Physiologia Plantarum 168: 77–87. 2020

ISSN 0031-9317

Why does Daphne pseudomezereum drop its leaves in thesummer? An adaptive alternative to surviving forest shade
Thomas Leia,* , Naoko Yamashitab , Takuya Watanabea , Takayuki Kawaharac and Tomiyasu MiyauraaaDepartment of Environmental Solution Technology, Ryukoku University, Otsu, 520-2194, Japan
Forest Ecology Group, Kansai Research Centre, Forestry and Forest Products Research Institute, Kyoto, 612-0855, Japanc Hokkaido Research Center, Forestry and Forest Products Research Institute, Sapporo, 062-8516, Japanb

Correspondence*Corresponding author,e-mail: tomlei@rins.ryukoku.ac.jp
Received 4 February 2018;
revised 11 March 2019
doi:10.1111/ppl.12972

Daphne pseudomezereum A. Gray (Dpm) appears to be the only woody
species in the north temperate forest that sheds its leaves in the summer
while remaining green over winter (i.e. wintergreen leaf habit). Yet, the reason
for this odd leaf habit has not been explored. To this end, we examined
the microclimatic settings and ecophysiological traits of Dpm and its three
native congeners in a ﬁeld study of eight natural populations. In addition, we
conducted a common garden experiment using Dpm plants where potential
carbon gain across the seasons was estimated, using actual ﬁeld microclimate
data. Together, these data tested the hypothesis that Dpm retained traits ofan open-grown upland ancestor, unable to adapt to the deep summer shade,it survived by becoming summer dormant and wintergreen. Our hypothesis
was supported by patterns of leaf ecophysiological traits and carbon gain
simulations in Dpm, consistent with the energetic feasibility of a summer
dormancy followed by an autumn leaf sprout. We also conclude that carbon
deﬁcit driven by low light and high respiration cost is the trigger for the leaf
habit of Dpm and assert that its phenological strategy represents a rare but
viable alternative strategy for persistence in the temperate understory.

Introduction
Given the primary function of a leaf as a photosyntheticorgan, it is not surprising that carbon gain and leaf life
span are intimately related (Chabot and Hicks 1982). Not
only is the timing and duration of leaf presence important to whole plant ﬁtness (e.g. Kikuzawa and Lechowicz
2011), their variations in a plant community can inﬂuence the invasive success of some species (Fridley 2012)
and impact forest dynamics in current and future climates (Kikuzawa et al. 2013, Reich et al. 2016). There
has been much discussion about the functional basis ofleaf longevity and leaf habits, as well as the driver forthe observed distribution of deciduous and evergreen

species (Chabot and Hicks 1982, Kikuzawa 1991, Reichet al. 1992, Givnish 2002, Wright et al. 2004, Kikuzawa
and Lechowicz 2011). However, among the existingtypes of leaf habits, Daphne psudomezereum (together
with its northern relative, Daphne jezoensis syn. Daphne
kamtschatica) stands out as an exception by being a summer deciduous shrub in the temperate forest (Kikuzawa
1984, Lei and Koike 1998).
Daphne pseudomezereum (Dpm), belonging to thefamily of Thymelaeaceae, is a small, ‘deciduous’ woody
shrub native to Japan. The genus Daphne is a groupof mostly forest understory shrubs with 92 accepted
species (theplantlist.org) distributed from the temperateregions of Europe to Asia, extending south to tropicalAbbreviations – LCP, light compensation point; LMA, leaf mass per area ratio; PAR, photosynthetically-active radiation; PPFD,
photosynthetic photon ﬂux density; Rd, respiration rate.

© 2019 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction
in any medium, provided the original work is properly cited.

77

Table 1. Location, leaf habit and forest environment of the four Daphne species in this study. WG, wintergreen; SG, summer green; EG, evergreen.
Forest type includes dominant overcanopy species present. Species name are based on accepted nomenclature (or alternatively, synonyms with highconﬁdence level) given in The Plant List http://theplantlist.org (accessed 2019/1/28).

SpeciesDaphne pseudomezereum A.
Gray

Daphne koreana
Nakai

Daphne kiusiana
Miq.

Daphnemiyabeana
MakinoLeaf habit

Location code

WG

Ibu

Mt. Ibuki, Shiga Pref.

35∘ 23′ 57′′ N,
136∘ 23′ 16′′ E (472)Ryo

Mt. Ryozen, Shiga Pref.

Gyo

Gyojagaeri, Nara Pref.

Tsu

Mt. Tsurugi, Tokushima Pref.

Kib

Kurama, Kyoto Pref.

Nag

Mt. Nagasaka, Hyogo Pref.

Mik

Mikuni, Kyoto Pref.

Hin

Mt. Ryuo, Shiga Pref.

