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\begin{document}

\title{Species-specific stoichiometric effects of leaf litter on algal growth,
production, and cell quotas}



\author[1]{Pei-Chi Ho}%
\author[1]{Suzuna Nakajima}%
\author[1]{Jotaro Urabe}%
\affil[1]{Tohoku University}%


\vspace{-1em}



  
  \date{March 14, 2025}


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\selectlanguage{english}
\begin{abstract}
Dissolved organic matter and nutrients released from forest leaf litter
are important cross-ecosystem resources that support freshwater
ecosystems. Dissolved organic matter released from leaf litter is one of
the allochthonous sources for heterotrophic organisms in freshwater
communities. However, the role of macro- and micronutrients released
from leaf litter in producing autochthonous organic matter in aquatic
ecosystems is not necessarily well understood. In this study, therefore,
we investigated how dissolved nutrients released from leaf litter affect
the algal growth, biomass production, and cell elemental quotas.
Specifically, we focused on the responses of the green algae to TDN,
TDP, and micronutrients released from the leaf litter of 11 temperate
tree species. We found that the algal growth rate increased with TDP
when micronutrients were amended. In contrast, the algal biomass
production increased with TDN, regardless of micronutrient amendment.
Micronutrient limitation of algal growth rate was found in the leaf
litter leachates from oak, Japanese elm, and Japanese hemlock. However,
algal biomass production was limited by micronutrients only in the leaf
litter leachate from Japanese hemlock. More importantly, leaf litter
leachates from different tree species altered algal cell quotas and
C:N:P ratios, which would affect secondary production. These results
suggest that forest vegetation change or succession affect the quantity
and quality of autochthonous organic matter and thus the mass transfer
efficiency in the aquatic community by changing the stoichiometry of
nutrient input from the leaf litter.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Dissolved organic and inorganic matter released from forest leaf litter
is an important cross-ecosystem resource that influences the communities
in freshwater ecosystems . Numerous studies have shown that, in lakes
and ponds, terrigenous dissolved organic carbon (DOC) can enhance
heterotrophic microbial production and reduce underwater light
intensity, thereby reducing the algal growth and primary production
rates . However, because leaf litter contains various elements other
than C, including nitrogen (N) and phosphorus (P), it is likely to
promote autochthonous production through algal growth in aquatic
ecosystems where leaf litter is introduced. Indeed, recent studies have
shown that leaf litter stimulates algal growth and increases energy
transfer from autochthonous organic matter to higher trophic levels
through the grazing food chain in lakes .

However, the effects of leaf litter on aquatic organisms through the
release of nutrient elements may vary among tree species because the
elemental content of leaves is highly species-specific . In addition,
the water-soluble fractions of nutrient elements in leaf litter also
vary among tree species . In fact, our previous study showed that the
concentrations of dissolved N and P in leaf litter leachates varied
widely among 11 tree species because the release efficiencies of these
nutrients differed among tree species (Ho et al. 2023). These results
suggest that the production of autochthonous organic matter is affected
by the change and succession of the surrounding terrestrial vegetation
through the changes in the inputs of various dissolved nutrient elements
from the leaf litter. To test this possibility, it is essential to
investigate the response of algae to leaf litter leachates from
different tree species.

In freshwater ecosystems, the algal production rate of is mainly limited
by the supply rate of dissolved P, although at certain times it may be
limited by N or co-limited by N and P . In addition, essential
micronutrients, such as iron, are known to affect algal production and
growth rates in aquatic ecosystems , because these trace metals are
essential core elements of mechanoenzymes that play various functional
roles in cell metabolism and catabolism . Therefore, if the relative and
absolute levels of these micronutrients released from leaf litter differ
among different tree species, algal production and growth rate may
change depending on the dominant tree species growing around the
freshwater ecosystems.

In autotrophs such as algae, the biomass production is related to its
photosynthetic activities, cell division rates, i.e., growth rate, and
cell quotas, i.e., cell-specific C, N, and P contents, are affected by
various cell processes other than photosynthesis . Accordingly, the
nutrient elements that affect algal production rates are not necessarily
the same as those that affect growth rates . More importantly, the
growth of herbivorous zooplankton is influenced by the cellular N and P
contents relative to the C content in the algae . This fact suggests
that not only the production rate of autochthonous organic matter but
also its mass transfer to higher trophic levels may be affected by
changes in the dominant tree species growing around the freshwater
ecosystems via macro- and micronutrients supply from fallen and
submerged leaf litter. However, although several studies have shown that
leaf litter can stimulate algal growth and increase energy transfer to
zooplankton , no study has yet examined whether such effects differ
among leaf litter from different tree species.

