Chapman H, Houliston GJ, Robson B, Iline I. A case of reversal: The evolution and maintenance of sexuals from parthenogenetic clones in Hieracium pilosella. Int J Plant Sci 164: 719-728

Abstract

We provide evidence for the origin of sexual individuals from parthenogenetic progenitors in natural populations. We demonstrate that this reversal has occurred independently in three geographically separated populations of the Asteraceous polyploid, Hieracium pilosella. We used chromosome counts and flow cytometry to determine ploidy and crossing experiments and flow cytometry to confirm sexuality. Inter-simple sequence repeat and allozyme markers demonstrated that the sexuals at each site were more closely related to their parthenogenetic neighbors than to sexuals at other sites. The same markers were used to estimate levels of ramet diversity, which were equally high among the parthenogens and sexuals. The observation that sexuals were always tetraploid is possibly explained by their having arisen through a rare sexual event, the fusion of two reduced (2x) gametes from pentaploid, facultatively apomictic parents. Such a reversal from almost total clonality to obligate sexual reproduction is unusual, and further work will determine whether the sexuals are in evolutionary equilibrium, are increasing at the expense of asexuals, or are simply surviving because of a lack of negative selection pressure.

Full text

A Case of Reversal: The Evolution and Maintenance of Sexuals from Parthenogenetic Clones in
Hieracium pilosella
Author(s): HazelChapman, GaryJ.Houliston, BethRobson, and IliaIline
Source:
International Journal of Plant Sciences,
Vol. 164, No. 5 (September 2003), pp. 719-728
Published by: The University of Chicago Press
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719
Int. J. Plant Sci. 164(5):719–728. 2003.
䉷2003 by The University of Chicago. All rights reserved.
1058-5893/2003/16405-0006$15.00
A CASE OF REVERSAL: THE EVOLUTION AND MAINTENANCE OF SEXUALS FROM
PARTHENOGENETIC CLONES IN HIERACIUM PILOSELLA
Hazel Chapman,
1
Gary J. Houliston, Beth Robson, and Ilia Iline
Department of Plant and Microbial Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand
We provide evidence for the origin of sexual individuals from parthenogenetic progenitors in natural pop-
ulations. We demonstrate that this reversal has occurred independently in three geographically separated
populations of the Asteraceous polyploid, Hieracium pilosella. We used chromosome counts and flow cytometry
to determine ploidy and crossing experiments and flow cytometry to confirm sexuality. Inter–simple sequence
repeat and allozyme markers demonstrated that the sexuals at each site were more closely related to their
parthenogenetic neighbors than to sexuals at other sites. The same markers were used to estimate levels of
ramet diversity, which were equally high among the parthenogens and sexuals. The observation that sexuals
were always tetraploid is possibly explained by their having arisen through a rare sexual event, the fusion of
two reduced (2x) gametes from pentaploid, facultatively apomictic parents. Such a reversal from almost total
clonality to obligate sexual reproduction is unusual, and further work will determine whether the sexuals are
in evolutionary equilibrium, are increasing at the expense of asexuals, or are simply surviving because of a
lack of negative selection pressure.
Keywords: apomixis, invasion biology, polyploidy.
Introduction
While the evolution of parthenogenetic clones from sexual
ancestors is relatively common, especially among plant and
invertebrate taxa (Bell 1982; Maynard Smith 1986; Dybdahl
and Lively 1995), the reverse situation (i.e., the origin of sex-
uals from parthenogenetic clones) is not. This paucity of in-
formation on the de novo origin of sex from asexuals is not
surprising. Theoretically, assuming all else is equal, the twofold
cost of sex (Maynard Smith 1978) should hinder newly evolved
sexuals from increasing in number in a population of well-
established clonal lineages. Parthenogenetic lineages in animals
can originate spontaneously within sexual populations from
single gene mutations (Innes and Hebert 1988) or, more com-
monly, through hybridization events (Vrijenhoek 1978; Foighil
and Smith 1995). In plants, the production of parthenogenetic
seed is termed “apomixis.” Several quite distinct mechanisms
of apomixis are recognized among plant genera (Nogler 1984;
Asker and Jerling 1992; Savidan 2000); here we refer to apos-
porous apomixis in particular because it is the mechanism
relevant to our study. In this type of apomixis in the developing
ovule, the products of meiosis are displaced and, typically,
destroyed by one or more embryo sacs arising directly from
the somatic cells of the nucellus (Asker and Jerling 1992).
Apospory usually arises through hybridization and in Hiera-
cium pilosella L. is controlled by a single dominant locus with
modifiers (Bicknell et al. 2000). Any obligate sexuals evolving
from apomictic lineages would therefore have to represent the
homozygous recessive genotype.
1
Author for correspondence; telephone 64-3-364-2987, ext. 7659;
fax 64-3-364-2590; e-mail h.chapman@botn.canterbury.ac.nz.
Manuscript received September 2002; revised manuscript received March 2003.
Most aposporous taxa are facultative (Bayer et al. 1990;
Asker and Jerling 1992); that is, the sexual pathway to seed
production is not lost, but under conditions conducive to apo-
mixis, the asexual embryo sac outcompetes the sexual one.
Exactly what determines which developmental pathway
“wins” is not yet understood. Apomixis is more common at
high latitudes and altitudes, and several studies indicate that
day length may be involved (Knox 1967). Recently, Espinoza
