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Article

Differential Effects of Desiccation on Hornworts with Contrasting Life Histories in Tropical Montane Forests: A Functional Trait—Based Perspective

by
Gabriel F. Peñaloza-Bojacá
1,*,
Tiago Vilas-Boas
2,
Juan C. Villarreal A
3,4 and
Adaíses S. Maciel-Silva
1
1
Laboratório de Sistemática Vegetal, Departamento de Botânica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, Brazil
2
Laboratório de Fisiologia Vegetal, Departamento de Botânica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, Brazil
3
Département de Biologie, Université Laval, Québec, QC G1V 0A6, Canada
4
Smithsonian Tropical Research Institute, Panama City 0843-03092, Panama
*
Author to whom correspondence should be addressed.
Forests 2023, 14(2), 255; https://doi.org/10.3390/f14020255
Submission received: 23 December 2022 / Revised: 14 January 2023 / Accepted: 26 January 2023 / Published: 29 January 2023
(This article belongs to the Special Issue Functional Traits of Forest Plant Communities—Limitations and Potentials)
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Abstract

:
Desiccation tolerance (DT) is the ability of an organism or structure to dry completely and subsequently survive in that air-dry state. Hornworts are excellent plant models to study desiccation effects as they have contrasting life histories which are likely associated with DT. We tested whether (1) epiphytic species had more efficient DT responses to drying and postrehydration than non-epiphytic species and whether (2) “green” spores were more sensitive than non-green spores to extreme drying. Hornwort species were collected from the Atlantic Forest of Rio de Janeiro, Brazil. We studied five species (gametophytes and spores: Dendroceros crispus, D. crispatus, Nothoceros vincentianus, Phaeoceros carolinianus; and only spores of Anthoceros lamellatus), using different relative humidity values, drying durations, and postrehydration conditions. All DT treatments affected the chlorophyll fluorescence (Fv/Fm) of gametophytes, with species-specific responses. D. crispatus and D. crispus (epiphytes) performed better than P. carolinianus and N. vincentianus, with fast recovery of Fv/Fm values postrehydration. The ability of non-green spores of P. carolinianus and A. lamellatus and green spores of D. crispus to support desiccation led us to reject our second hypothesis. The DT strategies of hornworts highlighted the trade-offs that are important in spore dispersal and plant establishment, such as fast colonization in Dendroceros spp. and potential spore soil banks in Phaeoceros and Anthoceros species.

