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Reproductive Biology |
School of Biological Sciences, University of Canterbury, P.O. Box 4800, Christchurch, New Zealand
Received for publication April 17, 2003. Accepted for publication August 14, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: apomixis • Asteraceae • compatibility analysis • Hieracium • ISSRs • recombination
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Menken et al., 1995
; Widén et al., 1995
; McLellan et al., 1997
; Lyman and Ellstrand, 1998
; van der Hulst et al., 2000
). Indeed, the majority of population studies using molecular markers demonstrate higher than expected levels of genotypic variation, assuming obligate apomixis. Two commonly cited explanations for this phenomenon are low levels of sexual reproduction and mutation (see Vrijenhoek, 1990
, for a review). Until recently, such explanations have remained largely speculative, because there has not been a reliable method by which to differentiate between them. Comparative approaches have been used to explain the origin of clonal plant variation but often lack the power necessary to draw strong conclusions in cases where one mechanism is not clearly dominant (see Richards, 1996
; Arnholdt-Schmitt, 2000
). These methods have proved to be useful in some circumstances, however. For example, in a study of apomictic Taraxacum F.H. Wigg., King (1993)
postulated that the population structure and apparent polyphyletic origin of some groups, detected from ribosomal and chloroplast DNA markers, was more consistent with sexual reproduction than mutation. Conversely, Schneller et al. (1998)
suggested that a single origin followed by subsequent mutations was the most likely mechanism for the low levels of variation detected in the apomictic fern Dryopteris remota (A. Braun. ex Döll) Druce with RAPD (random amplified polymorphic DNA) markers. While in cases like these it is often possible to determine the mechanism due to a clear pattern of variation that can be more readily assigned to either mutation or sex, in many cases a more robust method is required.
More recently, cladistic analysis has been used in the study of facultative apomicts to partition the genotypic variation observed in these populations, especially as detected with dominant markers, between recombination/genetic exchange and mutation (Mes, 1998
; van der Hulst et al., 2000
; Mes et al., 2002
). "Component compatibility analysis" is used to determine if differences between genotypes are best explained by the accumulation of mutations or whether the character patterns observed are more parsimoniously accounted for by genetic exchange. An incompatibility is illustrated by a comparison of potential patterns observed in pair-wise, binary characters; if mutation is the sole mechanism for the generation of variation, only three of the four possible combinations of characters can be achieved in a lineage, excluding the presence of back mutations. If all four possible character combinations are present, this is deemed an incompatibility and is most parsimoniously explained by genetic exchange. Compatibility analysis is applied to molecular methods that produce dominant markers by treating each band as an individual character. Mes (1998)
states that this is most likely an underestimate of incompatibility, as only pair-wise comparisons are made; if multiple characters were included simultaneously then estimates would be expected to be higher. The sum of all incompatibilities in an entire matrix can be used to heuristically gauge the amount of genetic exchange; the higher the number of incompatibilities, the greater the contribution of sexual reproduction to genotypic variation. Compatibility analysis is particularly useful in systems where sexual events are rare, as it does not require a high frequency of genetic exchange for detection (cf. Brookfield, 1992
). A further advantage is that it is not necessary to have an "allelic interpretation," making it applicable to polyploids and dominant data (van der Hulst et al., 2000
).
The present study is unique in that we have an empirical measure of the potential for sexual reproduction in a facultative apomict under field conditions in three populations (Houliston and Chapman, 2001
); we also have measures of genotypic variation in the same populations, based on dominant molecular markers. The combination of these data types has allowed us to examine whether a relationship exists between the potential for sexual reproduction and levels of population genetic variation. This measure of the potential for sexual reproduction is also compared to the results of the compatibility analysis to further attempt to confirm the role of sexual reproduction in apomictic populations. We hypothesize that as the rate of sexual reproduction increases, the amount of incompatibility would also increase, given that the populations have comparable histories and assuming there has not been strong selection on the recruitment of either sexual or asexually derived progeny.