et al. (2002) have demonstrated that in Paspalum notatum the
frequency of outcrossed individuals varies from 0% to 20%,
depending on time of pollination relative to anthesis. Here we
report the presence of mixed populations of obligate sexual
and apomictic H. pilosella and test the hypothesis that the
sexuals at different sites have originated independently from
coexisting apomicts.
All ecological theories for the maintenance of sex are based
on the premise that sexual offspring are different from one
another, in contrast to their clonal siblings. It was therefore
important for us to establish levels of genetic diversity in all
our ramets, both sexual and apomictic. We chose to use inter–
simple sequence repeat (ISSR) markers for this investigation
because they had already proven to be useful for the identi-
fication of clonal diversity in H. pilosella (Chapman et al.
2000, as Pilosella officinarum) and so would allow direct com-
parisons of clonal diversity. The same data sets were used to
investigate the origins of the sexual ramets; i.e., were they of
independent origins, each having evolved on-site, or were they
a single lineage having spread among sites? A similar approach
to determine the putative origin of asexual clones from sexual
ancestors was followed by Dybdahl and Lively (1995), using
allozymes in snails. We used allozymes to confirm the overall
patterns of genetic diversity identified by the ISSR data and to
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720 INTERNATIONAL JOURNAL OF PLANT SCIENCES
Fig. 1 Location of the three investigated sites, each of which had two coexisting populations of sexual and apomictic Hieracium pilosella
investigate the idea that only certain apomictic genotypes gave
rise to sexuals.
Material and Methods
Study Organism
Hieracium pilosella is a native European Asteraceous herb,
reproducing by either sexual or apomictic seed production or
stoloniferous spread (Tutin et al. 1976). It was first introduced
to New Zealand during the 1800s, most probably as a con-
taminant of imported grass seed. Because of its weedy nature,
New Zealand populations were surveyed during the 1980s for
ploidy and breeding system. Only apomictic pentaploids and
rare apomictic hexaploids were found (Makepeace 1981; Jen-
kins and Jong 1997). Since then, apomictic tetraploids, hexa-
ploids, heptaploids, and aneuploids have been recorded from
some of the same populations (Chapman and Lambie 1999,
as Pilosella officinarum), possibly indicative of evolution.
We have recently demonstrated that the frequency of recom-
binant offspring from aposporous mothers in New Zealand is
typically between 0.2% and 3% (Houliston and Chapman
2001). These rare recombinant offspring are usually them-
selves apomictic and either tetraploid or pentaploid (Houliston
and Chapman 2001). In contrast, some populations produce
up to 30% recombined seeds, and we show here that this is
from the presence of obligate sexual tetraploid individuals.
The mechanism by which such sexuals have evolved from
apomicts is understood for H. pilosella (Chapman and Bicknell
2000). Tetraploid sexuals resulting from crosses among pen-
taploid parents appear to involve the fertilization of a rare
reduced embryo sac (2x) by a reduced pollen nucleus (2x).
Sexual individuals will have the homozygous recessive con-
dition at the apomixis locus (Bicknell et al. 2000). We have
shown that apomictic H. pilosella produces abundant fertile
pollen (Chapman and Bicknell 2000).
Sampling and Identification of Sexuals
Typical New Zealand populations of H. pilosella are su-
perficially uniform, often comprising swards of visually indis-
tinguishable individuals. During a routine survey of over 20
populations of H. pilosella throughout the South Island, we
noticed that in three of them (fig. 1) some of the ramets were
smaller than usual and looked less robust than the majority.
Consequently, we collected forty ramets from each of these
populations for further observation, ensuring we collected
both typical and “diminutive” types. The distribution of di-
minutive ramets (scattered among typical ramets) varied at
each site. At Rakaia, they were present over ca. 0.5 ha of
pasture, while at Dracophyllum Flat (DF) and Lyndon, they
were confined to roughly circular patches of ca. 20 m diameter
in tussock grassland. Our scale of sampling reflected variation
in population size and was chosen to maximize the inclusion
of both typical and diminutive ramets from each population
and to minimize the sampling of vegetative clones. We sampled
along transects, at Rakaia collecting every meter, and at DF
and Lyndon every 30 cm. Ramets were transplanted to the
University of Canterbury greenhouses and maintained at a day
temperature of 25⬚–30⬚C and a night temperature of 15⬚–18⬚C.
High-pressure sodium vapor lamps were used to extend the
natural day length, providing a 16-h photoperiod to promote
floral induction (Yeung and Peterson 1971).
In the greenhouse the morphological differences among the
diminutive and typical ramets mainly disappeared. However,
after anthesis, the once-diminutive ramets from all three sites
produced no filled seed, while the typical-looking ones pro-
duced an abundance of presumably apomictic seed. The pres-
ence of apomixis in these latter individuals was confirmed by
the emasculation of immature capitula (Koltunow et al. 1995).
Any seed production would then have to be the result of apo-
mixis. An absence of any filled seed is likely indicative of ob-
ligate sexual plants (Chapman and Bicknell 2000).
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CHAPMAN ET AL.—REVERSAL OF ASEXUAL TO SEXUAL REPRODUCTION 721
Table 1
Summary of ISSR Bands Scored
Primer name Primer sequence
No. bands
per primer
No. polymorphic
bands
UBC900 5

