordination. Tissue traits were relativ-
ized by trait maximum due to differences in measure-
ment units and to equalize variable weights (McCune
et al. 2002). Then, we used the Bray-Curtis distance,
as it is more effective for heterogeneous data (McCune
et al. 2002) to run the NMDS. The best solution (i.e.,
lowest final stress) was found for a 3-dimensional
NMDS after 500 runs, each starting randomly (500
iterations per run), and evaluated with a Monte Carlo
test (250 runs with randomized data). Coefficients of
determination (r2) between original plot distances and
distances in the final ordination solution were calcu-
lated to assess how much of the biocrust tissue trait
variability was represented by the NMDS axes
(McCune et al. 2002). Climate and soil variables were
overlaid on the NMDS ordination as correlation vec-
tors (McCune et al. 2002). We also included the dis-
tance to the nearest coast as an environmental variable
to detect potential variation in the data explained by
the proximity to the sea (e.g., differences in tissue
traits due to increased salinity). Individual correlations
between these variables and NMDS site scores were
determined using Spearman’s rank correlations coeffi-
cients to account for potential nonlinearity in the rela-
tionships (correlations were considered significant for
P < 0.05). To test for significant grouping of biocrust
groups (i.e., lichens vs. mosses) according to trait
composition we used the Multi-response permutation
(MRPP) procedure, with groups considered signifi-
cantly different if P < 0.05. When significant grouping
was found, a post-hoc Mann–Whitney U test was
performed to determine differences in the individual
tissue traits between lichens and mosses. Mann–Whit-
ney U test was appropriate to our data due to non-
normality and small sample size.
To evaluate the relationships between individual tis-
sue traits and environmental variables at the site level,
we calculated Spearman’s rank correlation coefficients
for mosses and lichens separately.
 Plant Soil

Multivariate analyses (NMDS and MRPP) were per-
formed with PC-ORD version 6.08 (McCune and
Mefford 2011). Mann-Whitney U test and Spearman’s
rank correlation coefficients were calculated using R (R
Core Team 2015).
Results
Tissue trait composition in biocrusts
The NMDS ordination (P = 0.0020, final stress = 7.2,
final instability <0.0001) of tissue trait composition
discriminated between lichens and mosses in the
first axis; which accounted for most of the ex-
plained variation in data structure (63% of variation
explained, Fig. 1). We found no significant correla-
tion between the first axis and the studied environ-
mental variables (P > 0.05, results not shown). The
second axis (19% of variation explained) was sig-
nificantly correlated with the aridity index and sev-
eral temperature and precipitation related variables,
as well as with soil K, OC and pH (Fig. 1, Table 2).
There was no significant correlation between dis-
tance to the nearest coast and neither axes (results
not shown for first axis, see Table 2 for second
axis), so this variable was discarded from further
analysis. Within the lichen group, most of the points
corresponding to Cladonia sp. separated clearly
from the other species (Fig. 1), particularly in the
case of site IT8. The MRPP indicated significant
grouping in tissue trait composition for lichens and
mosses (A = 0.1644, P < 0.0001).
Table 2 Spearman correlation coefficients between NMDS ordi-
nation axis 2 and environmental variables
ρ P

Aridity index 0.25 *

Annual Mean T −0.35 **
Mean Diurnal Range −0.42 ***
Isothermality −0.52 ***
T Seasonality 0.18
Max T of Warmest Month −0.40 **
Min T of Coldest Month −0.16
T Annual Range −0.15
Mean T of Wettest Quarter 0.09
Mean T of Driest Quarter −0.32 **
Mean T of Warmest Quarter −0.33 **
Mean T of Coldest Quarter −0.27 *
Annual P 0.11
P of Wettest Month 0.13
P of Driest Month 0.32 **
Fig. 1 Non-metric multidimensional scaling (NMDS) ordination P Seasonality −0.29 **
of lichen (filled symbols) and moss (open symbols) tissue trait
(δ13C, δ15N, C and N) values. Vector overlays indicate the mag- P of Wettest Quarter 0.05
nitude and direction of significant correlations between environ- P of Driest Quarter 0.28 **
mental variables and the ordination (see also Table 2): AI: Aridity P of Warmest Quarter 0.46 ***
index; AMT = Annual Mean Temperature; ISO = Isothermality
P of Coldest Quarter 0.04
(BIO2/BIO7); MDR = Mean Diurnal Range (Mean of monthly
(max temp - min temp)); MTCQ = Mean Temperature of Coldest OC 0.26
Quarter; MTDQ = Mean Temperature of Driest Quarter; P −0.10 **
MTWM = Mean Temperature of Warmest Month; MTWQ = Mean K 0.37 *
Temperature of Warmest Quarter; PDM = Precipitation of Driest
Month; PDQ = Precipitation of Driest Quarter; TWM = Max pH 0.34 **
Temperature of Warmest Month; (■) Cladonia foliacea; (●) Distance to coast −0.15
Cladonia aff. rangiformis; (▲) Cladonia sp.; (□) Ptychostomum
capillare; (○) Pottiaceae sp.; (Δ) Pleurochaete squarrosa; (◊) *P < 0.05, **P < 0.01, ***P < 0.0001
Trichostomum spp.; (x) P. squarrosa, Trichostomum spp. N = 91. Significant P-values (<0.05) are in bold
Plant Soil

Lichen and moss isotope ratios and nutrient content

Biocrust δ13C values ranged from −29.6 to −22.4‰ in

lichens and from −30.7 to −23.9‰ in mosses, while δ15N
values ranged from −8.3 to −0.5‰ in lichens and from
−5.7 to 1.5‰ in mosses (Table S1, Fig. 2). Lichen C
content ranged from 26.3 to 42.8%, while C content in
mosses ranged from 26.4 to 47.3%. The N content ranged
from 0.4 to 1.2% in lichens and from 0.6 to 1.5% in mosses
(Table S1, Fig. 2). Lichens had significantly higher δ13C
values and lower δ15N values and N content than mosses
(Mann-Whitney U test, P < 0.05, Fig. 2), while C content
was not significantly different between lichens and mosses
(Mann-Whitney U test, P > 0.05, Fig. 2).

