Bull. Natl. Mus. Nat. Sci., Ser. B, 37(3), pp. 127–132, August 22, 2011

Geographical Differentiation in Leaf Thickness between
Coastal and Freshwater Populations of the Coastal Plant
Lathyrus japonicus (Fabaceae)
Tatsuo Ohtsuki* and Hiroaki Setoguchi
Graduate School of Human and Environmental Studies, Kyoto University,
Yoshida Nihonmatsucho, Sakyo-ku, Kyoto 606–8501, Japan* E-mail: t.otsuki@ks4.ecs.kyoto-u.ac.jp(Received 12 April 2011; accepted 22 June 2011)

Abstract The beach pea, Lathyrus japonicus, is a coastal plant that is also found along the shoreof Lake Biwa, an ancient freshwater lake in Japan. These plants occur in two fundamentally different types of habitats, seacoast and freshwater lakeshores, suggesting the possibility of ecological
adaptation to habitats (e.g., salt tolerance). We focused on morphological differentiation between
coastal and inland populations and measured leaf blade thickness in a total of ten Japanese populations of L. japonicus from Lake Biwa, Ise Bay on the Pacific Ocean side, and Wakasa Bay on theSea of Japan side. In addition, we compared transverse leaf sections between inland and coastal
populations using light microscopy. An analysis of variance with a post-hoc test revealed that theinland and coastal populations comprised three major groups, with the inland populations accounting for one group and the coastal populations being assigned to the other two groups regardless oflocality. Inland individuals tended to have thinner leaf blades, due to differences in the cell sizes ofthe palisade and spongy tissues. The succulence in L. japonicus at Lake Biwa may have been lost
since salinity tolerance was not essential for their survival in a freshwater environment.
Key words : coastal plant, geographical variation, isolation, Lake Biwa, Lathyrus japonicus, leaf
thickness, salinity stress.

Introduction
Coastal environments are often inhospitable,
and several factors, such as soil moisture, soil
nutrients, wind exposure, sand burial, salt spray,
and soil salinity, likely limit plant growth oncoastal sand dunes (e.g., Oosting and Billings,
1942; van der Valk, 1974; Barbour, 1978; Maun,
1994). The action of surf breakers along coasts
may result in a considerable amount of salt sprayon soil and plant surfaces, and deposition of airborne seawater causes damage to plant tissues(Barbour, 1954; Rozema et al., 1985). Thus, salt
spray permits the growth and survival of a limited number of tolerant species along the coast andindirectly controls plant communities and theirdevelopment (Oosting and Billings, 1942; Oost-ing, 1945; Barbour, 1954; Rozema et al., 1985;
Sykes and Wilson, 1988; Hesp, 1991; Maun,1994; Greipsson and Davy, 1996; Wilson andSykes, 1999).
The beach pea, Lathyrus japonicus Willd.
(Fabaceae), is a perennial coastal herb native totemperate coastal areas of Asia, Europe, andNorth and South America. The unusually extensive native range of this species is explained by
the ability of the seeds to remain viable while
floating in seawater for up to five years, enabling
the seeds to drift nearly worldwide (Brightmoreand White, 1963). Germination occurs when thehard outer seed coat is abraded by waves on sandor gravel (Kondo and Yamaguchi, 1999). L.
japonicus inhabits the zone near the storm crestof the beach or at the seaward margin of a fixed