35∘ 17′ 4′′ N,
136∘ 21′ 53′′ E (651)34∘ 11′ 38′′ N,
135∘ 56′ 39′′ E(1449)
33∘ 51′ 38′′ N,
134∘ 05′ 40′′ E(1728)
35∘ 07′ 46N,
135∘ 46′ 8′′ E (526)34∘ 44′ 39N,
135∘ 07′ 27′′ E (385)35∘ 19′ 16′′ N,
135∘ 47′ 42′′ E (929)35∘ 01′ 24′′ N,
136∘ 19′ 22′′ E (787)

SG

EG

EG

Location

Southeast Asia, Polynesia and Australia. Unlike any otherco-occurring plants in the temperate forest, Dpm andD. jezoensis (determined to be the southern and northern races, respectively, of a single species based onmolecular phylogeny; T. Kawakara unpublished data)
undergo summer senescence under dense canopy shade.
This is followed by a ﬂush of leaves in late summerthat remain green over winter when the predominantly
deciduous canopy is open (Table 1, Fig. 1; images of theDaphne species are provided as Supporting Information).
An additional leaf ﬂush occurs in the spring, in sync
with many winter dormant species, but both autumn andspring leaves are shed between July and September. Thisleaf habit is commonly known as wintergreen (Bell andBliss 1977). Our study is an attempt to shed light on thereason for this unusual leaf phenology, which appears to
be a shade-avoidance mechanism, from an ecophysiological and evolutionary perspective.
To understand the unusual phenological behavior of
Dpm, we begin with the classical interpretation of variations in leaf phenology as the consequence of the environment, affecting how leaves accrue their investment
cost and how much each leaf contributes to whole plant
carbon gain (Monk 1966, Chapin III 1980, Chabot andHicks 1982, Givnish 2002, Kikuzawa and Lechowicz
2011). Research has shown that a leaf can recover its costof investment in as little as 22 days after leaf emergence
under high light and 43 days in partial shade (Fragaria
78

Latitude/Longitude(elevation in m)

Forest type and dominant
species (deciduous
species in bold)
Mixed broadleaf forest, Carpinus
laxiﬂora, Quercus mongolica
subsp. crispula, Q. variabilis
Mixed broadleaf forest, Cornus
controversa, Zelkova serrata
limestone outcrop, scatteredtrees, Chamaecyparis obtusa,
Fagus crenata
limestone outcrop, low trees andshrubs, Abies veitchii, Betula
ermanii
Chamaecyparis obtusa plantation
forestCryptomeria japonica plantation
forestMixed broadleaf forest, Carpinus
laxiﬂora
Mixed broadleaf forest, Quercus
mongolica subsp. crispulaFig. 1. Leaf phenology of Daphne pseudomezereum population at theIbu site (Table 1). Tagged shoots were monitored over complete 2 yearsof growth (including three winters). The number of leaves per shoot isderived from the mean of 13 shoots ± SE, at one shoot per plant.

species, Jurik and Chabot 1986). Having accrued theinitial leaf cost, the leaf can accumulate carbon proﬁt,
minus the maintenance cost mainly as respiration). But
depending on the speed of decline in assimilative capacity (faster under high light), a quicker leaf turnover (i.e.
shorter leaf lifespan) may be more proﬁtable for the plant(Jurik and Chabot 1986, Kikuzawa 1991, Xu et al. 2017).
The longer payback time for shade leaves necessitates

Physiol. Plant. 168, 2020

longer leaf lifespan and because their photosynthetic performance declines more gradually, leaf longevity can
extend beyond the payback time and lead to more carbon beneﬁt for shade leaves (Jurik and Chabot 1986). It istherefore reasonable to expect individual leaf longevity
to be determined by its intrinsic carbon economy andphysiological status with additional regulation imposedby unfavorable seasonal conditions, such as cold or
drought on the whole plant leaf habit.
Relative to a more ‘disposable’ deciduous leaf, evergreen leaves are generally higher in leaf mass per area
ratio (LMA) and tend to occur in more shaded environments. Therefore the time needed to recover theirleaf construction cost is longer, resulting in longer leaf
lifespans (Wright et al. 2004, Valladares and Niinemets2008). During their longer payback period, evergreen
leaves often experience additional stresses such as
drought and cold that limit carbon gain, but they may
also beneﬁt from seasonal improvements in light, during open winter canopies in many temperate forests(Miyazawa and Kikuzawa 2005). Deciduous shrubs
and tree seedlings with early season bud-burst (Seiwa
1999, Augsperger et al. 2005) adopt a similar strategy
for carbon capture and delay leaf senescence pastthe ‘post-canopy’ period (Fridley 2012). For Dpm, by
ﬂushing leaves in the autumn and retaining them over
winter, carbon gain in both post-canopy and early spring
pre-canopy periods can be achieved. But paradoxically,by doing so, the cohort of ‘deciduous’ leaves must
survive the cold winter months that would normally
have triggered leaf fall.
When delving into leaf phenology, a clear distinction between individual leaf lifespan and whole plantleaf habit must be made, because they respond differently to intrinsic demands and environmental constraints. Regardless of individual leaf lifespan, which canrange between a few weeks to decades (Wright et al.
2004, Kikuzawa and Lechowicz 2006), leaf lifespan byitself does not deﬁne leaf habit. Only when lifespan of
all leaves is reached synchronously on the plant during a
12-month period does the deciduous habit arise (Chabot
and Hicks 1982). Some plants produce only leaves withlongevity less than 1-year, but because they senesce
asynchronously, the tree remains evergreen (Kikuzawa
and Lechowicz 2006). Independent of the complex regulation involved in individual leaf senescence (which
deﬁnes leaf longevity; Woo et al. 2013), the synchronous
leaf shedding (i.e. leaf habit) would further require a systemic and coordinated whole-plant response. While theprecise factors that trigger a deciduous leaf habit are
not known, it is assumed that the onset of plant level
carbon deﬁcit, be it winter freeze or seasonal droughtthat is likely to prevail over a signiﬁcant period, plays aPhysiol. Plant. 168, 2020

