plants
Article
The Habitat of the Neglected Independent Protonemal Stage of
Buxbaumia viridis
Ameline Guillet 1 , Vincent Hugonnot 2, * and Florine Pépin 2
1
2
*
Independent Researcher, 58 rue Georges Rissler, FR-63000 Clermont-Ferrand, France;
guillet.ameline@gmail.com
Independent Researchers, Le Bourg, FR-43380 Blassac, France; flopepin@gmail.com
Correspondence: vincent.hugonnot@wanadoo.fr
Abstract: Buxbaumia viridis is a well-known species of decaying deadwood, which is protected in
Europe. All previous studies dealing with the ecology of B. viridis rely on the sporophyte generation
because the gametophyte generation is allegedly undetectable. Recent advances have shown that the
protonemal stage, including gemmae, is recognizable in the field, thereby considerably modifying our
perception of the species’ range and habitat. In France, we demonstrate the existence of independent
protonemal populations, with the implication that the range of B. viridis is widely underestimated.
Sporophytes and sterile protonema do not share the same ecological requirements. The sporophyte
stage was found in montane zones, almost exclusively in coniferous forests, and on well-decayed
wood. The sterile protonemal stage extends to lower elevations, in broad-leaved forests, and on
wood in a less advanced state of decay. Our results suggest that the humidity could be one of the
most relevant explanatory variables for the occurrence of sporophytes. Opening of the canopy seems
to promote sporophyte development. Previous anomalous observations of B. viridis growing on
humus or bark might be explained by the presence of a protonemal population that is able to produce
sporophytes under rarely occurring but favorable climatic events.
Keywords: gemmae; decaying wood; dead-wood; ecological modelling; conservation
Citation: Guillet, A.; Hugonnot, V.;
Pépin, F. The Habitat of the Neglected
Independent Protonemal Stage of
Buxbaumia viridis. Plants 2021, 10, 83.
https://doi.org/10.3390/plants
10010083
Received: 15 November 2020
Accepted: 27 December 2020
Published: 2 January 2021
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
Buxbaumia viridis (Moug. ex Lam. et DC.) Brid. ex Moug. et Nestl. (Buxbaumiaceae) is
a hygrophilous, sciaphilous, and acidophilous species, widely distributed in the Northern
hemisphere, where it mainly colonizes decaying wood in wet, shaded coniferous montane
woodlands [1–3]. Ecological factors, that have been suggested as relevant in explaining the
occurrence of the species, are the amount of available deadwood, the degree of its decomposition, canopy openness, humidity, elevation, aspect, and deadwood composition [3–5].
As a species apparently linked to well-preserved forest stands with large accumulations
of bulky woody debris, it has been suggested that B. viridis is an indicator of ancient
woodland [5].
B. viridis is protected in Europe by Annex I of the Bern Convention [6], and Annex
II of the “Habitats-Fauna-Flora” Directive (Habitat Directive 92/43/EEC), since a large
proportion of its habitat has disappeared worldwide during the 20th century [7]. Over
recent decades the species has been assessed in the European Red-list for bryophytes as
“vulnerable”, and this has led to a disproportionate increase in the number of targeted studies [3,4,8,9]. Hence its status has changed to “least concern” in the most recent Red-List [10].
However, its conservation remains a matter of critical concern, for instance on account of
forest management practices that lead to a reduction in coarse wood debris (CWD), or to
clear-felling which permits light penetration and consequently drought [11,12].
Unlike other mosses whose haploid gametophyte is dominant and clearly visible,
B. viridis possesses a minute gametophyte which consists of a filamentous protonema
that produces much reduced female and male buds [4,13]. The distinctive protonema
Plants 2021, 10, 83. https://doi.org/10.3390/plants10010083
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μ been previously described in vitro [14]. It comprises colorless, occasionally branched
has
rhizoids, 15 µm in diameter with oblique cross-walls, and an upright, somewhat undulating
chloronematal filament system with relatively frequent anastomosis [13,14]. By contrast,
the sporophyte is disproportionately large and can be from 7 to 25 mm long [3].
Because the minute gametophyte of B. viridis is reputedly impossible to spot in the
field, all previous studies have been based on the sporophyte stage [3–5]. However, one
recent study has described protonemal gemmae (i.e., asexual propagules) in B. viridis [13].
