American Journal of Botany 88(12): 2243–2251. 2001.
EVIDENCE BIOLOGY OF
FOR A SWITCH IN THE REPRODUCTIVE
RUBUS ALCEIFOLIUS (ROSACEAE)
TOWARDS
APOMIXIS, BETWEEN ITS NATIVE RANGE AND ITS AREA OF INTRODUCTION1
LAURENT AMSELLEM,2,3 JEAN-LOUIS NOYER,2 MARTINE HOSSAERT-MCKEY3,4
AND
CIRAD, Centre de Coope´ration Internationale de Recherche Agronomique pour le De´veloppement, Avenue Agropolis, TA 74/0, 34 398 Montpellier Cedex 5, France; and 3Centre d’Ecologie Fonctionnelle et Evolutive, CEFE/CNRS, 1919 route de Mende, 34293 Montpellier Cedex 5, France
2
We compared the reproductive system of Rubus alceifolius in its native range in Southeast Asia, in Madagascar, where the plant was introduced apparently some centuries ago, and in La Re´union, an Indian Ocean island onto which R. alceifolius was introduced (from Madagascan source populations) around 1850. While tetraploidy makes it impossible to analyze variation in R. alceifolius using classical methods of population genetics, both the patterns of genetic diversity (as revealed by AFLP [amplified fragment length polymorphism] markers) and differences between half-sib progeny and their maternal parents (revealed by microsatellite markers) show that in the plant’s native range in southeast Asia, seeds are produced sexually. In contrast, in Madagascar sexual reproduction cannot alone account for the genetic patterns observed with microsatellite markers. Over 85% of the half-sib progeny resulting from open pollination gave multilocus genotypes identical to those of their respective maternal parents, despite the fact that the latter had alleles that were rare in the population. The other progeny differed in having an allele with one motif more or less than that of the maternal parent. Seeds thus appear to be produced mostly or exclusively by apomixis in Madagascar. We present findings suggesting that Madagascan populations result from hybridization of introduced R. alceifolius and native populations of R. roridus, a closely related species of Rubus subgenus Malachobatus, and suggest that apomixis was a consequence of this hybridization. In Reunionese populations of R. alceifolius (derived from Madagascan populations), seeds obtained in controlled pollination experiments were all genetically identical to maternal parents. While genetic variation (microsatellite markers) in Reunionese populations was low, it was sufficient to allow us to demonstrate that seeds could not have resulted from fertilization by the pollen donors chosen for controlled pollinations, or from autogamy, and were produced exclusively by apomixis. Key words: apomixis; cytogenetics; Indian Ocean islands; Madagascar; microsatellite markers; reproductive biology; Rosaceae; Rubus alceifolius; Southeast Asia; weeds; SSR.
Processes of invasion are very often associated with a reduction in the genetic diversity in populations of the invading species due to founder effects during colonization (Barrett and Richardson, 1986; Frankham, 1997). This reduction in genetic diversity is also caused by selection for certain traits in the mating system of the invading species, such as selfing or asexual reproduction. In fact, selfing species, or species with intense asexual reproduction, predominate among effective colonizers, as has been noted for plants (Allard, 1965; Baker, 1967; Brown and Marshall, 1981; Price and Jain, 1981; Husband and Barrett, 1991), as well as for animals, such as parthenogenetic geckos (Cuellar and Kluge, 1972), slugs (Selander, 1983), and snails (Doums, Perdieu, and Jarne, 1997; Viard, Justy, and Jarne, 1997; Ostrowski, Jarne, and David, 2000). Such traits are preadaptations that appear to facilitate colonization by species that already possess them, and in other species, island colonization has been suggested to be accompanied by shifts to higher levels of inbreeding or clonal growth Manuscript received 19 January 2001; revision accepted 29 May 2001. The authors thank T. Pailler (Universite´ de La Re´union) and N. Faure (INRA, Montpellier) for their able assistance in the field; T. Le Bourgeois (CIRAD, Montpellier) and A. Razafimahatratra (CNRADR, Madagascar) for sampling seeds in Vietnam and Madagascar, respectively; and D. McKey and M. C. Anstett (both of the CEFE, Montpellier), and B. Roy (ETH, Zurich) for helpful comments on the manuscript. This work was financially supported by La Re´union district (CIRAD collaborative contract REG/97/0307). 4 Author for reprint request (e-mail:
[email protected]). 1
