Annals of Botany 89: 145±156, 2002
doi:10.1093/aob/mcf020, available online at www.aob.oupjournals.org
Barriers to Sexual Reproduction in Polygonum viviparum: A Comparative
Developmental Analysis of P. viviparum and P. bistortoides
P A M E L A K . D I G G L E * , M A R G A R E T A . M E I X N E R ² , A M Y B . C A R R O L L ³ and
CHRISTIE F. ASCHWANDEN§
Department of Environmental, Population and Organismic Biology, University of Colorado, Boulder,
CO 80309-0334, USA
Received: 18 July 2001 Returned for revision: 12 September 2001 Accepted: 11 October 2001
Polygonum viviparum is widely distributed in arctic and alpine regions of the northern hemisphere. Fruit set has
never been observed in North American populations and has been reported only very rarely in Europe. Although
this species is extremely well studied, the impediments to successful fruit production are unknown. We investigated the sexual reproductive process in P. viviparum growing in the southern Colorado Rocky Mountains. For
comparison, we also examined this process in the sympatric congener P. bistortoides, in which reproduction is
exclusively sexual. Lack of viable fruit production in P. viviparum has no single developmental explanation;
defects occur in each of the processes and structures associated with sexual reproduction studied, yet, these processes and structures also appear to function normally in at least some ¯owers or individuals. Development is
abnormal in many ovules of P. viviparum, however, comparison with P. bistortoides shows that these abnormalities do not contribute to differences in seed production between the two species. The virtual absence of sexual
reproduction in P. viviparum appears to be due largely to a low rate of fertilization and to embryo/fruit abortion.
ã 2002 Annals of Botany Company
Key words: Asexual reproduction, embryo abortion, development, fruit abortion, pollen viability, Polygonaceae,
Polygonum viviparum, Polygonum bistortoides, polyploidy, seed abortion, sexual reproduction.
INTRODUCTION
The majority of ¯owering plants are capable of some form
of asexual reproduction by means of a diverse range of
structures and mechanisms. Clonal progeny may be produced by stolons, runners, rhizomes, tubers, buds on bulbs,
corms and roots, layering of stems, and agamospermous
seed (Grant, 1971). Despite the prevalence of asexual
mechanisms of propagation, most clonal species also
reproduce sexually (Stebbins, 1971; Silander, 1985;
Richards, 1997) and exclusively asexual species are rare.
Polygonum viviparum L. may be one of the few species that
relies solely upon vegetative reproduction throughout much
of its range (Fryxell, 1957; Callaghan, 1973; Engell, 1973;
Petersen, 1981).
Polygonum viviparum L. [= Bistorta vivipara (L.) S.
Gray] (Polygonaceae) is widely distributed in both arctic
and alpine regions of the northern hemisphere (Petersen,
1981; Law et al., 1983; Callaghan and Emanuelsson, 1985;
Bauert, 1993; Wookey et al., 1994). Although most
individuals of P. viviparum ¯ower profusely, fruit set has
never been observed in alpine populations of North America
and has been reported only very rarely in Europe (Bauert,
1993). P. viviparum is a very common and important
* For correspondence. Fax 001 303 492 8699, e-mail Pamela.
[email protected]
² Present address: 22 Forest Road, Delmar, NY 12054-3039, USA
³ Present address: Boreal Ecology Cooperative Research Unit,
University of Alaska, PO Box 756780, Fairbanks, AK 99775-6780, USA
§ Present address: 111 Bonanza Drive, Nederland, CO 80466, USA
component of tundra plant communities and has been the
subject of numerous ecological studies (Callaghan, 1973;
Petersen, 1981; Bauert, 1993; Crawford et al., 1993;
Wookey et al., 1994 and references cited therein), and
most workers comment on the lack of seed set. The only
published reports of sexual reproduction in P. viviparum
include: Porsild and Porsild (1920) in Greenland; SoÈyrinki
(1989) in Scandinavia; Bliss (1959) in the subalpine of the
Rocky Mountains of Wyoming; Murray and Miller (1982)
in interior Alaska; and Bauert (1993) in the central Swiss
alps. Sexual reproduction is clearly a rare event in this
species.