Graphical Abstract

1. Introduction

Desiccation tolerance (DT) is the ability of an organism or structure to air-dry completely and subsequently survive in this state [1,2,3,4]. This trait occurs mainly in cyanobacteria, lichens, green algae, and bryophytes [5,6,7], and may have played an important role during the colonization of land by plants [2,8,9,10]. DT is a key factor in plant life cycles, influencing the responses of both their reproductive (e.g., spores and seeds) and vegetative tissues [2,8,11,12,13]. Desiccation tolerance has been reported in less than 0.15% of all vascular plant species in regards to their ability to tolerate water contents (WC) near 30%–40% at c. 94%–95% relative humidity (RH) [8,14,15]. Conversely, DT in bryophytes (liverworts, mosses, and hornworts) has been characterized by gametophytes surviving ~10% WC, which can result from a range of RH values less than 50%, and then recovering their metabolic activity even after long periods of time [2,8,16,17].
Multiple factors are used to accurately assess DT in bryophytes: (1) the rate of drying (RoD); (2) water content at equilibration (equilibrating relative humidity, RHeq); (3) the duration dry; and (4) the rate of rehydration (RoR; for details see [2,18,19] . The desert moss Syntrichia ruralis, for example, was kept in its dried state at ~30% RH for 20 years and when subsequently rehydrated for 28 days it showed regenerated shoots and a good parameter of chlorophyll fluorescence, at c. 0.35 [10]. Although the DT has been investigated in multiple taxa of bryophytes in recent decades ([2,8] and references therein), including the responses of their gametophytes and spores, there is long-standing uncertainty regarding whether hornworts are desiccation-tolerant species. For instance, Wood (2007) classified the hornwort Dendroceros granulatus as a desiccation-tolerant plant based on a personal communication from Michael C. F. Proctor, but there is no experimental data focusing on the ability of hornworts to withstand desiccation. Moreover, this knowledge gap makes our understanding of the hornwort ecology in tropical forests quite shallow, consequently hindering conservation proposals for rare and endangered species.
Hornworts are a small group of land plants, comprising c. 250 species [20] with a dominant gametophyte phase, and they display various features that are unique among land plants. All hornwort species have symbiotic relationships with fungi and/or endophytic nitrogen-fixing bacteria [21,22,23] and can present a pyrenoid-based biophysical carbon-concentrating mechanism [24,25]. Hornwort phylogeny has recently been resolved, with one of their key morphological traits being their spore color and ornamentation (Figure 1A; [25,26,27,28]). Most recent phylogenies propose hornworts as a sister group to mosses and liverworts [29], as part of the bryophyte clade [30,31].
Hornworts grow in many varied niches, such as on soils and rocks (e.g., Anthoceros and Phaeoceros) and on rotten logs near rivers and streams (most Nothoceros species), and they can also be found as epiphytes on live trunks, twigs, and leaves (the genus Dendroceros; [31,32,33,34]). Epiphytism is commonly related to desiccation tolerance, as most epiphytic plants have adaptations to deal with drying [35,36,37] and many bryophyte epiphytes tend to curl inwards when in a dry state [38,39]. Following this idea, we expected that epiphyte hornworts would also display desiccation-tolerant traits.
Spore color and ornamentation have been suggested as features of protection against desiccation in hornworts. Yellow/dark spores with thick walls are supposedly more resistant than those with thin walls and a prominent plastid, thus appearing “green” [28,40,41,42]. Differential rates of metabolism, storage sources, and longevity have been recorded for the different spore types [28,36,43], with “green” spores assumed to germinate faster than darker spores, which are typically found in soil banks [44]. Although spores of Phaeoceros and Anthoceros are yellow, brown, or black (non-green spores), Nothoceros and Dendroceros have “green” spores [32,45], suggesting contrasting DT responses in these species. Hornwort spores also have different germination types, which are likely associated with DT strategies. For instance, unicellular spores (e.g., in Nothoceros, Anthoceros, and Phaeoceros; [28,46,47]) raise gametophytes directly in contact with the substrate, whereas multicellular spores develop sporelings located inside their walls and protected from desiccation (e.g., endospory in Dendroceros [28,36,48].
Our main goal was to test for DT in different hornwort species. As discussed above, hornworts present very contrasting life histories that may be associated with DT strategies, which prompts several questions. Are hornworts, in general, desiccation-tolerant? Does the species niche influence the DT of the plant? Do spore traits have differential effects on DT responses?
To that end, we investigated the gametophytes and spores of species of the genera Dendroceros, Nothoceros, Phaeoceros, and Anthoceros (only spores in the latter) and hypothesized that (1) Epiphytic species have a more efficient DT response to drying and postrehydration treatments than non-epiphytic species. We expected the gametophytes of the Dendroceros species, compared to the gametophytes of Nothoceros and Phaeoceros, to present higher or constant chlorophyll fluorescence during the different RHeq, dry duration, and postrehydration treatments. Conversely, we also hypothesized that (2) “green” spores would be more sensitive than non-green spores to extreme drying. We expected spores of Anthoceros and Phaeoceros to have higher survival and growth rates than those of Dendroceros and Nothoceros under the different desiccation treatments.

2. Materials and Methods

2.1. Species Descriptions

We examined the following five species of hornworts: (1) Dendroceros crispatus (Hook.) Nees: a gametophyte with galeate wings, a solid midrib, one chloroplast per cell, with pyrenoids, and rectangular cells in the capsule with nodular wall thickenings [27]. (2) Dendroceros crispus (Sw.) Nees: a flat undulate or crispate gametophyte with a solid midrib, one chloroplast per cell, with pyrenoids, and rectangular cells in the capsule with wall thickenings. Both of these epiphytic species produce multicellular green spores (Figure 1B) and are found in forests at 800–2000 m a.s.l. [27,32]; see Figure 1F,J. (3) Nothoceros vincentianus (Lehm.) J.C. Villarreal: a pinnately branched gametophyte with 1–2 chloroplasts per cell, the absence or the presence of pyrenoids, rectangular cells in the capsule, and unicellular spores that are green at maturity (Figure 1C). This species is terricolous or saxicolous in Neotropical forests above 400 m a.s.l., along streams [28,49]; see Figure 1G,K. (4) Phaeoceros carolinianus (Michx.) Prosk.: a gametophyte with a rosette shape with a smooth margin, one chloroplast per cell, with pyrenoids, rectangular cells in the capsule with stomata, and unicellular spores that are yellow at maturity (Figure 1D). This species has a worldwide distribution, growing on soils and exposed areas above 300 m a.s.l. [32,50,51]; see Figure 1H,L. (5) Anthoceros lamellatus Steph. A.: a gametophyte with a rosette shape, with crenulate margins, abundant dorsal lamellae, one chloroplast per cell, pyrenoids present, rectangular cells in the capsule with stomata, and unicellular spores that are dark brown to blackish at maturity (Figure 1E). It has a neotropical distribution, growing on soils and exposed areas above 1000 m a.s.l. [28,52]; see Figure 1I,M.

