Apomixis in Amelanchier laevis, Shadbush (Rosaceae, Maloideae

Apomixis in Amelanchier laevis, Shadbush (Rosaceae, Maloideae)
Christopher S. Campbell; Craig W. Greene; Benedict F. Neubauer; Jean M. Higgins
American Journal of Botany, Vol. 72, No. 9. (Sep., 1985), pp. 1397-1403.
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Amer. J. Bot. 72(9): 1397-1403. 1985.
APOMIXIS IN AMELANCHIER LAEVIS, SHADBUSH
(ROSACEAE, MALOIDEAE)
W. GREENE,~
CHRISTOPHER
S. CAMPBELL,
CRAIG
BENEDICT
F. NEUBAUER,
AND JEANM. HIGGINS~
Department of Botany and Plant Pathology, University of Maine, Orono, Maine 04469, and
ZCollegeof the Atlantic, Bar Harbor, Maine 04609
ABSTRACT
We studied ovule and megagametophyte development in tetraploid (n = 34) individuals of
Amelanchier laevis in Maine. Nomarski differential interference contrast microscopy of cleared,
whole ovules and conventional microscopy of sectioned, stained material show no clear evidence
for the successful completion of meiosis. Instead, the megasporocyte or its derivatives degenerate
and one to six nearby cells develop into aposporous initials. Usually more than one of these
divide to form eight-nucleate, Polygonum-type megagametophytes. The egg apparently forms
a proembryo parthenogenetically, but seed maturation requires pollination. This evidence for
apospory and pseudogamy, the first to be reported in Amelanchier, conforms to the general
pattern found in other apomictic genera of the Maloideae.
AMELANCHIER
contains 20 to 30 species oftrees
and shrubs of the North Temperate region;
many are widely distributed in eastern North
America (Robertson, 1974). This genus, along
with about another 20 genera, belongs to the
subfamily Maloideae, a group uniquely characterized by the pome type of fruit. The
subfamily also has the distinctive base chromosome number of 17, while the other three
subfamilies of the Rosaceae are mostly x = 7,
8, or 9 chromosomes. This higher base number
probably represents an ancient allopolyploidization event in the subfamily's ancestry (Sax,
1932; Stebbins, 1958; Challice, 1974).
In addition to their inferred role in the origin
of the subfamily, polyploidy and hybridization
are now widespread and conspicuous evolutionary phenomena in the Maloideae. In the
larger genera, such as Amelanchier, Cotoneaster, Crataegus, and Pyrus (including Aronia,
Malus, and Sorbus), triploids and tetraploids
are frequent (Robertson, 1974). Hybridization
occurs regularly within these genera. In Amelanchier, as in the other genera, hybridization
apparently follows habitat disturbance by fire,
deforestation, or the abandonment of farmland
Received for publication 4 October 1984; revision accepted 27 March 1985.
We acknowledge the Faculty Research Fund of the University of Maine at Orono for financial support of this
research, K. L. Chambers, T. A. Dickinson, and F. Hyland
for comments on a draft of this manuscript, Scott E.
Bergquist for technical assistance, and the Systematics class
of the College of the Atlantic.
Present address: Department of Biology, University of
California at Santa Cruz, Santa Cruz, CA 95054.
(Wiegand, ;9 12; Nielsen, 1939; Cinq-Mars,
197 1; Landry, 1975; Robinson, 1982). Cruise
(1964) amply demonstrated the existence of
morphologically intermediate, putative hybrids in New Jersey and Pennsylvania populations ofA. laevis Wiegand (the subject of this
report), A. arborea (Michaux f.) Fernald, and
A. canadensis (L.) Medicus. Fernald (19 50) reported crosses between A. laevis and seven other common species of eastern North America.