μ are multicellular,
μ
These gemmae
obloid, with a warty ornamentation, and their size varies
between 40 µm and 70 µm. They tend to form brownish, large and highly distinctive
aggregations that are relatively easy to locate in the field (see Material and Methods
section).
By conducting non-systematic searches all over France, far beyond the current known
range of the sporophytes of B. viridis, the authors were able to find many unexpected
protonemal populations, for instance in the Northern Oceanic and Mediterranean regions
of France. This led the authors to suspect that the species could be well-established at
numerous sites far removed from localities where the sporophytes are known, as previously
suggested [14].
In this paper, we investigate the vertical distribution of both stages of B. viridis in
France (i.e., sterile protonema + sporophyte), and we study the ecology of the protonemal
stage from a conservation perspective. Do the protonema and sporophyte share the
same ecological requirements at two different scales, namely habitat (forest stand) and
microhabitat (decaying deadwood)?
2. Results
2.1. Distribution of Buxbaumia viridis
Buxbaumia viridis was found in 79 forests spread over the 12 French departments
(Figure 1). In 50 of them the protonemal stage was found. Sporophyte stage was found in
29 of them, which was always accompanied by gemmae.
Figure 1. All 137 habitats (forest stands) surveyed. Black lines: French departments limits. © Google Terrain.
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2.2. Habitat
Definition of habitat is given in Materials and Methods section. The altitudinal
distribution of the protonema localities ranged from 421 m to 1452 m a.s.l (Table 1a).
Sporophytes were observed at elevations between 840 and 1452 m a.s.l. More than half of
the protonema localities were on north- (27%), northeast- (16%) or northwest-facing (10%)
slopes. Of coniferous forests 74, of mixed 78, and of broad-leaved forests 15% contained B.
viridis (Table 1b). Only protonemal colonies were found in broad-leaved forests. Coniferous
forests were dominated by Pseudotsuga menziesii, Abies alba or Picea abies. Mixed forests
often consisted of Fagus sylvatica associated with P. menziesii or P. abies. Broad-leaved forests
were dominated by F. sylvatica or Acer platanoides.
Table 1. Description of the set of environmental quantitative variables (a) and qualitative
variable (b) of all stands surveyed (137), stands containing Buxbaumia viridis (sporophyte +
sterile protonema) (79) and stands containing sporophytes (29). No: number.
(a)
Variable
Mean ± SD
Min
Max
Elevation
(m a.s.l)
All stands
With B. viridis
With sporophytes
869 ± 273
946 ± 276
1142 ± 135
313
421
840
1452
1452
1452
Slope
(degrees)
All stands
With B. viridis
With sporophytes
17 ± 13
18 ± 14
17 ± 14
0
0
2
50
50
45
Distance to the
nearest watercourse
(m)
All stands
With B. viridis
With sporophytes
366 ± 593
279 ± 418
224 ± 222
8
8
10
3330
3190
1156
Deadwood surface
(%)
All stands
With B. viridis
With sporophytes
14 ± 7
18 ± 6
19 ± 6
3
3
5
33
33
33
Northness
All stands
With B. viridis
With sporophytes
0.29 ± 0.67
0.40 ± 0.61
0.55 ± 0.48
−1
−1
−0.71
1
1
1
(b)
Forest Type
No
All stands
No
with B. viridis
No
with sporophytes
Broad-leaved
Mixed
Coniferous
Total
40
27
70
137
6
21
52
79
0
3
26
29
Elevation (Figure 2a) and forest type (Figure 2b) were the only variables differing significantly
between habitats with sporophytes, and those with the protonemal stage only.
The occurrence of B. viridis is best explained by the extent of deadwood surface and
the forest type (Table 2a), and sporophyte occurrence is best explained by elevation, the
extent of deadwood surface and Northness (Table 2b). The probability of finding B. viridis
increases in mixed and coniferous forests compared to broad-leaved forests and increases
with increasing extent of deadwood surface. The probability of finding the sporophyte stage
increases with increasing extent of deadwood surface, greater elevation, and a northward
aspect.
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Table 2. Logistic regression results after model simplification using stepwise selection procedure
(the model with the smallest AIC (Akaike information criterion) was chosen). The first model (a)
tests the relationship between the occurrence of Buxbaumia viridis (sterile protonema + sporophyte)
as a dependent variable, and environmental variables at habitat scale as independent variables
(predictors). Model (b) is similar, except that the dependent variable is the occurrence of sporophytes.
