Loss and recovery of Arabidopsis-type telomere repeat sequences 5′-(TTTAGGG)n-3′ in the evolution of a major radiation of flowering plants

Abstract

Fluorescent in situ hybridization and Southern blotting were used for showing the predominant absence of the Arabidopsis-type telomere repeat sequence (TRS) 5'-(TTTAGGG)(n)-3' (the 'typical' telomere) in a monocot clade which comprises up to 6300 species within Asparagales. Initially, two apparently disparate genera that lacked the typical telomere were identified. Here, we used the new angiosperm phylogenetic classification for predicting in which other related families such telomeres might have been lost. Our data revealed that 16 species in 12 families of Asparagales lacked typical telomeres. Phylogenetically, these were clustered in a derived clade, thereby enabling us to predict that the typical telomere was lost, probably as a single evolutionary event, following the divergence of Doryanthaceae ca. 80--90 million years ago. This result illustrates the predictive value of the new phylogeny, as the pattern of species lacking the typical telomere would be considered randomly placed against many previous angiosperm taxonomies. Possible mechanisms by which chromosome end maintenance could have evolved in this group of plants are discussed. Surprisingly, one genus, Ornithogalum (Hyacinthaceae), which is central to the group of plants that have lost the typical telomere, appears to have regained the sequences. The mechanism(s) by which such recovery may have occurred is unknown, but possibilities include horizontal gene transfer and sequence reamplification.

Full text

Loss and recovery of Arabidopsis-type telomere
repeat sequences 5'-(TTTAGGG)
n
-3' in the evolution
of a major radiation of ¯owering plants
S. P. Adams
1,2
{ ,T. P.V.Hartman
1
{,K.Y.Lim
1
{,M.W.Chase
2
,M.D.Bennett
2
,
I. J. Leitch
2
and A. R. Leitch
1*
1
School of Biological Sciences, Queen Mary, University of London, London E1 4NS, UK
2
Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, SurreyTW9 3DS, UK
Fluorescent in situ hybridization and Southern blotting were used for showing the predominant absence of
the Arabidopsis-type telomere repeat sequence (TRS) 5'-(TTTAGGG)
n
-3' (the `typical' telomere) in a
monocot clade which comprises up to 6300 species within Asparagales. Initially, two apparently disparate
genera that lacked the typical telomere were identi¢ed. Here, we used the new angiosperm phylogenetic
classi¢cation for predicting in which other related families such telomeres might have been lost. Our data
revealed that 16 species in 12 families of Asparagales lacked typical telomeres. Phylogenetically, these
were clustered in a derived clade, thereby enabling us to predict that the typical telomere was lost,
probably as a single evolutionary event, following the divergence of Doryanthaceae ca. 80^90 million
years ago. This result illustrates the predictive value of the new phylogeny, as the pattern of species
lacking the typical telomere would be considered randomly placed against many previous angiosperm
taxonomies. Possible mechanisms by which chromosome end maintenance could have evolved in this
group of plants are discussed. Surprisingly, one genus, Ornithogalum (Hyacinthaceae), which is central to
the group of plants that have lost the typical telomere, appears to have regained the sequences. The
mechanism(s) by which such recovery may have occurred is unknown, but possibilities include horizontal
gene transfer and sequence reampli¢cation.
Keywords: phylogeny; telomere; chromosomes; monocots; Ornithogalum
1. INTRODUCTION
Telomeres are the physical ends of chromosomes in
eukaryotes. They have a specialized and complex DNA
^
protein structure (see the review in Price 1999), thereby
enabling them to function in several critical cell processes
(see the review in Pryde et al. 1997). In particular, telo-
meres have a special role in overcoming the problem of
end replication. Conventional template-dependent DNA
polymerases do not replicate the ends of linear DNA
molecules and so chromosome ends are gradually lost
over successive cell generations. Telomere sequences,
however, are typically restored by telomerase, a ribo-
nucleoprotein that synthesizes DNA sequences onto
chromosome ends using an internal RNA template
(Greider & Blackburn 1985; Blackburn et al.1989).
