Effects of Forest Fragmentation on Seed Productionof the Understory Herb Trillium camschatcense
HIROSHI TOMIMATSU* AND MASASHI OHARA
Department of General Systems Studies, Graduate School of Arts and Sciences, The University of Tokyo,
Tokyo 153-8902, Japan

Abstract: We investigated the effects of forest fragmentation on reproduction of a common understory perennial, Trillium camschatcense, in Hokkaido, Japan. We measured seed production of 12 (1998) and 14 (1999)
populations, and we explored the relationships of seed production to the number of flowering plants ( population size) and to the surrounding landscape condition ( landscape type). We discriminated between two landscape types, isolated and continuous, to describe the quantity and distribution of forests around T. camschatcense populations. Population size may be correlated with attractiveness to pollinators and the availabilityof compatible mates, whereas landscape condition can affect the abundance of pollinators. The number ofseeds per flower was related to population size in 1999, but not in 1998. In both years, small populations of
50 flowering plants produced few seeds. Although the effect of landscape type was not significant, continuous populations with abundant forest series in their neighborhood tended to produce a higher number ofseeds than isolated populations located in small, isolated forest. We attribute the decrease in seed production
to pollen limitation because the stigmatic pollen load had a positive relationship to seed production and addition of pollen by hand increased seed production. Variation in population structure ( four stage classes)
among populations was better explained by population size than number of seeds produced, suggesting therelative importance of population size for the demography of T. camschatcense. Larger populations had ahigher proportion of seedlings, possibly because large populations had fewer edge effects, which reduce seedling recruitment and survival. Small populations with 220 flowering plants or less showed almost no seedlingrecruitment. The data on seed production and population structure suggest that continuous populations withat least 1000 flowering plants may be required to avoid the effects of fragmentation.
Efectos de la Fragmentación del Bosque en la Producción de Semillas de la Hierba de Sotobosque Trillium
camschatcense
Resumen: Investigamos los efectos de la fragmentación del bosque en la reproducción de una perenne
común en el sotobosque, Trillium camschatcense, en Hokkaido, Japón. La producción de semillas de 12(1998) y 14 (1999) poblaciones fue medida, y exploramos las relaciones de la producción de semillas alnúmero de las plantas florecientes (tamaño de la población) y a la condición del paisaje circundante (tipodel paisaje). Dos tipos de paisaje, aislados y continuos, fueron discriminados para describir la cantidad y la
distribución de bosques alrededor de poblaciones del T. camschatcense. El tamaño de la población se puede
correlacionar con la atracción de los polinizadores y con la disponibilidad de parejas compatibles, mientras
que la condición del paisaje puede afectar la abundancia de polinizadores. Se relacionó el número de semillas/flor con el tamaño de la población en 1999, pero no en 1998. En ambos años, las poblaciones pequeñasde  50 plantas florecientes produjeron escasas semillas. Aunque el efecto del tipo del paisaje no fue significativo, las poblaciones continuas con series abundantes de bosque a su alrededor tendieron a producir unmayor número de semillas que las poblaciones aisladas localizadas en bosques pequeños y aislados. Atribuimos la disminución de la producción de semillas a la limitación del polen porque la carga de polen estigmático tuvo una relación positiva con la producción de semillas y la adición manual de polen aumentó la*Current address: Laboratory of Ecology and Genetics, Graduate School of Environmental Earth Science, Hokkaido University,
Sapporo 060-0810, Japan, email tomi@ees.hokudai.ac.jp
Paper submitted October 2, 2000; revised manuscript accepted November 13, 2001.

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producción de semillas. La variación en la estructura de la población (quatro clases de etapa) entre poblaciones se explicó mejor por el tamaño de la población que por el número de semillas producidas, sugiriendola importancia relativa del tamaño de la población para la demografía del T. camschatcense. Poblaciones
más grandes tenían una mayor proporción de plántulas, posiblemente porque las poblaciones grandes
tenían pocos efectos de borde que reducen el reclutamiento y la supervivencia de la plántula. Poblaciones
pequeñas con 220 plantas florecientes o menos casi no mostraron reclutamiento de plántulas. Los datos sobre la producción de semillas y la estructura de la población sugieren que se pueden requerir poblaciones
continuas con por lo menos 1000 plantas florecientes para evitar los efectos de la fragmentación.