Relationships between environmental variables

and tissue traits

As we found significant differences between lichen and

moss tissue traits, we calculated Spearman correlation
coefficients for lichens and mosses separately. Correla-
tions between individual traits and environmental vari-
ables are summarized in Table 3. In lichens, δ13C cor-
related positively with temperature diurnal range,
isothermality and summer temperature. The δ15N corre-
lated negatively with temperature seasonality, and sum-
mer temperature and precipitation. Both δ15N and N
content correlated positively with the aridity index and
other precipitation-related variables. The C content cor-
related positively with temperature seasonality and an-
nual range, and summer precipitation. This trait also
correlated negatively with winter temperature (Table 3).
Additionally, δ13C and C content correlated negatively
with soil K. The C content was also negatively correlat-
ed with soil P. Also, both δ15N and N content correlated
negatively with soil OC and pH (Table 3).
In mosses, we found a more consistent pattern in
tissue traits in relation to environmental variables. Both
δ13C and δ15N correlated positively with several
temperature-related variables and precipitation season-
ality. On the contrary, these traits correlated negatively
with the aridity index and summer precipitation. Addi-
tionally, δ15N was positively correlated with winter and
summer temperature. The C content correlated negative-
ly with annual mean temperature, winter temperature
and precipitation seasonality. In contrast, this trait was Fig. 2 Boxplots showing biocrust tissue traits by group (lichens
positively correlated with summer precipitation. The N and mosses). Boxes show the median, 25th and 75th percentiles;
content correlated positively to annual mean tempera- vertical lines show the minimum and maximum values that fall
ture, annual diurnal range and summer temperature, within 1.5 times the height of the box
 Plant Soil

Table 3 Spearman correlation coefficients between biocrust tissue traits (δ13C, δ15N, C and N) and environmental variables

Lichens Mosses

δ13C δ15N C N δ13C δ15N C N

Aridity Index −0.23 0.66*** 0.03 0.65*** −0.33** −0.42** −0.02 −0.36**
Annual Mean T 0.15 −0.09 −0.36 −0.17 0.43* 0.71** −0.32* 0.29*
Mean Diurnal Range 0.40* −0.29 0.22 −0.23 0.33* 0.36** 0.07 0.32*
Isothermality 0.49** 0.13 −0.12 −0.07 0.51*** 0.60** −0.22 0.24
T Seasonality −0.03 −0.43* 0.40* −0.18 −0.22 −0.31* 0.14 −0.11
Max T of Warmest Month 0.39* −0.39* 0.25 −0.35 0.30* 0.51*** 0.08 0.38**
Min T of Coldest Month 0.03 0.43* −0.40* 0.18 0.25 0.47*** −0.29* 0.12
T Annual Range 0.32 −0.19 0.44* −0.16 −0.02 −0.01 0.23 0.09
Mean T of Wettest Quarter 0.15 −0.09 −0.36 −0.17 −0.24 −0.16 0.06 0.18
Mean T of Driest Quarter 0.52** −0.41* 0.04 −0.50** 0.21 0.56*** −0.14 0.34**
Mean T of Warmest Quarter 0.51** −0.34 −0.02 −0.47** 0.23 0.58*** −0.14 0.35**
Mean T of Coldest Quarter 0.07 −0.04 −0.41* −0.10 0.45** 0.61*** −0.34** 0.19
Annual P −0.28 0.61** −0.02 0.63** −0.04 −0.10 −0.23 −0.34**
P of Wettest Month −0.12 0.79*** −0.13 0.58** −0.12 −0.23 −0.20 −0.32*
P of Driest Month −0.05 −0.46** 0.38* −0.28 −0.45** −0.49*** 0.38** −0.20
P Seasonality 0.03 0.64*** −0.15 0.47** 0.40** 0.39** −0.37** 0.01
P of Wettest Quarter −0.28 0.61** −0.02 0.63** 0.03 −0.02 −0.23 −0.24
P of Driest Quarter −0.15 0.09 0.36 0.17 −0.44** −0.56*** 0.35** −0.23
P of Warmest Quarter −0.12 0.05 0.38* 0.09 −0.55*** −0.55*** 0.17 −0.33**
P of Coldest Quarter −0.28 0.61** −0.02 0.63** 0.08 −0.01 −0.19 −0.29*
OC −0.16 −0.49** −0.28 −0.40* −0.15 −0.28* 0.07 −0.16
P −0.20 −0.33 −0.40* −0.19 0.22 0.07 0.01 0.23
K −0.52** −0.08 −0.48** 0.06 −0.17 −0.04 −0.22 −0.26*
pH 0.01 −0.59** −0.18 −0.54** −0.18 −0.28* −0.13 −0.20

Lichens; N = 30, Mosses; N = 61. Significant P-values (P < 0.05) are in bold
*P < 0.05, **P < 0.01, ***P < 0.0001

