Plant Ecol
https://doi.org/10.1007/s11258-022-01286-x
Freezing resistance and xylem anatomy in low and high
elevation populations of Senecio formosus Kunth
in the tropical Andes
Meiby Araujo · Fermin Rada · Francisca Ely
Received: 23 June 2022 / Accepted: 16 December 2022
© The Author(s), under exclusive licence to Springer Nature B.V. 2022
Abstract High mountain ecosystems are subjected
to frequent freeze–thaw events all-year round, con-
sequently plants have developed freezing resistance
mechanisms to cope with extreme low temperatures.
Additionally, these events have also been correlated
with the risk of cavitation so that plants need to adapt
their water transport system. Information on freez-
ing resistance and xylem vessel characteristics along
elevation gradients is scarce for neotropical high ele-
vation species. In this study we aboard two specific
questions: 1. Are there intraspecific differences in
freezing resistance between low and high elevation
populations of Senecio formosus Kunth? 2. Could an
increase in freeze–thaw cycle frequency and lower
Communicated by Paul Ramsay.
M. Araujo
Departamento de Biología, Facultad de Ciencias,
Universidad de Los Andes, Mérida, Venezuela
Present Address:
F. Rada (*)
Laboratorio de Ecología y Fisiología Vegetal, Universidad
de Los Andes, Bogotá, Colombia
e-mail: f.rada@uniandes.edu.co
F. Rada
Instituto de Ciencias Ambientales y Ecológicas (ICAE),
Universidad de Los Andes, Mérida, Venezuela
F. Ely
Instituto Jardín Botánico de Mérida, Universidad de Los
Andes, Mérida, Venezuela
freezing temperatures at higher elevations determine
differences in xylem conduit traits between low and
high elevation S. formosus populations? We expect
greater freezing resistance and a safer water trans-
port system, mainly shaped by narrower tracheary
elements in higher elevation populations compared
to lower ones. Freezing resistance (avoidance and
tolerance) and tracheary elements were studied in S.
formosus at its lower (3100 m) and upper (4200 m)
distributional limits in the Venezuelan paramo. Freez-
ing resistance was determined through injury and
freezing temperature determinations; whereas xylem
conduit characteristics dealt with were: vessel ele-
ment and tracheid diameters, % conducting area and
vessel element density. S. formosus increased freez-
ing resistance and presented narrower vessel element
diameters under more extreme thermal conditions
(4200 m). Increasing evidence of intraspecific plant
trait variations under different environmental gradi-
ents will aid to determine the outcome of individual
species and their effects on ecosystem functioning
under a changing climate.
Keywords Cavitation risk · Freezing avoidance ·
Freezing tolerance · Paramo · Tracheary elements ·
Vessel element diameter
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Introduction
High mountain ecosystems are subjected to freez-
ing temperatures all-year round, a determinant fac-
tor influencing fitness and species distribution along
elevation gradients (Sakai and Larcher 1987). Conse-
quently, alpine plants have developed different freez-
ing resistance strategies to cope with these extreme
low temperatures and as indicated by Körner (2021),
this is the first filter plants must confront in order to
survive. These strategies may be divided into two
basic broad categories, frost avoidance and/or freez-
ing tolerance (Sklenář et al. 2012, 2016; Ladinig
et al. 2013; Pescador et al. 2016; Schulze et al. 2019).
Frost-avoidance refers to the ability of a plant to pre-
vent the formation of ice nuclei in its tissues, e.g. by
supercooling (the ability of plant tissues to main-
tain water in a liquid state at temperatures below the
freezing point) or by the presence of insulating struc-
tures, important in a limited number of plant species
since these are considered mostly poikilothermic or
limited homeotherms (Michaletz et al. 2015). Alter-
natively, freezing tolerance refers to the capacity to
resist and survive the formation of ice crystals, i.e.,
frost occurrence in the intercellular spaces (Azócar
and Rada 2006; Pescador et al. 2016; Sklenář et al.
2016). The formation of ice nuclei in the intercellu-
lar spaces immediately generates a gradient in which
intracellular water moves outward and freezes in the
extracellular spaces, preventing injury from the rup-
ture of membranes and organelles by ice crystals
within the cytoplasm (Rada et al. 2019). Cells under
this freezing process undergo significant dehydration,
thus freezing tolerant plants must also be very toler-
ant to water deficit.
Additionally, a vast number of references have
correlated freeze–thaw events with risks of cavita-
tion (Carlquist 1966; Davis et al. 1999; Hacke and
Sperry 2001; Sperry et al. 2006; Choat et al. 2011;
Hacke et al. 2017). These physiological risks derived
from freezing events may be prevented or reduced
by adjustments in the water transport system. Xylem
structure studies have examined the existing relation-
ship between xylem hydraulic efficiency and safety
from embolism and have determined that vessel
diameter is one of the most informative xylem traits
(Lens et al. 2013; Hacke et al. 2017; Percolla et al.
2021). Vessel density is also known to decrease per
unit area with increasing vessel diameter (Choat et al.
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Plant Ecol
2011). Narrow xylem vessels have a higher resist-
ance to embolism formation and cavitation compared
to wider vessels, both in woody (Cochard and Tyree
1990; Davis et al. 1999; Sperry et al. 2006) and in
herbaceous perennials (Hernández-Fuentes et al.
2015). However, this reduction in vessel diameter
results in lower hydraulic conductivity, giving rise to
a dilemma, the trade-off between water transport effi-
ciency and safety. Again, due to the high frequency
of freeze–thaw events that occur at high elevations,
plants must confront this dilemma between efficiency
and safety in the transport of water from roots to
leaves.
In contrast to temperate high mountains where
seasonal temperature patterns are important and
determine plant responses related to growth and sur-
vival (Larcher et al. 2010; Ladinig et al. 2013), in
tropical mountains these are not as critical and 24 h
daily cycle conditions become more relevant. On the
other hand, the frequency of night frosts increments