2.2. Field Sampling

We carried out field collections in January and February 2020 in the Atlantic Forest in the state of Rio de Janeiro, Brazil. We collected samples (c. 8 cm2) of the hornwort species (except A. lamellatus) in the forest located in the Serra da Bocaina National Park, 22°46′04″ S and 44°36′34″ W, 1237–1524 m a.s.l., 17 °C–23 °C, with a mean rainfall of 1700–3000 mm.year−1 [53]. Anthoceros lamellatus samples were collected along the roadside next to the forest on the way to the Três Picos State Park, 22°20′04″ S, 42°42′04″ W, 1250–1368 m a.s.l., 17 °C–21 °C, with a mean rainfall of 1000–2000 mm year [54]. All samples presented gametophytes with rhizoids and mature sporophytes. Plants were transported in hermetically sealed plastic pots to the laboratory, thus maintaining a high internal relative humidity (>98% RH) and preventing their acclimation (acclimation to desiccation occurs during a dry-down or partial drying event; [10]).

2.3. Deacclimation

To assess desiccation tolerance and avoid variations due to unknown field hardening effects, we deacclimated plants for 15 days (effective time in Sphagnum species; [10,55]) using a 12 h photoperiod, at 21 °C, 74 µmol of photons m−2 s−1, and RH > 98% in a culture room. Deacclimation is a process in which plants collected in the field are subjected to uninterrupted hydration under non-stressful conditions, overcoming any physiological hardening to desiccation that may have been obtained in the field [10]. In the same way, a period of 15 days can help stimulate plant growth under non-stressful conditions, ensuring complete deacclimation (Figure 2). A shorter period of time may not ensure deacclimation, as in Exormotheca Holstii or Crossidium, in which signs of hardening still persisted after 7 days [10,56]. This process ensured that suprasaturation conditions were applied to gametophytes and spores before the experiments began. Twenty-four hours before the end of the deacclimation period, we selected adult gametophytes from each species (here defined as plants with (a) male and female gametangia, (b) cyanobacteria colonies, (c) lengths greater than 2 cm, and (d) mature sporophytes (with signs of apical dehiscence)). Those plants were cleaned while viewing them under a stereomicroscope with the help of needles and forceps, washing them several times with deionized water until all excess unwanted material was removed. Subsequently, they were kept in deionized water until the completion of the deacclimation period.
To control the rate of drying, we followed the methodology proposed by [2,10,13], with modifications, using cell culture well plates with two sheets of filter paper (18 mm2, 3 µm porosity), 20 µL of sterilized water, a digital thermohygrometer (Htc-2A) to monitor the temperature and internal humidity of the desiccation chambers, and HR 85%. We standardized the rate of drying based on measurements conducted over 24 h, that is, from full turgor (blotted) to plant curling. The volume of water and the rate of drying period had been previously tested in the laboratory (unpublished data—for details RoD see Stark 2017 and references). Deacclimated gametophytes and sporophytes were gently passed through filter paper to remove excess surface water, before being placed into the well plates. We used a total of 27 gametophytes per species, with three replicates and one individual plant per well. We used a total of 81 sporophytes per species, with three replicates and three mature sporophytes per well (Supplementary File S1). Finally, we applied an equilibrium time period, in which the plates with gametophytes or sporophytes and covers were placed in the desiccation chambers for 24 h at different relative humidity treatments (RHeq [10]) and before the period referred to as the duration dry (Figure 2, see below). Once this period was complete, we removed the covers from the plates.