-ACTTCCCCACAGGTTAACACA-3

65
UBC822 5

-TCTCTCTCTCTCTCTCA-3

66
UBC845 5

-CTCTCTCTCTCTCTCTRG-3

10 10
UBC866 5

-(CTC)
6
-3

10 10
UBC895 5

-AGAGTTGGTAGCTCTTGATC-3

85
Herbarium Specimens
We examined all of the 50 available herbarium sheets (1921–
1998) of H. pilosella from the Canterbury District, where this
work was carried out, for specimens resembling the diminutive
ramets from DF, Rakaia, and Lyndon.
Ploidy Analysis
Ploidy level was determined using a combination of chro-
mosome counts and flow cytometry. Chromosome counts of
squashed root tip preparations followed the method of Kra-
hulcova´ and Krahulec (1999), using lactopropionic orcein
staining. For flow cytometry, isolation of nuclei from leaf tissue
followed the method of Galbraith et al. (1983) with some
modifications. Punched disks of fresh leaf tissue (24 mm
2
) were
placed together with the reference in a plastic petri dish. A few
drops of commercial isolation nuclei isolation buffer, CyStain
UV Precise T solution A (100 mL deionized water, 2.1 g citric
acid, 0.5 g Tween 20) (Partec, Mu¨nster) was added, and the
tissue was chopped finely with a stainless steel razor blade.
After ca. 90 s the sample was filtered through a 30-mm filter
and 2.0 mL of Partec CyStain Precise T solution B (100 mL
deionized water, 7.9 g dibasic sodium phosphate, 0.5 mL DAPI
stock [4.55 mg 4