In the present study, therefore, we investigated whether the algal
response is different depending on the tree species providing the leaf
litter to freshwater ecosystems. For this purpose, we prepared culture
media using leaf litter leachates from 11 common tree species in the
plain and mountainous regions of northeastern Japan. Then, we
investigated the cell growth rate, carbon biomass production, and the C,
N, and P cell quotas of a green alga \emph{Scenedesmus obliquus} . We
used \emph{Scenedesmus} because this group of green algae is common
freshwater phytoplankton and has been used in various experimental
studies . Our specific objectives are to clarify (1) if N and P nutrient
supply relative to algal requirements for growth and production differ
among the leaf litter leachates of the 11 tree species, (2) if the leaf
litter leachates provide sufficient micronutrients, and (3) if the cell
C, N, and P quotas of the algal species vary depending on the type of
leaf litter leachates. Based on these results, we discuss the
possibility that the quantity and quality of autochthonous organic
matter produced by algae and its mass transfer to higher trophic levels
in aquatic ecosystems are influenced by the changes in tree species
composition in the surrounding forests.

\textbf{Materials and methods}

\emph{Preparation of leaf litter leachates}

We collected the leaf litter of 6 broadleaf and 5 coniferous species
commonly found in the plains and mountainous of northeastern Japan.
Although we did not check when they fell, we collected leaf litter on
top of the leaf litter layer on the ground so that the litter did not
have direct contact with the soil. These fallen leaves were collected at
Zao (N38.122, E140.451) on October 3, 2020, at Kawatabi (N38.745,
E140.757) on October 15, 2020, and at Aobayama (N38.259, E140.837) on
November 19, 2020 in Miyagi Prefecture, Japan (Table 1).

Before use, leaf litter was dried at room temperature (20 \selectlanguage{ngerman}°C) and was
cut with scissors to mimic the early decomposition processes by
detritivores. For each tree species, we weighed the cut leaf litter and
placed it in two 1000 mL Nalgene bottles containing 800 ml of distilled
water. The weight of leaf litter in each bottle was adjusted to control
the final concentration of TDP to approximately 1-4 \selectlanguage{greek}µ\selectlanguage{english}M after 7 days of
the leaching process based on the release efficiency (Table 1) estimated
by Ho et al. (2023). The water in the two replicate bottles was aerated
in the dark for 7 days at 20 \selectlanguage{ngerman}°C and mixed before being used as leaf
litter leachates.

We used \emph{Scenedesmus obliquus}as the experimental algal species
because it has been frequently used in various studies . Prior to the
incubation of \emph{Scenedesmus} , the leaf litter leachate was filtered
through 40 \selectlanguage{greek}µ\selectlanguage{english}m mesh to remove litter particles. DOC and total dissolved N
(TDN) concentrations in the leaf litter leachate were measured using a
TOC/TNb analyzer (multi N/C 3100, Analytik Jena GmbH, Jena, Germany).
Total dissolved P (TDP) concentrations were measured using the ascorbic
acid-molybdenum blue method after persulfate oxidation .

\emph{Experiment}

For leaf litter leachate from each tree species, we prepared ``control''
and ``amended'' treatments using 200 mL Nalgene incubation bottles. The
control treatment consisted of 150 mL of filtered leaf litter leachate,
and the amended treatment consisted of 147 mL of filtered leachate and 3
mL of basal COMBO medium, which contained macroelements (K, Ca, Mg, B),
trace elements (Fe, Mn, Cu, Zn, Co, Mo, Se, and V), and vitamins (B12,
biotin, and thiamin), but no inorganic N and P nutrients . We referred
these chemicals as micronutrients. In each leaf litter leachate, we set
up three replicates for each treatment.