Understanding the role and functioning of telomeres and
their associated proteins has also proved to be extremely
important in assessing the potential of cloned animals
such as `Dolly' to live their normal life spans, the immor-
talization of cell lines and tumorogenesis (Price 1999).
This has led to telomere length being used in the assess-
ment of ageing and apoptosis (Harley & Villeponteau
1995; Karlseder et al. 1999).
As telomeres have so many vital functions, it is perhaps
not surprising that their nucleotide sequence is highly
conserved across a broad phylogenetic spectrum. For
example, there is only a single base pair di¡erence
between the telomeric sequence of all investigated
vertebrates, the fungus Neurospora and the protozoan
Trypanosoma, which share (TTAGGG)
n
and nearly all
plants previously studied, which have the sequence
(TTTAGGG)
n
. Most arthropod telomeres have (TTAGG)
n
repeats, whereas all ciliate protista studied are either
(TTGGGG)
n
or (TTTTGGGG)
n
(Murray 1990). These
`typical' telomeres are thought to be stabilized by an inva-
sion of the 3'-single-stranded telomeric overhang into its
proximal duplex telomeric repeat array, which forms the
so-called T-loop (Gri¤th et al.1999).
The typical telomere sequence in plants was ¢rst char-
acterized in Arabidopsis thaliana (Richards & Ausubel
1988). This Arabidopsis-type telomere sequence has since
been found in many plant species investigated, including
bryophytes (Pellia), lycopods (Selaginella), gymnosperms
(Pinus and Zamia) and most angiosperms studied (Cox et
al.1993;Fuchset al. 1995). The typical plant telomere
sequence was thought to be ubiquitous until Allium and
two other genera in Alliaceae (Nothoscordum and Tu l b a g h i a )
were reported to lack these sequences (Fuchs et al.1995;
Pich et al. 1996a,b). Recently, we showed that it was also
lacking in Aloe (Asphodelaceae) (Adams et al. 2000). This
result led us to look at the new angiosperm classi¢cation
(Chase et al.2000;Fayet al. 2000), which places both
Allium and Aloe in Asparagales, a large group of 28
monocot families containing ca. 28 000 species (ca.11%of
all angiosperms). We used the phylogenetic data of Fay
et al. (2000) for predicting and selecting representative
species from Asparagales families in order to evaluate the
hypothesis that the loss of typical telomere sequences was
Proc. R. Soc. Lond. B(2001)26 8,1541^1546 1541 & 2001 The Royal Society
Received 11 January 2001 Accepted 27 April 2001
doi 10.1098/rspb.2001.1726
*
Author for correspondence (a.r.leitch@gmw.ac.uk).
{These authors contributed equally.

a unique event occurring early in the evolution of Aspara-
gales (estimated to be nearly 90 million years ago)
(Bremer 2000). In order to test for the presence/absence
of Arabidposis-type telomere sequences, we used £uores-
cent in situ hybridization (FISH) and dot
^
blot approaches
using established methods for demonstrating the absence
of these sequences in Aloe (Adams et al. 2000).
2. MATERIAL AND METHODS
(a) Pla nt material
Plant materials and their sources are listed in Appendix A.
(b) In situ hybridization and Southe rn sl ot blotting
The methods for FISH followed Leitch et al. (1994) and
Adams et al. (2000), with minor modi¢cations. Brie£y,
digoxigenin-11-dUTP-labelled (Roche Diagnostics Ltd, Lewis,
UK) or biotin-11-dUTP-labelled (Sigma-Aldrich Company Ltd,
Gillingham, UK) DNA probes were hybridized to root
^
tip
metaphase spreads and sites of probe hybridization detected
using anti-digoxygenin-£uorescein isothiocyanate and avidin-
Cy3 (Vector Laboratories, Burlingame, CA, USA), respectively.
In order to detect the Arabidopsis-type telomeric DNA, the
telomere repeat sequence (TRS) was prepared by polymerase
chain reaction (PCR) concatenation using TTTAGGG primers
and methods described in Cox et al.(1993).Asapositivecontrol
for the FISH technique, either digoxigenin-labelled pTa71 (9 kb
EcoRI fragment of the wheat 18^25S ribosomal DNA (rDNA)
unit) (Gerlach & Bedbrook 1979) or digoxigenin-labelled
pTZ19-R (120 bp fragment of the Nicotiana rustica 5S rDNA unit)
(Venkateswarlu et al. 1991) were used.