(Barrett and Richardson, 1986; Barrett and Husband, 1990). However, few studies have documented major changes in mating systems of invading species in areas of introduction. Such a shift in mating system was suggested by previous information available on a serious weed in several Indian Ocean Islands, the bramble Rubus alceifolius. A comparative study using AFLP (amplified fragment length polymorphism) markers of the genetic diversity of R. alceifolius (Amsellem et al., 2000), through out the plant’s native range (from northern Vietnam to Java) and in its areas of introduction (in the Indian Ocean islands of Madagascar, Mayotte, La Re´union, and Mauritius, and in Queensland, Australia), showed a considerable reduction in genetic diversity in all areas of introduction, with the exception of Madagascar. In Madagascar, Vaughan (1937) considered the Rubus on the island to include an endemic species, which he named Rubus roridus, that differed from R. alceifolius in stipules and hair morphology. Later, other botanists ranked R. roridus as a simple ecotype of R. alceifolius (Owadally, 1960; Rivals, 1960; Kalkmann, 1993). Within the Indian Ocean Islands, each population sampled was characterized by a single clone, and each clone was closely related to a subset of individuals from Madagascar. Successive nested founder events thus appear to have resulted in cumulative reduction in genetic diversity followed by the spread by asexual means of a single principal genotype in each invaded island. However, plants on these islands produce large numbers of fruits and seeds (plants do not set any seeds if
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pollen is excluded, L. Amsellem, personal observation), and areas occupied by the plant are characterized by a dense soil seed bank (The´baud, 1989; Lobet and Triolo, 1999). Variation in reproductive characters and AFLP markers suggest that in the invaded islands, a shift may have occurred from sexually to apomictically produced seeds. This hypothesis is supported by the fact that apomixis has been already documented in Rubus (Thomas, 1940; Gustafsson, 1943; Einset, 1951; Pratt and Einset, 1955; Aalders and Hall, 1966; Nybom, 1988, 1995). In the present study, we investigated whether such shifts in the reproductive system have occurred during the invasion of Indian Ocean islands by R. alceifolius. Microsatellite markers were developed (Amsellem, Dutech, and Billotte, 2001) to investigate the reproductive biology of this weed, because they are multiallelic, codominant, and particularly adapted to discriminate among closely related genotypes (Plaschke, Ganal, and Ro¨der, 1995). We conducted experiments to test for the occurrence of apomixis in R. alceifolius both in its native range and in areas of introduction, through comparison of genotypes of half-sib progenies with those of their maternal parents. Moreover, as apomictic species are known to have a large proportion of empty pollen grains due to disruptions during pollen meiosis (Nybom, 1985, 1988, 1995; Richards, 1986), cytological observations of pollen grains were also performed within populations in the area of introduction to confirm our observations. MATERIALS AND METHODS Studied species—Rubus alceifolius Poir. (Rosaceae, subgenus Malachobatus Focke) is a simple-leafed bramble of Southeast Asia that has become a serious weed in Indian Ocean islands, particularly in La Re´union (Sigala and Lavergne, 1996). The precise origin and time of introduction of R. alceifolius into La Re´union and Mauritius are still unknown, but date of introduction is thought to be about 1850. Populations studied—In the native range—We studied one population, located near Lang Son (228059 N, 1068389 E), in northeastern Vietnam. In this population, several fruits from open pollination were collected from different ramets on the same individual, from which leaves were also collected to determine the maternal genotype. Nine half-sib seedlings obtained from these seeds were grown in a greenhouse. Seeds from other individuals were collected, but unfortunately had to be discarded because no leaves of these other individuals were sampled by the plant collector. Although this limited sample allows no estimation of population level variation of sexual reproduction in relation to other modes, if differences in genotypes between maternal parent and half-sib progeny are in fact observed, this finding would at least demonstrate that sexual seed production occurs for this individual. In the area of introduction—In six different localities from the eastern coastal lowlands of Madagascar, we sampled 1–5 individuals and seeds produced by them in 1–5 populations per locality, each population being ;5 km from other populations in the same locality. A total of 87 individuals were sampled. These were screened to identify individuals with rare alleles that would facilitate comparison of maternal genotypes with those of half-sib progeny. Three Madagascan individuals and their half-sib progenies were selected. Two of these individuals were chosen in the locality of Bengaly, from populations ;15 km distant from each other (individuals B13 and B51). The third individual was chosen in the locality of Ivoloina (individual I12) . Sample sizes for the half-sib progenies of B13, B51, and I12 were n 5 30, 29, and 22 seeds, respectively. For each maternal parent, half-sib progeny consisted of a mixture of seeds taken from several fruits harvested from different branches. In La Re´union, we performed experimental pollination in a population on