2.4. Experimental Design and Desiccation

Deacclimated gametophytes and sporophytes were used in all our assays (Figure 2). During the DT experiments, the plants were maintained under constant temperature and light conditions (21 °C, 74 µmol of photons m−2 s−1) in a culture room with a 12 h photoperiod. To evaluate the effects of desiccation times and intensities for each species (as described above), we prepared desiccation chambers (airtight plastic boxes of 38 × 56 × 37 cm and, internally, an Htc-2A digital thermohygrometer), with saturated salt solution in Petri dishes to provide constant internal RHs. Equilibrating relative humidity treatments (RHeqs) were achieved using the following saturated saline solutions: RHeq 56% (Ca [NO3]2), RHeq 74% (NaCl), and RHeq ~99% with deionized water.
Our experimental design for the measurement of DT in gametophytes was as follows: four species with three replicates each × three RHeqs × three dry durations × three postrehydration times. For sporophytes: five species with three replicates each × three RHeqs × three duration dry × one postrehydration time. The duration dry periods were 1 day (1D), 3 days (3D), and 9 days (9D). Postrehydration was applied to all replicates, with three different times: 24 h for gametophytes and sporophytes and 72 h and 216 h for gametophytes. These Postrehydration times were observed in the same number of replicates for gametophytes. One period of 8 h of pre-hydration (in which desiccated gametophytes and sporophytes were submitted to a relative humidity of ~99% RHeq) was applied in all postrehydration treatments before adding liquid water [10,57]. The plants and sporophytes were subsequently rehydrated by adding 2 mL of deionized water directly onto the filter paper, keeping them immersed until the completion of the different postrehydration times [10,13].
To assess gametophyte responses to DT, we measured the maximum photochemical quantum yield of PSII (Fv/Fm), using the equation: Fv/Fm = (Fm − Fo)/Fm, where Fm is the maximum fluorescence and Fo is the fluorescence in the absence of actinic light [58,59]. Fv/Fm measurements were taken in the morning, before the start of the light period, so that the plants had been under 12 h of dark adaptation. The latter is related to PSII reaction centers, which are open after dark adaptation and which decrease under dehydration stress; thus, this can be used as a measure of survival under drought conditions in bryophytes [17,60,61]. Chlorophyll fluorescence emissions were assessed using a pulse-amplitude modulation fluorometer (model MINI-PAM, WALZ, Effeltrich, Germany) with a light saturation pulse of ~5000 μmol m−2 s−1.
To assess DT in spores, we collected spores from the capsule apex (sporogenesis is asynchronous in these hornworts and the capsule apex contains mature spores) from the sporophytes under different treatments. Spores from three capsules were mixed and cleaned with deionized and sterilized water and plated for 30 days in Petri dishes on solid culture medium (Knop II with 0.5% agar, pH 5.6; [62], using a 12 h photoperiod at 21 °C and 74 µmol of photons m−2 s−1. We measured spore survival (n = 100) and sporeling size (50) per replicate. Survival was measured as viable spores or germinated spores when they showed signs of germination after 30 days (i.e., the development of a rhizoid or germ tube or several green cells inside the spore wall), or, to the contrary, non-viable spores after 30 days (no signs of development, being hyaline or dark brown, with no green chloroplasts and no rhizoid or germ tube). Sporeling size was analyzed using BEL Capture Application software (400× magnification), assessing their lengths (in mm). Additionally, after deacclimation and before beginning our experiments, we determined Fv/Fm reference values for the gametophytes, as well survival and growth values for spores of the study species at RHeq 56%, 74%, and RHeq ~99% (Supplementary Figure S1).

2.5. Statistical Analyses

We generated generalized linear models (GLMs) to test our hypotheses and verify our predictions. The effects of “species”, “RHeq”, “dry duration”, and “postrehydration” treatments on the “Fv/Fm” values were tested with the GLM (Gaussian distribution and identity link function; model details provided in Supplementary File S2). The effects of “species”, “RHeq”, and “dry duration” on “spore survival” and “sporeling size” were analyzed with a model equivalent to the one above. Since the response variable “sporeling size” had spores as a random factor nested in each replicate, we attempted to include that factor in our previous model. Because of singularity effects, however, we preferred to exclude it and to use the simplest and most powerful (Akaike’s information criteria, AIC) model for our analyses. Additionally, analyses of deviance for GLMs and post hoc comparisons (Tukey test) were applied. The latter test was used to evaluate all pairwise differences at α = 0.05. All analyses and graphs were implemented in R [63] using RStudio V.3.6.2 software [64], with the ggplot2 [65] and MASS packages [66].

3. Results

3.1. Desiccation Effects on Hornwort Gametophytes

All DT treatments (RHeq, dry duration, postrehydration) had significant effects on the Fv/Fm of gametophytes of the study species; significant interactions of these treatments with “species” indicated that the Fv/Fm in hornwort species exhibited very distinct behaviors under different desiccation treatments (Table 1; Figure 3). In general, the species with the highest Fv/Fm values were D. crispatus (mean ± se: 0.515 ± 0.012) and D. crispus (0.443 ± 0.018), as compared to P. carolinianus (0.188 ± 0.025) and N. vincentianus (0.144 ± 0.020; Supplementary Figure S2, File S3). RHeq treatments of 56% and 74% had similar negative effects on the gametophytes, compared to 99% RHeq, generating damage mostly in the gametophytes of P. carolinianus and N. vincentianus. We observed signs of regeneration in the gametophytes of P. carolinianus at 99% RHeq after three days of desiccation (Figure 4, red arrows). Although several plants recovered their photosynthetic capacities, the chlorophyll fluorescence of the gametophytes in our experiment did not reach the reference values of recently deacclimated plants.
In terms of the dry duration, the Fv/Fm values of hornwort gametophytes decreased from 1D (0.360 ± 0.022) to 3D (0.371 ± 0.023) and to 9D (0.023 ± 0.020) (Supplementary Figure S2). The significant interactions we observed between “dry duration” and “species” and “RHeq” were mostly the result of the gametophytes of D. crispus and D crispatus being less affected by drying than those of P. carolinianus and N. vincentianus. These latter species had the lowest Fv/Fm values among the different treatments (Figure 3).
We observed a tendency of the gametophytes to recover their Fv/Fm values after all the tested postrehydration times, with better recovery rates recorded after the postrehydration times of 24 h (0.25 ± 0.021), 72 h (0.32 ± 0.024), and 216 h (0.40 ± 0.020) (Supplementary Figure S2). The Fv/Fm values in D. crispatus and D crispus increased similarly after 24 h and 72 h of postrehydration for all RHeqs values, and after dry durations of 1D and 3D. At 216 h, the Fv/Fm of both species increased for all RHeqs values only under 1D of drying (except for D. crispus at 74% RHeq and for D. crispatus at 56% Rheq), however, indicating an effect of duration dry on species’ responses after postrehydration. Chlorophyll fluorescence in N. vincentianus and P. carolinianus showed an inverse pattern, with low values at 24 h and 72 h that increased after 216 h for all dry durations and RHeqs treatments (Figure 3).

