Science of the Total Environment 850 (2022) 157908

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Age-specific and species-specific tree response to seasonal drought in

tropical dry forests
⁎
Emily A. Santos a, , Xavier Haro-Carrión b, Jasper Oshun c
a
University of California, Davis, Davis, CA 95616, United States of America
b
Macalester College, St Paul, MN 55105, United States of America
c
U.S. Fulbright Scholar and Visiting Professor at the Universidad de Ingeniería y Tecnología, Lima, Peru

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Dry season subsurface moisture is held at

greater tensions in disturbed forests.
• Regenerating forests access rock mois-
ture/groundwater to meet dry season
demands.
• Native Tropical Dry Forest species have
distinct dry season water use strategies.

A R T I C L E I N F O A B S T R A C T

Editor: Manuel Esteban Lucas-Borja Millions of people depend on ecosystem services provided by Tropical Dry Forests (TDFs), yet their proximity to pop-
ulation centers, seasonally dry climate, and the ease at which they are converted to agriculture has left only 10 % of
Keywords: their original extent globally. As more TDFs become protected, basic information relating TDF age to subsurface
Hydrogeology water resources will help guide forest recovery. Severe deforestation and recent reforestation around Bahía de
Forest ecology
Caráquez, Ecuador produced a mosaic of different successional stages ideal for exploring relationships between TDF
Tropical dry forests
Reforestation
age, subsurface water availability and species-specific responses to seasonal drought. Over one year, we measured
Stable isotopes gravimetric water content, predawn and midday leaf water potential, and the stable isotope composition of xylem
Plant-water uptake and source waters in two regenerating and one primary forest. Over the transition from wet to dry season, we discov-
ered a sharper decrease in predawn water potential in younger successional forests than in the primary forest. Growing
in degraded subsurface environments under increased competition, successional forest trees accessed deeper sources
of moisture from unsaturated weathered bedrock and groundwater through the dry season; however, different species
employed distinct water use strategies. Ceiba trichistandra maintained midday water potentials above −1.27 MPa
through a drought avoidance strategy dependent on groundwater. Sideroxylon celastrinum tolerated drought by lower-
ing predawn and midday water potential through the early dry season but took up greater proportions of saprolite
moisture and groundwater as the dry season progressed. Contrastingly, Handroanthus chrysanthus maintained access
to shallow soil and saprolite moisture by dropping midday water potential to −4.30 MPa, reflecting drought tolerance.

⁎ Corresponding author at: 3006 Concord Place, Davis, CA 95618, United States of America.
E-mail address: eadsantos@ucdavis.edu (E.A. Santos).

