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The Effects of Heat on Spore Viability of Lygodium
microphyllum and Implications for Fire Management
Nicole Sebesta1,*, Jennifer Richards1, and Jonathan Taylor2
Abstract - The vining fern Lygodium microphyllum (Old World Climbing Fern), which is
native to the Old World tropics, has invaded central and southern Florida, disrupting native
habitats, reducing biodiversity, and altering fire-line intensity and behavior. Prescribed fire,
one of several methods used to manage Old World Climbing Fern infestations, reduces the
fern’s above-ground biomass over large areas, but its effects on spore viability are unknown.
To determine the heat tolerance of spores, we exposed spores to temperatures ranging from
50 °C to 300 °C for durations of 5 sec to 1 h, then assessed their germination on agar in
Petri plates. Temperatures of 50 °C had little effect; 300 °C killed spores for all durations.
Results indicate that spore viability decreases with increasing temperature and duration of
heat exposure, and that spores are killed at relatively low tem peratures (≥100 °C).
Introduction
Lygodium microphyllum (Cav.) R. Br. (Old World Climbing Fern, hereafter
OWCF), native to the subtropics of Africa, Asia, and Australia, has become a major
invasive exotic species in central and southern Florida. Since its introduction
to Palm Beach, FL, in the late 1950s (Pemberton and Ferriter 1998), OWCF has
infested more than 49,000 ha of central and southern Florida, forming dense rachis
mats that smother and shade native vegetation, damage natural habitats, and
alter fire-line intensity and behavior (Ferriter and Pernas 2006, Lott et al. 2003,
Stocker et al. 2008). In the invaded range, it is found in wetlands and uplands,
including sawgrass marshes, pinelands, hardwood hammocks, cypress stands,
bayheads, and mangrove communities (Pemberton and Ferriter 1998). The fern
has been designated one of Florida’s most serious invasive species by the Florida
Exotic Pest Plant Council (FLEPPC) because of the severe ecological damage
it has caused; the species even threatens the success of Everglades restoration
(Hutchinson et al. 2006). Prescribed fire is one of several methods currently
used to manage OWCF infestations (Hutchinson et al. 2006, Stocker et al. 2008).
Despite the use of this management tool, the species is still spreading and infestations
in southwestern Everglades National Park (ENP) are expanding (Rodgers
et al. 2014); thus, efforts to manage and control OWCF need refinement. Three
components to consider in OWCF management are fern biology, invasive species
characteristics, and fire relations.
Although occasionally regarded as the only genus within the family Lygodiaceae,
Lygodium is increasingly treated as a genus within the family of Schizaeaceae
(Gandolfo et al. 2000; Lott et el. 2003; Madeira et al. 2008; Mueller 1982a, 1982b,
1Department of Biological Sciences, Florida International University, Miami, FL 33199. 2Everglades
National Park, Homestead, FL 33034. *Corresponding author - nsebe001@fiu.edu.
Manuscript Editor: Richard Baird
Everglades Invasive Species
2016 Southeastern Naturalist 15(Special Issue 8):40–50
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2016 Vol. 15, Special Issue 8
1983; Pemberton 1998; Wikstrӧm et al. 2002). Recently, Christenhusz and Chase
(2014) have reaffirmed its placement in Schizaeaceae based on molecular phylogenetic
analyses. OWCF is a homosporous fern composed of a dichotomously
branching rhizome with adventitious roots and climbing leaves (fronds; Mueller
1982a). The primary leaves are determinate and usually less than 10 cm long, while
secondary leaves are indeterminate and twining, sometimes reaching over 30 m
in length (Mueller 1982a). The indeterminate twining fronds make this plant very
unusual morphologically, and produce the characteristic and easily recognizable
climbing habit. Climbing leaves have alternate pinnae, each possessing opposite
pinnules that are further subdivided into alternate pinnulules and a resting leaf bud,
which can resume indeterminate growth if the leaf apex becomes damaged (Mueller
1982a, 1983). Pinnae are sexually dimorphic with fertile and sterile pinnae often
occurring on the same climbing leaf. Fertile pinnulules form sorophores of revolute
leaf tissue (Gandolfo et al. 2000), which contain sporangia on the abaxial surface. A
typical pinnulule may produce more than 28,000 spores, which are wind-dispersed
and can be produced throughout the year (Volin et al. 2004). A single spore can give
rise to a new sporophyte through intragametophytic selfing (Lott et al. 2003); this
ability likely contributes greatly to OWCF’s long-range dispersal and colonization
ability (de Groot et al. 2012).
