bs_bs_banner

Plant Species Biology (2012) 27, 233–240 doi: 10.1111/j.1442-1984.2011.00345.x

NOTES AND COMMENTS

Morphological and anatomical analyses of rheophytic

Rhododendron ripense Makino (Ericaceae) psbi_345 233..240

RYOSUKE UEDA,* YUKIO MINAMIYA,† AYA HIRATA,‡ HIROSHI HAYAKAWA,*† YUKO MURAMATSU,‡
MICHIHIRO SAITO* and TATSUYA FUKUDA*
*Faculty of Agriculture, Kochi University, Monobe 200, Nankoku, Kochi 783-8502, Japan, †United Graduate School of
Agricultural Sciences, Ehime University, Kochi University, Monobe 200, Nankoku, Kochi 783-8502, Japan, and ‡Graduate School
of Integrated Arts and Sciences, Kochi University, Monobe 200, Nankoku, Kochi 783-8502, Japan

Abstract
The comparative morphology and anatomy of the leaves of the rheophytic Rhododendron
ripense and the closely related inland species Rhododendron macrosepalum were exam-
ined. The leaf of R. ripense is thinner than that of R. macrosepalum, with leaf length to
width ratios (leaf index) of 2.92 and 1.91, respectively. Moreover, the leaf of R. ripense
consists of fewer cells than the leaf of R. macrosepalum, suggesting stenophyllization of
R. ripense caused by the decreased number of cells. In addition, leaf thickness and the
number of stomata per leaf of R. ripense were significantly greater than those of R. mac-
rosepalum, but the density of the short glandular pilose hairs on the leaf of R. ripense was
lower. The observed morphological differences between the two species may be
explained by certain aspects of the riparian environment, such as high irradiation and
frequent flooding after heavy rainfall, to which R. ripense is exposed.
Keywords: anatomy, leaf, morphology, rheophyte, Rhododendron ripense.
Received 31 May 2010; revision received 18 February 2011; accepted 16 May 2011

Introduction the narrow leaves in rheophytes. For example, Osmunda

lancea Thunb., a rheophytic species of Osmundaeae, has a
Plants adapt to unfavorable environments by acclimatiz-
smaller cell width along the leaf median-lateral axis than
ing to surrounding environmental factors, such as tem-
its dryland relative Osmunda japonica Thunb. (Imaichi &
perature, soil, nutrients and light. Plants can even invade
Kato 1992). In angiosperms, a decreased cell number
the area along rivers or streams, where they are easily
along the leaf width contributes to the narrow form in
disturbed by fluctuating water levels and high irra-
the rheophyte Farfugium japonicum (L. fil.) Kitam. var.
diation. Such plants are called rheophytes and they
luchuense (Masam.) Kitam. (Compositae) (Usukura et al.
have many anatomical and morphological characteri-
1994). A decreased number of whole leaf cells could also
stics suitable for extreme environments compared with
trigger stenophyllization, as reported in the speciation of
closely related species (van Steenis 1981). Rheophytes
rheophytic Rhododendron indicum (L.) Sweet f. otakumi T.
include more than 1000 species from 60 families
Yamaz. (Ericaceae) from R. indicum f. indicum (Setoguchi
from bryophytes to angiosperms (van Steenis 1981; Kato
& Kajimura 2004). Tsukaya (2002) indicated that variation
1999).
in leaf width in the rheophyte Dendranthema yoshinagan-
A number of comparative studies of the leaves of
thum (Makino ex Kitam.) Kitam. (Compositae) involves
rheophytes and related species have been conducted to
both the size and the number of leaf cells.
understand morphological modifications accomplished
Rhododendron ripense Makino (Ericaceae) is a semi-
by rheophytes. These studies indicate that the size and the
evergreen shrub endemic to Japan and is distributed
number of leaf cells could contribute to the evolution of
in western Honshu, Shikoku and northeastern Kyushu
(Fig. 1a). This species is a putative obligate rheophyte and
Correspondence: Tatsuya Fukuda is considered to be a rheophytic land plant because it only
Email: tfukuda@kochi-u.ac.jp grows on exposed rocks at river edges, where it is subject