http://dx.doi.org/10.1016/j.scitotenv.2022.157908
Received 4 January 2022; Received in revised form 3 August 2022; Accepted 4 August 2022
Available online 6 August 2022
0048-9697/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
E.A. Santos et al.
Our results show that limited subsurface water resources in regenerating TDF's lead to species-specific adaptations re-
liant on deeper sources of moisture. The recovery of soil and saprolite hydrologic properties following disturbances is
likely to exceed 100 years, highlighting the importance of forest conservation.
1. Introduction
Tropical Dry Forests (TDFs), found between 23.5° North and South in
areas where annual rainfall ranges from 250 to 2000 mm with a marked
dry season of at least five to six months (Murphy and Lugo, 1986; Bullock
et al., 1995; Miles et al., 2006), are adapted to uniquely challenging
environmental conditions. TDFs occur under the ‘Aw’ and ‘BSh’ Köppen-
Geiger classification, and the limited amount and seasonality of rainfall
strongly influence species distribution, tree phenology, structure, and
species-specific adaptations to survive in water limited environments
(Borchert, 1994; Schwinning and Ehleringer, 2001; Kennard, 2002;
Borchert et al., 2004; Pennington et al., 2009; Sanchez-Azofeifa et al.,
2013; Horstman, 2017). These drought adaptations fall along a spectrum
from drought avoidance to drought tolerance (e.g., Borchert and
Pockman, 2005), and include deciduousness (Hoffmann et al., 2011), inter-
nal water storage (e.g., Butz et al., 2018), the capacity to withstand ex-
tremely negative leaf water potentials, or to reduce stomatal conductance
(Poorter and Markesteijn, 2008; Chaturvedi et al., 2021), and the invest-
ment in greater root to shoot growth (e.g., Bullock and Solis-Magallanes,
1990) to develop complex rooting systems to access deep sources of
water (Jackson et al., 1995; Meinzer et al., 1999; Johnson et al., 2016;
Querejeta et al., 2006; Querejeta et al., 2007; Hasselquist et al., 2010;
Estrada-Medina et al., 2013). In some cases, drought coping strategies are
linked to nutrient availability as species exhibit lower hydraulic safety mar-
gins (HSM) and faster rates of recovery in nutrient rich soils, prioritizing
growth over safety (Oliveira et al., 2021).
TDFs, which account for 42 % of tropical forests (Miles et al., 2006),
have experienced historically high rates of resource exploitation due to fac-
tors that make them ideal areas to deforest for short-cycle crop agriculture
and livestock farming (Janzen, 1988; Portillo-Quintero and Sánchez-
Azofeifa, 2010; Hansen et al., 2010; Gillespie et al., 2012; Portillo-
Quintero et al., 2015). The rate of deforestation in the TDF biome has
been the third highest globally (Hansen et al., 2010), resulting in a global
decrease to 10 % of their original extent (Banda-R et al., 2016). Remaining
TDFs are highly fragmented and among the least protected and most
vulnerable ecosystems on Earth (Banda-R et al., 2016). Few studies have
described the processes of degradation in TDFs, documented how past
land use might influence the timespan of recovery (Griscom and Ashton,
2011), or explored how sensitive TDFs might be to changes in future
climate (e.g., Allen et al., 2017). For example, deforestation of TDFs for
cattle production is likely to compact the soil, decrease bulk density, and re-
duce water infiltration (Holl, 1999), leading to a loss in subsurface soil
water storage and tree water availability (Zimmermann et al., 2006,
Germer et al., 2010), and decreased ecosystem resilience (Hatfield et al.,
2001). Reforestation and natural forest succession can lead to the recovery
of the regulatory and provisional hydrologic services provided by TDFs
through enhanced soil organic matter, increased infiltration and groundwa-
ter recharge, reduced evaporation and increased subsurface storage
(Frankie et al., 2004; Chazdon, 2008; Ilstedt et al., 2016; Jones et al.,
2017; Portillo-Quintero and Smith, 2018; Wright et al., 2018; Quijas
et al., 2019). Despite the hydrologic importance of a healthy TDF for plants
and animals dependent on rare dry season moisture (e.g., Haro-Carrión
et al., 2021), there remains large uncertainty in how TDFs in general and
recovering TDFs in particular will respond to seasonal or longer-term
droughts (e.g., Oliveira et al., 2021). This outstanding uncertainty limits
effective forest conservation and regeneration (Quesada et al., 2009;
Espinosa et al., 2012; Calvo-Rodriguez et al., 2017; Escribano-Avila et al.,
2017; Wright et al., 2018; Chitra-Tarak et al., 2021; Oliveira et al., 2021;
Fremout et al., 2021).
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Science of the Total Environment 850 (2022) 157908
Similar to global trends, the TDF's of coastal Ecuador have been heavily
altered, resulting in fragmented forests of varied ages and levels of distur-
bance. This mosaic of forest ages provides a valuable setting to study the
seasonal patterns in water use across forests of varying age and between
different native species. We took advantage of this mosaic to ask: where
do native species in regenerating and old growth forests derive their
water over wet and dry seasons and what strategies are used to cope with
seasonal drought? We explored the following hypothesis: shallow sources
of tree available moisture would be limited in regenerating secondary for-
ests (<100 years old), requiring younger trees to employ species-specific
strategies to access water through the dry season. We conducted high fre-
quency measurements of subsurface moisture dynamics, predawn and mid-
day water potential, and the stable isotope composition of soil, weathered
bedrock and tree water xylem across primary (>100 years), late succes-
sional (30–100 years), and early succession enriched (10–30 years) TDFs.
The results of this study can inform community efforts to resist forest
fragmentation and guide best practices in forest regeneration in one of
the few remaining TDFs in the world.
2. Methods
2.1. Regional climate and seasonality differences across forest age
This study was conducted in the Cordillera del Bálsamo, a local
landowner-managed bio-corridor consisting of approximately 9.64 km2 of
protected TDF, near the city of Bahía de Caráquez (0°25′60″S 80°25′31″
W), Manabí province, coastal Ecuador (Fig. 1a). The underlying geology
is composed of massive sandstones from the upper Miocene-Pliocene
Bourbon Formation overlain by Quaternary terraces, alluvial deposits,
and silty loam soil (National Geological Map of the Republic of Ecuador,
1982). Only 2 % of coastal Ecuador's TDFs remain (Plan de Desarrollo y
Ordenamiento Territorial 2015–2019, n.d.). Even in the Cordillera del
Bálsamo, few intact stands of TDF remain, and primary TDF is found only
in the most inaccessible areas. Because of these tremendous and exponen-
tial rates of loss, organisms that once were common in these forests now
face extinction for lack of habitat (Horstman, 2017). Of the remaining forest
patches, some are selectively logged while others are well preserved
(Asociación Cerro Seco Reservistas Privados de la Cordillera del Bálsamo,
2015). In the last 40 years, local and international organizations have
planted over 45,000 native trees across the Cordillera del Bálsamo.
Mean annual temperature is 25 °C and relative humidity generally ex-
ceeds >75 % year-round. Moisture is generally slightly higher on slopes fac-
ing the coast due to fog rising off the coast (referred to locally as ‘brisas’)
(Dodson and Gentry, 1991). Data from Climate Hazard Group InfraRed
Precipitation with Station Data (CHRPS) indicates that Bahía receives an
annual average rainfall of approximately 492 mm, about 70 % of which
falls between January and March (wet season). A low precipitation period
(dry season) can exceed eight months from late April throughout early De-
cember (Fig. 1b, SM Appendix A). Patterns in precipitation drive the timing
of tree growth, green-up, and senescence in the Cordillera Bálsamo. To ex-
amine the seasonal variation of greenness patterns across our study sites,
we used Moderate Resolution Imaging Spectroradiometer (MODIS) derived
Enhanced Vegetation Index (EVI) for each site. EVI peaks as forests put on
new growth in response to both solar radiation and water. However, in
the Cordillera del Bálsamo water, or water availability, is more likely to
control peak EVI because solar radiation remains relatively constant
(Fig. 1c, SM Appendix B). We considered these trends including the timing
of peak EVI and the steady decline as the landscape dries to design our
2017–2019 sampling campaigns (SM Appendix C).
E.A. Santos et al.
Fig. 1. Study Area: a) study sites and vegetation seasonality (i.e. ratio between Normalized Difference Vegetation (NDVI) during wet and dry seasons); Towns and built areas
are indicated in black; b) 2016–2019 average monthly precipitation for the study area (Derived from Climate Hazard Group InfraRed Precipitation with Station Data
(CHRPS), Funk et al., 2015); c) 2010–2020 averaged MODIS/MCD43A4 remotely sensed Enhanced Vegetation Index (EVI) surface reflectance composites for Sites 1, 2,
and 3. Sampling dates of 2017–2019 field campaign indicated with black arrows.
To estimate the geographic extent of the characteristic forest type of
each study site, we used Sentinel-2 derived Normalized Difference Vegeta-
tion Index (NDVI). With significantly finer (i.e. pixel size) spatial resolution
than MODIS, Sentinel-2 NDVI data allowed us to quantitatively differenti-
ate study sites in terms of vegetation seasonality. For each site, we obtained
NDVI data for the wet and dry season and calculated their difference.
Forests with lower seasonality have more similar NDVI values in both sea-
sons resulting in a difference closer to zero. Forests with high seasonality
would tend to be substantially greener during the wet season compared
to the dry season, which would be manifested by higher NDVI difference
(SM Appendix D).
This “Seasonality” metric indicates that Site 1 is the least seasonal
(NDVI wet – dry difference = 0.06). About 16.3 % of the study area is cov-
ered by forests similar to Site 1, most occurring in higher elevation and less
accessible areas. Sites 2 and 3 are significantly more seasonal than Site 1,
with 2 (NDVI wet – dry difference = 0.30) slightly less seasonal than
3 (NDVI wet – dry difference = 0.44). Sites 2 and 3 each cover about
37.3 % of the landscape (Fig. 1a; SM Appendix D).
2.2. Site characterization
We selected three sites to capture differences in forest age while control-
ling for topography and long-term moisture availability (Fig. 2) and mea-
suring similarities and differences in soil characteristics. While similar soil
types exist across the Cordillera del Bálsamo, the tectonic convergence pro-
duces a compressive tectonic regime, forming high-relief valleys and hills.
Site 1, one of the only primary (>100 year old) forests in the Cordillera
del Bálsamo, is located at an elevation of 303 m.a.s.l. and due to its remote
and rugged location was only accessible in the dry season. Prior to and dur-
ing the early 1900's the more accessible areas of the Cordillera del Bálsamo
were deforested for agricultural use, livestock and firewood. Today, these
areas hold a mix of old trees, such as C. trichistandra, deemed unsuitable
for logging, and over 20,000 trees planted within sections of recovering
TDF. Site 2 is a well-established late successional (30–70 years) forest at
an elevation of 65 m.a.s.l. Closer to Bahía's city center nearly all of the old
growth trees have been harvested for lumber and firewood, and replaced
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Science of the Total Environment 850 (2022) 157908
with cash crops and pasture land for animals, save some smaller efforts
of reforestation. Site 3 was recently reforested (10–30 years) located at
35 m.a.s.l. adjacent to an agricultural sector. A similar distribution of native
species and dry season canopy cover was found across all three sites.
To control for the influence of site topography on soil moisture
availability we calculated the Topographic Wetness Index (TWI; Beven
and Kirkby, 1979) at each site using a 30-m digital elevation model (SM
Appendix E). Although TWI varied from 2.9 to 16.4 across the region
shown in Fig. 1a (SM Appendix F), the values of our three study sites
were relatively similar (Fig. 2), falling between 6.0 and 6.5.
2.3. Sampling of liquid waters and soil and saprolite
Rainwater samples (n = 27) were collected as often as possible during
field campaigns using a plastic funnel, a glass collector and a ping pong ball
to prevent evaporation. Groundwater samples (n = 9) were taken from 1 m
below the water table in two different wells (SM Appendix G). One well was
located between Site 1 and Site 2 (5 samples), and the other well was lo-
cated adjacent to Site 3 (4 samples). Surface water samples (n = 3) were