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dune but generally does not grow inland, implying that this plant grows under salinity stressfrom airborne salt and higher soil osmotic pressure. In Japan, L. japonicus is a typical coastal
plant, but also occurs at Lake Biwa, an ancient
freshwater lake in central Japan.
Lake Biwa, which was formed approximately
four million years ago, is one of the world’s few
ancient lakes. The lake was initially located approximately 40 km southeast of its present position and this predecessor lake gradually moved
northwest to its present position as a result offault subsidence (Takaya, 1963; Yokoyama,
1984; Kawabe, 1989, 1994; Meyers et al., 1993).
Many typically coastal plants inhabit the Lake
Biwa area, including Calystegia soldanella (L.)
Roem. et Schult. (Convolvulaceae), Vitex rotundifolia L., Arabis kawasakiana Makino (Cruciferae), Raphanus sativus L. var raphanistroides
Makino (Cruciferae), Dianthus japonicus Thunb.
(Caryophyllaceae), and Pinus thunbergii Parl.
(Pinaceae) as well as L. japonicus (Kitamura,1968). It is assumed that these plants migrated tothe lake from coastal populations during the period when the lake was adjacent to the sea, andthat the lake populations subsequently became
isolated from the coastal populations (Takaya,1963; Kitamura, 1968). Thus, these isolated Lake
Biwa plants are assumed to corroborate longterm isolation of the inland populations andintraspecific differentiation.
Furthermore, these plants occur in two fundamentally different types of habitat, seacoasts andfreshwater lakeshores, suggesting the possibilityof ecological adaptation, e.g., salt tolerance. Bothnuclear and cytoplasmic markers indicate that C.
soldanella and L. japonicus at Lake Biwa are isolated from coastal populations (Noda et al.,2011; Ohtsuki et al., unpublished). Isolated or
fragmented habitat types are expected to promote
the potential for local adaptation, and leaf succulence is correlated with salinity stress exposurein coastal plants (Boyce, 1951; Mayr, 1963;
Hesp, 1991; Maun, 1994). Landlocked plants at
Lake Biwa have been found to be morphologically differentiated from coastal populations; Se-toguchi et al. (2009, 2010) found that leaf bladesof C. soldanella and V. rotundifolia were thinner
in inland populations than in coastal populations,indicating adaptation to a different habitat. Thus,
given the geographic history of inland populations, we hypothesized that L. japonicus may also
exhibit allopatric differentiation in morphology
in relation to adaptation to its habitat. As with C.
soldanella and V. rotundifolia, inland populationsof L. japonicus have thinner leaf blades. In thisstudy, we examined morphological differentiationin L. japonicus leaf-blade thickness between inland and coastal individuals. We measured theleaflet blade thickness of 410 individuals from atotal of ten populations located along the shore ofLake Biwa and the coast, and discuss the effectsof ecological factors and genetic background on
morphology. In addition, we compared the structural differences in the transverse leaf sections ofthe inland and coastal populations using lightmicroscopy.
Materials and Methods
To assess mature leaves, 410 Lathyrus japonicus individuals were measured in June 2010 innatural habitats on the shore of Lake Biwa (fourpopulations), the Pacific coast (Ise Bay, threepopulations), and the Sea of Japan coast (WakasaBay, three populations). The localities and theircoordinates are summarized in Table 1.
All voucher specimens were deposited in
National Museum of Nature and Science, Japan(TNS).
Inland populations at Lake Biwa have been
decreasing in number, and only five populations
currently remain as small patches. We used electronic calipers to measure leaf blade thickness of
10 fully matured leaves from each individual.
Because leaves of beach pea are compound, leaf
thickness measurements were conducted onleaflets: the average thickness of each leaf wasestimated based on measurements from the third
proximal leaflets on either side of the rachis
within a compound leaf. Additionally, we remeasured that leaflet on one side of the rachis to cor-Leaf Thickness between Coastal and Freshwater Populations of Lathyrus japonicus
Table 1.

Sampling localities, coordinates and number of individuals for Lathyrus japonicus
Cordinates

RegionLike Biwa

Pacific(Ise Bay)Sea of Japan(Wakasa Bay)

Table 2.

129

No.
1
2
3
4
5678910

Number ofindividuals

Location
OmimaikoHiragawa
Shinkaihama
Imajuku
Matsunase
Higashioizu
Muramatsu
Sugahama
Sakajiri
Wada

LatitudeLongitude

35°13N35°13N35°13N35°09N34°36N34°33N34°33N35°39N35°37N35°36N135°57E135°56E136°07E135°56E136°34E136°40E136°41E139°58E135°58E135°56E

41
41
41
41
41
41
41
41
41
41

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

Morphologicalcharacteristic

Source ofvariation

SSdF

MSFP

Leaf thickness

Among groups
Within groups
Total

430028.653
403184.751
833213.404

9400
409

47780.961
1007.962

47.404

0.001

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

rect for measurement error. Mean values werecalculated for each individual, and the dataset of41individuals from each population was subjected to a one-way analysis of variance (ANOVA) totest for differences in leaf blade thickness among
the populations. Tukey’s HSD post-hoc test for
multiple comparisons was used to test for significant differences between populations. Statisticalanalyses were performed using PASW Statistics
17.0 (WinWrap Basic). In addition, to analyze
morphological structure, fully expand and matureleaflets (Biwa and Coastal population) were collected. Cross sections of those leaflets wereinvestigated with a light microscope (BX-51
OLYMPUS). Photographs of the observed crosssections were taken with digital camera (C4040ZOOM OLYMPUS) attached to a light microscope. Fresh and mature leaflets were sectioned with a razor blade. Cross sections of 200350 m m in thickness were used for the observations.