as elevation increases (Rada et al. 2019). In any case,
plants at lower elevations may show absence or low
to moderate freezing resistance compared to higher
elevations where frequency of frosts increases and
minimum temperatures are lower. Bucher and Ros-
bakh (2021) describe a strong positive relationship
between freezing resistance and elevation in a large
number of vascular species in the German Alps, sug-
gesting a strong environmental filter which removes
species with low freezing resistance from higher
elevations. Neuner et al. (2020) report a higher freez-
ing resistance for different plant growth forms in their
upper limit of their elevational range compared to the
same species at lower elevations in the Austrian Alps.
Morales et al. (2020) found that low-elevation annual
species were more frost sensitive than other annuals
or perennials growing at higher elevations in the cen-
tral Chilean Andes. Squeo et al. (1996) and Sierra-
Almeida et al. (2009) support these findings to some
extent, i.e., high elevation species from the Chilean
Andes rely on higher freezing resistance compared to
lower elevation species, however, these authors also
report other species which do not change their freez-
ing resistance along elevation gradients. Rosbakh
et al. (2020) report no significant differences in frost
tolerance between low and high elevation seedlings of
nine alpine species.
Many authors have reported a clear association
between xylem conduit diameter and decreasing
Plant Ecol
temperatures along elevation gradients (Zhang et al.
2013; Morris et al. 2018; Neuner et al. 2020). Zhang
et al. (2013) report a positive correlation between
xylem conduit diameter and temperature in a study
of more than 300 angiosperm species. García-Cer-
vigón et al. (2020) describe a reduction in vessel
diameter with low temperature at higher elevations in
Nothofagus pumilio (Poepp. & Endl.) Reiche in the
Chilean Andes, while Fisher et al. (2007) report this
same result for Metrosideros polymorpha J.R.Forst.
ex Hook. f., a Hawaiian tree species. Interestingly,
these two studies also emphasize that this reduction
in xylem conduit vessel diameter is not only due to
a temperature effect, but to a combined effect which
includes moisture gradients, i.e., correlations between
vessel diameter and freezing and/or drought resist-
ance. However, Olson and Rossell (2013), Olson
et al. (2018) and Fajardo et al. (2019) argue that the
narrower vessels observed in woody species of drier
communities (both warm or cold) are not necessarily
correlated with soil moisture nor stem size, but are
because dryland plants are on average smaller, com-
pared to those of humid ecosystems.
The humid tropical high Andes, referred to as ‘par-
amos’ hereafter, are of great ecological importance
because they hold the highest floristic diversity and
the largest number of endemic species of any alpine
ecosystem (Sklenář et al. 2014; Pouchon et al. 2018)
including many of the world’s biodiversity hotspots
(Spehn et al. 2010; Trew and Maclean 2021). In order
to have a clearer insight of their future under today’s
changing climate, it is crucial to identify the diversity
of plant responses to their particular environmental
conditions. Data and information on freezing resist-
ance and xylem vessel characteristics along elevation
gradients is scarce. Goldstein et al. (1985) describe
well-defined trends of increasing freezing resistance
along elevation gradients in different giant rosette
species in the Venezuelan paramos. Conversely,
Márquez et al. (2006) did not find any correlations
between freezing resistance and elevation for many
paramo grasses and concluded that microhabitat con-
ditions may have a greater effect than elevation dif-
ferences. Most research on freezing resistance along
elevation gradients has focused on different species
from varied plant growth forms, but a reduced num-
ber of studies have been directed towards intraspe-
cific differences in freezing resistance along elevation
gradients (Neuner et al. 2020). Few studies report
increases in freezing resistance at higher elevations,
e.g. Espeletia schultzii Wedd., a caulescent giant
rosette of the tropical Andes (Rada et al. 1987). While
Rada et al. (2008) describe an interspecific increase in
freezing resistance when they compare a lower eleva-
tion herbaceous species, Lupinus meridanus C.P.Sm.,
with a higher elevation species, Lupinus eromono-
mus C.P.Sm. However, these authors did not find any
intraspecific differences in either of these two species
within their own elevation gradients.
With respect to xylem conduit characteristics
along elevation gradients in the tropical Andes, stud-
ies are even more limited. Few have shown a direct
relationship between vessel diameter and increasing
elevation. Briceño et al. (2000) reported interspecific
differences (L. meridanus had wider vessels at lower
elevations (2200–3550 m) compared to L. eromono-
mus at higher elevations (3750–4300 m)), yet no
intraspecific elevational differences. León-Hernández
and Gámez (2018) described wider vessel diameters
in low-elevation (2468 m) compared to high elevation
species (4302 m) of Pentacalia Cass. (Asteraceae) in
the tropical Andes. Soukup et al. (2021) support these
other authors’ findings in a study of various species
of Senecio L. (Asteraceae) growing along an eleva-
tion gradient in the Ecuadorian Andes. To our knowl-
edge, other references on intraspecific relationships
between vessel diameter and elevation have not been
reported for neotropical high mountains.
In this study we aboard two specific questions: 1
Are there intraspecific differences in freezing resist-
ance between low-elevation and high elevation pop-
ulations of Senecio formosus Kunth (Asteraceae),
a widely distributed perennial herb of the tropical
Andes? We expect greater freezing resistance in high
elevation populations due to the more extreme low
temperature conditions present. 2 Will xylem conduit
traits differ between low-elevation and high elevation
populations of S. formosus, due to an increase in the
frequency of freeze–thaw cycles and the presence
of lower freezing temperatures at higher elevations?
We hypothesize that higher elevation plants will dis-
play a safer water transport system, mainly shaped
by narrower vessels compared to lower elevation
individuals.
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Materials and methods
