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\begin{document}

\title{Anatomical traits explain comparative drought response of seedlings from
wet tropical forests}



\author[1]{Rishiddh Jhaveri}%
\author[1]{Lakshmipriya Cannanbilla}%
\author[2]{K. S. Arpitha Bhath}%
\author[3]{Mahesh Sankaran}%
\author[1]{Meghna Krishnadas}%
\affil[1]{Centre for Cellular and Molecular Biology CSIR}%
\affil[2]{Bangalore University}%
\affil[3]{National Centre for Biological Sciences}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Water availability regulates plant community dynamics but the drought
response of seedlings remains poorly known despite their vulnerability,
especially for the Asian tropics. In particular, discerning how
functional traits of seedlings mediate drought response can aid
generalizable predictions of tree responses to global environmental
change. We assessed interspecific variation in drought response
explained by above- and below-ground seedling traits. We conducted a
dry-down experiment in the greenhouse using 16 tree species from the
humid forests of Western Ghats in southern India, chosen to represent
differences in seasonality affiliations--sites of high and low seasonal
drought. We compared seedling survival, growth, and photosynthetic
performance under drought and well-watered conditions, and assessed the
extent to which species responses were explained by seasonality
affiliation and 12 traits of root, stem and leaf. We found that the
species from seasonally dry forest reduced photosynthetic rate in
drought compared to well-watered conditions, but seasonality affiliation
did not explain differences in growth and survival. Performance changes
in drought vs well-watered conditions were best explained by anatomical
traits of xylem, veins and stomata. Species with larger xylem reduced
their growth and photosynthesis to tolerate desiccation. In drought,
species with smaller stomata showed reduced survival even though
photosynthetic activity decreased by a larger extent with larger
stomata. Our study shows that anatomical traits related to xylem and
stomata, directly related to water transport and gas-exchange, played a
more prominent role than commonly used soft traits (e.g., specific leaf
area, wood density) in explaining species response to drought, and may
offer a better proxy for physiological traits related to drought
tolerance of seedlings.%
\end{abstract}%



\sloppy


\textbf{Anatomical traits explain comparative drought response of
seedlings from wet tropical forests}

\emph{Rishiddh Jhaveri, Lakshmipriya Cannanbilla, K. S. Arpitha Bhat,
Mahesh Sankaran, Meghna Krishnadas*}

\emph{Email: meghna@ccmb.res.in; meghna@csirccmb.org}

\textbf{Abstract}

Water availability regulates plant community dynamics but the drought
response of seedlings remains poorly known despite their vulnerability,
especially for the Asian tropics. In particular, discerning how
functional traits of seedlings mediate drought response can aid
generalizable predictions of tree responses to global environmental
change. We assessed interspecific variation in drought response
explained by above- and below-ground seedling traits. We conducted a
dry-down experiment in the greenhouse using 16 tree species from the
humid forests of Western Ghats in southern India, chosen to represent
differences in seasonality affiliations--sites of high and low seasonal
drought. We compared seedling survival, growth, and photosynthetic
performance under drought and well-watered conditions, and assessed the
extent to which species responses were explained by seasonality
affiliation and 12 traits of root, stem and leaf. We found that the
species from seasonally dry forest reduced photosynthetic rate in
drought compared to well-watered conditions, but seasonality affiliation
did not explain differences in growth and survival. Performance changes
in drought vs well-watered conditions were best explained by anatomical
traits of xylem, veins and stomata. Species with larger xylem reduced
their growth and photosynthesis to tolerate desiccation. In drought,
species with smaller stomata showed reduced survival even though
photosynthetic activity decreased by a larger extent with larger
stomata. Our study shows that anatomical traits related to xylem and
stomata, directly related to water transport and gas-exchange, played a
more prominent role than commonly used soft traits (e.g., specific leaf
area, wood density) in explaining species response to drought, and may
offer a better proxy for physiological traits related to drought
tolerance of seedlings.

\textbf{Keywords}

Tropical tree species; Seedling drought response; Above-and-below ground
plant traits; Seasonality affiliation; Greenhouse experiment.