gathered during the wet season from a small valley channel adjacent to
the first well from a 15 cm immersion depth. Samples were collected in
plastic scintillation vials, and sealed with a stopper and Parafilm (Bemis
Co., Neenah, WI) to prevent evaporation prior to vacuum distillation, and
then stored in a freezer (−20 °C) until stable isotope analysis.
On each sampling date, we augured one hole per site to measure gravi-
metric moisture content and stable isotope composition. Holes were made
below trees monitored for water potential and stable isotope analyses. We
collected bulk soil and saprolite samples with a bucket auger at 10 cm inter-
vals to a maximum depth of 100 cm, where subsurface density and auger
length prevented deeper sampling. Depth, color, field texture, observable
structure, and the presence or absence of roots was recorded for each sam-
ple. Immediately upon collection, bulk soil and saprolite samples were
placed inside a plastic scintillation vial and sealed with a stopper and
Parafilm. All solid samples were stored in a cooler until brought to the lab
where they were kept in a −20 °C freezer. We extracted water from bulk
soil, bulk saprolite and tree xylem via cryogenic distillation (see below).
E.A. Santos et al.
Fig. 2. Cordillera del Bálsamo above and belowground metrics reveal similarities across sites as well as key differences resulting from land use.
Before and after extraction, we measured the mass of the bulk soil and bulk
saprolite samples to determine gravimetric moisture content. We also mea-
sured dry season infiltration rates by conducting falling head tests at Sites 2
and 3 using a bottomless graduated cylinder. We began with an initial
volume of 2000 ml and recorded differences in volume at minute intervals
for 30 min.
2.4. Measurements of plant water potential
We measured ‘predawn’ leaf water potential (Ψpd) in 26 trees at times
between 0300 and 0500 h. Eight trees were measured at Site 1, thirteen
trees at Site 2, and six trees at Site 3 (SM Appendix H). Measurements
were made using a portable pressure chamber (Model 1000, PMS Instru-
ment Company, Albany, OR, USA). We used O2 gas, as N2 gas was not avail-
able in Bahía. Prior to field work, we conducted comparisons using O2 and
N2 and found no statistical difference between the two sets of measure-
ments (SM Appendix I).
To explore species-specific responses to seasonal drought, we measured
both predawn and midday (Ψmd) leaf water potentials at Site 2. Here we
focused on three common native species: Ceiba trichistandra, Sideroxylon
celastrinum, and Handroanthus chrysanthus. The three individuals measured
were spaced about 10 m apart, forming a triangle. C. trichistandra are typi-
cally among the tallest tropical forest species, with large umbrella-shaped
canopies, bulbous green trunks, and enormous, buttressing above-ground
roots. As TDFs are slashed and burned, C. trichistandra remain untouched
and are often the oldest and most established species in reforested areas.
Therefore, the C. trichistandra sampled at Site 2 is much larger and
estimated to be much older (>100 years) than the other two species
sampled. We followed the same procedures as described for the predawn
measurements, but conducted midday water potential measurements
between 1100 and 1300 h.
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Science of the Total Environment 850 (2022) 157908
2.5. Xylem collection and isotope analysis
We sampled tree xylem across the three sites for isotope analyses. Xylem
was sampled by clipping stems with a pole pruner (some samples in
December 2017 and all samples from March 2018 onwards), or by using
an incremental tree borer to sample the bole 1–1.5 m above the ground
(some samples in December 2017, only). Immediately upon extraction of
a core, all tissue external to the xylem was removed to the first 5 cm of
xylem tissue. Cores and stems were immediately placed inside plastic scin-
tillation vials and sealed with a stopper and Parafilm and placed in freezer
upon arriving from the field. The bark of some TDF species is exceptionally
hard, such that trees with both tall canopies and impenetrably hard bark
were excluded from this study.
Water was extracted from plant xylem and bulk soil/saprolite samples
at the Center for Stable Isotope Biogeochemistry at the University of
California at Berkeley using a cryogenic vacuum distillation line (100 °C,
1 h) (Ehleringer et al., 2000) following the procedure of West et al.
(2006). Samples were analyzed using a hot chromium reactor unit
(H/Device™) interfaced with a Thermo Delta V Plus mass spectrometer
and a Thermo Gas Bench II interfaced to a Thermo Delta V Plus mass spec-
trometer to generate hydrogen (δD) and oxygen (δ18O) in permil notation
relative to Vienna Standard Mean Ocean Water, VSMOW.
2.6. Determination of plant water source(s) from stable isotope composition
We conducted a semiquantitative analysis on the stable isotope data to
determine xylem source waters at each site and in the three species moni-
tored at Site 2. We measured the isotope composition of three potential
water sources: bulk soil moisture, bulk rock moisture (from the saprolite,
only), and groundwater. On each date for which data were available
(May 3–July 5, 2018), we calculated mean δ18O and mean δD for soil
E.A. Santos et al.
water and for saprolite water, respectively. For the third potential water
source, groundwater, we calculated the mean δ18O and mean δD for all
subsurface and surface water samples taken over the monitoring period
(n = 12). To determine the source water(s) at each site or the source
water(s) for the three species at Site 2, we conducted the following analysis
on all dates for which xylem water and source water data were available.
First, if the mean xylem water isotope composition plotted within the
standard deviation of any of the three sources in dual isotope space, we
determined that the site/species derived all its water from that source.
Second, if the mean xylem water plotted within a triangle bounded by the
three sources, we applied a three-end-member isotope linear mixing
model (Rothfuss and Javaux, 2017; von Freyberg et al., 2020; Hahm
et al., 2020). We determined the relative proportions of source water de-
rived from soil, saprolite, and groundwater by solving the system of three
equations:
δ18 Oxylem ¼ δ18 Ogw f gw þ δ18 Osm f sm þ δ18 Orm f rm (1)
δDxylem ¼ δDgw f gw þ δDsm f sm þ δDrm f rm (2)
f gw þ f sm þ f rm ¼ 1 (3)
where the subscripts gw, sm, rm, and xylem refer to groundwater, bulk soil
moisture, bulk rock moisture, and xylem water, respectively.
Finally, if the mean xylem water plotted between two sources in dual
isotope space, we applied a two-end-member isotope linear mixing model
for both δ18O and δD:
δ18 O, or δDxylem ¼ δ1 f 1 þ δ2 f 2 (4)
f1 þ f2 ¼ 1 (5)
If the results of f1determined by the two-end-member mixing models
using δ18O and δD were within 0.1, we accepted the results. We then com-
bined the results of our semi-quantitative analysis with our water potential
data to interpret site- and species-specific source waters over the monitor-
ing period.
2.7. Statistical analysis
Statistical analyses were done on the following data using JMP
(SAS Institute, Cary, NC, United States). Soil and saprolite moisture data
and water potential data were expressed as average ± standard error.
Differences in stem water potential data were tested by one-way ANOVA.
We used Shapiro-Wilk goodness of fit tests to check or to confirm the
assumption that data were normally distributed. When this assumption
was violated, we used Kruskal-Wallis tests to detect significant differences
among groups. We used Levine and Bartlett tests to test the assumptions
of equal variance among groups. When this assumption was violated, we
used Welch tests to detect significant differences among groups. If signifi-
cant differences among groups were found, Tukey's HSD multiple means
comparisons were used to determine how groups differed. For these tests,
values of P < 0.05 were considered statistically significant. Stable isotope
data were expressed as average ± standard error and as raw data in dual
isotope space.
3. Results and interpretations
3.1. Subsurface material properties and moisture dynamics
Whereas the general soil texture was similar across the three sites, we
found several important differences (SM Appendix J). We observed the
greatest leaf litter layer at Site 1, and the organic content in soil was greater
at Sites 1 and 2 than at Site 3. Clay content, however, was found in greatest
concentrations in Site 3 soils.
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Science of the Total Environment 850 (2022) 157908
Soil gravimetric moisture content steadily decreased at Sites 1, 2, and 3
from late March 2018 to July, but then leveled off through the end of the
year (Fig. 3a). Saprolite gravimetric moisture content decreased steadily
at sites 2 and 3, but leveled off at Site 1. Gravimetric moisture content in
soil and saprolite was consistently lower and declined most rapidly at Site
2 through the dry out (March through July 2018). Variability with depth
was large on most sampling dates (Fig. 3b).
In the middle of the wet season (March 2018), average Ψpd was similar
at Sites 2 in 3 in March 2018 (Site 1 was inaccessible). Through the dry
season, average Ψpd decreased at each site (Fig. 4); however, we observed
statistically significant differences between sites (SM Appendix K). The de-
crease in site average Ψpd was most pronounced at Site 3, reaching mini-
mum values of and −2.80 ± 0.57 MPa, while the average Ψpd at Site 1
never fell below −1.00 MPa. Whereas there was no observed rainfall dur-
ing the study period, we observed dew forming on leaves at Site 1 on
May 19th and May 30th. At Site 2, average Ψpd declined rapidly in early
summer to a minimum value −1.57 ± 0.35 MPa, but then was constant
for nearly a month before increasing slightly. The average Ψpd at Sites 2
and 3 increased to nearly mid wet season values in January 2019.
3.2. Patterns in stable isotopes and predawn water potential across TDFs of
different ages
Rainfall samples collected from December 2017 to May 2018 and in
January 2019 averaged −2.18 ± 0.14 ‰ in δ18O and −5.36 ± 0.73 ‰
in δD (SM Appendix L). Using our rainfall data, we calculated a (limited)
Local Meteoric Water Line (LMWL), which differed in slope and intercept
from the Global Meteoric Water Line (GMWL).
Groundwater samples averaged −2.75 ± 0.16 ‰ in δ18O and
−11.10 ± 0.62 ‰ in δD. Over the length of the study, groundwater
δ18O varied from −2.5 ‰ to −4 ‰ and δD varied from −8 ‰ to
−15 ‰. At all three sites, the mean isotopic composition of bulk soil mois-
ture (≤60 cm) was enriched relative to bulk saprolite moisture (≥70 cm)
(SM Appendix L), and generally showed a wider range of variability (SM
Appendix M). There were no significant differences in isotope composition
of bulk soil or bulk saprolite between sites.
Isotopic analyses of xylem water showed seasonal patterns in source
water uptake that differed across the three sites (Fig. 5, SM Appendix N).
Whereas the seasonal trends in δ18O values at all three sites were remark-
ably similar, the average δD and d-excess values were very different
(SM Appendix O).
The isotope compositions of Site 1 xylem values fall along a trend line
with a slope of approximately 4.5, with δD delta values negatively offset
approx. 10 ‰ and average d-excess remaining <0 ‰ for the entire dry sea-
son. Xylem values generally plot below bulk soil and saprolite values.
Xylem water δ18O and δD both decreased as the dry season progressed
(Fig. 5a).
At Site 2, the stable isotope composition of xylem water plots below the
GWML in the early part of the dry season with average d-excess <0 ‰.
From March 5, 2018–May 31, 2018 (wet-early to dry season) xylem
water values were similar to first bulk soil and then bulk saprolite moisture
(Fig. 5b). On June 13, xylem water δD increased suddenly with d-excess >0
‰ and as high as 15 ‰. As the dry season progressed, both δ18O and δD
increased steadily as the isotope composition of xylem water moved closer
to that of groundwater.
The stable isotope composition of Site 3 xylem water formed a trendline
above the GMWL, with an average d-excess >7 ‰. As the dry season prog-
ressed, the average stable isotope composition of xylem water decreased,
although there is a large amount of variation across sampled individuals.
Although the results of the semi-quantitative analysis are limited, they
show important site-specific trends (SM Appendix P, SM Appendix Q).
Overall, xylem water shifts from a dominantly bulk soil moisture source
to deeper sources of moisture (bulk saprolite and groundwater) as the sea-
son progresses. In mid to late May, xylem water at Site 1 reflects bulk soil
moisture, while xylem water at Sites 2 and 3 contains greater contributions
from deeper moisture sources.
E.A. Santos et al. Science of the Total Environment 850 (2022) 157908