In contrast to the rarity of fruit set by P. viviparum,
asexual reproduction occurs readily via the production of
bulbils, vegetative axillary buds borne within in¯orescences
(Troll, 1937; Engell, 1973; Diggle, 1997). After bulbils are
dispersed, they germinate and grow under moist conditions
(Callaghan, 1973; Engell, 1973, 1978; Petersen, 1981) and
establish new, physiologically independent plants that are
genetically identical to the parent.
The impediments to successful fruit set in P. viviparum
are unknown. Any of the many stages of development of
male and female gametophytes, gametes, embryo and
endosperm, or the processes of pollination and fertilization,
might pose a barrier to sexual production of progeny in this
species. Preliminary embryological studies of P. viviparum
have been published (Edman, 1929; Schnarf, 1931; Engell,
1973); however, the causes of sterility were not identi®ed.
In order to explain the lack of fruit production, we
ã 2002 Annals of Botany Company
146
Diggle et al. Ð Sexual Reproduction in Polygonum
investigated the sexual reproductive process of P. viviparum
growing in the alpine tundra of the southern Colorado
Rocky Mountains. Our study focused on ovule and female
gametophyte development, pollen viability and early
embryo/seed development. For comparison, we also examined these processes in the sympatric congener, P.
bistortoides. P. bistortoides is very common at the study
site, produces large numbers of seed and has no means of
vegetative reproduction.
MA TE R IA L S A N D ME T H O D S
Study site
Study sites were located in the alpine tundra of Niwot
Ridge (elevation 3750 m) in the Front Range of the
eastern
Colorado
Rocky
Mountains
(40°03¢N,
105°35¢W). Niwot Ridge is managed cooperatively by
the University of Colorado Mountain Research Station
and the United States Forest Service as an ecological
reserve and is the site of the Niwot Ridge Long-Term
Ecological Research Program. Polygonum viviparum and
P. bistortoides were sampled haphazardly in a dry
meadow community.
Species description
Polygonum viviparum is widely distributed in both
arctic and alpine regions of the northern hemisphere
(Petersen, 1981; Law et al., 1983; Callaghan and
Emanuelsson, 1985; Bauert, 1993; Wookey et al.,
1994). Within the study area, P. viviparum is common
in all plant communities except in areas of very late
melting snow (May and Weber, 1982). Polygonum
bistortoides Pursh. (= Bistorta bistortoides (Pursh)
Small), is sympatric with P. viviparum in the southern
Rocky Mountains and is also widely distributed across
all alpine communities, including snowbeds (Walker
et al., 1995). Both species are herbaceous perennials.
The main axis of each is a monopodial unbranched
rhizome that grows plagiotropically, with little internode
elongation, below the soil surface. Rhizomes mature
several preformed leaves and axillary in¯orescences each
year (Diggle, 1997; P.K.D., pers. obs.). P. bistortoides is
larger than P. viviparum in most respects: rhizomes are
longer and of greater diameter; leaves are longer, wider
and more numerous; and in¯orescences are taller. The
reproductive characters of the two species are very
similar. In¯orescences are condensed panicles bearing
numerous white ¯owers. Flowers are small, approx. 3 mm
in length. The perianth consists of ®ve white tepals
[Laubengayer (1937); Ronse Decraene and Akeroyd
(1988), review interpretations of the perianth of the
polygonaceous ¯ower]. Stamen number is variable and the
gynoecium consists of a three-angled, uniovulate ovary
with three styles and stigmas. The two species differ in
one critical aspect of in¯orescence structure (and consequently, life history): P. viviparum may bear bulbils in the
proximal region of the in¯orescence whereas P. bistortoides bears only ¯owers and reproduces exclusively by
seed (Mooney, 1963). Both species ¯ower in July and
mature fruits of P. bistortoides are dispersed in August. P.
viviparum is polyploid with 2n = 96, x = 12 (LoÈve and
LoÈve, 1948, 1974, 1975; Wcislo, 1967; Engell, 1973;
LoÈve, 1988). P. bistortoides is diploid with 2n = 24
(Mooney, 1963).
Ovule and female gametophyte development
All ¯oral organs are initiated in the growing season prior
to ¯ower maturation. The preformed in¯orescence-bearing
¯oral primordium undergoes dormancy below ground.