Species and study site descriptions
Senecio L. (Asteraceae) is one of the most character-
istic genera in the paramos. This genus is made up of
5 species in the Venezuelan paramos (Briceño and
Morillo 2002). One of the best represented species in
this genus is Senecio formosus (Fig. 1a, b), a peren-
nial herb restricted to the paramos of Colombia and
Venezuela. In the Venezuelan paramos, this species is
20–60 cm tall (Vivas and Ubiergo 2010) and distrib-
uted from 3000 to 4350 m a.s.l. (Briceño and Morillo
2002).
For this study, individuals of S. formosus were
selected at the lower and upper limits of their dis-
tribution, Paramo de San Jose (PSJ) at 3100 m a.s.l.
(8º 19′ 43′′ N, 71º 18′ 14′′ W) and Paramo de Piedras
Blancas (PPB) at 4200 m a.s.l. (8º 51′ 37′′ N, 70º 49′
09′′ W) in the Cordillera de Merida in the Venezuelan
Andes (Fig. 1c, d). PSJ presents a bimodal precipita-
tion regime with an annual rainfall of 1100 mm with
Fig. 1  Specimens of
Senecio formosus (a).
Detail of the inflorescence
(b). Paramo de San José at
3100 m (c). Páramo de Pie-
dras Blancas at 4200 m (d)
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maximums in the months of May and October and a
mean annual air temperature of 8 °C. The vegetation
corresponds primarily to a grassland formation with
dominant populations of shrubs (Hypericum L.) and
caulescent rosettes (Espeletia Mutis ex Humb. and
Bonpl.) (Ataroff 2004). PPB presents an unimodal
precipitation regime with a mean annual of 760 mm,
a dry season between the months of December and
March and a wet season between April and November
and a mean annual air temperature of 3.9 °C (Rada
et al. 2019). The vegetation consists of a higher stra-
tum dominated by tall caulescent rosettes of the genus
Coespeletia Cuatrec. and a lower stratum composed
of small perennial herbs and dwarf shrubs: Sene-
cio, Hinterhubera Sch. Beep ex Wedd, Lachemilla
(Focke) Rydb.; acaulescent rosettes: Hypochaeris
L., Calandrinia Kunth., Oenothera L., Malvastrum
A. Gray and Draba L.; and cushion plants: Azorella
Lamb, Arenaria L., Aciachne Benth, Lucilia Cass. In
contrast with PSJ which presents a high percentage
of vegetation cover, between 50 and 90% of bare soil
and rocks characterizes PPB.
Plant Ecol
Microclimatic measurements at the two elevations
Portable 4-channel dataloggers (HOBO-ONSET,
USA) were installed at each of the study sites. Air
temperature ­(Ta), total daily radiation (TR) and soil
water content (SWC) were measured. The ­Ta sen-
sors, screened from direct sunlight, were placed
25 cm above the soil surface, so that measurements
reflected the temperature plants were subjected to.
The TR and SWC sensors were placed 50 cm above
and 5 cm below the soil surface, respectively. The
dataloggers were programmed to record every
10 min from November 18 to April 23 for PSJ and
from December 12 to April 23 for PPB. Measure-
ments included both wet and dry seasons. Unfor-
tunately, the PPB logger malfunctioned during the
month of January. Nonetheless, available data give
a clear picture of the environmental conditions at
both paramos.
Freezing temperature (FT) determinations
Five individuals from each PSJ and PPB were cut
at ground level, placed in containers with water and
cut again below the water in order to maintain con-
tinuity in the xylem transport system. This material
was covered with polyethylene bags and transported
to the laboratory and left overnight in growth cham-
bers (ca. 5 °C) before the tests began. Stem sec-
tions, 4 cm in length, and whole leaves from each
of the individuals were selected and placed inside
test tubes in contact with copper-constantan ther-
mocouples and sealed with rubber stoppers. These
test tubes were placed inside a low temperature
cooling bath (Model RTE-111, NESLAB, Thermo
Electron Company-USA) containing an isopropyl
alcohol-water mixture and temperature was lowered
from 5 to − 20 °C at a rate of 6.5 °C/h. All thermo-
couples were connected to a data acquisition card
and a software program (Planta-ICAE) was used to
retrieve the information. This program allowed us to
view changes in temperature for all sample tissues
contained within the test tubes. FT were obtained
from the exotherms formed during the freezing
process, i.e., the liquid to solid phase change in
the studied tissues releases energy resulting in a
temperature rise which is detected by the installed
thermocouples.
Injury temperature (IT) determinations
IT were determined by a modified electrolyte release
method (Lindén 2002). The method consisted in
determining the temperature at which an increase in
the electrical conductivity (EC) of a deionized solu-
tion containing the plant tissue samples occurred.
This increase in EC resulted from the release of elec-
trolytes by the tissues due to cell wall breakdown. The
IT is established at a 50% of electrolyte loss. Stem
sections (4 cm in length) and five leaf lamina 0.5 cm
diameter discs, extracted from the leaves per test tube,
were subjected to a gradual temperature decrease
from 5 to − 20 °C, in the same manner as previously
described for the FT determinations. As temperature
decreased, five test tubes were removed from the bath
at 5 °C intervals and introduced into polyethylene
vials containing 15 ml of deionized water, stored in a
refrigerator at 4 °C for 48 h with a control which only
contained deionized water. After this time period,
the EC (μS/cm2) of the solution containing the sam-
ples was measured with a digital conductivity meter
(ExStik EC 500, Extech Instruments Corp-USA).
Cell injury was evidenced through the increase in EC
of the solution with the sample, which initially had a
value close to 0 μS/cm2. 48 hours after measuring the
EC of all the samples subjected to the thermal gradi-
ent, these were submerged for a few seconds in liquid
N in order to induce rupture of all the cell walls of
the samples. Subsequently, these samples were again
placed in containers for 48 h at 4 °C in a refrigera-
tor and after this time had elapsed, the EC was again
recorded. This value corresponded to 100% loss of
electrolytes in the samples. Relative electrolyte con-
ductivity (%) was obtained for each target tempera-
ture using the following equation:
EC for each temperature
REC = × 100
EC for each temperature after induced rupture
Afterwards, REC was plotted against temperature