while it correlated negatively with the aridity index and
other precipitation-related variables (Table 3). In
mosses, only δ15N and N content correlated, negatively,
with soil variables; soil OC and pH, and soil K, respec-
tively (Table 3).
Discussion
Tissue traits differ between lichens and mosses
We found support for our first hypothesis that tissue
traits reflect differences in climate and soil properties.
However, when examining tissue traits of biocrusts
along the gradient, group identity accounted for most
of the variation in trait composition, pushing apart
mosses and lichens in the NMDS ordination. Also,
different isotope ratios and N content characterized
these two groups.
We expected differences in δ13C between lichens and
mosses due to group-specific carbon relations. Although
δ13C of both green-algae lichens and mosses fall in the
range of C3 plants, δ13C value is generally more negative
in mosses (Lakatos et al. 2007; Proctor et al. 1992; Rundel
et al. 1979; Teeri 1981, but see Lee et al. 2009). This may
be due to the existence of a CO2-concentrating mechanism
to accumulate inorganic carbon in specific cell structures of
many lichens (i.e., with Trebouxia as the photosynthetic
partner), which allows them to continue photosynthesizing
in situations of limited CO2 uptake (Lakatos et al. 2007).
Plant Soil
Lower δ13C in mosses also suggests that they are less
prone to desiccation –or dehydration is more gradual-
while lichens suffer more from hydric stress. This may
be explained by group-specific water relations. In dry-
lands, water absorption is generally higher in mosses
while lichens tend to retain water longer (Concostrina-
Zubiri et al. 2016; Mallen-Cooper and Eldridge 2016).
However, this pattern is highly dependent on specific
lichen and moss morphological attributes (Larson 1981)
and growth habits; e.g., scattered stems vs. dense
patches in mosses. In the present study, we found
mosses growing in more or less dense cushions and in
more shaded areas (i.e., close to vascular vegetation),
while lichens were usually in more open areas and
predominantly had a squamulose-foliose growth form
(i.e., Cladonia spp). This may imply that, on one hand,
mosses experience lower hydric stress and physiological
damage (Colesie et al. 2012; Cruz de Carvalho et al.
2015, 2017; Stark et al. 2013) and, on the other, that they
may incorporate a higher proportion of respired CO2
than lichens growing on more open microsites (Proctor
et al. 1992; Kohn 2010), explaining the more negative
δ13C in the mosses. Specific water relations may also
contribute to differences in δ13C, since tissue water
determines photosynthetic performance by regulating
CO2 diffusion and conductance processes (Royles
et al. 2014; Williams and Flanagan 1996).
In line with our predictions, we also found differences
in δ15N between lichens and mosses, likely related to
group-specific nitrogen sources and uptake preferences
(Bates 1992; Brown and Bates 1990; Dahlman et al.
2004). Due to the lack of active mechanisms for soil
nutrient uptake, lichens and mosses incorporate N in their
tissue predominantly from atmospheric sources; such as
NH3, NO3− and NH4+ from dry and wet deposition. How-
ever, the lower δ15N lichens may be reflecting a higher
influence of N dry deposition likely dominated by NH3
derived from cattle (δ15N values ranging from from −56 to
23‰) (Pinho et al. 2017), which is one of the main
activities in the region. In contrast, mosses seem to receive
a higher N input from NH4+ and NO3− (δ15N values
ranging from c. -10 to 10‰) found in wet deposition
(Moore 1974). Also, mosses have the capacity to passively
uptake N from soil via rhizoids (Ayres et al. 2006), while
there is no evidence that soil lichens derive significant N
quantities from the soil (Ellis et al. 2004). Thus, the typi-
cally higher δ15N in soil (δ15N values up to c. -5 to +25‰)
(Moore 1974) due to the presence of organic N forms such
as aminoacids, and the preference of Cladonia species for
inorganic N forms (i.e., Dahlman et al. 2004) such as soil
NH4+ (δ15N values up to c. 8‰) may be also contributing
to the higher δ15N in mosses. Potential associations be-
tween mosses and endophytic or epiphytic cyanobacteria
may explain higher δ15N and also higher N content as well
due to transfer of fixed atmospheric N (i.e., N2 has a δ15N
close to zero) to the host plant (Berg et al. 2013; Deane-
Coe and Sparks 2016).
Contrasting responses of lichen and moss traits
to environmental factors
Tissue traits differed between lichens and mosses, and
its response to environmental variables was more or less
similar depending on the trait and the group of variables
on focus (Fig. 3). Interestingly, δ13C in both lichens and
mosses was positively related to temperature range and
isothermality, suggesting that temperature temporal pat-
terns act as an environmental filter to biocrust tissue
traits. Also, δ13C increased with summer temperature,
so our results are in agreement with earlier studies
reporting increases in δ13C of epiphytic and saxicolous
lichens during the warmest and driest months (Riera
2005; Shomer-Ilan et al. 1979). In warmer conditions,
net carbon gain can decrease due to higher respiration
rates (Palmqvist 2000), which is likely to increase δ13C,
particularly in lichens since they contain a fungal partner
(i.e., fungal respiration). However, Deane-Coe et al.
(2015) reported no changes in moss δ13C after 5 years
of experimental warming (i.e., soil warming), suggest-
ing that δ13C response to temperature is also dependent
on the temporal scale. We found no relationship between
lichen δ13C and precipitation variables, although earlier
studies have shown δ13C decrease (Batts et al. 2004) and
increase (Cuna et al. 2007; Krouse & Herbert cited in
Batts et al. 2004) in epiphytic lichens with precipitation.
We did find a negative relationship between moss δ13C
and the aridity index and precipitation in the driest and
warmest periods. This suggests that water availability
during periods of high evaporative losses (i.e., high
temperature and solar radiation in summer) exerts a
negative effect on moss photosynthetic activity, even
when they might be less vulnerable to desiccation than
lichens, as discussed above. This may be explained by
the fact that under these conditions, mosses are photo-
synthetically active and, thus, can suffer more than
lichen which might remain inactive. Furthermore, it
has been shown that smaller but more frequent precip-
itation events resulted in a rapid mortality of desert