Fig. 3. a) Gravimetric Water Content in soil and saprolite averaged across 10 cm intervals to depths of 1 m (error bars represent standard error). The absence of Site 1 soil and
saprolite moisture prior to May 19, 2018 and post July 6, 2018 was due to site inaccessibility during the wet season; b) Variability in gravimetric water content across
sampling dates and with depth in the subsurface.

All sites show a similar seasonal range in average xylem water δ18O,
however we observed differences in the relationship between subsurface
water availability (average Ψpd) and δ18O (Fig. 6a–c). At Site 1, seasonal
drying resulted in progressively negative xylem water that coincided with
decreasing predawn water potential. As the subsurface dried, average
Ψpd decreased from −0.45 to −0.76, and average δ18O decreased from
−2.49 ‰ to −4.53 ‰.
Seasonal drying at Site 2 resulted in an initial decrease in average Ψpd.
On June 13th, average xylem water δ18O reached a minimum value of
−4.70 ‰ and average Ψpd reached a minimum value of −1.57 MPa.
Through the remainder of the dry season, average values of δ18O and
Ψpd steadily increased.
At Site 3, decreases in average xylem water δ18O coincided with a steep
decline in average Ψpd to −2.80 MPa. We observed that both δ18O and

Fig. 4. Predawn tree leaf water potential (Ψpd) averaged across (n) individuals at each site in replicates of 3 leaves per tree. Data is plotted on a non-linear x-axis. Error bars
represent standard error. Absence of Site 1 Ψpd on March 5, 2018 and January 7, 2019 was due to site inaccessibility during the wet season.