Introduction
Habitat fragmentation caused by human activities, suchas road construction and development of agricultural
fields, dramatically reduces plant population sizes andincreases their spatial isolation ( Wilcove et al. 1986;
Saunders et al. 1991; Rathcke & Jules 1993). Because
plants are sessile, it is generally accepted that habitat
fragmentation will alter both the ecological and genetic
properties of plant populations (e.g., Ellstrand & Elam
1993; Rathcke & Jules 1993; Schemske et al. 1994;
Young et al. 1996). In the case of outcrossing of entomophilous plants, for instance, population size is closely
associated with the attraction to pollinators and shouldhave a significant influence on reproductive success. Because small populations may be less attractive to pollinators than large populations (Sih & Baltus 1987 ), the reduction in population size results in decreased fruit orseed production due to insufficient pollen transfer (e.g.,Jennersten 1988; Ågren 1996). Reduction in seed production is one of the major threats to plant life-history
processes and may increase the probability of extinctionof populations and species ( Rathcke & Jules 1993; Olesen & Jain 1994).
The availability of pollinators may be affected not only
by population size itself but also by the landscape thatsurrounds a focal population. Even if two populations ofa plant species are the same size and have the same extent of floral display, pollinator availability necessarily
will be different depending on the potential pollinatorabundance, which may be related to the amount of available resources for pollinator species. Because pollinators continuously need flowering plant species overtheir entire life and often have specific nesting requirements (Kearns & Inouye 1997), a limited quantity of habitat available for pollinators may decrease pollinatorabundance and activity and may consequently result in
low reproductive output in plants. Moreover, when thespatial isolation among habitats by fragmentation becomes greater than the foraging range of pollinators,
pollination services also will decrease considerably.
Trillium camschatcense is a common understory
spring herb distributed mainly in Hokkaido and in north-Conservation Biology
Volume 16, No. 5, October 2002

ern Honshu, Japan. In the Tokachi region of eastern
Hokkaido, populations of T. camschatcense have beenhighly fragmented as a result of recent agricultural development. Populations in this region are self-incompatible,
and seed production results from obligatory outcrossing
by insect pollination (Ohara et al. 1996). We examinedthe effects of population size and surrounding landscapeconditions on the reproduction and demography of T.
camschatcense populations. We addressed the following questions: (1) Do large populations produce a significantly higher number of seeds than small populations?
(2) Do populations located in a forest-abundant landscape produce a higher number of seeds? (3) What is therelative importance of the effects of population size andsurrounding landscape conditions? (4) How does seedproduction relate to stage-class population structure?

Methods
Study Species and Study Sites
Trillium camschatcense Ker Gawler ( Trilliaceae), a corrected name for T. kamtschaticum Pall. (Fukuda et al.
1996), has four major life-history stages, as do other Trillium species: seedling, one-leaf sterile, three-leaved sterile, and flowering (Ohara & Kawano 1986b). It takes
more than 10 years for a seedling to go through thestages to become a flowering plant. When the stems orleaves of the flowering plant are damaged, however, theplant often returns to its three-leaved sterile stage in thenext year, possibly because of impaired photosynthesis(Ohara & Kawano 1986b). Trillium camschatcense reproduces exclusively by seeds (Ohara & Kawano
1986a). Most reproductive plants have one or two flowering stems, although some have three or four. In the
study area, T. camschatcense flowers in mid or late May,and the flowers remain in anthesis for about 1 week.
Flowers are visited by a wide range of insect species,
mostly belonging to Diptera, Coleoptera, and Hymenoptera (Ohara et al. 1991; H. T. et al., unpublisheddata). In study populations, members of Nitidulidae, Melandryidae, Oedemeridae (Coleoptera), Scatophagidae,

Tomimatsu & Ohara

Table 1.

Effects of Forest Fragmentation on Trillium

1279

Study sites of Trillium camschatcense populations.

