Bull. Natl. Mus. Nat. Sci., Ser. B, 36(2), pp. 39–42, May 22, 2010

Leaf Blade Thickness Differentiation between Coastal and Freshwater
Populations of the Coastal Plant Vitex rotundifolia (Verbenaceae)
Hiroaki Setoguchi*, Nobuchika Ishibashi, Koichi Fujita, Hisashi Matsubara,
Keiko Terada and Haruki Yagi
Graduate School of Human and Environmental Studies, Kyoto University,
Yoshida Nihonmatsu-cho, Sakyo-ku, Kyoto, 606–8501 Japan* E-mail: seto@botany.mbox.media.kyoto-u.ac.jp(Received 24 November 2009; accepted 24 March 2010)

Abstract Vitex rotundifolia (Verbenaceae) is one of the coastal plants that commonly grows onsandy seashores, but also occurs at Lake Biwa, a freshwater inland lake. To assess morphologicaldifferentiation between coastal and inland individuals with regard to the leaf thickness, we measured nine populations of Vitex rotundifolia from Lake Biwa, Ise Bay in Pacific Ocean side andWakasa Bay in Sea of Japan side. We measured leaf blade thickness and applied one-way analysisof variance (ANOVA) to test the difference. Post-hoc multiple comparison resulted in four majorgroups recognizing all inland populations from Lake Biwa as a distinct group while affiliation ofcoastal populations was assigned to other three groups regardless to localities. Inland individuals
tended to have thinner leaves, implying intraspecific differentiation due to adaptation to habitatenvironments.
Key words : coastal plant, geographical variation, isolation, Lake Biwa, leaf thickness, salinity
stress, Vitex rotundifolia.

Introduction
Vitex rotundifolia L.fil. (Verbenaceae) is a deciduous creeping tree, which grows on sandy
seashores in the temperate-tropic zone including
Japan. The seed is covered with spongy pericarp
and its dispersal is accomplished by seawater forlong distance. V. rotundifolia inhabits frontline ofcoastal vegetation and never grows in inland,
implying this plant grows under salinity stress byairborne salt and higher osmotic pressure of soil.
Tolerance to airborne saltwater is critical to thesurvival of the coastal populations; in coastal
plant communities, the distribution of species
can sometimes be determined according to theirtolerance to airborne saltwater (Oosting andBillings, 1942; Oosting, 1945; Boyce, 1954; Barbour, 1978; Barbour et al., 1985; Rozema et al.,
1985; Sykes and Wilson, 1988; Hesp, 1991;
Maun, 1994; Greipsson and Davy, 1996; Wilsonand Sykes, 1999). However, in Japan, this plantalso inhabits the sandy lakeshore in Lake Biwa, afreshwater lake located in central Japan.
Lake Biwa is an ancient lake that was formed
about four million years ago (Yokoyama, 1984;
Kawabe, 1989, 1994; Meyers et al., 1993), andharbors many other coastal plants that commonly
inhabit the seashore, including Calystegia soldanella (L.) Roem. et Schult. (Convolvulaceae),Lathyrus japonicus Willd. (Leguminosae),
Arabis kawasakiana Makino (Cruciferae),Raphanus sativus L. var. raphanistroides Makino(Cruciferae), Dianthus japonicus Thunb.
(Caryophyllaceae), and Pinus thunbergii Parl.
(Pinaceae) (Kitamura, 1968). These plants are assumed to have migrated to the inland lake fromcoastal populations during the period when Lake
Biwa had been adjacent to the seashore, andinland lake populations might have later become
isolated from the coastal populations (Takaya,40

Hiroaki Setoguchi et al.

1963; Kitamura, 1968).
The landlocked plants at the freshwater lake
may have become morphologically differentiatedwith regard to long-term isolation and/or salt-tolerance as a result of adaptive evolution to thespecific habitat over the long term. In particular,
leaf succulence is correlated to exposure to salinity stress in coastal plants (Boyce, 1951; Hesp,1991; Maun, 1994). Noda et al. (2009) assessed
physiological differentiation between coastal andinland individuals of Calystegia soldanella withregard to the response to salinity stress, andfound that inland individuals decrease leaf blade
thickness than those of coastal ones.
Coastal plants which inhabit both in Lake
Biwa and coast are appropriate example to determine the allopatric differentiation in morphology
in relation with adaptation to habitat environment. In this study, we aimed to examine morphological differentiation in leaf blade thicknessof coastal plant Vitex rotundifolia between theinland and coastal habitats. We have measured
the leaf blade thickness for 183 individuals fromnine populations that located in Lake Biwa andseashore, and discussed ecological factors andgenetic background that may affect morphology.