We have chosen Hieracium pilosella L., a perennial, herbaceous weed as a model species. Although it is predominantly apomictic, it is also known to have the potential to reproduce sexually under field conditions (Houliston and Chapman, 2001
). Additionally, populations possess high levels of genotypic variation, almost equivalent to that found in outcrossing species (Chapman et al., 2000
). By combining empirical measures of the rate of sexual reproduction with compatibility analysis, we hope to identify the relative contribution of sex and mutation to the genotypic variation that is observed in these populations.
The populations of H. pilosella selected for this study have the advantage of having only recently colonized the three sites examined, most likely from very few founders. The species has only been recorded in New Zealand in the last 120 yr, having been inadvertently introduced as a contaminant of grass seed, and at this point had a very restricted distribution. The range expansion of this species has only been significant since the 1950s, making most populations of recent origin (Connor, 1992
). There is also evidence that in some cases a proportion of the population variation that is observed has arisen on site (see Chapman et al., 2003
). This gives an excellent opportunity to observe the change that this species has undergone at the population level and to identify the most likely mechanism for the generation of genotypic variation in this facultative apomict.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The use of this technique is possible because H. pilosella is self-incompatible (Krahulcová et al., 1999
). Hieracium aurantiacum ramets from each of a triploid (2n = 3x = 27) and an aneuploid (2n = 3x + 4 = 31) accession, respectively, were multiplied in the greenhouse at the University of Canterbury, Christchurch, New Zealand, to provide a "bank" of approximately 30 plants that were used as pollen donors. These particular accessions were chosen because they were known to produce high levels of functional pollen (R. A. Bicknell, Crop and Food Research, Lincoln, New Zealand, personal communication). Flowering was induced under greenhouse conditions to coincide with flowering of H. pilosella in the field.
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Seed was surface sterilized in a 1% solution of sodium hypochlorite for 50 min and sown on an agar medium containing MS salts and vitamins (Murashige and Skoog, 1962
) with 3% sucrose (Bicknell, 1994
). Once germinated the F1 progeny were propagated in a mist propagation unit for approximately 8 wk and the seedlings then transferred to the glasshouse for characterization.
The potential for sexual reproduction was assayed in each of three seasons for the Chilton Valley and Little River sites (1998/1999, 1999/2000, 2000/2001) and over two seasons (1998/1999, 2000/2001) for the Cave Stream site due to the failure of seed set during 1999/2000 due to drought conditions. While this method provides an estimate of the potential for sexual reproduction, for several reasons this may under- or overestimate the actual frequency of sex. The limitations of this method are discussed in detail in Houliston and Chapman (2001)
.
DNA isolation and polymerase chain reaction (PCR)
Individual rosettes were sampled at each of the three field sites and DNA extracted following the CTAB method described in Chapman et al. (2000)
. At each site selected rosettes were at least 500 mm apart and within the boundaries of the existing field sites of Houliston and Chapman (2001)
. Samples at Little River and Chilton Valley were all collected from an area 5 x 5 m. Due to the patchy distribution of plants at Cave Stream, plants were collected from a 10 x 10-m area.
Inter-simple sequence repeat (ISSR) PCR was carried out in 25-µL reactions (2.5 µL 10x Taq polymerase PCR buffer [Boehringer Mannheim, Mannheim, Germany], 1.25 µL 10 mmol/L dNTPS, 3.0 µL 25 mmol/L magnesium chloride, 1.0 µL 10 mmol/L Primer, 0.2 µL Taq DNA polymerase [Qiagen, Hilden, Germany], 16.05 µL dH20, 1.0 µL suspended DNA product). Primers were selected from the University of British Columbia Biotechnology Laboratory Microsatellite primer set 9 (Table 1) on the basis of a previous survey with H. pilosella (Chapman et al., 2000
). Reactions were amplified in a PTC-200 Thermal Cycler (MJ Research, Watertown, Massachusetts, USA) using the protocol: 4 min at 93°C, followed by 41 cycles of 93°C for 20 s, 48°C for 60 s, and 72°C for 20 s. The PCR was completed using a final extension of 72°C for 4 min and halted by a constant 4°C. The PCR products were separated on a 2% Tris acetate gel (3:1 NuSieve agarose; FMC Bioproducts, Rockland, Maine, USA), stained with ethidium bromide, and photographed under UV illumination. Only bands that were clear and repeatable were included in the analysis.
