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Mating Structure and Inbreeding and Outbreeding Depression in The Rare Plant Gentianella Germanica (Gentianaceae)
Mating Structure and Inbreeding and Outbreeding Depression in The Rare Plant Gentianella Germanica (Gentianaceae)
MATING
AND
DIETHART MATTHIES
Institut fur Umweltwissenschaften, Universitat Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland
Isolation and small size of populations as a result of habitat destruction and fragmentation may negatively affect plant
fitness through pollinator limitation and increased levels of inbreeding. To increase genetic variation in small populations
of rare plants artificial gene flow has been suggested as a management tool. We investigated whether pollinator limitation
and inbreeding depression could reduce fitness in Gentianella germanica, an endangered biennial of increasingly fragmented
calcareous grasslands in Central Europe. We experimentally excluded pollinators and generated progenies by hand-pollinating
flowers with pollen from different distances. G. germanica was highly selfing. Pollinator exclusion strongly reduced seed
set, indicating that pollinator limitation could potentially reduce plant fitness. Germination rate as well as number of leaves
and rosette size of progeny from 10-m crosses was higher than that of progeny from open pollinations, self-, 1-m, and
interpopulation crosses. After 6 mo of growth differences in the number of surviving plants persisted, whereas differences
in plant size did not. The results suggest that inbreeding depression may reduce plant performance in G. germanica.
Outbreeding depression in the performance of progeny from interpopulation crosses indicates that caution is necessary in
using artificial interpopulation gene flow as a management tool.
Key words: Gentianaceae; Gentianella germanica; hand pollination; inbreeding depression; mating system; outbreeding
depression; pollen quality; reproduction.
average plant fitness, in the short term because of inbreeding depression, which may be enhanced further by
the accumulation of mildly detrimental mutations (mutational meltdown, Lande, 1994; Lynch, Conery, and
Burger, 1995), and in the long term because the decrease
in genetic variation reduces the potential to adapt to
changing environmental conditions.
Pollen transfer as well as seed migration between populations seems to be very rare in many species (Slatkin,
1985). Nevertheless, for the maintenance of a sufficient
level of genetic diversity and the prevention of increasing
inbreeding levels occasional gene transfer between isolated populations is crucial (Ellstrand and Elam, 1993).
Increased fitness of offspring from interpopulation crosses has been found in a number of studies and the artificial
increase of gene flow among rare plant populations has
been suggested as a management tool (Van Treuren,
1993; Oostermeijer, Altenburg, and den Nijs, 1995).
However, large-distance gene transfer is not always beneficial. If genes in a local population have adapted to the
genetic environment defined by other genes (intrinsic
coadaptation) or if different populations have become
locally adapted, gene flow might result in outbreeding
depression (Price and Waser, 1979; Templeton, 1986;
Waser and Price, 1989; Lynch, 1991; Waser, 1993). However, there are hardly any studies that have actually investigated the possible effects of increased gene flow between populations of rare plants.
Genetic considerations are probably most important for
declining species with short generation times, i.e., annuals and biennials. For these species pollinator limitation
is also of particular importance, because their persistence
largely depends on seed production. In a previous study
on the demography of the formerly common biennial
Gentianella germanica (Gentianaceae), which is now rare
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Test for apomixis and seed set of emasculated flowersIn September 1994, we selected 22 plants of G. germanica in a population of 660
flowering plants near Kleinlutzel, 25 km southwest of Basel, Switzerland. On each plant we marked two flowers that were about to open.
To test the importance of own pollen for seed set we esmaculated one
flower by removing the anthers with a pair of fine forceps. Between
treatments we sterilized the forceps in a flame. We additionally caged
half of the emasculated flowers. Unless there is pseudogamy, this allows
to test for ability to set apomictic seeds. Around these flowers we placed
small bags made from pollen-proof fabric and carefully closed them
with gardening wire. To stabilize the manipulated flowers we fixed the
bags to small poles. After 4 wk we collected the fruits and counted the
number of ovules and seeds in each fruit.
For the estimation of offspring fitness we used two independent performance measures: the germination rate and the rosette diameter on
day 79. We took the fitness estimate for the offspring from 10-m crosses
as an estimate of woutcrossed. We calculated the standard error of s from
the standard errors of the two performance parameters using standard
error propagation formulas (Bronstein and Semendjajew, 1981).
Effects of pollinator exclusion and interparental distanceIn September 1993, we randomly selected and marked 90 plants in a population of 1400 flowering plants near Langenbruck, 35 km south of Basel,
Switzerland. We took five measures of plant size: numbers of branches,
leaves, and flowers, plant height, and widest diameter.