,6

-diamidino-2-phenylindole, 10 mL deion-
ized water]) was added. Samples were then analyzed for DNA
content after at least 90 s of staining. For this, the Partec PA-
II Particle Analyzing System was employed, using filter com-
binations of UG 1, TK420, TK590, and GG435 and a mercury
arc lamp (HBO 100 W/2). Internal standards were a tetraploid
H. pilosella and a diploid Bellis perennis L. We used B. per-
ennis because it was particularly stable and has a diploid ge-
nome content very close to H. pilosella.
To determine if the diminutive individuals were sexuals, we
looked for evidence of hybridization in their offspring. Four
capitula from plants from each of the three sites (DF, Lyndon,
and Rakaia) were hand-pollinated at full anthesis with pollen
from a known accession of the closely related Hieracium au-
rantiacum (Houliston and Chapman 2001). The orange flower
color of H. aurantiacum, in contrast to the yellow of H. pi-
losella, acts as a readily observable marker for hybrid offspring
(Houliston and Chapman 2001). Seed was collected, surface-
sterilized in a 1% solution of sodium hypochlorite for 50 min,
and sown onto an agar-solidified medium containing MS salts
and vitamins (Murashige and Skoog 1962) and 3% sucrose.
To confirm that all seed produced by these ramets was a con-
sequence of sexual reproduction rather than apomixis, the re-
sulting seedlings were checked for the hybrid traits of inter-
mediate flower color and leaf size.
ISSRs
Fresh leaf tissue was used for total genomic DNA isolation
following the procedure in Chapman et al. (2000). The ISSR
primers (Zietkiewicz et al. 1994) were supplied from the Uni-
versity of British Columbia Biotechnology Laboratory as
primer set 9. They were amplified by the modified PCR pro-
cedure of Williams et al. (1990). Five ISSR primers were
screened over 20 samples. PCR was performed in a 25-mL
reaction mixture per sample (1#Taq polymerase PCR buffer,
400 mM dNTPs, 6 mM magnesium chloride, 0.2 mM of primer,
2.5 Units of Taq DNA polymerase [Roche], and 100 ng of
genomic DNA). The amplification was done in a PTC-200
Thermal Cycler (MJ Research). Initial denaturation was for 4
min at 94⬚C, followed by 40 cycles of 90 s at 94⬚C, 30 s at
48⬚C, 1 min at 72⬚C, and a final extension of 4 min at 72⬚C.
The PCR products were separated electrophoretically on 2%
agarose gels in 1#TAE buffer and stained with ethidium
bromide. The presence or absence of bands was scored under
UV illumination. Five primers (table 1) were selected that gave
clear and consistent banding patterns for the analysis of the
complete sample set.
From this, bands were scored based on their reproducibility
and consistency to determine the ISSR phenotype for each in-
dividual sampled. Duplicates were run for most primer/indi-
vidual plant combinations, and a negative control was included
in each gel.
Allozyme Electrophoresis
Allozyme electrophoresis was carried out on four loci, which
preliminary screening had shown were polymorphic to differ-
ences among sexuals and apomicts. Fresh leaves were ground
in each of three extraction buffers: (a) 40 mM pH 7.5 sodium
phosphate, 1 mM EDTA (tetrasodium salt), 3 mM DTT, 5
mM sodium ascorbate, 3 mM sodium metabisulfite, 6 mM
diethyldithiocarbamate, 5% PVP-40, 5% sucrose, and 0.1%
2-Mercaptoethanol, with a final pH of 7.3 (Rothe 1994); (b)
0.2 M tris-HCl buffer, pH 7.5, containing 0.2 M sodium tetra-
borate, 20 mM diethyldithiocarbamate, 10% PVP-40, 0.25 M
sodium ascorbate, 20 mM sodium metabisulfite, 1% bovine
serum albumine, and 7% sucrose (Weeden and Wendel 1990);
and (c) 0.2 M tris-HCl buffer, pH 7.5, containing 29 mM
sodium tetraborate, 17 mM sodium metabisulfite, 0.2 M so-
dium ascorbate, 16 mM diethyldithiocarbamate, 0.28 M 2-
Mercaptoethanol, 7% sucrose, and 15 mg polyvinylpolypyr-
rolidone per 0.5 mL of buffer (Cosner and Crawford 1994,
with small modifications).
Vertical polyacrylamide electrophoresis (6%–10% acrylam-
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722 INTERNATIONAL JOURNAL OF PLANT SCIENCES
Table 2
Summary of Number of Ramets Analyzed (in Parentheses) and Band Characteristics at Each Site
Total no.
bands No. nonvariable No. variable No. private
No. bands fixed
in apomicts,
absent in sexuals
DF (22) 34 12 22 0 2
Sexual (14) 30 17 13
Apomictic (8) 35 13 22
Lyndon (24) 37 9 28 0 0
Sexual (7) 33 12 21
Apomictic (17) 28 12 16
Rakaia (13) 30 11 19 2 0
Sexual (7) 30 11 19 2
Apomictic (6) 15 14 1 1
Fig. 2 UPGMA dendrogram of all the 59 ramets of Hieracium pilosella from each of the three sites (DF, Lyndon, and Rakaia), and six
populations (sexual and apomictic from each site). Open squares, Rakaia sexual; filled squares, Rakaia apomictic; open triangle, Lyndon sexual;
filled triangle, Lyndon apomictic; open circle, DF sexual; filled circle, DF apomictic.
ide gels) was conducted on protein extracts of leaf tissue in
the several buffer systems, the best results being obtained by
using Ornstein-Davis system (Rothe 1994).
The following enzymes were assayed in all populations:
NADH diaphorase (DIA, E.C. 1.8.1.4.), phosphoglucomutase
(PGM, E.C. 5.4.2.2.), shikimate dehydrogenase (SKD, E.C.
1.1.1.25), and phosphogluconate dehydrogenase (PGD, E.C.
1.1.1.44). Extraction buffer awas used for DIA and PGM and
extraction buffer bfor SKD and PGD. Histochemical stains
for specific enzymes were used as described by Weeden and
Wendel (1990) and Murphy et al. (1996), with some modifi-
cations.
When more than one locus was detected for a particular
enzyme system, the most anodal was designated as locus 1,
