Structure-activity studies of semiochemicals from Caladenia
plicata for sexual deception
BJORN BOHMAN1,2,3*, AMIR KARTON1, GAVIN R FLEMATTI1, ADRIAN
SCAFFIDI1, AND ROD PEAKALL1,2
1School of Molecular Sciences, The University of Western
Australia, Crawley WA 6009, Australia
2Research School of Biology, The Australian National University,
Acton ACT 2601, Australia
3Research School of Chemistry, The Australian National
University, Acton ACT 2601, Australia
[email protected]
Abstract− Sexually deceptive orchids attract specific
pollinators by mimicking insect sex pheromones. Normally this
mimicry is very specific and identical compounds have been
identified from orchids and matching females of the pollinators. In
this study, we conduct a detailed structure-activity investigation
on isomers of the semiochemicals involved in the sexual attraction
of the male pollinator of the spider orchid Caladenia plicata. This
orchid employs an unusual blend of two biosynthetically unrelated
compounds, (S)-β-citronellol and 2-hydroxy-6-methylacetophenone, to
lure its Zeleboria sp. thynnine wasp pollinator. We show that the
blend is barely attractive when (S)-β-citronellol is substituted
with its enantiomer, (R)-β-citronellol. Furthermore, none of the
nine-possible alternative hydroxy-methylacetophenone regioisomers
of the natural semiochemical are active when substituted for the
natural 2-hydroxy-6-methylacetophenone. Our results were surprising
given the structural similarity between the active compound and
some of the analogues tested, and results from previous studies in
other sexually deceptive orchid/wasp systems where substitution
with analogues was possible. Interestingly, high-level ab initio
and density functional theory calculations of the
hydroxy-methylacetophenones revealed that the active natural
isomer, 2-hydroxy-6-methylacetophenone, has the strongest
intramolecular hydrogen bond of all regioisomers, which at least in
part may explain the specific activity.
Key Words− Caladenia, sexual deception, pollination,
structure-activity, isomers, hydrogen bonding.
Acknowledgements: BB, GRF, and RP acknowledge the Australian
Research Council for funding (DE160101313, FT110100304, LP130100162
and DP150102762). The authors acknowledge the facilities, and the
scientific and technical assistance of the Australian Microscopy
& Microanalysis Research Facility at the Centre for Microscopy,
Characterisation & Analysis, The University of Western
Australia, a facility funded by the University, State and
Commonwealth Governments. Alyssa Weinstein is thanked for field and
laboratory assistance. RP was hosted as a visiting fellow by UWA
while completing this study.
INTRODUCTION
Sexual deception is a highly specialised pollination strategy
where insect pollinators are sexually lured to flowers by pheromone
mimicry. This strategy is most commonly found among the Orchidaceae
(Ayasse et al. 2011; Borg-Karlson 1985; Franke et al. 2009; Gaskett
2011; Johnson and Schiestl 2016; Phillips et al. 2014; Schiestl et
al. 200) where it has evolved independently in many unrelated
genera (Ayasse et al. 2011; Gaskett 2011; Phillips et al. 2014,
Bohman et al. 2016). Despite comprehensive early studies of the
pollination biology of some sexually deceptive orchids in Europe
and Australia (Coleman 1929; Kullenberg 1950), chemical ecology
investigations leading to identification of the semiochemical(s)
involved commenced much later (Bohman et al. 2012a; Bohman et al.
2012b; Bohman et al. 2014; Bohman et al. 2017a; Borg-Karlson 1987;
Borg-Karlson 1990; Cuervo et al. 2017; Franke et al. 2009; Gervasi
et al. 2017 ; Schiestl et al. 1999; Schiestl et al. 2000; Schiestl
et al. 2003; Xu et al. 2017).