Population(abbreviation)
Shibusan (SS)Obihiro (OH)
Motoimatsu ( MM)Toyonishi (TN)Kiyokawa (KK)
Hirono-kita (HK)Hirono-minami (HM)
Pepekiki (PK)
Churui (CR)
Moewa (MW )Toberi (TB)
Rekifune (RF)Monbetsu ( MB)
Hiroo (HI)

LatitudeLongitude

Population
area (m2)425255 N
425323 N
424818 N
424811 N
424522 N
424554 N
424544 N
424217 N
423355 N
423046 N
423018 N
422756 N
422635 N
421857 N

1425913 E
143 841 E
143 643 E
143 99 E
143 652 E
143 27 E
143 036 E
143 65 E
1432034 E
1431843 E
1432523 E
1432137 E
1431941 E
1431953 E

550
1050
18700
3400
7700
1400
5500
1450
400
2600
190
60000
480
50000

Forestarea (ha)Floweringplantdensity a

Population
size

Landscape
typeb

0.19
0.39
1.9
0.34
1.0
1.7
1.2
0.23
0.80
240
0.019
31
0.048
30

10
13
24
16
33
15
25
20
0.25
0.07
6
64
18
63

220
550
18000
2200
10200
900
5500
1200
4
7
46
153600350
126000

C
C
I
I
I
C
I
C
C
C
I
C
I
C

Average number of flowering plants in 25 m2 was calculated by randomly placing a 5  5 m quadrat five times within each population.
Two landscape types, isolated ( I ) and continuous (C), were discriminated according to the abundance and distribution of the forest aroundthe populations. See text for details.
ab

and Bibionidae (Diptera) were often observed ( H.T. etal., unpublished data).
We conducted field studies on 14 populations locatedin the Tokachi region of eastern Hokkaido in 1998 and1999 ( Table 1). The overstories of the study populations
were mostly broad-leaved deciduous trees, dominatedmainly by Ulmus davidiana var. japonica , Quercus
mongolica var. grosseserrata, and Fraxinus mandshurica var. japonica, except in the HK and HI populations,which included Larix leptrepis plantations. (Definitions
for population abbreviations are provided in Table 1.)

multiplying fruit set (1) and number of seeds per mature
fruit (2). We examined 12 populations (excluding OH
and CR) in 1998 and all 14 populations in 1999. In 1998
we conducted supplemental hand-pollination experiments in 11 populations (excluding OH, CR, and MW )to assess the possibility of pollen limitation. In each population, flowers (3–37; total n  244) were artificiallypollinated with pollen from at least three distant individuals. We measured the number of seeds produced per
flower for each population after the fruits were collected in July and compared them with open-pollinatedcontrols established at each site (6–35; total n  259).

Fruit and Seed Production
To assess the level of seed production, we randomly selected flowers (4–35) from each population before anthesis in early May and left them in an open-pollinatedcondition (266 and 277 flowers in total for 1998 and
1999, respectively). Individuals with only one floweringstem were sampled for this study, except when we
could not obtain sufficient sample size in small populations. No significant differences in the number of seeds
produced per flower were found, however, between
plants with different numbers of flowering stems (oneway analysis of variance [ANOVA], R2  0.008, n  41,
F  0.03, p  0.86; H. T. & M. O., unpublished data). All
fruits were collected in July, and the number of seeds
was counted for each fruit under a stereomicroscope.
Three indices were calculated to characterize fruit andseed production for each population: (1) fruit set, a population measure described by the percentage of flowersthat fruit; (2) number of seeds per mature fruit, a population measure described by the mean of the number ofseeds that mature fruits contained; and (3) number ofseeds per flower, a population measure calculated by