\textbf{Introduction}

Water availability drives the distribution and dynamics of plant
species, even within relatively wet biomes (Engelbrecht \emph{et
al.}2007, Comita \& Engelbrecht 2009, Esquivel-Muelbert \emph{et al.}
2019, Krishnadas \emph{et al.} 2021). Quantifying plant response to
water stress is imperative given that global environmental change will
alter patterns of precipitation and seasonal drought. For closed canopy
forests, much attention has focused on the response of mature trees to
drought (Suresh \emph{et al.} 2010, Anderegg \emph{et al.} 2015,
Rowland\emph{et al.} 2015, Hartmann \emph{et al.} 2018), but less is
known of seedling responses. Seedlings can be highly vulnerable to
drought because their shallow roots do not allow access to deeper water
layers during dry periods. Moreover, the light-limited forest understory
may impose carbon starvation during drought (Slot \& Poorter 2007,
Amissah\emph{et al.} 2015) such that even short dry spells can result
in substantial seedling mortality in tropical wet forests
(Engelbrecht\emph{et al.} 2007). Seedling responses to water
availability can therefore regulate regeneration and persistence of
species in a changing climate and shape community structure of later
life-stages (Comita \& Engelbrecht 2009, 2014).

Interspecific variation in drought response can be mediated by species'
traits---anatomical, morphological and physiological characteristics
associated with differences in strategies of resource use (Slot \&
Poorter 2007, Poorter \& Markesteijn 2008). For example, wood density
and leaf mass per area can correlate positively with drought tolerance
of mature trees (Muscarella \emph{et al.} 2016, O'Brien \emph{et
al.}2017), but the extent to which these relationships hold true for
seedlings is not known. Notably, the role of root traits in mediating
species' drought responses remains poorly explored, despite increasing
calls to include below ground traits in assessing plant functional
strategies and whole-plant response to stressors (Freschet \emph{et
al.}2021, Laughlin \emph{et al.} 2021, Weemstra \emph{et al.} 2023). In
addition, understanding variation in anatomical features of stomata and
xylem, which help regulate water balance and response to dry conditions,
may serve as stronger proxies for hydraulic function than commonly used
traits such as specific leaf area (Bartlett \emph{et al.} 2016).

Species of evergreen habit may respond to seasonal droughts either by
avoiding or tolerating desiccation, with functionally distinct
strategies arising from different trait combinations. Desiccation
avoiders try to maintain water status through a drought. Due to higher
metabolic demands, these species may be resource-acquisitive with their
higher rates of photosynthesis and growth selecting for thinner, cheaper
leaves, large stomata and xylem to move adequate water, and deep roots
to maintain access to water (Chitra-Tarak \emph{et al.} 2021, Reich
2014). Alternatively, species may tolerate desiccation by decreasing
photosynthesis to reduce water loss. This strategy impedes growth but
reduces the chance of embolism or hydraulic failure (Anderegg \emph{et
al.} 2015, 2016, Adams \emph{et al.} 2017, Skelton \emph{et al.} 2017).
To prevent carbon starvation, desiccation tolerant species should be
resource-conservative, reflected in traits such as low specific leaf
area (SLA), high leaf dry matter content (LDMC), smaller xylem and
stomata, and relatively lower investment in below-ground tissue to
forage for water compared to desiccation avoiders. Alternatively, traits
may not be coordinated and different trait combinations may offer
equivalent responses to drought.

Easily measurable so-called `soft' traits may help categorize species as
avoiding or tolerating desiccation based on an integrated whole-plant
evaluation of photosynthesis, growth and survival, and offer a helpful
adjunct to hydraulic traits (Bartlett \emph{et al.} 2012, Sun \emph{et
al.} 2020). With that in mind, we assessed the relationship between
traits and species' performance in dry vs. well-watered conditions for
seedlings of 16 tree species from a wet tropical forest region in a
global biodiversity hotspot in southern India, the Western Ghats. Across
the region, most seedlings establish during the monsoon, but then have
to withstand dry periods varying from 3-9 months to persist. The species
were chosen to represent a gradient in their affinity to seasonal
climatic water deficit during the dry season (henceforth seasonality)
based on a previous analysis of tree species distributions in the
Western Ghats (Krishnadas \emph{et al.} 2021). We asked:

Do species from less vs. more seasonal forests vary in their drought
response?

We expected that species associated with greater seasonality would
tolerate drought better than species from less seasonal forests, i.e.,
show smaller decline in survival and/or growth in drought vs.
well-watered conditions.

How well do individual traits explain interspecific variation in drought
response?

We expected that species with higher SLA, lower LDMC, higher root mass
fraction (RMF) or deeper roots, larger stomata and wider xylem would
maintain better function (photosynthesis and growth) early in drought,
but survive less than species with lower SLA, higher LDMC, and lower RMF
or shallow roots, smaller stomata, and narrower vessels.

Do distinct trait combinations characterize species that avoid or
tolerate desiccation?