3.2. Desiccation Effects on Hornwort Spores

Although the hornwort species differed significantly in terms of their spore survival rates, no main effect of RHeq values and dry durations was observed on the spore survival of the hornworts (Table 1 and Figure 5A). In general, spore survival differed among hornwort species, as follows: Phaeoceros carolinianus (84.5% ± 1.56%), followed by D. crispus (82.0% ± 1.02%), A. lamellatus (69.4% ± 3.67%), N. vincentianus (61.3% ± 4.55%), and D. crispatus (53.0% ± 3.68%). We observed significant interactions of “species” with “RHeq” (Table 1) in A. lamellatus and P. carolinianus, with better spore survival in lower RHeqs treatments. In contrast, D. crispus, D. crispatus, and N. vincentianus exhibited their best spore survival at higher RHeqs (Figure 5A). P. carolinianus and A. lamellatus spores exhibited similar survival rates after desiccation treatments as compared to recently collected plants.
Sporeling sizes varied in response to RHeq treatments, with species exhibiting decreasing sizes from 56% RHeq (0.40 mm ± 0.007), followed by 74% (0.296 mm ± 0.005) and 99% (0.298 mm ± 0.005; Figure 5B). Phaeoceros carolinianus, A. lamellatus, and D. crispus had smaller sporelings with 74% and 99% RHeqs treatments. Dendroceros crispatus and N. vincentianus produced small sporelings in all treatments. Significant interactions were observed between “dry duration” and “species” (Table 1), with the longer the dry duration, the larger the sporelings of P. carolinianus, A. lamellatus and D. crispus; whereas D. crispatus and N. vincentianus had the smallest sporelings at 3D as compared to 1D and 9D. The sporelings of P. carolinianus, A. lamellatus, and D. crispus reached the reference values of sporeling size after the 9D desiccation treatment with 56% and 74% RHeqs (Supplementary Figure S1).

4. Discussion

We corroborated our first hypothesis: the gametophytes of epiphytic species (D. crispatus and D. crispus) were more tolerant of desiccation than the non-epiphytic species, and Phaeoceros carolinianus and N. vincentianus gametophytes suffered greater physiological and morphological damage. Our second hypothesis was rejected, as P. carolinianus and A. lamellatus (non-green spores) and D. crispus (green spores) showed better spore survival and sporeling sizes in desiccation treatments, distinct from D. crispatus and N. vincentianus (green spores), of which the same parameters were the lowest.

4.1. Desiccation Tolerance of Hornwort Gametophytes

The gametophytes of the epiphytic species studied here exhibited less morphological injury and damage to photosystem II (Figure 2 and Figure 3), as was suggested earlier for Dendroceros species [2,36]. Epiphytism, unique plastid traits, and a star-shaped chloroplast with a complex pyrenoid are assumed to provide protection for the photosynthetic apparatus of Dendroceros during drying periods [2,36,67]. A relationship between DT and epiphytism has been widely observed in other tropical plants, including the mosses Prionodon densus and Dicranoloma fragiliforme, the liverwort Frullania peruviana, and the fern Microgramma reptans, of which the gametophytes do not show morphological or physiological damage after drying periods with RHeqs values lower than 50% [2,35,37,68].
Additionally, the failure of N. vincentianus and P. carolinianus gametophytes to restore normal morphological and physiological parameters after prolonged drying regimes at RHeqs <99% expressed their low DT capacities (Figure 2 and Figure 3). The niches of both species may be linked to their DT responses, as N. vincentianus is commonly found in very humid and shady areas (e.g., along rivers and streams or inside forests) and P. carolinianus usually grows on moist soil in exposed sites (e.g., roadsides and ravines). These niches contrast greatly, with the latter usually being colonized by bryophytes that are resistant to occasional dry periods [8,69]. We assume, therefore, that the lower DTs of N. vincentianus and P. carolinianus gametophytes, even if they are only for short periods of time, are related to protective mechanisms, such as their sugar contents or constitutive cellular protection by abscisic acid (ABA; [2,8,10,70]).