Despite the successful elucidation of the chemistry of sexual
deception in a growing number of cases (see the recent review of
Bohman et al. (2016) for a summary), these represent just a
fraction of the several hundred orchids using this pollination
strategy. Nonetheless, it is already evident that an extraordinary
diversity of semiochemicals is involved, and that more often than
not, the chemical cues consist of unusual compounds, or even new
classes of compounds on their initial discovery (see references
above). Furthermore, so far, the orchid semiochemicals involved
precisely match constituents of the sex pheromone of the
pollinators (Bohman et al. 2016). Such examples include Ophrys
sphegodes (Schiestl et al. 1999; Schiestl et al. 2000), Ophrys
speculum (Ayasse et al. 2003), Chiloglottis trapeziformis (Peakall
et al. 2010; Schiestl et al. 2003), Drakaea glyptodon (Bohman et
al. 2014), Caladenia crebra (Bohman et al. 2017a) and Caladenia
plicata (Xu et al. 2017).
In all documented cases so far, precise matches between insect
sex pheromones and floral pollinator attractants have been observed
(Bohman et al. 2016, Bohman et al. 2017, Xu et al. 2017), raising
the question as to whether this tight specificity is a general
characteristic of sexual deception. Scope for substitution of
natural semiochemicals with synthetic structural analogues in
sexually deceptive pollination systems has previously been tested
in two cases of Australian hammer orchids (Bohman and Peakall 2014;
Bohman et al. 2015). In the first case involving a Catocheilus wasp
pollinator of Drakaea livida, it was shown that
3-(3-methylbutyl)-2,5-dimethylpyrazine, a close analogue to the
floral pollinator attractant
2-(3-methylbutyl)-3,5,6-trimethylpyrazine, was equally active in
attracting the pollinator. In the second case,
2-hydroxymethyl-3,6-diethyl-5-methylpyrazine, one of two components
of the sex pheromone of Zaspilothynnus trilobatus and the
attractant from Drakaea glyptodon, was substituted with a set of
structural analogues. One analogue,
2-hydroxymethyl-3,5-dimethyl-6-ethylpyrazine, exhibited comparable
activity to the natural product. These two studies illustrate that
in these particular wasp-semiochemical interactions some changes in
the positions of methyl and ethyl substituents around an aromatic
ring can be tolerated with little or no change in the biological
activity. Such active homologues could function as parapheromones
and may have advantages for the producer, in this case the orchid,
over the natural product in terms of ease of synthesis or improved
physicochemical properties. From an evolutionary perspective, less
structural specificity may allow evolutionary flexibility and
increased sustainability.
Despite Caladenia being a very large orchid genus comprising
>350 species, with many of these species sexually deceptive
(Bohman et al. 2017b), the pollination chemistry was until
recently, unknown. In C. crebra and C. attingens,
(methylthio)phenols are now confirmed to be the chemical cues for
pollinator attraction (Bohman et al. 2017a, Bohman et al. 2017b).
Caladenia plicata, employs an unusual combination of two
biosynthetically unrelated compounds, (S)-β-citronellol and
2-hydroxy-6-methylacetophenone, to sexually lure the males of a
single thynnine wasp species (Zeleboria sp.) as pollinator. A 1:4
blend elicits the strongest sexual response with attempted
copulation rates equivalent to that observed at flowers, when
compared with 1:1, 20:1 and 1:20 blends (Xu et al. 2017).
In this present study, we investigate in detail the
semiochemical requirements of this interaction between Caladenia
plicata and its Zeleboria sp. pollinator. First, the requirement of
enantiomeric specificity of β-citronellol was evaluated by
comparing pollinator attraction between the (R)- and
(S)-enantiomers in blends with the natural
2-hydroxy-6-methylacetophenone. Second, by synthetically preparing
all ten possible regioisomers of hydroxy-methylacetophenone, the
effects of the position of all ring substituents were evaluated by
blending with (S)-β-citronellol. Unexpectedly, none of the
alternative acetophenones were biologically active. Similarly,
(R)-.β-citronellol was significantly less attractive compared to
the naturally occurring enantiomer. To better understand the strict
specificity requirements for the acetophenone compounds , we
estimated the structural and energetic characteristics of each
regioisomer generated by high-level ab initio and density
functional theory calculations to provide clues towards
understanding the basis of this extreme structural specificity of
the antennal receptor.
