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Article
Vitality and Growth of the Threatened Lichen Lobaria
pulmonaria (L.) Hoffm. in Response to Logging and
Implications for Its Conservation in Mediterranean
Oak Forests
Elisabetta Bianchi 1, Renato Benesperi 2, Giorgio Brunialti 3, Luca Di Nuzzo 2,
Zuzana Faˇckovcová4, Luisa Frati 3, Paolo Giordani 5, Juri Nascimbene 6, Sonia Ravera 7,
Chiara Vallese 6and Luca Paoli 8,*
1Department of Life Science, University of Siena, 53100 Siena, Italy; elisabetta.bianchi@unisi.it
2Department of Biology, University of Florence, 50121 Florence, Italy; renato.benesperi@unifi.it (R.B.);
luca.dinuzzo@stud.unifi.it (L.D.N.)
3TerraData environmetrics, Spin-offCompany of the University of Siena, 58025 Monterotondo Marittimo,
Italy; brunialti@terradata.it (G.B.); frati@terradata.it (L.F.)
4Plant Science and Biodiversity Centre, Slovak Academy of Sciences, 84523 Bratislava, Slovakia;
zuzana.fackovcova@savba.sk
5DIFAR, University of Genoa, 16148 Genoa, Italy; giordani@difar.unige.it
6Department of Biological, Geological and Environmental Sciences, University of Bologna, 40126 Bologna,
Italy; juri.nascimbene@unibo.it (J.N.); vallese.chiara@gmail.com (C.V.)
7
Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo,
90123 Palermo, Italy; sonia.ravera@unipa.it
8Department of Biology, University of Pisa, 56126 Pisa, Italy
*Correspondence: luca.paoli@unipi.it
Received: 7 August 2020; Accepted: 11 September 2020; Published: 16 September 2020
Abstract: Forest logging can be detrimental for non-vascular epiphytes, determining the loss of key
components for ecosystem functioning. Legal logging in a Mediterranean mixed oak forest (Tuscany,
Central Italy) in 2016 heavily impacted sensitive non-vascular epiphytes, including a large population
of the threatened forest lichen Lobaria pulmonaria (L.) Hoffm. This event offered the background for this
experiment, where the potential effects of logging in oak forests are simulated by means of L. pulmonaria
micro-transplants (thallus fragments <1 cm). Our working hypothesis is that forest logging could
negatively influence the growth of the thalli exposed in logged stands compared to those exposed in
unlogged stands. One hundred meristematic lobes and 100 non-meristematic fragments are exposed
for one year on 20 Turkey oak trees (Quercus cerris), half in a logged and half in an unlogged stand.
Chlorophyll (Chl) afluorescence emission and total chlorophyll content are used as a proxy for the
overall vitality of the transplants, while their growth is considered an indicator of long-term effects.
Generally, vitality and growth of the transplants in the logged stand are lower than in the unlogged
stand. Both vitality and growth vary between the meristematic and non-meristematic fragments,
the former performing much better. Hence, irrespective of forest management, meristematic fragments
show higher growth rates (0.16–0.18 cm
2
year
−1
) than non-meristematic ones (0.02–0.06 cm
2
year
−1
).
Considering that a conservation-oriented management for this species should be tailored at the
habitat-level and, especially, at the tree-level, our results suggest that for appropriate conservation
strategies, it is necessary to consider the life cycle of the lichen, since the probability of survival of the
species may vary, with meristematic fragments having more chance to survive after logging.
Keywords:
biodiversity conservation; chlorophyll fluorescence; epiphytic macrolichens; forest
management; growth rates; indicator species
Forests 2020,11, 995; doi:10.3390/f11090995 www.mdpi.com/journal/forests
Forests 2020,11, 995 2 of 15
1. Introduction
Biodiversity is increasingly threatened by several anthropogenic factors [
1
–
5
]. Among them,
five main pressures have been recently pinpointed [
6
], including the overexploitation of species;
the introduction of invasive alien species; pollution from industrial, mining and agricultural activities;
changes in land use; climate change. Due to these pressures, some models predict that up to 50% of
the species are expected to become extinct in the next 50 years [
7
–
9
]. Pollution, land use (including
forest management) and climate change cause habitat loss and fragmentation, changes in species
range, population size and abundance, vitality and reproductive capacity [
10
–
12
]. Epiphytes may
be particularly at risk due to their dependence on trees for their entire life cycle [
13
]. Specifically,
growing on other plants they depend on their host for physical support and on tree host-specific
throughfall chemistry to satisfy their nutrient requirements [
14
,
15
]. Non-vascular epiphytes fulfill
various ecological functions in forests. They contribute to water and nutrient cycling by intercepting
and retaining nutrients from atmospheric humidity, with some also adding nitrogen by nitrogen
fixation [
16
–
18
]. Moreover, they provide resources and microhabitats for bark-dwelling invertebrates,
birds, and mammals [19,20].
Intensive logging and forest fragmentation can be particularly detrimental for epiphytic lichens,
causing a break in the availability of their primary habitat [
21
,
22
], especially for species with low
dispersal capacity (e.g., [
23
]). The foliose lichen Lobaria pulmonaria (L.) Hoffm. is considered an
“umbrella” species requiring spatial and temporal continuity of the forest habitat to maintain viable
populations [
24
–
27
]. It is a tripartite species in which the fungus is associated with both a green alga
(phycobiont) and a nitrogen-fixing cyanobacterium (cyanobiont). It is often accompanied by other
rare or endangered lichens [
28
–
31
] and sensitive to abrupt changes in light conditions (especially
in the dry state [
32
]), such as those occurring after forest logging. Its vulnerability to logging may
be exacerbated in dry environments, such as in the Mediterranean region, where oak-dominated
forests represent its main habitat (e.g., [
33
]). Actually, the results of a recent work carried out in
Italy suggest that oak (Quercus sp.pl.)-dominated forests provide more suitable habitat conditions
for L. pulmonaria than montane mixed forests, with chestnut forests in an intermediate position [
34
].