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this island in order to test directly for apomixis, using parents with unique patterns of microsatellite markers not found in the rest of the population. These experiments were conducted in the district of Grand-Etang (480 m above sea level, 218049580 S, 558409300 E), near Saint-Benoıˆt. This population is large; flowers are plentiful and the flowering season is prolonged. In a first survey, 20 individuals were sampled along a transect following the road; each individual was separated by a minimum distance of 3–4 m from the previous one. Genotypes of the parents were determined at eight microsatellite loci (Amsellem, Dutech, and Billotte, 2001). In addition to the one dominant monomorphic microsatellite pattern observed in this patch, nine individuals clearly showed variation either at a single locus at which genetically variant individuals were homozygous or at two loci (individuals bearing a homozygous mutation at one locus and a heterozygous mutation at another one). In order to trace their diagnostic alleles in half-sib progenies, double genetically variant individuals were used only as pollen receivers, whereas nonvariant and simple genetically variant individuals were used both as pollen donors and receivers. Flowers of individuals chosen as parent individuals were then bagged at the bud stage to prevent any uncontrolled pollination or contamination of anthers with unwanted pollen before experiments. Each inflorescence of R. alceifolius includes ;12 hermaphroditic flowers. Each flower possesses ;100 separate styles and stigmas, which are much longer than the stamens disposed around them. During 10 d, bags enclosing flower buds were inspected every morning, and flowers were pollinated just after opening, using freshly opened flowers as pollen sources. Preliminary studies (T. Pailler, Universite´ de la Re´union, unpublished data) show that flowers are receptive as soon as they open. Male flowers were obtained by cutting styles with scissors at the height of stamens. These flowers were then taken and carefully rubbed onto the styles of the flowers chosen to receive pollen. One sepal of each pollinated flower was then painted on its external face with acrylic paint, to mark the manipulated flowers within the inflorescence (sepals are persistent). Pollinated flowers were then bagged again to prevent any uncontrolled pollination after manipulation. A total of 44 flowers were manually pollinated. After each individual flowering period, flowers that were still on the maternal parent were then bagged in mosquito net sleeves. Fruits were collected once ripe. Germinated seeds were grown in a greenhouse and seedlings were harvested 1.5 mo later. Their microsatellite profiles were obtained as described in Amsellem, Dutech, and Billotte (2001) and were compared to those of the maternal parent and the pollen donor. Microsatellite markers—Rubus alceifolius is tetraploid (Amsellem, Chevallier, and Hossaert-McKey, in press). Because it is not known whether the plant is allo- or autotetraploid, we were not able to analyze the microsatellite profiles we obtained using classical methods of population genetics. Indeed, in the absence of information on the type of tetraploidy, it is impossible to link alleles to one or the other homeologous loci observed. The analysis was thus restricted to a comparison of genotypes of half-sib progeny to that of their maternal parent, to determine whether sexuality occurs, by the presence or not of alleles not found in the maternal parent. For all localities studied, maternal genotypes and genotypes of their halfsib progeny were determined for the same eight polymorphic microsatellite markers studied by Amsellem, Dutech, and Billotte (2001). AFLP markers—In Madagascar we also conducted an AFLP analysis on the half-sib progeny of individual B13, in order to have an overview of its genetic diversity and for comparison with previous results (Amsellem et al., 2000). We used commercial AFLP kits (GIBCO-BRL) and followed methods used by Amsellem et al. (2000). The four primer pairs used were E-AAC/MCAA, E-AAC/M-CAT, E-AAC/M-CTA, and E-AAC/M-CTT. Pollen aspect—Pollen viability can be a good indicator of disruptions during meiosis in pollen development of apomictic species, as has been shown in other species of Rubus (Nybom, 1985, 1988, 1995; Richards, 1986). If such disruptions occur, a fraction of pollen grains will be empty of cytoplasm and nonfunctional, whereas functional pollen will be full of cytoplasm. To ex-
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ET AL.—APOMIXIS IN
R. ALCEIFOLIUS
2245
TABLE 1. Comparison of microsatellite genotypes of a Vietnamese half-sib progeny with their maternal genotype. Alleles are coded by size (base pair, bp). For each locus, half-sib profiles different from that of their mother plant are indicated in boldface type. New alleles, i.e., alleles not in the maternal parent, are indicated in italic boldface type.