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E.A. Santos et al. Science of the Total Environment 850 (2022) 157908

Fig. 5. Dual isotope plots showing seasonal trends in the site average xylem water. Also shown is the Global Meteoric Water Line (GMWL; equation: δD = 8*δ18O + 10), as
well as bulks soil and saprolite moisture measured at each site. A limited number of precipitation samples (n = 27) roughly plot along a LMWL. Panels a, b, and c correspond
to Site 1, Site 2, and Site 3, respectively. Absence of Site 1 data on March 5, 2018–May 3, 2018 was due to site inaccessibility during the wet season.

Ψpd increased towards the observed wet season values (March 2018) at the
beginning of the wet season (January 2019).
3.3. Species specific differences in water potential and source water
At Site 2, seasonal drying resulted in distinct patterns in predawn (Ψpd)
and midday (Ψmd) water potentials and the stable isotope composition of
xylem water among three deciduous native species: C. trichistandra,
S. celastrinum, and H. chrysanthus.
From May through early July, C. trichistandra Ψmd decreased from
−0.33 ± 0.15 MPa to −1.27 ± 0.21 MPa (Fig. 7 and SM Appendix
R) tracking modest declines in Ψpd. Over this period, the stable isotope
composition of C. trichistandra xylem water was consistently enriched com-
pared to neighboring species, plotting close to groundwater in dual isotope
space. By January 7, 2019 Ψpd and Ψmd had risen to −0.43 ± 0.12 MPa
and −0.80 ± 0.20 MPa, respectively, reflecting an increase in subsurface
moisture availability relatively to the previous July. However, the stable
isotope composition of C. trichistandra xylem water still reflected that of
groundwater.
In the early dry season spanning March 5 to June 13, S. celastrinum Ψmd
decreased steadily from −1.31 ± 0.36 MPa to −3.50 ± 0.26 MPa,
tracking decreases in Ψpd. These decreases in leaf water potential coin-
cided with decreases in xylem water isotope composition to minimum
values in δ18O and δD on June 2. Over this initial drying, S. celastrinum
xylem water resembled the isotope composition of bulk soil and saprolite
water. Beginning on June 23, Ψmd followed slight increases in Ψpd by
increasing to −2.43 ± 0.06 MPa, and further to −2.04 ± 0.01 MPa on
July 4 (Fig. 7, SM Appendix S). This shift in Ψpd and Ψmd closely followed
increases in xylem water δ18O and δD, which began on June 13. Through
the remainder of the dry season, increases in Ψpd and Ψmd suggest access
of deeper, and less tightly held water resources. The stable isotope compo-
sition of S. celastrinum xylem water plotted closer to groundwater in dual
isotope space through the remainder of the dry season.
H. chrysanthus Ψmd and Ψpd declined steadily throughout the dry sea-
son. From March 5 to July 4, Ψmd decreased from −1.60 ± 0.10 MPa to
−4.30 ± 0.90 MPa (Fig. 7, SM Appendix T). These dry season values of
Ψmd were the most negative water potentials measured among Site 2 spe-
cies. By January 7, 2019, H. chrysanthus Ψpd and Ψmd had returned to
values similar to those measured in March 2018. H. chrysanthus xylem
water stable isotope composition matched bulk soil and bulk saprolite for
the first part of the dry season, but δ18O began to increase on June 13th,
and δD began to increase on June 24th. Whereas the water potential data

Fig. 6. Site average xylem water δ18O plotted against site average predawn leaf water potential. Symbol color reflects the date of xylem and leaf water potential
measurements. Error bars represent the standard error of all measurements (SM Appendix N) indicating variability across the species. Panels a, b, and c correspond to Site
1, Site 2, and Site 3, respectively. Absence of Site 1 data on March 5th, 2018 and January 7th, 2019 was due to site inaccessibility during the wet season.