The methods for Southern slot blotting followed Adams et al.
(2000). Brie£y, 1 mg of total genomic DNA was loaded into the
slot blot and probed ¢rst with the TRS probe and then reprobed
with pTa71 for rDNA in order to control for the possibility of
poor DNA adhesion during Southern transfer. The Gene
Images
1
random prime labelling and detection modules
(Amersham Pharmacia Biotech, Little Chalfont, UK) were used
for probe labelling and detection following the manufacturer's
instructions.
Plant species from families known or expected to have
Arabidopsis-type telomeres were used as positive controls for the
TRS probe in both FISH and Southern slot blots (see the
`outgroups' in Appendix A).
3. RESULTS
Recently, Fay et al. (2000) produced a well-supported
phylogeny of the 28 Asparagales families (¢gure 1). This
places Alliaceae and Asphodelaceae, which both contain
species shown to lack the Arabidposis-type telomeres, in
more derived positions than Orchidaceae, the only other
Asparagales family studied and shown to contain a
species (Paphiopedilum insigne) with the typical telomere
sequences (Cox et al. 1993). We selected 27 representative
taxa from 16 Asparagales families (see Appendix A) using
the angiosperm phylogeny in a predictive manner in
order to evaluate the hypothesis that loss of typical telo-
mere sequences was a unique event occurring early in the
evolution of Asparagales.
The presence of Arabidopsis-type telomeres can be
detected using FISH and the probe TRS which typically
appear as paired £uorescent dots at or near the termini of
the chromosomes (¢gure 2). No species investigated had a
TRS signal at an interstitial or centromeric location. All
species labelled appropriately with 5S and/or 18^26S
rDNA probes as a positive control (as well as those in the
outgroups, including Tradescantia, Commelinaceae) (see
¢gure 2 and Appendix A). Our results showed that only
11 of the Asparagales taxa examined generated a FISH
signal using TRS probes. The remaining 16 species lacked
aTRS in situ hybridization signal and by inference the
typical telomeric sequence. Southern slot blot analysis
corroborated these data (see ¢gure 3a and Appendix A).
Proof of appropriate DNA transfer to the membrane was
con¢rmed by reprobing the membrane with the 18^26S
rDNA probe. The uneven intensity of signal using the
telomere and rDNA probes re£ects di¡erent copy
numbers of the sequence rather than uneven loading of
the membrane with genomic DNA.
4. DISCUSSION
The results strongly suggest that many species in
Asparagales lack the Arabidopsis-type telomere sequence.
We discuss how reliable a negative result is likely to be
and then describe the phylogenetic distribution of species
that appear to lack the sequence. We discuss mechanisms
that might replace the Arabidopsis-type telomere sequence
and explain how some species appear to have secondarily
regained these sequences.
(a) Demonstrating an abse nce of
Arabidopsis-type telomeres
Our results showed that only 11 of the 27 Asparagales
taxa examined contained the typical telomeric sequences.
The remaining 16 taxa appeared to lack this sequence.
1542 S. P. Adams and others Loss and gain of telomeres in monocots
Proc. R. Soc. Lond. B(2001)
Agapanthaceae
absent
Arabidopsis-type telomere
genus-dependent
present
loss of
'typical'
telomere
sequence
Orchidaceae
Commelinales/
Asparagales
+
+
+
+
+
+
+
+/−
−
−
−
−
−
−
−
−
−
−
Zingiberales
Liliales
Pandanales
Blandfordiaceae
Lanariaceae
Hypoxidaceae
Asteliaceae
Boryaceae
Ixioliriaceae
Tecophilaeaceae
Doryanthaceae
Iridaceae
Xeronemataceae
Hemerocallidaceae
Xanthorrhoeaceae
Asphodelaceae
Laxmanniaceae
Convallariaceae
Asparagaceae
Themidaceae
Aphyllanthaceae
Hyacinthaceae
Agavaceae
Anthericaceae
Herreriaceae
Behniaceae
Anemarrhenaceae
Alliaceae
Amaryllidaceae
Figure 1. Phylogenetic tree for Asparagales and related
outgroups (taken from Fay et al. 2000) showing which
families contained the typical telomere sequences and the
predicted node at which the typical telomeric sequences
were lost during the evolution of Asparagales. The plus
and minus signs indicate the presence and absence of typical
telomere sequences in the families investigated, respectively.