and IT determined, i.e., the temperature at which
a 50% release of electrolytes occurred. In order to
determine the freezing resistance strategy (avoid-
ance or tolerance), FT and IT were compared, if FT
and IT did not show significant differences it was
assumed that freezing resulted in injury (avoidance),
while if IT were significantly lower than FT, it meant
that there was a thermal margin in which plant tissues
were frozen before injury occurred (tolerance).
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Anatomical studies
Xylem conduit determinations: vessel elements
and tracheid diameter
For the studies of xylem composition, fresh stems
were collected in the field and fixed in an FAA solu-
tion (8:1:1 ethanol 70%, glacial acetic acid 100% and
formaldehyde 37% (Johansen, 1940) for 48 h. Fixed
stems were sectioned into smaller portions and rinsed
thoroughly until all of the fixing solution was elimi-
nated. Sections of stems were then heated at 70 °C
in a solution of 50% aqueous glycerin until tissues
softened. Part of this material was used to obtain
free hand 20–30 µm cross sections and the remain-
ing material was macerated to separate tracheary
elements from other tissues. Both cross-section and
macerated preparations were stained with alcian-blue-
safranine 0.5% (7:3) and mounted in 50% aqueous
glycerin. The preparations were observed at 40 × in an
optical microscope (Zeiss, Axioscope) equipped with
a graduated eyepiece (μm scale), making 30 diameter
measurements, 6 for each individual (vessel elements
and tracheids) for both elevations.
Measurements of conducting area and vessel element
density
For the measurement of the conducting area (%CA),
5 photomicrographs of the best preparations (differ-
ent cross-sectional fields) were taken with a SONY
digital camera attached to the optical system of a
microscope (Zeiss, Axioscope). These photomicro-
graphs, together with the photograph with the μm
scale, were processed with AutoCAD software. To
obtain the stem %CA, the proportion of xylem area
for all vascular bundles with respect to the entire stem
cross-sectional area was determined and presented as
a % (n = 5 for each elevation). Vessel element density
(VED) was established as the number of vessel ele-
ments per area of the photograph (Number of ves-
sel elements/mm2 of vascular bundle cross-section,
n = 5).
Statistical analysis
One-way analysis of variance (ANOVA) and t-tests
were conducted to assess the differences between ele-
vations in all studied variables (freezing temperature,
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injury temperature, vessel element and tracheid diam-
eters, xylem conducting area and vessel element den-
sity) and to determine differences between freezing
and injury temperatures in order to define freezing
resistance strategies. The data required no transforma-
tions to meet the assumptions of normality or homo-
geneity of variance (Shapiro–WilK and Levene’s test,
p > 0.05). All statistical tests were performed in RStu-
dio version 4.0.2 (RStudio Team 2020) at a signifi-
cance level of 0.05.
Results
Low and high elevation microclimatic conditions
Wet and dry seasons may be differentiated for both
PSJ and PPB from SWC results (Fig. 2). In the case
of PSJ, the decreasing trend begins in the middle of
December until the end of February, with a period in
the month of January in which precipitation events
increased the SWC. For PPB, as mentioned in the
methods section, unfortunately we were not able to
have data for the month of January so we do not know
if any precipitation occurred, however, the decreas-
ing trend in SWC observed for PSJ repeats for PPB.
Additionally, SWC for PPB were lower than in PSJ
the entire measurement period. Daily temperature
amplitude is greater for the dry months compared
to the wet season for both paramos, and at the same
time, this amplitude is greater for PSJ compared to
PPB (Fig. 2). The largest 24 h amplitude occurred
on January 20 for PSJ (33 K) and on February 4 for
PPB (24 K). With respect to daily total radiation
(TR), even though it is variable during both wet and
dry seasons for both paramos, as expected, days with
lower TR occurred during the wet season (Fig. 2).
Measured microclimatic variables are summarized
in Table 1. For all variables there were significant dif-
ferences between seasons in both paramos, higher max-
imum temperatures (­ Tmax) and TR, and lower minimum
temperatures ­(Tmin) and SWC during the dry season. It
is interesting to note that both paramos presented freez-
ing temperatures, the minimum absolute temperatures
were − 3.4 °C and − 6.4 °C for PSJ and PPB, respec-
tively. However, in terms of the number of days with
below zero temperatures, PSJ showed none during the
wet and only 16% for the dry season; contrasting with
PPB in which most of the days during the wet season
Plant Ecol
Fig. 2  Mean daily total radiation (TR), temperature range
(daily maximum and minimum) and soil water content (SWC)
for the two study sites from November to April. This measure-
ment period includes both wet and dry seasons (wet season
Nov 18-Dec 12 and February 24-April 23; dry season Dec
(60%) and practically every day in the dry season (97%)
presented freezing temperatures. Absolute maximum
air temperatures were obtained for both paramos in the
dry season, 34.9 °C for PSJ and 20.9 °C for PPB.
Low and high elevation freezing and injury
temperatures for S. formosus
By definition, S. formosus presented different freez-
ing resistance strategies when we compare specimens
13-February 23). Paramo de San Jose (3100 m) on left side
panels and Piedras Blancas (4200 m) on right side panels.
SWC correspond to the 07:00 h measurements. Arrows corre-
spond to beginning and end of the dry season
of PSJ with PPB (Table 2). At the lower elevation,
there were no significant differences between FT
and IT, suggesting that this species avoids freez-
ing. However, the extremely high FT and IT values
(close to 0 °C) rather indicate that this species does
not rely on a well-defined freezing resistance strategy.
On the other hand, S. formosus at the higher eleva-
tion undoubtedly presents tolerance to freezing as its
survival strategy, i.e., significant differences between
FT and IT for both leaves and stems. Additionally, a
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 Plant Ecol

