Characterisation of pseudogamous apospory in the reproductive

Acta Bot. Gallica, 2002, 149 (2), 217-224.
Characterisation of pseudogamous apospory in the reproductive biology of the invasive weed Rubus alceifolius (Rosaceae) in its area of introduction
by Laurent Amsellem(1,2), Thierry Pailler(3), Jean-Louis Noyer(1) and Martine HossaertMcKey(2)
(1) CIRAD, Centre de Coopération Internationale de Recherche Agronomique
Développement, Avenue Agropolis, TA 7410, F-34398 Montpellier Cedex 5
(2) Centre d'Écologie fonctionnelle et évolutive, CEFEICNRS, 1919 route de Mende, F-34293
Montpellier Cedex 5
(3) Laboratoire de Biologie et Physiologie végétales, Université de La Réunion, 15 avenue René
Cassin BP 7151, F-97715 Saint Denis Cedex 9
arrivé le 18 septembre 2001, accepté le 25 octobre 2001
Abstract- A previous study on reproductive biology of Rubus alceifolius showed that this invasive weed reproduces asexually via apomixis, with however a
residual sexuality, in the Indian Ocean islands where it has been introduced,
whereas it reproduces only sexually in its South-eastern Asian native range. To
determine which type of apomixis occurs in the area of introduction, we performed hand-pollinations on flowers of individuals from La Réunion island to test
whether pollination is necessary for seed set. exhibit evidence of pollination by
showing three nuclei. Pollination was found to be necessary for seed set, cells
clearly exhibit three nuclei, showing that fertilisation took place. These data
strongly suggest that R. alceifolius reproduces by pseudogamous apospory in its
area of introduction. By preserving and multiplying a single well-adapted genotype, this trait may contribute to the invasiveness of it where it was introduced.
Key-words : Rubus alceifolius - weed - apomixis - apospory - pseudogamy.
Résumé .-
Une étude précédente sur la biologie de la reproduction de Rubus
alceifolius a montré que cette plante envahissante se reproduit asexuellement
via apomixie, avec cependant une sexualité résiduelle, dans les îles de l'Océan
Indien où elle a été introduite, tandis qu'elle se reproduit sexuellement dans son
aire d'origine sud-est asiatique. Pour déterminer quel type d'apomixie est pratiqué dans l'aire d'introduction, nous avons effectué des pollinisations manuelles
sur les fleurs d'individus originaires de l'île de La Réunion, afin d'évaluer si la pollinisation est nécessaire à la formation de graines. La pollinisation apparaît
nécessaire pour la formation de graines et les cellules d'albumen montrent clairement trois noyaux, preuve qu'une fécondation a eu lieu, ce qui suggère fortement que R. alceifolius s'y reproduit par aposporie pseudogame. En préservant
et multipliant un seul génotype bien adapté, ce trait peut contribuer au fait qu'il
soit envahissant dans les îles sur lesquelles il a été introduit.
Mots-clés :
pseudogamie.
Rubus alceifolius - plante envahissante - apomixie - aposporie -
218
1. INTRODUCTION
In the genus Rubus (Rosaceae), apomixis is well documented, both by cytological and
embryological observations (Crane, 1940; Thomas, 1940; Gustafsson, 1943; Jennings et
al., 1967; Czapik, 1981, 1983). Apomixis usually occurs by the formation of unreduced
embryo-sacs from sporogenous (diplospory) or other archeosporal cells (apospory) (Pratt
& Einset, 1955; Czapik, 1983). Among the 12 subgenera of the genus Rubus, apomixis prevails in the subgenus Rubus, only in polyploid taxa, under the form of pseudogamous apospory. In this type of apomixis, asexual reproduction takes place, but the endosperm may
require fertilisation to develop (Crane, 1940; Thomas, 1940; Gustafsson, 1943; Jennings et
al., 1967; Czapik, 1981, 1983; Nybom, 1985, 1988, 1995; Richards, 1986; Weber, 1996;
Kollmann et al., 2000; van Dijk & van Damme, 2000). Apomixis also occurs in polyploid
species from other subgenera of Rubus (Petrov, 1939; Pratt & Clausen, 1958; Nybom,
1988). Apospory, diplospory, and reduced embryo sacs commonly occur together in the
same species. Usually, reduced embryo sacs do not develop until fertilized, and unreduced
embryo sacs develop parthenogenetically (Nybom, 1988, 1995). However, exceptions to
both phenomena occur, thereby rendering reproduction in the genus Rubus extremely versatile.