Only significant variables were retained in the final model (Wald, p < 0.05).
Predictor
(a) Buxbaumia viridis
(Sterile Protonema + Sporophyte)
β
SE
p (Wald Test)
0.946
0.790
0.699
0.049
(b) Sporophyte
β
(Intercept)
Mixed forest
Coniferous forest
Deadwood surface
Elevation
Northness
−5.282
3.426
3.432
0.253
Observations
R2 Tjur
Hoslem–Lemeshow p
(Goodness-of-fit test)
135
0.555
135
0.418
0.190
0.854
<0.001
<0.001
<0.001
<0.001
SE
−10.023 1.853
0.096
0.007
1.250
0.043
0.001
0.522
p (Wald Test)
<0.001
0.026
<0.001
0.017
Figure 2. The number of occurrences of the protonemal stage of Buxbaumia viridis (light gray) and the sporophyte stage
(hatched) classified by elevation (a) and forest type (b). Dark gray: total stands surveyed. The two variables were significantly
different between habitats with sporophytes, and those with protonemal stage only (Kruskal–Wallis chi-squared = 9.7036,
–
df = 1, p < 0.05 for elevation, and χ2 test, p <χ 0.05 and absolute residual value >2 for forest type); * p ≤ 0.05. ≤
2.3. Microhabitat
A definition of microhabitat is given in Materials and Methods section. A total of 275
microhabitats were sampled, and 217 contained B. viridis (sterile protonema + sporophyte).
Gemmae occupancy had an average value of 30 ± 23 cm2 (min = 3, max = 135). Sporophytes
were found in 61 microhabitats with an average number of 7 ± 6 sporophytes (min = 1,
max = 34). Most of the microhabitats were logs because they were more numerous than
stumps.
The mean microhabitat diameter where B. viridis (sterile protonema + sporophyte)
occurred was 19.0 ± 11.8 cm (Table 3a). Canopy opening was on average 11 ± 4.6%, with a
minimum of 2.5% for microhabitats containing B. viridis. It was on average 12.8 ± 4.3%
for microhabitats containing sporophytes, and never went below 5.5% (Table 3a). Most
sporophytes developed on wood with a decomposition stage of 4 and 5, and only 3% on
wood with a decomposition stage of 3, whereas gemmae were found a little more on the
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latter (14%) (Table 3b). The species was never found on wood with a decomposition stage
of 2.
Table 3. Description of the set of microenvironmental quantitative variables (a) and qualitative
variable (b) of all microhabitats sampled (275), microhabitats containing Buxbaumia viridis (sterile
protonema + sporophyte) (217) and microhabitats containing sporophytes (61). No: number.
(a)
Variable
Mean ± SD
Min
Max
Canopy opening (%)
All substrates
With B. viridis
With sporophytes
12.0 ± 4.7
11.0 ± 4.6
12.8 ± 4.3
2.5
2.5
5.5
24.5
22.5
22.5
Slope (◦ )
All substrates
With B. viridis
With sporophytes
35 ± 29
34 ± 29
32 ± 30
0
0
0
90
90
90
Northness
All substrates
With B. viridis
With sporophytes
0.28 ± 0.66
0.29 ± 0.66
0.34 ± 0.61
−1
−1
−0.92
1
1
1
Bryophyte cover (%)
All substrates
With B. viridis
With sporophytes
26 ± 21
28 ± 21
30 ± 22
0
0
0
98
88
88
Diameter (cm)
All substrates
With B. viridis
With sporophytes
18.5 ± 11.2
19.0 ± 11.8
16.4 ± 6.4
10.0
10.0
10.0
90.0
90.0
90.0
(b)
Wood Decomposition
No
All substrates
No
with B. viridis
No
with sporophytes
Stage 2
Stage 3
Stage 4
Stage 5
Total
25
52
134
64
275
0
31
124
62
217
0
2
42
17
61
The main co-occurring species were Dicranum scoparium, which was present on more
than half of the microhabitats containing B. viridis (sterile protonema + sporophyte) (57%),
followed by Hypnum andoi (43%), Hypnum jutlandicum (41%), Herzogiella seligeri (37%),
species of the genus Cephalozia s.l. (30%), and Brachythecium rutabulum (25%).
Only canopy opening (Figure 3a) and the stage of wood decomposition (Figure 3b)
differed significantly between microhabitats with sporophytes, and those with protonemal
stage only.