Table 1  Mean maximum ­ (Tmax) and minimum temperature for Paramo de San Jose (3100 m) and Paramo de Piedras Blan-
­(Tmin), % of days with freezing temperatures (%DFT), total cas (4200 m) during wet and dry seasons
radiation (TR, 06:00–18:00 h) and soil water content (SWC)
Site Tmax (°C) Tmin (°C) %DFT TR ­(Wm−2) SWC ­(m3m−3)

San Jose
Wet season 20.9 ± 0.5a (27.5) 5.4 ± 0.2a (1.6) 0 200.7 ± 7.3a 0.415 ± .004a
Dry season 25.4 ± 0.5b (34.9) 2.6 ± 0.3b (− 3.4) 16 238.0 ± 8.7b 0.378 ± .004b
Piedras Blancas
Wet season 13.2 ± 0.5a (19.2) − 0.4 ± 0.2a (− 3.8) 61 298.6 ± 16.6a 0.346 ± .005a
Dry season 16.3 ± 0.4b (20.9) − 2.6 ± 0.3b (− 6.4) 97 408.8 ± 15.8b 0.281 ± .006b

Absolute maximum and minimum temperatures in parenthesis. Different superscript letters indicate significant differences (p < 0.05)
between seasons for each of the measured variables

Table 2  Mean freezing (FT, corresponds to ice nucleation sus at the two study sites: Paramo de San Jose (3100 m) and
temperature) and injury (IT) temperatures for leaves and stems, Paramo de Piedras Blancas (4200 m)
and prevailing freezing resistance strategy (FRS) for S. formo-
Site Leaves Stems FRS
FT (°C) IT (°C) FT (°C) IT (°C)

San Jose − 3.7 ± 0.41a − 2.6 ± 0.41a − 3.4 ± 0.21a − 4.5 ± 0.91a Avoidance*
Piedras Blancas − 4.8 ± 0.31b − 7.6 ± 0.52b − 4.1 ± 0.21b − 8.4 ± 0.72b Tolerance

Different superscript numbers correspond to significant differences (p < 0.05) between FT and IT for leaves and stems for each eleva-
tion. Different superscript letters correspond to elevational significant differences (p < 0.05) in FT and IT
*By definition, no significant differences between FT and IT corresponds to an avoidance strategy, however, due to the relatively
high injury temperatures (close to 0 °C) we may consider that this species does not display freezing resistance mechanisms at this
elevation

more negative IT for this species at the higher eleva- 4200 m) compared to those growing at the lower one
tion indicates that freezing resistance increments with (PSJ, 3100 m) (Table 3, Fig. 3c, d).
elevation.