We inoculated algal cells into each experimental bottle at an initial
incubation cell density of 2.2\selectlanguage{ngerman}×10\textsuperscript{4} cells
mL\selectlanguage{english}\textsuperscript{-1}. The algae were incubated for 7 days in the
bottles, which were placed in an incubator at a temperature of 20 degC and
light intensity of 100 \selectlanguage{greek}μ\selectlanguage{english}E m\textsuperscript{-2} s\textsuperscript{-1}.
The bottles were manually shaken every day to resuspend the cells. After
7 days, we preserved 20 mL of culture medium in Lugol's solution for
estimating the cell number by microscopic enumeration. We also filtered
5 mL of culture medium on two combusted GF/F filters (Whatman), which
were dried for further analyses of the C, N, and P contents
of\emph{Scenedesmus obliquus} .

\emph{Measurements of algal growth rates and elemental contents}

We enumerated the number of algal cells at the end of incubation using
the Fuchs-Rosenthal counting chamber and an optical microscope (Olympus,
Tokyo, Japan; eyepiece magnification 10x, objective magnification 10x)
(cells mL\textsuperscript{-1}). Then, the algal growth rate was
calculated from the difference between the cell number at the start
(\emph{N} \textsubscript{0}) and the end of incubation
(\emph{N\textsubscript{t}} ) as follows,

\(\text{GR}=\frac{\ln\left(N_{t}\right)-ln(N_{0})}{t}\) (1),

where \emph{t} = 7 days.

The C and N contents of algal cells collected on GF/F filters (\selectlanguage{greek}μ\selectlanguage{english}g
mL\textsuperscript{-1}) were measured using a CHN elemental analyzer
\selectlanguage{english}(2400 Series II CHNS/O Elemental Analyzer, Perkin Elmer, Massachussets,
USA). The P content of algal cells was measured by the ascorbic
acid-molybdenum blue method after potassium persulfate oxidation . The
C, N, and P cell quotas of the algal cell were calculated as:

\(Q_{E}=\selectlanguage{greek}\frac{\text{Scenedesmus\ }\mathrm{C,\ N,\ or\ P\ biomass}\ (\mathrm{\text{μg}}\ \mathrm{\text{mL}}^{-1})}\selectlanguage{english}{\text{Scenedesmus}\mathrm{\text{\ density\ }}(\mathrm{\text{cells\ m}}\mathrm{L}^{-1})}\)(2).

\emph{Statistical analyses}

We used ANOVA and Tukey's multiple comparison test to examine if the
algal growth rate, C biomass, and C, N, and P cell quotas were
significantly different among the control treatments prepared from the
leaf litter leachates of 11 tree species. We also tested for the algal
response to micronutrient amendment in each leaf litter leachate using
Student's \emph{t} -test. These analyses were performed using
functions\emph{aov} , \emph{TukeyHSD} , and \emph{t.test} in the R ver.
4.2.2 . We examined the effects of TDN and TDP on the algal growth rate
and C biomass by the segmented linear regression model using
functions\emph{lm} and \emph{segmented} in package \emph{segmented} in
the R ver. 4.2.2 .

\textbf{Results}

\emph{Growth rate and carbon biomass}

The growth rate (\emph{F} = 59.65, \emph{p} \textless{} 0.001) and C
biomass (\emph{F} = 286.9, \emph{p} \textless{} 0.001)
of\emph{Scenedesmus} were significantly different among the culture
media prepared from 11 types of leaf litter leachates in the control
treatment (Fig. 1a). The addition of micronutrients to the culture
medium had a significant positive effect on the algal growth rate of in
the leaf litter leachates of oak, Japanese elm, and Japanese hemlock.
However, the micronutrient amendment had a negative effect on growth in
the culture medium prepared from the leaf litter leachate of hinoki
cypress. For the algal C biomass, a significant positive effect of
micronutrient amendment was detected only in the culture medium prepared
from the leaf litter leachate of Japanese hemlock (Fig. 1b). However,
algal C biomass was not affected by the micronutrient amendment in
culture media prepared from other leaf litter leachates.

Because TDN and TDP concentrations varied among the different types of
leaf litter leachates, we examined whether variations in the algal
growth rate and C biomass of \emph{Scenedesmus} were related to these
nutrient concentrations in the culture media prepared from the leaf
litter leachate. Due to the large variation of growth rate at TDN
concentrations \textless{} 5 \selectlanguage{greek}μ\selectlanguage{english}M, the growth rate was not related to TDN
concentration regardless of the micronutrient amendment (Fig. 2a and b).
However, the algal growth rate tended to increase with TDP concentration
in the amended treatment but not in the control, although the growth
rate also varied widely and decreased when TDP concentration was
\textless{} 2 \selectlanguage{greek}μ\selectlanguage{english}M (Fig. 2c and d).