Until now, the reproductive biology of tropical Rubus species has been poorly investigated. According to Nybom (1986), species of the East Asian subgenus Malachobatus
Focke are polyploid (Nybom, 1986; Thompson, 1997), and reproduce only sexually. Other
studies confirm this conclusion for native Asian populations (Busemeyer et al., 1997;
Amsellem et al., 2000; Amsellem et al., 2001 a, Amsellem et al., 200lb).
Rubus alceifolius Poir. (subgenus Malachobatus) is a simple-leafed bramble of southeast Asia that is an invasive weed in Indian Ocean islands where it has been introduced. A
study of the genetic diversity of R. alceifolius showed a great genetic variability within
populations and among geographically close populations in its Asian native range
(Vietnam, Thailand, Sumatra, Java, Laos). In its area of introduction, R. alceifolius has
lower genetic variability in Madagascar, and is apparently monoclonal in the other islands
where it has been introduced (Mayotte, La Réunion, Mauritius) and in Queensland
(Amsellem et a!., 2000). Successive nested founder effects appear to have resulted in
cumulative reduction in genetic diversity, from Asia to Madagascar (which appears to have
been the first stage of R. alceifolius in its migratory route), and from Madagascar to the
other islands.
To explain the monoclonal genetic patterns observed on those islands, despite the production of huge numbers of fruits and seeds, a comparative study of the reproductive biology of R. alceifolius was conducted in its native range and in its area of introduction.
Microsatellite profiles of progenies from Vietnam, Madagascar and La Réunion were compared with those of their respective mother-plants (Amsellem et al., in press b). Our results
showed that a switch from sexual reproduction towards apomixis occurred between the
native range and the area of introduction. This constitutes the first record of apomixis for
species of the subgenus Malachobatus.
In this study, we analysed whether pollen fertilisation was necessary to trigger development of a fruit set by individuals from populations in La Réunion. We also made both
embryological and cytological observations on embryo sacs of individuals of R. alceifolius
from La Réunion populations, to characterise the type of apomixis of this weed in its area
of introduction.
219
II. MATERIALS AND METHODS
A. Material
Comparison of fruit set and abortion rates between hand-pollinated and non-pollinated
flowers
This experiment was conducted in La Réunion island, in the population of Grand-bang
(alt. 480 m, 21°04'58" S, 55°40'30" E).
Because pollination of Rubus alceifolius is entomophilous (for La Réunion: Thébaud,
1989; S. Maurice & D. Strasberg, University of La Réunion, pers. comm.), we tested the
ability of R. alceifolius to develop fruit without any vector of pollen. We compared proportions of flowers which aborted and gave ripe fruits, between hand-pollinated flowers
and non-pollinated flowers. Non-pollinated flowers correspond to flower buds that were
bagged and left to mature without any further intervention. A total of 44 hand-pollinated
flowers were manipulated on 9 individuals, and 26 non-pollinated flowers were studied on
9 individuals.
Growth of pollen tubes and evidence for fertilisation of the embryo sac
Some hand-pollinated flowers were harvested 24 and 48 hours after manipulation (2 flowers for each harvesting time), and fixed in FAA (10% formalin, 80% ethanol, and 10%
glacial acetic acid). For each harvesting time, observations of styles allowed us to estimate growth of pollen-tubes, and whether or not they reached the ovules and the embryo sacs.
Observations were made on 70 and 64 styles of the flowers harvested 24 and 48 hours after
pollination, respectively. For each observation, we noted the position of the growing pollen tube in the style (distal or basal) or in the ovary.
The number of nuclei was registered in endosperm cells at indicator of fertilisation (3
nuclei) or its absence (2 nuclei), in the flowers collected at 24 and 48 hours after pollination (two flowers for each harvesting time). Nuclei were counted in five cells randomly
chosen in three embryo sacs for each harvesting time.
B. Methods
Comparison of fruit set and abortion rates between hand-pollinated and non-pollinated
flowers
Flowers used as pollen receptors were bagged at the bud stage to prevent any uncontrolled pollination. Non-pollinated flowers were also bagged at the bud stage, and remained non-manipulated until the end of maturation.
Inflorescences of R. alceifolius contain around twelve hermaphroditic flowers. The
gynoccium is apocarpous with about a hundred separate carpets, with styles and stigmas
much longer than the stamens, so that the pollen cannot be deposited on styles except by a
vector. During 10 days flowers were pollinated just after opening, using freshly opened flowers as pollen sources. Preliminary studies (unpublished data) show that flowers are receptive as soon as they open. On flowers chosen as pollen donors, styles were first cut with
scissors at the height of stamens. These "male" flowers were then taken and carefully rubbed onto the styles of the flowers chosen to receive pollen. One of the persistent sepals of
each pollinated flower was then painted on its external face with acrylic paint, to mark the
pollinated flowers within the inflorescence. Pollinated flowers were then bagged again to
prevent any uncontrolled pollination.