Fig. 3 Summary of lichen (filled symbols) and moss (open symbols) trait responses to environmental variables (temperature, precipitation
and soil properties). Only significant responses are shown
mosses due to a negative carbon balance (Reed et al.
2012). Our findings are consistent with those reported
by Matos (2016), who found that temperature, rather
than precipitation, and seasonality, rather than mean
value, was the main predictor of other functional traits
such as type of photobiont, growth form and size in
epiphytes along a wider aridity gradient in the
Mediterranean.
Similarly, δ15N responded to temperature and precip-
itation seasonality together with temperature in the
coldest periods in both groups. In mosses, more positive
δ15N has been found under increased precipitation fre-
quency and temperature (Zelikova et al. 2012), likely
resembling a shift in available N sources mediated by
changes in microbial activity. In vascular plants, δ15N
(i.e., foliar δ15N) shows a positive relationship with
temperature and a negative relationship with precipita-
tion, and this pattern is mostly explained by N availabil-
ity in soil (Amundson et al. 2003; Craine et al. 2009).
This may be partially explained by the positive effect of
higher temperature on soil nitrification rates. Thus, our
results suggest that mosses respond in a similar manner
to that of vascular plants, reflecting the δ15N of the
micro-environment where they grow (i.e., soil N
sources). By contrast, lichens might be reflecting other
local attributes or group-specific responses (e.g., ammo-
nia assimilation capacity) (Hauck 2010).
When examining tissue traits and soil properties, we
found negative relationships between lichen δ13C and C
content and soil nutrients, and between lichen δ15N and
Plant Soil
N content and soil OC and pH. Moss N content also
responded negatively to soil K. On the other hand, both
mosses and lichens showed a negative response to soil
OC and pH in δ15N values. These results suggest that
both groups similarly reflect soil nutrient content and
availability in tissue traits. Our findings partially agree
with those reported by Asplund and Wardle (2015)
showing that lichen δ13C and δ15N responded negative-
ly to soil mineral P, although this study was carried out
in boreal islands. In contrast, Delgado-Baquerizo et al.
(2015) found a positive relationship between lichen-
biocrust C and N content and soil OC in a continental
Mediterranean grassland, which they attributed to
species-specific effects exerted on soil properties.
Biocrust tissue traits as ecological indicators
in the Mediterranean
The suitability of biocrust tissue traits as indicators of
global change has not been explored largely before. To
our knowledge, only a few studies have proposed iso-
tope ratios in lichens and mosses as tracers of climate
(Cuna et al. 2007; Deane-Coe et al. 2015; Lakatos et al.
2007; Ménot and Burns 2001; Royles et al. 2014, 2016),
mostly in high latitudes. Some others have reported
changes in lichen and moss isotope ratios related to
substrate type, resources sources, nutrient availability
and symbiotic relationships (Asplund et al.
2014; Asplund and Wardle 2014, 2015; Beck and Mayr
2012; Deane-Coe and Sparks 2016; Delgado et al. 2013;
Plant Soil
Izquieta-Rojano et al. 2016; Lakatos et al. 2007; Máguas
et al. 1993; Zwolicki et al. 2016). Indeed, this is the first
work to characterize biocrust isotope ratios and nutrient
content along climate and soil gradients in drylands.
Although we cannot establish causal relationships based
on the observations presented here, our results suggest
that isotope ratios and nutrient content of biocrusts are
tightly linked to the value, range and variability of
temperature and precipitation in the Mediterranean re-
gion, as well as to soil properties.
In this study, the replacement of dominant lichen spe-
cies along the gradient limits the interpretation of our
results, since we had a low number of sites for each species
(i.e., one site for Cladonia aff. rangiformis and two sites
for Cladonia sp.). Also, the separation of Cladonia sp.
points in the ordination space suggest that this species
responds in a particularly distinct way of the other lichen
species. This may be due to its different morphology (i.e.,
crustose vs. foliose or fruticose), which may imply differ-
ent water relations and, thus, physiological and chemical
activity, or to a different nitrogen metabolism, as suggested
by its relatively higher δ15N (Table S1). Nevertheless, we
found a general trend in these lichens responding to climate
and soil properties (Fig. 4). On the other hand, the higher
sampling size of mosses (i.e., total and for most of the
studied species) and the way species were distributed along
the gradient allow us to suggest that there is a strong
relationship between moss tissue traits and climate. In
particular, lichen tissue traits seem to be potential indicators
of soil K (δ13C and C content), precipitation of wettest
month (δ15N) and aridity (N content), while moss traits
seem to reflect differences in summer precipitation (δ13C
and C content) and both mean annual and summer tem-
perature (Fig. 4). For mosses, we observed similar trends at
the group level and at the species level in the case of
P. squarrosa and Trichostomum spp. (Fig. 4). Notwith-
standing, we are aware that other potential sources of
variation which we did not evaluate in this study, such as
atmospheric N deposition, could be influencing our results.
For example, species-specific values of δ15N in mosses
have been found to be more negative when NHx forms
prevail while more positive values have been related to
NOx dominance (Bragazza et al. 2004; Delgado et al.
2013; Izquieta-Rojano et al. 2016; Solga et al. 2005).
The presence and abundance of several species (e.g.,
C. foliacea, C. coniocraea, T. crispulum; results not
shown) with low tolerance to N eutrophication (Hill et al.
2007; Nimis 2016) throughout the aridity gradient suggests
that atmospheric N input is likely to be, at least, similar
among sites. Also, due to the limitations of this work in
terms of sampling size, our findings should be interpreted
with caution. Thus, we suggest that lichen tissue traits may
be interesting as ecological indicators of processes occur-
ring in the soil, while moss tissue traits are likely to be better
indicators of both soil processes and climate change. Fur-
ther work should include a higher sampling size for biocrust
individual species along wider environmental gradients to
account for interspecific variations in tissue traits. Indeed, it
would be interesting to test if tissue traits of lichen and moss
biocrusts can track changes in climate and soil processes
regardless of species identity (i.e., responses at the group
level). Then, the character of biocrust tissue traits as eco-
logical indicators would be more universal. Also, future
research conducted under controlled experimental condi-
tions (e.g., microcosm studies) would allow us to disentan-
gle the species effect from the environmental factors effect
(e.g., N deposition, soil N sources) on tissue traits and better
understand the their role as ecological indicators of global
change (e.g., Liu et al. 2016, 2017). It would be interesting
also to identify which specific attributes (e.g., ecological,
physiological) contribute the most to make biocrusts good
indicators, and determine if biocrusts show a faster or
greater response to global change drivers than other eco-
system components such as vascular plants.
In conclusion, we demonstrated that the δ13C, δ15N and
C and N content of biocrusts responded significantly to a
wide array of climate related variables and soil properties,
highlighting their potential as ecological indicators of glob-
al change drivers. Moreover, biocrust tissue traits can be
regarded as interesting ecological tools to understand and
predict future changes in ecosystem properties and pro-
cesses in drylands: biocrusts are widespread in drylands,
and their tissue traits are cost-effective measurements
which provide an integrated information of a wide array
of environmental conditions.
In most cases, lichens and mosses responded oppositely
to climate variables. However, they showed a similar
pattern when evaluating trait responses to soil nutrients
and pH. This suggests that mosses can act as better indi-
cators of global change at regional and local scales than
lichens; i.e., they respond to differences in climate and soil,
respectively. At the biocrust level (i.e., shared responses for
lichens and mosses) only differences in soil properties
could be hypothetically inferred. This suggests that
biocrusts as a composite ecosystem component can be a
good indicator of soil processes, due to their tight link to
nutrient cycling and soil chemistry. Overall, the δ15N in
mosses was the most responsive trait to climate, suggesting
 Plant Soil

