Springer-VerlagTokyo102650918-94401618-086030669031Journal of Plant ResearchJ Plant Res12110.1007/s10265-003-0121-7

J Plant Res (2004) 117:13–18
Digital Object Identifier (DOI) 10.1007/s10265-003-0121-7© The Botanical Society of Japan and Springer-Verlag Tokyo 2003

ORIGINAL ARTICLE
Hiroshi Tomimatsu • Akihiko Hoya • Hideki Takahashi •Masashi Ohara

Genetic diversity and multilocus genetic structure in the relictual endemic
herb Japonolirion osense (Petrosaviaceae)

Received: March 3, 2003 / Accepted: August 12, 2003 / Published online: September 20, 2003

Abstract Plant clonality may greatly reduce effective population size and influence management strategies of rareand endangered species. We examined genetic diversity andthe extent of clonality in four populations of the monotypic
herbaceous perennial Japonolirion osense, which is one of
the most rare flowering plants in Japan. Allozyme analysis
revealed moderate levels of genetic variation, and the
proportion of polymorphic loci (P = 66.7%) was higher
than the value for species with similar life-history traits.
With four polymorphic loci, 19 multilocus genotypes wereobserved among 433 aerial shoot samples and 10 (52%)
were found only in single populations. The proportion of
distinguishable genotypes (PD = 0.10) and Simpson’s index
of diversity (D = 0.52) also exhibited moderate levels of
genotypic diversity compared to other clonal plants, withgenotype frequencies at Hardy-Weinberg equilibrium. The
distributions of genotypes were often localized and they
were mostly found within a radius of 5 m. Spatial autocorrelation analysis showed that shoot samples located 4 mapart were expected to be genetically independent. The
results suggest that the spatial extent of genets was relatively narrow and thus the clonality was not extensive.
Key words Allozyme · Clonal structure · Genetic diversity· Japonolirion osense · Rare and endemic species · Spatial
autocorrelation analysis

H. Tomimatsu · M. Ohara (*)
Laboratory of Ecology and Genetics, Graduate School ofEnvironmental Earth Science, Hokkaido University, Sapporo
060–0810, Japan
Tel. +81-11-7064525; Fax +81-11-7064525
e-mail: ohara@ees.hokudai.ac.jp
A. Hoya
Department of General Systems Studies, Graduate School of Arts andSciences, The University of Tokyo, Tokyo, Japan
H. Takahashi
The Hokkaido University Museum, Hokkaido University, Sapporo,Japan

Introduction
Japonolirion is a monotypic and endemic genus of Japan,
and J. osense is a relictual species that is endemic to serpentine areas in Honshu and Hokkaido, Japan (Nakai 1930;
Tatewaki 1931; Hara and Kanai 1959). In Honshu the plant
occurs in Mt. Shibutsu and Mt. Tanigawa (Gunma Prefecture), while in Hokkaido the plant occurs only in the Teshio
region. Based on this rarity and restricted disjunctive distribution, J. osense is listed as vulnerable (VU) under the Red
Data Book of Japanese plants (Environmental Agency ofJapan 2000). Despite the need for conservation, little is
known of the life history characteristics of J. osense.
Although it has been reported that this species reproduces
both sexually and vegetatively (Hara 1935), there is no
study on sexual reproduction. As for vegetative reproduction, Tsukui and Takahashi (1995) only describe the architecture of underground rhizomes by excavating two clonalfragments and suggest that its clonal growth forms andlong-lived rhizomes may contribute to extensive vegetative
spread on unstable sandy serpentine slopes. The present
study focuses on the clonal diversity and structure of thisspecies.
Clonal growth can significantly influence the conservation of rare and endangered species. First, the number ofgenetically distinct individuals (genets) may be much lessthan the ostensible number of individuals (ramets). At theextreme, each population may consist of one or several
fragmented genets (e.g., Swensen et al. 1995; Sydes andPeakall 1998). Genetically small populations are likely to
be influenced by stochastic environmental changes including emergence of novel pathogens (Shaffer 1981; Schemske
et al. 1994), and sexual reproduction among few genets may
result in inbreeding depression (Barrett and Kohn 1991;
Ellstrand and Elam 1993). If the species has strong selfincompatibility, restricted pollen transfer within a clone
may further limit seed production (Handel 1985). Therefore, genet abundance and its structure may influence
population persistence. Second, the extent of clonality
should be taken into account in reserve design for in situ

14conservation and in sampling strategies for ex situ conservation or re-establishment projects (Frankham et al. 2002).
Conservation plans should be designed to maximize genetic
diversity in order to maintain the long-term evolutionary
potential of the species (Frankel et al. 1995). To achieve this,knowledge of clonal diversity and its spatial arrangement
within and among populations is essential.
In this study, we examined clonal diversity and structureof four populations of Japonolirion osense in the Teshio
region, northern Hokkaido, by collecting leaf samples to beanalyzed by allozyme electrophoresis. Allozyme analysis
requires only a small amount of plant material and is therefore appropriate for studies on rare and endangered species
where extensive survey and sampling are impractical.