However, demonstrating an absence of a sequence beyond
all doubt is di¤cult. Based on the following discussion,
we estimate that our methods are sensitive enough to
detect low numbers of the (TTTAGGG)-type telomere in
the genome and, thus, those species that did not generate
a signal with the TRS probe may indeed lack functional
Arabidopsis-type telomeric sequences.
(i) In a previous study by Adams et al. (2000) we
carried out asymmetric PCR on Aloe DNA. Like
Allium, genomic DNA from Aloe failed to generate a
DNA product using single-direction (TTTAGGG)
7
primers, clearly indicating that these telomeric
sequences were either absent or present in low
numbers. In positive controls using Nicotiana sylvestris
DNA (a species known to possess typical telomeres)
as the template, single-direction (TTTAGGG)
7
primers successfully generated the expected product.
(ii) It has previously been shown in Southern blotting
that telomeric probes will hybridize to non-identical
but related sequences under high stringency
(Allshire et al. 1988). We therefore conclude that
those species for which DNA failed to hybridize to
the TRS probe in Southern slot blots not only lack
the Arabidopsis-type telomere but also related telo-
meric sequences.
(iii) In previously reported Southern blotting experi-
ments (Adams et al. 2000), we serially diluted
N. sylvestris DNA (which contains typical telomeric
sequences) with Allium cepa DNA (which lacks
typical telomeric sequences) keeping the same total
DNA concentration in each mixture. These dilution
mixtures contained ever fewer typical telomeric
repeats as the amount of Allium DNA increased.
These mixtures were bound to a nylon membrane.
We then probed the blots with TRS and observed
that we could still generate a hybridization signal
when N. sylvestris DNA was diluted 10 000-fold with
Allium DNA. Assuming that each chromosome arm
of N. sylvestris (2n  24) has 0.2^1.3 kb of telomeric
repeats (Fajkus et al. 1996), a 10 000-fold dilution of
N. sylvestris DNA would be predicted to reduce the
abundance of telomeric repeats to less than one copy
per chromosome arm, yet we could still detect TRS
probe hybridization at this dilution. Despite this
sensitivity, we recognize that the TRS probe will not
hybridize e¤ciently to short lengths of a (TTTA-
GGG)-type repeat, and so very low numbers of the
repeat might still be present but undetected.
(iv) Theoretically, even if a few copies of the telomeric
repeat are present at the chromosome ends, the
repeats are unlikely to be maintained because they
would fail to serve as e¤cient primers for telomerase.
Furthermore, end degradation of the lagging strand
at each round of DNA replication causes tens of 5'-
(CCCTAAA)-3' units to be lost (50^200 bp). Short
stretches of the TTTAGGG repeat would require
immediate telomerase-generated synthesis of the
leading strand and then lagging strand replication to
replace the loss. If this failed to happen at each cell
cycle, then the telomere sequence would immediately
erode.
(v) Allium cepa is the only member of Asparagales in
which DNA sequencing of the chromosome ends has
Loss and gain of telomeres in monocots S. P. Adams and others 1543
Proc. R. Soc. Lond. B(2001)
Figure 2. FISH of root
^
tip metaphase chromosome preparations: (a) Tradescantia purpurea, (Commelinaceae),
(b) Kniphophia uvaria (Asphodelaceae) and (c) Ornithogalum umbellatum (Hyacinthaceae). Double labelling was performed in
each experiment using a TRS probe (red signal) and either 5S rDNA (cyan signal) (arrows in a,b)or18^26SrDNA
(cyan signal) (arrows in c) as a positive control. Note that the typical telomeric signal is only seen in (a) Tradescantia and
(c) Ornithogalum.Scalebar 10 mm.