We randomly selected one flower on each plant that was about to
open, but with both stigma and anthers still closed. We subjected the
selected flowers to one of six different treatments (15 replicates per
treatment): (1) no experimental manipulation (open pollination), (2)
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TABLE 1. Skeleton ANOVA for the effect of different pollination treatments. Pollination modes were open pollination, exclusion of pollinators
and hand-pollination. The hand-pollination treatment consisted of hand-pollinations with four different interparental distances.
Source of variation
df
Mean squares
Pollination mode
Hand-pollination mode nested within pollination mode
Maternal plant (seed family) nested within two upper levels
Residual variation among pots
2
3
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394
MSP
MSH
MSF
MSI
MSP/MSF
MSH/MSF
MSF/MSI
RESULTS
Test for apomixis and seed set of emasculated flowersEmasculation and caging had no effect on the mean
(61SE) number of ovules per fruit (70.7 6 4.16, N 5
38), but significantly affected the number of seeds per
fruit. The 12 flowers that had been both emasculated and
caged produced no seeds at all. Unless the species is
pseudogamous, this indicates that there is no apomixis in
the study population. Emasculation without caging reduced seed set by 66% compared with open pollination
(P , 0.001; Fig. 1), indicating that pollination within the
same flower is an important part of natural pollination.
Effects of pollinator exclusion and interparental distancePlant size did not affect seed production per fruit
in G. germanica. There was a weak negative relationship
between the number of flowers of the maternal plant and
the number (N 5 72, r 5 20.16, P , 0.18) and mass of
seeds per fruit (N 5 72, r 5 20.16, P , 0.17). The
position of a fruit along the inflorescence did not affect
its number of seeds. The size of the paternal plant also
had no influence.
The exclusion of pollinators by cages reduced the number of seeds per fruit by 55% (Fig. 2) and seed mass per
fruit by 65% compared with open pollination (Tables 2,
3). This indicates that pollinators contribute considerably
to natural pollination, and that pollinator limitation could
potentially reduce fitness of G. germanica. In contrast,
there were no significant differences in these reproductive
Figs. 23. The effect of six pollination treatments on (2) the mean
number of seeds per fruit and (3) the germination rate in Gentianella
germanica. Flowers were either caged to exclude pollinators, open-pollinated, or hand-pollinated after emasculation. Hand-pollination was
carried out with self pollen and with pollen from 1 m, 10 m (same
population), and 25 km (different population) distances. Error bars indicate 1 SE.
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Pollination mode
Interparental
distance
Seed family
14.8
10.5
3.50
0.0389
0.193
1.15
1.97
3.80
3.20
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TABLE 3. The effect of (A) different pollination modes and (B) different interparental distances in the hand-pollination treatment on
reproduction and offspring fitness in Gentianella germanica. Flowers were either caged to exclude pollinators, open-pollinated, or
hand-pollinated after emasculation. Hand-pollination was carried
out with self pollen and with pollen from 1 m, 10 m (same population), and 25 km (different population) distances. The fitness estimate w1 is the number of surviving offspring per fruit, w2 is the
product of w1 and the mean cumulative leaf length. Data on germination, survival, and multiplicative fitness w1 were angular-transformed prior to analysis; data on leaf length and w2 were log transformed. Back-transformed means are given in the table. Values in
a line with different superscripts are different at the 5% level (Tukeys HSD procedure).
A)
Pollination mode
Day 79
Number of leaves
Rosette diameter (mm)
Cumulative leaf length (mm)
2.57
1.32
2.14
7.15
4.14
5.95
1.25
2.12
1.99
Day 175
Surviving plants per seed
Number of leaves
Rosette diameter (mm)
Cumulative leaf length (mm)
w1
w2
0.376
0.491
2.03
1.94
8.60
8.56
3.51
0.604
2.03
0.373
3.34
4.17
2.78
2.78
1.46
1.99
Caged
Open
23.5a
3.28a
0.137a
52.4b
9.28b
0.192b
60.8b
9.88b
0.161ab
Day 79
Germination rate
Number of leaves
Rosette diameter (mm)
Cumulative leaf length (mm)
0.507
6.22
21.8
69.5
0.531
6.30
23.5
78.2
0.542
6.65
24.9
86.7
0.424
11.55
36.41
214
5.12a
92.1a
0.439
11.15
39.85
227
16.3b
299b
0.491
11.08
38.77
219
25.1b
446b
Parameter
ences in pollen viability between different hand-pollination treatments are unlikely. In contrast, the germination
rate of seeds resulting from far crosses within a population was much higher than that of seeds resulting from
the other treatments. The germination rate of seeds resulting from pollinations with pollen from a distance of
10 m was . 40% higher than that of seeds from selfed
flowers, from flowers pollinated with pollen from a distance of 1 m, or from flowers pollinated with pollen from
a different population (Fig. 3). This indicates inbreeding
depression after short-distance crosses and outbreeding
depression after interpopulation crosses.