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Figure 3. The number of occurrences of the protonemal stage of Buxbaumia viridis (light gray) and the sporophyte stage
(hatched) classified by canopy opening (a) and stage of wood decomposition (b). Dark gray: total stands surveyed. The
two variables were significantly different between microhabitats with sporophytes, and those with protonemal stage only
(Kruskal–Wallis chi-squared
= 31.254, df = 1, p-value < 0.05 for canopy opening, and χ2 test, pχ< 0.05 and absolute residual
–
value > 2 for stage of wood decomposition); * p ≤ 0.05.
≤
Gemmae occupancy was best explained by decomposition stages 3, 4, and 5 and
canopy opening (Table 4a). Gemmae occupancy increased with the stages of decomposition
(3, 4, 5) and slightly with reduced canopy opening. The number of sporophytes was best
explained by decomposition stages 4 and 5 and canopy opening (Table 4b). It increased
with advanced stages of decomposition (4 and 5) and with increased canopy opening.
Table 4. Results of model regression after model simplification using stepwise selection procedure
the (model with the smallest AIC was chosen). The first model (a) tests the relationship between
gemmae occupancy as a dependent variable, and environmental variables at microhabitat scale
as independent variables. Model (b) is similar, except that the dependent variable is the number
of sporophytes, and the model is a negative binomial regression. Only significant variables were
retained in the final model (t-test, p < 0.05).
Predictor
β
(a) Gemmae Occupancy
β
SE
p (t-Test)
(Intercept)
Decomposition stage 3
Decomposition stage 4
Decomposition stage 5
Canopy opening
0.465
1.539
2.654
2.862
−0.034
Observations
R2 Nagelkerke
275
0.602
0.348
0.317
0.287
0.310
0.017
<0.001
<0.001
<0.001
<0.001
0.045
(b) Number of Sporophytes
SE
p (t-Test)
β β
0.020
1.025
<0.001
15.288
14.457
1.164
0.940
0.956
0.031
0.004
0.006
<0.001
217
0.487
−
3. Discussion
3.1. The Independent Protonemal Populations
Our study provides clear evidence of the existence of independent protonemal populations of B. viridis and reveals that the species is certainly much more widespread in
France than previously realized. Gemmae were found more often than sporophytes, which
confirms that the currently national known range of B. viridis is obviously and widely
underestimated.
B. viridis is apparently a unique case in bryophytes, possessing a morphologically
distinct, independent, and persistent protonemal stage. In other bryophyte species where
persistent protonema are known (Ephemerum, Tetrodontium, Pogonatum), they are apparently
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never observed independently from sporophytes, but represent a transitory filamentous
phase. The independent protonemal stage of B. viridis is facultative in the sense that it may
or may not grow separately from sporophytes, as in the fern species Pleurosoriopsis makinoi [15]. As suggested by Duckett et al. [14], it is also similar to another fern, Vandenboschia
speciosa, which occurs as perennial, gemmiferous gametophytes that have been found in
many sites far from localities where the sporophytes are known [16].
Generally, species inhabiting small and transient microhabitats rely more on gemmae
formation to persist than species of larger and stable microhabitats [17]. The optimal
substrate for B. viridis is estimated to last for a few decades on logs, depending on tree
species, size (the smaller fragments lasting the shortest time), and humidity [4,18]. Usually
spores disperse over long distances by wind, and gemmae disperse predominantly over
short distances [2,19,20]. Our results suggest that gemmae are probably more efficiently
dispersed than realized. Pohjamo et al. [21] and Rumsey et al. [22] also assumed that asexual
propagules as well as spores may contribute to long-distance dispersal, with reference to
Crossocalyx hellerianus, and Vandenboschia speciosa, respectively. Our field observations of
frequently trampled and pecked logs suggest that gemmae could be carried efficiently by
mammals or birds [23] over considerable distances, or by other means. Nonetheless, it is
not known whether independent protonemal populations originate from the germination
of spores or gemmae and this should be studied further.
3.2. Habitat and Microhabitat Comparison between Protonemal and Sporophyte Sites
Deadwood surface was an explanatory variable shared by both the sporophyte and
protonemal stages: the two generations need a certain amount of deadwood to occur.