We expected that traits associated with conservative water-use or safe
hydraulic strategy, e.g., thicker leaves, denser stem, smaller stomata
and xylem, would correspond with responses characteristic of desiccation
tolerance -- decreased photosynthesis and growth. Species with thinner
leaves and larger stomata or xylem would avoid desiccation by
functioning at similar levels in drought compared to watered conditions.
We also expected that species with higher SLA, lower LDMC, deeper roots
and higher RMF would avoid desiccation. Species with lower SLA, higher
LDMC, shallow roots and lower RMF were expected to tolerate desiccation.

\textbf{Methods}

We conducted a dry-down experiment in the greenhouse to gauge the
response of seedlings to seasonal drought. Hence, our experiment
represented a scenario where seedlings growing in a limited volume of
soil were exposed to sudden drought and results should be interpreted
accordingly. Seeds of 16 different tree species (Appendix Table A1) were
obtained from two locations, Valparai in southern Western Ghats and
Sirsi in northern Western Ghats, representing less and more seasonal
climates respectively. Both sites have similar mean annual rainfall but
differ in their climatic water deficit (Sirsi: Mean annual precipitation
(MAP) = 3425 mm/year, number of dry months: 6, CWD = -826.9 mm/year;
Valparai: MAP = 2169.3 mm/year, number of dry months: 4, CWD = -396.51
mm/year, Source: Chave et al. 2014; Fick and Hijmans 2017) that
characterizes seasonal drought conditions (Krishnadas \emph{et
al.}2021).

\emph{Greenhouse experiment}

Between July and November 2019, seeds were germinated in germination
trays containing a 2:1 mixture of all-purpose soil and cocopeat at the
greenhouse at the National Centre of Biological Sciences (Bengaluru,
India). Because species fruited at different times, had recalcitrant
seeds, and germination times varied among species, we planted species
for germination whenever seeds became available. Seeds were watered
every alternate day and monitored for germination. Once germinants lost
their cotyledon leaves and developed at least four mature leaves they
were transplanted into PVC pipes of diameter 15 cm and depth of 45 cm.
The PVC columns were filled with a prepared soil mixture (all-purpose
soil: sand: cocopeat: compost = 1:1:1:0.1) and randomly assigned to six
blocks within the greenhouse. Seedlings within a block were randomly
assigned to drought and control treatments. Each treatment per block had
an equal number of individuals of a species, but numbers differed among
species depending on seedling availability. To maintain a consistent
post-germination age at which seedlings were subject to drought, species
were included in the experiment as their seedlings became available. All
new seedlings were allowed to acclimate in their pots for two weeks and
watered every two days to avoid transplant shock. Seedlings that
perished during this period were replaced and included after
acclimation. In total, we had 434 seedlings in the experiment with 1-3
individuals per species per treatment per block.

After the acclimation period, we stopped watering the seedlings in the
drought treatment for each block. Seedlings in the control treatment
were given 75-100 ml of water twice weekly to ensure a consistent level
of soil moisture. The greenhouse was maintained at approximately 65\%
relative humidity and 26\selectlanguage{ngerman}°C temperature. Initially, we planned the
drought treatment to last 16 weeks to simulate the post-monsoon seasonal
drought that occurs from January through April in the Western Ghats.
However, monitoring and measurements were disrupted due to the COVID-19
lockdown. For 12 of 16 species, we implemented the experiment for 16
weeks. For the remaining four species, control plants were watered by
the greenhouse staff but data collection was not possible due to
restricted access to the facility. We harvested these plants in the
12\textsuperscript{th} week of their drought experiment when we got
permission to access the facility. These 12 weeks still give us a
reasonable idea of interspecific variation in response to seasonal
drought conditions.

For growth, stem height, leaf length and width (for the four largest
leaves) and survival was recorded weekly for the first 8 weeks and then
once a month till the plants were harvested. We calculated the growth
rate at the end of 12 weeks for all individuals as follows: (final
height -- initial height)/time in weeks. Seedling final height was
strongly correlated with initial height (\emph{r} = 0.7, t =
20.8,\emph{p} \textless{} 0.001), hence we calculated relative growth
rates, standardized by initial height as follows: (final height --
initial height)/(time in weeks*initial height). Photosynthesis was
measured monthly using a LiCOR 6400XT on seedlings of all species from
control and drought treatments. Photosynthesis rates were measured with
a 2x3 cm\textsuperscript{2} leaf chamber. The chamber conditions were
maintained at PAR 1500 \selectlanguage{greek}µ\selectlanguage{english}molm\textsuperscript{-2}s\textsuperscript{-1},
65\% humidity, 400 \selectlanguage{greek}µ\selectlanguage{english}mols\textsuperscript{-1} flow rate, fan speed at
10,000 RPM. Measurements were taken between 0700 - 1200 hours.