signiﬁcant role in initiating the process. However, insteadof freezing or drought, the coordinated leaf shedding of
Dpm appears to concur with low light and high temperature occurring in mid-summer. This seems to be
unique among forest understory species, because they
commonly persist through summer by physiological andmorphological means (Valladares and Niinemets 2008).
This leads to the hypothesis that Dpm lacks sufﬁcientphysiological shade adaptation, and instead, has evolved
an alternative (summer deciduous) strategy to avoid thisperiod of environmental stress.
To test this hypothesis, we examined key ecophysiological traits of Dpm and assessed whether summer dormancy is related to their lack of leaf shade adaptation.
To put the leaf properties of Dpm in context, we compared it with three of its evergreen and summergreen
congeners to highlight its phenological anomaly as partof an alternative functional design. Congeners include
two evergreens, Daphne miyabeana and Daphne kiusiana, differing in their preference for mixed forests andevergreen conifer forests, respectively, and the summergreen D. koreana, occurring mainly on limestone outcrops greater than 1000 m in elevation (Table 1). Daphne
koreana, while sharing a particularly close genetic afﬁnity with Dpm (T. Kawahara, unpublished), exhibits adiametrically opposite leaf phenology. Based on botha ﬁeld study of the four species and growth chamber
simulation experiments, we found evidence supporting
Dpm’s unique leaf phenology as a viable adaptive strategy (Marks and Lechowicz 2006).

Materials and methods
Study site
Within the naturally occurring populations of the four
species of Daphne in western Japan, two populations
per species were selected for this study. Their locations
and the prevailing forest type and common canopy tree
species are given in Table 1.
Environmental attributes of the Daphne habitat
At each location, we assessed the long-term microclimate changes in irradiance, air temperature, soil temperature and soil moisture by using data loggers and sensors
from Onset Computer Corp, (Borne, MA). The sensors used for irradiance were Hobo PAR smart sensors
mounted on posts between 0.5 and 1 m above ground(adjusted to just above the canopy height of adjacent
Daphne); for air temperature, Tidbit v2 Temp Loggers
were mounted at ca. 0.8 m aboveground; for soil temperature, 12-Bit Temp Smart Sensors were inserted belowground at a depth of ca. 15 cm and for soil moisture,
79

Soil Moisture Smart Sensors (20 cm-long probe) werepositioned vertically from ca. 3 cm below the soil surface. The sensors (excluding the Tidbit v2 Temp Loggers)
were connected to the Hobo Micro Station Data Loggers. To obtain an average condition of the location,
three complete sets of sensors were placed at each siteroughly 10 m apart. Sensor readings taken at 2 min intervals were continuously recorded and maintained for atleast 2 years. In addition to the photosynthetically-active
radiation (PAR) sensor, forest light was also assessedusing canopy photographs. These were taken with a
Nikon Coolpix 990 digital camera and a Nikon Fisheye Converter FC-E8 0.21× lens with the camera lensplaced at the canopy height of Daphne. Between 17
and 32 images were taken at each location under overcast sky. The images were analyzed using CanopOn2
by A. Takenaka (http://takenaka-akio.org/etc/canopon2/,
in Japanese). The software (presently with instructionsin Japanese only) was developed based on Anderson(1964) with minor modiﬁcations (A. Takenaka, pers.
comm.). Canopy openness (%) was derived from theimage analysis.

Ecophysiological attributes of Daphne
To assess the carbon assimilation capacity of the species,
we measured the gas exchange of ﬁeld plants at each
population on multiple occasions (between 3 and 9
measurement dates) during the growing season between
2005 and 2010 (dates are provided in Table S1). All measurements were taken between 09:00 am and 15:00 pm.
The number of plants sampled from each location was
between 5 and 15. Fully expanded leaves, generally
from the 6th to 12th node position from the shoottip, were selected for measurements. For each leaf, aphotosynthetic light response curve was taken usinga photosynthesis system (LI-6400; LI-COR) coupled
to a leaf chamber with a leaf area of 2 cm2 and anlight-emitting diode light source (6400-40). Measurements were taken under ambient temperature, reference
CO2 was set to 380 μmol mol−1 and vapor pressuredeﬁcit was kept within the range of 0.9–1.5 kPa. A light
response curve was taken for each leaf, measurements
started with photosynthetic photon ﬂux density (PPFD)
matching ambient levels and raised incrementally until
light saturation to obtain maximum photosynthetic rate
(Amax ), photosynthesis readings were recorded after rates
have stabilized at each PPFD). PPFD was then returnedto ambient level, conﬁrming a return to initial rates
before incrementally decreasing it to zero and dark
respiration rate (Rd) was taken when rates stabilized.
Relevant parameters were derived from the data using
80