Polyploidy and hybridization are often associated with gametophytic apomixis in angiosperms (Gustafsson, 1946, 1947a, b; Nygren, 1967; Asker, 1979). Such asexual seed
production may provide an escape from sterility accompanying polyploidy and hybridization (see Marshall and Brown, 198 1, for a
discussion of the adaptive significance of apomixis). It is not surprising then that apomixis
occurs in many polyploid taxa of Cotoneaster,
Crataegus, and Pyrus (Dermen, 1936; Liljefors, 1953; Hjelmqvist, 1957, 1959, 1962; MuniyammaandPhipps, 1979,1984a). All known
apomicts in the Maloideae are aposporous with
the exception of diplosporous Crataegus dissona (Muniyamma and Phipps, 1984b). One
or more cells adjacent to the megasporocyte
develop into eight-nucleate megagametophytes with the unreduced chromosome number. The egg cell divides parthenogenetically
to produce an embryo. In almost all cases pseudogamy-fertilization of the polar nuclei to
form endosperm-is necessary for embryo
maturation.
The occurrence of polyploidy and hybridization in Amelanchier and the prevalence of
1398
AMERICAN JOURNAL OF BOTANY
[Vol. 72
apomixis in related genera suggest that apomixis might also be present in Arnelanchier.
Apomixis has not been previously reported in
Arnelanchier, although McVaugh (1946) noted
it as a possibility. Robinson (1982) rejected it
but without studying megagametogenesis. We
present the first evidence for apospory and
pseudogamy in Arnelanchier.
a minimum of 200 pollen grains per sample
from randomly selected flowers.
Flowers were emasculated prior to anthesis.
Racemes with emasculated flowers or with undisturbed flowers were covered with fabric bags
to exclude pollinators. Maturing fruit was collected to determine seed set and whether or
not more than one embryo develops per seed.
AND METHODS- Material for this
MATERIALS
study comes from three trees of Arnelanchier
laevis in Maine (Campbell 4352, Orono, Penobscot County, and Greene 1213, 1223, Bar
Harbor, Hancock County; vouchered specimens at MAINE). For our study of megagametogenesis, flowers were collected at 1- or
2-day intervals from the beginning of bud enlargement in April to the shedding of petals in
May 1983 and 1984. Prior to fixation, we cut
flowers with a razor blade to allow rapid entry
offixative. Tissue was fixed in FPA,, (formalin,
propionic acid, 50% ethanol; 5:5:90 by vol)
and immediately put under vacuum with a field
pump. After 24 hr at 20 C the material was
transferred to 70% ethanol for storage at 4 C
until use.
We examined cleared and sectioned ovules.
With cleared material, which can be processed
relatively rapidly, we examined about 2,000
ovules of various ages. Ovules were isolated
and cleared in Herr's fluid (85% lactic acid,
chloral hydrate, phenol, clove oil, xylene; 2:2:
2:2:1 by wt) for 24 hr at 20 C (Herr, 1971).
Integuments were removed from older ovules
to improve clearing. Ovules or megasporangia
(nucelli) were mounted in Herr's fluid on Raj
slides. Two No. 1 coverslips were set on a
microscope slide about 5 mm apart; these supported a third coverslip above the ovules to
prevent their being crushed. Development was
studied under Nomarski differential interference contrast using a Zeiss standard microscope. Sections of about 1,000 ovules facilitated observation of cell walls. Ovaries for
sectioning were dehydrated in an ethanol-xylene series, embedded in paraffin, sectioned at
12 pm, stained with either safranin and fast
green or iron hematoxylin (Sass, 1958), and
examined under brightfield.
Meiotic studies were made of microsporocytes fixed in either modified Carnoy's solution
(100% ethanol, chloroform, acetic acid; 6:3:1
by vol) or Farmer's solution (100% ethanol,
acetic acid; 3: 1 by vol) for 24 hr at 20 C, transferred to 70% ethanol, and stored at 4 C until
use.
Pollen stainability by cotton blue in lactophenol was taken as a measure of pollen viability. Percent stainabilitywas determined from
OBSERVATIONS
AND RESULTS-Thegynoecium of Arnelanchier laevis usually consists of
five carpels, and the ovary contains five locules,
each with two ovules. The ovules are crassinucellate and anatropous, and the micropyle,
which is formed by the inner integument alone,
faces the base of the. ovary (Fig. 1).