7
E.A. Santos et al.
Fig. 7. Predawn (Ψpd) and midday (Ψmd) leaf water potential for 3-replicate leaves averaged from Site 2 single species C. trichastandra, S. celastrinum and H. chrysanthus.
Error bars represent standard error. Lower plot shows species specific xylem water δD and δ18O plotted in time series with Site 2 groundwater and subsurface moisture.
showed consistent decreases throughout the dry season, the stable isotope
composition of xylem water revealed a mid dry season shift towards more
enriched subsurface water sources.
Limited results of our semi-quantitative analysis reflect important tem-
poral and species-specific patterns in water use (SM Appendix U, SM Ap-
pendix V). First, there is a site-wide shift towards deeper sources of
moisture as the dry season progresses. C. trichistandra source water shifted
from 91 % groundwater on May 31 to 100 % groundwater through the
rest of the dry season (and even January 2019 via direct inference).
S. celastrinum, used exclusively soil in late wet season (May 3, 2018), but
shifted to 46 % bulk soil moisture, 44 % bulk saprolite moisture, and
11 % groundwater on May 31, and 46 % bulk saprolite and 54 % groundwater
on June 24. The semi-quantitative analysis yielded only one result for
H. chrysanthus (May 31) showing 87 % bulk soil moisture and 13 % bulk
saprolite moisture. In January 2019, C. trichistandra relied exclusively on
groundwater, and S. celastrinum used a mix of groundwater (68 %) and
bulk saprolite moisture (33 %). Although the mixing model failed
for H. chrysanthus, the data suggest approximately 70 % of source water
was derived from groundwater with the remaining 30 % coming from
bulk saprolite.
4. Discussion
4.1. Water resource availability in primary and regenerating forests
Whereas wet season predawn water potentials are similar across Sites 2
and 3 (March), and similar to late wet season predawn water potentials at
Site 1 (May), dry season predawn water potentials are more negative in
younger successional forests. This discovery supports the first part of our
hypothesis that subsurface water resources are limited in regenerating for-
ests (Fig. 4).
While tree water potential may remain high due to the presence of
fog in cloud forests or coastal environments (e.g., Bruijnzeel, 2001;
8
Science of the Total Environment 850 (2022) 157908
Bruijnzeel, 2004; Johnson and Smith, 2006; Breshears et al., 2008;
Reinhardt and Smith, 2008; Eller et al., 2013; Goldsmith et al., 2013;
Dawson and Goldsmith, 2018), we observed dew only at Site 1 only on
May 19th and May 30th, and thus conclude there were no significant mois-
ture inputs during the dry out period.
We did, however, find significant differences in the structure of the shal-
low subsurface that may partially explain the differences in water availabil-
ity. Although all three sites were classified as silty loams, Site 3 soils showed
much lower organic matter content and much higher concentrations of
clay. Additionally, infiltration capacity was smaller at Site 3 than at Site
2, consistent with a loss of organic matter and greater compaction due to
land use (e.g., Holl, 1999). Although we were unable to measure infiltration
capacity at Site 1, the presence of a litter layer and a high organic matter
content would both contribute to larger infiltration rates and larger mois-
ture storage (at more accessible water potentials) in the organic rich soil.
Our data show that accessing water resources requires greater energy
expenditure in the youngest forest (Site 3), intermediate energy expendi-
ture in the intermediate aged forest (Site 2), and lowest energy expenditure
in the primary forest (Site 1). Limited water resources in the regenerating
TDFs of coastal Ecuador have thus required trees to develop adaptations
to survive seasonal drying, as has been observed in other TDFs
(e.g., Brown and Lugo, 1990; Lebrija-Trejos et al., 2010; Alvarez-Añorve
et al., 2012; Lohbeck et al., 2013; Becknell and Powers, 2014; Paz et al.,
2015). Below we discuss the differences in tree water use we documented
across forests of different age, explore possible explanations for negative
offsets in δD xylem water, and highlight species-specific adaptations to sea-
sonal drying in a regenerating forest.
4.2. Observed forest water use in primary and regenerating forests
At Site 1 (primary forest age >100 years), decreases in Ψpd coincide
with modest decreases in average xylem water δ18O (Fig. 6a). Site 1 δD,
however, was typically more negative than the GMWL, with d-excess
E.A. Santos et al.
between −10 and 0 through the summer (SM Appendix O) and more neg-
ative than bulk soil and saprolite moisture (Fig. 5a). The negative offset in
xylem water δD from the GMWL presents a challenge to isotope mixing
analyses, however, the results of our semi-quantitative analysis show
Site 1 trees to use bulk soil moisture in the wet season, and shift to greater
contributions of bulk saprolite moisture through the dry season
(SM Appendix P).
With the exception of C. trichistandra (discussed in detail below), the sta-
ble isotope composition of xylem waters at Site 2 (late successional forest
age 30–70 years) decreased through the first part of the dry season,
reflecting uptake of bulk soil moisture and then bulk saprolite moisture
(Fig. 5b, SM Appendix N). Similar to Site 1, xylem water δD was more neg-
ative than the GMWL, however the d-excess was larger, approximately
equal to 0 through the early dry season (SM Appendix O). At Site 2,
xylem water reflected bulk soil and saprolite moisture in the early dry sea-
son, but shifted mid dry season to larger d-excess source water. This shift
corresponded with a leveling off and then a slight increase in Site 2 average
Ψpd (Figs. 4, 6b). For the remainder of the dry season, the stable isotope
composition of xylem water increased, moving towards the groundwater
source (Fig. 5b). In the early dry season (May 3rd), Site 2 trees use exclu-
sively soil water. As the subsurface dries, Site 2 trees use greater propor-
tions of saprolite and then groundwater. Although the two-end-member
mixing analysis failed for dates in mid-June to early July, there is a clear
trend in decreasing uptake of bulk saprolite moisture and increasing uptake
of groundwater (SM Appendix P). In January 2019, the 2-end-member
mixing yielded a result of 76 % groundwater, possibly reflecting uptake
of recent precipitation, which had an isotope composition slightly more
enriched (−1.93, −4.75) than our groundwater source endmember and
resulted in an increase in soil and saprolite gravimetric water content
(Fig. 3). The water potential and stable isotope data are consistent in show-
ing Site 2 trees rely more heavily on deeper sources of moisture (rock mois-
ture and groundwater) to sustain hydraulic function through the mid to
latter part of the dry season.
At Site 3 (early succession enriched age 10–30 years), the stable isotope
composition of xylem water resembled groundwater in the early dry sea-
son. As the dry season progressed, the stable isotope composition of
xylem water decreased steadily (Fig. 5c). Over this period, Ψpd also
steadily decreased (Fig. 6c). Similar to Site 2, C. trichistandra showed a dif-
ferent trend as xylem water stable isotope composition increased, suggest-
ing sustained groundwater use. Mean Site 3 xylem waters reflected
dry season uptake of groundwater and bulk saprolite moisture (Fig. 5c,
SM Appendix P).
Collectively, our observations show trees at all sites rely on deeper
water sources as the dry season progresses, however this shift occurs earlier
in younger forests where shallower water resources are limited and compe-
tition is greater. The increased spatial distribution of roots in early succes-
sional forests (e.g., Addo-Danso et al., 2018) can affect the physical
properties of the subsurface, including predawn water potential (Paz
et al., 2015; Brown and Lugo, 1990; Aide et al., 1995; Kennard, 2002;
Lebrija-Trejos et al., 2011), which may require individuals in younger
forests to rely more heavily on deeper water sources to survive the dry sea-
son. Our data are consistent with previous studies showing younger forests
invest more heavily in the development of deeper root systems (Paz et al.,
2015), whereas in more established forests, species tend to be more shal-
lowly rooted, perhaps in part due to a greater capacity to store water within
plant tissues (Pineda-García et al., 2013; Paz et al., 2015).
4.3. Potential stable isotope effects associated with uptake
We acknowledge the growing body of evidence documenting isotope
offsets, most commonly a negative offset in δD, between xylem water and
subsurface water sources (e.g., Cernusak et al., 2005; Ellsworth and
Williams, 2007; Brooks et al., 2010; Ellsworth and Sternberg, 2015; Zhao