Materials and methods
Species and study sites
Japonolirion osense Nakai (Petrosaviaceae) is a diploid(2n = 24; Sato 1942), long-lived herbaceous perennial,
which is one of the most rare flowering plants in Japan.
Japonolirion had been classified as Liliaceae thus far, but
recent molecular phylogenetic analysis suggested that the
species should be placed in Petrosaviaceae along with Petrosavia (Fuse and Tamura 2000). Aerial shoots often aggregate (10–200 shoots·m-2) and form patches, which can easily
be distinguished in space. Rhizomes creep undergroundwith an annual elongation of ≤90 mm and persist for at least
24 years (Tsukui and Takahashi 1995). Erect aerial shoots
sprout from the tips of the rhizomes of the previous yearand grow for only one season. One to five new buds areformed at the base of the shoots and some of them elongate
as rhizomes (Tsukui and Takahashi 1995) so that the plants
can acquire horizontal space effectively via frequent rhizome branching. Flowering scapes sprout from the tips ofsome of the previous year’s shoots (Hara 1935; Tsukui and
Takahashi 1995). The density of flowering scapes is ≤10 m-2.
The inflorescence is an erect raceme with many yellowish,
hermaphrodite flowers. The fruit is a greenish capsule withsmall (~0.8 mm), wingless seeds (Utech 1984).
The study was conducted in four populations (Table 1).
In these sites, the species occurs as small populations andgrows in exposed riverside slopes. Because the sites aremoist, serpentine sands with a steep slope, they are suscep-tible to landslide. The Kikusui-A population is close to the
Kikusui-B population with a separation of several hundred
meters, while the other populations were separated by 3–
10 km.
Leaf sampling and allozyme electrophoresis
In 1999, young leaves were sampled from 433 aerial shootsin the four populations (Table 1). In each population, leaf
samples were collected from almost all the shoot patches.
Within each patch, between 1 and 21 samples were randomly collected and the positions sampled were recorded.
Sample sizes depended on the actual density of plants andthe size of patches. Leaf samples were transported in ice tothe laboratory and kept at -80°C until allozyme electrophoresis. Enzyme extraction and polyacrylamide verticalgel electrophoresis were carried out as described byTomimatsu and Ohara (2003). We preliminarily tested 16
enzymes; subsequently a total of six enzyme systems thatconsistently showed interpretable banding patterns were
examined: aspartate aminotransferase (AAT; E.C. 2.6.1.1; 1
locus), leucyl aminopeptidase (LAP; E.C. 3.4.11.1; 1 locus),
phosphoglucomutase (PGM; E.C. 5.4.2.2; 1 locus), 6-phosphogluconate dehydrogenase (6PGDH; E.C. 1.1.1.44; 1
locus), alcohol dehydrogenase (ADH; E.C. 1.1.1.1; 1 locus),and sorbitol dehydrogenase (SODH; E.C. 1.1.1.14; 1 locus).
We resolved six putative loci and assumed Mendelian inheritance of all loci based on banding patterns given the
expected enzyme unit substructures.
AnalysisStandard measures were used to quantify genetic variation:
proportion of polymorphic loci (P) and mean number ofalleles per locus (A). Genotypic diversity within populations was evaluated using the following measures: thenumber of detected genotypes (G), the proportion of
distinguishable genotypes (PD), and Simpson’s index of
genotypic diversity (D). PD was calculated as G/N, where
N is the number of samples (Ellstrand and Roose 1987).
This measure corresponds to the probability that the next
sample will be a different genotype. Simpson’s index ofdiversity was calculated as:ni ( ni - 1)i N ( N - 1)

D = 1-Â

(1)

Table 1. Locations and genetic diversity of four populations of Japonolirion osense. N Sample size, P proportion of polymorphic loci (%),
A mean number of alleles per locus, G number of multilocus genotypes, PD proportion of distinguishable genotypes, D Simpson’s index of
genotypic diversity
Population

LatitudeLongitude

Area (m2)