As the integuments begin to grow up around
the base of the ovule primordium, the archesporium develops, usually with one conspicuous megasporocyte (Fig. 3). We have not observed meiosis directly in the megasporocyte.
We have examined several hundred ovules of
the appropriate stage, however, and in no case
have we seen clearly the presence of linear or
T-shaped tetrads or triads of megaspores indicative of meiosis. Instead, before integuments reach the tip of the megasporangium,
we see a narrow line or small mass of degenerated tissue, which stains darkly in sectioned
material (Fig. 2) and has a distinctive appearance in cleared material (Fig. 4, 5). Usually
lateral or micropylar to this degenerated tissue,
one to six expanding cells with conspicuously
large nucleoli appear (Fig. 4, 5). One or more
of these cells divide mitotically, passing through
two-nucleate (Fig. 6) and four-nucleate stages
before maturing into Polygonurn-type, eightnucleate megagametophytes (Fig. 7,9). The egg
apparatus includes the egg, with a conspicuous
nucleus and abundant starch in its cytoplasm,
and two synergids lying between the egg and
micropyle (Fig. 8). In mature synergids, the
nuclei are inconspicuous or absent, and the cell
walls sometimes show a filiform apparatus (Fig.
8). Two unfused polar nuclei are prominent in
the central cytoplasm (Fig. 9, 10). The three
antipodals occupy the chalaza1 end of the mature megagametophyte and degenerate as it
matures.
The majority of mature ovules contain two
mature megagametophytes (Fig. 9). In a sample
of 129 ovules with mature megagametophytes,
39% contained one megagametophyte, 5 1%
two, 9% three, and 1% four. Sometimes an
eight-nucleate and developmentally younger
megagametophytes are found together (Fig. 7).
Interpretation of ovules with more than two
megagametophytes is often difficult.
Figure 10 shows a portion of a megaspo-
September, 19851
CAMPBELL ET AL.
-APOMIXIS IN AMELANCHIER
KSY TO LABELING: a = aposporous initial; d = degenerating tissue; e = egg cell; ii = inner integument; m
sporangium; oi = outer integument; pe = proembryo; pn = polar nuclei; s = synergid.
= mega-
Fig. 1,2. Brightfield photomicrographs of sectioned, stained young ovules. 1. Young anatropous ovule with expanding
gametocyte. Micropyle pointed toward base of ovary. x 100. 2. Ovule with two expanding aposporous initials toward
micropyle from mass of degenerating tissue (arrow). Inner integument approaching tip of megasporangium. x 530.
rangium with one mature megagametophyte
containing a developing proembryo but two
unfertilized polar nuclei. The apparent absence
of a pollen tube suggests parthenogenesis. Pollen tubes were visible entering other ovules of
open-pollinated flowers. While pollen is not
necessary for embryo initiation, it is essential
for fruit production. Bagged, unemasculated
flowers generally set fruit, but bagged, emasculated flowers produced no fruit. In a sample
of 50 fruits from open-pollinated flowers, a
mean of only 3.04 seeds per fruit (SD = 1.14)
contained well-developed embryos. There was
at least one well-developed embryo per fruit
in all but three fruits.
From meiotic figures of microsporocytes, we
have determined that all three of the trees studied embryologically are tetraploid (n = 34).
Microsporogenesis appears to be normal, and
pollen viability is over 83% in all samples.
DISCUSSION-Gametophyticapomixis entails two separate processes: the formation of
an unreduced megagametophyte and the parthenogenetic development of the egg into an
embryo (Gustafsson, 1946; Asker, 1979). The
unreduced megagametophyte may arise in two
ways. The first, diplospory, comes about when
a megasporocyte circumvents meiosis and divides mitotically to form a megagametophyte.
Apospory involves the derivation of the megagametophyte from a nucellar cell adjacent to
the archesporium. The presence of a multi-
cellular archesporium in many Rosaceae (Gustafsson, 1946) and Maloideae (Liljefors, 1953;
Hjelmqvist, 1957) renders the distinction between these two possibilities somewhat arbitrary (Nygren, 1967). The important difference
seems to be whether or not meiosis was initiated. The apparent degeneration of the megasporocyte in Amelanchier laevis makes the apomictic process aposporous. The maturation of
the apomictically derived embryo depends
upon the production of endosperm for which
fertilization of the polar nuclei may be necessary (pseudogamy). Most aposporic taxa are
pseudogamous (Nygren, 1967).