Regarding Mediterranean Italy, the climatic niche of L. pulmonaria widely overlaps (>70%) with that
of oak dominated forests [
35
], therefore the conservation of forest habitats with suitable ecological
conditions (e.g., [36]) is important.
Due to its heterothallic nature, self-incompatibility, poor dispersal capacity, long generation
cycles (up to 25 years) and susceptibility to environmental parameters (such as air pollution) [
37
–
39
],
this sensitive species is decreasing across Europe [
40
]. Considering all the above reasons, it is deemed
(and often used as) a model species to assess the response of epiphytic lichens to multiple environmental
factors [
41
]. Current forest management practices can hardly sustain future viable populations of
this species [
42
,
43
]. Additionally, air pollution still may limit recolonization of potentially suitable
forest habitats [
44
]. However, despite being declining and threatened in Southern Europe [
43
,
45
–
47
],
the species is not often recognized in conservation policies in Mediterranean regions [48].
This research began in 2016 with a legal logging in a Mediterranean mixed oak forest in Tuscany,
Central Italy, that heavily impacted a large population of L. pulmonaria. It was estimated that 40% of
L. pulmonaria biomass (8.5–12.3 kg ha
−1
) was lost (in the mostly colonized area, up to 1.8 kg 100 m
−2
),
including large and fertile thalli [
48
]. More than one year later, the analysis of chlorophyll afluorescence
emission revealed a significant reduction of the vitality of the thalli left on retained-isolated trees [
49
].
Here, the potential effects of logging on L. pulmonaria in this oak forest have been simulated by means
of micro-transplants (thallus fragments <1 cm). Our working hypothesis is that forest logging could
negatively influence the growth of the thalli exposed in logged stands compared to those exposed in
unlogged stands, with potential consequences for the conservation of the species. Since the viability of
L. pulmonaria populations in relation to forest management often depends on the regenerative capacity
of the thalli, we focus our attention on the behavior of thalli (fragments) with meristematic (young) and
non-meristematic (adult) properties. Hence, healthy young and adult fragments of L. pulmonaria are
Forests 2020,11, 995 3 of 15
transplanted for one year to a logged and an adjacent unlogged mixed oak stand, two and half years
after the conclusion of logging. Afterwards, ecophysiological responses (vitality and growth) of the
species are recorded. To optimize survival and growth, the response to logging is tested under the most
suitable conditions for lichen growth (north side of the trunk, breast height) [
34
,
50
]. The following
questions were addressed: (i) does forest management influence the growth capacity of the model
species in logged and unlogged stands? (ii) do vitality and growth vary between meristematic and
non-meristematic thalli?
2. Materials and Methods
2.1. Study Sites
The study was carried out in two forest sites, a logged and an adjacent unlogged stand dominated
by Quercus cerris L., Q. ilex L. and Q. pubescens Willd. (Tuscany, Central Italy, WGS84: N 43.1851
◦
;
E 11.3602◦) (Figure 1).
Forests 2020, 11, x FOR PEER REVIEW 3 of 16
logged and an adjacent unlogged mixed oak stand, two and half years after the conclusion of
logging. Afterwards, ecophysiological responses (vitality and growth) of the species are recorded.
To optimize survival and growth, the response to logging is tested under the most suitable
conditions for lichen growth (north) side of the trunk, breast height [34,50]. The following questions
were addressed: (i) does forest management influence the growth capacity of the model species in
logged and unlogged stands? (ii) do vitality and growth vary between meristematic and
non-meristematic thalli?
2. Materials and Methods
2.1. Study Sites
The study was carried out in two forest sites, a logged and an adjacent unlogged stand
dominated by Quercus cerris, Q. pubescens and Q. ilex, (Tuscany, Central Italy, WGS84: N 43.1851°; E
11.3602°) (Figure 1).
Figure 1. Map of the study sites. Taken from Paoli et al., [48] (modified).
Both study sites were located along a narrow valley with comparable orientation (North), soil
type, tree age (average around 40 years, with scattered older trees) and composition, moisture, and
distance from the closest stream. The density of the stems in the unlogged area was about 1100 ha−1,
decreasing to 165 ha−1 in the logged stand, with a consequent increase of sun irradiance all around
retained-isolated trees (from 130–1100 to 900–1550 μmol m−2 s−1 PAR at noon) [49]. The logged stand
(about 4.4 ha) was part of a local hotspot of L. pulmonaria, which had a patchy distribution and
colonized more than 1000 trees. Prior to logging, the overall biomass of L. pulmonaria in the study
Figure 1. Map of the study sites. Taken from Paoli et al., [48] (modified).
Both study sites were located along a narrow valley with comparable orientation (north), soil type,
tree age (average around 40 years, with scattered older trees) and composition, moisture, and distance
from the closest stream. The density of the stems in the unlogged area was about 1100 ha
−1
,
decreasing to 165 ha
−1
in the logged stand, with a consequent increase of sun irradiance all around
retained-isolated trees (from 130–1100 to 900–1550
µ
mol m
−2
s
−1
PAR at noon) [
49
]. The logged
stand (about 4.4 ha) was part of a local hotspot of L. pulmonaria, which had a patchy distribution and
Forests 2020,11, 995 4 of 15
colonized more than 1000 trees. Prior to logging, the overall biomass of L. pulmonaria in the study area
was 15.8–19.6 kg ha
−1
[
48
]. Oak forests in Tuscany are mainly managed by a coppice system with
standards and rotation cycles of 18–20 years [
51
]. Some stands with low management intensity have a
longer logging cycle, which dates back to more than 40 years ago, as in our case. Since L. pulmonaria is
not protected by law in Italy, logging operations did not take into account the presence of the species.