Our observations show that flowers fall in a period of 2 to 3 days after their opening if
they were insufficiently or not pollinated. If pollination is successful, styles necrose and
220
Table I.- Fruit set rates of non-pollinated and hand-pollinated flowers, for individuals from
the population of Grand-Etang (La Réunion island).
Tableau I.- Taux de fructification des fleurs manuellement pollinisées et non pollinisées,
pour des individus de la population de Grand-Étang (île de La Réunion).
Non-pollinated flowers
Indiv. No.
1
2
3
4
5
6
7
8
14
16
18
Total number
Percentage
Number of
flowers
1
Number aborted
Number of fruits set
1
0
9
2
3
2
7
2
2
2
2
0
1
0
4
1
3
1
1
0
3
1
3
1
0
0
26
19
73.1
7
26.9
Hand-pollinated flowers
Number of
Number of fruits
Number aborted
flowers
set
2
10
8
9
1
2
4
1
3
1
2
0
1
0
1
7
7
7
1
1
4
7
1
5
0
2
1
44
13
29.5
31
70.5
the calyx closes in I to 2 days after pollination. Flowers that were still on their mother-plant
10 days after their closing were likely to form mature fruits. One week to 10 days after pollination, inspection of bags containing hand-pollinated and non-pollinated flowers permitted us to count flowers fallen in the bag, and to estimate the rate of abortion. After this
period, flowers which were still on the mother-plant were then bagged in mosquito-net
sleeves. When ripe, fruits fell into in the sleeve and were collected.
We compared the proportion of flowers aborted between non-pollinated and hand-pollinated flowers with a χ2 test.
Growth of pollen tubes and evidence for fertilisation of the embryo sac
Fluorescence microscopy was performed on styles of pollinated flowers preserved in
FAA and put between slides and cover slips, according to Martin (1959). Observations of
fluorescence in aniline blue were made under an ultraviolet light of wavelength 340-380
nm. Endosperm cells of embryo sacs were observed under a microscope with light under
contrast phase.
III. RESULTS
A. Lower abortion rates of pollinated flowers
Proportions of non-pollinated and hand-pollinated flowers that gave ripe fruits are registered in Table l. Of the 26 non-pollinated flowers, 19 aborted and 7 gave fruits (73.1% and
26.9%, respectively), whereas of the 44 hand-pollinated flowers, 13 aborted and 31 gave
fruits (29.5% and 70.5%, respectively). Difference in abortion rate between the two treatments was highly significant (χ2 = 12.42; df = 1; P = 0.0004).
221
Table II.- Observations of pollen tubes in styles and ovaries from hand-pollinated flowers of Rubus
alceifolius in La Réunion island, 24 and 48 hours after pollination.
Tableau Il.- Observations de tubes polliniques et d'ovaires de fleurs de Rubus alceifolius
manuellement pollinisées à l'île de La Réunion, 24 et 48 heures après pollinisation.
On
In the distal half of the In the basal half of the Reaching the
stigmata
style
style
ovule
(apical)
Within the
ovule
Embryo sac is
developed
24h after pollination
(n = 70)
Total number
0
0
5
6
59
70
Proportion
0
0
0.07
0.09
0.84
1
48h after pollination (n= 64)
Total number
0
0
10
20
34
64
Proportion
0
0
0.16
0.31
0.53
1
B. Growth of pollen tubes and evidence for fertilisation of the embryo sac
Fluorescence microscopy observations of elongation of pollen-tubes in the styles showed that they entered the embryo sac and freed their spermatic cells 24 hours after pollination (Table Il and Fig. 1).
Endosperm cells with three nuclei were clearly visible in multi-cellular embryo sacs 24
hours after pollination (Fig. 2), as classically described after fertilisation (two nuclei originate from the embryo sac, and one nucleus from a spermatic cell of the pollen).
IV DISCUSSION
A. Type of apomixis occurring in introduced R. alceifolius
Our results show that flowers of R. alceifolius, although apomictic, require pollination
to set fruit, and that pollination requires a pollen vector. This is in accord with the usually
lower efficiency of hand-pollination compared to non-pollination of flowers (Guitian &
Fuentes, 1992; Holderegger, 1996; Kollmann et al., 2000).