a curve-ﬁtting software (AQ Curve Analysis, Photosyn
Assistant; Dundee Scientiﬁc).
For LMA determination and leaf nitrogen analysis, leaf
samples from each location were collected, packed in
ziplock bags and kept cool until measurements. Theseleaves included those used for gas exchange measurements as well as additional leaves from similar shootpositions of other plants at each site. We determined
LMA from the dry mass of 8.5 mm diameter leaf disks,oven-dried for 48 h at 70∘ C). Similar prepared dried leafdiscs were used to determine leaf nitrogen content as %leaf dry mass using a nitrogen and carbon analyzer (Sumigraph NCH-22F; SCAS). A calibration curve was madefrom an acetanilide standard (Wako Chemicals) to convert the spectrometer outputs into nitrogen mass.
Estimating monthly carbon gain
To understand how potential carbon gain of Dpm mightvary across the seasons, we estimated its carbon gainusing actual variations in PAR and temperature fromthe Ibu site (Table 1 and Fig. 2) and plants raised in acommon shaded garden at Ryukoku University (at 6.2%
canopy openness, similar to those of Dpm habitats).
Using ﬁve nearly mature (2–3-year-old) individuals, weﬁrst determined light- and temperature-dependent photosynthetic responses in the laboratory. To do this, wemeasured each plant by placing it in a growth chamber(Conviron PGR15) set to one of six measurement temperatures: 5, 10, 15, 20, 25 and 30∘ C. A single fully
expanded leaf, generally from the 6th to 10th node position from a shoot tip, was selected for measurements
using LI-6400 [at 380 ppm CO2 , 50% relative humidity and one of the six preset chamber temperatures]. The
leaf and LI-6400 chamber assembly were enclosed in thegrowth chamber with measurement commands operated
from outside the chamber. The plant was allowed 1 h
to stabilize to each temperature before a light response
curve was taken (with PAR at 0, 10, 30, 50, 100, 300.
500, 700, 900 and 1100 μmol m−2 s−1 ) at the set temperature. The procedure is repeated for the next temperature setting. By making the measurements in Octoberusing recently ﬂushed autumn leaves, we could estimate peak physiological performance of Dpm. In deriving the photosynthetic response surface to temperatureand PAR, we noticed that the response pattern under low
light (i.e. 0–75 μmol m−2 s−1 ) was qualitatively different to that at higher light levels (75–1100 μmol m−2 s−1 )and required different best ﬁtting regression models as
described below. The response surfaces composed ofphotosynthetic rates (Pn, z) generated from the six temperatures (Temp, x) x PAR (y) was ﬁtted to 3D regression functions using SigmaPlot (Systat Software Inc.). The

Physiol. Plant. 168, 2020

model that best ﬁtted the low light response data was aparaboloid model of the following form:() ()
Pn = k– a x Temp + b x PAR()()+ c x Temp2 – d x PAR2
(1)
where k, a, b, c and d are derived from the Paraboloid
regression analysis. And the best ﬁt model for the higher
light response data was a Lorentzian model of the following form:(((() )2 )
Pn = a∕ 1 + Temp-k ∕b((() )2 ))(2)x 1 + PAR-l ∕cwhere a, k,b, l and c are derived from the Lorentzian
regression analysis.
The R2 value of the individual response surface ranged
between 0.52 and 0.97 (median = 0.90 and 0.87 forParaboloid and Lorentzian, respectively), and the ANOVA(df = 4, 19), corrected for the mean of the observations,
all showed signiﬁcant values of P < 0.005. The model
equations were then applied to actual microclimate data(from 10th to 15th day of the month) at 2-min intervals from Ibu and Ryo (Table 1, Fig. 2). Carbon gain was
summed over the 6 days and a mean daily carbon gain
was calculated. Our models occasionally produced values that are inconsistent with the input (for example,
a positive photosynthesis value when PAR = 0), in thatcase, we replaced it by extrapolating its likely value fromthe array of data adjacent to the error with similar input
values. We also estimated the effect of leaf aging on thesimulated carbon gain. This was done using both ﬁeld
gas exchange data and laboratory temperature response
measurements to derive a correcting factor for leaf aging
where a 5–10% monthly decline in photosynthetic performance was applied (detailed method is given in Supplemental Information).
Statistical analysis
Means and differences in traits such as LMA, percent leaf
nitrogen and gas exchange measurements among sites
and species were determined using a nested two-way
ANOVA (R Core Team 2013) with a sample size of between
5 and 15 per site. The difference between the regression
slopes of Amax − Rd between Dpm and D. koreana wastested using a multiple regression with dummy variables:
Rd = b0 + b1 x Amax + b2 x d1 + b3 x Amax x d1

(3)

where b0, b1, b2 and b3 are coefﬁcients and d1 is adummy variable where Dpm = 0 and D. koreana = 1.
The signiﬁcance of the term Amax x d1 tests the null
hypothesis Ho: slope of Dpm = slope of D. koreana.