the next as 2, and so on. Similarly, at each polymorphic loci
coding for the most anodally migrating allozyme was desig-
nated a, the next b, and so on.
Data Analysis
The interpretation of allele frequency data of dominant
markers must be approached with caution because statistical
methods are based on assumptions of Hardy-Weinberg equi-
librium (Lynch and Milligan 1994). Here we use them only as
a broad indicator of population genetic structure and com-
plement them with the phenetic analysis of molecular variance
(AMOVA) (Excoffier et al. 1992), based on the analysis of
pairwise genetic distances (Excoffier et al. 1992). AMOVA is
now routinely used in the analysis of RAPD and ISSR data
(Huff et al. 1998; Bartish et al. 1999; Kimball et al. 2001).
We also use the multivariate phenetic approach of cluster anal-
ysis to visualize the data.
Descriptive statistics on the allele (ISSR fragment) frequency
data were used to provide an indication of the genetic differ-
entiation among sites (F
ST
values), average heterozygosities (H)
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CHAPMAN ET AL.—REVERSAL OF ASEXUAL TO SEXUAL REPRODUCTION 723
Table 3
Nei’s (1972) Mean Genetic Difference among Sexuals and Apomicts within and among the Six Populations
DF sexuals DF apomicts Lyndon sexuals Lyndon apomicts Rakaia sexuals Rakaia apomicts
DF sexuals …
DF apomicts 0.1420 …
Lyndon sexuals 0.1808 0.2018 …
Lyndon apomicts 0.1814 0.2317 0.1900 …
Rakaia sexuals 0.4465 0.3030 0.2455 0.3352 …
Rakaia apomicts 0.4391 0.3687 0.4553 0.2931 0.1965 …
Fig. 3 Dendrogram of mean genetic distance among the six pop-
ulations (sexual and apomictic) from the three sites, DF, Lyndon, and
Rakaia. Numbers on branches indicate bootstrap support. Only boot-
strap values of 150% are shown.
and percent polymorphic loci (P) for each population. They
were estimated at the 95% criterion using the program “Tools
for Population Genetic Analyses” (TFPGA) (Miller 1997), em-
ploying the Lynch and Milligan (1994) option for analysis of
dominant data. F
ST
values were estimated over all polymorphic
loci and averaged over loci, and confidence intervals at the
99% confidence level were generated by bootstrapping the loci
over 1000 iterations. The percentage polymorphic loci esti-
mates were based on the percentage of loci not fixed for one
allele.
AMOVA (Excoffier et al. 1992) was used to estimate vari-
ance components for the ISSR phenotypes, partitioning the
variation among individuals/within populations, among pop-
ulations/within sites, and among sites. The resulting coeffi-
cients of subdivision, f
CT,
f
SC
, and f
ST
, are analogous to
Wright’s (1965) Fstatistics, but they differ in their assumptions
of heterozygosity. Significance values were assigned to variance
components on the basis of a set of null distributions generated
by a permutation process that draws 1000 individual samples
from the raw matrix and randomly assigns individuals to one
of the six populations (Excoffier et al. 1992).
A salient point was the partitioning of variation among the
sexual and apomictic populations at each site; they could only
be considered populations in their own right if f
ST
was high.
To test this, an AMOVA was run on populations within each
site independently.
The multivariate technique of phenetic cluster analysis was
used to visualize the relationships among all the 59 individual
ramets. A Jaccard similarity matrix, calculated from presence
and absence of bands was used to construct a UPGMA den-
drogram, using MVSP version 3.1 (Kovach Computing Ser-
vices, Pentraeth, UK, 1998). The relationships among sexuals
versus asexuals within and among populations was interpreted
through a cluster analysis. A UPGMA dendrogram of mean
genetic distances for each population was computed using
TFPGA version 1.3 (Miller 1997) and Nei’s (1972) mean ge-
netic distance with the Lynch and Milligan (1994) option for
analysis of dominant data. We chose Nei’s unbiased genetic
distance because the evolutionary model underlying this mea-
sure allows for both neutral mutation and drift among line-
ages. A Mantel test was carried out to determine if there was
a significant correlation between Nei’s (1972) genetic distance
and geographic distance (km), again using TFPGA version 1.3
(Miller 1997).
Results
Breeding System
Strong evidence for obligate sexuality in our diminutive ra-
mets came from the crossing experiments. All of their offspring
showed morphological characteristics intermediate to each
parent; flower color was typically light orange and leaves were
of intermediate shape. While this result could arise from ad-
dition hybrids (the fertilization of an unreduced egg), which
some would argue is not true sexual reproduction, this was
not the case. Flow cytometry of hybrid offspring using ma-
ternal plants from the same populations showed that 100%
of hybrid progeny were 4x, indicative of 2x+2x hybrids and,
therefore, meiosis in both parents (Houliston 2002).
Ploidy Level
All apomictic plants were pentaploid (5xp45). The sexuals
were typically tetraploid (4xp36), although several were of
intermediate DNA mass, equivalent to Ⳳ40 chromosomes.
One of these, from the Lyndon site, was karyotyped and found
to be 4xp36 plus an extra chromosomal fragment (A. Kra-
hulcova´, personal communication). All the hybrid progeny an-
alyzed were tetraploid (4xp36), indicative of reduction divi-
sion in both parents.
Population Structure and Clonal Diversity
The five ISSR primers produced a total of 40 clear and re-