Our conclusion that Amelanchier laevis reproduces apomictically rests on two pieces of
evidence. First, the apparent absence of megaspores in the vast majority of ovules indicates
that meiosis does not occur. We cannot rule
out the possibility that reduced megagametophytes might occasiondly be formed; chromosome behavior in dividing megasporocytes
remains to be studied. Moreover, in a few ovules
we observed a large cell, possibly a functional
megaspore, chalazal to a line of degenerating
tissue that could represent remanants of micropylar megaspores. These rare observations
may indicate that in exceptional cases sexual,
reduced megagametophytes may be formed.
However, in most cases megagametophytes develop from aposporous initials that are lateral,
not chalazal, to degenerating archesporial tissue. In Malus, apomixis can be facultative,
1400
AMERICAN JOURNAL OF BOTANY
[Vol. 72
September, 19851
CAMPBELL ET AL. -APOMIXIS IN AMELANCHIER
1401
Fig. 9, 10. Nomarski DIC photomicrographs of cleared, mature megasporangia, integuments removed. 9. Megasporangium with two mature megagametophytes. Note two eggs and two pairs of unfused polar nuclei. ~ 2 3 0 10.
.
Megasporangium with single megagametophyte. Note proembryo, unfused polar nuclei (arrows), and absence of pollen
tube at micropylar end. x 210. See page 1399 for QY TO LABELING.
with both reduced and unreduced megagametophytes being formed (Olden, 1953;
Hjelmqvist, 1957). Our interpretation of these
expanding cells as aposporic initials parallels
similar observations in other apomictic maloid
genera, all of which are aposporous (Dermen,
1936; Liljefors, 1953; Hjelmqvist, 1957, 1959,
1962; Muniyamma and Phipps, 1979).
The second piece of evidence is the frequent
development of more than one megagametophyte per ovule. It is possible that these arise
from different megasporocytes of the multicellular archesporium. "Accessory" and "secondary" megasporocytes have been reported
in Sorbus (Liljefors, 19 53) and Cotoneaster
(Hjelmqvist, 1962),but these are rare and usually degenerate. Apospory is often characterized by the production of multiple megagametophytes (Davis, 1966). In maloid genera
with apomictic species, the sexual species that
have been studied produce only one megagametophyte per ovule, while their aposporic rel-
Fig. 3-8. Nomarski DIC photomicrographs of cleared ovules of various ages. 3. Young ovule with single megasporocyte (arrow). Note its single large nucleolus in granular nucleoplasm. Inner integument just starting to differentiate.
x 650. 4. Expanding aposporous initial lateral to degenerating megasporocyte. Inner integument about half as long as
megasporangium. x 350.5. Three expanding aposporous initials adjacent to degenerating megasporocyte. Two of these
initials are multinucleolate. Inner integuments almost as long as megasporangium. x 300. 6. Two two-nucleate megagametophytes, each with central vacuole and with long axis parallel to long axis of megasporangium. Integuments
removed. x 620. 7. Immature eight-nucleate megagametophyte with four of five micropylar nuclei in focus. Arrows
indicate nuclei of three chalazal antipodal cells. Note pair of aposporous initials chalazal to eight-nucleate gametophyte.
x 7 10. 8. Egg apparatus in micropylar end of mature megagametophyte. Note conspicuous nucleus and plastids in egg
and rippled micropylar wall, the filiform apparatus of synergid on left (arrow). ~ 6 0 0 See
. page 1399 for KEY TO
LABELING.
1402
AMERICAN JOURNAL OF BOTANY
[Vol. 72
atives frequently develop more than one (Der- vious study. Robinson and Partanen (1980)
men, 1936; Liljefors, 1953; Hjelmqvist, 1957, published two diploid counts for this species.
1959, 1962; Muniyamma and Phipps, 1979).
The only other study concerning apomixis
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