Development resumes in the spring and the in¯orescence
begins to emerge above ground. Sampling of developing
in¯orescences began when the tips were ®rst visible above
the soil surface (®rst week in July, 1995) and continued until
¯owers reached anthesis. All in¯orescences were ®xed in
formalin±acetic acid±alcohol (Berlyn and Miksche, 1976).
Flowers and ¯ower buds were sampled from basal positions
of the short (two or three ¯owered) branches of the
paniculate in¯orescences. The gynoecium was dissected
from mature ¯owers and large buds. Small buds were
processed whole. Gynoecia and buds were dehydrated to
95% EtOH, embedded in JB4 methacrylate resin, serially
sectioned on a Microm microtome at 4 mm, stained with
toluidine blue (O'Brien and McCully, 1981) and observed
with a Zeiss Axioskop.
Pollen viability
Mature in¯orescences were collected, placed in sealed
microcentrifuge tubes, and transported to the laboratory on
ice. In 1992 (year 1), in¯orescences from 150 individuals of
P. viviparum were collected. In 1993 (year 2), in¯orescences were collected from 20 P. viviparum and 47 P.
bistortoides individuals. All ¯owers were examined within
12 h of collection.
The FCR (¯uorochromatic) test (Heslop-Harrison and
Heslop-Harrison, 1970; Shivanna et al., 1991) was used to
evaluate pollen viability. [See Thomson et al. (1994) and
Stone et al. (1995) for discussions of the reliability of FCR
as a measure of pollen viability.] The FCR test may give
false negative results due to pollen drying (Heslop-Harrison
et al., 1984; Shivana and Johri, 1985; Knox et al., 1986;
Thomson et al., 1994). To prevent desiccation, ¯owers were
kept in the humid atmosphere of the microfuge tubes until
the test was performed.
Three to ®ve ¯owers per in¯orescence were examined.
All anthers were removed from a single ¯ower and were
placed on a glass slide. Pollen grains were teased from the
anthers with ®ne forceps. Each slide was ¯ooded with a
solution of ¯uorecine diacetate (FDA; 0´02 g FDA/10 ml
acetone in 2 ml of 10 % sucrose) for 5±10 min. Slides were
examined with a Zeiss Axioskop equipped for epi¯uorescence. Bright®eld was used to count all pollen grains
present. The same slide was then examined under ultraviolet
light. Stainability was calculated as the number of
¯uorescing grains divided by the total number of grains
present. The stainability of all ¯owers from the same
in¯orescence was averaged, and this mean was used in
Diggle et al. Ð Sexual Reproduction in Polygonum
147
F I G . 1. Early ovule development in P. bistortoides (A, C and E) and P. viviparum (B, D and F). A, B, Nucellus surrounded by ovary wall. A
megasporocyte is evident in B. C, D, Megasporocyte(s) entering prophase of meiosis I. The inner integument has been initiated. E, F,
Megasporocyte(s) in meiosis, the outer integument has been initiated. B, Nucellar beak; ii, inner integument; M, megasporocyte; N, nucellus; O, ovary
wall; oi, outer integument. Bars = 25 mm.
148
Diggle et al. Ð Sexual Reproduction in Polygonum
F I G . 2. Megaspore formation in P. bistortoides (A, C) and P. viviparum (B, D). A, End of meiosis II. The four spores that will form a tetrad
are evident but the last walls have not yet formed. B, Linear tetrad. The chalazal-most spore is on an adjacent section; its position is indicated by
the arrow. C, D, The chalazal-most spore (arrows) of each tetrad is functional, the remaining spores degenerate. A hypostase begins to differentiate
at the base of the nucellus. Note multiple megaspores in P. bistortoides (C). ii, Inner integument; oi, outer integument; H, hypostase.
Bars = 25 mm.
subsequent statistical analyses. Pollen stainability was
analysed with StatView for Macintosh. Stainability data
were arcsine square root transformed prior to analysis.
R E SU L T S
Pre-fertilization ovule development
Ovule development is nearly identical in the two species,
and the description that follows applies to both with
exceptions as noted.
Flowers of both species are uniovulate, and initiation of
the ovary and nucellus had already occurred in all material
collected. In¯orescence and ¯oral primordia are preformed,
and these events probably occur in the year preceding
in¯orescence maturation. In the youngest ¯oral primordia
sampled, the nucellus was already enclosed by the ovary
wall (Fig. 1A, B). The large nucellus has an epidermal layer
and one parietal layer. In ovules of both species, the
epidermis of the nucellus is often elaborated into an apical
`beak' (e.g. Fig. 1C, D, F).