N

P

A

G

PD

D

Kikusui-A
Kikusui-B
Shiunbashi
Hassenzawa
Mean
Overall

44°56¢N
44°56¢N
44°57¢N
45°00¢N

142°11¢E
142°11¢E
142°14¢E
142°02¢E

500
120
10
700

168
55
11
199

66.7
33.3
16.7
66.7
45.9
66.7

1.83
1.33
1.17
1.83
1.54
1.83

11
4
2
16
8.3
19

0.07
0.07
0.18
0.08
0.10

0.78
0.39
0.18
0.72
0.52

15

where ni is the number of sampled shoots of the ith genotype (Pielou 1969). Geneotypic diversity among populations was measured as the number of local genotypes(genotypes found only in a single population) and the number of widespread genotypes (genotypes found in 75% ormore of the populations) (Ellstrand and Roose 1987). These
measures can be compared to other studies of clonal plants(Ellstrand and Roose 1987; Widén et al. 1994).
Observed heterozygosity (Ho) and gene diversity (He)were calculated with each multilocus genotype treated as anindividual. Because there were few observed genotypes ineach population, these indices were determined only at theoverall population level. Mating system was assessed by
calculating the inbreeding coefficient (FIS) only for populations where greater than ten multilocus genotypes were
found, using the estimate of Weir and Cockerham (1984).
Other fixation indices (FIT and FST) were not calculated dueto the low number of genotypes. The analysis was performed using the program FSTAT version 2.9.1 (Goudet
2000), and the level of significance was obtained by randomization procedures (see Goudet 2000 for details). The
probability that identical genotypes arise through sexual
reproduction by chance was determined within each population for each genotype assuming random mating and linkage equilibrium among loci:Êˆ
Pgen = Á ’ pi qi ˜ 2 h
Ë i
¯

(2)

where pi and qi are the frequencies of the two alleles at the
ith locus and h is the number of loci that are heterozygous(e.g., Parks and Werth 1993).
To investigate fine-scale spatial genetic structure, we conducted spatial autocorrelation analyses (Sokal and Oden
1978; Smouse and Peakall 1999; Kalisz et al. 2001) for the
Kikusui-A and Hassenzawa populations where a large number of samples were obtained. In the Hassenzawa population, the site consisted of two subpopulations divided by asmall stream. Thus the data of the larger subpopulation
were used for analysis. We followed the method of Smouseand Peakall (1999), who developed a multilocus estimate of
autocorrelation coefficient (r). Distance classes of 1 m wereused and significance was tested for each distance class bycomparing the observed value of r with those obtained from999 randomizations. The significance of the correlogram asa whole was then assessed by T 2-test (see Smouse andPeakall 1999 for details). Analysis was conducted using the
program written by H. Tomimatsu in S language (Lucent
Technologies, Murray Hill, N.J.) on the software S-PLUS
version 3.3 (Insightful, Seattle, Wash.).

Results
Genetic and genotypic diversity
Two of the six loci resolved (Aat and Lap) were monomorphic. Four loci were polymorphic in at least one of the
populations examined: Pgm exhibited three alleles and6Pgdh, Adh, and Sodh exhibited two alleles. P and A at the
species level were 66.7% and 1.83, respectively (Table 1). A
total of 19 multilocus genotypes were found (Table 2); G
per population averaged 8.3, ranging from 2 (Shiunbashi)
to 16 (Hassenzawa) (Table 1). Although 162 genotypes(= 6 ¥ 3 ¥ 3 ¥ 3) were possible with the four polymorphic
loci, a much lower number of genotypes was observed. PD
exhibited relatively low values, with a mean of 0.10. Large
populations (Kikusui-A and Hassenzawa) were more genotypically diverse, with relatively large numbers of genotypes
and high D values. At the overall population level, Ho was
0.133 and He was 0.247. Inbreeding coefficients in two large
populations were somewhat elevated but did not differ significantly from Hardy-Weinberg expectations (FIS = 0.211
for Kikusui-A; FIS = 0.207 for Hassenzawa). Among the 19
genotypes observed, 10 (52%) were local genotypes andfound only in single populations. Only four (21%) werewidespread genotypes (genotypes 8, 13, 15, and 18; Table 2).
This was partly explained by the fact that two alleles (Adha and Sodh-a; Table 3) were common in the Hassenzawa
population, while in the other populations only a single
genet hold the alleles as heterozygotes (genotype 7;
Table 2). These results suggest that the populations were
genetically differentiated.
In the case of the local genotypes, Pgen values were low
enough to reject the null hypothesis (Pgen < 0.05) that identical genotypes were generated through sexual reproduction by chance, indicating that aerial shoots with the same
multilocus genotype were highly likely to be members of
the same clone. However, Pgen of the other genotypes
including those that were widespread were sometimes not
significant (Pgen > 0.05) and exceeded 0.1 in six cases (datanot shown). In such cases, it is probable that two or moregenets with the same multilocus genotype arose throughsexual reproduction.
Table 2. Nineteen multilocus genotypes with the four polymorphic locidetected for four populations of Japonolirion osense. Pgm Phosphoglucomutase, 6Pgdh 6-phosphogluconate dehydrogenase, Adh alcohol
dehydrogenase, Sodh sorbitol dehydrogenase
Multilocus
genotype