et al., 2016; Vargas et al., 2017; Poca et al., 2019; Barbeta et al., 2020;
Zhao and Wang, 2021; Vega-Grau et al., 2021; Chen et al., 2021; Barbeta
et al., 2022; Duvert et al., 2021). Often, xylem water δ18O resembles that
9
Science of the Total Environment 850 (2022) 157908
of accessible moisture in the soil/weathered bedrock but δD is negatively
offset 10–20 ‰ (e.g., Brooks et al., 2010; Gierke et al., 2016; Geris et al.,
2017; Barbeta et al., 2019). Proposed mechanisms for this offset include
fractionation associated with transport through symbiotic mycorrhizal
fungi (e.g., Fuchs and Haselwandter, 2004; Moreira et al., 2007; Poca
et al., 2019), intra-bole isotope effects (Barbeta et al., 2022), fractionation
from source values along the root-to-crown xylem stream (Vega-Grau
et al., 2021), as well as artefacts of cryogenic extraction (Chen et al.,
2020). Below ground, tightly bound moisture is isotopically negative rela-
tive to mobile water in soils (e.g., Brooks et al., 2010; Gaj and McDonnel,
2019; Barbeta et al., 2022; Chen et al., 2021) and in weathered and fresh
bedrock (Oshun et al., 2016), yet it remains challenging to identify the iso-
tope composition of available subsurface waters because an unknown pro-
portion of bulk water will be held at water potentials below the minimum
achievable water potential of trees (e.g., Barbeta et al., 2020; Berry et al.,
2018; Oerter et al., 2019). Recently, a number of studies have used soil
grain size distribution, and in particular the fraction of silts and clays, to
empirically predict the ‘unbound’ fraction of bulk soil moisture (Chen
et al., 2016; Duvert et al., 2021; Barbeta et al., 2022). Empirical corrections
have also been developed to correct xylem δD to a computed ‘source line’ or
best fit trendline of all potential sources (Barbeta et al., 2019; Hahn et al.,
2021; Li et al., 2021; Duvert et al., 2021). We decided not to apply this em-
pirical correction to our subsurface data because of the very low sand frac-
tion at our sites (5–29 %) compared to the reported range in previous
studies (60–100 %) and because of the TDF's observed dry season reliance
on saprolite and weathered bedrock moisture, for which empirical correc-
tions have yet to be developed.
In the Cordillera del Bálsamo, the range of xylem water δ18O was
approx. −2 to −8 ‰ and spanned the combined ranges of groundwater
δ18O (−2.23 to −3.81 ‰) and bulk soil and saprolite moisture (−3.13
to −6.44 ‰). Xylem water δD, however, was offset from the GMWL at
Site 1 (d-excess of −10 to 0 ‰), and at Site 2 in the early dry season (d-ex-
cess of approximately 0 ‰). Xylem data at Site 3 plotted along the GMWL.
The greatest and most consistent offset in δD occurred in the primary forest
(Site 1), which may be a result of isotope effects associated with reliance on
shallow roots and, potentially, greater internal water storage (e.g. Paz et al.,
2015). Conversely, at Site 2, the mid-dry season shift to deeper sources of
moisture as evidenced by the slight increase in average predawn water po-
tential and a large increase in δD, eliminated the δD offset between the
GMWL and xylem water.
Although we were unable to identify the mechanism for the δD offset at
Site 1 and at Site 2 in the early dry season, we applied a δD correction for
possible cryogenic extraction following the formula introduced in Chen
et al. (2020) and based on Evaristo et al. (2015) (SM Appendix W, SM
Appendix X). This analysis decreased the Site 1 offset, resulted in a positive
δD offset for the majority of Site 2 xylem water samples, and resulted in a
positive δD offset from the GMWL at Site 2 for all xylem water samples
(SM Appendix Y). The δD offsets, and the potential for isotope effects in
δD do not affect our interpretations of site specific or species specific trends
in water use through the dry season.
4.4. Species-specific water use strategies in a regenerating forest
Although the three native species we measured at Site 2, C. trichistandra,
S. celastrinum, and H. chrysanthus, are drought deciduous, we observed all to
be fully foliated through July 4th. The dry out from March to July resulted
in all species taking up greater proportions of groundwater. However, pat-
terns in Ψpd, Ψmd, and the stable isotope compositions of xylem water re-
veal distinct dry season water use strategies among these three native
species. We interpret these three strategies using the spectrum of drought
avoidance to drought tolerance (e.g., Borchert and Pockman, 2005; Butz
et al., 2018) of species exploiting different ecological niches within a
regenerating TDF (Oliveira et al., 2021).
Although C. trichistandra Ψmd and Ψpd decreased through the dry sea-
son, values remained greater than −0.57 and −1.27 MPa, respectively.
Our semi-quantitative analysis showed C. trichistandra to be using primarily
E.A. Santos et al.
or exclusively groundwater throughout the dry season. The relatively high
water potential and the stability of xylem water isotope composition are
consistent with a drought avoidance water use strategy (Fig. 8) and low re-
sistance to cavitation (e.g., Méndez-Alonzo et al., 2012).
C. trichistandra is a stem succulent species with very low wood density
(0.26 g/cm3) (Butz et al., 2018), greater amounts of paratracheal paren-
chyma cells (e.g., Borchert and Pockman, 2005; Choat et al., 2005;
Jacobsen et al., 2007; Pratt et al., 2007; Pineda-García et al., 2011), and
greater internal water storage capacity (Butz et al., 2018, Holbrook,
1995). Residence times of stored water in mature C. trichistandra were mea-
sured to be between 18 and 25 days (Graefe et al., 2019), and daily water
use to be over 200 kg/day (Butz et al., 2018; Graefe et al., 2019). Sapflux
density is sensitive to subsurface drying, and stored water may be used to
maintain high rates of transpiration (Butz et al., 2018), although it is likely
replenished through continued uptake, albeit at decreased rates. At Site 2,
the isotope composition of xylem water reflected a groundwater source,
and although we did not measure root depth, groundwater in the valley
at an elevation of approximately 40 m, was >2 m below the ground surface.
Use of groundwater at Site 2 would require deep roots (Fig. 8), likely
greater than five meters, which is consistent with estimates in the TDFs of
coastal Ecuador (Canadell et al., 1996; Fan et al., 2017).
S. celastrinum Ψpd and Ψmd decreased steadily through the early dry
season to minimum values of −1.70 and −3.50, respectively, on June
13th. For the remainder of the dry season, however, Ψpd and Ψmd in-
creased slightly (Fig. 7). We observed no precipitation during this time pe-
riod, which suggests the increase in Ψpd and Ψmd resulted from a shift in
water source. Our semi-quantitative analysis revealed S. celastrinum to shift
from bulk soil moisture to a mixture of bulk soil, bulk saprolite, and ground-
water, to bulk saprolite and groundwater as the dry season progressed.
From May 31 to June 24, the proportion of groundwater in S. celastrinum
Fig. 8. At Site 2, measurements of subsurface moisture, predawn and midday water potentials, and stable isotope compositions illustrate distinct water use strategies of native
TDF species: C. trichistandra likely has deep roots that enable access to consistent sources of relatively high water potential groundwater; S. celastrinum likely has a dimorphic
rooting system with access to deep moisture and groundwater; H. chrysanthus may possess active shallow subsurface roots, as well as a deep tap root which may allow for
simultaneous access to a consistent water source and shallow nutrients.
10
Science of the Total Environment 850 (2022) 157908
xylem water increased from 11 to 54 %, coincident with a slight relaxation
in tree water status.
We were unable to find any published studies on S. celastrinum, how-
ever, our data suggest the presence of a dimorphic rooting system
(e.g., Tyree and Sperry, 1989; Dawson and Pate, 1996; Fan et al., 2017)
composed of shallow, lateral roots which obtain nutrients from soil layers
and a deep taproot which accesses groundwater as the water potential of
shallow water sources declines and groundwater becomes an energetically
favorable source (Fig. 8). We therefore interpret S. celastrinum to employ a
hybrid strategy of early dry season drought tolerance, and late dry season
drought avoidance.
From mid-May to early July, H. chrysanthus Ψpd and Ψmd decreased to
values more negative than any other Site 2 species (Fig. 7). The water po-
tential data suggest H. chrysanthus, unlike S. celastrinum and
C. trichistandra, lowered Ψmd to continue to access water from the shallow
subsurface as it dried. The isotope composition of H. chrysanthus xylem
water matched bulk soil and bulk saprolite for the first part of the dry sea-
son, but shifted towards the GMWL on June 13th. Our semi-quantitative