The species differ in the number of megasporocytes per
nucellus. In P. bistortoides, each nucellus develops one
(rarely) to several megasporocytes (Fig. 1C). In P.
viviparum there is typically only one megasporocyte per
nucellus (Fig. 1D) although occasionally there are two. In
both species, as megasporocyte differentiation occurs, the
nucellus elongates, and the inner integument of the ovule is
initiated (Fig. 1C, D).
The megasporocyte (P. viviparum) or megasporocytes (P.
bistortoides) undergo meiosis (Figs 1E and 2B) and each
forms a linear tetrad (Fig. 2B). The chalazal-most spore of
each tetrad becomes the functional megaspore while the
remaining three spores degenerate (Fig. 2C, D). In P.
bistortoides there may be several functional megaspores per
ovule, each the product of a separate meiotic event (Fig.
2C). A hypostase begins to differentiate at about the time of
spore formation (Fig. 2C, D).
Diggle et al. Ð Sexual Reproduction in Polygonum
149
F I G . 3. Mature ovules of P. bistortoides (A and B) and P. viviparum (C). Part B shows the section adjacent to that in part A showing the position of
the fusion nucleus (arrow) relative to the egg. C, Arrow indicates position of the fusion nucleus. E, Egg; ii, inner integuments; N, nucellus; oi, outer
integuments; S, synergids. Bars = 50 mm.
At, or just prior to meiosis of the megasporocyte(s), the
outer integument of the ovule is initiated (Figs 1E and 2B).
As megagametophyte development begins, the integuments
elongate and enclose the nucellus. The micropylar region of
the inner integument expands, becoming several cell layers
thick, and forms the micropyle. The outer integument
remains two cell layers thick throughout development and
does not contribute to the micropyle.
One (P. viviparum) to several (P. bistortoides) gametophytes begin to develop within the nucellus. During this
time, the gametophytes elongate only slightly. Nucellar
elongation is primarily in the chalazal region; there are no
further cell divisions in the parietal layers of the apical
region of the nucellus, and the existing layer may be
crushed. At the completion of mitosis to yield eight nuclei in
seven cells, the female gametophyte elongates dramatically.
The chalazal portion of the mature gametophyte is `wedge'
shaped, appearing very narrow in one dimension, and
¯attened in the perpendicular plane. No ovules of either
species were observed to contain more than one mature
gametophyte.
The mature ovules of both species are orthotropic and
crassinucellate. The polar nuclei of the female gametophyte usually fuse prior to fertilization so that the
mature gametophyte comprises seven nuclei in seven
cells (Fig. 3A±C). The two synergids are densely
cytoplasmic and each has a distinct ®liform apparatus.
The egg cell is vacuolate and the large nucleus is
located in the chalazal end of the cell (Figs 3A, C). The
fusion nucleus, formed from the two polar nuclei, is
located in the micropylar end of the gametophyte, either
adjacent to the egg or near the wall of the central cell
(Fig. 3B, C). The central cell is large and vacuolate.
The antipodal cells are much smaller than the other
cells of the gametophyte and typically persist beyond
anthesis. The hypostase of mature ovules is pronounced
(Fig. 3C) and the blue-green staining with toluidine blue
O suggests that the cell walls contain lignin.
150
Diggle et al. Ð Sexual Reproduction in Polygonum
TA B L E 1. Numbers and percentages of total
parentheses) of mature ovules of differing classes.
Ovule type
(in
P. bistortoides
P. viviparum
14 (35´9)
16 (41)
30 (76´9)
9 (23´1)
39
22 (52´4)
9 (21´4)
31 (73´8)
11 (26´2)
42
Unfertilized
Fertilized (with zygote or embryo)
Total functional
Aborted
Total ovules
Development of abortive ovules
Some ovules of both species fail to complete development. The relative frequency of such aborted development
can be inferred from analysis of mature ovules. If those
ovules that contain a mature female gametophyte and those
containing a zygote or embryo are classi®ed as `functional',
then 30 of 39 (76´9 %) mature P. bistortoides ovules were
apparently functional, whereas nine (23´1 %) contained
obviously malformed gametophytes (Table 1). Similarly, 31
of 42 (73´8 %) mature P. viviparum ovules appeared
functional and 11 (26´2 %) were malformed. The frequency
of functional ovules did not differ signi®cantly between the
two species (c2 = 0´226, P > 0´1). (Clearly, the ovule
becomes an immature seed upon fertilization, however, the
term `ovule' is retained here for simplicity.)