Locus
Maternal genotype
Half-sib progeny 1
2
3
4
5
193 191 255
193
193 191 255
193 191
193 191 255
193 191
225
225
225
225
200
200
192
192 252
mRaCIRRI1D3 mRaCIRRI1G3
mRaCIRRI1H3
208 192
225
mRaCIRRI2B5
198 252
208 198 192 252
230 214 mRaCIRRIV2A8 171 169
mRaCIRRV1C10
151 149 192 188
mRaCIRRV2F4
230 214
230 214
171
171
169 155 192 188
230 214
151 192
225 212 208
8
9
193
193 191
255 225
208 198 192 252
198
230 214
230 214
230 214
230 214
230 214
171
171
171 169
171
151 149
151
151 149 192
151
192 188
188
169
192 188
208 188
188
293 291
293 291 4 6
188
mRaCIRRIV1E8
196 188 311
188 311
188
196 188 311
188 311
309 NLNAa NLDCAb a b
291 2 4
291 4 7
225
250
169 151
149 192 188 210
191 255
192
250 236 230 214 175
7
195 193 255 251
208 198 192
6
291 3 6
291 3 6
291 4 6
291 4 6
149 192 188 208
188
188
309 293 291 3 5
309 293 291 3 5
NLNA 5 number of loci presenting new alleles compared to the maternal parent. NLDCA 5 number of loci presenting different combination of alleles compared to the maternal parent.
amine condition of pollen grains from Reunionese individuals, they were harvested from six flowers, each flower being haphazardly chosen from one individual during manual pollinations. Pollen grains were stained with cotton blue (lactophenol) and observed with an optical microscope. We counted 143–179 pollen grains per flower, determining the fractions of pollen grains empty and full of cytoplasm for each of the six flowers. A total of 971 pollen grains was counted. To determine whether the proportions of the two types were significantly different from each other we applied a x2 test on the whole sample of pollen grains.
RESULTS Reproductive biology in the native range of R. alceifolius—Genotypes of half-sibs and their maternal parents are given in Table 1. Half-sibs showed variable allelic segregation and possessed alleles absent from the maternal parent. Among the 8 screened loci, each half-sib individual showed genetic variation at 4–7 loci and possessed 2–4 alleles not represented in the maternal parent. These results suggest that all half-sibs were produced through allogamy and that apomixis did not occur in the production of these seeds. Reproductive biology of R. alceifolius in its area of introduction—In Madagascar—Details of polymorphism found in the six Madagascan localities are given in Table 2.
Genotypes of maternal parents compared to those of their halfsib progenies are given in Table 3. A total of 12 individuals among the 81 screened in the three half-sib progenies (14.8%) showed variant microsatellite profiles compared to that of their mother plant, involving differences in four loci among the eight tested (mRaCIRRI1D3, mRaCIRRI1G3, mRaCIRRV2F4, and mRaCIRRIV1E8). Genetic diversity revealed with AFLP markers showed that all of the 30 individuals of the half-sib progeny of B13 were monomorphic for the same 491 band levels detected by Amsellem et al. (2000), except for two individuals (which were also among the 12 variant individuals detected with microsatellite markers): descendant B13–15 (polymorphic with primer pair E-AAC/M-CTA) and descendant B13–23 (polymorphic with primer pair E-AAC/M-CAA). Those differences consisted of one band level out of the total of 491 levels for each descendant. No segregation was found among the eight microsatellite loci between the individual B51 and its half-sib progeny, although this mother plant possessed unique variation in the homozygous state on locus mRaCIRRIV1E8, compared to the rest of the population. Of the half-sib progeny of individual I12, four individuals were found to have genotypes different from that of their
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TABLE 2. Details of the microsatellite screening of the putative maternal parents of half-sib progenies of R. alcefolius in Madagascan populations, sampled in six localities of the eastern coast of Madagascar. For each locality and population, the number of variant individuals (VI) are indicated. For each of these, the name of the variant microsatellite locus and the number of different motifs (NDM) are compared with the Reunionese standard type. Cells in boldface type represent individuals I12, B13, and B51, from top to bottom, respectively, chosen to study their half-sib progenies, with their associated microsatellite variation profile compared to the Reunionese standard type. Locality
Population no.