A
G
M
P
J
N
Q
C
I
L
F
O
E
K
H
B
A
G
M
P
J
DD
N
Q
C
I
L
F
O
E
K
H
B
(a)(b)
Figure 3. (a)SouthernslotblotprobedwithTRSand
(b) stripped and reprobed with 18^26S rDNA in order to
ensure that genomic DNA transfer to the membrane had
occurred. One microgram of the following genomic DNAs
were loaded onto the membrane: (a) Aspidistra lurida,
(b) Hosta hybrid, (c) Hemerocallis hybrid, (d ) Albuca altisima,
(e) Hippeastrum hybrid, ( f ) Zephyranthes candida,(g) Iris
tectorum,(h) Chlorophytum tetraphylum,(i) Tecophilaea violi£ora,
( j) Blandfordia nobilis,(k) Scilla cooperi,(l ) Hypoxis occidentalis,
(m) Kabeyia hostifolium,(n) Milligania densi£ora,
(o) Aloe tenuior,(p) Allium cepa and (q) Strelitzia reginae.

been conducted. Here, end cloning failed to reveal
(TTTAGGG) or similar DNA sequences (Pich &
Schubert 1998).
(b) Phylogenetic distribution of the
Arabidopsis-type telomere
All but one of the 11 Asparagales species with
Arabidopsis-type telomeres were clustered in families
occupying the ¢rst three nodes of the Asparagales tree
(¢gure 1). The remaining 16 species lacking typical telo-
mere sequences all belong to families clustered in a
derived clade that diverged after the separation of
Doryanthaceae (¢gure 1), which is estimated to have
occurred perhaps 80 million years ago. The most parsi-
monious interpretation of these results is that there was a
single evolutionary event when the Arabidopsis-type telo-
mere sequence was lost in an early progenitor of most
families of Asparagales, such that now up to 6300 species
(ca. 2.5% of angiosperms) are predicted to lack typical
telomeres. Interestingly, none of the species investigated
that lacked Arabidopsis-type telomeres had interstitial
telomeric signals that could be a relic from ancient
chromosome fusion/rearrangement events. Such signals
are known in a number of plant species (e.g. Vicia, Petunia,
Pinus and Gibasis)(Fuchset al. 1995). Loss of typical telo-
mere sequences has also been observed in some arthro-
pods (including dipterans and an arachnid) (Sahara et al.
1999).
(c) Regain of the Arabidopsis-type
telomere in Hyacinthaceae
Although Scilla cooperi and Albuca altisima lacked the
Arabidopsis-type telomeres as predicted, four species in
Ornithogalum (Ornithogalum umbellatum, Ornithogalum virens,
Ornithogalum montana and Ornithogalum arabicum)andprob-
ably Scilla siberica (D. Schweizer, personal communica-
tion) were shown to possess the typical telomere (all
species in Hyacinthaceae) (e.g. ¢gure 2c). Hyacinthaceae
has a central position within the group of Asparagales
that we predict should lack typical telomeres (¢gure 1).
The most parsimonious explanation is that, after the loss
of the typical telomeres in Asparagales, Ornithogalum
recovered them secondarily. However, this recovery is
clearly very restricted since J. Manning, M. F. Fay and
M. W. Chase (personal communication) and Pfosser &
Speta (1999) have shown that, phylogenetically, Albuca
and Ornithogalum are interdigitated and Albuca has been
shown to lack typical telomeres.
Secondary acquisition could have occurred through
one of several mechanisms, any of which would be novel
for telomeres.
(i) Horizontal gene transfer. The typical plant telomere
sequence and/or enzyme machinery for its synthesis
may have been reintroduced by horizontal gene
transfer from another species, as has been implicated
in Nicotiana tabacum for the acquisition of rep from
geminiviruses and rolC from Agrobacterium (Bejarano
et al.1996).
(ii) Sequence inactivation. A mutation (e.g. point or
frame shift) or epigenetic change (e.g. DNA methyl-
ation or histone acetylation) in typical telomere-
negative species could have inactivated telomerase or
associated enzymes essential for Arabidopsis-type telo-
mere synthesis, but in some species this change has
reverted. Such reversions may be possible over time-
frames of 0.6^6 million years (Marshall et al.1994).