Although apomixis is the main mechanism by which seeds of R. alceifolius are produced in the islands where it has been introduced, a residual sexuality seems to occur
(Amsellem et al., in press b). Moreover, our experiments showed that auto-incompatibility mechanisms do not occur during pollen tube growth in the styles. Thus, the small proportion of fruits obtained from non-pollinated flowers could have originated from
autogamy, caused by pollen grains within the bags (due to movement by wind, rain or any
other disturbance). Floral buds of R. alceifolius are very fragile, so that any attempt to
manipulate them at this stage resulted in their death after a few hours (pers. obs.). It was
thus impossible for us to remove stamens before bagging buds, for a non-pollination experiment. The fruits produced from bagged buds could also have resulted from apomixis. As
the mother-plant genotypes were homozygous at the eight microsatellite loci we obtained
(unpublished data), it was not possible to obtain any segregation of alleles in their offspring
to determine whether seeds produced by these bagged buds resulted from apomixis and !
or autogamy.
In Madagascar and La Réunion, R. alceifolius produces on the same plant fruits resulting predominantly from apomixis, sexual reproduction being rare (Amsellem et al., in
press). Apospory seems to be the most likely type of apomixis occurring. Apospory can
222
Fig. 1.- Pollen tubes reaching an ovule 24h after pollination. Diffusion of fluorescence in the embryo sac
-2
strongly suggests that spermatic cells of pollen tubes had been freed in it (bar scale represents 10 cm).
Fig. 1.- Tubes polliniques atteignant un ovule 24h après la pollinisation. La diffusion de fluo-rescence dans le
sac embryonnaire suggère fortement que les cellules spermatiques des tubes polliniques y ont été
libérées (la barre d’échelle représente 10-2 cm).
DF: diffusion of fluorescence in the embryo sac, diffusion de fluorescence dans le sac embryonnaire; ES:
embryo sac, sac embryonnaire; M: micropyle; O: ovule; PT: pollen tube, tube pollinique; V: vessel,
vaisseau; S: style.
explain the predominantly clonal reproduction due to the parthenogenetic development of
an unreduced embryo, as well as the occurrence of sexuality, through fertilisation of a few
reduced embryo sacs occurring on the same plant (Richards, 1986; Nybom, 1988;
Kollmann et al., 2000).
B. Evidence of pseudogamy for R. alceifolius in its area of introduction
We have provided evidence that pollen tubes grow until reaching embryo sacs where
they free their spermatic cells. Moreover, we observed that endosperm cells require fertilisation to develop. Apomixis thus appears to involve pseudogamy in R. alceifolius from La
Réunion island. The strong contrast between abortion rates of hand-pollinated flowers and
non-pollinated flowers may indicate that pollination is necessary for fruit development, as
expected for a pseudogamous species (Jennings et al., 1967; Jennings, 1975; Kollmann et
al., 2000).
Though only apospory has been demonstrated for R. alceifolius in Madagascar
(Amsellem et al., in press), and pseudogamous apospory in La Réunion, the latter phenomenon may occur generally in the area of introduction of R. alceifolius. This weed is mono-
223
Fig. 2.- Multi-cellular embryo sacs exhibiting tri-nucleate endosperm cells, 24 hours after pollination (bar
scale represents 2,5.10-3 cm).
Fig. 2.- Sacs embryonnaires pluri-cellulaires montrant des cellules d’albumen tri-nucléees, 24h après la
pollinisation (la barre d’échelle représente 2,5.10-3 cm).
: nucleus in an endosperm cell, noyau dans une cellule d’albumen.
clonal in its whole area of introduction except in Madagascar. If R. alceifolius on other
Indian Ocean islands was introduced from Madagascar (Amsellem et al., 2000), pseudogamous apomictic R. alceifolius may also occur in Madagscar.
V. CONCLUSION
The pseudogamous apospory occurring in R. alceifolius in its area of introduction is the
same type of apomixis as that described in European species of the subgenus Rubus
(Thomas, 1940; Gustafsson, 1943; Nybom, 1988, 1995; Weber, 1995; Kollmann et al.,
2000).
To determine whether fruit-set from non-pollinated flowers results from apomixis or
autogamy, individuals with heterozygous microsatellite loci are necessary (Amsellem et
al., 2001a). Examining whether alleles at these loci segregate in the offspring would be a
rapid and accurate means to determine whether fruits are issued from autogamy or apomixis (Roy, 1995).
Further experiments are necessary to confirm our results, through genetic studies of
controlled pollinations, and cytological and embryological observations in Madagascar and
other islands where R. alceifolius has been introduced.
224
Acknowledgements - The authors thank N. Fauve (INRA, Montpellier) for her kind assistance in the field and J.
Escoute (CIRAD, Montpellier) for his help in coloration of pollen tubes and observations at the microscope. D. McKey
(CEFE-CNRS, Montpellier) and C. Lefebvre (ULB Brussels) gave helpful comments on the manuscript. This work
was financially supported by La Réunion district (CIRAD collaborative contract REG/97/0307).
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