Pgm

6Pgdh

Adh

Sodh

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19

aa
ab
ab
ab
ab
bb
bb
bb
bb
bb
bb
bc
bc
bc
bc
bc
cc
cc
cc

bb
aa
ab
bb
bb
aa
aa
aa
ab
ab
bb
aa
aa
ab
ab
bb
aa
aa
bb

bb
bb
bb
ab
bb
aa
ab
bb
ab
bb
bb
ab
bb
ab
bb
bb
ab
bb
bb

bb
bb
bb
ab
bb
aa
ab
bb
ab
bb
bb
ab
bb
ab
bb
bb
ab
bb
bb

16
Table 3. Allele frequencies at four polymorphic loci in four populations of Japonolirion osense, calculated with each multilocus genotype treatedas an individual
Locus

Pgm
a
b
c
6Pgdh
a
b
Adh
a
b
Sodh
a
b

Populations

Overall

Kikusui-A

Kikusui-B

Shiunbashi

Hassenzawa

0.227
0.636
0.136

0.000
0.500
0.500

0.000
0.500
0.500

0.063
0.594
0.344

0.106
0.591
0.303

0.500
0.500

0.875
0.125

1.000
0.000

0.625
0.375

0.636
0.364

0.045
0.955

0.000
1.000

0.000
1.000

0.250
0.750

0.136
0.864

0.045
0.955

0.000
1.000

0.000
1.000

0.250
0.750

0.136
0.864

Multilocus genetic structure
The distribution of multilocus genotypes is shown in Fig. 1.
The results for the Kikusui-A and Kikusui-B populations
are representatively included in the figure. Many shootpatches possessed multiple genotypes. In Kikusui-A, forexample, the distributions of genotypes were often localized
and the majority of genotypes were found within a radiusof 5 m. Some genotypes were widely distributed across the
populations (e.g., genotypes 8 and 10; Fig. 1), although thesewere widespread genotypes that may consist of several genets (e.g., Pgen = 0.09 for genotype 8, Pgen = 0.17 for genotype10). It is also noteworthy that although the Kikusui-A andKikusui-B populations are closely located only several hundred meters apart along the Kikusui River, their composition of multilocus genotypes was largely different.
Figure 2 shows the correlograms of spatial autocorrelation analyses for two large populations, Kikusui-A and
Hassenzawa. Both populations showed significant multilocus genetic structure (P < 0.01); r remained significantly
positive out to 4 m in the Kikusui-A and 2 m in the Hassenzawa populations. The coefficients were significantly negative at around 10 m.

Discussion
Genetic diversity and the extent of clonality
Although rare species typically show low levels of genetic
variation (Hamrick and Godt 1996; Gitzendanner and Soltis
2000), Japonolirion osense maintained moderate allozyme
variation (Table 1). The proportion of polymorphic loci atthe species level (Ps = 66.7%) was larger than the value(48.1%) for endemic long-lived perennial species (Hamrickand Godt 1996). The measures of genotypic diversity (G,PD, D) were slightly lower than values for 45 clonal species
in which sexual recruitment is rare (G = 11.1, PD = 0.27,