\par\null

Unlike the growth rate, the algal C biomass increased significantly with
TDN in the control treatment, although the increasing trend was
attenuated when TDN exceeded 5 \selectlanguage{greek}μ\selectlanguage{english}M, as shown by the segmented regressions
with breakpoints at 5.31 \selectlanguage{greek}μ\selectlanguage{english}M (Fig. 2e and f). In the amended treatment,
the relationship between the algal C biomass and TDN was not improved.
It was almost the same as in the control treatment, as indicated by
similar values in the segmented regressions (Fig. 2e and f). Similarly,
algal C biomass tended to increase with TDP regardless of the
micronutrient amendment, although the increasing trend was not
significant due to low C biomass values relative to TDP in some culture
media, such as those prepared from leaf litter leachates of Siebold's
beech, Japanese elm and Japanese red pine (species ID 5, 6, 9 in Fig. 2g
and h).

\emph{Cell quotas and stoichiometry}

The C, N, and P cell quotas in \emph{Scenedesmus} varied significantly
among the culture media from different types of leaf litter leachates in
the control treatment (Fig. 3; \emph{F} = 33.24, \emph{p} \textless{}
0.001 for C quota; \emph{F} = 40.27, \emph{p} \textless{} 0.001 for N
quota; \emph{F} = 16.36, \emph{p} \textless{} 0.001 for P quota). The
micronutrient amendment of the culture media had a significant negative
effect on the C cell quota in the leaf litter leachates of oak and
Japanese hemlock, and a significant positive effect was detected in the
leaf litter leachate of hinoki cypress (Fig. 3a). The micronutrient
amendment had a negative effect on the N cell quota only in the leaf
litter leachate of Japanese hemlock (Fig. 3b). Similarly, the negative
effect of the micronutrient amendment on the P cell quota was detected
in the leaf litter leachate of Japanese hemlock, and a significant
positive effect was found in the leaf litter leachates of Japanese red
pine and hinoki cypress (species ID 9 and 11; Fig. 3c).

The molar C:N cell quota ratios of \emph{Scenedesmus} varied from 6.6:1
to 21:1, and was less than 12:1 except for Japanese maple, oak, Japanese
hemlock, Japanese red pine, and Japanese larch (species ID 1, 4, 7, 9,
10 in Fig. 4a). The molar C:P ratios of the algae varied from 106:1 to
300:1, except for cells in grown in the culture medium prepared from the
leaf litter leachate of Japanese maple (species ID 1), which reached C:P
= 600:1 (Fig. 4b). The molar N:P ratio of the algae was distributed
between 10:1 and 30:1, and was lower than 16:1 in the leaf litter
leachates of Siebold's beech, Japanese elm, Japanese red pine, and
hinoki cypress (species ID 5, 6, 9, 11 in Fig. 4c).

\textbf{Discussion}

The effect of leaf litter leachates on the growth, production, and cell
stoichiometry of the green alga \emph{Scenedesmus} varied significantly
among the leaf litter leachates of different tree species. As nutrient
concentrations were not the same among the culture media, growth rates
in different types of leachates could not be directly compared with each
other. Therefore, we plotted the C biomass accumulated during the 7-day
incubation period against TDN and TDP in the leachate. The result showed
that the accumulated C biomass increased with increasing TDN in the
leachate regardless of the micronutrient amendment, although the
increasing trend was attenuated at higher TDN concentrations. In
addition, C biomass was highly limited in the leachates of Siebold's
beech, Japanese elm and Japanese hemlock regardless of high TDP
concentrations (species ID 5, 6, 7 in Fig. 2g and h). These results
suggest that the variation in C biomass among the different culture
media from leaf litter leachate was mainly caused by TDN concentration.
In a previous study, Ho et al. (2023) suggested that nitrogen is the
most deficient element in the leaf litter leachates relative to
phytoplankton demand, because the percentage of release relative to leaf
litter elemental content was lowest for TDN compared to DOC and TDP in
all 11 tree species studied. The present study confirmed the validity of
this suggestion for phytoplankton production.