Physiol. Plant. 168, 2020

Results
Microclimate of the Daphne
The prevailing light environment of Dpm during summer was characterized by heavy forest canopy shadeat canopy openness of 6.41 ± 1.56%, n = 17 for Ibu
and 9.60 ± 1.81%, n = 28 for Ryo with ambient PAR
at their lowest in July and early August, coinciding
with their summer dormancy (Fig. 1). For example, at
Ibu in August, we recorded PAR below the light compensation point (LCP) of 9.2 μmol m−2 s−1 in 89% of thetime when low daytime irradiance and night time werecombined, so if leaves were present at that time (estimated using simulated response data), this would leadto a respiration cost 6.2 times that of photosynthetic
gain (i.e. when PAR is greater than LCP). Even for thebrighter Ryo site, 60% of August PAR was less than LCP
which would incur a cost of respiration nearly equaling(91%) that of the potential gain in carbon. It is important to note that concurrent with the deep shade, Dpm
also experienced the highest temperature during thesemonths (Fig. 2). Following leaf senescence of the deciduous canopy in autumn, canopy openness reached levelsof about 30%. PAR sensors located near individual plants
revealed that, while overall light levels were low during
the winter months, some plants experienced periods ofhigh PAR (>800 μmol m−2 s−1 ) while other plants may be
temporarily snow-covered (T. Lei, personal observation).
Compared to Dpm sites, canopy openness at D. koreanasites on limestone outcrops were higher at 22.6 ± 4.2%,
n = 29 (Gyo, refer to Table 1 for site description) and
32.4 ± 6.5%, n = 25 (Tsu) in mid-summer. The light environment of the two evergreen species was generally dimmer, in particular that of D. kiusiana. The sugi-hinoki
forests (Kib and Nag sites, Table 1) cast a persistentshade on the order of 10% canopy openness. The mixed
forest canopy of D. miyabeana provided an improvement in winter PAR similar to that in Dpm sites (Fig. 2).
Soil moisture in the top 20 cm of soil largely remained
above 10% by volume throughout the year but was lowest during the months of September and October (datanot shown). Rainfall data for the region that includes
the Dpm habitats of Ibu and Ryo (30-year average for
Hikone and Nagahama, Japan Meteorological Agency,
www.data.jma.go.jp, last accessed Mar. 6, 2019) show
a peak of 200 mm in July, declining to 100, 160 and
110 mm in August to October, respectively, indicating
the absence of a clear dry season.
Ecophysiological variations among Daphne
Dpm presented itself as a high-light adapted plant
through a number of attributes. First, in Amax vs dark
81

Fig. 2. Cumulative monthly PAR (closed dots) and monthly mean air temperature (open dots) of the four Daphne species by two populations, basedon long-term (>2 years) microclimate data (at 2-min intervals) collected from three replicated sites at each location. Location codes correspond to
those given in Table 1.

respiration (Rd), Dpm showed a signiﬁcant range ofvalues that largely overlap those of D. koreana growing in bright upland habitats (Fig. 3). The regression
ﬁt for Dpm at Rd = 0.2201 + 0.0503 𝜒 Amax is closely
matched by that of D. koreana at Rd = 0.4048 + 0.0504
𝜒 Amax (with virtually identical slopes, P = 0.99). The
offset in the slopes is associated with a signiﬁcantdifference (Student’s t-test, P = 0.005) in Rd between
D. koreana (0.816 ± 0.036 μmol m−2 s−1 ) and Dpm(0.667 ± 0.037 μmol m−2 s−1 ) suggesting a better carbon
use efﬁciency in Dpm (Fig. 3). The two evergreen species(D. miyabeana and D. kiusiana) exhibited signiﬁcantly
lower means of Amax and Rd than the deciduous species(Table 2) but with similar regression slopes (Fig. 3). It
is also clear that there were, on the whole, greater
differences between sites for Dpm. Speciﬁcally, gas
exchange of Dpm at Ibu was signiﬁcantly lower thanthose in the Ryo population (Table 2A). This difference
appears to be associated with the Ibu site being a moreshaded location during the summer (Fig. 2). Secondly,
there is a signiﬁcant cohort by site variation in LMA.
Between cohorts, the autumn (over-wintering) leavesof Dpm can be as high as its evergreen congeners(Site Ryo: 55.8 g m−2 ) while the spring leaves tended
to be low (31.1 g m−2 ) on the same order as the winterdeciduous D. koreana (Table 2A). But at the Ibu site,
the same difference between cohorts was not evident
as LMA of both spring and autumn leaves were about
31 g m−2 . Thirdly, consistent with other leaf-level traits
expressing high-light-adapted properties, Dpm leaves
also have higher percent leaf nitrogen relative to its threecongeners (Table 2).
82

Fig. 3. Photosynthetic properties of the four Daphne species native toJapan from multiple ﬁeld measurements at each site. Daphne pseudomezereum (Dpm) exhibited a large range of gas exchange rates overlapping and exceeding D. koreana, despite its shadier habitat. Regression
curve results are given in table with Dpm (red) and D. koreana (green)nearly identical in slope.

Simulated patterns of daily carbon gain
Initially, we used the same photosynthetic response data
derived from newly matured leaves to estimate monthly
carbon gain without accounting for leaf aging. This analysis allows us to ask what potential optimum carbon
gain might Dpm achieve given the microclimate conditions where it grows. With this simulation, we founda pattern of daily carbon gain ranging between −25
and 109 mmol m−2 day−1 with increased carbon gains inautumn and spring and possible negative carbon balance

Physiol. Plant. 168, 2020

Table 2. Key leaf traits of the four Daphne species native to Japan are shown in A. All measurements taken on recently matured leaves in early summer(D. koreana, Daphne kiusiana and Daphne miyabeana) or late autumn (Daphne psuedomezereum, after autumn leaf ﬂush). Differences between species
and sites for each trait are indicated by different letters following the mean values. Values are given as mean ± SE. Results of differences between species
and between sites within species of traits are shown in B. Data were analyzed using nested ANOVA and Tukey HSD test.
(A)
SpeciesDaphne pseudomezereum (Dpm)
D. koreana (kor)Daphne kiusiana (kiu)Daphne miyabeana (miy)