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). Jaccards similarity was used in all cases as this method takes into account only positive matches between bands, as missing bands in ISSR profiles can occur for several different reasons (Weising et al., 1994
). A UPGMA dendrogram was constructed using a Jaccards similarity matrix. The UPGMA was selected as it gives equal weight to each point in each cluster. This assumes that the clusters in the data are of approximately equal size, and no hierarchical sampling has occurred, with no one cluster being sampled more or less than others. It treats the cases as independent, random samples, as is appropriate for the sampling method used (Kovach Computing Services). Genotypic diversity was plotted against the number of markers included to determine the amount of resolution we had to detect clones in the data set.
Data comparison
The mean percentage of progeny produced sexually per cross was calculated for each of the three sites over the three field seasons recorded. This was regressed against the marker heterozygosity at the site determined from the ISSR profiles, as a measure of population variation, to determine if a relationship existed between the two.
Compatibility in each of the population ISSR data sets was measured by calculating both the total number of incompatible character combinations and the probability of incompatibility. Total incompatibility was calculated as the sum of all character incompatibilities in the data set, where an incompatibility was defined as the presence of all four pairwise combinations of character states in a binary comparison of two characters. The probability of incompatibility was calculated following the method of Le Quesne (1969)
; the distribution of characters was compared to random permutations of characters at the frequencies present in the data set, to try to identify conservatively evolving characters. Markers that identify less incompatibility than is expected from their frequency at random also indicate the amount of recombination that has occurred; the higher the frequency of these conserved markers, the less contribution from recombination to population structure. Furthermore, if it can be shown that conservative characters in a population are responsible for incompatibilities in other populations, this is further evidence for recombination (Mes et al., 2002
).
Matrix incompatibility for each of the sites was calculated using PICA version 4.0 (Wilkinson, 2001
). The "Jactax.exe" function was used to calculate the incompatibilities present in the data set due to all genotypes and to sum the total for the data set. The genotypes responsible for the greatest number of incompatibilities were successively deleted from the data set, following the method of Mes et al. (2002)
. The analysis was then repeated with the next most conflicting genotype removed until all compatibility was resolved. This allowed the calculation of the number of genotypes in the population that could be explained by compatibility and the number having to be removed to achieve this. The probability that a particular ISSR marker had no less incompatibility with other characters in the data set than a random character was examined using the LQPROB.EXE of PICA version 4.0 (Wilkinson, 2001
). This indicates whether marker distributions are significantly more conservative than random permutations and therefore are more indicative of clonal reproductive modes (Mes et al., 2002
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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).
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).
Population genotypic variation
Twenty-seven samples from Chilton Valley and Cave Stream and 28 from Little River were amplified by the five primers. The ISSR primers produced a high degree of scorable bands, although the proportion of polymorphic bands per primer was variable (Table 1). Only one primer (866) was monomorphic over the three populations examined. Genetically identical (clonal) individuals were found at all sites, although no genotypes were shared among sites (Fig. 2). All individuals from the same population clustered together with the exception of two, one each from Chilton Valley and Cave Stream that clustered closer to the Little River group (Fig. 2).
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The potential for sex and genotypic variation
Regression between the average percentage of seed produced sexually under field conditions over the 3 yr (2 yr for Cave Stream) and ISSR marker heterozygosity at the three sites produced a significant, positive relationship (R2 = 0.58, P = 0.029). Seasonal mean percentages of sexual seed per cross were used in the regression as a measure of the proportion of progeny produced sexually at each of the sites. Arcsine transformation of the proportion of progeny produced via sex did not increase the normality of the data set and very slightly decreased the fit of the regression, indicating that data transformation was not desirable (Zar, 1996
).
Compatibility analysis
The Little River sample had high incompatibility, with 59% of the genotypes having to be removed to allow complete compatibility of the data set (Fig. 3). The other two sites had much lower levels of initial incompatibility and required the removal of fewer genotypes to gain complete compatibility (Fig. 3). The continuous decline of matrix incompatibility as genotypes were successively removed indicated that no groups of genotypes were more or less incompatible with each other than any other groups or individuals (van der Hulst et al., 2000
).