However, the algal growth rate, estimated from temporal changes in cell
abundance, differed from that of C biomass. In the present study, it
ranged from 0.2 to 0.3 d\textsuperscript{-1}in for most of the leaf
litter leachates, except for Japanese elm and Japanese hemlock, where
the growth rate was \textless{}0.2 d\textsuperscript{-1}. The result
suggests that the nutrient conditions in the present study were moderate
according to the growth rate of \emph{Scenedesmus} under different
culture conditions in previous studies . However, the growth rate was
not closely related to either TDN or TDP in leaf litter leachate,
especially when micronutrients were not amended. The result suggests
that the factors regulating growth rate, i.e., cell division rate, and
net C biomass accumulation rate, i.e., organic carbon production rate,
are different in this algal species.

It should be noted that when micronutrients were added, the algal growth
rate significantly increased in the leaf litter leachates of oak,
Japanese elm and Japanese hemlock, although the opposite trend was found
in the leachate of hinoki cypress. The results contrasted with the
response of C biomass, which showed a significant difference between the
culture medium with and without micronutrient amendment only in Japanese
hemlock. These results suggest that the fitness of the present algal
species in the terms of cell division rate is more sensitive to
micronutrient supply than the net production rate. In fact, the algal
growth rate was significantly correlated with TDP but not TDN when
micronutrients were additionally supplied. This fact implies that the
variation of algal growth rate among different types of leaf litter
leachates was mainly regulated by P supply in concert with
micronutrients, whereas that of net production rate was more regulated
by N supply. The relative importance of P rather than N may be related
to the fact that P is an essential element of DNA and RNA and can
strongly limit cell division .

Micronutrients are known to control the enzyme activity that regulates
the N and P use efficiency of phytoplankton . Indeed, in leaf litter
leachates from Japanese red pine and hinoki cypress, a significant
increase in N and P cell quotas was found when micronutrients were
additionally supplied. In these types of leaf litter leachates,
micronutrients may play a role in P and N use efficiency. In addition,
micronutrients appear to play different roles in algal growth and C
biomass accumulation. Several studies have shown that the factors
affecting algal cell division rates are different from those affecting
the photosynthetic production rates . For example, Li et al. showed that
in the dinoflagellate \selectlanguage{english}\emph{Amphidinium carterae} , the cell division
cycle was halted but the photosynthesis continually accumulated C in
cells under P deficiency. On the contrary, Negi et al. found that in the
green alga \selectlanguage{english}\emph{Chlorella sorokiniana} , N deficiency did not change
the cell density, but decreased the photosynthetic rate.

As mentioned above, the addition of micronutrients tended to reduce the
growth rate of \emph{Scenedesmus} incubated in a culture medium from the
leaf litter leachate of hinoki cypress. The result suggests that the
leaf litter leachate of hinoki cypress may contain excessive amounts of
some micronutrients that are harmful to \emph{Scenedesmus} . Rueter et
al. showed that Al and Cu tended to reduce the growth rate
of\emph{Scenedesmus} when supplied in excess. Because we added a mixture
of micronutrients, we could not determine which micronutrients and trace
elements were harmful when supplied in excess. However, the present
study indicates that the water-soluble content of micronutrients and
trace elements varies widely among the leaf litter of different tree
species as suggested by Pourhassan et al. .

Studies have shown that decomposition and nutrient release rates
somewhat differ between coniferous and broadleaf tree species . However,
we could not determine whether leaf litter leachates from broadleaf or
coniferous trees had a better nutrient composition for the growth rate
of \emph{Scenedesmus} due to the large variation of growth rate within
each of these two groups. In addition, we could not find significant
difference in the C biomass production rate of this green alga between
the culture media from broadleaf and coniferous leaf litter leachates
due to the large variations in the rate within these tree species. These
results suggest that the effect of leaf litter on aquatic organisms
cannot be generalized across broad taxonomic tree groups.