Developmental analyses show that similar types of
abnormalities occur in both species and that cessation of
ovule development is not stage-speci®c. Some aborted
ovules of each species appear to be at the megasporocyte or
megaspore stage, i.e. the nucellus is small (Fig. 4B) or has
not yet been enclosed by the two integuments (Fig. 4A), yet
there is no evidence of a megasporocyte or spore (e.g.
compare Fig. 4A with Fig. 2A, C).
Development of ovules can also cease at later stages (Fig.
4C±F). In some, the nucellus had begun to elongate but did
not contain evidence of a female gametophyte. In other
ovules, some, but not all, cells of a mature female
gametophyte are present but the nucellus had not elongated
to mature dimensions (Fig. 4C, D). In still others, the
integuments and nucellus are indistinguishable from those
of ovules containing a normal female gametophyte except
that the nucellus is solid tissue and the central area is
occupied by collapsed cells (Fig. 4E, F). It appears that a
female gametophyte began to develop in these ovules and
then collapsed.
Post-fertilization ovule development
Although the frequency of apparently functional ovules
did not differ between the species, a signi®cantly greater
proportion of gametophytes of P. bistortoides had been
fertilized (Table 1). Sixteen of the 30 (53´3 %) apparently
functional ovules of P. bistortoides contained zygotes or
embryos compared with nine of 31 (29 %) ovules of P.
viviparum (c2 = 7´353, P < 0´01).
Embryo development of the two species was not studied
in detail. Ovaries that contained zygotes and embryos were
collected from ¯owers at anthesis at the same time as those
containing mature female gametophytes. Presumably,
fertilization and the initiation of embryo development
takes place very rapidly following anthesis. The embryos
were in either the ®lamentous or globular stage and were
surrounded by free nuclear endosperm. Development of one
embryo of P. viviparum had progressed to the initiation of
cotyledons and the endosperm had cellularized. Mature
embryos were not observed in collected material of either
species.
Pollen viability
Average pollen stainability for P. viviparum in year 1 was
20´6 % and ranged from 0± 75 %. In approx. half (54 %) of
the individuals, less than 20 % of pollen grains stained
positively with FCR, and stainability was greater than 70 %
in only two individuals (Fig. 5A). In year 2, average
stainability for P. viviparum pollen was 17´4 % and ranged
from 0´66±43 %. Stainability was less than 20 % in the
majority of individuals (65 %) (Fig. 5B). Stainability of P.
viviparum pollen was not signi®cantly different between
years (Mann±Whitney U = 719; P = 0´5361). Average
pollen stainability for P. bistortoides in year 2 was 56´6 %
with a range of 13±83 % (Fig. 5C). Percent stainability
differed signi®cantly between the species in 1993 (t = 11´9,
P < 0´0001).
D I SC U S S IO N
Lack of viable seed production in P. viviparum has no single
developmental explanation. Defects occur in each of the
processes and structures associated with sexual reproduction studied, yet these processes and structures also appear
to function normally in at least some ¯owers or individuals.
Malformed ovules are common in P. viviparum; however,
comparison with P. bistortoides shows that these abnormalities do not contribute to differences in seed production.
The virtual absence of sexual reproduction in P. viviparum
appears to be due largely to a low rate of fertilization and to
embryo/fruit abortion.
Pre-fertilization development
Beginning with the studies of Strasburger (1879), the
origin and development of the female gametophyte in
members of the Polygonaceae have become classic
examples of `normal' processes (Degeon, 1918).