Xylem conduit system characteristics
Significant differences (p < 0.05) between elevations
were found for vessel element diameters, proportion
of secondary xylem and of conductive tissue (% CA)
that evidence greater cambial activity in the stems
of plants of S. formosus growing at 4200 vs 3100 m
(Fig. 3a, b), while no differences were observed for
tracheid diameter nor vessel element densities (VED)
(Table 3). Individuals of the lower elevation (PSJ,
3100 m) presented wider vessel elements and larger
piths, the latter due to a higher proportion of paren-
chyma tissues, compared to the high elevation popu-
lation, whereas the proportion of conducting stem
area and fiber-tracheids increased significantly in the
individuals growing at the higher elevation (PPB,
Discussion
Differences in freezing resistance strategies and
in xylem conduit characteristics described in this
study represent adjustive responses to the contrast-
ing environmental conditions between low and high
elevation populations of S. formosus in the tropical
Andes. Low or absent freezing resistance is found at
the low-elevation while at the high elevation this spe-
cies depends on moderate freezing tolerance to sur-
vive the harsh conditions of the high tropical Andes.
Additionally, xylem conduit system characteristics
also illustrate how low-elevation species have wider
vessel elements compared to the higher elevation,
indicating that S. formosus at lower elevations, where
freeze–thaw cycles are much less frequent and freez-
ing temperatures are not as extreme, rely on more

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Plant Ecol

Fig. 3  Transverse sec-

tions of S. formosus stems
collected at Páramo de
San José (3200 m) (a) and
Páramo de Piedras Blancas
(b), showing details of
cortex, secondary xylem
and parenchyma tissues of
the pith. Details of the sec-
ondary xylem and tracheary
elements of plants growing
at 3200 m (c) and at 4200 m
(d). Notice the narrower
vessels and greater develop-
ment of fiber-tracheids in
the latter

Table 3  Mean vessel element and tracheid diameter, % of
xylem conducting area (%CA) and vessel element density
(VED, number of vessel elements/mm2) for stems of S. formo-
sus from the two study sites: Paramo de San Jose (3100 m) and
Paramo de Piedras Blancas (4200 m)
Site Diameter %CA VED
Vessel Tracheids
elements (μm)
(μm)
San Jose 27.7 ± 1.4a 16.6 ± 0.9a 22.0 ± 1.9a 174 ± ­17a
Piedras Blan- 20.2 ± 0.9b 15.0 ± 0.6a 51.1 ± 2.3b 155 ± ­12a
cas
Different superscript letters correspond to significant differ-
ences (p < 0.05) between elevations for each of the xylem com-
ponents
efficient transport characteristics. On the contrary,
this species at high elevations, presents narrower ves-
sel element diameters in a more severe thermal sce-
nario, which emphasizes safety over efficiency.
Low and high elevation microclimatic characteristics
temperatures, the occurrence of night frosts which
increase in frequency at higher elevations and large
daily temperature oscillations. The elevation boundary
at which freezing temperatures occur is ca. 3000 m for
the Venezuelan paramos (Rada et al. 2019). PSJ pre-
sents a low frequency of freezing events during the dry
season while plants at PPB are subjected to freezing
temperatures on a high proportion of days of the wet
and practically every day during the dry season. Daily
temperature oscillations were greater for PSJ compared
to PPB, with absolute maximums above 30 °C and
20 °C, respectively. Even though it is out of the scope
of this study, these results point to the fact that not only
low temperatures shape plant responses at high eleva-
tions, but the direct effects of high temperatures and
its consequences on plant function need also be con-
sidered. PPB presents a lower SWC compared to PSJ
which determines harsher conditions, and together with
lower temperatures and more frequent freeze–thaw
cycles will define plant responses in terms of freezing
resistance and xylem conduit characteristics as will be
discussed ahead.