Similar to the growth and C biomass production rates, leaf litter
leachates from different tree species had different effects on the C:N:P
ratio of the algae. It is well known that the elemental contents of
algae are important determinants of the quality of food for herbivorous
zooplankton . In this study, the molar C:N and C:P ratios
of\emph{Scenedesmus} varied from 6.6 to 21 and 106 to 600, respectively,
depending on the leaf litter leachate. In general, algal cells with C:N
and C:P ratios close to Redfield ratios are good quality food for
zooplankton growth . In the leaf litter leachates of Siebold's beech,
Japanese elm, Japanese red pine and hinoki cypress, the molar C:N and
C:P ratios ranged from 6.6 to 12 and 106 to 160, respectively, which are
close to the Redfield ratios. Thus, algae grown in lentic waters
receiving nutrients from leaf litter of these species would be suitable
food for herbivorous zooplankton. However, in the leaf litter leachates
from Japanese maple and Japanese cedar, the C:P ratio
of\emph{Scenedesmus} cells was greater than the threshold elemental
ratio (\textgreater{}300) above which the growth rate of zooplankton
such as\emph{Daphnia} is limited by phosphorus . Urabe and Watanabe also
suggest that algal cells with C:N ratios greater than 15 are suboptimal
food for zooplankton. In this study, the C:N ratio of
\emph{Scenedesmus}cells in the leaf litter leachates of Japanese maple
and Japanese hemlock was greater than this value. In addition, depending
on the leaf litter leachate of different tree species, algae may alter
the cellular content of other chemical substances, such as fatty acids
and amino acids, that affect food quality for herbivores. Together with
such a possibility, the present results suggest that the mass transfer
from algae to herbivorous zooplankton in a lake may be influenced by the
vegetation composition in the watershed via subsidies of nutrient inputs
from leaf litter.

\textbf{Conclusion}

The present study showed that algal growth rate, biomass production, and
elemental contents varied depending on the leaf litter leachates from
different tree species, due not only to differences in N and P
concentrations but also to those of micronutrients. These results
suggest that the stoichiometry and composition of leaf litter leachate
can cause substantial changes not only in algal fitness and primary
production, but also in mass transfer from algae to herbivores. Although
we conducted this study using only a single algal species, the responses
to the changes in leaf litter leachate may differ between algal species
and other heterotrophic organisms that utilize organic matter in leaf
litter leachate. This possibility is worth investigating in future
studies, as the effects of the change and succession in the surrounding
vegetation may have extended to the composition of the entire community
in the freshwater ecosystems. In this study, we investigated the
short-term and direct effects of leaf litter using leaf litter leachate
for 7 days of incubation. In nature, the effects of leaf litter on
aquatic ecosystems may last longer through decomposition in soils and
sediments. Therefore, to fully understand the effects of vegetation
changes on the aquatic community, long-term monitoring studies are
needed in aquatic ecosystems where the catchment vegetation has been
changed.