Stigmatic Pollen LoadTo estimate the outcross pollen load of the study populations, flowers (4–10) of each population were emasculated before anthesis and the stigmas were collected latein the flowering period (52 and 80 flowers in total for1998 and 1999, respectively). The samples were stored
in 70% ethanol until examination, and we used the glycerin-jelly method to count the number of outcross pollen grains on the stigmas ( Beattie 1971; Kearns & Inouye
1993). Although we cannot ignore the possibility of underestimating the number of pollen grains that may havealready grown down the style during the experiment,
this procedure can provide a good estimation of the outcross pollen load. The experiment was conducted for 11
populations (excluding OH, CR, and MW ) in 1998 andfor 12 populations (excluding CR and MW ) in 1999.
Factors Affecting Seed Production
Population size and surrounding landscape type were
evaluated as factors affecting seed production of theConservation Biology
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study populations. Population size, defined here as theestimated number of flowering plants in each population, was calculated by multiplying the population
area by the flowering plant density (Table 1). Population size was log-transformed for analysis. The surrounding landscape type was evaluated by classifying
the study populations into two types, isolated and continuous (referred to as I and C, respectively), based on
the abundance and the distribution of the forest
around the focal T. camschatcense populations ( Table1). Populations classified as I were surrounded by pastures or agricultural fields in all directions and weredistant (at least 200 m) from other large forests. In
contrast, C populations were located within windbreak forests or surrounded by several adjacent patchy
forests, both of which were much larger than the focal
Trillium population itself. In either case, no other Trillium populations were found in the focal forest fragment. We used two variables to characterize the twolandscape types: (1) a ratio of forest area to population
area (log-transformed data, mean 0.08, range, 0–0.34
for I and 1.02, 0.20–2.97 for C, t  2.18, p  0.05) and(2) the proportion of the forest within a radius of 500
m from the center of the focal population (mean 0.06,
range 0.02–0.11 for I and 0.20, 0.06–0.43 for C, t 
2.18, p  0.05). Both variables represented themarked differences between the two landscape types.
To determine forest areas and their related variables,
we optically scanned aerial photographs on a Macintosh computer and analyzed them using the NIH Image Program (version 1.62, developed at the U.S. National Institutes of Health and available from http://
rsb.info.nih.gov/nih-image/).
Trillium camschatcense is one of the dominant understory herbs in this region. It appears that formerly
huge T. camschatcense populations were divided into
small and isolated pieces with the fragmentation of theforests. Therefore, we presume that the remnant population sizes and their landscape types reflect the history of forest fragmentation. All study populations
were distant from other T. camschatcense populations, and we did not consider the effect of population
isolation.

Statistical Analyses

Stage-Class Population Structure

Three indices were determined for each population tocharacterize fruit and seed production (i.e., fruit set,
number of seeds per mature fruit, and number of seeds
per flower) (Table 2). We could not obtain data for the
RF population in 1999 because of the extremely low recovery of fruits from the marked individuals which resulted from intensive grazing, possibly by Yeso sika deer
(Cervus nippon var. yesoensis). A mature fruit produced, on average, 77.8 seeds in 1998 and 81.3 seeds in
1999. Each flower had 169.7  56.9 ovules (mean  SD;n  253), and approximately half the ovules developed
into seeds. Fruit set and mean number of seeds per ma-

In May 2000, we investigated population structures
based on life-history stages in six populations (SS, TN,
KK, MW, TB, and HI ). We randomly established 10, 1 
1 m quadrats on the forest floor of SS, TN, KK, and HI
populations. We established one quadrat in the MW
population and four quadrats in the TB population because of their small population size. All of the individuals in each quadrat were classified into one of the fourstage classes (i.e., seedling, one-leaf sterile, three-leavedsterile, or flowering).

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Volume 16, No. 5, October 2002

Fruit set, number of seeds per mature fruit, and numberof seeds per flower of each population were tested for
differences between the 2 years by paired t test. This ttest was conducted based on the mean values of each
population studied over 2 years. We tested the effect of
supplemental-hand pollination by nested analysis of variance by comparing the number of seeds of hand-pollinated flowers with open-pollinated controls. The dependent variable was the number of seeds produced, andthe effect of hand pollination was nested in that of population to distinguish between the variations among populations from the effect of artificial pollen addition. Relationships between outcross pollen load and seedproduction were explored by least-square linear regressions. We used analysis of covariance (ANCOVA) to test
the effects of population size and landscape type on thenumber of seeds produced per flower of each population. The dependent variable in this model was the number of seeds per flower. The main effects were the effectof population size (continuous variable) and of landscape type (categorical variable).
To analyze the population structure, we assessed theextent of heterogeneity of stage-class frequencies among
the populations examined through a Monte Carlo simulation in which a chi-square value was compared withthat estimated from 1000 randomizations by permuting
the original matrix (6  4 matrix) with the constraintthat row and column totals remain constant (Roff &Bentzen 1989). To explore the relationships between
each stage-class frequency in each population and population size, or the number of seeds per flower, we used
Kendall’s coefficient of rank correlations. All statisticalanalyses, except for the randomization test in the studyof population structure, were performed with the software SAS version 6.12 (SAS Institute 1997 ) or JMP version 4.0 (SAS Institute 2000).