Coordinates
Ranomafana
188569 S; 488259 E
Ampasimadinika
188589 S; 498139 E
Fe´ne´rive Est Ivoloı¨na
178429 S; 498369 E 188449 S; 488249 E
Fanandrana
188279 S; 498229 E
Bengaly
198129 S; 488779 E
Total
Sample size
VI
Locus name
NDM
1 2 3 4 5 1
5 5 3 5 4 3
1 1
mRaCIRRI1D3 mRaCIRRI1D3
1 3
2 3 4 5
4 4 5 5
1
mRaCIRRI1D3 mRaCIRRIV1E8 mRaCIRRIV1E8 mRaCIRRI1D3 mRaCIRRIV1E8
1 21/11 21 23 11/0
1 3
mRaCIRRIV1E8 mRaCIRRI1D3 mRaCIRRI1D3 mRaCIRRI1D3
21 1 21 21
1 1 2 1 2 3
1 5 1 2 3 3
1
mRaCIRRI1D3
21
2
4 5
3 5
1 2
1 2 3 4 5
5 4 4 4 4 87
1 1
mRaCIRRI1D3 mRaCIRRI1D3 mRaCIRRI1D3 mRaCIRRI1D3 mRaCIRRI2B5 mRaCIRRIV1E8 MRaCIRRI1G3
21 21 21 21 21 1 22
mRaCIRRIV1E8
1
1 20
Locus name
2 3
mRaCIRRIV1E8
21
mRaCIRRI1G3
0/22
mRaCIRRI1G3
21
mRaCIRRI1G3
0/21
TABLE 3. Comparison between microsatellite genotypes of the three Madagascan half-sib progenies and those of their respective maternal parents. Only individuals with a genotype different from that of their maternal parent, for the loci of interest, are represented. For each half-sib, variation in comparison with its mother plant is represented in boldface type. Locus
Individuals
MGa VGb
B13 B13-2 B13-9 B13-12 B13-15 B13-20 B13-23 B13-27 B13-28
MGc
B51
MG VG
I12 I12-8 I12-20 I12-21 I12-22
mRaCIRRI1D3
0 0 0 0 0 0 0 0 12/0 0
21 21 21 21 21
mRaCIRRI1G3
mRaCIRRV2F4
0/21 0/22 0/22 0/22 0/21 0/21 0/22 0/22 0/21
0 0 0 0 0 0 0 0 0
0
0
0/22 0/22 0/22 0/22 0/22
0 0/21 11/0 11/0 11/0
MG 5 Maternal genotype. VG 5 Half-sib progeny individuals showing a variant genotype compared to that of their maternal parent. c There was no variant individual in the half-sib progeny compared to the mother plant. a
b
mRaCIRRIV1E8
1 1 1 12/11 12/11 12/11 1 1 1
No. of variants in half-sib progeny
8/30
1
0/29
0 0 0 0 0
4/22
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Fig. 1. Autoradiography of microsatellite locus mRaCIRRI1G3 for individual I12 and its half-sib progeny. Two band levels can be easily distinguished, corresponding to homeologous loci and not to alleles. The ‘‘high’’ locus (noninformative in this study) was not taken into account. This figure shows that no allelic segregation occurs among the 22 descendants of I12, although the maternal parent is heterozygous at the ‘‘low’’ locus (0/22); allele ‘‘22’’ is diagnostic for this individual in its population.
maternal parent, only at locus mRaCIRRV2F4. No segregation was found either at locus mRaCIRRI1G3, for which the maternal parent was found to be heterozygous, or at locus mRaCIRRI1D3. To illustrate that the heterozygosity considered here is ‘‘allelic’’ and not a fixed heterozygosity (band levels would then represent homeologous loci and not alleles of a single physical locus, as could plausibly be expected in an allotetraploid), Fig. 1 shows the autoradiography of the microsatellite locus mRaCIRRI1G3 for individual I12 and its half-sib progeny. Variation in the ‘‘low’’ locus between the maternal parent and the half-sib progeny clearly represents alleles of a single physical locus and not homeologous loci. In this figure, it is also clear that no Mendelian segregation occurred among the offspring studied. In La Re´union—Details of genetic polymorphism in the
patch of Grand Etang are given in Table 4, which also presents the choice of the parents used in experimental pollinations as a function of their genotypes. From the manually pollinated flowers, we obtained 896 seeds from 38 harvested fruits. After scarification, only 71 seeds germinated (7.9%), and 10 seedlings (1.1%) survived after 1 mo. This spectacularly low germination rate and high seedling mortality rate were never observed before in previous efforts to grow R. alceifolius (from native populations). Surviving seedlings were screened with the eight microsatellite loci, and their genotypes compared to those of their maternal parents and pollen donors. Because of the low sample size obtained for Reunionese progenies, our microsatellite results cannot be regarded as conclusive. However, genotypes of all progenies were strictly identical to those of their respective maternal parents (data not presented), showing that fertilization could not have taken place. No segrega-
TABLE 4. Detail of the microsatellite polymorphisms observed in the Reunionese population studied. Reference numbers of individuals indicate their position along a transect. For each individual, the name of the variant microsatellite loci and the number of different motifs (NDM) compared with the Reunionese standard type are indicated. Cells in boldface type represent individuals that were selected as parents in manual pollinations. Reference no.