© 2012 The Society for the Study of Species Biology

234 R. UEDA ET AL.

Fig. 1 Flowers and leaves of (a) Rhododendron macrosepalum and (b) Rhododendron ripense. Bar = 1 cm. (c) Diagram of the leaf measure-
ments. 1, angle of the leaf base; 2, leaf length; 3, leaf width. (d) Measured parts of the leaf for epidermal cells. I, central part; II, proximal
part; III, distal part; IV, marginal part. The short glandular pilose hairs of (e) R. ripense and (f) R. macrosepalum. Bar = 0.5 mm.

to flooding (Yamanaka & Takezaki 1959), and has narrow
lanceolate leaves. Rhododendron ripense belongs to the
series Scabra of this genus with five other species: Rhodo-
dendron boninense Nakai, Rhododendron amanoi Ohwi,
Rhododendron scabrum G. Don, Rhododendron macrosepalum
Maxim. and Rhododendron yedoense Maxim. ex Regel
(Yamazaki 1993). Of these, R. macrosepalum is a closely
related species to R. ripense, even though it grows in a
different environment (Yamazaki 1989). Moreover, the
morphology of R. ripense is similar to R. macrosepalum
(Fig. 1b); for example, the branchlets and petioles have
spreading short glandular pilose hairs mixed with
ascending glandular hairs (Fig. 1c,d) (Yamazaki 1993).
However, a comparative morphological and anatomical
analysis of R. ripense and R. macrosepalum has not yet been
conducted. In the present study, we compared the leaf
anatomies and morphologies of R. ripense and R. macrose-
palum to determine how stenophyllization is achieved in
the former species. In particular, we focused on the size
and number of leaf cells. We then compared our results to
those obtained from other species to further illustrate the
stenophyllization process in rheophytes.
Materials and methods
Plant materials
The distribution of R. ripense and R. macrosepalum partly
overlaps in eastern Kochi Prefecture, but they are not
usually distributed close together in other areas. There-
fore, we found a few localities where both species grow in
close proximity, but we collected leaves from all over the
prefecture to exclude locality-specific characteristics
(Table 1; Fig. 2). Leaves were collected from 55 individuals
of 16 populations for R. ripense and from 51 individuals of
16 populations for R. macrosepalum.

© 2012 The Society for the Study of Species Biology Plant Species Biology 27, 233–240
 L E A F A N AT O M Y O F R H O D O D E N D R O N R I P E N S E 235