Three major characteristics of the paramo environ-

ment that mold plant responses are: low nighttime

Vol.: (0123456789)
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Freezing resistance for low and high elevation S.
formosus
Senecio formosus was found to be freezing toler-
ant at the upper limit of the elevation gradient, how-
ever, in its lower limit it seems that it did not exhibit
a response mechanism against freezing temperatures.
These results coincide with many others which con-
clude that freezing tolerance in vegetative and repro-
ductive tissues is the most common freezing resist-
ance strategy at higher elevations where conditions
are harsher, both in tropical, Mediterranean and tem-
perate climates (Squeo et al. 1996; Sierra-Almeida
et al. 2009; Larcher et al. 2010; Ladinig et al. 2013;
Pescador et al. 2016; Rada et al. 2019; Körner 2021)
and support Sakai and Larcher (1987) who suggested
freezing tolerance over avoidance under more extreme
thermal conditions. Our results indicate that freezing
resistance is determined by the thermal conditions to
which plants are subjected to, validating our hypothe-
sis: individuals of S. formosus in its lower distribution
limit would present less developed freezing resistance
mechanisms compared to the upper limit populations.
A 4–5 K increase in freezing resistance for
stems and leaves of S. formosus from low to high
elevation (equivalent to a − 0.45 K/100 m) coin-
cides with the variation in mean minimum tem-
perature (5.2 K) between sites, which is equivalent
to a − 0.47 K/100 m. A range between 0.16 K and
0.25 K/100 m intraspecific increases in freezing
resistance have been reported for extratropical eleva-
tion gradients (Sierra-Almeida et al. 2009; Neuner
et al. 2020). Rada et al. (1987) describe a 0.4 K/100 m
in freezing resistance for the caulescent rosette
Espeletia schultzii, along a 2600–4200 m gradient in
the Venezuelan paramos. According to Sklenář et al.
(2012), the biogeographical origin of plants adapted
to high elevation ecosystems determines their freez-
ing resistance mechanisms; genera of tropical and
south temperate origin tend to avoid freezing (super-
cooling), as opposed to genera of north temperate ori-
gin that tolerate freezing. However, exceptions to this
trend were observed by these authors in herbaceous
perennials of tropical origin (e.g. Lasiocephalus spp
and Senecio nivalis) that were frost-tolerant, whereas
both frost-avoidance and tolerance mechanisms were
observed in species of the genera Valeriana and
Lupinus. They suggest that different frost-resistance
mechanisms may evolve, regardless of the geographic
Vol:. (1234567890)
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Plant Ecol
origin, as a result of the selective pressures imposed
by the environmental conditions and the evident
improvement in freezing resistance has determined
their success at conquering higher elevations. How-
ever, this is not always the case as some studies have
reported neither intra nor interspecific changes in
freezing resistance along elevation gradients in adult
stages (Márquez et al. 2006; Rada et al. 2008; Sierra-
Almeida et al. 2009; Sklenář et al. 2012) or in seed-
lings (Rosbakh et al. 2020).
Xylem vessel characteristics for individuals of S.
formosus growing at low and high elevations
The critical xylem conduit element diameter (vessel
element or tracheid) above which cavitation risks
greatly increase under freeze–thaw events is in the
order of 40–44 μm (Davis et al. 1999; Pittermann
and Sperry 2003). Smaller conduit diameters are
more resistant to embolism formation by freezing.
But on the other hand, narrower vessels are less effi-
cient in the transport of water than wide ones (Tyree
and Ewers 1991), posing the dilemma of trad-
ing-off between safety and efficiency in transport
(Hacke et al. 2006; Choat et al. 2011; García-Cer-
vigón et al. 2020). S. formosus’ conduit diameters
(20.2 μm and 27.7 μm for vessels and 15 μm and
16.6 μm for tracheids from low and high elevations,
respectively) were considerably under this critical
specified conduit diameter, emphasizing safety over
efficiency at both elevations, but with significantly
narrower vessel diameters at the highest elevation.
In the particular case of the Venezuelan paramos,
León-Hernández and Gámez (2018) found that for
8 Pentacalia spp (Asteraceae) distributed along an
elevation gradient, P. imbricatifolia (Sch.Bip. ex
Wedd.) Cuatrec. growing at the highest elevations
(> 4000 m a.s.l.) presented mean vessel diameters
of 16.8 μm, other 6 spp distributed along middle
elevations (3100–3500 m) with values between
22 and 40 μm, while P. reflexa (Kunth) Cuatrec.,
a cloud forest species which is never exposed to
freezing temperatures displayed mean diameters of
104 μm. Narrow vessels are a common adaptation
in other high elevation Asteraceae species growing
above the treeline, Monticalia imbricatifolia (Sch.
Bip. ex Wedd.) C.Jeffrey (24 μm) and Hinterhubera
imbricata Cuatrec. and Aristeg. (13 μm), growing
at 4050 m in a Venezuelan paramo (A Escalona,
Plant Ecol
unpublished work). Likewise, narrow vessel ele-
ments have been described also in herbaceous
dicots such as Salvia mellifera Greene (20–30 μm)
(Hargrave et al. 1994). Briceño et al. (2000)
recorded vessel diameters of 36–40 μm for Lupi-
nus meridanus at lower (3000–3500 m) compared
to 20–22 μm for L. eromonomus at higher eleva-
tions (3750–4200 m). Similar trends were observed
in alpine Rhododendron species, characterized by
narrow as well as distinctly short vessels elements,
which very likely are an adaptation to the frequent
freeze–thaw cycles that characterize the Nepalese
mountains (Noshiro and Suzuki 2001). Soukup
et al. (2021) find a clear relationship between ele-
vation and vessel elements in 9 Senecio spp grow-
ing from 3600 to 4600 m in the Ecuadorian Andes.
Hacke et al. (2017) describe inter and intraspecific
variations in vessel diameters in more than 100
shrub and tree spp across different biomes. They
find that vessel diameter was always below 30 μm in
regions where frosts occurred, while it was variable
for other biomes, e.g. tropical rainforests. Interest-
ingly enough, vessel diameter has been associated
with freeze–thaw embolism vulnerability, according
to the studies conducted by Hargrave et al. (1994)
in Salvia mellifera, which were later confirmed by
Medeiros and Pockman (2014) in Larrea Cav. spe-
cies growing under different temperature regimes.
However, these authors considered latitudinal rather
than elevational gradients, but again it corroborates
the view that xylem conduit traits are determined
by freeze–thaw events and that safety overrides effi-
ciency under freezing temperature regimes.
Despite the fact that this study did not consider
plant water relations at the two elevations, moisture
conditions probably enhanced the effect on both
freezing resistance and xylem conduit traits in S.
formosus. PPB (4200 m) is one of the driest paramos
in the tropical Andes; therefore, these drier as well as
colder conditions may have accentuated its responses,
since both low temperatures and drought operate in
the same direction with respect to freezing resistance
and conduit size. The anatomical plasticity observed
in terms of xylem development and structure could
explain the broad elevation range of S. formosus
in the Venezuelan paramos and coincides with the
intraspecific xylem plasticity observed in perennial
herbs exposed to different moisture conditions in the
Alpine tundra of the Colorado Rocky Mountains (von
Arx et al. 2012). More extreme nighttime freezing
temperatures occur under drier conditions, having an
effect on freezing resistance strategies; and the con-
sequences of drought on xylem conduit characteris-
tics have been well-documented (Olano et al. 2013;
Medeiros and Pockman 2014; Dória et al. 2019;
García-Cervigón et al. 2020, 2021).
Tracheid development is another anatomical
trait that appears to be influenced by elevation in
high mountain species. The proportion of tracheids
and fiber-tracheids over vessel elements appears to
increase at higher elevations, as a consequence of
growing in colder and drier environments, due to
a greater cambial activity, which is the case of S.
formosus at 4200 m. Tracheid development may pre-
dominate over vessel development in species growing
in colder and/or drier environments, since tracheids
provide safer conduction and reduced risks of cavita-
tion according to previous studies of xylem anatomy
in high elevation species (Carlquist 1988). Motomura
et al. (2007) attribute the loss of vessels in Nepalese
populations of Ephedra pachyclada growing from
2560 to 4820 m a.s.l. to a decrease in water supply
with increasing elevation. According to these authors,
drier conditions inhibit lateral expansion of the stems,
modifying cambial activity by suppressing vessel
development and favoring tracheid formation instead.
Percolla et al. (2021) emphasize the importance of
tracheids during periods of water stress or following
freeze–thaw stress due to their narrower diameters,
reducing the risk of hydraulic failure in comparison
with vessel elements.
A lower xylem conductivity efficiency due to
reduced vessel diameter displayed by higher eleva-
tion plants may be compensated to some extent by
an increase in vessel density (Sperry et al. 2008).
These narrow vessel diameter-higher vessel density
patterns have been reported previously in different
species growing along elevational gradients (Fisher
et al. 2007; Pandey et al. 2021). Even though vessel
element density did not show significant differences
between elevations, the increased stem conducting
area in S. formosus plants from the higher elevation
may be considered a compensating strategy to reduce
the less efficient narrower vessel diameters.
Although we did not find differences in the den-
sity of vessel elements in plant stems of S. formosus
growing at the lower and upper limits of its distribu-
tion range, we did observe a greater development of
Vol.: (0123456789)
13