Typical traits associated with invasive species include fast growth-rate, exceptional
propagule pressure, tolerance of variable habitat (Jose et al. 2013), and in
some cases, allelopathy. OWCF has all of these characteristics, which facilitate its
displacement of native plants (Lott et al. 2003, Pemberton and Ferriter 1998, Wang
et al. 2014). OWCF’s ability to produce spores year-round combined with the potential
for multi-year spore viability (Hutchinson et al. 2006) may also contribute
to its ability to invade new sites. These traits should be addressed in management
strategies, for example, when determining the length of time to monitor a site posttreatment
to ensure that spores in the soil do not germinate and allow OWCF to
re-infest the area.
Although prescribed fire is used to reduce the above-ground biomass of OWCF
infestations (Hutchinson et al. 2006, Stocker et al. 2008), the fern, in turn, affects
fire-line intensity and behavior in habitats that it invades. Pinus elliottii Engelm.
(Slash Pine), which is adapted to ground fires, can be severely affected by OWCF
infestations (Lodge 2010, Pemberton and Ferriter 1998). Fire generally burns
through only the lower vegetation in these pineland habitats, while the pines’ sensitive
meristematic and reproductive tissues remain protected in the canopy above the
height of the fire (Whelan 1995). OWCF’s climbing leaves produce vertical rachis
mats that function as a fuel ladder, linking the understory vegetation to the forest
canopy and lifting the fire into the vulnerable crown, often resulting in tree death
(Osborne et al. 2010).
Fire also affects OWCF’s growth—in its native range, this species resprouts
from the rhizome after fire (Goolsby et al. 2006). Although prescribed fire has been
used successfully in fire-adapted ecosystems to reduce above-ground fern biomass,
it is unknown what effects fire has on sporulation or spore viability. Fire-created
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updrafts may aid spore dispersal (Stocker et al. 2008), although there is some evidence
to the contrary (Osborne et al. 2010).
Fire behavior is complex and depends on many factors, including fuel, soil
moisture, temperature, relative humidity, heat released, duration, and other variables
(Bond and van Wilgen 1996, Whelan 1995). A heterogeneous horizontal and
vertical distribution of fuels tends to produce a patchy fire, while more evenly
distributed fuels produce more homogeneous fires (Whelan 1995). A patchy prescribed
fire, which mimics natural fire systems, is often a desirable management
outcome because it creates refugia for native plant propagules, facilitating their
reestablishment in the burned areas. However, reproductive OWCFs may also survive
in unburned patches, producing spores and furthering invasive spread. Fires
create convective airflows, and mature spores might be caught in an updraft and
dispersed to new sites. Alternatively, a passing fire may expose spores to common
fire temperatures that damage the plant or kill the spores even without ignition, thus
reducing dispersal concerns (Whelan 1995).