The exposure lasted from March 2019 to March 2020. During the experimental (1-year) period, the study
area was characterized by an average temperature of 14.1
◦
C, with the hottest period between June and
August (average of daily maximum temperature 32
◦
C), the coldest in January and February (average
of daily minimum temperature 0
◦
C); precipitation was about 920 mm, distributed over 72 rainy days
(precipitation ≥1 mm), 18 of which occurred in November.
2.2. Experimental Design
A graphical representation of the experimental design is presented in Figure 2.
Forests 2020, 11, x FOR PEER REVIEW 4 of 16
area was 15.8–19.6 kg ha−1 [48]. Oak forests in Tuscany are mainly managed by a coppice system
with standards and rotation cycles of 18–20 years [51]. Some stands with low management intensity
have a longer logging cycle, which dates back to more than 40 years ago, as in our case. Since L.
pulmonaria is not protected by law in Italy, logging operations did not take into account the
presence of the species. The exposure lasted from March 2019 to March 2020. During the
experimental (1-year) period, the study area was characterized by an average temperature of 14.1
°C, with the hottest period between June and August (average of daily maximum temperature 32
°C), the coldest in January and February (average of daily minimum temperature 0 °C);
precipitation was about 920 mm, distributed over 72 rainy days (precipitation ≥1 mm), 18 of which
occurred in November.
2.2. Experimental Design
A graphical representation of the experimental design is presented in Figure 2.
Figure 2. Graphical representation of the experimental design. n = 10 number of trees; n = 100
number of thallus fragments for each type; T0—time zero, beginning of the experiment; TF—final
time, harvesting of the samples; FV/FM =the maximum of the quantum yield of primary
photochemistry.
A total of 200 thallus fragments (half meristematic and half non-meristematic) were randomly
cut from a batch of about 100 healthy thalli of L. pulmonaria randomly selected from a nearby oak
forest (Figure 1). Particularly, the former are upward-growing young lobes with intact apical
meristems, and the latter are fragments of the inner sorediate or non-sorediate parts of the
thallus, lacking apical growth [52,53]. The source habitat for collected fragments has the same
characteristics as the unlogged stand, being adjacent to the study sites and extending on a
hillside with a N slope, where Quercus cerris L., Q. ilex L. and Q. pubescens Willd. are the most
common trees colonized by L. pulmonaria. To minimize the harvesting of material from the native
population, meristematic fragments were cut respecting the natural shape of the meristematic lobes
(dimensions 0.55–0.95 cm for most of the samples). Non-meristematic fragments were obtained
with a hole-puncher by selecting discs of approximate diameter (0.50–0.65 cm) from internal adult
parts of healthy adult thalli. Lobaria pulmonaria was exposed using a specific transplant device
(“barella”) composed of a sterilized bandage supported by a plastic net (10 × 2 cm). Concerning
practical reasons, each device brought five meristematic or five non-meristematic fragments of the
thalli tied on the bandage using cotton threads, in a way that each lichen fragment did not overlap
with the others. The following experimental conditions were considered (explanatory variables) to
infer the implications of logging on the conservation and distribution of the model species in
Mediterranean oak forests: forest management (logged versus unlogged stand) and type of thallus
fragment (meristematic versus non-meristematic). Two hundred thallus fragments (each
representing a sample) were transplanted on the north side of twenty randomly selected Turkey
oak trunks (Q. cerris) (reciprocal distance >10 m), at about 100 cm from the ground, half in the
logged and half in the unlogged stand: 50 meristematic and 50 non-meristematic fragments in each
Figure 2.
Graphical representation of the experimental design. n=10 number of trees; n=100
number of thallus fragments for each type; T0—time zero, beginning of the experiment; TF—final time,
harvesting of the samples; FV/FM=the maximum of the quantum yield of primary photochemistry.
A total of 200 thallus fragments (half meristematic and half non-meristematic) were randomly cut
from a batch of about 100 healthy thalli of L. pulmonaria randomly selected from a nearby oak forest
(Figure 1). Particularly, the former are upward-growing young lobes with intact apical meristems,
and the latter are fragments of the inner sorediate or non-sorediate parts of the thallus, lacking
apical growth [
52
,
53
]. The source habitat for collected fragments has the same characteristics as the
unlogged stand, being adjacent to the study sites and extending on a hillside with a north slope,
where Q. cerris,Q. Ilex and Q. pubescens are the most common trees colonized by L. pulmonaria.
To minimize the harvesting of material from the native population, meristematic fragments were
cut respecting the natural shape of the meristematic lobes (dimensions 0.55–0.95 cm for most of
the samples). Non-meristematic fragments were obtained with a hole-puncher by selecting discs of
approximate diameter (0.50–0.65 cm) from internal adult parts of healthy adult thalli. Lobaria pulmonaria
was exposed using a specific transplant device (“barella”) composed of a sterilized bandage supported
by a plastic net (10
×
2 cm). Concerning practical reasons, each device brought five meristematic
or five non-meristematic fragments of the thalli tied on the bandage using cotton threads, in a way
that each lichen fragment did not overlap with the others. The following experimental conditions
were considered (explanatory variables) to infer the implications of logging on the conservation and
distribution of the model species in Mediterranean oak forests: forest management (logged versus
unlogged stand) and type of thallus fragment (meristematic versus non-meristematic). Two hundred
thallus fragments (each representing a sample) were transplanted on the north side of twenty randomly
selected Turkey oak trunks (Q. cerris) (reciprocal distance >10 m), at about 100 cm from the ground,
half in the logged and half in the unlogged stand: 50 meristematic and 50 non-meristematic fragments
Forests 2020,11, 995 5 of 15
in each forest type. The selection of the Turkey oak was justified by its presence and distribution after
logging. Growth rates, chlorophyll (Chl) afluorescence emission and total chlorophyll content were
assessed. The vitality of the samples was measured monthly in terms of chlorophyll afluorescence
emission and seasonally (every three months) for chlorophyll content. Pre-exposure and final values
were considered for the purpose of this article. Final values for chlorophyll afluorescence emission were
represented by the average of the last three months (winter season), to account for monthly fluctuations.