Category
1
Double genetically variant individual
2
Double genetically variant individual
3
Double genetically variant individual
4
Double genetically variant individual
5 6 7 8 9 10 14 15 16 17 18 19 20
Nonvariant individual Nonvariant individual Nonvariant individual Simple genetically variant individual Simple genetically variant individual Simple genetically variant individual Nonvariant individual Simple genetically variant individual Simple genetically variant individual Nonvariant individual Nonvariant individual Nonvariant individual Nonvariant individual
Locus
NDM
mRaCIRRI1G3 mRaCIRRIV1E8 mRaCIRRI1G3 mRaCIRRIV1E8 mRaCIRRI1G3 mRaCIRRIV1E8 mRaCIRRI1G3 mRaCIRRIV1E8
11/0 11 11/0 11 11/0 11 11/0 11
mRaCIRRIV1E8 mRaCIRRIV1E8 mRaCIRRIV1E8
11 11/0 11
mRaCIRRI1D3 mRaCIRRI1D3
11 11
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tion for the heterozygous loci is seen in the offspring, suggesting that autogamy did not take place, although our sample size for Reunionese half-sibs does not allow us to unequivocally exclude this alternative. Pollen aspect—Of the 971 pollen grains counted, 488 grains were full of cytoplasm and 483 grains were empty, representing 50.3% and 49.7% of the total, respectively. A x2 test showed that in the flowers we studied, nonfunctional grains and functional ones occur in equal proportions (x2 5 0.03; df 5 1; P 5 0.86). DISCUSSION Our results show that Madagascan half-sib progenies and Reunionese offspring from controlled pollination are predominantly produced asexually. In contrast, based on the results of this study and those of Amsellem et al. (2000), sexual reproduction seems to be the exclusive method of reproduction in the native range of R. alceifolius. Moreover, in La Re´union, our observations of pollen grain viability showed that half of the pollen grains within stamens are empty of cytoplasm. These results strongly suggest that R. alceifolius has switched from sexual reproduction to apomixis during the colonization of the Indian Ocean islands. In fact, among the 12 subgenera commonly recognized in the genus Rubus, apomixis is frequent in the European subgenus Rubus, but only in polyploid taxa (Gustafsson, 1943; Nybom, 1986; Weber, 1995). The reproductive biology of tropical species of the genus Rubus has been only briefly investigated. According to Nybom (1986) and Thompson (1997), Asian species of the subgenus Malachobatus, to which R. alceifolius belongs, are mostly polyploid and produce seeds exclusively sexually. However, R. alceifolius was not represented in these studies. Proportions of fully functional pollen grains of the species already investigated (60–95%) are higher than those we found in the present study. The highest values for this biological trait were described for R. moluccanus (95%), which is considered to be the sister species of R. alceifolius. Busemeyer et al. (1997) showed that natural populations of R. moluccanus in the Philippines are genetically very diverse and that gene flow within and among populations is high. Similar conclusions are suggested for R. alceifolius in its native range by a previous study of genetic diversity of the plant (Amsellem et al., 2000). The results on the half-sib progeny of the Vietnamese individual obtained in the present study (all seeds were strictly produced through allogamy), combined with a brief study of genetic diversity in Vietnamese and Thai populations using microsatellite markers (Amsellem, Dutech, and Billotte, 2001) point in the same direction. However, these results must be confirmed with experiments employing controlled crosses and conducted in a number of different populations with large numbers of descendants. Evidence for a switch in the reproductive biology of R. alceifolius in the area of introduction—Madagascan half-sib progenies showed very little variation from the genotypes of their respective maternal parents (12 variant individuals of the 81 considered in the 3 half-sib progenies, or 14.8%), even though plants chosen as maternal parents bore microsatellite alleles that were relatively rare in their populations. Variations observed between maternal parents and their progeny could result either from sexual reproduction or somatic mutations.