Although we had aimed to measure above and below-ground biomass
allocation for all individuals in both drought and control, we lost many
samples in an oven fire. Moreover, due to COVID-19 related restrictions
on accessing the facility, \textasciitilde{}20\% of individuals could
not be harvested on time. Hence, we were unable to assess relative
differences in above- vs. below-ground allocations in relation to water
availability.

\emph{Trait measurements}

For leaf trait measurements (listed in Appendix Table A2), 1-3 leaves
each were collected from selected seedlings and immediately stored in a
ziplock bag between moist tissue paper to make sure the leaves were
fully saturated with water (Cornelissen \emph{et al.} 2003). Five
seedlings from the control group were selected for each species for
trait measurements.

To measure SLA (cm\textsuperscript{2} g\textsuperscript{-1}) and LDMC
(\selectlanguage{greek}µ\selectlanguage{english}m mg\textsuperscript{-1}) (Cornelissen \emph{et al.} 2003), the fresh
leaf area was measured using the CI-202 leaf scanner (CID bio-science).
After measuring fresh weight, leaves were placed in paper bags and
air-dried for a week, following which they were dried at 60\selectlanguage{ngerman}°C for 3 days
to get dry leaf mass. SLA was calculated as the ratio of leaf area to
its dry biomass. LDMC was calculated as the ratio of dry leaf biomass
and fresh leaf biomass.

To measure stomatal traits, the leaf surface was wiped thoroughly to get
rid of dust and excess moisture. Nail polish imprints were prepared and
scanned under 40X magnification in a light microscope. For stomatal
density (SD, number per cm\textsuperscript{2}), four fields of vision
were imaged from each imprint. The number of stomatal apertures were
counted and the image area was measured using the rectangle tool in
ZEISS ZEN2 software (v1.0, RRID: SCR\_013672). We chose three of the
clearest stomates from each of the images and estimated stomatal size
(SS, \selectlanguage{greek}µ\selectlanguage{english}m\textsuperscript{2}) by drawing an ellipse around the stomata and
stomatal length (SL, \selectlanguage{greek}µ\selectlanguage{english}m) using ZEISS ZEN2 (v1.0, RRID: SCR\_013672). We
then calculated stomatal area fraction (SAF) for cm\textsuperscript{2}of
leaf by taking a product of SD and SS (SD * SS *
10\textsuperscript{-8}). Model results for SAF are similar to SD
(Appendix Figure A3) and therefore we decided to use SD for further
analysis and interpretation.

To measure vein density (VD), leaves were immersed in petri dishes with
cleaning bleach and monitored daily. Once the leaves lost all the
chlorophyll, they were washed with distilled water and stained with
methylene blue diluted 1:100 with distilled water. The samples were put
on a clean slide and imaged under 20X magnification. Xylem diameter (XD,
\selectlanguage{greek}µ\selectlanguage{english}m) was measured using an inch-long section of the stem that was cut off
and sectioned immediately into thin slices with a stainless-steel blade.
Five to ten of the thinnest sections were observed under the 40x light
microscope and the diameter of 8-10 of the largest xylem vessels were
measured using the line and circle tools of the software. All
microscopic measurements were performed using a Primostar, Carl Zeiss
microscope and images were taken using the camera accessory Axiocam-105
mounted on the microscope. All images were analyzed using the Zen (3.0
Blue edition) software.

To measure root traits, harvested roots were stored in a freezer until
scanning. The root samples were taken out just before the scan and
cleaned thoroughly to remove any traces of soil and dirt. The roots were
scanned using an Epson Regent LA2400 scanner. The scans were analyzed
using WinRHIZO (Regent Instruments Inc., Canada) for total root length
(RL, mm.), diameter and root surface area (RA, mm\textsuperscript{2}).
Specific root length (SRL, cm. g.\textsuperscript{-1}) was calculated as
the ratio of the total root length and the root's dry biomass. Root mass
fraction (RMF, g g\textsuperscript{-1}) was calculated as the fraction
of root dry mass and total plant dry biomass. Seasonality index (SI) of
each species was derived from a previous analysis that estimated
species' affinity to seasonal drought conditions based on their
distribution across the Western Ghats (Krishnadas \emph{et al.} 2021).
Positive value of seasonality index indicates that the species prefers
less seasonal conditions, i.e., wet associated.

\emph{Statistical Analysis}

We used mixed-effect models with appropriate error structures (see
below) to test the impact of drought on survival, growth and
photosynthesis. Preliminary models for growth showed that trends did not
vary between growth rate (Appendix Table A4) or relative growth rate
(Appendix Table A7) and we used the latter for interpretation since
relative growth rates were standardized by initial height.