4.2. Postrehydration Effects

Epiphytic species responded more effectively to short postrehydration periods of 24 h and 72 h (with a pre-hydration period of 8 h for all of the treatments used here) than the non-epiphytic species did, which can be explained by the rate at which bryophytes rehydrate—as the highest performance plants commonly have high surface areas and low weights [10,71]. Phaeoceros and Nothoceros plants are thick, with several layers of cells in their thalli, whereas Dendroceros species are thinner and have uniseriate wings. We observed different responses among the gametophytes of Dendroceros species after a postrehydration period of 216 h; Dendroceros crispus responded better than D. crispatus with longer rehydration (Figure 3). Although D. crispatus is a galeate gametophyte with many macro-perforations and pores in its wings, D. crispus is a crispate gametophyte (with a higher surface area) with few and irregular pores [27].
The responses of the N. vincentianus and P. carolinianus gametophytes, which showed better recovery after 216 h of postrehydration, suggest the need for longer rehydration times. The desiccated cell membrane in those species may require greater exposure to a saturated atmosphere and free water to recover and mitigate the injuries caused by drying, compared to Dendroceros species [10,72]. Another important and unprecedented factor in hornworts is the strategy used by P. carolinianus at an RHeq of 99% with a postrehydration time of 216 h for all drying treatments (mainly 1D and 3D) for gametophyte regeneration (Figure 4). This strategy has been reported for other groups of bryophytes (e.g., Octoblepharum, Orthodontium, and Dicranoweisia), with asexual propagule or vegetative regeneration being associated with habitat drying and directly influencing dispersal and establishment processes in these species [8,73,74,75].

4.3. Desiccation Tolerance of Hornwort Spores

Phaeoceros carolinianus and A. lamellatus exhibited higher spore survival and larger sporeling sizes under the different desiccation treatments, as was expected. Both species have thicker spore walls than those found in green spores, which apparently provides protection against desiccation [28,76]. Additionally, the inner spore walls in Phaeoceros and Anthoceros contain callose [77], an important polymer for the construction and maintenance of wall integrity [78]. Spores with callose may deal well with desiccation as that polymer provides a protective matrix [77,79,80], although that trait alone is not enough to explain all of the differences observed in our study (e.g., Nothoceros vs. Dendroceros).
Phaeoceros and Anthoceros spores also contain oils and starch as storage compounds, which can help provide greater longevity and drought resistance [28,36]. However, unlike green spores, they require longer times to mobilize their storage compounds and to develop protonemata (e.g., early gametophytes [28,41,43,81]). Phaeoceros and Anthoceros, as well as other hornworts with non-green spores [82], produce gametophytes with short lifespans that are able to resist dry seasons as spores in soil banks [44].
The DT responses of green and multicellular spores (e.g., endosporic germination) of Dendroceros species are likely associated with endospory. This trait is present in other bryophytes and assumed to be an adaptation to environments that experience periodic desiccation [36,83,84]. The differential responses of the spores of the two Dendroceros species could explain why D. crispus is widely distributed compared to D. crispatus [85]. The spores of N. vincentianus demonstrated the lowest performance due to their exosporic germination, which exposes the delicate protonema to drought conditions. The green spores of Dendroceros crispus, D. crispatus, and N. vincentianus also lack storage space for large amounts of oil [28,77,86]. This is unlike the unicellular green spores of some ferns, such as Osmunda regalis, which present lipids with the ability to protect the integrity of the membrane during desiccation [87].

4.4. Ecological Strategies of DT in Hornworts

We observed complex trade-offs of DT responses among the different life phases of hornworts, with contrasting life strategies linked to niche requirements, including (1) gametophytes that were sensitive and spores that were strongly tolerant of drying (P. carolinianus and A. lamellatus), (2) gametophytes and spores that were tolerant of dry periods (D. crispus), (3) tolerant gametophytes and sensitive spores (D. crispatus), and (4) both gametophytes and spores that were sensitive to drying (N. vincentianus). The above responses are similar to those described in the DT continuum hypothesis proposed for bryophytes [1], which states that plant life history phases (e.g., protonemata, juvenile shoots, adult shoots, spores, and others) can have different DT strategies, even within the same species and along an inducible or constitutive tolerance gradient [1,88].
This is the first study to highlight these trade-offs in hornworts, in line with similar differential DT strategies reported in both vascular and non-vascular plants. Sporophytes of the desert moss Tortula inermis, for example, were found to be more sensitive to rapid drying than their gametophytes [89]. The protonemata vs. juvenile shoots in Bryum argenteum [13] and the asexual propagules vs. shoots of Syntrichia pagorum also exhibited different DT responses. Equally, the gametophytes of some fern species (e.g., Asplenium auritum and Polystichum retroso-paleaceum) are more tolerant than their sporophytes [68,88,90]. The differential life strategies of hornworts may be important evolutionary triggers for spore dispersal and the maintenance of gametophytes in the field.
Finally, we have highlightd the importance of hornworts in tropical montane cloud forests, especially Dendroceros species as potential indicators of climate change. This genus presents unique ecological characteristics, such as its altitudinal distribution range (700–1000 m in Brazilian forests and up to 2300 in Colombian forests), its epiphytic niche, the height of the phorophyte (20–200 cm), and the degree of DT (gametophytes vs. multicellular spores). The aforementioned characteristics make this plant group particularly sensitive to climate change, as reported for vascular epiphytic species [91,92,93]. In addition, these unique characteristics of Dendroceros can be related to the seasonality and conservation status of cloud forests and indicate possible alterations in their ecosystems [94]. Climate change may directly affect the climatic conditions of the forest, together with the diversity, distribution, establishment, and spread of Dendroceros and other hornwort species [95,96,97].