(iii) Sequence reampli¢cation. The methods reported
here which fail to detect typical telomere sequences
in the majority of Asparagales investigated indicate a
loss of these sequences in these species. However, it is
possible that very low numbers of the telomeric
sequence may still be present in at least some of the
species. If true, this opens up the possibility that,
under certain conditions, telomeric sequences could
be reampli¢ed and once again form typical, func-
tional telomeres. If this is the explanation then it
seems likely that other species with typical telomeres
will also be discovered, occurring sporadically
throughout the Asparagales clade that predomi-
nantly lacks the typical telomeres.
(d) Alternative mechanisms for
resynthesizing telomeres
It is clearly of major importance to discover what DNA
sequences have replaced the typical telomeric DNA in
the majority of Asparagales families and whether all
taxa lacking the sequence have the same replacement
mechanism. The typical telomeric sequences in Drosophila
have been replaced by retroelements (HeT-A and TA RT ),
which balance terminal loss of DNA (Danilevskaya et al.
1994; Mason & Biessmann 1995; Biessmann & Mason
1997). In A. cepa (onion) di¡erent chromosome ends may
be terminated by di¡erent DNA sequences such as those
related to En/Spm-transposable elements or rDNA (Pich
et al.1996a,b; Pich & Schubert 1998). Perhaps some
Asparagales families have similarly replaced the typical
telomere with such elements. If so, the elements were
probably already present in the ancestral Asparagales
genome and able, perhaps by competition, to replace the
typical telomere at the point of loss.
Alternatively, it has been shown that chromosome ends
can be synthesized de novo by a mechanism involving gene
conversion (Mikhailovsky et al. 1999; Kass-Eisler &
Greider 2000) and there is growing recognition that this is
an important mechanism in eukaryote telomere
maintenance (Blackburn 2000). Asparagales include many
species with large genomes (Bennett, M. D., Cox, A.V. and
Leitch, I. J. 1998 Angiosperm DNA C-values database.
http://www.rbgkew.org.uk/cval/database1. html/). Perhaps
chromosome elongation by gene conversion is operating
both to increase genome size and to synthesize and stabilize
chromosome ends. If gene conversion or (retro)trans-
position are involved we would expect the DNA sequence
at the chromosome ends to re£ect the `£avour' of that
species' chromatin and the sequences would not necessarily
be restricted to chromosome termini. Alternatively, it may
be that the species shares a common sequence that di¡ers
from the Arabidopsis-consensus sequence at the ends of their
chromosomes and this sequence carries out the essential
functions of the telomere.
Whichever model for Arabidopsis-type telomere replace-
ment is correct, most families within Asparagales have
evolved a novel way of stabilizing their chromosome ends.
It will be important to discover the nature of these alter-
native mechanism(s) and the DNA sequences involved.
1544 S. P. Adams and others Loss and gain of telomeres in monocots
Proc. R. Soc. Lond. B(2001)

The authors wish to thank Dr J. David, Dr C. Pires, Mr R.
Ahmed and Mr J. Kavanagh for help. This work was funded by
the Natural Environment Research Council and the Leverhulme
Trust. We thank Chelsea Physic Gardens for support.
APPENDIX A
Table A1 shows the taxa investigated, listed alphabeti-
cally by family and then by species. The results in the
Southern slot blotting and FISH columns show either the
absence or presence of typical TRS (TTTAGGG) as
detected with a TRS probe. Double labelling with the
TRS probe and either 5S or 18S^5.8S^26S rDNA was
performed for each experiment as a control. A positive
control species was used for each run of experiments in
order to ensure the TRS probe was hybridizing and
detectable.
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Adams, S. P., Leitch, I. J., Bennett, M. D. & Leitch, A. R. 2000
Aloe L.
ö
a second family without (TTTAGGG)
n
telomeres.
Chromosoma 109, 201^205.
Allshire, R. C., Gosden, J. R., Cross, S. H., Cranston, G., Rout,
D., Sugawatara, N., Szostak, J. W., Fantes, P. A. & Hastie, N.