For the first question concerning differences in species response to
drought in relation to their affinity for less vs. more seasonal areas,
we modeled species' performance as a function of seasonality index,
drought treatment, and their interaction. For question 2, we tested the
role of each trait in governing drought response. We implemented
separate models per trait, where species' performance was modeled as a
function of their trait values, drought treatment, and drought-trait
interaction. In all models, we used species-level trait means. Species
ID and block ID were included as random intercepts. Survival was modeled
using a Bernoulli error structure and Gaussian errors were assumed for
growth and photosynthesis. Because species differed in the extent of
variation in their response, we added a weighting term to separately
model the variance per species. Trait models for SRL were implemented
with and without the species \emph{Actinodaphne malabarica} , which had
small seedlings with very light roots and high SRL values that were an
outlier compared to other species.

Model structure in Wilkinson-Rogers notation:

Performance variable \textasciitilde{} trait + treatment +
trait:treatment + (1\textbar{}species) + (1\textbar{}block)),
weights=varIdent(form=\textasciitilde{}1\textbar{}species)

Finally, to understand the impact of the overall trait-based phenotype
on performance during drought (question 3), we used a two-step approach.
First, we performed a pairwise correlation among traits (Appendix Figure
A5) and a principal component analysis (PCA) on traits to obtain
composite phenotypes defined by trait combinations. Then, we modeled
individual survival, growth, and photosynthesis in control vs. drought
plants using GLMM with species-level intercepts and slopes. From these,
we extracted each species' baseline performance (survival, growth,
photosynthesis) in well-watered conditions and the change in performance
with drought and conducted a PCA on these six variables to get species'
composite response to drought in relation to their baseline differences
in growth, survival and photosynthesis. For both PCAs, we performed
varimax rotation to simplify loadings for each factor. In the second
step, to explore the correspondence between traits and performance, we
performed Procrustean superimposition on the two PCA, using the axes
that explained \textgreater{}75\% of the variation in each PCA.
Procrustes analysis tests the strength of association between two
ordinations for a common object (species in our case) with each other
(Peres-neto \& Jackson 2001, R\selectlanguage{ngerman}üger \emph{et al.} 2018), i.e., how well
species positions in trait\selectlanguage{english} space matched their positions in performance
space. Significance of the Procrustes correlation was assessed using
permutation tests with 10,000 random iterations.

All analyses were performed using R v 3.4. We used packages nlme
(Pinheiro \emph{et al.} 2022)\selectlanguage{english} for mixed effects models with Gaussian
error structure lme4 (Bates \emph{et al.} 2015)\selectlanguage{english} for models with
Bernoulli error, FactoMineR (Le \emph{et al.} 2008)\selectlanguage{english} for PCA and vegan
(Oksanen \emph{et al.} 2020)\selectlanguage{english} for Procrustes analysis.

\textbf{Results}

\emph{Seasonality affiliation and drought response}

Species' seasonality affiliation did not affect seedling survival (\selectlanguage{greek}β \selectlanguage{english}=
-0.89, CI = -2.38 to 0.61, z = -1.16, P =0.25, Figure 1a) but overall
seedling had lower survival in drought vs well-watered conditions (\selectlanguage{greek}β \selectlanguage{english}=
-2.17, CI = -2.95 to -1.39, z = -5.47, P \textless{} 0.05, Figure 1a).
However, photosynthesis rates for species from less seasonal forests,
i.e., larger values of seasonality index experienced larger decline
under drought condition (\selectlanguage{greek}β \selectlanguage{english}= -0.45, CI = -0.82 to -0.08, n=51, t =
-2.46, P = 0.02, Figure 1c). Seasonality index did not explain
interspecific variation in growth for plants in drought vs well-watered
conditions (\selectlanguage{greek}β \selectlanguage{english}= -0.001, CI = -0.003 to -0.002, n=339, t = -0.64, P =
0.52, Figure 1b).

Of the 12 traits we measured, traits related to leaf structural
investment, anatomy of stomata and xylem best-explained variation in
drought response of seedlings. Species with larger xylem had lower
survival in drought conditions as compared to well-watered conditions (\selectlanguage{greek}β
\selectlanguage{english}= -1.3, CI = -2.14 to -0.45, n = 412, z = -3.0, P = 0.002, Figure 2a).
Smaller stomata correlated with poorer survival in well-watered
conditions (\selectlanguage{greek}β \selectlanguage{english}= 1.94, CI = 0.11 to 3.76, n = 412, z = 2.08, P = 0.04,
Figure 2b), and survival also declined more steeply for species with
smaller stomata in drought (\selectlanguage{greek}β \selectlanguage{english}= -3.29, CI = -5.08 to -1.5, n = 412, z =
-3.61, P \textless{} 0.001, Figure 2b). Although survival in
well-watered conditions decreased with higher LDMC, this difference
disappeared under drought conditions (\selectlanguage{greek}β \selectlanguage{english}= 1.2, CI = 0.41 to 2.0, n =
412, z = 2.97, P = 0.003, Figure 2c).