Results
Fruit and Seed Production

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Effects of Forest Fragmentation on Trillium

Table 2. Fruit and seed production (mean  SD) for all Trillium
camschatcense populations examined for each year of the study.*
Year

Fruit set (%)
Number of seeds
per mature fruit
Number of seeds
per flower

Paired t test

1998

1999

t

p

44.5  19.5
(14.3–72.4)
77.8  28.5
(34.0–120.7)
34.8  19.9
(6.7–70.2)

52.9  20.0
(22.2–79.2)
81.3  27.8
(15.5–133.9)
41.8  19.1
( 7.8–66.9)

0.89

0.40

0.79

0.45

1.20

0.26

*Each variable was tested for the difference between years by pairedt test. Ranges are in parentheses.

ture fruit varied markedly among the populations examined. Therefore, mean number of seeds produced per
flower, which was employed as an index of total seedproduction, differed greatly among populations, ranging
from 6.7 (MW ) to 70.2 (HI) in 1998 and from 7.8 (CR) to
66.9 (TN ) in 1999. None of the three indices differed between years ( Table 2).
Pollen Load and Effect of Hand Pollination
The mean number of outcross pollen grains on stigmas
varied among populations from 22.8  12.6 (means SD; TB, n  4) to 148.5  60.5 (HI, n  5) in 1998 andfrom 16.0  11.9 (TB, n  4) to 209  93.4 (HI, n  8)
in 1999. Data from the SS population were excluded
from the analyses in both years because the samples
contained many pollen grains from several other species
that could not easily be distinguished from those of T.

Figure 1. Relationship between mean outcross pollenload and mean number of seeds produced per flower
of Trillium camschatcense populations in (a) 1998
and (b) 1999. Data are given for isolated (open circles) and continuous (closed circles) populations. Linear-regression lines are indicated in those cases where
the slope of the line was significantly different from
zero.

1281

camschatcense. The mean number of seeds per flower
was significantly related to the outcross pollen load in1998 ( Fig. 1a; regression coefficient, b  0.45, R2 
0.79, n  10, t  5.55, p  0.001), but not in 1999 (Fig.
1b; R2  0.21, n  9, t  1.34, p  0.22). The relationship in 1999, however, seemed to be similar to that in1998 except for one outlier ( HI; Fig. 1b). We therefore
used the regression line obtained in 1998 ( y  7.70 +0.45x, where x and y are the number of pollen grains
and the number of seeds per flower) to predict the number of seeds per flower that we should have found in
1999. With this procedure, we found no significant difference between the predicted and observed number ofseeds in 1999 (paired t test; n  9, t  1.12, p  0.30),
indicating a significant relationship between stigmaticpollen load and seed production in 1998 and the dataobtained in 1999. The mean number of seeds per flower
pollinated by hand was 57.9  21.8 (mean  SD), which
was higher than open-pollinated controls (37.4  21.8).
Artificial hand pollination significantly increased thenumber of seeds produced (nested analysis of variance[ANOVA]; df  11, MS  6674.81, F  2.08, p  0.05),
whereas the difference among populations was highly
significant (df  10, MS  16647.02, F  5.19, p 
0.0001). The mean number of seeds per flower after
hand pollination ranged from 25.3 (MB) to 104.3 (HI ).
Effects of Population Size and LandscapeType on Seed Production
In 1999 the mean number of seeds per flower was significantly and positively related to the log of population
size (regression coefficient, b  9.65, R2  0.43, n 
13, t  2.91, p  0.01; Fig. 2). In contrast, the mean
number of seeds per flower in 1998 had no relationshipto the log of population size. Analysis of covariance also
detected a significant effect of population size in 1999,
whereas no effect was observed in 1998 ( Table 3). Populations smaller than 50 flowering plants consistently
showed low seed production in both years ( Fig. 2).
There was no consistent trend for the effect of landscape type on seed production ( Table 3), but C populations produced more seeds than I populations inTable 3. Effects of population size (size), landscape type (type),
and their interaction (size  type) on the mean number of seeds
per flower observed for all Trillium camschatcense populations
examined (analyzed with analyses of covariance).
1998
MS