5. Conclusions

Our study highlights the existence of a DT continuum among hornwort species, with different life strategies associated with life phases (spores vs. gametophytes), niches, genera, and species. We corroborated the observed DT behaviors of Dendroceros gametophytes, which were linked to their epiphytic niche in montane forests. The endospory of Dendroceros spores conferred a moderate DT response, especially in D. crispus, although the high performance of the non-green spores of Phaeoceros and Anthoceros indicated their excellent potential to form soil banks in open habitats. The high desiccation sensitivities of both the spores and gametophytes of Nothoceros vicentianus are likely associated with the very humid niches in which that species lives. Differential DT strategies may increase the chances of spore dispersal and fast colonization (e.g., Dendroceros), or even spore bank formation, by Phaeoceros and Anthoceros species. Finally, rehydrating cycles suggest that long-term studies will provide evidence for a more complex DT continuum across many species.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f14020255/s1, File S1—Descriptions of the materials used in the desiccation tolerance tests of five hornwort species; File S2—Summary of the generalized linear models of the desiccation tolerances of the gametophytes and spores of the hornwort species tested; File S3—Summary of the post hoc Tukey honest significant difference (HSD) test of the gametophytes and spores of the hornwort species; Figure S1—Reference values for DT experiments with hornworts in this study. (A) Means of the Fv/Fm chlorophyll fluorescence parameters of hornwort gametophytes. (B,C) Hornworts cultured for 30 days. (B) Percentage spore survival. (C) Sporeling reference sizes. Error bars represent ±1 SE; Figure S2—Means of the Fv/Fm chlorophyll fluorescence parameters of hornwort gametophytes. (A) Balanced relative humidity treatments; (B) Dry durations; (C) postrehydration periods. Error bars represent ±1 SE.

Author Contributions

Conceptualization and methodology, G.F.P.-B. and A.S.M.-S.; collected data, T.V.-B. and G.F.P.-B.; data curation, validation, and statistical analysis, G.F.P.-B., J.C.V.A. and A.S.M.-S.; writing—original draft preparation, G.F.P.-B., J.C.V.A. and A.S.M.-S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded and supported by the Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior, Brasil (CAPES- Finance Code 001); Emerging Leaders in the Americas Program–ELAP (called 2018–2019); International Association for Plant Taxonomy (IAPT—Research Grant 2019); and the Rufford Foundation (Small Grants 2018–2019).

Data Availability Statement

All data for this study are available in Supplemental Materials.