METHODS AND MATERIALS
Bioassays
To assess the level of pollinator sexual attraction when
(S)-β-citronellol was replaced by (R)-β-citronellol, 1a was
replaced by 1b at the optimal 1:4 ratio, in field bioassays that
followed Xu et al. (2017). In short, spiked dummies made up of
black plastic dressmakers pins (4 mm diameter pinhead) attached to
bamboo skewers (25 cm in height) were prepared for sequential
experiments. Each experiment consisted of a series of two-phase
trials. In phase one, the test blend of 1b and 2a was presented for
3 min. Phase 2 commenced with the addition of the control blend of
1a and 2a on a second dummy ca. 1 m from the test blend dummy,
perpendicular to the wind direction. Four replicate experiments
each with new dummies and chemical preparations, and consisting of
four or more trials, were conducted over two successive days, with
each day including one morning and one afternoon experiment.
To evaluate the wasp response to the various regioisomers of the
hydroxy-methyl acetophenone, 2a was replaced with one of 2b-2j or 3
(Fig 1) in the blend with 1a in a series of field bioassays. We
initially began tests of the regioisomers in 2014 at blend ratios
of 1:1, before we had experimentally established that the optimal
blend ratio was 1:4 (1a:2a). For these experiments, up to four
different test blends and a control blend were made up
simultaneously each morning or afternoon. Subsequently, sequential
two-phase trials (as above) were conducted, with a single test
blend evaluated for 3 min, followed by the second phase choice test
involving the addition of the control blend (~ 1 m away) for a
further 3 min. Each of the test blends were randomly allocated to
successive trials, ensuring each test blend was evaluated twice
over the course of either a morning or afternoon session.
None of the regioisomers (other than the natural product) were
found to be active at the 1:1 ratio. However, following our
discovery that the optimal blend ratio was 1:4, in 2015 and 2016 we
re-tested the regioisomers at this ratio to ensure the lack of
response was not due to a suboptimal blend. By this point in time,
we had also firmly established that the wasps respond rapidly
(within 1 min) to the optimal blend. Therefore to expedite the
number of experiments that could be conducted, the second phase
choice test was limited to just one minute.
All field bioassays were conducted at a study site in natural
heathland near the small township of Peaceful Bay (S35.025°,
E116.935°) where male Zeleboria sp. wasps were known to be locally
abundant (Xu et al. 2017) in September-October 2014-2016. The
experiments were restricted to warm (>18°C) and sunny conditions
with limited wind between the hours of approximately 10 am and 2
pm, when male thynnine wasps commonly are known to be actively
patrolling for mates (Peakall 1990). These stringent weather
conditions considerably restrict the number of days available for
conducting bioassays during the short peak flying period of the
wasp (~3-4 weeks), particularly in this southern coastal location
with highly variable spring weather. The number of wasps that
approached, landed and attempted to copulate were recorded for each
phase and treatment. Individual trials were aborted as unsuccessful
if there was no wasp response to the control blend in phase 2.
Given the typical rapid decline in pollinator responses over the
3 min experiments, and results from mark-recapture studies
demonstrating that other thynnine wasps do not revisit baits on the
same day, the risk of pseudo-replication, within or between trials,
is considered unlikely (Peakall 1990; Bohman et al. 2014; Whitehead
and Peakall 2014). Consequently, as in previous studies, the total
number of approaches, lands and attempted copulations were pooled
across trials for each treatment, with G-tests performed in GenAlEx
6.5 (Peakall and Smouse 2006; Peakall and Smouse 2012) to compare
the proportion of total responses among control and treatment in
phase 2 of the sequential trials.
General chemical procedures. The two enantiomers of
β-citronellol (1a and 1b) and 2-hydroxyacetophenone (3) were
purchased from Sigma Aldrich (Australia).