analysis yielded only one result (May 31) for H. chrysanthus, reflecting
87 % bulk soil moisture and 13 % bulk saprolite moisture. Although the re-
sults of the two end-member mixing analysis between bulk saprolite and
groundwater revealed different results when using δ18O and δD, there
was a consistent decline in the proportion of saprolite, and therefore an in-
crease in the proportion of groundwater as the dry season progressed. This
result suggests that although Ψpd and Ψmd continue to decline due to
maintained shallow water uptake, H. chrysanthus begins to use groundwater
in mid-June and the proportion of groundwater increases through the dry
season.
H. chrysanthus (formerly classified as Tabebuia chrysantha), is a slow
growing hardwood species with thick bark (Gonzáles Estrella et al., 2005;
E.A. Santos et al.
Günter et al., 2009) and high wood density of 1.1 g/cm3 (Borchert, 1994).
Typically, H. chrysanthus show high growth rates from the wet season
through the mid dry season, and lose their leaves through the latter part
of the dry season (Homeier, 2004). Stem diameter has been shown to fluc-
tuate daily (Borchert and Pockman, 2005) and to decrease steadily through
the leaf-on dry season (Volland-Voigt et al., 2011). H. chrysanthus has low
water storage capacitance in its dense wood, which results in a strong
response in leaf water potential to the use of stored water and signals
the wood tissues are tolerant of desiccation to low water potentials
(Borchert and Pockman, 2005). Our stable isotope data suggest that if
H. chrysanthus was diurnally replacing stored stem water, the source of
this water was groundwater in mid to late dry season.
Maintaining active shallow roots would allow H. chrysanthus to rehy-
drate rapidly (e.g., Borchert and Pockman, 2005) in response to the first
rains of the wet season. We observed H. chrysanthus to produce a full
flush of flowers at the start of the rainy season, similar to in other TDFs
(Volland-Voigt et al., 2011; Homeier, 2004), a trait that would benefit
from shallow roots that captured the first rains of the season as well as
maintained access to nutrients (e.g., Cornejo et al., 1994; Schüßler et al.,
2016). We find H. chrysanthus to be the most drought tolerant of the three
measured species at Site 2.
In summary, we observed all species at Site 2 to depend on deep sources
of moisture, however, there were key, species-specific differences in water
use. The oldest and largest tree, C. trichistandra, is a drought avoiding, stem
succulent with large water storage capacitance reliant on groundwater.
S. celastrinum shows drought tolerance through the first part of the dry
season by lowering its water potential to access bulk soil and saprolite mois-
ture, but switches to groundwater in a drought avoidance strategy in the
late dry season. H. chrysanthus is a drought tolerant species with the lowest
predawn and midday water potential and a small hydraulic safety margin
(e.g., Markesteijn et al., 2011), however H. chrysanthus also relies on
groundwater, albeit while maintaining access to shallow bulk soil water
in anticipation of the first rains.
4.5. Regional implications
Compared to older forests in this region, subsurface water resources in
regenerating forests are limited. We observed lower leaf litter layer, greater
compaction (lower infiltration rates) and greater clay content in the
regenerating forests. Additional factors include more vigorous competition
in young forests (e.g., Paz et al., 2015; Brown and Lugo, 1990; Aide et al.,
1995; Kennard, 2002; Lebrija-Trejos et al., 2011) and lower canopy cover
which leads to increased soil evaporation. The remotely sensed data show
early to late successional forests similar to sites 2 and 3 cover 74.6 % of
the Cordillera del Bálsamo, whereas primary forests similar to Site 1
cover only 16.3 %. In contrast to previous studies suggesting the subsurface
water availability in regenerating forests reaches primary forest conditions
in 12–15 years (Lebrija-Trejos et al., 2010, 2011; Pineda-García et al.,
2013), or a return to primary forest canopy cover in 23 years (Kennard,
2002), our data suggest a recovery time in the subsurface of >100 years.
In the interim, successional forests, on average, invest more in root growth
(e.g., Brown and Lugo, 1990), to access tightly held shallow water or deeper
water to survive the dry season.
Of the current TDFs worldwide, 97 % of them are deemed at risk for
proximate and indirect loss, jeopardizing the ecosystem services they pro-
vide (Miles et al., 2006). Moving forward, we suggest there is an urgent
need to quantify the effects of successional forest age on subsurface water
storage and water availability, evapotranspiration, groundwater recharge,
and forest water yield to sustainably manage regenerating forests and
plan for local and regional water security. Recently, the Bosque Seco Bio-
sphere Reserve (BSBR) has led to an increase in protected TDF along the
Ecuadorian coast from 4 to 8 % of the existing 6000 km2 (Escribano-Avila
et al., 2017). Our results suggest intensive monitoring of native species
can be paired with remotely sensed data to scale up ecohydrological find-
ings and predict water yield across a broad range of forest to inform and
add justification to ongoing conservation and forest regeneration.
11
Science of the Total Environment 850 (2022) 157908
5. Conclusion
We used wet and dry season measurements of gravimetric water con-
tent, predawn and midday leaf water potential, and stable isotope measure-
ments of subsurface moisture and xylem water to quantify differences in
subsurface moisture dynamics, and tree source water across regenerating
and old growth TDF. During the transition from wet season to dry season,
steady declines in subsurface moisture resulted in decreases in average
Ψpd at each site. However, these decreases were far sharper in successional
forests (sites 2 and 3), showing that regenerating forests harbor limited
subsurface water availability. A return of subsurface water potentials to
that of primary forests is expected to take at least 100 years. Within the
regenerating TDF, we identified specific water use strategies across three
native deciduous species through the seasonal drought. Whereas the stem
succulent C. trichistandra avoided drought by relying on relatively accessi-
ble groundwater, S. celastrinum followed a drought tolerance strategy in
early dry season through maintained access to shallow water sources, but
shifted to greater proportions of groundwater in the late dry season in a
drought avoidance strategy. As the subsurface dried, H. chrysanthus showed
drought tolerance by maintaining access to shallow water sources via very
low water potentials and a smaller hydraulic safety margin, however
H. chrysanthus also accessed groundwater as the dry season progressed.
We hope our results inform future studies investigating tree water source
(s) in the TDF and directly aid local TDF conservation and management ef-
forts in Bahía.
CRediT authorship contribution statement
Emily Santos: Conceptualization, Methodology, Resources, Investiga-
tion, Data Curation, Visualization, Writing-Original draft preparation.
Xavier Haro-Carrión: Data curation, Writing-Reviewing and Editing.
Jasper Oshun: Conceptualization, Supervision, Methodology, Resources,
Writing-Reviewing and Editing.
Data availability
Data will be made available on request.
Declaration of competing interest
The authors declare that they have no known competing financial inter-
ests or personal relationships that could have appeared to influence the
work reported in this paper.
Acknowledgements
Thank you to those whose generosity made this study possible finan-
cially: The Consortium of Universities for the Advancement of Hydrologic
Science, Inc. (CUAHSI) for the 2019 Instrumentation Discovery Travel Grant
and the 2019 Graduate Student Travel Grant; Humboldt State University
Department of Geology for the 2019 Geology Field Work Summer Assistance
Award and the 2018 Geology Opportunities Fund Award; Andrea and Don
Tuttle for the 2019 Tuttle Climate Conference Scholarship; The California
State University Program for Education & Research in Biotechnology
(CSUPERB) for the 2018 Graduate Student Travel Grant; Humboldt State
University and Dr. Bud Burke for the 2017–2018 Bud Burke Scholarship.
Thank you to Lucy Kerhoulas and Andrew Stubblefield for their guidance
and support through the early stages of this manuscript. Infinite thanks to
all the Corredores de Conservación de Biorregiones collaborators, Ramón
Cedeno Loor and Blas Loor, and Kathrine Castillo, Haley Sellers, Amanda
Donaldson, Lucas Oshun, Dr. Stefania Mambelli and Dr. Wenbo Yang.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2022.157908.
E.A. Santos et al.
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