producible bands, 36 (90%) of which were polymorphic (table
1). Table 2, a summary of band characteristics at each site,
shows that only two private bands were recorded, both from
Rakaia. One of these was fixed in the population, while the
other was present in two sexual ramets only. The populations
varied in their levels of genetic diversity, which were H: 0.25,
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724 INTERNATIONAL JOURNAL OF PLANT SCIENCES
Table 4
Descriptive Statistics of the Sexual and Apomictic
Subpopulations at Each Site
DF Lyndon Rakaia
Sexual Apomictic Sexual Apomictic Sexual Apomictic
n13 9 8 16 7 6
H 0.08 0.16 0.19 0.16 0.17 0.09
P 20 57 65 40 52.5 27.5
Note. n, number of individuals sampled; H, average heterozygosity
(direct count); P, % polymorphic loci (95% criterion).
P: 60 (Rakaia); H: 0.24, P: 57.5 (Lyndon); and H: 0.20, P:
47.5 (DF). The estimated F
ST
averaged over all polymorphic
loci was 0.36 (95% C.I. 0.46–0.25), and 20 of the 36 poly-
morphic loci (56%) had F
ST
values of 10.2.
A UPGMA dendrogram, showing relationships among all
of the 59 ramets sampled from the three populations illustrates
a high level of ramet diversity at each site (fig. 2). All 13 ramets
from Rakaia had unique ISSR phenotypes. At Lyndon (np
) only two clones, comprising two apomictic ramets each,24
were identified. Three clones were found at DF ( ), andnp22
the largest of these, comprising seven ramets, was sexual. The
second largest clone comprised five apomictic ramets, some
well separated in space, and others only 30 cm apart. Ramet
diversity among the apomictic clones is as high as among the
sexual individuals. The results of the Mantel test to determine
whether there was a significant correlation between genetic
distance and geographic distance (km) over the three sites was
not significant ( ).
rp0.48
The genetic distance data (table 3) and the UPGMA den-
drogram, bootstrapped over all loci with 1000 permutations
(fig. 3), illustrate relationships among populations and among
sexuals versus apomicts. The DF and Lyndon populations are
more closely related to each other than to Rakaia, and within
each population the sexual individuals are more closely related
to apomicts in the same population than to the sexuals from
other populations. Most nodes on the dendrogram are strongly
supported by the bootstrap analysis (fig. 3).
F
ST
averaged over all polymorphic loci was 0.60 (95% CI
0.70–0.50) for the sexual subpopulations and 0.59 (95% CI
0.70–0.46) for the apomicts.
Descriptive statistics for the sexual and apomictic subpop-
ulations at each site are summarized in table 4. The genetic
diversity H (as described by observed heterozygosity) at the
DF site is quite different from Lyndon and Rakaia in that the
sexual subpopulation has lower heterozygosity and a lower
percentage polymorphic loci (P) than the apomicts. At the
other two sites the opposite is true, with the sexuals having
higher levels of H and P than their respective coexisting apo-
mictic clones.
The AMOVA based on populations corroborates the allele
frequency statistics in demonstrating highly significant genetic
differences among them, ( ), and amongFp0.59 P!0.001
ST
the subpopulations within them, ( ) (tableFp0.42 P!0.001
SC
5). Another AMOVA, based on the analysis of sexual versus
apomictic subpopulations at each site separately, also dem-
onstrated high genetic structuring: DF, ( );Fp0.5 P!0.001
ST
Lyndon, ( ); Rakaia, (Fp0.36 P!0.001 Fp0.39 P!
ST ST
).0.001
Allozyme Electrophoresis
The four enzymes analyzed (SKD, DIA, PGD, and PGM)
produced a total of five variable and interpretable loci, and
15 alleles (table 6). All loci were polymorphic. More genotypes
were recorded in the sexuals than the apomicts at Lyndon and
Rakaia, but fewer genotypes were recorded in the sexuals than
the apomicts at DF (table 6). There were major differences in
some allele frequencies among sexuals and apomicts. For ex-
ample, at the SKD locus, the aallele was present in all the
sexual individuals at each population, but it was rare in the
apomicts from DF and Lyndon (occurring with a frequency of
8% and 27%, respectively) and absent from all five apomicts
sampled at Rakaia. In contrast, the SKD dallele was present
in 92% of the apomicts at DF but was not recorded from the
sexuals at this site. A similar pattern was seen at Lyndon, so
for both of these sites, the rare apomictic genotype a2c3 is the
most similar to the coexisting sexuals. At the PGM locus in
the DF ramets, allele cwas present in 73% of the apomicts
but absent from the sexuals. At Lyndon another difference was
noted; the aallele was present in 77% of the sexuals but in
only 13% of the apomicts. At Rakaia, the aallele was in 45%
of the sexuals but absent from the small sample of apomicts
surveyed.
Discussion
Our results demonstrate the presence of obligate sexual
Hieracium pilosella at three geographically separated sites in
New Zealand: DF, Lyndon, and Rakaia (fig. 1). They support
our hypothesis that they have originated on-site from different
apomictic lineages.
Evolution On-Site
Evidence for an independent, local origin of the sexuals
comes from three main sources: (1) the genetic distance data
(table 3; fig. 3), which illustrate that the sexuals are genetically
more similar to their coexisting apomictic neighbors than to
sexuals from other sites; (2) the distribution of unique ISSR
bands (table 2): the only two “private” bands in the study
were from Rakaia, and one of these was fixed in the population
in both sexuals and apomicts; and (3) the high F
ST
value (0.36)