Table 1 List of samples and localities used in the present study

Species No. Locality Latitude and Longitude

Rhododendron 1 Ehime Prefecture Minamiuwa-Gun, Ainan-Cho, Souzu River N 33 0′31″; E 132 36′24″
ripense 2 Kochi Prefecture Sukumo City, Matsuda Town, Matsuda River N 33 3′11″; E 132 41′39″
3 Kochi Prefecture Agawa-Gun, Niyodogawa-Cho, Tyouja-Hei, Tyouja River N 33 29′19″; E 133 7′30″
4 Kochi Prefecture Agawa-Gun, Niyodogawa-Cho, Takenotani, Doi River N 33 37′17″; E 133 10′12″
5 Kochi Prefecture Agawa-Gun, Ino-Cho, Nagasawa, Yoshino River N 33 43′31″; E 133 18′4″
6 Kochi Prefecture Agawa-Gun, Ino-Cho, Oomorigawa River N 33 42′34″; E 133 18′47″
7 Kochi Prefecture Agawa-Gun, Ino-Cho, Kamiyakawa, Edagawa River N 33 39′4″; E 133 20′31″
8 Kochi Prefecture Tosa-Gun, Ookawa Villege, Inokawa, Yoshino River N 33 47′34″; E 133 23′53″
9 Kochi Prefecture Nagaoka-Gun, Ootoyo-Cho, Hiura, Yoshino River N 33 46′4″; E 133 41′26″
10 Kochi Prefecture Aki City, Ioki, Aki Rievr N 33 35′10″; E 133 58′58″
11 Kochi Prefecture Aki-Gun, Kitagawa Villege, Shima, Nahari River N 33 31′46″; E 134 7′55″
12 Kochi Prefecture Aki City, Hatakeyama, Aki River N 33 36′47″; E 133 54′53″
13 Kochi Prefecture Aki-Gun, Kitagawa Villege, Kannoue, Ogawa River N 33 33′19″; E 134 10′54″
14 Tokushima Prefecture Miyoshi City, Nishiiyayama Villege, Tuchihiura, Yoshino River N 33 51′30″; E 133 47′2″
15 Tokushima Prefecture Miyoshi City, Ikeda-Cho, Kawasaki, Yoshino River N 33 56′21″; E 133 45′43″
16 Tokushima Prefecture Naka-Gun, Naka-Cho, Kaikawa, Kaigawatani River N 33 44′47″; E 134 16′12″
Rhododendron 17 Kochi Prefecture Konan City, Noichi Town, Ohtani N 33 33′54″; E 133 42′30″
macrosepalum 18 Kochi Prefecture Konan City, Noichi Town, Higashisako N 33 35′39″; E 133 43′36″
19 Kochi Prefecture Kami City, Tosayamada Town, Sakagawa N 33 36′13″; E 133 44′38″
20 Kochi Prefecture Kami City, Kahoku Town, Birafu N 33 38′36″; E 133 46′47″
21 Kochi Prefecture Konan City, Yasu Town, Kamiyasu N 33 32′57″; E 133 46′36″
22 Kochi Prefecture Aki City, Ogawa N 33 34′14″; E 133 53′55″
23 Kochi Prefecture Aki City, Hatakeyama N 33 37′16″; E 133 55′9″
24 Kochi Prefecture Kami City, Monobe Town, Tonjo N 33 40′47″; E 133 52′22″
25 Kochi Prefecture Kami City, Kahoku Town, Inono N 33 42′33″; E 133 50′15″