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Variations detected in Madagascan half-sib progenies are consistent with the stepwise mutation model (Schlo¨tterer and Tautz, 1993). However, the large proportion of variant individuals found in the half-sibs (23.3% and 18.2% of variants presenting this type of variation in half-sib progenies of B13 and I12, respectively) is not compatible with the mutation rates of microsatellite markers described in the literature, ranging from 1026 to 5.1024 (Tachida and Izuka, 1992; Valdes, Slatkin, and Freimer, 1993). Such a hypothesis about the origin of variations observed in Madagascan half-sibs, if plausible, seems thus not to be exclusive. Variation seems thus to result largely from sexual reproduction. The results are not compatible with production of seeds by autogamy, because in this case there would have been M endelian segregation at heterozygous loci in maternal parents. This pattern was not found at such loci for half-sibs produced by B13 and I12 (as seen in Fig. 1 for individual I12). The most plausible explanation is that the observed variation resulted from allogamous crosses involving pollen bearing variant alleles. This variant pollen could come, for example, from an individual (in the same population or a neighboring population) not sampled during the screening. However, the respective contributions of mutation and recombination in producing the observed variation are unknown. Our observations of pollen grains empty of cytoplasm suggest a high degree of disruption in meiosis during pollen development. Disruptions accompanying apomixis are known to include formation of multivalent, univalent, and sometimes inversion bridges (Thomas, 1940; Gustafsson, 1943; Craig, 1960; Bammi and Olmo, 1966; Nybom, 1988). There may be a link between cytology and the degree of sexuality: The less similar the homeologous genomes of an allopolyploid species, the more regular meiosis is likely to be, and thus the higher the degree of sexuality realized (Thomas, 1940; Nybom, 1988). However, apomixis may also occur even when meiosis is not disrupted (Gustafsson, 1943). The low proportion of Madagascan half-sibs showing variation compared to their maternal parents, the clonal pattern obtained for a Madagascan half-sib progeny with AFLP markers, the apparent absence of fertilization in Reunionese progeny, and the large proportion of empty pollen grains in Reunionese plants (indicating irregular meiosis) all strongly suggest that in the islands where R. alceifolius has been introduced, a very large proportion of seeds are produced asexually and that sexual reproduction is infrequent (Madagascar) or perhaps even absent (La Re´union). The lack of analogous data on numbers of functional and nonfunctional pollen grains in flowers from the Asian native range means we cannot definitively conclude that a switch in that biological trait occurred during or following introduction of R. alceifolius into the Indian Ocean islands. Rubus alceifolius was found to be monoclonal within all Indian Ocean islands other than Madagascar, the source of these secondary introductions (Amsellem et al., 2000). The hypothesis of a sexual reproductive biology associated with the short period of time that had elapsed since the introduction of R. alceifolius in these islands (;150 yr ago for La Re´union and Mauritius [in Lavergne, 1978] and 50 to 100 yr ago in Queensland [E. Bruzzeze and A. Kirk, CSIRO, personal communication]), and the possibility of several independent introductions, would not allow fixation of a genotype.
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Possible scenarios to explain a switch towards apomixis in the area of introduction—Owing to our low sample size of half-sib progeny in the species’ native range, we cannot firmly conclude that a switch in this biological trait occurred in the area of introduction. Thus, it is possible that the plants introduced into the Indian Ocean Islands may have originated from unsampled apomictic lines from the species’ native range. However, if a proportion of the offspring of a sexual plant carries some genes for apomixis, the offspring of an obligate apomict plant only carries apomixis genes. Thus, population genetic models show that if all else is equal, an apomict plant always replaces its outcrossing sexual variety after a few generations (Marshall and Brown, 1981), and prior studies of genetic diversity, using both AFLP and microsatellite markers (Amsellem et al., 2000; Amsellem, Dutech, and Billotte, 2001), didn’t indicate apomixis in the plant’s native range. Other factors, such as difficulty in pollination or in finding mates, might have favored the capacity for asexual seed production. Also, in an ecosystem without the adapted enemies occurring in its native range, Red Queen pressures favoring sexuality are likely to have been relaxed (Jaenick, 1978; Lively, 1987; Ebert and Hamilton, 1996). The higher level of genetic diversity found in Madagascar suggests that genetically different individuals may have been introduced on that island. Pollination is entomophilous (pollinators such as honey bees, Apis mellifera, and a wasp, Polistes hebraeus, are present on these Indian Ocean islands and visit numerous individuals of R. alceifolius [The´baud, 1989, for La Re´union]), and R. alceifolius is potentially autogamous (S. Maurice and D. Strasberg, University of La Re´union, personal communication; L. Amsellem, unpublished data). Thus, difficulty in finding