Fig. 4 Relationship between lichen (left) and moss (right) tissue rangiformis; (▲) Cladonia sp.; (□) Ptychostomum capillare; (○)
traits (mean ± SE) and most correlated environmental variables Pottiaceae sp.; (Δ) Pleurochaete squarrosa; (◊) Trichostomum
(see also Table 3). (■) Cladonia foliacea; (●) Cladonia aff. spp.; (x) P. squarrosa, Trichostomum spp
Plant Soil
that this trait integrates processes occurring in the atmo-
sphere and soil best, and thus, arises as a promising tool in
global change research in drylands. Notwithstanding, fur-
ther studies are needed to investigate causal relationships
between biocrusts and environmental factors explaining
our results. This will help to elucidate if the lower respon-
siveness that we found in lichens is due to a lower sam-
pling size and a less continuous sampling gradient. Also,
the characterization of N sources, not taken into account in
the present study, would improve the interpretation of
biocrust tissue traits response to climate and soil properties.
Since climate and soil properties may also determine the
proportion of lichens to mosses in drylands, the overall
effect of environmental variables on biocrust abundance
and tissue traits also has potential implications on ecosys-
tem functioning, due to differential biocrust inputs to de-
composition rates and nutrient cycling. Thus, biocrust
tissue traits can be considered as response-effect traits
allowing a simultaneous assessment of global change
drivers and their subsequent impact on ecosystem proper-
ties (Lavorel and Garnier 2002). This is particularly rele-
vant in a climate change scenario where temperature and
precipitation regimes are expected to be altered, especially
in drylands (IPCC 2007, 2014).
Acknowledgements LCZ was supported by a Marie Curie IEF
grant from European Commission’s FP7 (BCSES-GA 628406).
PM was supported by FCT-MEC through: project PTDC/AAG-
GLO/0045/2014. Special thanks to C. Tejada, M. Köbel, A.
Nunes, M. Lo Cascio, L. Morillas, and S. Mereu for help in the
field, T. Roovers for the assistance in the laboratory and R. Maia
for the isotope and elemental analysis. Special thanks also to
Professor M. Aleffi (Bryology Laboratory & Herbarium,
Camerino University) for his assistance on moss species identifi-
cation. We also thank P. Pinho who provided valuable comments
on the manuscript. M.A. Bowker and other anonymous reviewers
provided helpful comments and discussion that improved an ear-
lier version of the manuscript.
References
Aerts R, Chapin FS (1999) The mineral nutrition of wild plants
revisited: a re-evaluation of processes and patterns. Adv Ecol
Res 30:1–67
Amundson R, Austin AT, Schuur EA et al (2003) Global patterns
of the isotopic composition of soil and plant nitrogen. Glob
Biogeochem Cycles 17:1031
Asplund J, Wardle DA (2013) The impact of secondary com-
pounds and functional characteristics on lichen palatability
and decomposition. J Ecol 101:689–700
Asplund J, Wardle DA (2014) Within-species variability is the
main driver of community-level responses of traits of
epiphytes across a long-term chronosequence. Funct Ecol
28:1513–1522
Asplund J, Wardle DA (2015) Changes in functional traits of the
terricolous lichen Peltigera aphthosa across a retrogressive
boreal forest chronosequence. Lichenologist 47:187–195
Asplund J, Sandling A, Kardol P, Wardle DA (2014) The influence of
tree-scale and ecosystem-scale factors on epiphytic lichen com-
munities across a long-term retrogressive chronosequence. J Veg
Sci 25:1100–1111
Ayres E, Van der Wal R, Sommerkorn M, Bardgett RD (2006) Direct
uptake of soil nitrogen by mosses. Biol Lett 2:286–288
Bates JW (1992) Mineral nutrient acquisition and retention by
bryophytes. J Bryol 17:223–240
Batts JE, Calder LJ, Batts BD (2004) Utilizing stable isotope
abundances of lichens to monitor environmental change.
Chem Geol 204:345–368
Beck A, Mayr C (2012) Nitrogen and carbon isotope variability in
the green-algal lichen Xanthoria parietina and their implica-
tions on mycobiont-photobiont interactions. Ecol Evol 2:
3132–3144
Berg A, Danielsson Å, Svensson BH (2013) Transfer of fixed-N
from N2-fixing cyanobacteria associated with the moss
Sphagnum Riparium results in enhanced growth of the moss.
Plant Soil 362:271–278
Bohuslavová O, Šmilauer P, Elster J (2012) Usnea Lichen com-
munity biomass estimation on volcanic mesas, James Ross
Island, Antarctica. Polar Biol 35:1563–1572
Bowker MA, Belnap J (2008) A simple classification of soil types
as habitats of biological soil crusts on the Colorado plateau
USA. J Veg Sci 19:831–840
Bowker MA, Belnap J, Davidson DW, Goldstein H (2006)
Correlates of biological soil crust abundance across a contin-
uum of spatial scales: support for a hierarchical conceptual
model. J Appl Ecol 43:152–163
Bowker MA, Belnap J, Chaudhary VB, Johnson NC (2008)
Revisiting classic water erosion models in drylands: the
strong impact of biological soil crusts. Soil Biol Biochem
40:2309–2316
Bragazza L, Tahvanainen T, Kutnar L, Rydin H, Limpens J, Hájek
M, Iacumin P (2004) Nutritional constraints in ombrotrophic
sphagnum plants under increasing atmospheric nitrogen de-
position in Europe. New Phytol 163:609–616
Branquinho C, Matos P, Pinho P (2015) Lichens as ecological
indicators to track atmospheric changes: future challenges.
In: Lindenmayer D, Barton P, Pierson J (eds) Indicators and
surrogates of biodiversity and environmental change. CSIRO
Publishing, Melbourne, CRC Press, London, pp 77–87
Bremner JM (1996) Nitrogen-total. In: Sparks DL et al (eds)
Methods of soil analysis part 3-chemical methods. Soil sci-
ence Society of America Inc, Madison, pp 1085–1121
Brown DH, Bates JW (1990) Bryophytes and nutrient cycling. Bot
J Linn Soc 104:129–147
Chamizo S, Cantón Y, Rodríguez-Caballero E, Domingo F (2016)
Biocrusts positively affect the soil water balance in semiarid
ecosystems. Ecohydrology 9:1208–1221
Colesie C, Scheu S, Green TA, Weber B, Wirth R, Büdel B (2012)
The advantage of growing on moss: facilitative effects on
photosynthetic performance and growth in the cyanobacterial
lichen Peltigera rufescens. Oecologia 169:599–607
Combs SM, Nathan MV (1998) Soil organic matter. In: Brown JR
(ed) Recommended Chemical Soil Test Procedures for the