Although burning can kill OWCF (if the rhizome is sufficiently damaged), the
effects of lower temperatures vary. Plant cells are damaged by heat via several
mechanisms, including protein denaturation (Whelan 1995), which typically occurs
between 40 °C and 65 °C (Hopkins and Hüner 2004). Temperatures resulting in
cell death, however, vary depending on the duration of heat exposure and hydration
of the cells (Whelan 1995). The cells of common mesophytes die at temperatures
between 50 °C and 55 °C (Hare 1961), but dehydrated organs can tolerate higher
temperatures (Bond and van Wilgen 1996). Non-green spores, like those of OWCF,
are relatively dehydrated, with water comprising just over 20% of their total weight,
as opposed to other plant cells, where water accounts for over 90% of plant weight
(Tryon and Lugardon 1991); thus, the temperature at which OWCF spores become
non-viable could exceed the 50–55 °C range. The purpose of our study was to experimentally
determine the effects of temperature and duration of heat exposure
on spore germination. We hypothesized that spore viability would decrease with
increasing temperatures and exposure durations.
Methods
Material
ENP personnel collected sporulating OWCF fronds from sites in Everglades
National Park, FL, in August 2014. Park staff double-bagged and transported to
Florida International University (FIU) samples collected under Florida Department
of Agriculture and Consumer Services permit no. 2013–022. In the lab, we
placed the fronds in a plant press to dry and release spores. We used a Mettler
AE240 analytical balance (accuracy = 0.01 mg) (Mettler Toledo, LLC, Columbus,
OH) to weigh out 1.00-mg spore samples which we placed in small tin cups (5-
mm diameter, 8-mm deep; Costech Analytical Technologies, Inc., Valencia, CA),
pinched and folded closed, and stored individually in 16 mm x 50 mm glass vials
at room temperature.
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Experimental conditions
Heat alone can damage plant cells; thus, it may not be necessary to combust
spores in order to reduce their viability. In our experiments, we examined a range
of temperatures that we thought might damage the spores without actually igniting
them.
We performed a series of preliminary experiments to establish growth conditions,
appropriate experimental protocols, and temperature ranges and durations,
followed by 2 final experiments, the results of which are reported here. The first
of these final experiments utilized temperatures below 100 °C and heat durations
from 5 min to 60 min. Results from the preliminary experiments indicated that these
durations were relevant to viability at temperatures within the protein-denaturation
range. The final experiment employed temperatures of at least 100 °C and short
durations of less than 1 min, which are conditions typically found in a passing fire.
During our preliminary experiments, spore viability decreased with increasing
spore age faster than expected from the literature, which reported that spores remain
viable for 4 y (Hutchinson et al. 2006). Although OWCF spores have been reported
to drop in viability (as indicated by germination rates) from 30% at 3.8 years of
age to less than 3% after 5.8 years (Hutchinson 2010), viability of untreated control
spores in our preliminary experiments was found to decline from 45% to 2% after
only 2 years (Sebesta 2015). In our 2 final experiments, we used spores from a
single collection; they were 4 months old at the time of the first experiment, and 7
months old at the time of the second experiment. There was some apparent loss of
viability from 4 to 7 months of age, so we standardized germination rates for these
experiments to their respective controls; thus we report data as percent of control
germination. In both of these experiments, we assigned each 1.00-mg sample to a
treatment or control for each temperature and duration combination, and replicated
the control 3 times.
We sowed both heated and control spores on the same day that heat treatments
were applied. We suspended spores in 1.0 ml of distilled water for 10 min, then pipetted
0.5 ml of the suspension onto 20-cm Petri dishes with 0.8% agar medium
containing Parker-Thompson basal nutrients (PhytoTechnology Laboratories,
Shawnee Mission, KS) and sealed them with Parafilm (American Can Company,
Greenwich, CT). During preliminary counts, we determined that each 0.5 ml of
suspension contained 800–900 spores. We cultured the plated spores in a growth
chamber (Environmental Growth Chambers model GC8-2H, Chagrin Falls, OH) under
a 13/1l-h light/dark cycle with temperatures set to 26/24 °C (light/dark) (Philippi
and Richards 2007). We placed temperature loggers in the chamber to monitor temperature
fluctuations, which were minimal during the course of the experiments. After
2 weeks, we determined percent spore germination in each Petri dish by dividing each
dish into 4 equal quadrants and using a dissecting microscope to scan each quadrant
for spores. We scored the first 50 spores encountered in each quadrant scan as germinated
or ungerminated. Germinated spores had a green, filamentous prothallus
protruding from a cracked spore (level 4 in Philippi and Richards 2007). We estimated
percent germination from the sum of the quadrants (number germinated/200).