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) of the marker distributions was also compatible with sexual reproduction being responsible for the population structures observed. Informative characters for incompatibility at Cave Stream and Chilton Valley had Le Quense probabilities substantially higher than P = 0.05, indicating that distributions of these markers were not significantly different to the results of 1000 random permutations. Of the 20 informative characters from Little River, only five had Le Quense probabilities of P < 0.05 (bands 9, 12, 26, 28, 32). Band 9 was informative for Chilton Valley with a Le Quense probability of 0.947, and band 26 was informative for Cave Stream (P = 0.875). Bands 12, 28, and 32 were not informative for either of the two remaining sites. Different markers were found to contribute to incompatibility in each of the three populations, which is most parsimoniously explained by sex (Mes et al., 2002
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Regression of the proportion of potential sexual reproduction at each site with population genetic variation (marker heterozygosity) produced a positive, significant relationship. Approximately 58% of the total variation in marker heterozygosity was predicted by the level of residual sexual reproduction (P = 0.029). While this indicates that population variation depends on the level of residual sexual reproduction, the limitations of regression prevents us from inferring a deterministic or causal relationship (Howell, 1992
; Zar, 1996
). It should also be noted that other factors such as mutation or gene flow can contribute to heterozygosity, and it should not be inferred from the regression that recombination or sex are the sole contributors to this in these populations.
Several factors suggest there is little if any gene flow between the populations we studied. All genotypes are restricted to only one site, and all three populations have private markers (Table 3). Private markers may indicate sorting of genotypes during establishment of the original populations at each of the sites, different origins for each of the populations, or divergence following founder events. The fact that two bands were conservative informative characters (van der Hulst et al., 2000
) in the Little River population, but not conservative in the other two populations, is similarly consistent with strong population divergence. It should be noted, however, that the low number of informative characters at Cave Stream and Chilton Valley reduce the chances of discovering "conservative, informative" characters.
Compatibility analysis
The use of compatibility analysis can possibly differentiate between the role of sexual reproduction and mutation in determining population structure (Mes, 1998
). The low number of genotypes (three) that had to be removed from Chilton Valley to achieve compatibility indicates that sexual reproduction has not made a large contribution to genotypic variation at this site. Conversely, the high proportion of genotypes having to be removed from Little River indicates that sexual reproduction has played a more substantial role in the structuring of this population.
The level of incompatibility we have demonstrated in Hieracium pilosella is less than Mes et al. (2002)
and van der Hulst et al. (2000)
detected in their studies of Taraxacum. This may indicate that sex has played a lesser role in H. pilosella in generating genotypic diversity, although the present study encompasses a much smaller geographic scale than either Mes et al. (2002)
or van der Hulst et al. (2000)
. Additionally, Mes et al. (2002)
and van der Hulst et al. (2000)
both employed amplified fragmant length polymorphisms (AFLPs), which generate many more genetic markers than ISSRs, and therefore provide greater opportunity to create character incompatibilities. Van der Hulst et al. (2000)
also included over twice as many samples as the present study.
Studies that combine compatibility analysis with an empirical measure of the potential for sexual reproduction in facultative taxa, as in our study, give an improved understanding of the population dynamics. Due to possible recruitment mechanisms, a measure of sexual potential cannot be directly extrapolated to population genotypic variation. If sexual progeny are being recruited at a rate different to the proportion of total progeny they comprise, then it becomes difficult to use this measure as an indication of their importance. Additionally, if seed production is not the only mechanism by which new plants can be recruited, as is the case in H. pilosella, it is difficult to determine the longevity of individuals and to accurately estimate population demographics. However, a measure of the potential for sexual reproduction does at least allow a better understanding of its potential role in the generation of population variation. The ability to confirm the potential for one of the most commonly cited mechanisms for the generation of variation is useful in that it is possible to determine if this mechanism is indeed responsible for the variation that is so often observed. Having confirmed the potential for sexual reproduction under field conditions in H. pilosella allows for more than just speculation about sex being a factor in these populations. Although the logical extension of this is to try to compare the potential for sexual reproduction to the amount of incompatibility present in the populations, there are several reasons why this comparison should not be made. Most importantly, unless the demographics of the populations are very well understood, the influence of recruitment and survivability (see Rose and Frampton, 1999
) is likely to prove to be of greater influence than the rate of sexual reproduction per se. For conclusions to be reached on the amount of incompatibility expected in populations of a species based on the potential for sexual reproduction, even as measured over multiple seasons as in this work, there must be "all else equal" assumptions drawn. Due to the laborious nature of estimating the potential for sexual reproduction, it is not practicable to carry out studies over temporal scales appropriate for the lifespan of the organism in question, even in the case of relatively short-lived plants such as H. pilosella.