(B)
Trait
LMA (g m−2 )%NAmax (μmol m−2 s−1 )Dark respiration (μmol m−2 s−1 )Location code

LMA (g m−2 )%NAmax (μmol m−2 s−1 )Dark respiration (μmol m−2 s−1 )

Ibu
Ryo
Gyo
Tsu
Nag
Kib
Hin
Mik

31.7 ± 0.9c
55.8 ± 4.0a
33.1 ± 2.4c
40.5 ± 4.1bc
52.9 ± 3.1ab
56.5 ± 6.5ab
53.6 ± 2.3ab
58.2 ± 2.2a5.27 ± 0.24a
3.98 ± 0.10b3.45 ± 0.26bc
2.94 ± 0.28c
2.86 ± 0.16c
2.77 ± 0.27c
2.85 ± 0.13c
ND

6.44 ± 0.42c
10.48 ± 0.65a
7.10 ± 0.41b9.11 ± 0.50ab
3.50 ± 0.23d
3.42 ± 0.22d
5.44 ± 0.64cd
6.60 ± 0.82bcd

0.51 ± 0.04c
0.77 ± 0.05ab
0.74 ± 0.05ab
0.88 ± 0.05a
0.45 ± 0.03c
0.46 ± 0.03c
0.36 ± 0.05c
0.48 ± 0.05bc

Species

Site (Species)

Diff. in Species means (P < 0.05)

***
***
***
***

**
**
***
***

kiu = miy > Dpm = kor
Dpm > kor = kiu = miy
kor = Dpm > miy > kiu
kor > Dpm > miy = kiu

in the summer (Fig. 4). Next, we estimated what effectleaf aging may have on the carbon gain pattern. We usedﬁeld gas exchange measurements (Fig. 3) and estimatedthe likely rate of decline in photosynthesis and respiration, independent of seasonal changes in temperature(Fig. 5, Fig. S1). Based on the calculations, we estimatedthat gas exchange performance declined from 100% in
October to 30% in the following August, applied the
5–10% a month decline to the simulated carbon gain
(Fig. 4).

Discussion
Functional attributes of D. pseudomezereum(Dpm)
In the temperate forest of western Japan, Dpm sharesthe forest understory with summergreen species (including hardwood seedlings of Acer and Quercus), and evergreen species (such as Aucuba japonica, Eurya japonica,
Quercus glauca seedlings, and in some locations, D. kiusiana) that do not senesce in the summer. So, why isthis plant behaving so differently from the others? Our
results indicate that unlike the others, Dpm does notappear to possess the prerequisite shade-adaptive traits
for the forest understory as it shares many trait properties, such as LMA, Amax and leaf N, with D. koreana, its summergreen relative growing in much brighter
locations (Figs 2 and 3; Table 2A,B). The LMA of Dpm
showed a range of values depending on site and season
but the autumn-ﬂushed leaves can attain values comparable to the evergreen Daphne and signiﬁcantly greater

Physiol. Plant. 168, 2020

Fig. 4. Simulation of leaf carbon gain in Daphne psudomezereum using
photosynthetic response equations derived from newly matured leaves
measured under different light and temperature settings. Response
equations were applied to actual ﬁeld microclimate data of Ryo and Ibu
to derive mean daily carbon gain for each month (mean of ﬁve plants ±SD) accounted for the effect of leaf aging (solid lines, red for Ibu, blackfor Ryo). The dashed line for each site represents the response pattern
not corrected for aging (error bars not shown). We also superimposed
Dpm leaf phenology taken from a portion of Fig. 1 (gray line).

than D. koreana (Table 2A, B). Leaves ﬂushed in spring,and those grown in more shaded locations (such as Ibu),
have lower LMA of 20–40 g m−2 , a range more similar
to other understory plants (Lei and Lechowicz 1998).
The signiﬁcant site differences in LMA also suggest thatsome Dpm populations, i.e. grown in Ibu, are expressing more shade tolerant attributes. Critical for Dpm is the
production of a durable cohort of over-winter leaves that83

Fig. 5. Field gas exchange measurements of Dpm showing leaf temperature (open circles) and Amax (blue dots) in the upper panel,
and Rd (black dots) in the lower panel across season. Leaf temperature is shown with ﬁtted equation (dotted line): Leaf temperature = −12.05 + 0.346 julian day (JD) – 0.0008 JD2 (R2 = 0.80). Amax
and Rd were grouped as spring and autumn data on account of the
midsummer dormancy (shown in gray). The regression lines (dashed)for each group are: Amax (spring) = 15.31–0.044 JD (R2 = 0.10); Amax
(autumn) = 29.71–0.059 JD (R2 = 0.09); Rd (spring) = 0.711–0.0004 JD(R2 = 0.002); Rd (autumn) = 3.811–0.0097 JD (R2 = 0.35).