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The first experiment examined the effect of temperatures below 100 °C and heat
durations from 5 to 60 min. We heated spore samples in their tin cups in a muffle
furnace (Fisher Scientific Isotemp model 550–12, Hampton, NH) at 50 °C, 65 °C,
80 °C, or 95 °C for durations of 5, 15, 30, or 60 min; each temperature/duration
combination had 3 replicates. For each run, we used 3 temperature-logging iButton
sensors (Maxim Integrated, San Jose, CA) to track internal oven temperatures for
all but the 95 °C treatment, which was outside the range of the iButton. The iButtons
time-stamped and logged the temperature every minute so that the measurements
could be accurately matched to the treatments. We averaged readings from
the iButtons to obtain the mean, standard error, and range of true temperatures in
the furnace. For the controls (n = 4 Petri dishes with 0.50 ml spore suspension in
each), we used spores that were identically stored but not heat ed.
Although the muffle furnace provided a wide range of suitable temperatures, its
internal sensor was sensitive to dips in temperature from brief opening of the furnace
door, which triggered the heating mechanism to produce a temperature spike
and slow cooling back to the set point, slightly skewing the exposure temperatures,
especially during the 80 °C treatments. We could not control humidity in the furnace
and this variable may have af fected spore viability.
In the second experiment, we exposed spores to 100 °C for 5 or 30 sec or 300
°C for 5 sec; each temperature/duration combination, as well as the controls, had 5
replicates. We heated spore samples (1.00 mg) in their tin cups by placing them into
a hotplate-preheated 19 mm x 65 mm glass vial equipped with a TJ36 Series type
K thermocouple (Omega, Westlake Village, CA). The thermocouple was connected
to a CR23X Micrologger (Campbell Scientific, Inc., North Logan, UT) to verify
exposure temperature every second. We removed samples from the vials quickly to
halt residual continued heating. Control samples were from the same stored collection
of spores but were unheated. Following treatments, we suspended the spores in
1.0 ml as before, pipetted 0.5 ml of the suspension onto prepared agar plates, and
determined germination as in the first experiment.
Although the hotplate used in this experiment maintained a relatively constant
temperature, it was difficult to remove the glass vials quickly from the stand. We
considered holding the tin cups by tweezers over the hot plate, but verifying exposure
temperature would have been very difficult in this scenario. Although the
tin (melting point: 231.9 °C) cups were damaged during the 300 °C treatments,
we heated another group of spores from the same collection in aluminum (melting
point: 660.3 °C) cups, and then sowed the spores and assessed germination; these
had the same charred appearance and germination rates as spores heated in tin cups.
We resealed and allowed to continue development for an additional week in
the growth chamber all treatments that had zero germination after 2 weeks in the
growth chamber (spores exposed to 95 °C for all durations, and all the treatments
at ≥100 °C ); germination was then reassessed. These secondary counts were more
exhaustive—we counted and scored at least 100 spores in each quadrant. Of these,
the spore samples exposed to 95 °C for 60 minutes were assayed a third time at 5
weeks with counts exceeding 800 spores per plate.
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Data analysis
To analyze the overall trends for combined durations within each temperature
group, we used a generalized linear model (GLM) assuming a binomial distribution
of the dependent variable and logit link. We compared each temperature/duration
combination to the control using the GLM, then compared durations within each
temperature using Tukey tests from the multcomp package in R (Hothorn et al.
2008, R Core Team 2014).
Results
In experiment 1, temperature inside the muffle furnace varied from 1 °C to 8 °C
from the set points (Table 1). Mean temperatures in the furnace were 0.6 °C to 4.8
°C greater than the target temperatures (Table 1). Similarly, in experiment 2, temperature
varied across the bottom of the glass vial above the hotplate from 0.6 °C to
3.5 °C (with 1 exception of 99 °C below the 300 °C target) from the target temperatures.