D. 1988 Telomeric repeat from T. thermophila cross hybridises
with human telomeres. Nat u re 332, 656^659.
Bejarano, E. R., Khashoggi, A., Witty, M. & Lichtenstein, C.
1996 Integration of multiple repeats of geminiviral DNA into
the nuclear genome of tobacco during evolution. Proc. Natl
Acad. Sci. USA 93, 759^764.
Biessmann, H. & Mason, J. M. 1997 Telomere maintenance
without telomerase. Chromosoma 106, 63^69.
Blackburn, E. H. 2000 Telomere states and cell fates. Na t u re 408,
53^56.
Loss and gain of telomeres in monocots S. P. Adams and others 1545
Proc. R. Soc. Lond. B(2001)
Table A1 Occurrence of Arabidopsis-type telomere sequence in Asparagales and related monocots
TRS detection positive
rDNA
Southern FISH control
Asparagales
Hosta hybrid (1) Agavaceae absence absence 5S/18S^26S
Allium cepa (2) Alliaceae absence
ö
18S^26S
Hippeastrum hybrid (3) Amaryllidaceae absence
ö
18S^26S
Zephyranthes candida (1) Amaryllidaceae absence
ö
18S^26S
Chlorophytum comosum (3) Anthericaceae
ö
absence 5S/18S^26S
C. tetraphylm (3) Anthericaceae absence
ö
18S^26S
Asparagus sprengeri (1) Asparagaceae
ö
absence 18S^26S
Aloe various (2) Asphodelaceae absence absence 5S
Knipho¢a uvaria (3) Asphodelaceae absence
ö
5S
Milligania densi£ora (2) Asteliacaceae presence
ö
18S^26S
Blandfordia nobilis (2) Blandfordiaceae presence
ö
18S^26S
Aspidistra lurida (2) Convallariaceae absence absence 18S^26S
Doryanthes excelsa (4) Doryanthaceae
ö
presence 18S^26S
Hemerocallis hybrid (1) Hemerocallidaceae absence absence 18S^26S
Albuca altisima (2) Hyacinthaceae absence
ö
18S^26S
Ornithogalum montana (4) Hyacinthaceae
ö
presence 18S^26S
O. arabicum (4) Hyacinthaceae
ö
presence 18S^26S
O. virens (4) Hyacinthaceae
ö
presence 18S^26S
O. umbellatum (4) Hyacinthaceae
ö
presence 18S^26S
Scilla cooperi (2) Hyacinthaceae absence absence 18S^26S
Hypoxis occidentalis (2) Hypoxidaceae presence
ö
18S^26S
Iris tectorum (3) Iridaceae absence absence 5S/18S^26S
I. winkleri (3) Iridaceae
ö
absence 18S^26S
Cordyline australis (1) Laxmanniaceae
ö
absence 18S^26S
Kabeyia hostifolium (2) Tecophilaeaceae presence
ö
18S^26S
Tecophilaea cyanocrocus (5) Tecophilaeaceae
ö
presence 18S^26S
T. violi£ora (2) Tecophilaeaceae presence
ö
18S^26S
outgroup: commelinoids/liliales
Tradescantia purpurea (3) Commelinaceae
ö
presence 5S
Rhoeo discolor (3) Commelinaceae
ö
presence 18S^26S
Strelitzia reginae (1) Strelitziaceae presence
ö
18S^26S
Lilium hybrid (1) Liliaceae
ö
presence 18S^26S
outgroup: dicots
Tanacetum parthenium (1) Asteraceae
ö
presence
18S^26S
T. vulgare (1) Asteraceae
ö
presence 18S^26S
Nicotiana sylvestris (3) Solanaceae presence presence 5S/18S^26S
Nicotiana various (3) Solanaceae presence presence 5S/18S^26S
Rosa canina (3) Rosaceae
ö
presence 18S^26S
Podophyllum hexandrum (4) Berberidaceae
ö
presence 18S^26S
The source of plant material is indicated after species designation: (1) Bardill's Garden Centre, Nottingham, (2) Royal Botanic Gardens,
Kew, (3) Queen Mary, University of London, (4) Chelsea Physic Gardens, London, and (5) Royal Horticultural Society, London.

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