1999
F

p

Source

df

Size
Type
Size  typeError

1 582.87 1.76 0.22
1 1004.31 3.03 0.11
1
5.24 0.02 0.90
8 331.04

df

MS

F

p

1 1837.24 7.92 0.05
1 377.10 1.62
0.23
18.32 0.04
0.85
9 232.12

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Effects of Forest Fragmentation on Trillium

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Figure 2. Relationship between the log of population
size (estimated number of flowering plants) andmean number of seeds produced per flower of Trillium
camschatcense populations in (a) 1998 and ( b) 1999.
Data are given for isolated (open circles) and continuous (closed circles) populations. Solid line in 1999 represents regression line for all 13 populations where regression coefficient was significantly different fromzero.

both years ( Fig. 2). In particular, only some C populations with over approximately 1000 flowering plants(PK, HK, and HI ) were consistently successful in producing a certain number of seeds (e.g., 40 seeds)(nearly average numbers of seeds per flower, cf. Table2) in both years.
Stage-Class Population Structure
In the study of population structure, we surveyed1865 T. camschatcense individuals in total (Table 4).
No seedlings were observed in MW and TB, whichhave extremely small population sizes. We also found
few seedlings in the relatively small SS population ( Table 4), although a substantial number of seeds were
produced (the mean number of seeds per flower was
60.4 in 1998 and 38.0 in 1999). The relative frequencyof each stage class differed significantly among popula-

Table 4.

tions ( 2  478.3, rank  1/1000, p  0.001). The
proportion of seedlings had a higher correlation withpopulation size (  0.966, p  0.01) than with thenumber of seeds per flower in 1998 (  0.690, p 
0.058) and in 1999 (  0.690, p  0.058). In large
populations (TN, KK, and HI), the proportions of
three-leaved steriles were slightly lower than those offlowerings, whereas small populations had 0.83 (TB)
to 2.48 (SS) times more three-leaved steriles than flowerings. The proportion of three-leaved steriles was alsosignificantly and negatively correlated with population
size (  1.000, p  0.01), but not with the numberof seeds per flower (1998:  0.600, p  0.09; 1999: 
0.200, p  0.57 ).

Discussion
We studied the effects of forest fragmentation on thepollination, seed production, and demography of T.
camschatcense populations displaying different population sizes and occurring in different landscape conditions for 2 consecutive years. Seed production differedmarkedly among populations examined but not between years. The positive relationship between outcrosspollen load and seed production suggested that reduced
seed production could be attributed to pollen limitation.
In 1999 but not 1998, we found a positive relationshipbetween population size and seed production. Althoughno significant effect of landscape type was observed onseed production, C populations tended to produce ahigher number of seeds than I populations. Stage-class
population structure was better explained by population size rather than the number of seeds produced. The
proportion of seedlings was significantly and positively
related to population size. On the contrary, the proportion of three-leaved steriles was negatively related topopulation size but was uncorrelated with seed production. Collectively, these results point to the importanceof population size in promoting consistent reproductionin this species.

Stage-class population structure in six Trillium camschatcense populations observed in 2000.*
No. of individuals per 1 m2

Population

No. ofquadrats
examinedflowering

three-leavedsterileone-leaf
sterileseedling

1
4
10
10
10
10

2.0 (0.40)
1.8 (0.50)
2.7 (0.12)
7.8 (0.19)
5.5 (0.16)
7.5 (0.09)

3.0 (0.60)
1.5 (0.42)
6.7 (0.30)
5.2 (0.12)
2.5 (0.07)
4.9 (0.06)

0.0 (0.00)
0.3 (0.08)
12.7 (0.56)
12.0 (0.29)
12.1 (0.35)
13.3 (0.16)

0.0 (0.00)
0.0 (0.00)
0.6 (0.03)
17.0 (0.40)
14.9 (0.43)
59.2 (0.70)

MW
TB
SS
TN
KK
HI

* The mean number of T. camschatcense individuals per 1-m2 quadrat is listed for each stage class. Proportions of each stage class within populations are shown in parentheses.