The mean logged temperatures during the 100 °C 5-sec and 30-sec targets
were 103.1 °C and 102.5 °C, respectively. The 300 °C target averaged 286 °C due
the single replicate with a 99 °C variation in treatment; excluding this sample, the
mean logged temperature was 295.3 °C.
Spore germination at ≤95 °C
OWCF spore-germination decreased with increasing temperatures (Fig. 1), and
germination of controls was 31 ± 3.4 %. All temperature groups, which combined
durations within each temperature treatment, differed from controls and from each
other (P < 0.0001). No spores heated to 95 °C for any of the durations had germinated
after 2 weeks, so we excluded this treatment from further statistical analysis.
Although when counted at 2 weeks, all spores treated for 5 min or more at 95 °C remained
ungerminated and were presumed dead, low levels of germination occurred
over time in these plates. At 3 weeks, mean germination-counts per 800 spores
(and percent germination) were: 5-min exposure, 4.33 (0.5%); 15-min, 10 (1.3%);
Table 1. Target temperatures and durations (Dur), measured temperatures of treatments (Mean Temp,
SE, Range), and difference (Diff.) between target and mean measured temperatures for experiment 1
treatments. No data for 95 °C treatment.
Target (°C) Duration (min) Mean Temp (°C) SE Range (°C) Diff. (°C)
50 5 54.81 0.11 55 4.81
50 15 54.11 0.22 52–55 4.11
50 30 52.39 0.04 52–53 2.39
50 60 51.54 0.23 42–53 1.54
65 5 67.64 0.03 68 2.64
65 15 67.50 0.21 66–68 2.50
65 30 66.13 0.04 66 1.13
65 60 67.29 0.16 66–71 2.29
80 5 81.78 0.46 81–83 1.78
80 15 81.69 0.14 81–82 1.69
80 30 81.46 0.09 80–82 1.46
80 60 80.64 0.03 80–81 0.64
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30-min, 0.67 (0.08%); 60-min, 0 (0%). We assayed only the 60-min group again
at 5 weeks; some germination occurred and reached 10 (1.3%). The gametophytes
from these treatments were stunted, but green.
Each temperature/duration combination differed significantly from the control
(all P < 0.0001) except for 50 °C at 5 min (Fig. 2). Spores exposed to 50 °C
for 5 min showed no difference in viability compared to controls. All longer durations
at 50 °C differed significantly from the 5-min treatment, but did not differ
from each other, and averaged 70% germination of controls. All of the durations
at 65 °C differed significantly from the controls, but did not differ from each
other, and averaged 60% germination of controls. All of the durations at 80 °C
differed significantly from the controls and from each other. The 15-min duration
had significantly lower germination than the 5- or 30-min durations, but did
not differ from the 60-min duration, and averaged 15% germination compared
to controls. The 5- and 30-min durations did not differ from each other but had
significantly greater germination than the 60-min duration and averaged 39%
germination compared to controls.
Spore germination at ≥100 °C
When spores were exposed to temperatures ≥ 100 °C for durations ≤ 30 sec,
no spores had germinated for any of the treatments after 2 weeks in the growth
Figure 1. Final experiment 1: Lygodium microphyllum spore germination expressed as percent
of germination of controls for each temperature treatment; heat duration of 5–60 min
combined at each temperature. amb = ambient temperature for controls. All temperature
treatments differed significantly from the control and from each other ( P < 0.0001).
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chambers, while germination of controls was 21 ± 4.5%. After 5 sec at 300 °C,
spores were brown, appeared charred, and none germinated. After the initial assay,
we resealed all of the 100 °C and 300 °C samples and placed them in the
growth chamber for another 10 days. When reassayed, average germination
reached only 0.23% for spores exposed to 100 °C for 5 sec; the other treatments
remained at zero germination.