26 Kochi Prefecture Aki-Gun, Yasuda Town, Higashishima N 33 27′23″; E 133 59′31″
27 Kochi Prefecture Aki-Gun, Yasuda Town, Ogawa N 33 30′52″; E 134 1′6″
28 Kochi Prefecture Aki-Gun, Kitagawa Villege, Kashiwagi N 33 27′59″; E 134 3′34″
29 Kochi Prefecture Aki-Gun, Kitagawa Villegem Wada N 33 28′11″; E 134 4′43″
30 Kochi Prefecture Aki-Gun, Umaji Villege, Umaji N 33 33′59″; E 134 4′26″
31 Kochi Prefecture Aki-Gun, Kitagawa Villege, Kuki N 33 33′49″; E 134 6′53″
32 Tokushima Prefecture Naka-Gun, Naka-Cho, Kaikawa N 33 45′43″; E 134 15′11″

Morphological and anatomical analyses leaves were cut in a horizontal direction at 8 mm length
using a rotary microtome and the size of the mesophyll
Laminar length and width of fully expanded leaves were
cells was measured at the widest part of the lamina under
measured for each individual using a digital caliper. The
a light microscope.
angle of the leaf base was also measured.
Statistical analyses were carried out using a t-test to
Fully expanded leaves collected from each individual
compare the characteristics of two species and a one-way
were fixed in FAA (50% ethanol–formaldehyde–acetic
anova for cell size at four sites on a leaf.
acid at 18:1:1 v/v) overnight. Cell numbers on the leaf
surfaces were conducted based on the peeled cuticles,
which were prepared by Suzuki’s Universal Micro-
Printing (SUMP) method. The number of cell prints along Results
the midrib was counted and the size measured after clas-
Morphological measurements of R. ripense and
sifying them into epidermal and stomatal cells. The
R. macrosepalum
number of stomata was also counted. A replica (1 cm2) was
made of each leaf to measure the density and size of Rhododendron ripense generally has a shorter leaf length
glandular hairs (Fig. 3). Each SUMP image was analyzed than R. macrosepalum (35.00 ⫾ 8.32 vs 55.29 ⫾ 11.97 mm)
10 times for each trait using a light microscope. (Table 2), but the size difference is more conspicuous
For measurement of epidermal and mesophyll tissue in the leaf width (9.57 ⫾ 2.25 in R. ripense and
thickness, leaves were dehydrated in ethanol and tertiary 22.57 ⫾ 5.49 mm in R. macrosepalum). Therefore, the mean
butyl alcohol series and embedded in histparaffin. The size is estimated to be 1.75 ⫾ 0.79 cm2 for R. ripense and