viduals were conducted in late July of 2008 and2009 in natural habitats in Lake Biwa (three populations), Pacific Ocean side (Ise Bay, three populations) and Sea of Japan side (Wakasa Bay,three populations). Localities and their coordination are summarized in Table 1. All voucher
specimens were deposited in National Museumof Nature and Science, Japan (TNS). Inland populations in Lake Biwa have been decreased thenumber of populations, and only the three populations are remained as small patches. We usedelectric callipers for measurement of leaf blade
thickness. The average thickness of each leaf wasestimated based on three measurements from different part within a leaf, and then ten fully matured leaves were examined for each individual.
Mean value was calculated for each individual,and data set of the ten individuals from each population was subjected to One-way ANOVA to test
the difference in the leaf blade thickness among
the populations. Post-hoc multiple comparison by
Tukey HSD test was used to determine the significant difference between each population. The
statistical analyses were performed using SPSS
version 10 (SPSS, Chicago).
Results and Discussion

Materials and Methods
Vitex rotundifolia is a deciduous tree and develops new leaves in early May and blooms inlate July in Kinki district, Japan. To assess mature leaves, measurements of V. rotundifolia indiTable 1.

The leaf blade thickness was normally distributed, and values were significantly different
among populations (ANOVA, p0.001; Table 2).
Leaf blade thickness varied among populations
ranging from 0.3020.002 mm (meanstandard

Sampling localities, coordinates and number of individuals for Vitex rotundifolia
Coordinates

RegionLake BiwaPacific(Ise Bay)Sea of Japan(Wakasa Bay)

No.
12
345
6789Locality
MaiamiShinkaihama
Sabae
Matsunase
Kawajiri
Yoshizaki
Sakajiri
MatsubaraWada

latitudelongitude

35°0824N
35°1656N
35°0847N
34°3622N
34°3555N
34°5500N
35°3719N
35°3636N
35°3700N

135°5947E
136°0106E
136°0116E
136°3442E
136°3715E
136°3840E
135°5758E
135°5529E
135°5625E

Number ofindividuals
20102530
29
27
21
1011

Leaf Thickness Differentiation in Vitex rotundifolia
Table 2.

41Result of one-way analysis of variance (ANOVA) of leaf thickness among populations

Morpholoicalcharacteristic

Source ofvariation

SSdFMSF value

p value

Leaf thickness

Among groups
Within groups
Total

0.127
0.099
0.226

8174
182

0.016
0.001

27.762

0.001

SS, Sum of Squares ; dF, degree of Freedom; MS, Mean Squares; F, F-statistics; p, p value for the null hypothesis.

error; 1. Maiami, Lake Biwa) to 0.3830.007
mm (8. Matsubara, Wakasa Bay) (Fig. 1). Theleaf blade thickness suggested a geographicaltrend, where inland populations from Lake Biwa
showed a thinner thickness than seashore populations (Fig. 1, Turkey HSD). Post-hoc multiple
comparison resulted in four groups recognizing
all inland populations (populations 1–3) as a distinct group while affiliation of seashore populations was assigned to other three groups regardless to their localities (Ise Bay on the Pacific sideor Wakasa Bay on the Sea of Japan side).
Thus, inland individuals inhabited in Lake
Biwa populations have significant thinner leaf
blade. This result implies the landlocked coastal
plants of Vitex rotundifolia in Lake Biwa may
have adapted to the habitat environment, i.e.,
freshwater lakeshore without salinity stress. Leaf
succulence in coastal plants is induced by salinity
stress (airborne salt loading on the leaves andbranches), resulting in salt-induced hypertrophy
and the doubling or tripling of leaf thickness(Boyce, 1951). Noda et al. (2009) detected physiological differentiation between inland andcoastal individuals of Calystegia soldanella interms of their response to salinity stress. Inland
individuals at Lake Biwa showed significant decrease of photosynthetic ability as a result of saltwater sprayed onto leaf blades, corresponding tostomatal closure, whereas coastal individuals
showed an insignificant decrease in the same parameter. Additionally, coastal individuals tended
to have thicker leaf blades than those of inland
individuals even after freshwater irrigation. Conversely, inland individuals developed thinner leaf
blades than those of coastal ones even after saltwater irrigation and saltwater spray onto leaf

Fig. 1. Means and standard errors of dimensionsof the leaf thickness in Vitex rotundifolia. Different abbreviation above the vertical barmeans significant difference in post-hoc Tukey
HSD test.

blades. Thus, the inland plants have adapted tofreshwater environment in terms of physiologicalresponses to surrounding environment in termsof genetic background. These findings suggestthat the same responses to salinity stress would
be also expected in Vitex rotundifolia.
Recent molecular phylogeography revealed theheterogeneous genetic structure between theinland and coastal populations, corroborating thelong-term isolation of coastal plants at Lake
Biwa (Noda et al., unpublished). Vitex rotundifolia plants at Lake Biwa may have caused a morphological differentiation in leaves in terms ofloss of leaf succulence along with loss of salinity
tolerance during long-term isolation within
inland. Further studies including transplanting

42

Hiroaki Setoguchi et al.

experiments and photosynthetic responses tosalinity stress should be examined for understanding the physiological background.
Acknowledgements
We thank Hajime Ikeda (Kyoto University) for
assisting statistic analyses. This study was supported by a Grant-in-Aid from the Japan Society
for the Promotion of Science to HS (13575011).
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