secondary xylem composed mostly of tracheids in
the plants growing at 4200 m. Another interesting
finding was the difference in proportions of paren-
chyma tissues against conducting tissues between
plants of low and high elevations. The larger piths
composed of parenchyma tissue observed in plant
stems of S. formosus growing in PSJ (3100 m)
imply a higher proportion of water-storing tissues;
yet conversely, it increases the susceptibility to
extracellular ice nucleation induced by nighttime
frosts. However, since these events are rare and of
very short duration at this elevation, a greater devel-
opment of parenchyma tissues associated to wider
vessels should provide a more rapid and efficient
conduction under moister conditions.
In conclusion, S. formosus responded to the con-
trasting environmental conditions from low to high
elevation by: 1. Increasing its freezing resistance
in order to survive the frequent freezing nighttime
temperatures of the paramos, and 2. Modifying its
xylem vessel diameters to avoid the increasing risks
of cavitation under continuous freeze–thaw cycles
that occur at higher elevations. Responses and adap-
tations to changing climate conditions are essential
to understand plant distribution shifts along eleva-
tion gradients, shifts which are evident from world-
wide tropical and temperate alpine reports (Moru-
eta-Holme et al. 2015; Koide et al. 2017; Liang
et al. 2018; Auld et al. 2022). Increasing evidence
of intra and interspecific plant trait variations under
different environmental gradients will aid to deter-
mine the outcome of individual species and their
effects on ecosystem functioning under climate
change. And as suggested by Rixen et al. (2022)
from the well-defined trait-environment relation-
ships of alpine plants, “species’ elevational pref-
erences and range sizes may be useful proxies for
inferring functional trait responses to environmental
gradients globally”.
Acknowledgements This project was funded by the Consejo
de Desarrollo Científico, Humanístico, Tecnológico y Arte of
the Universidad de Los Andes (ULA, Merida-Venezuela) (Pro-
ject Nº C-1731-11-01-F). F. Rada is grateful to the Open Soci-
ety University Network (OSUN) and the Universidad de Los
Andes (UniAndes, Bogota-Colombia) for their sponsorship and
essential support.
Author contributions All authors planned the research and
designed the experiments. MA and FR conducted freezing
resistance measurements, MA and FE conducted anatomical
Vol:. (1234567890)
13
Plant Ecol
studies. All authors analyzed the data. FR wrote the original
draft, MA and FE reviewed and edited the manuscript.
Funding This work was suported by the Consejo de Desar-
rollo Científico, Humanístico, Tecnológico y Arte of the Uni-
versidad de Los Andes (ULA, Merida-Venezuela) (Project No.
C-1731–11-01-F). FR received funding through an Open Soci-
ety University Network (OSUN) fellowship.
Data availability All data are available if required.
Declarations
Competing interest The authors declare that they have no
conflict of interest. Authors have no relevant financial or non-
financial interests to disclose.
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