However, the other variables in their respective models differ: while forest type (mixed and
coniferous) was the other explanatory variable for the protonemal stage, the sporophyte
was best explained by Northness and elevation. Our results agree with those of Spitale
and Mair [3], who also found Northness and the volume of necromass to be environmental
predictors for sporophyte occurrence.
Sporophytes of B. viridis were found at a narrower range of elevation in the montane
zone, almost exclusively in coniferous forests (90%), and on wood in a more advanced
state of decomposition (stages 4 and 5). These results are supported by previous studies
(in ferns) which demonstrated that the sporophyte generation has a narrower ecological
habitat than the gametophyte [17,18]. The wood of coniferous trees (Abies or Picea) has
lower pH values in comparison to broad-leaved trees (Fagus) [24], which might seem
favorable for the development of B. viridis sporophytes [25]. Indeed, the study of Goia and
Gafta [26] demonstrated a negative correlation between beech wood and the occurrence
of B. viridis sporophytes. Another explanation of the supposed affiliation of B. viridis to
coniferous forests could be different management methods applied between both types of
forest [4,27]. Even though we found no significant correlation between deadwood surface
and forest type, the average deadwood surface area was lower in broad-leaved forests than
in mixed and coniferous forests. In 54% of broad-leaved forests, 10% or less of their surface
area was covered by deadwood. Müller et al. [27] also found lower numbers of trunks and
stumps in broad-leaved forests.
Elevation is an indirect variable influencing moisture, temperature, precipitation, and
solar irradiance [28], thus pointing towards a strong relationship between sporophyte
development and microclimatic variables. A detailed climatic study would certainly cast
some light on this most relevant issue. Even though no significant correlation was found
between the occurrence of B. viridis and the distance to the nearest watercourse, moisture
can be provided by other variables such as rainfall [3], or microtopography. Northness,
which was an explanatory variable for the occurrence of sporophytes, could also provide
cooler conditions and greater humidity. The late stages of decomposition in wood occupied
by B. viridis provide more stable humidity [18,29]. In addition, the species was almost
always found on small logs and stumps which are directly in contact with the soil, thus
enhancing water intake by capillarity and water retention [29]. At microhabitat scale, the
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protonemal stage can become established on less decomposed wood (stage 3 versus 4–5 for
sporophyte stage) that is more prone to desiccation.
Interestingly, another difference between the sporophyte and protonemal generations is their relation to canopy openness. The probability of finding a high number of
sporophytes increases as canopy opening increases, whereas gemmae occupancy follows
a reverse trend and increases as canopy opening decreases. We assume that the species
occurs frequently as protonema only, and that a slight opening of the canopy could be
one of the factors initiating the development of sporophytes. Observations of sporophytes
in B. viridis are mostly sporadic and scarce [9,30,31], and they could be explained by the
generally suboptimal availability of light at forest scale. Our results need to be further
investigated as the microtopography of the microhabitat could affect light availability,
and thus response to canopy opening [32]. Additionally, shadier localities could favor the
development of shade-loving bryophyte species that could outcompete B. viridis.
The protonemal stage may provide a biological explanation of observations of B.
viridis growing on unusual substrates, e.g., on humus [4,7,33], on a disintegrated Sphagnum
hummock [34], on bark at the base of trees [35], and at very low elevations [7,33]. Protonemal populations may occasionally differentiate into sporophytes as a consequence of
temporarily favorable ecological parameters (decreased temperature, high precipitation,
local opening of the canopy, etc.). The distribution and rarity of male plants could also
account for relative rarity of the sporophyte stage compared with gametophytic one but
this has not been addressed here.
3.3. Conservation Implications
In France, and probably at a European scale also, the range of the sporophyte stage
of B. viridis is currently well-known, but the same is not true for the protonemal stage,
whose distribution remains to be clarified. The conservation status of the species is likely
to change in coming years, with increasing knowledge of the protonemal stage.
B. viridis is often associated with old-growth forests as they tend to be more humid,
and to contain larger amounts of coarse wood debris [36]. However, over recent years,
increasing doubts have been raised about the status of B. viridis as a characteristic species
of ancient or natural forests [5,9]. Published studies and our own observations have
demonstrated the occurrence of the species in strongly managed forests [5,9], often in
young Douglas-fir or spruce plantations.
B. viridis is sometimes considered as an umbrella species (i.e., a species whose conservation entails the conservation of notable co-occurring species), and sometimes associated
with rare species such as Buxbaumia aphylla [33]. It would be worth reconsidering this
proposition, as we have consistently found B. viridis in assemblages of mundane species.