Similarly, after 79 d of growth the number of leaves,
the rosette diameter, and the cumulative leaf length were
highest for the progeny of the 10-m treatment. The differences in plant size decreased with time. On day 175
survivorship of progeny of the 10-m treatment and that
of other treatments was still significantly different,
whereas the size of the surviving plants did not significantly differ between treatments any more. The survival
rate per pot (after angular transformation) from day 79
until day 175 was positively correlated with the mean
number of leaves (r 5 1 0.169, F 5 10.01; P , 0.002)
and the mean diameter of the plants in a pot (r 5 1
0.139, F 5 6.75; P , 0.01) on day 79. This indicates
that mainly small plants died during this period, which
diminished the differences in plant size between treatments.
Multiplicative fitness was lowest in the caged treatment
and highest in the hand-pollination treatment with the
intermediate outcrossing distance of 10 m (Table 3).
These differences were significant for day 175. This indicates consistent inbreeding depression after short-distance crosses and outbreeding depression after interpopulation crosses until day 175.
Overwinter mortality from day 175 to day 420 was
Day 175
Surviving plants per planted
seed
Number of leaves
Rosette diameter (mm)
Cumulative leaf length (mm)
w1
w2
Hand
B)
Interparental distance
Parameter
Selfed
1m
Day 79
Germination rate
Number of leaves
Rosette diameter (mm)
Cumulative leaf length (mm)
0.490a
0.513a
0.714b 0.453a
6.08a
6.43ab
7.10c
6.98bc
22.9a
23.0a
28.5b
25.4ab
72.4a
78.4a
104.3b
91.5ab
0.413a
11.2
36.4
208
18.9a
330a
0.473a
11.0
39.2
220
25.4ab
455ab
64.4
11.5
0.18
25 km
59.8
10.6
0.176
Day 175
Surviving plants per planted
seed
Number of leaves
Rosette diameter (mm)
Cumulative leaf length (mm)
w1
w2
60.9
8.93
0.142
10 m
0.700b
10.7
40.9
220
40.9b
792b
57.9
8.45
0.145
0.373a
11.4
38.6
224
17.2a
277a
December 1997]
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estimates are based on data from different years and different populations. Outcrossing rates may vary among
populations (Schemske and Lande, 1985), among years,
and among seasons (Molau et al., 1989). Nevertheless,
both independently derived estimates suggest high selfing
rates. Self compatibility might allow G. germanica to establish a new population from a single seed after longdistance dispersal (Baker, 1955, 1967). A high selfing rate
is also of advantage for species whose populations show
large annual fluctuations in the number of individuals
(Stebbins, 1957), as reported for G. germanica (Runge,
1963). Inbred offspring of G. germanica suffered on the
other hand from reduced fitness, and selfing should therefore not be favored. The findings are in line with the
interpretation of mixed mating systems as a result of the
conflict between moderate levels of inbreeding depression that would favor selfing, and the genetic association
of outcrossing and high fitness, which favors some outcrossing (Uyenoyama, Holsinger, and Waller, 1993).
In a previous study we found significant genetic variation among 25 populations of G. germanica and reduced
levels of genetic variation in smaller populations (Fischer,
1996). Significant genetic variation among isolated populations also has been found in several other rare species
(Waller, OMalley, and Gawler, 1987; Brauner, Crawford,
and Stuessy, 1992; Raijmann et al., 1994). This suggests
that the average degree of relatedness of parent plants
used in this study was lower for parents of interpopulation crosses than for within-population crosses.
The most unusual aspect of our study is the detection
of significant outbreeding depression after interpopulation crosses. There are several mechanisms by which
gene transfer into a population could lead to reductions
in fitness: through disruption of coadapted gene complexes (Price and Waser, 1979; Templeton, 1986; Dudash,
1990), or by the disruption of local adaptations (Price and
Waser, 1979), a phenomenon that in plants has even been
observed within distances of 30 m (Waser and Price,
1994). Reproductive isolation in plants could also evolve
as a direct result of adaptive differentiation in sympatry
(MacNair and Christie, 1983).
Botanists have been reluctant to accept the phenomenon of outbreeding depression (Waser, 1993). In contrast,
zoologists have been aware already a decade ago of the
problem of simultaneously minimizing detrimental effects of inbreeding and outbreeding depression in breeding programs or management of natural populations
(Templeton 1986). In principle, enhanced gene flow
among isolated populations could be beneficial for average plant fitness in the short term through heterosis
effects (van Treuren et al., 1993; Hauser and Loeschke,
1994) and in the long term because of enhanced genetic
variation in the population (Ouborg and van Treuren,
1994; van Treuren et al., 1994). For example, in the rare
gentian G. pneumonanthe seed set and several measures
of offspring fitness, e.g., adult mass, increased with increasing outcrossing distance (Oostermeijer, Altenburg,
and Den Nijs, 1995). This was mainly the result of low
fitness of selfed offspring and high fitness of offspring
resulting from interpopulation crosses. Similarly, crosses
between individuals of different populations substantially
increased measures of offspring fitness such as biomass
and adult survivorship in Scabiosa columbaria (Van
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quences of small population size: implications for plant conservation. Annual Review of Ecology and Systematics 24: 217242.