Plant Species Biology 27, 233–240 © 2012 The Society for the Study of Species Biology
236 R. UEDA ET AL.
Kochi Pref.
Shikoku
Fig. 2 Sampling localities used in the present study. See Table 1 for the names of the numbered sites. Black circles indicate sampling
localities of Rhododendron ripense. White circles indicate sampling localities of Rhododendron macrosepalum.
6.53 ⫾ 3.00 cm2 for R. macrosepalum when the leaf length
is multiplied by leaf width. These traits were significantly
different between the two species (P < 0.01). The leaf
thickness of R. ripense and R. macrosepalum was 135 ⫾ 9.58
and 101 ⫾ 15.0 mm, respectively. We also calculated the
leaf index value as the ratio of leaf length to leaf width, as
specified by Tsukaya (2002). Data plots show significant
variation (P < 0.01) in the leaf index (2.92 in R. ripense and
1.91 in R. macrosepalum). The average angle of the leaf base
was 41 ⫾ 10° for R. ripense and 78 ⫾ 17° for R. macrosepa-
lum. In most cases the values for R. ripense were less
than 80°.
Epidermal cells of R. ripense and R. macrosepalum
We confirmed that there were no significant differences
in the epidermal cell size between R. ripense and R. mac-
rosepalum at four sites on a leaf (Table 2). In addition, we
could not find any significant differences in the epider-
© 2012 The Society for the Study of Species Biology
Japan
mal cell size at the four sites on the leaf within a species,
even if the width of the epidermal cell in the central part
was wider than the other parts of the leaf. Therefore, the
size and number of epidermal cells was calculated using
the central part of the leaf, excluding the horizontal epi-
dermal cell numbers, which were used to measure the
width of the epidermal cells in the central (Min.) and
other parts (Max.) of the leaf. Epidermal cell size was
not significantly different between the two species at
23.5 ⫾ 3.6 ¥ 21.7 ⫾ 2.6 mm (length ¥ width) in R. ripense
and 22.9 ⫾ 3.2 ¥ 22.3 ⫾ 2.4 mm in R. macrosepalum. The
mean epidermal cell size was calculated using the length
and width of a cell measured from the SUMP samples of
R. ripense and R. macrosepalum from all localities exam-
ined. The mean cell size was 503 ⫾ 111 mm2 for R. ripense
and 503 ⫾ 94.8 mm2 for R. macrosepalum; this difference is
not significant.
The epidermal cell number was estimated to be
approximately 406 000 ⫾ 154 000 for R. ripense and
Plant Species Biology 27, 233–240
 L E A F A N AT O M Y O F R H O D O D E N D R O N R I P E N S E
Fig. 3 Suzuki’s Universal Micro-Printing method (SUMP) replicas of (a) Rhododendron ripense and (b) Rhododendron macrosepalum.
Bar = 25 mm.
approximately 1 540 000 ⫾ 582 000 for R. macrosepalum by
dividing the leaf dimension by the mean cell dimension
(Table 2). The total cell number is significantly lower in
R. ripense. Based on the longitudinal and horizontal direc-
tion of the leaf and cell, we calculated the estimated lon-
gitudinal and horizontal epidermal cell numbers of a leaf
to be approximately 1620 ⫾ 330 and 462–490, respectively,
for R. ripense, and 2670 ⫾ 595 and 1080–1120, respectively,
for R. macrosepalum. We also found significant differences
in the mean length and density (No./mm2) of the short
glandular pilose hairs between the two species (R. ripense:
0.86 ⫾ 0.26 mm and 2 662 ⫾ 735; R. macrosepalum:
0.66 ⫾ 0.29 mm and 6 332 ⫾ 1 887). Guard cell size
was found to be 645 ⫾ 114 mm2 for R. ripense and
556 ⫾ 148 mm2 for R. macrosepalum, and the difference
between the cells was not significant. However, stomatal
density (333.5 ⫾ 60.12) of R. ripense was significantly
higher than that of R. macrosepalum (226.3 ⫾ 47.41).
The abaxial epidermis looked thinner in R. macrosepa-
lum than in R. ripense (Table 2). In fact, the mean thickness
of the abaxial epidermal cells is significantly different
(19.7 ⫾ 3.57 mm for R. ripense and 12.3 ⫾ 1.63 mm for
R. macrosepalum). However, the difference in adaxial epi-
dermal cells was not so different (R. ripense: 11.0. ⫾
1.28 mm; R. macrosepalum: 12.0 ⫾ 1.84 mm). Although we
found significant differences in the thickness of the
palisade cells between R. ripense and R. macrosepalum
(43.2 ⫾ 7.07 and 37.1 ⫾ 7.59 mm, respectively), the thick-
Plant Species Biology 27, 233–240
237
ness of the spongy tissues was not significantly diffe-
rent (65.9 ⫾ 8.0 for R. ripense and 46.5 ⫾ 12.0 mm for
R. macrosepalum).
Discussion
Stenophyllization of R. ripense leaves
Previous studies have shown similarities in the leaf mor-
phology and anatomy of rheophytes and closely related
species (e.g. Imaichi & Kato 1997). Speciation of a rheo-
phyte from an inland species appears to be associated
with stenophyllization, and van Steenis (1981) noted that
rheophytes are found in various taxa from bryophytes to
angiosperms. In the present study, we found that R.
ripense has a significantly narrower leaf in comparison
with R. macrosepalum, indicating that R. ripense is a rheo-
phytic species. Moreover, our results indicate that a
decrease in leaf cell number contributed to stenophylliza-
tion of R. ripense, which is similar to previous anatomical
studies of R. indicum f. otakumi (Setoguchi & Kajimura
2004). Rhododendron indicum f. otakumi and R. ripense
belong to the same genus, implying that rheophytic taxa
in this genus originated independently with the same
morphological process. Multiple origins of rheophytic
species may be caused by a small number of mutations
because this evolution has occurred rapidly and indepen-
dently in different lineages. Studies of how genes control
© 2012 The Society for the Study of Species Biology
238 R. UEDA ET AL.