Measurements were taken in the morning to minimize the variability of external conditions during
the day. One measurement was taken for each sample (one measurement per visit, with one visit per
month).
2.3. Vitality of the Lichen Photobiont
Photosynthetic parameters were previously used to assess the vitality of L. pulmonaria [
54
–
59
].
Here, chlorophyll (Chl) afluorescence emission and total chlorophyll content were used as a proxy for
the overall vitality of the transplants. The measurements of chlorophyll afluorescence were carried out
by a Plant Efficiency Analyzer Handy PEA (Hansatech Ltd., Norfolk, UK). Thalli were kept hydrated
(sprayed with mineral water) and dark-adapted for at least ten minutes (covered with a black velvet
cloth) before the measurements. Each sample was illuminated using the clip for 1 s with a saturating
excitation pulse (3000
µ
mol(photon) s
−1
m
−2
) of red light (650 nm) from a LED into the fluorometer
sensor. All fluorescence induction curves were recorded up to 1 s. The condition of the samples
was expressed by the maximum of the quantum yield of primary photochemistry as inferred from
fluorescence data: F
V
/F
M
=(F
M−
F
0
)/F
M
, where F
v
=(F
m−
F
0
) is the variable fluorescence, F
0
is the
calculated basal fluorescence and Fmis the maximum Chl afluorescence.
The chlorophyll content of the samples, expressed as total chlorophyll per m
2
of biological material
(mg m
−2
), was measured by a Chlorophyll Content Meter-300 (Opti-Sciences CCM-300, Hudson, NH,
USA), which gauged the chlorophyll content based on reflectance and/or absorbance of radiation by
chlorophyll molecules. The method provided accurate readings also in lichens, comparable to those
obtained using the classical dimethyl sulphoxide (DMSO) extraction method [60].
2.4. Lichen Growth
Lichen growth was used as an indicator of potential long-term effects. We are aware that growth
rates may vary on a seasonal basis, depending on several environmental factors; however, for the
purpose of this research, the attention was focused on the annual growth rate of the thallus. Therefore,
both at the beginning and at the end of the transplant experiment, each thallus fragment was fully
hydrated with mineral water and carefully flattened to avoid the folding of the lobes before scanning
by Canon i-SENSYS MF4320d (Canon Inc., Tokyo, Japan). The area (A) of the meristematic and
non-meristematic fragments was assessed using Photoshop CS6 extended (Adobe Systems, San Jose,
CA, USA). The lichen growth comparing the same samples before and after the exposure was quantified
as percentage increases A (%A) =[(area T
F−
area T
0
) area T
0−1
]
×
100. After one year, the growth of
the surface of each individual thallus was assessed by subtracting the initial area from the respective
area at harvest. All transplant devices remained attached to the bark during the transplant period.
Only a few samples (both meristematic and non-meristematic) detached, likely due to the presence
of wildlife. The loss was similar in the two stands (unlogged and logged). Thus, the final sample
size for statistical analyses comprised 90 meristematic fragments (40 in the logged stand and 50 in the
unlogged one) and 97 non-meristematic ones (49 in the logged stand and 48 in the unlogged one).
2.5. Data Analysis
The datasets of meristematic and non-meristematic transplanted fragments were processed
separately, accounting for the irregular shape of meristematic lobes and possible dimensional differences
with non-meristematic ones. The non-parametric rank-sum Wilcoxon–Mann–Whitney test was used
to test the significance of the differences in the pairwise comparisons between samples transplanted
Forests 2020,11, 995 6 of 15
respectively in the logged and in the unlogged stand. A Principal Component Analysis (PCA) was
used as an explorative unsupervised multivariate analysis to study the relationships among variables.
Multiple linear regression models were applied to fit the relationship between the above-mentioned
predictors (delta Chl, delta F
V
/F
M
and forest management) and the response variable ‘growth rate’.
Two models were performed accounting separately for meristematic and non-meristematic thallus
fragments. Regarding each model, R2, adjR2and F statistics were considered.
The following variables were considered for data processing:
•Growth rate =(area TF−area T0) area T0−1, where:
area TF: area of the transplanted thallus fragment at the end of the exposure.
area T0: area of the transplanted thallus fragment at the beginning of the experiment.
•Delta fluo =[(FV/FM)TF −(FV/FM)T0)] (FV/FM)T0−1, where:
(FV/FM)TF: FV/FMof the transplanted thallus fragment at the end the exposure.
(FV/FM)T0: FV/FMof the transplanted thallus fragment at the beginning of the experiment.
•Delta Chl =(ChlTF −ChlT0) Chl T0−1, where:
Chl
TF
: chlorophyll content of the transplanted thallus fragment at the final time of the exposure.
Chl
T0
: chlorophyll content of the transplanted thallus fragment at the beginning of the experiment.
The R package Stats was used for all the analyses [61].