The amount of compatibility in all three of the populations indicates that mutation has played a role in the structuring of these populations. In particular, the low level of incompatibility in combination with moderate genotypic variation at Chilton Valley and Cave Stream may indicate that mutation has contributed significantly to these populations.
The findings of the compatibility analysis for the combined data set are interesting in that it was possible to find compatibility whilst retaining genotypes from all sites. This may indicate that the three sites have had a common genetic origin. The same genetic founders may have established at each site and subsequently undergone differentiation. It is also possible that the three sites have been colonized by different but closely related genotypes, with subsequent generation of further genotypic variation via sex and mutation. Alternatively, there may have been gene flow from neighboring populations contributing to the variation at each of the sites. The clustering of the individuals from Little River furthest from those from the other two sites in the UPGMA dendrogram and its geographic isolation is consistent with a longer temporal isolation from the other populations and is perhaps evidence for the contribution of sexual reproduction to genotypic variation at this site. The identification of patterns indicative of sex, and perhaps mutation, demonstrates the dynamic nature of these populations and is further evidence of the differentiation at the population level that has been observed in H. pilosella (see Chapman et al., 2000
; Chapman and Brown, 2001
).
Evolutionary potential of Hieracium pilosella
The levels of genotypic variation detected in this study for the three populations are not unusual in apomictic taxa (Ellstrand and Roose, 1987
; Widén et al., 1995
). The most commonly cited explanation for high genotypic variation in these groups is facultative sex (Asker and Jerling, 1992
; Mogie, 1992
); we have confirmed that this is the case with Hieracium pilosella and have also demonstrated the presence of sexual potential in this species. The findings of the compatibility analysis indicate that sexual reproduction is of different importance in different populations. This is most likely to be controlled by the frequency of sexual reproduction, in association with the amount of seed recruitment and the population size. Some works have suggested that "somatic recombination" may play a role in generating variation in apomictic taxa, primarily those of the diplosporous type (Mogie, 1992
). We do not consider this to be a likely source of variation in the aposporous Hieracium subgen. Pilosella as the origin of the aposporous initial may preclude such mechanisms (Koltunow, 1993
).
Apomictic species appear to have considerable ability to generate genotypic variation. The partitioning of this variation between residual sexual reproduction and mutation events can be tentatively achieved by compatibility analysis. The application of this method as a general step in the analysis studies of clonal plant species may further develop an understanding of these mechanisms. The findings of this work and others (see Ellstrand and Roose, 1987; Widén et al., 1995
; van der Hulst et al., 2000
; Mes et al., 2002
; for reviews) challenge the idea that these species are evolutionary dead ends (Stebbins, 1950
; Burt, 2000
). Further application of techniques such as compatibility analysis may allow the mechanisms for the generation of variation in these groups to be determined at the population level and the evolutionary potential of these species to be better understood.
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FOOTNOTES |
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2 hazel.chapman{at}canterbury.ac.nz
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Asker S. E. L. Jerling 1992 Apomixis in plants. CRC Press, Boca Raton, Florida, USA
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Schneller J. R. Holderegger F. Gugerli K. Eichenberger E. Lutz 1998 Patterns of genetic variation detected by RAPDs suggest a single origin with subsequent mutation and long-distance dispersal in the apomictic fern Dryopteris remota (Dryopteridaceae). American Journal of Botany 85: 1038-1042[Abstract]
Stebbins G. L. 1950 Variation and evolution in plants. Columbia University Press, New York, New York, USA
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