sustains the necessary carbon gain through winter, evenunder relatively low air temperatures (Figs 2–4). The
ability for Dpm to photosynthesize in low temperatures
is evident in ﬁeld measurements where Amax in December was 9.1 ± 0.9 μmol m−2 s−1 at air temperatures of
14.9 ± 0.6∘ C (Fig. 5). These early winter rates were comparable to those of D. koreana measured in mid-summer(Table 2). Similar low temperature responses were seenin D. jezoensis grown in northern Japan where highest
photosynthetic rates (ca. 6 μmol m−2 s−1 ) were obtained
at 15∘ C, and even at 10∘ C, rates of 3–5 μmol m−2 s−1
were observed (N. Yamashita, unpublished). Percent leaf
nitrogen (Table 2) is another trait that distinguished Dpm
from typical shade-adapted species (Lei and Lechowicz
1998), but despite the higher N content, Dpm does notappear to sustain substantial insect damage (T. Lei, personal observation). It is tantalizing to speculate that dormancy could also play a role in escaping predators in
mid-summer.
Dark respiration, together with LMA, Amax and leaf
N, is considered an attribute of the ‘cardinal’ leaf
84

design, linking the timing and duration of foliar presence
to whole plant carbon gain (Reich et al. 1997, 1999,
Wright et al. 2004, Kikuzawa and Lechowicz 2011).
Leaf respiration received particular attention as a key
trait in explaining leaf longevity because it appears tocorrespond well to a plant’s shade tolerance (Wrightet al. 2006, Baltzer and Thomas 2007). Using Baltzer
and Thomas’s data for tropical seedlings, Reich (2014)
showed that leaves with higher dark respiration (Rd) not
only demanded higher light environment for survival but
are also associated with dramatically shorter leaf lifespans. We can infer from these results that for a shadeplant, a lower Rd is critical for maintaining a lower wholeplant LCP (Reich 2014) and those unable to do so, could
face an unsustainable carbon deﬁcit. Furthermore, studies have shown that shade tolerant plants can survive
under low light not because they have higher photosynthetic rates or a better light capture efﬁciency thanless tolerant plants, rather it is because they can maintain carbon gain by minimizing respiratory losses (Walters and Reich 2000, Craine and Reich 2005). Excessive respiratory cost has also been attributed as thedriver for the initiation of dormancy in drought deciduous arid species (Mooney et al. 1975). These ﬁndings
together suggest that, for Dpm, initiating summer dormancy at a time of high respiratory losses is a plausible response strategy. Under the combination of hightemperature and low PAR, if Dpm plants had leaves inAugust, they would spend between 60 and 89% of thetotal time respiring and Rd would render daily carbon
gain near 0 or even negative (Fig. 4). We also estimatedthat even a small difference in Rd could mean the difference between a summer month where carbon gain ispositive (at Rd = 0.50 μmol m−2 s−1 ) or one with a carbon
deﬁcit (at Rd = 0.54 μmol m−2 s−1 ).
Simulated patterns of seasonal carbon gain
by DpmOur simulations clearly show that retaining leaves over
winter contributes to achieving yearly net carbon gain
in a typical Dpm environment (Fig. 4). It is also clear thatsuch carbon balance is possible even if Dpm were toretain leaves over the summer, but it would be difﬁcult
without the boost in physiological performance initiatedby the autumn ﬂush. With or without accounting for leaf
aging, the overall pattern did no differ qualitatively, withboth showing a mid-summer carbon deﬁcit which wascritically for Dpm, coincides with the period of summer dormancy in the ﬁeld (Fig. 4). Simulation allowed
us to visualize the summer carbon deﬁcit, when in fact
leaves are absent, and points to the stress Dpm would
have to endure by spending most of the time in the dark

Physiol. Plant. 168, 2020and respiring at high rates (Fig. 5, Fig. S1). By sprouting its major cohort of leaves in autumn, Dpm is also
unique among temperate woody species in deploying
leaves that ﬁrst experience a decreasing air temperature and daylength and then a reversal in spring prior
to leaf shedding. Because of this seasonal effect, Dpm
leaves ﬁrst show an end-of-year decline in Amax whenleaf aging is in sync with lowering temperatures andthen continues to decline in the spring, but under rising temperatures. Newly sprouted spring leaves have ashorter lifespan where leaf carbon gain declines quickly
into summer. Retaining leaves over winter offers Dpm arare carbon gain opportunity under an open deciduous
canopy that resembles evergreen saplings, where a large
fraction of their lifetime carbon gain occurs during thistime (Miyazawa and Kikuzawa 2005). The evergreen D.
miyabeana also beneﬁts from similar microclimate conditions and can, for example, amortize leaf costs sooner
than D. kiusiana, growing mainly under a persistently
evergreen canopy (Fig. 2). From this, we would expect alonger leaf lifespan for D. kiusiana than D. miyabeana,
which is conﬁrmed by our ﬁeld phenology measurements where D. kiusiana has a mean leaf lifespan of
2.7–4.0 years, while that for D. miyabeana is 2.0 years(N. Yamashita, unpublished data). Although the summer
dormancy of Dpm is unique in the temperate forest, thesummer deciduous habit is common in arid regions as a
response to extended drought (Mooney and Dunn 1970).
Drought stress can also be exacerbated by shade, as
the evergreen Heteromeles arbutifolia nearly ceases carbon gain in mid-summer (Valladares and Pearcy 2002).
Despite these similarities, Dpm and drought deciduous
plants differ fundamentally in the primary drivers of theirleaf response.
Payback time and contribution to whole plant
carbon gain
Relevant to the phenology of Dpm is whether summer
dormancy is energetically compatible with the paybackof its leaf cost and carbon return to the plant. We knowthat despite the large variation in leaf longevity and thediverse environments that leaves are exposed to, the costof leaf construction is remarkably stable at approximately 1.5 g g−1 glucose (Williams et al. 1989, Villar
and Merino 2001, Kikuzawa and Lechowicz 2011) andthe lifetime carbon contribution by a leaf is consistently
about 4 g g−1 (G-net lifetime carbon gain in Kikuzawa
and Lechowicz 2006). What this suggests, is that thelongevity of a leaf may be determined by when the common set point in carbon gain per gram of leaf is reached.
Given the wide range of leaf morphology, physiological performance and lifespan, the time needed to reachPhysiol. Plant. 168, 2020