Acknowledgments

This research was carried out with permission from the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio/SISBIO, license number 58259-8). The first author thanks the park rangers and Diniz, V.S, Magalhaes, M.D, and Tomasi, M.O. for their help in the field, as well as Oliveira, M.F., for his invaluable help with the experiments and recording the information.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Diversity of spores and niches of hornwort species. (A) Phylogenetic diagram of the hornwort genera with green and non-green spores; (B,C) types of green spores—(B) multicellular spores; (C) unicellular spores; (D,E) unicellular non-green spores. (F–I) General view of hornwort gametophytes and sporophytes. (J–M) Different niches of hornwort species. (J) Epiphytic (on the bark of live trees and shrubs), Dendroceros genus. (K) Saxicolous (directly on rock surfaces), Nothoceros vincentianus. (L,M) Terricolous (on soils), (L) Phaeoceros carolinianus and (M) Anthoceros lamellatus.
Figure 1. Diversity of spores and niches of hornwort species. (A) Phylogenetic diagram of the hornwort genera with green and non-green spores; (B,C) types of green spores—(B) multicellular spores; (C) unicellular spores; (D,E) unicellular non-green spores. (F–I) General view of hornwort gametophytes and sporophytes. (J–M) Different niches of hornwort species. (J) Epiphytic (on the bark of live trees and shrubs), Dendroceros genus. (K) Saxicolous (directly on rock surfaces), Nothoceros vincentianus. (L,M) Terricolous (on soils), (L) Phaeoceros carolinianus and (M) Anthoceros lamellatus.
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Forests 14 00255 g002 550
Figure 2. Summary of the desiccation experimental design for gametophytes and sporophytes of hornworts.
Figure 2. Summary of the desiccation experimental design for gametophytes and sporophytes of hornworts.
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Forests 14 00255 g003 550
Figure 3. Means of the Fv/Fm chlorophyll fluorescence parameters of hornwort gametophytes with equilibrating relative humidity treatments (RHeq) of 99%, 74%, and 56%. Experiments were conducted with the different species placed under drying treatments with differing durations in terms of days (1D, 3D, and 9D) and postrehydration times, in terms of hours (24 h, 74 h, and 216 h); error bars represent ±1 SE.
Figure 3. Means of the Fv/Fm chlorophyll fluorescence parameters of hornwort gametophytes with equilibrating relative humidity treatments (RHeq) of 99%, 74%, and 56%. Experiments were conducted with the different species placed under drying treatments with differing durations in terms of days (1D, 3D, and 9D) and postrehydration times, in terms of hours (24 h, 74 h, and 216 h); error bars represent ±1 SE.
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Figure 4. Effects of drying on hornwort gametophytes in the three treatments consisting of differing RHeq values (Left) and dry durations (right), with a postrehydration time of 216 h. The first line displays gametophytes with deacclimation for 15 days and without drying effects. Scale-bar: 1 mm. Red arrows: gametophyte regeneration.
Figure 4. Effects of drying on hornwort gametophytes in the three treatments consisting of differing RHeq values (Left) and dry durations (right), with a postrehydration time of 216 h. The first line displays gametophytes with deacclimation for 15 days and without drying effects. Scale-bar: 1 mm. Red arrows: gametophyte regeneration.
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Figure 5. Spore survival percentages and sporeling sizes of hornworts cultured for 30 days. (A,B) after RHeq and 24 h postrehydration before cultivation—(A) spore survival with RHeq treatment; (B) sporeling size with RHeq and duration dry treatments. Error bars represent ±1 SE.
Figure 5. Spore survival percentages and sporeling sizes of hornworts cultured for 30 days. (A,B) after RHeq and 24 h postrehydration before cultivation—(A) spore survival with RHeq treatment; (B) sporeling size with RHeq and duration dry treatments. Error bars represent ±1 SE.
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Table
Table 1. Generalized linear model of the effects of desiccation tolerance in gametophytes and spores of hornworts species (GLM with Gaussian distribution and identity link function; bold indicates statistical significance).
Table 1. Generalized linear model of the effects of desiccation tolerance in gametophytes and spores of hornworts species (GLM with Gaussian distribution and identity link function; bold indicates statistical significance).
SourceDegree of Freedom (d.f.)DevianceResid. DfResid.DevFPr(>F)
Chlorophyll fluorescence parameters for hornwort gametophytes
Null model 323184.989
Species382.696320102.2932.130.616<0.001
RHeq221.85131880.443844.457<0.001
Duration dry212.20331668.240471.616<0.001
Postrehydration212.28931455.951474.911<0.001
Species:RHeq612.78230843.169164.665<0.001
Species:duration dry60.210030241.06927.0540.014
Species:postrehydration60.277329638.29635.723<0.001
Model: AIC: −460.44; Residual deviance: 3.8296; 296 degrees of freedom (df)
Survival spores
Null model 13455441
Species419447.313035993190.645<0.001
RHeq2967.21283502618.9630.154
Duration dry29.7126350160.01890.981
Species:RHeq84295.71183072121.0560.041
Species:dry duration82668.61102805213.0800.246
Model: AIC: 1155.5; Residual deviance: 28052; 110 degrees of freedom (df)
Sporeling Size
Null model 6749580.75
Species4198.5476745382.211.132.689<0.001
RHeq217.9946743364.21205.306<0.001
Duration dry222.9086741341.30261.377<0.001
Species:RHeq826.0786733315.2374.386<0.001
Species:duration dry820.5236725294.7058.540<0.001
Model: AIC: −1928.8; residual deviance: 294.70; 6725 degrees of freedom (df)
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Peñaloza-Bojacá, G.F.; Vilas-Boas, T.; Villarreal A, J.C.; Maciel-Silva, A.S. Differential Effects of Desiccation on Hornworts with Contrasting Life Histories in Tropical Montane Forests: A Functional Trait—Based Perspective. Forests 2023, 14, 255. https://doi.org/10.3390/f14020255

AMA Style

Peñaloza-Bojacá GF, Vilas-Boas T, Villarreal A JC, Maciel-Silva AS. Differential Effects of Desiccation on Hornworts with Contrasting Life Histories in Tropical Montane Forests: A Functional Trait—Based Perspective. Forests. 2023; 14(2):255. https://doi.org/10.3390/f14020255

Chicago/Turabian Style

Peñaloza-Bojacá, Gabriel F., Tiago Vilas-Boas, Juan C. Villarreal A, and Adaíses S. Maciel-Silva. 2023. "Differential Effects of Desiccation on Hornworts with Contrasting Life Histories in Tropical Montane Forests: A Functional Trait—Based Perspective" Forests 14, no. 2: 255. https://doi.org/10.3390/f14020255

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