In well-watered conditions, growth rates increased with increasing xylem
diameter (\selectlanguage{greek}β \selectlanguage{english}= 0.01, CI = 0.003 to 0.022, df = 13, t = 2.97, P = 0.01,
Figure 2d) and higher stomatal density (\selectlanguage{greek}β \selectlanguage{english}= 0.01, CI = 0.005 to 0.025,
df = 14, t = 3.29, P \textless{} 0.05, Figure 2e). However, in drought
condition, species with larger xylem and higher SD showed larger
declines in growth but the effect sizes were weak (\textbf{XD} : \selectlanguage{greek}β \selectlanguage{english}=
-0.01, CI = -0.008 to -0.004, df = 321, t = -5.09, P \textless{}
0.01;\textbf{SD} : \selectlanguage{greek}β \selectlanguage{english}= -0.004, CI = -0.009 to -0.0002, df = 339, t =
-2.09, P = 0.04, Figure 2d, 2e). Species with lower LDMC and VD grew
less under drought condition, however with increasing values for both
the traits, growth rates became more similar in well-watered and drought
conditions (\textbf{LDMC} : \selectlanguage{greek}β \selectlanguage{english}= 0.003, CI = 0.001 to 0.004, df = 321, t
= 3.37, P \textless{} 0.05, Figure 2f; \textbf{VD} : \selectlanguage{greek}β \selectlanguage{english}= 0.01, CI =
0.003 to 0.009, df = 339, t = 4.31, P \textless{} 0.05, Figure 2g).
Species with larger RD showed greater decline in growth under drought (\selectlanguage{greek}β
\selectlanguage{english}= -0.003, CI = -0.006 to -0.0001, df = 296, t = -2.04, P = 0.04, Figure
2h).

Physiological function (photosynthesis) increased with larger xylem (\selectlanguage{greek}β \selectlanguage{english}=
1.14, CI = 0.08 to 2.19, df = 13, t = 2.3, P = 0.04, Figure 3a) and
greater stomatal density (\selectlanguage{greek}β \selectlanguage{english}= 1.6, CI = 0.65 to 2.49, df = 14, t = 3.65,
P \textless{} 0.05, Figure 3b) as expected, and both slopes decreased in
drought conditions (\textbf{XD} : \selectlanguage{greek}β \selectlanguage{english}= -0.8, CI = -1.01 to -0.60, df =
49, t = -7.73, P \textless{} 0.05; \textbf{SD} : \selectlanguage{greek}β \selectlanguage{english}= -0.83, CI = -1.22
to -0.45, df = 51, t = -4.37, P \textless{} 0.05, Figure 3a, 3b). SLA
did not explain overall differences in photosynthetic rates of species
in well-watered conditions, but in drought, photosynthesis rates
increased with SLA (\selectlanguage{greek}β \selectlanguage{english}= 0.53, CI = 0.17 to 0.89, df = 49, t = 2.98, P
\textless{} 0.05, Figure 3c), as well aswith increasing LDMC (\selectlanguage{greek}β \selectlanguage{english}= 0.4,
CI = 0.15 to 0.64, df = 49, t = 3.2, P \textless{} 0.05, Figure 3d).
Drought led to larger proportional decline in photosynthesis for species
with lower vein density (\selectlanguage{greek}β \selectlanguage{english}= 0.79, CI = 0.40 to 1.18, df = 51, t = 4.1,
P \textless{} 0.05, Figure 3e). Additionally, photosynthesis decreased
with larger and longer stomata in dry conditions (\textbf{SS} : \selectlanguage{greek}β \selectlanguage{english}=
-0.55, CI = -0.86 to -0.24, df = 49, t = -3.6, P \textless{} 0.001,
Figure 3f; \textbf{SL} : \selectlanguage{greek}β \selectlanguage{english}= -0.37, CI = -0.72 to -0.01, df = 49, t =
-2.08, P = 0.04, Figure 3g).