Fig. 6. Estimated time (in days) to pay back leaf construction cost andthe number of days required to contribute 4 g of carbohydrates (CHO)
per g leaf CHO to the plant, as determined by mean daily carbon gain
for leaves of different LMA (10, 30 and 60 g m−2 are shown). The same
curves apply to both the payback time (left axis) and the time required
to accrue additional 4 g of CHO per g leaf CHO (right axis). Calculations
are derived from leaf morphology, microclimate data and results fromthe simulated carbon budget experiment shown in Fig. 4. The arrows
point to values relevant to the discussion.

the set point would depend largely on the mean realized photosynthetic rate, leaf size and leaf tissue density.
No leaf should live beyond a year, unless they are large,
dense or are constrained by lower realized carbon gain(such as low PAR) that necessitates a longer payback time
(Villar and Merino 2001). We estimated that deciduous
leaves can repay their leaf construction cost in 46 dayson average (range 13–112 days) and evergreen leaves in
219 days (range 92–427 days) using potential instantaneous photosynthesis rates (data provided in Kikuzawa
and Lechowicz 2006) and assuming that these rates canbe sustained for on average of 5 h per day. These payback times are consistent with earlier estimates of requiring 3 weeks under high light to 6 weeks in the shade
for wild strawberry (Jurik and Chabot 1986). For Dpm,
we can estimate the payback time based on the simulated carbon budget given in Fig. 6 at a range of LMA(serving as a proxy for leaf construction cost). Given that
autumn leaves have an LMA in the range of 60 g m−2 ,and a daily carbon assimilation of 40 mmol m−2 (Fig. 4),
it would require 51 days to payback the construction
cost of such a leaf (Fig. 6), consistent with the deciduous averages above. Thus, even when we estimate thepayback time using the actual daily carbon budget (andnot just the mean A during daytime or Amax ), an autumn
leaf can accrue carbon for 200 additional days during
its lifespan, after paying back the leaf construction cost.
This compares well with our estimate of the 204 days
required to gain an additional 4 g g−1 (Fig. 6). Even aspring leaf with LMA of 30 g m−2 and a short lifespan85

of 60–80 days can payback its leaf construction cost in
17 days at 60 mmol m−2 (March, Fig. 4) and contributeclose to 4 g g−1 to whole plant carbon gain with an additional 68 days. (Fig. 6). It is also clear that Dpm produces
about three times more autumn leaves than spring leaves(T. Lei, unpublished data) suggesting that on a wholeplant level, the leaf phenology deployed by Dpm is energetically viable in its habitat and consistent with the
expected leaf functional longevity.
In conclusion, our ﬁndings support the view that Dpm
is constrained by a suite of ecophysiological traits better
suited to a higher light environment. But, by its ingenious
ﬂip in the timing of leaf deployment and leaf dormancy, it
circumvented the fate of being a shade-intolerant species(Craine and Reich 2005). For Dpm, the imperative of its
summer dormancy and autumn ﬂushing in maintaining
carbon balance and enabling reproduction highlights
is a novel adaptive response in a common selective
landscape (Marks and Lechowicz 2006).

Author contributions
T.L. conceived and designed the study and wrote
the manuscript. T.L. and N.Y. jointly carried out the ﬁeld
study and data analysis. T.W. conducted the carbon
gain simulation experiment. T.K. inferred the phylogeny
of Daphne based on chloroplast and nuclear genomic
analysis. T.M carried out the vegetation survey of theﬁeld sites.
Acknowledgements – We thank Makiko Owashi for assisting with the simulation experiment. Funding for this work
was provided in part by JSPS KAKENHI Grant No. 20380093
and by Scientiﬁc Collaboration Research Grant of Ryukoku
University. Valuable suggestions from Dr. J. Flexas, Dr. J.
Rodríguez-Calcerrada and two anonymous reviewers havehelped to improve the interpretation and clarity of this study.

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Supporting Information
Additional supporting information may be found online
in the Supporting Information section at the end of thearticle.
Appendix S1. Procedure for estimating leaf aging of
Dpm.
Table S1. Dates of ﬁeld gas exchange measurements.
Fig. S1. Temperature responses of Amax and Rd in
pot-grown Dpm.
Fig. S2. Dpm in snow taken in late February at Ibu. The
naked terminal buds and ﬂowers are visible.
Fig. S3. Dpm in winter, showing its growth preference forbases of trees and boulders.
Fig. S4. Dpm during summer dormancy (July 22). New
leaf ﬂush has appeared on the right shoot.
Fig. S5. The evergreen Daphne kiusiana with fruit, taken
on June 11.
Fig. S6. D. koreana growing among limestone boulders,also preferred by Dpm, showing signs of leaf senescence(end of August).
Fig. S7. A typical Dpm habitat (Ryo) among limestones
under a closed forest canopy in late June.

Edited by J. Flexas

Physiol. Plant. 168, 202087