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Tomimatsu & Ohara

Effects of Fragmentation on Plant Reproductive
Output and Its Mechanisms
The results of many previous studies concerning the effects of fragmentation on plant reproductive successsuggest that seed production is reduced in small, fragmented populations in both self-incompatible and selfcompatible plant species ( Jennersten 1988; Lamont etal. 1993; Aizen & Feinsinger 1994; Gigord et al. 1999;
Molano-Flores & Hendrix 1999; Kéry et al. 2000). Although the consequences of fragmentation are potentially diverse, such as increased spatial isolation between
populations and changes in the abiotic and biotic conditions of habitats suitable for plants and pollinators, studies
on factors other than size are limited. For instance, Yasaka
et al. (1994) found that surrounding green area positively
correlates with fruit set of populations of Corydalis ambigua, a bumblebee-pollinated understory spring herb.
In Clarkia concinna, population size and spatial isolation have an interaction effect on pollination and seedproduction (Groom 1998).
The effect of population size on seed production in T.
camschatcense was observed in one of the study years,
and small populations of 50 flowering plants produced
few seeds in both years (Fig. 2). As in many studies (e.g.,Jennersten 1988; Ågren 1996), the reduced number ofseeds could be attributed to pollen limitation. The outcross pollen load had a positive relationship with seedproduction (Fig. 1), and hand pollination significantly increased seed production, indicating that the amount of
outcross pollen could be a constraint for the seed production of self-incompatible populations of this plant.
Thus, the reduced seed production in small populations
must be a result of low quantity and quality of pollination services. Small populations may be less attractive topollinators, resulting in low pollination success (Sih &Baltus 1987). In some small populations with low flowering plant density ( Table 1), the density-related decline
in pollination efficiency may be more important because
pollinators will deposit other species’ pollen throughtheir long travel via nonconspecifics (Kunin 1993). Also,in populations of extremely small sizes, low genetic variation at the self-incompatibility locus (S locus) decreases
mate availability ( Byers & Meagher 1992; Byers 1995)
and consequently seed production, regardless of how
many pollinators and resources are available.
Differences in landscape conditions can also affect theavailability of pollinators in various ways (see also Kearns& Inouye 1997 ). Although some C populations apparently
produced a higher number of seeds than I populations,the effect of landscape type was not significant in either
year. The result may have been complicated by an outlier because the largest C population (RF ) received only
a few pollen grains on stigmas in both years (27.5 for1998 and 23.8 for 1999; the data for 1999 do not appear in
Fig. 2 because of fruit predation). Flower visitors of T.

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1283

camschatcense, small insect species belonging to Diptera
and Coleoptera, can travel only relatively short distances
and are thought to forage within only one or between
several closely linked forests. Because their foraging
ranges are still unknown, however, scale of the landscape analysis in our study may not accurately reflect
the scale of insect-visitor movements. Therefore, we attempted to design better models by adding other landscape variables (i.e., forest area and a proportion of forest within the radius of a certain distance [125 m, 250
m, 500 m, 1 km] from the center of the focal population) to the present ANCOVA model and incorporatingthem instead of landscape type; however, none of thesemodels satisfactorily described the variations observedin the number of seeds per flower in 1998. It is also possible that landscape variables were not significant because each pollinator species may have different requirements for nesting and food for larvae. The interactions
between population size and other variables illustratecomplex phenomena (Kearns et al. 1998), and further
studies are required to gain more knowledge about theeffects of fragmentation.
Our study seems to suggest that pollen limitation isjust one of several to many factors influencing reproductive output in fragmented populations. Seed production
was inconsistent between years, and seed production after supplemental hand pollination varied among populations. In such a fragmented landscape, other factors,
such as environmental deterioration (Oostermeijer et al.
1998), pesticide use (Batra 1982), and inbreeding depression (Gigord et al. 1998), may also be responsible
for reduced pollination services or seed production.
These factors may explain the high variance of seed production and the exceptionally low seed production of
the large, continuous RF population.
Consequences of Reduced Seed Production
The study of stage-class population structure indicatesthat population size rather than number of seeds produced has a high correlation with the relative frequencyof seedlings and three-leaved steriles, suggesting that pollen limitation is not the most important factor determining demographic processes. Few seedlings and many
three-leaved steriles were observed in small populations.
In this context, population size has more relative importance for demographic processes than does surrounding
landscape condition. Although T. camschatcense shows
continuous growth from seedling to flowering, with a
gradual yearly increase in leaf size, flowering plants
sometimes return to the three-leaved sterile stage in thesubsequent year (Ohara & Kawano 1986b). This stepback in life-history stage could be due to accidental damage to stems or leaves in the previous year (Ohara &Kawano 1986b) or as a result of deterioration in environmental conditions. Thus, the high number of three-leaved