4. Materials and Methods
4.1. Practical Recognition of Buxbaumia viridis Non-Sporophytic Stage
The gametophytic stage of B. viridis can be recognized in the field (Figure 4) by the
combination of a whitish-grey film immersed in wood fibers (protonema) and discrete
chocolate-brown granular masses of protonemal gemmae. A confirmation is provided by a
microscopic examination, which shows characteristically multicellular, obloid and warty
gemmae (Figure 5) attached to undulating-anastomosing protonema (Figure 6) [14].
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Figure 4. Gametophytic stage of Buxbaumia viridis growing on wood-fibers.
Figure 5. Buxbaumia viridis gemma.
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Figure 6. Germinating gemma of Buxbaumia viridis, attached to protonema (note anastomosis on the left).
4.2. Study Area
This study took place in France in 12 departments from the oceanic Limousin (Corrèze,
Creuse) to the alpine regions (Isère, Haute-Savoie, Savoie), via the Massif Central (Allier,
Ardèche, Cantal, Haute-Loire, Loire, Puy-de-Dôme, Rhône).
Forest stands were selected, taking into account accessibility, occurrence of sufficient
amount
of dead wood, and representativity. Sufficient amount of dead wood corresponds
≥
to ≥
3%
of soil surface covered by dead wood. The work was undertaken at two spatial
≥
scales: habitat and microhabitat (Figure 7).
Figure 7. Representation of the two spatial scales used in the study. 1: Habitat (500 m2 ). 2: Microhabitat (300 cm2 ).
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4.3. Habitat Scale
Habitat was defined by means of forest stands, each covering 500 m2 and encompassing an ecologically homogeneous forest zone, thus excluding topographic intrusions
such as screes, rivulets etc. 137 habitat stands were selected in order to capture a relatively
large array of environmental factors. A similar sampling time was allocated in each of
the stands. We recorded GPS coordinates (Garmin, eTrex Vista HCx), forest type (i.e.,
coniferous, mixed, broad-leaved), dominant tree species (i.e., tree species covering more
than 50% of the stand surface) and aspect (using a compass). We visually estimated slope
(in degrees), and the area of soil surface occupied by pieces of deadwood with a diameter
>5 cm (% deadwood/500 m2 ), as a proxy for the amount of deadwood. The occurrence
of the protonemal stage and sporophytes was recorded. Elevation and distance from
the nearest watercourse (as a proxy for humidity) were assessed (in meters) off-site from
topographic maps on Geoportail and on Qgis (3.10.5).
4.4. Microhabitat Scale
Microhabitat has been restricted to logs and stumps, as a preliminary survey demonstrated that the gametophytic stage was very rare on other substrates (soil, base of trunks
etc.). Microhabitat was defined by the presence of favorable woody debris over an area
of 300 cm2 (Figure 1). 5 microhabitats colonized by B. viridis were sampled within each
of 55 habitat stands (275 microhabitats recorded). A grid of 30 cm × 10 cm was placed
on the woody debris (Figure 8), within which the cover of gemmae (%/300 cm2 ) and the
number of sporophytes (young, old, and eaten) were recorded. The type of deadwood
(i.e., log or stump) was noted and its diameter was measured (in centimeters with help
of a caliper). Diameter was sometimes impossible to measure when microhabitat was
completely crumbled away on soil. Slope was visually estimated (in degrees), and aspect
was recorded (using a compass). The degree of decomposition of the wood was determined
according to the 5 decay classes proposed [37], going from stage 1 (hard texture, intact bark,
round shape, branch present) to stage 5 (soft and powdery texture, no branch, no bark,
partly sunken on ground). See Bunnel and Houde Figure 1 for more details [37]. We also
noted the dominant type of fungi associated with the decay of the wood (i.e., red or white).
Canopy openness was measured using a convex densiometer (Forestry Suppliers, Spherical
Crown Densiometer, Model A) (% of sky). Four readings facing North, East, South, and
West were taken about a microhabitat and averaged. Finally, we identified co-occurring
bryophyte species within the grid, with a visually estimate of their cover (%).
Figure 8. A grid of 30 cm × 10 cm placed on a decaying wood. Groups of Buxbaumia viridis gemmae correspond to the
small black dots whose main areas are circled in red.