Discussion
In this study, we determined the lower limit of temperatures and durations
required to significantly reduce OWCF spore viability. A temperature of 50 °C,
when applied for durations ≥ 15 min, significantly reduced spore viability as
compared to controls; exposure to temperatures ≥ 100 °C for only 5 sec resulted
in nearly complete loss of viability. Thus, although desiccated and therefore tolerant
of higher temperatures than hydrated (vegetative) tissue, OWCF spores are
vulnerable to relatively low temperatures given that temperatures in a fire often
reach 700 °C or more (Whelan 1995). Spore germination was reduced to less than
2% by a 5-sec exposure to temperatures of at least 100 °C, suggesting that spores
dispersed following exposure to these temperatures would likely have greatly
reduced viability. Osborne et al. (2010) reported similarly encouraging results
when examining uninvaded Cladium jamaicense (Crantz) Kük. (Jamaica Swamp
Figure 2. Final experiment 1: Lygodium microphyllum spore germination expressed as
percent of germination of controls for each temperature and heat duration treatment. amb =
ambient temperature for controls.
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Sawgrass) plots adjacent to burned OWCF-infested plots. Following a prescribed
burn, Osburne et al. (2010) found only 1 in 10 of the uninfested plots were colonized
by OWCF. Although fire alone may not be sufficient to manage OWCF
infestations (Hutchinson et al. 2006), its use in combination with herbicides or
with biocontrols shows promise (Hutchinson and Langeland 2010, Stocker et al.
2008). Our findings show that relatively low temperatures reduce spore viability;
this result suggests that fire-aided dispersal of viable spores is likely not as great
a threat as has been hypothesized (Hutchinson et al. 2006). Thus, our data support
the use of fire as part of a management strategy for OWCF infestations, particularly
if temperatures near sporulating fronds can be raised to 100 °C.
Spore longevity in our experiments declined quickly, compared to other
reports of viability over time (Hutchinson 2010, Hutchinson et al. 2006).
Germination rates examined by Hutchinson (2010) remained between 30% and
45% for up to 3.8 y and then dropped to below 3% for spores older than 5.8 y.
Germination rates for the younger spores used as controls in our experiments
were similar to most rates reported by Hutchinson (2010). Differences in spore
longevity may be influenced by storage conditions or attributed to differences
among populations. All of the spores used in our experiments were from a single
population in ENP, but some differences in germination have been reported
among populations across the state (Hutchinson 2010). After germinating the
spores from 12 different populations, Hutchinson (2010) found a north–south
gradient of increasing rates of germination, ranging from 19% to 46%. Our collection
was from a more southern location than all of those used in Hutchinson’s
germination evaluations; thus, we would expect those spores to have somewhat
higher germination rates if the north–south gradient he observed can be extrapolated.
Both the 31% and 21% germination rates obtained for our controls
fall within the published range for OWCF germination rates. However, they are
lower than would be expected according to population or age, suggesting that
these unexplored factors also affect longevity. Additional evaluations of germination
rates and long-term viability for more southerly populations may inform
monitoring and retreatment protocols relevant to ENP, particularly if germination
rates for these populations normally follow the trends shown by Hutchinson
(2010). Additionally, experiments that explore whether viability declines for
spores exposed to temperatures and humidity levels found in the field would
be helpful for understanding spore longevity in vivo. This information would be
especially useful in deciding whether a spore bank exists and how to adjust management
practices accordingly.
Acknowledgments
Funding was provided by NSF-FL-GA Louis Stokes Alliance for Minority Participation
Bridge to Doctorate Program FIU HRD #1301998, and Everglades National Park CESI
#P12AC11125. We thank Hillary Cooley, Steve Oberbauer, Evelyn Gaiser, Lorenzo Menzel,
Franco Tobias, Suzanne Koptur, and Michael Ross for their various contributions. We
are also grateful to 3 reviewers who provided invaluable feedba ck on this manuscript.
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