Table 2 Morphological and anatomical

Rhododendron Rhododendron characteristics of Rhododendron ripense and
Trait ripense macrosepalum Significance Rhododendron macrosepalum
Morphological characteristics
Leaf length (mm) 35 ⫾ 8.3 55 ⫾ 12 **
Leaf width (mm) 9.6 ⫾ 2.3 23 ⫾ 5.5 **
Thickness of the leaf (mm) 135 ⫾ 9.58 101 ⫾ 15.0 **
Leaf size (cm2) 1.75 ⫾ 0.79 6.53 ⫾ 3.00 **
Anatomical characteristics
Angle of the leaf base (°) 41 ⫾ 10 78 ⫾ 17 **
Epidermal cell length (mm)
I. Central 23.5 ⫾ 3.66 a 22.5 ⫾ 3.15 a n.s.
II. Proximal 24.5 ⫾ 2.35 a 24.3 ⫾ 1.18 a n.s.
III. Distal 24.3 ⫾ 2.00 a 23.6 ⫾ 1.14 a n.s.
IV. Marginal 23.6 ⫾ 2.22 a 24.0 ⫾ 1.36 a n.s.
Epidermal cell width (mm)
I. Central 21.7 ⫾ 2.58 a 22.3 ⫾ 2.43 a n.s.
II. Proximal 20.0 ⫾ 1.90 b 20.4 ⫾ 1.66 b n.s.
III. Distal 20.8 ⫾ 2.56 b 20.8 ⫾ 1.21 b n.s.
IV. Marginal 19.9 ⫾ 1.74 b 20.2 ⫾ 1.55 b n.s.
Epidermal cell size (mm2)
I. Central 503 ⫾ 111 a 503 ⫾ 94.8 a n.s.
II. Proximal 496 ⫾ 46.4 a 491 ⫾ 73.7 a n.s.
III. Distal 493 ⫾ 36.3 a 505 ⫾ 75.8 a n.s.
IV. Marginal 486 ⫾ 57.6 a 472 ⫾ 72.8 a n.s.
Epidermal cell numbers 406 000 ⫾ 154 000 1 540 000 ⫾ 582 000 **
Longitudinal epidermal cell 1620 ⫾ 330 2670 ⫾ 595 **
numbers
Horizontal epidermal cell 462–490 1080–1120 **
numbers
Guard cell size (mm2) 645 ⫾ 114 556 ⫾ 148 n.s.
Stomatal density (N/mm2) 333.5 ⫾ 60.12 226.3 ⫾ 47.41 **
Epidermal cell thickness on the 19.7 ⫾ 3.57 12.3 ⫾ 1.63 **
abaxial side (mm)
Epidermal cell thickness on the 11.0 ⫾ 1.28 12.0 ⫾ 1.84 n.s.
adaxial side (mm)
Palisade cell thickness (mm) 43.2 ⫾ 7.07 37.1 ⫾ 7.59 n.s.
Spongy tissue thickness (mm) 65.9 ⫾ 8.0 46.5 ⫾ 12.0 **
Short glandular pilose hair 0.86 ⫾ 0.26 0.66 ⫾ 0.29 **
length (mm)
Short glandular pilose hair 2662 ⫾ 735 6322 ⫾ 1887 **
density (N/mm)

*P < 0.05; **P < 0.01; n.s., P > 0.05 (t-test). Values are mean ⫾ standard deviation. Values
followed by the same lowercase letter within each trait are not significantly different at the
5% level, determined by one-way anova.

morphological development can potentially help clarify
how the evolution of genes and genetic systems can result
in morphological differentiation. A crucial link between
genetic studies in model systems and the evolution of
morphological diversity lies in the study of comparative
developmental morphology (Tsukaya 1995). It is difficult
to specify the genetic basis of morphological traits when
there is a presumption that a morphological difference
between species reflects a large number of genetic
changes and that such differences evolve through the
accumulation of small mutations over the course of plant
diversification. However, morphological changes may
arise from a relatively small number of genes (Hilu
1983; Gottlieb 1984). For instance, narrow-leaf or leaflet
mutants, which resemble rheophytic leaves, are con-
trolled by single genes (Bassett 1981; Jaynes 1981). Recent
studies in a model plant, Arabidopsis thaliana, indicate that
leaf expansion is governed by some genetic pathways,
such as angustifolia, rotundifolia 3 and aintegumenta genes
(Tsuge et al. 1996; Mizukami & Fischer 2000; Tsukaya &
Machida 2000; Tsukaya 2000, 2010). Future evolutionary
developmental studies using these species will be of