Development of the female gametophytes of P. bistortoides
and of P. viviparum is similar and conforms to reports of
other species of Polygonum and close relatives (summarized
in Davis, 1966; also Stevens, 1912; Degeon, 1918; Schnarf,
1931; Mahony, 1935). Seemingly unusual features such as
multiple megasporocytes and the conspicuous nucellar beak
observed in both P. viviparum and P. bistortoides are
common among other species of Polygonum but are not
ubiquitous (Davis, 1966). Interestingly, developmental
abnormalities of the ovule and female gametophyte are
frequently observed among the species of Polygonum that
Diggle et al. Ð Sexual Reproduction in Polygonum
151
F I G . 4. Abnormal ovules of P. bistortoides (A, C and E) and P. viviparum (B, D and F). A and B, Ovule with both outer and inner integuments but no
evidence of megasporocyte or megaspore formation. Compare with Fig. 2. Bars = 25 mm. C and D, Ovules appear nearly mature in anatomy yet have
collapsed (A) or are incomplete (B) gametophytes. Part D shows a small gametophyte with synergids and an egg cell. Bars = 25 mm. E, F, Mature
ovules with solid nucellus and collapsed gametophytes. The ovules are similar in size to those containing normal female gametophytes. The collapsed
cells in the centre of the nucellus indicate that a gametophyte began, but did not complete, development. Bars = 50 mm. G, Gametophyte; ii, inner
integument; N, nucellus; oi, outer integument.
152
Diggle et al. Ð Sexual Reproduction in Polygonum
motivated by the absence of seed set in this species. Edman
(1929) and Schnarf (1931) found that irregularities in the
development of female gametophytes were common and
that degeneration could occur at any time following meiosis.
Our results show that in addition pre-meiotic failure can
occur. Engell (1973) did not note abnormalities, but was
unable to examine mature ovules and questioned whether P.
viviparum was capable of sexual reproduction.
While previous investigators have examined some
aspects of female gametophyte development in P. viviparum
in the context of low seed set, this study is the ®rst to include
an analysis of a `control' taxon. Comparison of the
development of P. viviparum ovules with that of P.
bistortoides, a sympatric congener with plentiful fruit set,
yields the unexpected conclusion that, although developmental irregularities are common in ovules of P. viviparum,
they are probably not responsible for the extremely low fruit
production. Malformed ovules occur with the same frequency in P. viviparum and P. bistortoides and ovule
development appears to abort at the same range of stages in
the two species. Clearly, the dramatic differences in fruit
production between the two species are not attributable to
abnormalities of pre-fertilization ovule development in P.
viviparum.
While the frequency of apparently functional ovules is
equivalent in the two species, pollen viability differs
signi®cantly. Average pollen viability (as assessed by the
FCR reaction) is far less in P. viviparum than in P.
bistortoides, and in both years a large proportion of ¯owers
had no viable pollen. Engell (1978) also found a high
frequency of small pollen grains in ¯owers of P. viviparum
collected from the Faroe Islands and suggested that these
were non-functional. Thus, low pollen viability is also
characteristic of northern European populations.
Fertilization
F I G . 5. Pollen viability as assessed by the FCR reaction.
have been studied and are not associated with low fruit
production.
There are no previous reports of ovule and female
gametophyte development for Polygonum bistortoides. In
contrast, there are least three published accounts of some
aspects of reproductive development in P. viviparum, each
Pre-fertilization ovule development of the two species is
nearly identical and apparently functional mature female
gametophytes occur with equal frequency, yet zygotes and
embryos were observed in only 29 % of functional ovules of
P. viviparum compared with 53 % in P. bistortoides.
Assuming that these are the result of fertilization (i.e. not
agamospermy), then the rate of fertilization is signi®cantly
lower in P. viviparum than in P. bistortoides. The difference
in fertilization rate between the two species may be
attributable to the large differences in pollen viability. If
pollen is not available in excess, then the probability of
fertilization occurring should be proportional to the frequency of viable pollen grains among potential donors.
Fertilization of P. viviparum ovules also may be limited by
pollinator service but pollen removal and deposition rates
were not examined.
A third potential explanation for differences in fertilization is self-incompatibility. Because of the prevalence of
asexual reproduction in P. viviparum, many potential pollen
donors could be genetically identical to the recipient (e.g.