North Central Region. NCR Research Publication,
Columbia, pp 53–58
Concostrina-Zubiri L, Martínez I, Rabasa SG, Escudero A (2014a)
The influence of environmental factors on biological soil
crust: from a community perspective to a species level ap-
proach. J Veg Sci 25:503–513
Concostrina-Zubiri L, Pescador DS, Martínez I, Escudero A
(2014b) Climate and small scale factors determine
functional diversity shifts of biological soil crusts in
Iberian drylands. Biodivers Conserv 23:1757–1770.
https://doi.org/10.1007/s10531–014–0683-9
Concostrina-Zubiri L, Molla I, Velizarova E, Branquinho C (2016)
Grazing or Not Grazing: Implications for Ecosystem Services
Provided by Biocrusts in Mediterranean Cork Oak Woodlands.
Land Degrad Dev. https://doi.org/10.1002/ldr2573
Cornelissen JHC, Lavorel S, Garnier E, Diaz S, Buchmann N,
Gurvich DE, Pausas JG (2003) A handbook of protocols for
15tandardized and easy measurement of plant functional
traits worldwide. Aust J Bot 51:335–380
Cornelissen JH, Lang SI, Soudzilovskaia NA, During HJ (2007)
Comparative cryptogam ecology: a review of bryophyte and
lichen traits that drive biogeochemistry. Ann Bot 99:987–1001
Cornwell WK, Ackerly DD (2009) Community assembly and
shifts in plant trait distributions across an environmental
gradient in coastal California. Ecol Monogr 79:109–126
Craine JM, Elmore AJ, Aidar MP, Bustamante M, Dawson TE,
Hobbie EA, Nardoto GB (2009) Global patterns of foliar
nitrogen isotopes and their relationships with climate mycor-
rhizal fungi foliar nutrient concentrations and nitrogen avail-
ability. New Phytol 183:980–992
Cruz de Carvalho R, Bernardes da Silva A, Soares R, Almeida
AM, Coelho AV, Marques da Silva, Branquinho C (2014)
Differential proteomics of dehydration and rehydration in
bryophytes: evidence towards a common desiccation toler-
ance mechanism. Plant Cell Environ 37:1499–1515
Cruz de Carvalho R, da Silva AB, Branquinho C, da Silva JM
(2015) Influence of dehydration rate on cell sucrose and
water relations parameters in an inducible desiccation tolerant
aquatic bryophyte. Environ Exp Bot 120:18–22
Cruz de Carvalho R, Catalá M, Branquinho C, Marques da Silva J,
Barreno E (2017) Dehydration rate determines the degree of
membrane damage and desiccation tolerance in bryophytes.
Physiol Plantarum 159:277–289
Cuna S, Balas G, Hauer E (2007) Effects of natural environmental
factors on δ13C of lichens. Isot Environ Health Stud 43:95–
104
Dahlman L, Persson J, Palmqvist K, Näsholm T (2004) Organic
and inorganic nitrogen uptake in lichens. Planta 219:459–467
Dawson TE, Mambelli S, Plamboeck AH, Templer PH, KP T
(2002) Stable isotopes in plant ecology. Annu Rev Ecol
Syst 33:507–559
Deane-Coe KK, Sparks JP (2016) Cyanobacteria associations in
temperate forest bryophytes revealed by δ15N analysis. J
Torrey Bot Soc 143:50–57
Deane-Coe KK, Mauritz M, Celis G, Salmon V, Crummer KG,
Natali SM, Schuur EA (2015) Experimental warming alters
productivity and isotopic signatures of tundra mosses.
Ecosystems 18:1070–1082
Delgado V, Ederra A, Santamaría JM (2013) Nitrogen and carbon
contents and δ15N and δ13C signatures in six bryophyte species:
Plant Soil
assessment of long-term deposition changes (1980–2010) in
Spanish beech forests. Glob Chang Biol 19:2221–2228
Delgado-Baquerizo M, Gallardo A, Covelo F, Prado-Comesaña A,
Ochoa V, Maestre FT (2015) Differences in thallus chemistry
are related to species-specific effects of biocrust-forming
lichens on soil nutrients and microbial communities. Funct
Ecol 29:1087–1098
Dı́az S, Cabido M (2001) Vive la difference: plant functional
diversity matters to ecosystem processes. Trends Ecol Evol
16:646–655
Elbert W, Weber B, Burrows S, Steinkamp J, Büdel B, Andreae MO,
Pöschl U (2012) Contribution of cryptogamic covers to the
global cycles of carbon and nitrogen. Nat Geosci 5:459–462
Eldridge DJ, Rosentreter R (1999) Morphological groups: a frame-
work for monitoring microphytic crusts in arid landscapes. J
Arid Environ 41:11–25
Ellis CJ, Coppins BJ (2006) Contrasting functional traits maintain
lichen epiphyte diversity in response to climate and autogenic
succession. J Biogeogr 33:1643–1656
Ellis CJ, Coppins BJ (2010) Integrating multiple landscape scale
drivers in the lichen epiphyte response: climatic setting pol-
lution regime and woodland spatial-temporal structure.
Divers Distrib 16:43–52
Ellis CJ, Crittenden PD, Scrimgeour CM (2004) Soil as a potential
source of nitrogen for mat-forming lichens. Can J Bot 82:
145–149
Farquhar GD, Ehleringer JR, Hubick KT (1989) Carbon isotope
discrimination and photosynthesis. Annu Rev Plant Phys 40:
503–537
Furr AK, Schofield CL, Grandolfo MC, Hofstader RA,
Gutenmann WH, John LES, Lisk DJ (1979) Element content
of mosses as possible indicators of air pollution. Arch
Environ Con Tox 8:335–343
Giordani P, Brunialti G, Bacaro G, Nascimbene J (2012)
Functional traits of epiphytic lichens as potential indicators
of environmental conditions in forest ecosystems. Ecol Indic
18:413–420
Giordani P, Incerti G, Rizzi G, Rellini I, Nimis PL, Modenesi P
(2014) Functional traits of cryptogams in Mediterranean
ecosystems are driven by water light and substrate interac-
tions. J Veg Sci 25:778–792
Hauck M (2010) Ammonium and nitrate tolerance in lichens.
Environ Pollut 158:1127–1133
Hevia V, Martín-López B, Palomo S, García-Llorente M, Bello F,
González JA (2017) Trait-based approaches to analyze links
between the drivers of change and ecosystem services: syn-
thesizing existing evidence and future challenges. Ecol Evol.
https://doi.org/10.1002/ece3.2692
Hijmans RJ, Cameron SE, Parra JL, Jones PG, Jarvis A (2005)
Very high resolution interpolated climate surfaces for global
land areas. Int J Climatol 25:1965–1978
Hill MO, Preston CD, Bosanquet SDS, Roy DB (2007) Bryoatt:
Attributes Of British And Irish Mosses, Liverworts And
Hornworts. Centre for Ecology & Hydrology, Huntingdon
Huckabee JW, Janzen SA (1975) Mercury in moss: derived from
the atmosphere or from the substrate? Chemosphere 4:55–60
IPCC (2007) Climate Change 2007: Synthesis Report, pp 73
IPCC (2014) Climate Change 2014: Synthesis Report
Contribution of Working Groups I II and III to the Fifth
Assessment Report of the Intergovernmental Panel on
Climate Change
Plant Soil
Izquieta-Rojano S, Elustondo D, Ederra A, Lasheras E,