© 2012 The Society for the Study of Species Biology Plant Species Biology 27, 233–240
 L E A F A N AT O M Y O F R H O D O D E N D R O N R I P E N S E
interest. Moreover, van Steenis (1981) noted that R. anna-
mense Rehder is another rheophytic species in this genus.
An analysis of the leaf of this species to determine if it has
followed the same process as closely related species is
needed.
Morphological and anatomical comparison of leaves
between R. ripense and R. macrosepalum
Our morphological data for R. ripense indicate that the
angle of the leaf base strongly correlates with the length
and the width of the leaf (Table 2). The decreasing angle of
the leaf base leads to a lanceolate leaf and, therefore, our
result indicates that stenophyllization of the leaves in
R. ripense occurred with the transition from an ovate to a
lanceolate shape. The general tendency of plants growing
close to streams to have leaves that are more lanceolate
than plants more distant is perhaps a result of habitat
selection pressure, with a gradual decrease in flooding
frequency from the streambed to the inland habitat
(Imaichi & Kato 1997). Lanceolate leaves would provide
R. ripense with a greater tolerance of swift-running
streams.
We found the leaf of R. ripense to be significantly thicker
than the leaf of R. macrosepalum, and this reflects the
increased size of the spongy tissue. In general, leaf thick-
ness and stomatal number correlate positively with mean
solar radiation during leaf expansion (e.g. Niinemets
1999, 2001). Rhododendron ripense grows mainly in river-
side habitats, on sunny moist rocks, and on riverbanks
and appears to prefer bright conditions, whereas R. mac-
rosepalum occurs along the forest margin. Therefore, the
high irradiance of riverside habitats may lead to increased
leaf thickness and stomatal density in R. ripense. Individu-
als of the rheophyte Farfugium japonicum var. luchuense
also have increased leaf thickness compared with inland
individuals of this species (Nomura et al. 2006). This sug-
gests that these characteristics represent a general ten-
dency in rheophytic leaf morphology because R. ripense
and F. japonicum var. luchuense have differentiated simi-
larly, despite the large phylogenetic distance between
them. To verify this tendency, further morphological and
anatomical analyses of other rheophytes are needed.
Both R. ripense and R. macrosepalum have short glandu-
lar hairs on the abaxial surface of their leaves, and the
length and density of these hairs differs significantly
between these two species. The arrangement, density, size
and shape of hairs on leaves vary widely across
angiosperms. Hairs on leaves have been implicated in
providing protection against various biotic and abiotic
stress conditions, including insect and pathogen attacks,
extreme temperature, and excessive light (e.g. Van Dam &
Hare 1998). The leaf hair of R. ripense is longer, but the
Plant Species Biology 27, 233–240
239
density is lower than the leaf hair of R. macrosepalum,
suggesting that leaves with dense hairs possibly offer
resistance to flash floods after heavy rains. However, the
function of the leaf hairs of R. ripense is unknown. To our
knowledge, this is the first study to indicate a difference in
leaf hairs between rheophytic and closely related inland
species. Further study is needed to confirm whether this is
a general tendency for rheophytes.
In summary, we analyzed the evolution of the rheophyte
R. ripense using morphological and anatomical data. Our
results identify key morphological and anatomical differ-
ences between two closely related species of Rhododendron,
one of which is specialized to a riparian habitat, and are
consistent with the findings of other studies that examine
trends in rheophyte evolution. Evolutionary developmen-
tal analysis using candidate genes and transgenic plants
will further illuminate the mechanisms by which rheo-
phytes differentiate from closely related species. It will also
reveal how these mechanisms evolved over time.
Acknowledgments
We thank Dr J. Yokoyama, Dr R. Arakawa, Dr J. Tsuka-
moto, Dr T. Ichie and Dr K. Ito for providing help with this
study. We would like to express our gratitude to Dennis
Murphy, who is a Professor of the United Graduate School
of Agricultural Sciences, Ehime University, for checking
our English. This study was partly supported by a