3. Results
3.1. Lichen Vitality
A summary of the results concerning lichen vitality is represented in Figure 3. Inferred from
fluorescence data, the lichens performed better in the unlogged area. The transplant experiment
induced a decrease of F
V
/F
M
in both stands (p<0.05) as compared with pre-exposure conditions
(F
V
/F
M
=0.749
±
0.040 for both meristematic and non-meristematic fragments). After one year of
exposure, a decrease in chlorophyll concentrations was observed both in the logged and unlogged
stand, compared with pre-exposure concentrations (Chl =403
±
96 mg m
−2
for meristematic and
437 ±106 mg m−2for non-meristematic fragments) (p<0.001).
Upon ending the experiment, according to the parameter F
V
/F
M
, meristematic fragments
performed better in the unlogged area (p<0.05). Concerning non-meristematic fragments, the difference
between F
V
/F
M
values in the logged and unlogged stands was not significant (p>0.05). Both types
of fragments in the unlogged stand were characterized by significantly higher (p<0.05) chlorophyll
contents compared with those in the logged stand. Notwithstanding the variability in shape and
dimensions existing between meristematic and non-meristematic fragments (which can limit a full
comparison between the two groups in the same forest stand), it was observed that meristematic lobes
had higher photosynthetic performances in the logged area than did the non-meristematic ones.
Forests 2020,11, 995 7 of 15
Forests 2020, 11, x FOR PEER REVIEW 7 of 16
Figure 3. Boxplots of the potential quantum yield of primary photochemistry (FV/FM) and the total
chlorophyll content (mg m−2) at the end of the transplant experiment as indicators of the vitality of
meristematic and non-meristematic fragments, in logged and unlogged stands. The average value is
shown at the side of each box. Regarding each type of fragment, different small letters indicate
significant differences according to forest management (p < 0.05).
Upon ending the experiment, according to the parameter FV/FM, meristematic fragments
performed better in the unlogged area (p < 0.05). Concerning non-meristematic fragments, the
difference between FV/FM values in the logged and unlogged stands was not significant (p > 0.05).
Both types of fragments in the unlogged stand were characterized by significantly higher (p < 0.05)
chlorophyll contents compared with those in the logged stand. Notwithstanding the variability in
shape and dimensions existing between meristematic and non-meristematic fragments (which can
limit a full comparison between the two groups in the same forest stand), it was observed that
meristematic lobes had higher photosynthetic performances in the logged area than did the
non-meristematic ones.
3.2. Thallus Growth
Following one year of exposure, the transplants in the unlogged stand (both non-meristematic
and meristematic) were characterized by larger surfaces and a comparable increase in the thallus
area (by 25% and 21%, respectively). Transplants exposed in the logged stand showed a
significantly lower (p < 0.05) growth in the case of non-meristematic fragments (their increase was
about 6%). The growth of meristematic fragments (14%) did not significantly differ between the
logged and unlogged stand. Irrespective of forest management, meristematic fragments showed
higher growth rates (surface increase 0.16–0.18 cm2 year−1) as compared with non-meristematic
Figure 3.
Boxplots of the potential quantum yield of primary photochemistry (F
V
/F
M
) and the total
chlorophyll content (mg m
−2
) at the end of the transplant experiment as indicators of the vitality of
meristematic and non-meristematic fragments, in logged and unlogged stands. The average value
is shown at the side of each box. Regarding each type of fragment, different small letters indicate
significant differences according to forest management (p<0.05).
3.2. Thallus Growth
Following one year of exposure, the transplants in the unlogged stand (both non-meristematic
and meristematic) were characterized by larger surfaces and a comparable increase in the thallus area
(by 25% and 21%, respectively). Transplants exposed in the logged stand showed a significantly lower
(p<0.05) growth in the case of non-meristematic fragments (their increase was about 6%). The growth
of meristematic fragments (14%) did not significantly differ between the logged and unlogged stand.
Irrespective of forest management, meristematic fragments showed higher growth rates (surface
increase 0.16–0.18 cm
2
year
−1
) as compared with non-meristematic fragments (0.02–0.06 cm
2
year
−1
).
The presence of regeneration lobules along the cut edges of meristematic fragments was not observed
(except for one case in the unlogged stand).
3.3. Principal Component Analysis (PCA)
A 3-dimensional solution was found both for meristematic and non-meristematic datasets,
explaining 100% of the total variance (Table 1). Figure 4shows the score plots of the first two axes,
that explained >80% of the variance. Regarding meristematic fragments, the first two principal
components (PC) cumulatively explained 80.44% of the total variance. PC1 (explained variance:
Forests 2020,11, 995 8 of 15
47.15%) showed an increasing gradient of vitality (delta fluo, R
2
=0.706) and chlorophyll content (delta
Chl, R
2
=0.705) in relation to the meristematic thalli that were transplanted in the unlogged stand
(26 out of the 35 samples, 74%). The growth was positively correlated with PC2 (explained variance:
33.29%), irrespective of forest management. Concerning non-meristematic fragments, the first two PC
cumulatively explained 80.86% of the total variance. The fragments transplanted in the unlogged stand
were distributed (26 out of the 33 samples, 79%) for negative values of PC1 (explained variance: 50.70%),
showing an increasing gradient of growth rate (R
2
=
−
0.639) and vitality (delta fluo, R
2
=
−
0.658),
while the values of chlorophyll content were highly related to the negative values of PC2 (explained
variance: 30.16%).
Table 1.
Matrix of variable loadings reporting the eigenvalues of the three principal components of the
PCA ordinations (explained variance in brackets), which were performed separately for meristematic
and non-meristematic thallus fragments. Values >0.5 are reported in bold.