Photosynthetic performance under drought was not explained by root area,
length and SRL. Yet, under well-watered conditions, photosynthesis
increased with greater root area, longer roots and higher SRL
(\textbf{RA} : \selectlanguage{greek}β \selectlanguage{english}= 1.36, CI = 0.47 to 2.26, df = 12, t = 3.32, P
\textless{} 0.05; \textbf{RL} : \selectlanguage{greek}β \selectlanguage{english}= 1.12, CI = 0.13 to 2.11, df = 12, t
= 2.48, P = 0.03; \textbf{SRL} : \selectlanguage{greek}β \selectlanguage{english}= -0.97, CI = -1.10 to 0.07, df = 11,
t = -2.06, P = 0.06; Appendix Table A8 h, A8 i, A8 k). Species with
thicker roots had higher photosynthetic rates in well-watered
conditions, but experienced larger proportional decline in
photosynthesis than species with thinner roots under drought stress (\selectlanguage{greek}β \selectlanguage{english}=
-0.89, CI = -1.28 to -0.50, df = 47, t = -4.6, P \textless{} 0.05,
Figure 3h).

\emph{Multi-trait phenotype and drought response}

The first two PCA axes explained 77.2\% of the variation in performance
among species-level coefficients for survival, growth and photosynthesis
(Appendix Figure A10 and Table A11). The first performance axis
separated species according to their survival under drought vs. control
conditions, with positive values indicating poor drought survival
(Appendix Table A11). As a whole, the PCA showed that species that grew
well also had higher photosynthetic rates and had higher survival in
both drought and well-watered conditions (Appendix Figure A9 and table
A10).

After varimax rotation, PCA for traits required four dimensions to
explain \textasciitilde{}80\% of the variation among species (Figure 4a,
Appendix Table A13). Most axes had at least two traits with loadings
\textgreater{} 0.5. The first PCA axis separated species based on their
stomatal, leaf structural and xylem traits and explained 29\% variation.
Positive values of axis 1 corresponded to lower SLA, larger stomates,
higher leaf structural tissue, smaller xylem, and larger root mass
fraction (Appendix Table S14). Axis 2 separated species based on water
acquisition traits, explaining \textasciitilde{}21\% variation. Species
with positive values on axis 2 had larger, longer and denser roots
(Appendix Table S14). However, the third and fourth PCA axes reveal
alternative combinations of measured traits. For instance, compared to
axis 1, axis 3 comprised species with higher LDMC that also had higher
SRL and smaller stomates. Axis 4 showed that, in contrast to axis 1, low
SLA species could have smaller xylem and higher vein density.

Procrustes analysis between the ordinations of species performance (two
axes) and traits (four axes) showed substantial concordance (sum of
squares = 38.24, Figure 4b, r=0.66, P = 0.03; permutation test for
significance at alpha 0.05). This indicates that species' performance in
drought conditions could be explained by phenotypes discerned from soft
traits.

\textbf{Discussion}

We found that drought reduced survival and growth for seedlings of 16
tree species from a wet tropical forest region in India (Western Ghats),
but species varied substantially in their responses. Interspecific
variation in survival and growth with drought was primarily explained by
anatomical traits that mediate water use. Generally, species with fewer
stomata and smaller xylem show lower decline in performance, growth and
survival under drought condition. Variation in photosynthetic
performance under drought stress correlated best with anatomical traits
related to water use and gas exchange (Figure 3). Growth constituted a
distinct axis as compared to survival and photosynthesis for variation
in species' drought response (Appendix Figure A9), but the overall
variation in performance among species was explained by composite
phenotypes determined by soft traits. Species from more seasonal sites
showed larger proportional decline in photosynthetic performance under
drought (Figure 1c), but species affinity to seasonality conditions did
not explain variation in drought response in terms of growth or
survival.

Different aspects of species' drought performance were regulated by
different traits, but stomata and xylem traits emerged as consistently
important. Seedling survival in well-watered conditions improved with
larger xylem and stomata (Figure 1a, b). Larger xylem also correlated
with faster growth (Figure 2d) in well-watered conditions indicating
that survival under well-watered conditions improves with traits that
allow moving larger volumes of water for carbon assimilation.
Accordingly, species with larger xylem also had higher photosynthetic
rates (Figure 3a). The benefit of larger xylem for survival eroded in
dry conditions (Figure 2a), indicating that species that move more water
through their conduits may be disadvantaged during drought, consistent
with larger xylem being more vulnerable to embolism and cavitation
(Hoffmann \emph{et al.} 2011, Skelton \emph{et al.} 2015,
Santiago\emph{et al.} 2018).

Greater stomatal density increased growth (Figure 2e) and photosynthesis
(Figure 3b) in well-watered conditions, highlighting that tighter
stomatal packing improved physiological function when resources were not
limited (Medeiros et al. 2019). However, in drought conditions, species
with larger, more densely packed stomates experienced substantially
larger declines in photosynthesis (Figure 3 b, f, g) and growth (Figure
2e). Since smaller stomatal size did not correlate with higher stomatal
density, stomatal size and density form different axes to regulate
water-use (Anderegg \emph{et al.} 2018). Stomatal area fraction (SAF =
SD*SS) showed results consistent with stomatal density (Appendix Figure
A3).