\textbf{References}

\textbf{Table 1.} Stoichiometry of the leaf litter used in the present
study. The standard deviation of the elemental contents from multiple
measurements is given in parentheses. Release efficiencies of dissolved
C, N, and P after 7 days of leaching are from Ho et al..\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllllll@{}}
\toprule
& \textbf{Tree species} & \textbf{Tree species} & \textbf{Tree species}
& \textbf{Tree species} & \textbf{Sampling site} & \textbf{Sampling
site} & \textbf{Sampling site} & \textbf{Leaf litter stoichiometry} &
\textbf{Leaf litter stoichiometry} & \textbf{Leaf litter stoichiometry}
& & &\tabularnewline
\midrule
\endhead
& Common name & Species name & Tree type & Leaf type & Area & Latitude &
Longitude & C (\selectlanguage{greek}µ\selectlanguage{english}g/mg) & N (\selectlanguage{greek}µ\selectlanguage{english}g/mg) & P (\selectlanguage{greek}µ\selectlanguage{english}g/mg) & C release \% & N release
\% & P release \%\tabularnewline
1 & Japanese maple & \emph{Acer amoenum} var.amoenum & Broadleaf &
Deciduous & Aobayama & N38.259 & E140.837 & 403.40 (8.07) & 9.56 (1.10)
& 0.29 (0.046) & 24.30 & 6.28 & 48.40\tabularnewline
2 & Zelkova & \emph{Zelkova serrata} & Broadleaf & Deciduous & Aobayama
& N38.259 & E140.837 & 389.16 (3.82) & 13.18 (2.07) & 0.44 (0.029) &
19.61 & 2.24 & 37.11\tabularnewline
3 & Erman's birch & \emph{Betula ermanii} & Broadleaf & Deciduous & Zao
& N38.129 & E140.463 & 489.34 (3.79) & 29.55 (1.48) & 0.73 (0.026) &
3.66 & 0.39 & 18.09\tabularnewline
4 & Oak & \emph{Quercus serrata} & Broadleaf & Deciduous & Zao & N38.122
& E140.451 & 458.88 (8.63) & 19.10 (1.59) & 1.11 (0.19) & 16.41 & 1.07 &
29.97\tabularnewline
5 & Siebold's beech & Fagus crenata & Broadleaf & Deciduous & Kawatabi &
N38.745 & E140.757 & 538.27 & 16.27 & 6.02 (0.60) & 18.76 & 3.23 &
34.17\tabularnewline
6 & Japanese elm & \emph{Ulmus davidiana} var. japonica & Broadleaf &
Deciduous & Kawatabi & N38.745 & E140.757 & 401.98 (5.08) & 20.38 (0.71)
& 9.79 (2.96) & 36.13 & 4.99 & 52.43\tabularnewline
7 & Japanese hemlock & \emph{Tsuga diversifolia} & Coniferous &
Evergreen & Zao & N38.122 & E140.451 & 500.32 (3.58) & 16.02 (1.36) &
0.78 (0.13) & 13.23 & 0.90 & 21.83\tabularnewline
8 & Japanese cedar & \emph{Cryptomeria japonica} & Coniferous &
Evergreen & Kawatabi & N38.745 & E140.757 & 489.19 (7.47) & 13.67 (0.52)
& 1.12 (0.42) & 14.83 & 1.01 & 22.50\tabularnewline
9 & Japanese Red pine & \emph{Pinus densiflora} & Coniferous & Evergreen
& Kawatabi & N38.745 & E140.757 & 498.30 (6.56) & 19.69 (3.09) & 1.26
(0.09) & 12.88 & 0.58 & 47.51\tabularnewline
10 & Larch & \emph{Larix leptolepis} & Coniferous & Deciduous & Kawatabi
& N38.745 & E140.757 & 453.12 (7.34) & 23.50 (1.87) & 1.52 (0.29) &
14.40 & 3.07 & 19.47\tabularnewline
11 & Hinoki cypress & \emph{Chamaecyparis obtusa} & Coniferous &
Evergreen & Kawatabi & N38.745 & E140.757 & 488.57 (2.12) & 18.68 (1.65)
& 1.90 (0.31) & 13.32 & 2.14 & 26.12\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1.} \emph{G} rowth rate (a), and C biomass (b) of algae
incubated in the leaf litter leachates of 11 tree species. White boxes
are incubations without micronutrient amendment (control treatment) and
gray boxes are incubation with micronutrient amendment (amended
treatment). Boxes are arranged according to the P content in leaf litter
biomass from low to high within broadleaf and coniferous tree groups as
in Table 1. Asterisks indicate that there is a significant difference
between control and amended treatments (\emph{p} \textless{} 0.05).
Different letters indicate a significant difference among control
treatments of different leaf litter leachates.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 2.} Algal growth rate (a-d) and accumulated C biomass
(e-g) plotted against total dissolved N (TDN) and total dissolved P
(TDP) concentrations in amended (a, c, e, and g) and control treatments
(b, d, f, and h). The significant segmented linear regression is shown
as solid lines. The numbers in the dots represent the tree species ID
shown in Table 1.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 3.} C (a), N (b), and P (c) cell quotas of algae
incubated in culture media prepared from leaf litter leachates of 11
tree species. White boxes are control incubations and gray boxes are
amended incubations. Boxes are arranged according to the P mass content
in leaf litter from low to high within broadleaf and coniferous groups
as in Table 1. Asterisks indicate that there is a significant difference
between control and amended incubations (\emph{p} \textless{} 0.05).
Different letters indicate a significant difference between control
treatments of different leaf litter leachates.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

\textbf{Figure 4.} Scatter plots of C and N ratios (a), C and P ratios
(b), and N and P ratios (c) of the algal cells. The molar C:N = 6.6:1,
12:1, 15:1 ratios (a), molar C:P = 106:1, 160:1, 300:1, and 600:1 ratios
(b), molar N:P = 10:1, 16:1, and 30:1 ratios (c) are shown as solid
lines. White dots represent control treatments and gray dots represent
amended treatments. The numbers in the dots represent the tree species
ID shown in Table 1.

\selectlanguage{english}
\FloatBarrier
\end{document}