PCA with Meristematic Dataset
PC1 (47.15%) PC2 (33.29%) PC3 (19.56%)
Growth rate 0.065 0.997 −0.034
Delta fluo 0.706 −0.022 0.707
Delta Chl 0.705 −0.070 −0.706
PCA with Non-Meristematic Dataset
PC1 (50.70%) PC2 (30.16%) PC3 (19.14%)
Growth rate −0.639 0.344 −0.688
Delta fluo −0.658 0.218 0.721
Delta Chl −0.398 −0.913 −0.087
Forests 2020, 11, x FOR PEER REVIEW 8 of 16
fragments (0.02–0.06 cm2 year−1). The presence of regeneration lobules along the cut edges of
meristematic fragments was not observed (except for one case in the unlogged stand).
3.3. Principal Component Analysis (PCA)
A 3-dimensional solution was found both for meristematic and non-meristematic datasets,
explaining 100% of the total variance (Table 1). Figure 4 shows the score plots of the first two axes,
that explained >80% of the variance. Regarding meristematic fragments, the first two principal
components (PC) cumulatively explained 80.44% of the total variance. PC1 (explained variance:
47.15%) showed an increasing gradient of vitality (delta fluo, R2 = 0.706) and chlorophyll content
(delta Chl, R2 = 0.705) in relation to the meristematic thalli that were transplanted in the unlogged
stand (26 out of the 35 samples, 74%). The growth was positively correlated with PC2 (explained
variance: 33.29%), irrespective of forest management. Concerning non-meristematic fragments, the
first two PC cumulatively explained 80.86% of the total variance. The fragments transplanted in the
unlogged stand were distributed (26 out of the 33 samples, 79%) for negative values of PC1
(explained variance: 50.70%), showing an increasing gradient of growth rate (R2 = −0.639) and
vitality (delta fluo, R2 = −0.658), while the values of chlorophyll content were highly related to the
negative values of PC2 (explained variance: 30.16%).
Table 1. Matrix of variable loadings reporting the eigenvalues of the three principal components of
the PCA ordinations (explained variance in brackets), which were performed separately for
meristematic and non-meristematic thallus fragments. Values >0.5 are reported in bold.
PCA with Meristematic Dataset
PC1 (47.15%) PC2 (33.29%) PC3
(19.56%)
Growth rate 0.065 0.997 −0.034
Delta fluo 0.706 −0.022 0.707
Delta Chl 0.705 −0.070 −0.706
PCA with Non-Meristematic Dataset
PC1 (50.70%) PC2 (30.16%) PC3
(19.14%)
Growth rate −0.639 0.344 −0.688
Delta fluo −0.658 0.218 0.721
Delta Chl −0.398 −0.913 −0.087
Figure 4. Score plots of the Principal Component Analysis obtained for meristematic (ordination on
Figure 4.
Score plots of the Principal Component Analysis obtained for meristematic (ordination
on the left) and non-meristematic fragments (on the right). Distribution of meristematic lobes in
logged and unlogged stand: 40 and 35 samples, respectively; non-meristematic fragments: 49 and
33 samples, respectively.
3.4. Multiple Linear Regression Models
Considering the growth of meristematic fragments as the response variable, a non-significant
model was obtained, thus showing that it was not affected by forest management and by photobiont
vitality and chlorophyll content (Table 2and Figure 5). The model for non-meristematic fragments
was significant (AdjR
2
: 0.197; p<0.001; Table 2), with an increasing gradient of growth with respect
Forests 2020,11, 995 9 of 15
to fragments transplanted in the unlogged stand (p<0.05), corresponding to an average of 0.142 cm
estimated higher linear growth. The growth also was positively related to higher values of delta fluo
(p<0.01; Figure 5).
Table 2.
Multiple Linear Regression Models describing the effects of logging and vitality on growth
rates both in meristematic and non-meristematic fragments. Estimates, Standard Errors, tvalues and
pvalues (* p<0.05; ** p<0.01) are reported. Summary statistics also are reported for each model
(Fstatistics and pvalues).
Type Estimate Std. Error tValue Summary Statistics
Meristematic
(Intercept) 0.055 0.131 0.419 RSE: 0.272 (71 df)
Multiple R2: 0.033
Adjusted R2:−0.008
F: 0.809 (3 and 71 df)
p-value: 0.493
Unlogged stand 0.119 0.077 1.537
Delta fluo −0.129 0.452 −0.285
Delta Chl content −0.077 0.124 −0.621
Non-meristematic
(Intercept) 0.184 0.099 1.854 RSE: 0.229 (78 df)
Multiple R2: 0.227
Adjusted R2: 0.197
F: 7.628 (3 and 78 df)
p-value: <0.001
Unlogged stand 0.142 0.067 2.109 *
Delta fluo 0.592 0.193 3.063 **
Delta Chl content −0.060 0.093 −0.650
Forests 2020, 11, x FOR PEER REVIEW 9 of 16
the left) and non-meristematic fragments (on the right). Distribution of meristematic lobes in logged
and unlogged stand: 40 and 35 samples, respectively; non-meristematic fragments: 49 and 33
samples, respectively.
3.4. Multiple Linear Regression Models
Considering the growth of meristematic fragments as the response variable, a non-significant
model was obtained, thus showing that it was not affected by forest management and by
photobiont vitality and chlorophyll content (Table 2 and Figure 5). The model for non-meristematic
fragments was significant (AdjR2: 0.197; p < 0.001; Table 2), with an increasing gradient of growth
with respect to fragments transplanted in the unlogged stand (p < 0.05), corresponding to an
average of 0.142 cm estimated higher linear growth. The growth also was positively related to
higher values of delta fluo (p < 0.01; Figure 5).
Table 2. Multiple Linear Regression Models describing the effects of logging and vitality on growth
rates both in meristematic and non-meristematic fragments. Estimates, Standard Errors, t values and
p values (* p < 0.05; ** p < 0.01) are reported. Summary statistics also are reported for each model (F
statistics and p values).