Grant-in-Aid for Scientific Research from the Ministry
of Education, Culture, Sports, Science and Technology of
Japan (to T. Fukuda).
References
Bassett M. J. (1981) Inheritance of a lanceolate leaf mutation in the
common bean. Journal of Heredity 72: 431–432.
Gottlieb L. D. (1984) Genetic and morphological evolution in
plants. American Naturalist 123: 681–709.
Hilu K. W. (1983) The role of single gene mutation in the evolu-
tion of flowering plants. Evolutionary Biology 16: 97–128.
Imaichi R. & Kato M. (1992) Comparative leaf development of
Osmunda lancea and O. japonica (Osmundaceae): hetero-
chronic origin of rheophytic stenophylly. Botanical Magazine,
Tokyo 105: 199–213.
Imaichi R. & Kato M. (1997) Speciation and morphological evo-
lution in rheophytes. In: Iwatsuki K. & Raven P. H. (eds).
Evolution and Diversification of Landplants. Springer-Verlag,
Tokyo, pp. 309–318.
Jaynes R. A. (1981) Inheritance of ornamental traits in mountain
laurel, Kalmia latifolia. Journal of Heredity 72: 245–248.
Kato M. (1999) Evolutionary Morphology of Plants. University of
Tokyo, Tokyo, pp. 131–226.
Mizukami Y. & Fischer R. L. (2000) Plant organ size control:
AINTEGUMENTA regulates growth and cell numbers during
organogenesis. Proceedings of the National Academy of Sciences
of the United States of America 97: 942–947.
© 2012 The Society for the Study of Species Biology
240 R. UEDA ET AL.
Niinemets Ü. (1999) Components of leaf dry mass per
area—thickness and density—alter leaf photosynthetic capac-
ity in reverse directions in woody plants. New Phytologist 144:
35–47.
Niinemets Ü. (2001) Global-scale climatic controls of leaf dry
mass per area, density and thickness in trees and shrubs.
Ecology 82: 453–469.
Nomura N., Setoguchi H. & Takaso T. (2006) Functional conse-
quences of stenophylly for leaf productivity: comparison of
the anatomy and physiology of a rheophyte, Farfugium japoni-
cum var. luchuence, and a related non-rheophyte, F. japonicum
(Asteraceae). Journal of Plant Research 119: 645–656.
Setoguchi H. & Kajimura G. (2004) Leaf morphology of the
rheophyte, Rhododendron indicum f. otakumi (Ericaceae). Acta
Phytotaxonomica et Geobotanica 55: 45–54.
Tsuge T., Tsukaya H. & Uchimiya H. (1996) Two independent and
polarized processes of cell elongation regulate leaf blade
expansion in Arabidopsis thaliana (L.) Heynh. Development 122:
1589–1600.
Tsukaya H. (1995) The genetic control of morphogenesis in Ara-
bidopsis and its relevance to the development of biodiversity.
In: Arai R., Kato M. & Doi Y. (eds). Biodiversity and Evolution.
The National Science Museum Foundation, Tokyo, pp. 253–
265.
Tsukaya H. (2000) Mutation of genes and species diversity. In:
Iwatsuki K. & Kato M. (eds). Biology of Plant Species (The
Botany of Biodiversity 3). Univiversity of Tokyo Press, Tokyo,
pp. 25–52.
© 2012 The Society for the Study of Species Biology
Tsukaya H. (2002) Leaf anatomy of a rheophyte, Dendranthema
yoshinaganthum (Asteraceae), and of hybrids between
D. yoshinaganthum and a closely related non-rheophyte,
D. indicum. Journal of Plant Research 115: 329–333.
Tsukaya H. (2010) Leaf development and evolution. Journal of
Plant Research 123: 3–6.
Tsukaya H. & Machida C. (2000) Leaf morphogenesis in dicoty-
ledonous plants. In: Okada K., Machida Y. & Matsuoka M.
(eds). Molecular Mechanisms of Plant Formations. Shujunsha,
Tokyo, pp. 103–116.
Usukura M., Imaichi R. & Kato M. (1994) Leaf morphology of a
facultative rheophyte, Farfugium japonicum var. luchuense
(Compositae). Journal of Plant Research 107: 263–267.
Van Dam N. & Hare J. D. (1998) Biological activity of Datura
wrightii glandular trichome exudates against Manduca sexta
larvae. Journal of Chemical Ecology 24: 1529–1549.
Van Steenis C. G. G. J. (1981) Rheophytes of the World. Sijthoff and
Noordhoff, Alpen aan den Rijn.
Yamanaka T. & Takezaki K. (1959) Distribution and ecology of
Rhododendron ripense Makino, with reference to the vegetation
and flora on rocky river-bank. Journal of Japanese Botany 34:
215–224.
Yamazaki T. (1989) Ericaeae. In: Satake Y., Hara H., Watari S. &
Tominari T. (eds). Wild Flowers of Japan Woody Plants Vol. II.
Heibonsha, Tokyo, pp. 122–156.
Yamazaki T. (1993) Ericaceae. In: Iwatsuki K., Tamazaki T., Bouf-
ford D. E. & Ohba H. (eds). Flora of Japan Vol. IIIa. Kodansha,
Tokyo, pp. 6–63.
Plant Species Biology 27, 233–240