FISCHER, M. 1996. Experimental population biology of the rare Gentianella germanica. PhD. dissertation, University of Basel, Basel.
HAIG, D., AND M. WESTOBY. 1988. On limits to seed production. American Naturalist 131: 757759
HAUSER T. P., AND V. LOESCHKE. 1994. Inbreeding depression and mating-distance dependent offspring fitness in large and small populations of Lychnis floscuculi (Caryophyllaceae). Journal of Evolutionary Biology 7: 609622.
HEGI, G. 1926. Illustrierte Flora von Mitteleuropa, vol. 5. Lehmanns,
Munchen.
HESCHEL, M. S., AND K. N. PAIGE. 1995. Inbreeding depression, environmental stress, and population size variation in scarlet gilia
(Ipomopsis aggregata). Conservation Biology 9: 126133.
JENNERSTEN, O., J. LOMAN, A. P. MLLER, J. ROBERTSON, AND B. WIDEN.
1992. Conservation biology in agricultural habitat islands. In L.
Hansson [ed.], Conservation biology by ecological principles, 396
425. Elsevier, London.
, AND S. G. NILSSON. 1993. Insect flower visitation frequency
and seed production in relation to patch size of Viscaria vulgaris
(Caryophyllaceae). Oikos 68: 283292.
JOHNSTON, M. O. 1991. Pollen limitation of female reproduction in
Lobelia cardinalis and L. siphilitica. Ecology 72: 15001503 .
KORNECK, D., M. SCHNITTLER, AND I. VOLLMER. 1996. Rote Liste der
Farn- und Blutenpflanzen (Pteridophyta et Spermatophyta)
Deutschlands. Schriftenreihe fur Vegetationskunde 28: 21187.
KUNIN, W. E. 1992. Density and reproductive success in wild populations of Diplotaxis erucoides (Brassicaceae). Oecologia 91: 129
133.
. 1993. Sex and the single mustard: population density and pollinator behavior effects on seed set. Ecology 74: 21452160.
LACY, R. C. 1987. Loss of genetic diversity from managed populations:
interacting effects of drift, mutation, immigration, selection, and
population subdivision. Conservation Biology 1: 143158.
LANDE, R. 1994. Risk of population extinction from fixation of new
deleterious mutations. Evolution 48: 14601469.
. 1995. Mutation and conservation. Conservation Biology 9:
782791
LANDOLT, E. 1991. Gefahrdung der Farn- und Blutenpflanzen in der
Schweiz mit gesamtschweizerischen und regionalen roten Listen.
Bundesamt fur Umwelt, Wald und Landschaft (BUWAL), Bern.
LEVIN, D. A. 1984. Inbreeding depression and proximitydependent
crossing success in Phlox drummondii. Evolution 38: 116127.
LLOYD, D. G., AND D. J. SCHOEN. 1992. Self- and cross fertilization in
plants. I. Functional dimensions. International Journal of Plant Sciences 153: 358369
LYNCH, M. 1991. The genetic interpretation of inbreeding depression
and outbreeding depression. Evolution 45: 622629.
, J. CONERY, AND R. BURGER. 1995. Mutation accumulation and
the extinction of small populations. American Naturalist 146: 489
518
MACNAIR, M. R., AND P. CHRISTIE. 1983. Reproductive isolation as a
pleiotropic effect of copper tolerance in Mimulus guttatus? Heredity 50: 295302.
MENGES, E. S. 1991a. The application of minimum viable population
theory to plants. In D. A. Falk and K.E. Holsinger [eds.], Genetics
and conservation of rare plants, 4761. Oxford University Press,
New York, NY.
. 1991b. Seed germination percentage increases with population
size in a fragmented prairie species. Conservation Biology 5: 185
164.
. 1992. Stochastic modeling of extinction in plant populations.
In P. L. Fiedler and S. K. Jain [eds.], Conservation biology: the
theory and practice of nature conservation, preservation and management, 253276. Chapman and Hall, New York, NY.
. HILL. 1989. Mating
MOLAU, U., M. CARLSSON, A. DAHLBERG, AND O
system and pollenmediated gene flow in Bartsia alpina. Oikos
55: 409419.
OBERDORFER, E. 1994. Pflanzensoziologische Exkursionsflora. 7. Auflage. Ulmer, Stuttgart.
OLESEN, J. M. AND S. K. JAIN. 1994. Fragmented plant populations and
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