Type Estimate Std. Error t Value Summary
Statistics
Meristematic
(Intercept) 0.055 0.131 0.419 RSE: 0.272 (71 df)
Multiple R2: 0.033
Adjusted R2:
−0.008
F: 0.809 (3 and 71
df) p-value: 0.493
Unlogged stand 0.119 0.077 1.537
Delta fluo −0.129 0.452 −0.285
Delta Chl
content −0.077 0.124 −0.621
Non-meristematic
(Intercept) 0.184 0.099 1.854 RSE: 0.229 (78 df)
Multiple R2: 0.227
Adjusted R2: 0.197
F: 7.628 (3 and 78
df)
p-value: <0.001
Unlogged stand 0.142 0.067 2.109 *
Delta fluo 0.592 0.193 3.063 **
Delta Chl
content −0.060 0.093 −0.650
Figure 5. Fitted modeled relationships between growth rate and delta fluo, according to the linear
models of Table 2, for meristematic (left; p > 0.05) and non-meristematic fragments (right; p < 0.01).
Bands represent 95% confidence intervals. Solid line = logged, dashed line = unlogged.
Figure 5.
Fitted modeled relationships between growth rate and delta fluo, according to the linear
models of Table 2, for meristematic (
left
;p>0.05) and non-meristematic fragments (
right
;p<0.01).
Bands represent 95% confidence intervals. Solid line =logged, dashed line =unlogged.
4. Discussion
After logging, forest lichens are exposed to a sudden increase in solar radiation and dry conditions,
which, if in excess of their ecological range, may negatively affect their photosynthetic activity and,
hence, their overall vitality [
54
]. A transplantation experiment with L. pulmonaria beginning during a
sunny and dry period induced extensive and irreversible bleaching after only 40 days [
32
]. These effects,
mostly studied in boreal forests (e.g., [
62
,
63
]), can be exacerbated in Mediterranean oak forests [
48
],
where high light and temperature (especially during summer), together with the decoupling of
moisture and light availability across an annual cycle, are supposed to influence the generation time
of L. pulmonaria in the long-term [
33
]. The results of our transplant experiment outline that the
exposure in a logged stand has negative effects on the growth capacity of non-meristematic fragments.
Regarding the case of meristematic fragments, higher photosynthetic performances and growth are
found irrespective of forest management, suggesting the potential for “meristematic tissues” under
suitable growth conditions (north side of the trunk, at about 100 cm from the ground) for maintaining
Forests 2020,11, 995 10 of 15
vital thalli of L. pulmonaria after logging. The positive correlation found between chlorophyll content
(as well as F
V
/F
M
) and the growth of our transplants fits with the indications of Gauslaa et al. [
55
].
They reported a positive correlation between chlorophyll content and growth of L. pulmonaria in boreal
forests and a negative relationship between total chlorophyll content (as well as F
V
/F
M
) and prolonged
high-light exposures in clear cuts, suggesting that excess high light induced chlorophyll degradation
and affected lichen growth [
55
]. Concerning the case of Mediterranean oak forests, a negative impact
by logging on the vitality and growth of L. pulmonaria is expected for the thalli exposed to high light,
such as on the south side of the boles (unpublished data). Comparative studies on growth rates of
L. pulmonaria (e.g., [
55
,
64
] and references therein) reveal a wide variability depending on several factors,
including micro- and macro-climatic parameters, forest structure and management. Further, our results
point to the importance of the regenerative capacity of “meristematic tissues” (and, hence, of young
healthy thalli) for maintaining vital populations of L. pulmonaria in relation to forest management.
Generally, the populations of L. pulmonaria are subject to sudden changes of environmental
conditions after coppicing events and, as a consequence of logging, lichen thalli can have different
fates:
•
They can be directly destroyed/lost due to the logging of the trunks for timber production,
as already occurred in the investigated oak forest (loss of 40% of L. pulmonaria biomass) [48].
•
They can remain attached to the bark of retained trees and face a gradient of more or less stressing
conditions according to their cardinal exposure, position on the trunk, and distance among
the trees.
Referring to the investigated forests, the fate of the thalli left after logging likely falls in one of the
following situations:
(a)
Some of the thalli show visible symptoms of damage consisting of discoloration and bleaching of
the surfaces. Curling can occur as a strategy to limit the damage [
65
]. The thalli can experience
a significant reduction of photosynthetic performance (decreased vitality) and the absence of
growth. They become thinner due to the significant reduction (by 35%) of the algal layer [
49
].
Most of them likely will be lost in the long term.
(b)
Some of the thalli try to acclimate to ongoing stresses by melanization of the fungal cortex and
reduction of the photosynthetic activity. To a certain extent, forest lichens are able to produce
melanin to screen excess solar radiation, they increase their thickness in situations with more
light, as well as their water holding capacity to acclimate for limited water availability and rare
occurrences of hydration events [56,66]. However, in the long term, they still can be at risk.
(c) Some of the thalli are not negatively affected; they maintain their vitality and growth rates thanks
to suitable microclimatic conditions that locally persist, even after logging. They can be preserved
in the long term. This is also the case for our transplant experiment (in particular for meristematic
fragments, likely due to their situation and the north side of the trunk).