Results and Discussion
Leaf blade thickness was normally distributed,
and the values differed significantly among populations (ANOVA, P0.001; Table 2), ranging
from 226.913.51 m m (meanstandard error) to315.054.43 m m (Fig. 1). Leaf blade thickness
suggested a geographical trend; inland populations from Lake Biwa tended to have thinner
blades than seashore populations (Fig. 1, Tukey’s
HSD). A post-hoc multiple comparison recognized all inland populations (1–4) as a single
group, whereas the seashore populations were assigned to the other two groups regardless of theirlocality (Ise Bay or Wakasa Bay). Transverse
sections of leaflets examined were shown in Fig.
2, which represented both inland and coastal
population’s leaflets. As has been suggested inFig. 1, thickness of the leaflets was greater incoastal individuals compared to those in inland
individuals. The difference in leaf thickness was
attributed to sizes of the palisade and spongy
parenchyma cells: leaves of inland individuals

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Tatsuo Ohtsuki and Hiroaki Setoguchi

have smaller parenchyma cells compared to thoseof coastal ones. On the other hand, the thicknessof epidermis and/or cuticle of the leaflets were
not obviously different between inland andcoastal individuals. Leaf succulence in plants
inhabiting coastal dunes is induced by salinity
stress (airborne salt loading on leaves andFig. 1. Means and standard errors of leaf thickness in Lathyrus japonicus. Different lettersabove the vertical bars indicate significant differences based on Tukey’s HSD post-hoc test.

branches), resulting in salt-induced hypertrophy
and the doubling or tripling of leaf thickness(Boyce, 1951). In addition, different effects ofsalinity in structure of leaf cells and other planttissues have been reported in several plantsspecies (Babber et al., 2000; Muthukumurasamy
et al., 2000; Sam et al., 2004). Thus, Lathyrus
japonicus in Lake Biwa may have caused the lossof leaflets succulence in the habitat environment.
Noda et al. (2009) detected physiological differentiation between inland and coastal individualsof Calystegia soldanella in terms of their response to salinity stress. Inland individuals atLake Biwa showed significantly decreased photosynthetic ability as a result of saltwater sprayedonto leaf blades, resulting in stomatal closure,
whereas coastal individuals showed an insignificant decrease following the same treatment.
Additionally, coastal individuals tended to havethicker leaf blades than those of inland individuals even after freshwater irrigation. Conversely,
inland individuals developed thinner leaf blades
than those of coastal individuals even after saltwater irrigation and saltwater spray onto leaf
blades (Noda et al., 2009). A preliminary study
also revealed physiological differentiation in L.

Fig. 2. Transverse leaflets sections illustrating the contrast between inland (a) and coastal populations (b). Scalebar100 m mLeaf Thickness between Coastal and Freshwater Populations of Lathyrus japonicus

japonicus between inland and coastal habitats;flavonoid content was significantly different between inland and coastal populations, implying
differentiation in physiological response, antioxidant, radical-scavenging functions, and UV-protective functions in response to changes in thesurrounding environment. Additionally, These indicate that the inland plants have adapted to a
freshwater environment in terms of physiologicalresponse to the surrounding environment. Thus,inland individuals from Lake Biwa had significantly thinner leaf blades, suggesting that L.
japonicus landlocked at Lake Biwa might haveadapted to the lack of salinity stress at the freshwater lakeshore accompanied with physiologicaldifferentiation.
In this study, we revealed a heterogeneous genetic structure between inland and coastal populations of L. japonicus, corroborating the isolation of inland populations at Lake Biwa as “landlocked” as determined using molecular phylogeographic analyses (Ohtsuki et al., unpublished). The same signature is exhibited by beachmorning glory, C. soldanella; populations atLake Biwa are genetically isolated from coastal
populations (Noda et al., 2011). With their lossof leaf succulence and salinity tolerance, L.
japonicus plants at Lake Biwa appear to havemorphologically differentiated from coastal
plants during their inland isolation. Further studies including transplant experiments and examination of photosynthetic responses to salinity
stress should be undertaken to better understandthe physiological basis for this adaptive response.
Acknowledgments
This study was supported by a Grant-in-Aidfrom the Japan Society for the Promotion of Science to HS (#21370036).
References
Babber, S., Sheokand, S. and Malik, S. 2000. Nodulestructure and functioning in chickpea (Cicer arietinum)
as affected by salt stress. Biological Plantarum 43:
269–273.