mates does not seem a plausible explanation for asexual seed production in Madagascar. These characteristics, as well as the short period since the introduction of R. alceifolius in Madagascar (from a few millennia to a few centuries ago), are not compatible with the ‘‘sudden’’ appearance of apomixis in Madagascar. The quasi-loss of sexuality of R. alceifolius in the islands where it has been introduced could be a consequence of interspecific hybridization. Its fixation might be also due to some selective advantage or simply represent a consequence of hybridization with no particular adaptive significance in these insular populations. It is interesting to note that some simple-leafed Madagascan Rubus were considered by Vaughan (1937) to constitute an endemic species, Rubus roridus (Lindl.). Alternatively, these may simply represent an ecotype (Owadally, 1960; Rivals, 1960; Kalkmann, 1993; Amsellem et al., 2000). This uncertainty is interesting. The subgenus Malachobatus, to which R. alceifolius belongs, is known only from Southeast Asia (Van Thuan, 1968, 1970; Kalkmann, 1993; Friedmann, 1997). Genetically differentiated populations belonging to a species from this subgenus in Madagascar could come from a remote introduction. Rubus roridus could represent populations differentiated from an ancient introduction of R. alceifolius (for example, carried by proto-Madagascan humans arriving ;3000 yr ago [Wigboldus, 1994]). Evolution and divergence of R. roridus on this island could have been insufficient to completely bar gene flow, but sufficient to create cytoplasmic incompatibility with recently introduced R. alceifolius in the event of hybridization between the two. This could have given rise to a genetically diversified stable hybrid taxon. Such stable hybrid taxa are known in the genus Rubus (Gleason and Cronquist, 1991; Weber, 1996). In this context, it is significant to
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note that many agamospermous plant species are derived through of hybridization (Manton, 1950; Stebbins, 1950; Kirchner and Stepanek, 1996; Holsinger, 2000). An alternative hypothesis is that individuals from several geographically, cytologically, and genetically divergent populations of R. alceifolius in the species’ native range in Asia were introduced in Madagascar. Hybridization between these divergent populations brought into contact in Madagascar could have led to a hybrid taxon with disruption of meiosis and appearance of apomixis. The paradoxical association in Madagascar of high genetic diversity (Amsellem et al., 2000) and occurrence of apomixis could support the hybridization hypothesis. In such a case, a hybrid swarm covering all the genetic intermediates between the two putative parents might be expected. This could explain the presence of several genetically distinct clonally reproducing lineages. Moreover, the low level of sexual reproduction suggested by our analysis of Madagascan half-sibs may be sufficient to maintain variation in a predominantly apomictic population (Watkinson and Powell, 1993; Kraft and Nybom, 1995; Kraft, Nybom, and Werlemark, 1996; Kollmann, Steinger, and Roy, 2000). Another possible scenario to explain monoclonality of R. alceifolius in other Indian Ocean islands is that several genotypes could have been first deliberately introduced into each island (for therapeutic properties, as ornamentals, or as botanical curiosities) several times independently. Then, among all the genotypes present, a particularly aggressive genotype for these insular environments (one in La Re´union and a very similar one in Mayotte, Mauritius, and Queensland [Amsellem et al., 2000]) could have been selected, apomixis permitting the conservation of favorable gene combinations. Conclusion—The evidence presented here of a switch in the reproductive biology of R. alceifolius between its native range and islands where it has been introduced—a switch from sexual seed production to apomixis—is the first evidence for asexual seed production for any Rubus species from the subgenus Malachobatus. The estimation that only ;15% (at most) of the seeds produced in uncontrolled crosses in Madagascan populations were sexually produced is consistent with another study of the reproductive biology of two pseudogamous aposporous European species of Rubus, in which estimated proportions of sexually produced seeds were 14 and 17% (Kollmann, Steinger, and Roy, 2000). To characterize the type of apomixis occurring in the introduced range of R. alceifolius, other experiments must be done, accompanied by both embryological and cytological observations. Apomixis may have allowed a particularly well-adapted and aggressive genotype of R. alceifolius to spread through these islands and to invade their native plant communities, which are known to be susceptible to invasions (Pickett, 1976; Mueller-Dombois, 1981). Our study suggests that the change in the reproductive biology of this weed took place in Madagascar, although more detailed studies on this island are necessary to validate this hypothesis. The hypothesis of the origin of an asexual hybrid in Madagascar must be confirmed, either by a fine taxonomic screening of individuals from wild populations or through controlled pollination experiments. LITERATURE CITED AALDERS, L. E., AND I. V. HALL. 1966. A cytotaxonomic survey of the native blackberries of Nova Scotia. Canadian Journal of Genetics and Cytology 8: 528–532.
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