Species with lower specific root length, i.e., ones that proportionally
invested more in root length than area, had higher survival when water
was plenty, but experienced larger decline in survival in drought
conditions (Appendix Table A9, l). Investing in below ground tissues may
help maintain growth during drought by improving access to water
(Joslin\emph{et al.} 2000, Poorter \& Markesteijn 2008, Paz \emph{et
al.} 2015, Chitra-Tarak \emph{et al.} 2018). The relationship between
drought response and organ-level allocation may be due to higher
photosynthesis in species that invest more above-ground, suggesting
coordination between root traits and stomata in regulating performance.
However, photosynthetic rates in well-watered conditions did not vary
with root mass fraction, or the length and area of roots. Moreover,
these relationships remained similar in drought conditions even as
average photosynthetic rates declined with water scarcity.

In general, trait values that supported better growth in well-watered
conditions resulted in larger declines in growth with drought,
suggesting a safety-efficiency trade-off with respect to water use
(Choat \emph{et al.} 2012). Species with larger xylem, suggesting
higher hydraulic conductivity, also decreased photosynthesis during
drought, but in this case, reduced photosynthesis translated to greater
mortality suggesting carbon starvation with drought. Variability of
trait effects on different aspects of performance was unsurprising when
considering that survival and photosynthesis comprised distinct axes as
compared to growth for variation among species (see PCA of species
coefficients for performance variables, Appendix Figure A10). Moreover,
different combinations of traits were possible and required to explain
interspecific variation in the overall drought response represented by
changes in survival, growth, and photosynthesis.

A few caveats temper interpretation of our results. Extrapolating from
pot experiments to performance in field conditions merits caution. Plant
size affects soil water depletion and transpiration rates when growing
in a limited volume of soil, influencing comparisons among species
(Comita \& Engelbrecht 2014). Initial height, however, did not predict
drought responses in our study. Secondly, in closed canopy forests,
shade may buffer seedlings from drought if shaded conditions decrease
evapotranspiration and/or maintain water in soil (Kupers \emph{et al.}
2019). In addition, larger trees often transport water from deeper
layers towards the surface (hydraulic redistribution) and increase water
availability for seedlings to buffer drought stress. Alternatively,
drought in natural settings may increase mortality due to other
stressors such as nutrient limitation or herbivory, which we did not
assess. Transplant experiments in the field will be needed to understand
`real world' implications of drought for seedling performance (Comita \&
Engelbrecht 2014).

\textbf{Conclusion}

We found that anatomical traits related to xylem and stomata played a
more prominent role than commonly used soft traits (e.g., SLA, wood
density) in explaining the drought response of tree seedlings from
seasonally wet humid forests of south Asia. These anatomical traits are
directly involved in water movement and gas exchange (Brodribb \&
Holbrook 2003, Brodribb \emph{et al.} 2003, Sterling 2005), which
highlights the need to link hydraulic performance and whole-plant
response of tree seedlings to drought in field conditions. Drought
conditions equalized performance differences seen in well-watered
conditions or advantaged some species. Species from more seasonal or
drier region may also induce carbon starvation by reduce photosynthetic
activity to survive drought (McDowell et al. 2008). The ability to
tolerate drought may thus influence tree species' regeneration and alter
species composition in humid tropical forests if global environmental
change increases the frequency of intra-seasonal droughts or severity of
the dry season (Chou \emph{et al.} 2013).

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\textbf{Author contributions}

Idea and concept development -- MK. Experimental set-up and data
collection -- MK, KSAB, LC, MS. Data analysis -- RJ, MK, LC.
Interpretation of results and manuscript writing -- RJ, MK, MS. All
authors have read the final version of the manuscript and agree to its
submission.

\textbf{Acknowledgements}

Seeds from Sirsi were provided by Raghavendra H. V. and from Valparai
were provided by Divya Mudappa, Srinivasan Kasinathan and others in the
Nature Conservation Foundation team, for which we are immensely
grateful. We thank Abhishek Gopal for help and guidance in initial data
analysis. We also thank the Greenhouse facility at The National Centre
for Biological Sciences (NCBS) for the use of their infrastructure and
personnel support. We acknowledge the support provided by NCBS, TIFR and
the Dept. of Atomic Energy, Government of India. Funding for this
project was provided by NCBS campus fellowship and National Geographic
explorer level-I.

\textbf{Data Availability Statement}

Data is provided in supplementary information

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