3-Hydroxy-4-methylacetophenone (2g) was purchased from Astatech Inc
(USA). All other isomers of hydroxy-methylacetophenones were
prepared according to literature procedures (2a (Seidel et al.
1990), 2b-2d, 2j (Wang et al. 2012), 2e-2f, 2h-2i (Lafleur et al.
2009)). NMR spectra were consistent with previously reported (2a,
2c-2d (Seidel et al. 1990), 2b, 2j (Fischer and Henderson 1981),
2e, 2h (Lafleur et al. 2009), 2f (Baldwin and Lusch 1982), 2i
(Fischer et al. 1978)).
Ab initio Calculations. Without an experimental structure of the
olfactory receptors of thynnine wasps, which can serve as a
starting point for theoretical simulations, we were unable to model
specific ligand-receptor interactions. Rather, to gain a better
chemical understanding of the observed strict structural
specificity, we have optimised the geometries of all the
methyl-hydroxyacetophenone isomers (Fig. 1), looking for structural
differences not obvious to the “naked eye”, which may explain the
otherwise incomprehensive observations. We show the there are some
structural differences between the isomers that may play a role in
their biological activity. In addition, we also carried out
high-level ab initio calculations in order to energetically
characterise the methyl-hydroxyacetophenone isomers. The high-level
ab initio and density functional theory calculations were performed
using the Gaussian 09 program suite (Frisch et al. 2009).
Geometries and vibrational frequencies were obtained at the
B3LYP/6-31G(2df,p) level of theory. Zero-point vibrational energy,
enthalpic, and entropic corrections have been obtained from such
calculations. The equilibrium structures were verified to have all
real harmonic frequencies. Gas-phase Gibbs free energies at 298 K
were obtained using the G4(MP2) thermochemical protocol (Curtiss et
al. 2007).
RESULTS
Bioassays: Neither (S)-β-citronellol (1a) nor
2-hydroxy-6-methylacetophenone (2a) could be substituted with their
enantiomer (1b) or regioisomers (2b-2j) and 3 respectively and
retain pollinator attraction comparable to the control blend (Fig.
2 and Table 1).
A total of 151 wasp responses were recorded across the 4
replicate experiments evaluating the wasp response to
(R)-β-citronellol (1b) as a substitute for (S)-β-citronellol (1a)
in the 1:4 optimal blend with 2-hydroxy-6-methylacetophenone (2a).
Of these responses, only 19 responses (13% of total) were recorded
to 1b in phase 1, while in the second phase with the test and
control choice, only 4 of the 132 visits (2.1%) were recorded to
the test blend (Fig. 1). While barely attractive, it is evident
that the presence of the (R)- enantiomer in the phase 2 choice test
is not inhibitory at the scale tested (~ 1 m from the (S)-
enantiomer blend). Further, it should be noted that commercial
samples of β-citronellol (99% purity) were used. Thus, traces of
the opposite enantiomer, difficult to detect by enantioselective
gas chromatography due to poor separation, may have attributed to
the weak attraction observed.
No wasp responses were recorded over the 3 min of phase 1 to any
test blend combination of 1a and any single regioisomers 2b-2j or 3
in either the 1:1 or 1:4 blend (Table 1). There was also no
response to these test blends observed in phase 2. By contrast, as
expected, wasps responded rapidly to the control in phase 2. For
example, for the 1:1 blend, at total of 23 responses were recorded
across the replicate trials for each of the 9 tests in the first
min (mean±se responses per replicate control for each
test=2.56±0.50), with a total of 89 responses over all replicate 3
min trials (mean±se responses per replicate control for each
test=9.89±0.92). For the 1:4 blend, a total of 49 responses were
recorded at the control (mean±se responses per replicate control
for each test=5.44±0.55) in the first min (Table 1).
Ab initio calculations. All acetophenone derivatives 2a-2j and 3
in (Fig. 1) appear structurally similar to each other, in
particular 2a-2d and 3, with the hydroxy positioned adjacent to the
carbonyl, yet only compound 2a is biologically active. High-level
calculations using the G4(MP2) thermochemical protocol were
performed to probe the structural and energetic differences between
compounds 2a–2j. Fig. 3 shows the optimised lowest-energy
conformations of the ten regioisomers.