among the three sites, which suggests a high level of genetic
structure because of low levels of gene flow (Wright 1965).
Although interpreting F
ST
values per se can be dangerous, it
has been suggested that values above 0.25 indicate great ge-
netic differentiation (review Balloux and Lugon-Moulin 2002).
Two alternative explanations could explain the presence of
obligate sexuals at the three sites; either they could have been
introduced into New Zealand from Europe and remained un-
detected until now or they could have evolved in New Zealand
at one site and spread to the others. The first of these alter-
native hypotheses is unsupported by either historical herbar-
ium specimens or ploidy counts. None of 50 herbarium spec-
imens examined from Canterbury (1921–1998) resembled the
diminutive sexuals we collected, and no tetraploids were re-
corded prior to the 1990s (Makepeace 1981; Jenkins and Jong
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CHAPMAN ET AL.—REVERSAL OF ASEXUAL TO SEXUAL REPRODUCTION 725
Table 5
Nested Analysis of Molecular Variance (AMOVA) for 59 Individuals Hieracium pilosella Using 40 ISSR Fragments
Source of variation df SSD MSD
Variance
component % total Fstatistics Pvalue
Among populations 2 136.7 68.37 2.25 30.69 F
ST
0.59 !0.001
Among subpopulations/within sites 3 63.6 21.2 2.11 28.76 F
SC
0.42 !0.001
Among individuals/within subpopulations 53 157.7 2.97 2.97 40.54 F
CT
0.036 0.036
DF:
Among subpopulations 1 21.34 21.34 2.02 49.69 F
ST
0.50 !0.001
Among individuals/within subpopulations 20 40.93 2.05 2.04 50.31
Lyndon:
Among subpopulations 1 22.9 22.9 1.96 36.45 F
ST
0.36 !0.001
Among individuals/within subpopulations 22 75.4 3.43 3.43 63.55
Rakaia:
Among subpopulations 1 19.35 19.35 2.41 39.16 F
ST
0.39 !0.001
Among individuals/within subpopulations 11 41.26 3.75 3.75 60.84
Note. The total data set contains individuals from three populations—DF, Lyndon, and Rakaia—each represented by two subpopulations
(sexual and apomictic). AMOVA was also performed for the two subpopulations at each site. Statistics include sums of the squared deviations
(SSD), mean squares deviations (MSD), variance component estimates, the percentage of the total variance (% total) contributed by each
component, Fstatistics, and the probability Pof obtaining a more extreme component estimate by chance alone (estimated from 1000 sampling
realizations).
1997). The second alternative hypothesis (their evolution at a
single site and subsequent radiation to others) is unsupported
by the genetic distance data, distribution of private alleles, and
high F
ST
values. The only other explanation for sexuals being
more closely related to apomicts at their site than to sexuals
at other sites would be that the sexuals share a common origin
but have subsequently moved into apomictic populations and
hybridized with their new neighbors. Once again, the distri-
bution of private alleles and high F
ST
values make this an un-
likely explanation.
Despite the coexisting sexual and apomictic individuals
within the same population being most closely related to each
other, both the ISSR and allozyme results indicate low levels
of gene flow among them. The F
ST
statistics (table 5) are high
among the sexual and coexisting apomicts, and there were
major differences in some allele frequencies among the sexuals
and apomicts, which most probably reflects phylogeny. The
allozyme data also suggest that sexuals have arisen from a few
rare clones. The apparent low levels of gene flow between the
pentaploid apomicts and the sexuals may be a consequence of
ecological differences between them, such as flowering phe-
nology. In the light of all our evidence, the most parsimonious
explanation for the obligate sexuals is one of independent or-
igin, each sexual lineage having evolved at each site from its
apomict neighbors.
Origin of the Obligate Sexuals
Under the environmental conditions encountered at these
sites apomictic H. pilosella has the potential to produce 0.2%–
3% recombinant seed in an otherwise apomictic capitulum
(Houliston and Chapman 2001) and abundant, reduced viable
pollen (Krahulcova´ and Krahulec 2000). It is therefore possible
that a rare cross will result in an even rarer tetraploid, ho-
mozygous recessive (sexual) offspring. Once established, such
a rare offspring could spread vegetatively by stolons, so that
a single outcrossing event could potentially lead to the pro-
duction of hundreds of sexual ovules and reduced (2x) pollen
carrying the aa genotype. In addition, although H. pilosella is
generally considered to be self-incompatible, selfing can occur,
especially under warm conditions (Krahulcova´ and Krahulec
2000), and this may be an avenue for more recombination. A
single, extremely rare event could thus act as a focus for pro-
moting recombination.
The fact that all of the obligate sexuals are tetraploid im-
mediately introduces the confounding effect of ploidy. Ploidy
alone may explain the size difference observed in the field
among the apomicts (usually pentaploid) and the sexuals (al-
ways tetraploid); high ploidy is frequently associated with high
vigor and a broad ecological amplitude (Stebbins 1950). For
example, de Kovel and de Jong (1999) demonstrated that in
Taraxacum diploids had shorter leaves than triploids under
shaded conditions.
Whether or not the sexuals are in evolutionary equilibrium
is not yet clear, but the fact that at Rakaia they are spread
across at least half a hectare indicates that they are maintaining
themselves, at least in the medium term. Each of the three