Santamaría C, Santamaría JM (2016) Pleurochaete
Squarrosa (Brid.) Lindb. As an alternative moss species for
biomonitoring surveys of heavy metal, nitrogen deposition
and δ15N signatures in a Mediterranean area. Ecol Indic 60:
1221–1228
Kappen L, Valladares F (2007) Opportunistic Growth and
Desiccation Tolerance: The Ecological Success of
Poikilohydrous Autotrophs. In: Pugnaire F, Valladares F
(eds) Functional Plant Ecology, 2nd edn. Taylor and
Francis, New York, pp 7–65
Kohn MJ (2010) Carbon isotope compositions of terrestrial C3
plants as indicators of (paleo) ecology and (paleo) climate.
Proc Natl Acad Sci 107:19691–19695
Lakatos M, Hartard B, Máguas C (2007) The stable isotopes δ13C
and δ18O of lichens can be used as tracers of microenviron-
mental carbon and water sources. Terr Ecol 1:77–92
Larson DW (1981) Differential wetting in some lichens and
mosses: the role of morphology. Bryologist 84:1–15
Lavorel S, Garnier E (2002) Predicting changes in community
composition and ecosystem functioning from plant traits:
revisiting the holy grail. Funct Ecol 16:545–556
Lavorel S, McIntyre S, Landsberg J et al (1997) Plant functional
classification: from general groups to specific groups based
on response to disturbance. Trends Ecol Evol 12:474–478
Lee YI, Lim HS, Yoon HI (2009) Carbon and nitrogen isotope
composition of vegetation on king George Island maritime.
Antarctic. Polar Biol 32:1607–1615
Liu YR, Delgado-Baquerizo M, Trivedi P, He JZ, Singh BK
(2016) Species identity of biocrust-forming lichens drives
the response of soil nitrogen cycle to altered precipitation
frequency and nitrogen amendment. Soil Biol Biochem 96:
128–136
Liu YR, Delgado-Baquerizo M, Trivedi P, He JZ, Wang JT, Singh
BK (2017) Identity of biocrust species and microbial com-
munities drive the response of soil multifunctionality to sim-
ulated global change. Soil Biol Biochem 107:208–217
Löbel S, Dengler J, Hobohm C (2006) Species richness of vascular
plants bryophytes and lichens in dry grasslands: the effects of
environment landscape structure and competition. Folia
Geobot 41:377–393
Maestre FT, Bowker MA, Cantón Y, Castillo-Monroy AP, Cortina
J, Escolar C, Martínez I (2011) Ecology and functional roles
of biological soil crusts in semi-arid ecosystems of Spain. J
Arid Environ 75:1282–1291
Mallen-Cooper M, Eldridge DJ (2016) Laboratory-based tech-
niques for assessing the functional traits of biocrusts. Plant
Soil 406:131–143
Máguas M, Griffiths H, Ehleringer J, Serodio J (1993)
Characterization of photobiont associations in lichens using
carbon isotope discrimination techniques. In: Ehleringer JR,
Hall AE, Farquhar GD (eds) Stable Isotopes and Plant Water
Relations. Academic Press, San Diego, pp 201–212
Marshall JD, Brooks JR, Lajtha K (2007) Sources of variation in the
stable isotopic composition of plants. In: Michener J, Lajtha K
(eds) Stable isotopes in ecology and environmentalnnn 2nd edn.
Blackwell, Oxford, pp 22–60
Matos P (2016) Development of ecological indicators of climate
change based on lichen functional diversity. Universidade de
Aveiro
Matos P, Pinho P, Aragon G, Martínez I, Nunes A, Soares AM,
Branquinho C (2015) Lichen traits responding to aridity. J
Ecol 103:451–458
McCune B, Mefford MJ (2011) PC-ORD multivariate analysis of
ecological data version 608. MjM Software, Gleneden Beach
McCune B, Grace JB, Urban DL (2002) Analysis of ecological
communities. MjM software design, Gleneden Beach
Ménot G, Burns SJ (2001) Carbon isotopes in ombrogenic peat
bog plants as climatic indicators: calibration from an altitu-
dinal transect in Switzerland. Org Geochem 32:233–245
Michel P, Payton IJ, Lee WG, During HJ (2013) Impact of distur-
bance on above-ground water storage capacity of bryophytes
in New Zealand indigenous tussock grassland ecosystems. N
Z J Ecol 37:114–126
Moore H (1974) Isotopic measurement of atmospheric nitrogen
compounds. Tellus 26:169–174
Nimis PL (2016) The Lichens of Italy. A Second Annotated
Catalogue. EUT, Trieste, pp 739
Nimis PL, Scheidegger C, Wolseley PA (2002) Monitoring with
lichens – monitoring lichens. Kluwer Academic Publisher,
Netherlands
Ochoa-Hueso R, Manrique E (2011) Effects of nitrogen deposition
and soil fertility on cover and physiology of Cladonia
Foliacea (Huds.) Willd., a lichen of biological soil crusts
from Mediterranean Spain. Environ Pollut 159:449–457
Palmqvist K (2000) Tansley review no 117. New Phytol 148:11–36
Pietrasiak N, Regus JU, Johansen JR, Lam D, Sachs JL, Santiago
LS (2013) Biological soil crust community types differ in key
ecological functions. Soil Biol Biochem 65:68–171
Pinho P, Branquinho C, Cruz C, Tang YS, Dias T, Rosa AP,
Máguas C, Martins-Loução MA, Sutton MA (2009)
Assessment of critical levels of atmospheric ammonia for
lichen diversity in cork-oak woodland Portugal. In: Sutton
MA, Reis S, Baker SMH (eds) Atmosheric ammonia.
Springer, Berlin, pp 109–119
Pinho P, Dias T, Cruz C, Sim Tang Y, Sutton MA, Martins-Loução
MA, Máguas C, Branquinho C (2011) Using lichen function-
al diversity to assess the effects of atmospheric ammonia in
Mediterranean woodlands. J Appl Ecol 48:1107–1116
Pinho P, Bergamini A, Carvalho P, Branquinho C, Stofer S,
Scheidegger C, Maguas C (2012) Lichen functional groups
as ecological indicators of the effects of land-use in
Mediterranean ecosystems. Ecol Indic 15:36–42
Pinho P, Llop E, Ribeiro M, Cruz C, Soares A, Pereira M,
Branquinho C (2014) Tools for determining critical levels
of atmospheric ammonia under the influence of multiple
disturbances. Environ Pollut 18:88–93
Pinho P, Barros C, Augusto S, Pereira MJ, Máguas C, Branquinho
C (2017) Using nitrogen concentration and isotopic compo-
sition in lichens to spatially assess the relative contribution of
atmospheric nitrogen sources in complex landscapes.
Environ Pollut 230:632–638
Porada P, Weber B, Elbert W, Pöschl U, Kleidon A (2013)
Estimating global carbon uptake by lichens and bryophytes
with a process-based model. Biogeosciences 10:6989–6989
Potzger JE (1939) Microclimate, evaporation stress, and epiphytic
mosses. Bryologist 42:53–61
Proctor MCF, Raven JA, Rice SK (1992) Stable carbon isotope
discrimination measurements in sphagnum and other bryo-
phytes: physiological and ecological implications. J Bryol
17:193–202