In the active pheromone (2a), the acetyl and hydroxyl groups are
in adjacent positions on the phenyl ring such that there is a
hydrogen bond between the hydrogen of the hydroxyl and the carbonyl
of the acetyl group. The length of this bond is 1.557 Å, indicating
it is a relatively strong hydrogen bond. It is possible to estimate
the strength of a similar intramolecular hydrogen bond by
considering the two conformations of 3 shown in Fig. 4. In one
conformation the acetyl and hydroxyl groups are pointing towards
each other forming a hydrogen bond (Fig. 4a), whilst in the other
conformation they point away from each other (Fig. 4b). On the
Gibbs-free energy surface at 298K (∆G298), the conformation with
the hydrogen bond is lower in energy than the one without the
hydrogen bond by 31.1 kJ mol–1. We note that the length of the
hydrogen bond in 3 is longer than that in compound 2a by 0.098 Å,
indicating that the hydrogen bond in compound 2a is slightly
stronger than in 3. We also note that the length of the hydrogen
bond in compound 2a is slightly shorter than that in the remaining
compounds with intramolecular hydrogen bonding capacity, 2b, 2c,
and 2d (see Fig. 4). This might arise from steric repulsion of the
acetyl methyl group with the adjacent aromatic methyl group in 2a,
in effect pushing the carbonyl oxygen closer to the hydroxyl
hydrogen.
Given that the intramolecular hydrogen bond in the active
pheromone 2a is estimated to be relatively strong, this structural
feature might be important for the interaction of the receptor with
compound 2a. In compounds 2e–2j, the acetyl and hydroxyl groups are
not positioned on adjacent carbons, so lack this hydrogen bonding
arrangement and are biologically inactive. However, compounds 2b-d
do contain this hydrogen bonding arrangement, but are also
biologically inactive. On the other hand, 2a is unique in that it
is the only compound with effectively adjacent methyl groups (Fig.
2), which might be important for binding in the receptor site.
DISCUSSION
Only the two natural product semiochemicals isolated from
Caladenia plicata and its Zeleboria sp. thynnine wasp pollinator,
acted as strong sexual attractants. It is perhaps not surprising
that the natural enantiomer of β-citronellol is required for
bioactivity, as the conformational space for these two enantiomers
would be rather different. Perhaps more surprisingly is that every
structural analogue to 2-hydroxy-6-methylacetophenone tested failed
to attract any male wasps. Thus, even very minor modifications in
the ring substitution pattern of this compound, such as the
movement or removal of a methyl group, appears to have completely
turned off any function as a semiochemical in this system. Such
strong specificity would seem to indicate a very well defined
active site of the receptor(s) in the wasp antennae (for example
see Brookes et al. 2009), although this remains to be determined in
the future.
To obtain more details about the specificity of interaction with
the unknown receptor, and whether the specificity was purely due to
steric factors or also electronic effects, we conducted some
theoretical studies. By calculating the energetic differences
between the series of analogues and determining the optimised
lowest-energy confirmations, we could observe a distinct difference
in the strengths of the internal hydrogen bonds among the compounds
analysed. It is well known that non-covalent C–H•••π interactions
can play important roles in catalytic processes in the gas-phase
(Wagner and Schreiner 2015; Neel et al. 2017). We therefore infer
that the presence of the methyl group in the position adjacent to
the acetyl makes the hydrogen bond stronger through steric
repulsion, which might play an important role for the biological
activity of compound 2a. Furthermore, the proximity of the two
methyl groups, resulting from the hydrogen bonding arrangement,
might also be important for the observed bioactivity, via increased
localized lipophilicity. This rationale is supported by the fact
that 2-hydroxyacetophenone (3) and analogues 2b-d are biologically
inactive.