Logging may affect the vitality of L. pulmonaria left on isolated oak trees much more than on
retained forest patches and unlogged oak stands [
49
]. Specifically, in a previous study [
48
] carried out
one year after logging, the thalli showed visible changes in 46% of the isolated trees. Such changes
consisted of melanization (in this case this could be seen rather as an acclimation than a stress response
to the new environment), or in the worst case (14%) evident discoloration, bleaching, up to extensive
necrosis in L. pulmonaria. Conversely, the remaining fraction (54%) still consisted of healthy thalli, with a
dominant greenish color and absence of discolorations and necrotic parts [
48
]. These observations
(based on native thalli) are consistent with the results of our transplant experiment, that the growth of
the transplants in the logged stand was lower than in the unlogged stand. Furthermore, despite our
not measuring melanin production, our field observations indicate that several fragments exposed in
the logged stand (irrespective if meristematic or non-meristematic) had a darker appearance when
dry compared to those in the unlogged area, as similarly reported by Coxson and Stevenson [
64
]
Forests 2020,11, 995 11 of 15
in boreal forests subjected to partial cuts. Noteworthy, L. pulmonaria transplanted into clear-cuts
in boreal forests showed that logging in winter was less harmful than logging in summer, being
associated with higher growth rates of the species than during the latter one [
63
] and that, in most
cases, such healthy thalli were N oriented [
63
,
67
] well reflecting the delicate balance between humidity,
light availability, and prevention of desiccation risk that influences the ecophysiology and distribution
of L. pulmonaria [
55
]. Except for one case, we did not find new regeneration lobules along the cut edges
of meristematic fragments (after one year), as the micro-transplants likely included only a narrow
apical zone. Considering intact Lobaria lobes, this rather thin portion is not attached to the bark and
undergoes curling during drying that contributes to protect it from photoinhibitory damage [
65
].
Conversely, in another experiment, we observed the presence of small regeneration lobules, already
after one year, in transplants of large thallus fragments saved from the logged area and exposed for
conservation purposes in three oak forests within nature reservations in Tuscany [44].
Lobaria pulmonaria is a sub-oceanic species strongly dependent on macro- and micro-climatic
conditions for its dispersal and establishment [
34
,
68
]. Concerning Italy, it meets its optimal climatic
suitability in areas characterized by small variations in seasonal temperature, high atmospheric humidity,
intermediate conditions of diffused light, and low or negligible air pollution [
69
]. Considering a local
scale, these microclimatic conditions could be altered by forest management, thus putting at risk
the health of the populations, and compromising their probability of survival [
49
,
55
]. Taking this
perspective, recent works also estimated that, due to the simultaneous loss of climatic suitability and
habitat availability, the distribution range of L. pulmonaria in Italy will decrease by 80% by 2060 [
35
,
43
].
A partial reduction of the spatial overlap between the climatic niche of L. pulmonaria and that of
its host tree species in the Mediterranean region, as well as the invasion of native woods by alien
species (e.g., black locust) are expected to further threaten L. pulmonaria populations [
35
]. Considering
such a complex risk framework, it becomes essential to understand the ecology of the species and its
microscale dynamics to be able to tailor and convey conservation strategies more effectively at all stages
of population development [
34
]. Taking a broader perspective, the conservation of suitable habitats by
maintaining tree species diversity in mixed stands and increasing the proportion of deciduous trees,
maintaining large trees and regeneration layers, and allowing heterogeneous light conditions within
the stands [36] appears important.
Our results support the view that effective conservation-oriented management for this species
should be tailored at the habitat-level and, especially, at the tree-level [
30
,
33
,
34
]. Indeed, the results
reveal that the probability of survival of the species is influenced by an interaction between abiotic
and biotic factors whose effects may vary during the life cycle of the lichen. Recent results indicated
that in oak (Quercus sp.pl.)-dominated forests the effect of habitat was significant only for adult thalli
of L. pulmonaria, while the early life stages of the lichen were habitat-independent and were strictly
associated with tree-level factors [
34
]. Meristematic fragments have more chance to establish new
individuals and survive in suitable conditions, such as the northern side of the trunk and at an adequate
height (e.g., about 100 cm), likely reflecting their high requirement for adequate solar radiation (under
humid conditions) to sustain photosynthesis. Under the same suitable conditions, non-meristematic
fragments did not show the same performances, likely due to a limited regenerative capacity of
“adult tissues” (compared to meristematic ones) to face the variation of environmental conditions that
suddenly occurs after forest logging.
5. Conclusions
The vitality and growth of L. pulmonaria in relation to forest management were tested in view of
possible conservation strategies of this sensitive and threatened species in Mediterranean oak forests.
Comparing transplants in logged and unlogged stands, the results pinpointed a lower growth of
L. pulmonaria in the logged stand with respect to the unlogged stand, with non-meristematic fragments
being more sensitive to the effects of forest management. It is suggested that a conservation-oriented
management should be tailored at the habitat-level and, especially, at the tree-level. The probability of
Forests 2020,11, 995 12 of 15
survival may vary during the life cycle of the lichen, with meristematic fragments having more chance
under suitable microclimatic conditions after logging.
Author Contributions:
Conceptualization, E.B., R.B., G.B., L.D.N., Z.F., L.F., P.G., S.R., C.V., L.P.; methodology,
E.B., R.B., G.B., L.D.N., Z.F., L.F., P.G., S.R., C.V., L.P.; investigation, data curation, validation and analysis E.B.,
R.B., G.B., L.D.N., Z.F., L.F., P.G., S.R., C.V., L.P.; writing—original draft preparation, E.B., R.B., G.B., Z.F., L.F., P.G.,
S.R., L.P.; writing—review and editing, E.B., R.B., G.B., Z.F., L.F., P.G., J.N., S.R., L.P.; supervision, L.P. This article
is part of the project “Effects of forest management on threatened macrolichens” developed by the Working Group for
Ecology of the Italian Lichen Society, coordinator L.P. All authors have read and agreed to the published version
of the manuscript.
Funding: This research received no external funding.
Acknowledgments:
We are grateful to Associazione Culturale di Murlo (www.murlocultura.com) for hosting the
meeting of the Working Group for Ecology of the Italian Lichen Society held in Murlo (Siena), 10–12 February 2020.
Conflicts of Interest: The authors declare no conflict of interest.
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