131

Barbour, M. G. 1954. The salt spray community. Ecological Monograph 24: 29–67.
Barbour, M. G. 1978. Salt spray as a microenvironmental
factor in the distribution of beach plants at Point Reyes,
California. Oecologia 32: 213–224.
Boyce, S. G. 1951. Salt hypertrophy in succulent dune
plants. Science 114: 544–545.
Brightmore, D. and White, P. H. F. 1963. Lathyrus japonicus Willd. (L. maritimus Bigel.). Biological Flora of theBritish Isles no. 459. Journal of Ecology 51: 795–801.
Hesp, P. A. 1991. Ecological precesses and plant adaptation on coastal dunes. Journal of Arid Environmentals
21: 165–191.
Kawabe, T. 1989. Stratigraphy of the lower part of the Kobiwako Group around the Ueno Basin, Kinki District,Japan. Journal of Geoscience of Osaka City University
32: 39–90.
Kawabe, T. 1994. The history of Lake Biwa. In: The
Research Group for the Natural History of Lake Biwa(ed.), The Natural History of Lake Biwa, pp. 25–72.
Yasaka Shoboo, Tokyo (in Japanese).
Kitamura, S 1968. Phytogeography of Shiga Prefecture.
Flora of Shiga Prefecture. Osaka: Hoikusha (in Japanese).
Kondo, T. and Yamaguchi, M. 1999. Germinnation characteristics of Lathyrus japonicas Willd. for landscaping
and vegetation restoration in seacoast. Journal of theJapanese Institute of Landscape Architecture 62:507–510 (in Japanese with English abstract).
MacArthur, R. H. & Wilson, E. O. 1967. The Theory ofIsland Biogeography. Princeton University Press,Princeton.
Maum, M. A. 1994. Adaptations enhancing survival andestablishment of seedlings on coastal dune systems.
Vegetatio 111: 59–70.
Mayr, E. 1963. Animal Species and Evolution. Harvard
University Press, Cambridge.
Meyers, P. A., Takemura, K. and Horie, S. 1993. Reinterpretation of late Quatenary sediment chronology ofLake Biwa, Japan, from correlation with marine glacial-interglacial cycles. Quatenary Research 39:
154–162.
Muthukumarasamy, M., Duttagupta, S. and Panneerselvam, R. 2000. Enahancement of peroxidase, polyphenol oxidase and superoxide dismutase activities by triadimefon in NaCl stressed Raphanus sativus L. Biological Plantarum 43: 317–320.
Noda, A., Nomura, N. and Setoguchi, H. 2009. Chlorophyll fluorescence in response to salt stress in thecoastal plant Calystegia soldanella: A comparison between coastal freshwater populations. Bulletin of theNational Museam of Nature and Science, Series B 35:
123–129.
Noda, A., Mitsui, Y., Ikeda, H., Setoguchi, H. 2011.

132

Tatsuo Ohtsuki and Hiroaki SetoguchiLong-term isolation of coastal plant Calystegia soldanella in an ancient lake Biwa, Japan. Biological Journal of the Linnean Society 102: 51–66.
Oosting, H. J. and Blings, W. D. 1942. Factors effecting
vegetational zobnation on coastal dunes. Ecology 23:131–142.
Oosting, H. J. 1945. Tolerance to salt spray of plants ofcoastal dunes. Ecology 26: 85–89.
Rozema, J., Bijwaard, P., Prast, G. and Bruekman, R.
1985. Ecophysiological adaptations of coastal halophytes from foredunes and salt marshes. Vegetatio 62:
499–521.
Sam, O., Ramírez, C., Coronado, M. J., Testillano, P. S.
and Risueño, M. C. 2004. Changes in tomato leaves induced by NaCl stress: Leaf organization and cell ultrastructure. Biological Plantarum 47: 361–366.
Setoguchi, H., Yamada, S., Sugimoto, Y. 2009. Geographical differentiation in leaf thickness in the coastal plant
Calystegia soldanella (Convolvulaceae): A comparison
between coastal and freshwater lakeshore populations.
Journal of Phytogeography and Taxonomy 57: 89–93
Setoguchi, H., Ishibashi, N., Fujita, K., Matsubara, H.,
Terada, K. and Yagi, H. 2010. Leaf blade thickness differentiation between coastal and freshwater populationsof the coastal plant Vitex roundifolia (Verbenaceae).
Bulletin of the National Museam of Nature and Science, Series B 36: 39–42.
Sykes, M. T. and Wilsion, J. B. 1988. An experimental investigation into the response of some New Zealandsand dune species to salt spray. Annals of Botany 62:
159–166.
Takaya, Y. 1963. Stratigraphy of the Paleo-Biwa groupand the paleography of Lake Biwa. Memoirs of theCollege of Science, University of Kyoto, Series B 30:
81–119.
van der Valk, A. G. 1974. Environmental factors controlling the distribution of forbs in Cape Hatteras National
Seashore. Canadian Journal of Botany 52: 1057–1073
Wilson, J. B. and Sykes, M. T. 1999. Is zonation oncoastal sand dunes determined primarily by sand burial
or salt spray? A test in New Zealand dunes. EcologicalLetters 2: 233–235.
Yokoyama, T. 1984. Stratigraphy of the Quatenary systems round Lake Biwa and geohistory of ancient Lake
Biwa. In: Horie, S. (ed.), Lake Biwa, Monographiae
Biologicae 54, pp. 43–138. Dr W. Junk Publishers, Dordrecht.