Weis and Hermanutz, 1993; Tangmitcharoen and Owens,
1997; NegroÂn-Ortiz, 1998). If the species is self-incompatible, these pollinations would not lead to successful
Diggle et al. Ð Sexual Reproduction in Polygonum
fertilization. Two facts argue against this explanation: (1)
although the compatibility system of P. viviparum is
unknown, this species is polyploid and polyploidy tends to
disrupt self-incompatibility (de Nettancourt, 1977;
Richards, 1997; Miller and Venable, 2000); and (2) a
genetic analysis of the study population found relatively
high levels of genotypic diversity (Diggle et al., 1997),
therefore, even if the species is self-incompatible, compatible genotypes should be available within populations.
Thus, low pollen viability or limited pollination are more
likely explanations of the differences in fertilization
between the two species.
Although the frequency of ovules with zygotes or
embryos is lower in P. viviparum than in P. bistortoides,
the observed fertilization rate does not explain the apparent
absence of fruit production. Embryo development was
initiated in 29 % of ovules of P. viviparum and ¯ower
number per in¯orescence at the study site ranged from 0
(some in¯orescences bear only bulbils) to 65, with a mean
of 16 (K. Stitt, unpubl. res.). If all fertilized ovules matured
into seeds, there should have been a detectable number of
fruit at the ®eld site.
Post-fertilization development
Although 29 % of P. viviparum ovules contained zygotes
or embryos, we observed neither a mature embryo in
sectioned material nor a mature fruit in the ®eld; all ¯owers
abscised from in¯orescences within a few days of anthesis.
Clearly, most fertilized ovules abort after initiation of
embryo development. Embryo/seed abortion followed by
abscission must occur rapidly because we did not observe
any apparently malformed (dying) embryos or endosperms
in sectioned material. Thus, in addition to a lower rate of
fertilization compared with P. bistortoides, abortion of new
sporophytes also must be extremely common in P.
viviparum.
Abortion of developing seeds and fruits is quite common
among angiosperms (reviewed in Stephenson, 1981; Lee,
1988; Sedgeley and Griffen, 1989; Diggle, 1995). Failure of
post-fertilization development has been ascribed to a variety
of factors including competition among seeds or fruits for
nutrients (Stephenson, 1981; Lee, 1988; Diggle, 1995 and
references therein; Shuraki and Sedgley, 1996), maternal
selection among embryos of varying genetic quality
(reviewed in Willson and Burley, 1983; Bawa and Webb,
1984; Guth and Weller, 1986; Briggs et al., 1987; Marshall
and Folsom, 1991; Akhalkatsi et al., 1999), late acting selfincompatibility (Seavery and Bawa, 1986), and genetic load
or other genetic abnormalities (Brink and Cooper, 1940,
1941, 1947; Cooper and Brink, 1940; Weins, 1984, 1987;
Charlesworth and Charlesworth, 1987; Charlesworth, 1989;
Andersson, 1993).
Competition for nutrients and maternal selection among
embryos implies that there are some `winners', i.e. although
abortion occurs some fruit should mature on each
in¯orescence or individual. This is clearly not the case for
P. viviparum. Late acting self-incompatibility also is not
likely to occur in P. viviparum for the reasons discussed
above for pre-fertilization self-incompatibility (see discus-
153
sion under `fertilization'). Given that pollen development is
affected so drastically in P. viviparum, and that fruit
production is uniformly negligible among individuals,
among years and among sites, genetic abnormalities are a
likely cause of embryo abortion.
Whereas P. bistortoides is diploid, P. viviparum is
reported to be an octoploid (2n = 96, x = 12; LoÈve and
LoÈve, 1948, 1974, 1975; Wcislo, 1967; Engell, 1973; LoÈve,
1988). Full or partial sterility is not uncommon among
polyploids and is often attributed to irregular meiosis (e.g.
Stebbins, 1971; Richards, 1997 and references therein).
Meiotic aberrations would result in low pollen viability as
observed in P. viviparum and could also explain embryo
abortions. Even when gametes or gametophytes appear
functional, they may have an unbalanced chromosome
content and fertilization would produce zygotes that ultimately abort. The association between polyploidy and meiotic
irregularity, however, is not well established (P. Soltis, pers.
comm.) and many polyploids have normal chromosome
pairing at meiosis (e.g. Grant, 1971; Law et al., 1983).
Further examination of meiosis in P. viviparum is warranted.