R Core Team (2015) R: A language and environment for statistical
computing. R Foundation for Statistical Computing, Vienna.
http://www.R-project.org/
Read CF, Duncan DH, Vesk PA, Elith J (2008) Biological soil
crust distribution is related to patterns of fragmentation and
land use in a dryland agricultural landscape of southern
Australia landscape. Landsc Ecol 23:1093–1105
Read CF, Duncan DH, Vesk PA, Elith J (2014) Biocrust
morphogroups provide an effective and rapid assessment tool
for drylands. J Appl Ecol 51:1740–1749
Reed SC, Coe KK, Sparks JP, Housman DC, Zelikova TJ, Belnap J
(2012) Changes to dryland rainfall result in rapid moss mortality
and altered soil fertility. Nat Clim Chang 2:752–755
Riera P (2005) δ13C and δ15N comparisons among different co-
occurring lichen species from littoral rocky substrata.
Lichenologist 37:93–95
Rosso A, Neitlich P, Smith RJ (2014) Non-destructive lichen
biomass estimation in Northwestern Alaska: a comparison
of methods. PloS ONE 9:e103739
Royles J, Horwath AB, Griffiths H (2014) Interpreting bryophyte
stable carbon isotope composition: plants as temporal and spatial
climate recorders. Geochem Geophys Geosyst 15:1462–1475
Royles J, Amesbury MJ, Roland TP, Jones GD, Convey P,
Griffiths H, Charman DJ (2016) Moss stable isotopes (car-
bon-13 oxygen-18) and testate amoebae reflect environmen-
tal inputs and microclimate along a latitudinal gradient on the
Antarctic peninsula. Oecologia 181:931–945
Rundel PW, Stichler W, Zander RH, Ziegler H (1979) Carbon and
hydrogen isotope ratios of bryophytes from arid and humid
regions. Oecologia 44:91–94
Shomer-Ilan A, Nissenbaum A, Galun M, Waisel Y (1979) Effect
of water regime on carbon isotope composition of lichens.
Plant Physiol 63:201–205
Solga A, Burkhardt J, Zechmeister HG, Frahm JP (2005) Nitrogen
content 15N natural abundance and biomass of the two
pleurocarpous mosses Pleurozium schreberi (Brid) mitt and
Scleropodium purum (Hedw) Limpr in relation to atmospher-
ic nitrogen deposition. Environ Pollut 134:465–473
Plant Soil
Stark LR, Greenwood JL, Brinda JC, Oliver MJ (2013) The desert
moss Pterygoneurum lamellatum (Pottiaceae) exhibits an
inducible ecological strategy of desiccation tolerance: effects
of rate of drying on shoot damage and regeneration. Am J Bot
100:1522–1531
Suding KN, Lavorel S, Chapin FS, Cornelissen JH, Diaz S,
Garnier E, Navas ML (2008) Scaling environmental change
through the community-level: a trait-based response-and-
effect framework for plants. Glob Chang Biol 14:1125–1140
Teeri JA (1981) Stable carbon isotope analysis of mosses and lichens
growing in xeric and moist habitats. Bryologist 84:82–84
Tilman D, Knops J, Wedin D, Reich P, Ritchie M, Siemann E
(1997) The influence of functional diversity and composition
on ecosystem processes. Science 277:1300–1302
van den Driessche R (1979) Proceedings. Forest Fertilization
Conference, University of Washington, WA, USA
Verheyen K, Honnay O, Motzkin G, Hermy M, Foster DR (2003)
Response of forest plant species to land-use change: a life-
history trait-based approach. J Ecol 91:563–577
Williams TG, Flanagan LB (1996) Effect of changes in water
content on photosynthesis transpiration and discrimination
against 13CO2 and C18O16O in Pleurozium and sphagnum.
Oecologia 108:38–46
Xiao B, Hu K, Ren T, Li B (2016) Moss-dominated biological soil
crusts significantly influence soil moisture and temperature
regimes in semiarid ecosystems. Geoderma 263:35–46
Zelikova TJ, Housman DC, Grote ED, Neher D, Belnap J (2012)
Biological soil crusts show limited response to warming but
larger response to increased precipitation frequency: implica-
tions for soil processes on the Colorado plateau. Plant Soil
355:265–282
Zomer RJ, Trabucco A, Bossio DA, van Straaten O, Verchot LV
(2008) Climate change mitigation: a spatial analysis of global
land suitability for clean development mechanism afforesta-
tion and reforestation. Agric Ecosyst Environ 126:67–80
Zwolicki A, Zmudczyńska-Skarbek K, Richard P, Stempniewicz L
(2016) Importance of marine-derived nutrients supplied by
planktivorous seabirds to high Arctic tundra plant communi-
ties. PLoS ONE 11:e0154950