D = 0.75 at the population level; Ellstrand and Roose 1987;
Widén et al. 1994). However, genotypic diversity is sensitive
to the number of diagnostic loci (Widén et al. 1994), andtherefore the estimates of J. osense with only four polymorphic loci may be underrated. Substantial genetic variations
may be those among populations because over half of the
genotypes were population-specific. The result also infers
low levels of pollen and seed dispersal. But within populations the species are not severely inbred, as suggested by
the genotype frequencies in Hardy-Weinberg expectations.
The clonal habit can be advantageous in the unstable andgenerally oligotrophic serpentine slopes where Japonolirion
osense grows, as plants can acquire space horizontally andminimize the effect of disturbances such as landslides(Tsukui and Takahashi 1995). Since a genet is physiologically integrated, plants can also buffer the effect of habitat
heterogeneity by sharing resources such as water and nutrients among ramets within a genet (e.g., Stuefer et al. 1996).
On the other hand, extensive clonality reduces effective
population size and may threaten its long-term persistence.
In general, the extent of clonality greatly differs among
species and therefore cannot be simply predicted by thelevel of sexual reproduction or spatial patterns of aboveground aerial shoots (reviewed by Ellstrand and Roose
1987; Widén et al. 1994; Hangelbroek et al. 2002). Hence,
genetic analyses with molecular markers are essential toclarify the present status of endangered clonal plants and todesign suitable management strategies.
In Japonolirion osense, the distribution of multilocus
genotypes revealed that the spatial extent of genets may berelatively narrow, mostly a radius of <5 m (Fig. 1). This view
was also supported by significant positive autocorrelationsof genotypes only within small distance classes (<4 m;
Fig. 2). That is, overall, aerial shoots located 4 m apart were
expected to be different genotypes. Although some widespread genotypes covered wide areas (Fig. 1), these seemed
to comprise multiple genets that were generated by sexual
reproduction. Therefore, the spatial extent of these genets
would be more restricted.

17Fig. 1. Map showing the
distribution of multilocus
genotypes at Kikusui-A andKikusui-B. The patches ofJaponolirion osense areindicated in gray. Numbers inparentheses are sample sizes

Kikusui-A
N
(6)

(5)

(6)

(10)

(10)

(10)

(6)

Genotypes
(5)

1
2
3
5
7
8
10
11
13
15
16

(8)
(12)

(10)
(21)
(9)
(3)

5m

(
(10)

(9)

(6)

(8)

) Sample size

(5)

(10)

Kikusui-B
N

(10)

(7)
(3)
(2)

(2)

(4)

Genotypes

(18)

8
13
15
18

(8)
1m

Fig. 2. Correlograms (solid lines)
resulting from spatial
autocorrelation analyses basedon six allozyme loci for two
populations of J. osense. The
dotted lines represent a 95% null
hypothesis confidence region
with no genetic structure based
on 1,000 randomizations(1)() Sample size

Kikusui-A

0.4

Hassenzawa

0.4

N = 168, P < 0.01

r

N = 142, P < 0.01

0.2

0.2

0

0

−0.2

−0.2

−0.4

−0.4
0

2

4

6

8

10

12

Distance class (m)Implications for conservation
Genotypic diversity of Japonolirion osense was not low
compared to other clonal plants, and the spatial extent ofgenets was relatively narrow. These results suggest that the

14

16

0

2

4

6

8

10

12

14

Distance class (m)clonality was not so extensive as to extremely limit the
number of genets within populations. To preserve the populations, long-term monitoring of population sizes is necessary, because the habitat of J. osense is restricted to small
serpentine areas and disturbances such as landslides can

18further reduce population size. The results of this study alsoemphasize the role of sexual reproduction in the dynamics
of populations. The frequency of successful seedling recruitment needs to be quantified and the factors limiting recruitment should be considered if this frequency is extremely
low. Unfortunately, almost no information is available forsexual reproduction, and even the level of seed production and the breeding system (whether the plant is selfcompatible or incompatible) are not known for this species.
The substantial levels of genotypic diversity among populations suggest that efforts should be made to conserve as
many populations as possible across the species’ distribution range.
This species was roughly estimated to have declined by
20% during the past decade owing to damage by mountainclimbers and collection for horticultural purposes (Environmental Agency of Japan 2000). Under the circumstances, ex
situ conservation and re-establishment of populations may
be needed in the future. The intercept of the correlograms
can also be used to establish efficient sampling strategies forthese purposes (Diniz-Filho and Telles 2002). The results
indicate that samples at a distance equal or greater than theintercept (2–4 m; Fig. 2) are genetically independent, andcollection of plants or seeds at intervals of this distancecan sample most of the genetic variation (including clonalvariation) with less replications of samples of correlated
genotypes.
Acknowledgments The authors thank S. Uematsu for locating study
populations, T. Ishibuchi for field assistance, and M.N. Tamura forvaluable comments. Thanks are also due to Teshio Experimental
Forest, Hokkaido University and Nakatonbetsu Office, Hokkaido
Regional Forest Office for permitting us to conduct this study. This
study was financially supported by Grants-in-Aid from the Japan
Society for the Promotion of Science (JSPS) for Scientific Research(13440226, 13640637) to M.O. and Research Fellowships of the JSPS
for Young Scientists to H. Tomimatsu.

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