Low pollen viability and embryo abortion also may be
due to accumulation of mutations in these very long lived
clonal organisms (Muller's ratchet; Muller, 1964; Gabriel
et al., 1993; Andersson and Hughes, 1996; Eckert et al.,
1999). Many mutations have been identi®ed in crop and
model species that speci®cally affect meiosis (e.g. Kaul and
Murthy, 1985). In addition, other mutations affect pollen
viability (e.g. Kennell and Horner, 1985; Kaul, 1988;
Benavente et al., 1989; Chaudhury, 1993), ovule development (Reiser and Fischer, 1993; Christensen et al., 1998;
Schneitz, 1999; Yang and Sundareasan, 2000) or embryo
development (reviewed in Mayer et al., 1991; Uwer et al.,
1998; Albert et al., 1999; Heckel et al., 1999). Such
mutations can be perpetuated by vegetative reproduction,
and could accumulate over the lifespan of a genet,
particularly if the mutations have no negative pleiotropic
effects on survival (Eckert et al., 1999). In addition, embryo
abortion could be the result of genetic load if the new
sporophytes are products of self-fertilization (Weins, 1984,
1987; Klekowski, 1988).
The absence of any clear developmental correlate of
embryo abortion is not unusual. There has been
considerable developmental analysis of seed and fruit
abortion, particularly in economically important species
(reviewed in Brink and Cooper, 1940, 1941; Sedgeley
and Griffen, 1989; Fernando and Cass, 1997). Most
commonly, abortion is not stage-speci®c and no visible
evidence of the cause of abortion can be identi®ed (e.g.
Persica: Sedgeley, 1980; Pistacia: Shuraki and
Sedgeley, 1996; Butomus: Fernando and Cass, 1997;
Melilotus: Akhalkatsi et al., 1999; many tree crops
including cherry, citrus, teak, mango and plum:
Stephenson, 1981). In other cases, clear developmental
abnormalities precede abortion. These include proliferation or death of the endosperm (Medicago: Cooper
et al., 1937; Oxalis: Guth and Weller, 1986) proliferation of integuments (Asclepias: Moore, 1946), or
pronounced development of the hypostase (Pisum:
154
Diggle et al. Ð Sexual Reproduction in Polygonum
Briggs et al., 1987). Although high rates of fruit
abortion occur regularly in all of these species, the
ultimate cause of abortion was rarely identi®ed conclusively.
Why is fruit production so low in P. viviparum?
Although irregular ovule development is common in P.
viviparum, it is no more frequent in this species than in the
sympatric P. bistortoides. In fact, multiple developmental
abnormalities are reported to be common throughout the
genus Polygonum and may be normal for the genus (an odd
situation for the `type' genus for female gametophyte
development!). Abortive ovule development does decrease
maximum seed production in P. viviparum, but is not
responsible for its absence. In contrast to the similarity of
ovule development in the two species, the incidence of
abnormal pollen is far greater in P. viviparum than in P.
bistortoides and may be associated with differences in
fertilization rates. Even low fertilization rates, however,
cannot account for the rarity of fruit production. Abortion of
young sporophytes must be the ®nal phase of reproductive
development that reduces fruit maturation to undetectable
levels.
Although fruit set in P. viviparum has not been observed
in the study population, it is likely that it does occur
occasionally. Genotypic diversity in the study populations is
equivalent to that of clonal species known to have regular
fruit/seed maturation (Diggle et al., 1997; see also Bauert,
1993, 1996 for European populations). The most likely
explanation of the existence of multiple genotypes in this
population is that viable seeds are produced, albeit rarely.
Theoretical models suggest that a very low rate of seedling
input into established populations is suf®cient to maintain
genetic diversity (Soane and Watkinson, 1979; Watkinson
and Powell, 1993). The causes of low fruit production in P.
viviparum are not absolute, and occasionally two normal
gametes may unite to form a viable new sporophyte.
A C K N O W L ED G EM EN T S
The authors thank Tara Doughty, Rachel Kaplan, Anne
Klein, Amber Moody, Mingon Macias and Kristine Stitt for
laboratory assistance, and Larry Hufford for comments on
the manuscript. Support was provided by NSF DEB9357076, Apple Computers, Inc., the Niwot Ridge LongTerm Ecological Research Program (NSF DEB-9211776)
and the University of Colorado Mountain Research Station
(NSF BIR-9115097).
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