populations is currently a mixture of apomictic and sexual
individuals and has the potential for outcrossing, inbreeding
(if self-incompatibility breaks down), and asexual reproduc-
tion. Other species known to sometimes have mixed popula-
tions of apomicts and sexuals include Dichanthium annulatum
(de Wet 1967), Antennaria parlinii (Bayer and Stebbins 1983),
Antennaria media (Bayer et al. 1990), and Taraxacum section
Ruderalia (Menken et al. 1995). Traditional ecological argu-
ments explain the maintenance of sexual individuals among
clones by their being “different,” e.g., the Tangled Bank hy-
pothesis (Williams 1975; Maynard Smith 1978) and the Red
Queen hypothesis (Jaenike 1978; Hamilton 1980; Lively
1996). Asker and Jerling (1992) have reviewed other ecological
mechanisms that have been proposed as allowing coexistence
between sexual and apomictic lineages. These include equilib-
rium theories (frequency dependence and niche differentiation)
and nonequilibrium theories (identical conditions but differ-
ences in competitive ability between sexuals and apomicts).
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726 INTERNATIONAL JOURNAL OF PLANT SCIENCES
Table 6
Frequency of Each Genotype in Each of the Sexual and Apomictic Populations Included in the Study
Locus
and
genotype
DF Lyndon Rakaia
Sexual Apomictic Sexual Apomictic Sexual Apomictic
Skd (18) (13) (7) (22) (14) (5)
a4 0.29
a3b 0.07
a3c 0.06 0.14 0.07
a2b2 0.07
a2bc 0.94
a2c3 0.07 0.27
a2c2 0.57
a2cd 0.29
ab3 0.07
ab2c 0.14
abc2 0.07
abcd 0.14
ac3 0.07
bc3d 0.85 0.73 1
b2cd2 0.77
Pgd2 (19) (14) (16) (27) (19) (5)
a5 0.29 0.22 0.20
a4 0.05 0.81 0.79
a4b 0.15
a4c 0.71 0.63 0.80
a3b 0.05
a2b2 0.06
a2c2 0.95 0.13 0.16
Dia1 (17) (9) (12) (19) (11) (4)
a5 0.67 0.53 0.50
a4 0.17 0.46
a4b 0.22 0.47 0.50
a3b 0.08 0.09
a2b3 0.11
a2b2 0.24 0.59 0.46
ab3 0.35 0.17
b4 0.41
Pgm1 (15) (11) (9) (15) (11) (6)
a4 0.22
a2b2 0.18
a2bc 0.11
a2c2 0.11
ab3 0.33 0.09
ac4 0.13
ac3 0.18
b5 0.27
b4 1 0.11 0.27
b4c 0.55 0.67 0.50
b3c2 0.09 0.20 0.50
b2c3 0.09
bc3 0.11
c4 0.27
Note. Values in parentheses indicate number of individuals sampled.
For frequency dependence theories, any advantage of sex
should be reduced if clonal genotypic diversity is high (Vri-
jenhoek 1978; Parker 1979; Bell 1982; review Lively 1996).
Genetic diversity within all of our populations (table 4) is high
relative to seed plants in general. For example, Deshpande et
al. (2001), using ISSRs, record values of H and P for the small
trees Symplocos laurina and Eurya nitida of 0.18 and 57.4,
and 0.18 and 49.4, respectively. This must reduce the likeli-
hood of frequency dependence playing an important role here.
Different success of sexuals and asexuals in various habitats
may lead to niche differentiation and allow coexistence. The
sexuals in this study are not, morphologically, at least, similar
to their apomictic progenitors. All sexuals are tetraploid, while
all the apomicts described here are pentaploid. Worth noting
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CHAPMAN ET AL.—REVERSAL OF ASEXUAL TO SEXUAL REPRODUCTION 727
is that in our original collections some of the ramets were
tetraploid apomicts, but these all died before analysis. Sexual
plants are diminutive in stature in the field, their ovules take
longer to develop (Bicknell et al. 2000), and they produce fewer
filled seeds (Houliston 2002) than the apomicts. Superficially,
these characteristics imply that the sexuals have a lower fitness,
yet they persist. It may be that some other difference coun-
terbalances the apparent disadvantages and enables the sexuals
in some way to exploit a different ecological niche, thereby
leading to the coexistence of both. For example, Ceplitis (2001)
found that seasonal variation in reproductive output in Allium
vineale was maintaining a mixed mating system because of
different rates of fecundity for seed or bulbil production. In
H. pilosella we have found strong temporal variation in seed
production between the sexuals and apomicts.
However, little is currently known about the dynamics of
such mixed populations, and further study is warranted for
the H. pilosella system described here. It may even be that rare
sexuals survive because of an absence of selection rather than
the presence of it, and because they are constantly generated.
Moreover, as this particular change in breeding system depends
on both the presence of facultative apomixis and the simple
mode of inheritance known to occur in Hieracium subgenus
Pilosella, we cannot generalize about the applicability of this
phenomenon to other apomictic taxa. In order to better un-
derstand this phenomenon and understand the dynamics of
these mixed populations, we will need to determine the fitness
differences among the sexuals and apomicts and measure the
rate of spread and age of the sexual subpopulations.
Acknowledgments
We would like to thank Norm Ellstrand and Ashley Sparrow
for insightful discussion and Mary Morgan-Richards, Steve
Trewick, and Jerry Coyne for informal review of the manu-
scripts and useful suggestions. We also thank two anonymous
reviewers for comments and suggestions. The herbarium sheets
were loaned to us from the Landcare Herbarium, Lincoln,
Canterbury; the Wellington Museum; and the Department of
Plant and Microbial Sciences Herbarium, University of Can-
terbury. This work was supported in part by grants from the
University of Canterbury and the Hellaby Indigenous Grass-
lands Research Trust.
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