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4.5. Statistical Analysis
A map of the distribution of B. viridis was prepared using Qgis (version 3.10.5).
The aspect variable was converted to ‘Northness’ according to the formula: Northness = cos((aspect in degrees × π)/180), to simplify the analysis [3]. A value close to 1
corresponds to a northward aspect.
Normality was tested using the Shapiro–Wilk test. Since the data do not follow
a normal distribution, we used the Kruskal-Wallis non-parametric test to compare the
quantitative environmental variables between sporophyte and protonemal sites (habitats
and microhabitats). Comparisons of qualitative variables were made using a χ2 test. If the
p value was significant for the χ2 test, it was followed by an analysis of the residuals to
determine which modalities of the categorical variables were “significant”: residual with
absolute value >2 is considered significant [38].
To test the influence of the environment on the occurrence of B. viridis, multiple logistic
regressions were performed between the binary dependent variables (“presence/absence”
of the species (sterile protonema + sporophyte), and “presence/absence” of sporophytes),
and the independent environmental variables (forest type, elevation, slope, Northness,
deadwood cover on soil, and distance to the nearest watercourse) as predictors. A stepwise
selection was made by simultaneously adding and dropping predictors in the model
until it led to the best model (i.e., the one with the smallest AIC). The significance of
explanatory variables confirming that they play a role in the model was assessed using
Wald test (p value < 0.05). Multicollinearity between independent variables was tested
by checking for the variance inflation factor (VIF). Goodness of fit was estimated using
Hosmer–Lemeshow test (p value > 0.05). Imperfection detectability (i.e., the inability to
detect a species despite its presence) was not considered in our model. This should not
be an issue according to Spitale and Mair [3], who demonstrated that the model ignoring
imperfection detectability was sufficient to identify the most important environmental
factors for species distribution.
Following the same steps as explained above (except for the Hosmer–Lemeshow test),
multiple linear regressions were performed to test the influence of microenvironment
(using canopy openness, slope of the substrate, bryophyte cover, stage of wood decomposition, and Northness of the substrate as the independent variables) on the dependent
variables (1) gemmae occupancy (log-transformed), and (2) number of sporophytes. For
the latter, negative binomial regression was preferred to Poisson since the data presented
overdispersion. Microhabitat diameter was not included in the models since the analysis
concerns gemmae occupancy and number of sporophytes within the 300 cm2 . All statistical
analyses were conducted on R (version 3.5.1).
5. Conclusions
Many questions about the ecology and distribution of this species remain to be investigated further. B. viridis could be dynamically expanding its range by means of gemmae
dispersal, in recent coniferous plantations that are maturing into a favorable condition for
a deadwood-dwelling species. In fact, in France, many plantations are nowadays several
tens of years of age, the amount of time necessary to begin to accumulate decaying woody
pieces. Diachronic monitoring could be conducted to evaluate population dynamics. The
sporophytes of B. viridis are generally considered weak competitors [5], but this may not be
the case for the protonemal stage, as our preliminary observations suggest that it is capable
of long-term durability even in closed bryophyte communities (very often behaving as
bryo-epiphytic).
Cultivation experiments could answer some biological questions: is the independent
protonemal generation of B. viridis isolated by a failure to reproduce sexually, or only by
limiting environmental variables? Is there a similar independent protonemal stage in other
species of the genus Buxbaumia?
Plants 2021, 10, 83
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Author Contributions: Conceptualization, V.H., F.P.; methodology, V.H., F.P. and A.G.; formal
analysis, A.G. and V.H.; investigation, A.G., V.H. and F.P.; writing—original draft preparation, A.G.,
V.H.; writing—review and editing, A.G., V.H. and F.P.; project administration, F.P. All authors have
read and agreed to the published version of the manuscript.
Funding: This research received no funding.
Institutional Review Board Statement: The study was conducted according to the guidelines of
the Declaration of Helsinki, and approved by the Direction régionale de l’Environnement, de
l’Aménagement et du Logement, arrêté préfectoral 07-2020-05-15-001
Informed Consent Statement: Not applicable.
Data Availability Statement: The Data are not publicly available as they are an essential part of
further publications under preparation.
Acknowledgments: Tom Blockeel offered many corrections and suggestions that helped us very
much to improve this text. Comments of reviewers were highly appreciated.
Conflicts of Interest: The authors declare no conflict of interest.
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