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Effects of Forest Fragmentation on Trillium

steriles in small populations may be caused by an increased step-back change from flowering to three-leavedsterile.
Reduced seed production must in part contribute to
low seedling recruitment in small populations, but environmental deterioration, such as “edge effects” causedby fragmentation, may also have significant effects on
population structure. Our other study of edge effects on
T. camschatcense showed similar results. The forest
edges in the KK population had a lower number of seedlings and a higher number of three-leaved steriles than
the forest interiors, and these effects of edge formation
penetrated more than 70 m from the edges into the forest (H. T. & M. O., unpublished data). Forest fragments
are exposed to drastic changes in microclimatic conditions, and abiotic and biotic environments are greatly altered near forest edges ( Lovejoy et al. 1986; Murcia
1995; Laurance & Bierregaard 1997). The extent of edge
effect should be correlated with the size and shape ofthe focal forest. In the study populations, the distancefrom the center of the population to the nearest forestedge was highly correlated with population size ( 
0.729, p  0.001; H. T. & M. O., unpublished data).
Therefore, small populations are expected to suffer fromstrong edge effects in addition to the effects of reduced
seed production. Even small changes in light availability
and soil-water conditions could affect the demography(especially seedling establishment) of forest plant species growing in a relatively stable environment.
Implications for Conservation
Our study does offer some information on the specifichabitat size and landscape conditions that must be
conserved. Small populations of 50 flowering plants
consistently showed low seed production, whereas C
populations with more than approximately 1000 flowering plants ( PK, HK, and HI ) were successful in producing constant and high seed production (Fig. 2).
The demographic study suggested that C population
with 220 flowering plants (SS) had almost no seedlingrecruitment (Table 4). It therefore appeared that C
populations with more than 1000 flowering plants
were essential to reproduction. However, seed production clearly was not the only factor determining
subsequent demography. Further studies combinedwith long-term observations of demographic changes
are required (e.g., Groom 1998; Jules 1998; Menges &Dolan 1998), and population viability analysis (Shaffer
1990; recently reviewed by Menges 2000), by meansof matrix projections, can provide population-extinction probabilities and evaluate minimum viable populations (Shaffer 1981). Landscape conditions should beanalyzed in detail and partitioned into some quantitative factors to lead to more concrete propositions forconservation.

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Tomimatsu & Ohara

Acknowledgments
We are grateful to T. Ishibuchi, A. Tazawa, K. Shiojiri, S.
Maezaki, M. Imamura, M. Oda, and S. Shimura for assistance in the field; K. Hiei for technical advice on pollencounting; T. Yahara for comments on statistical analyses;
and C. Kremen and three anonymous reviewers for critical comments on the manuscript. We also acknowledge
the Hiroo town office for offering the use of their facility. This work was funded in part by the Sasakawa Scientific Research Grant from the Japan Science Society, theHokscitec Foundation, Grants-in-Aid from the Japan Society for the Promotion of Science ( JSPS) for Scientific Research (10440230, 13640637), and research fellowshipsof the JSPS for Young Scientists.
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