The requirement of highly specific semiochemicals, or blends of
semiochemicals, is well documented in the literature, even if the
number of examples is limited. Such examples include the butterfly
Pieris napi, which employs citral as the male sex pheromone. Here
it was shown that females only showed acceptance behaviour in the
presence of citral but not to the individual components, geranial
and neral, demonstrating the importance of the specific blend
(Larsdotter-Mellström et al. 2016). The pine sawfly Neodiprion
sertifer (Geoffroy) uses the acetate or propionate of
(2S,3S,7S)-3,7-dimethyl-2-pentadecanol (diprionol) as pheromone
components. Anderbrant et al. (2010) tested the attraction of males
to the acetates of three analogues of diprionol, each missing one
methyl group. None of the analogues alone, or in combination with
diprionol acetate, were attractive in Sweden, while in Japan, the
acetate of (2S,3S)-3-methyl-2-pentadecanol attracted males when
tested in amounts 10–20 times higher than the acetate pheromone
component, showing that even geographic variation in sex pheromone
specificity is possible (Anderbrant et al. 2010). Perhaps the most
striking example of sex pheromone specificity is in the honey bee.
Blum et al. tested 19 structural analogues to the sex pheromone
9-oxo-trans-2-decenoic acid, without detecting activity for any
compound but the natural product (Blum et al. 1971).
All these findings can be compared with the results from
previous structure-activity studies of sexually deceptive Drakaea
orchid semiochemicals. In those studies, structural analogues of
the biologically active hydroxymethylpyrazine in D. glyptodon and
D. livida, in contrast, were biologically active (Bohman and
Peakall 2014; Bohman et al. 2015). It is noteworthy that when
similar calculations were performed on the pyrazines evaluated as
attractants to Zaspilothynnus trilobatus, the pollinator of Drakaea
glyptodon, the energetic differences were negligible, perhaps
providing evidence as for why the natural semiochemicals could be
substituted with other synthesised analogues and still retain
similar activity. The results from the Drakaea studies correlated
well with the results from studies in insects such as the citrus
mealybug Planococcus citri (Dunkelblum et al. 1987), the astigmatid
mites Aleuroglyphus ovatus and Acarus immobilis, (Shibata et al.
1998), and the lichen moth (Muraki et al. 2014), where structural
pheromone analogues were active.
In conclusion, we report on the highly structurally specific
requirements for the Zeleboria sp. wasp sex pheromones and C.
plicata pollinator attractants. Our results reveal for the first
time in a sexually deceptive orchid-pollination system that extreme
pollinator specificity is finely controlled by chemical
structure.
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Fig. 1 Pollinator attractants (1a and 2a) and structural
analogues (2b-2j and 3) to the female sex pheromone of the thynnine
wasp Zeleboria sp.
Fig. 2. Outcomes of sequential bioassays comparing the
pollinator wasp response to (R)-β-citronellol (1b) as a substitute
for (S)-β-citronellol (1a) at the optimal 1:4 ratio. Phase 1
consisted of the presentation of compounds 1b and 2a for 3 min.
This was followed by phase 2, where both the initial blend and the
control blend of 1a and 2a was presented for 3 min. Pollinator
responses are shown as mean proportions of the total (± se) across
replicate experiments, further partitioned into approach A, land L
and attempted copulation C (left panel). The total count across
experiments is shown separately for phase 2 (right panel).
Fig 3 B3LYP/6-31G(2df,p) optimized structures of the
lowest-energy conformations of compounds 2a–2j (atomic colour
scheme: H, white; C, grey; O, red). The single dashed lines
represent hydrogen-bond interactions (H-bond distances are given in
Å).
Fig. 4 B3LYP/6-31G(2df,p) optimised structures of two
conformations of 2-hydroxyacetophenone (3) (atomic colour scheme:
H, white; C, grey; O, red). The single dashed line represents a
hydrogen-bond interaction and its distance is given in Å.
Regioisomer R
#Wasp ResponsesTest (1a + R)3 MinPhase 1
#Wasp ResponsesTest (1a + R)1st MinPhase 2
#Wasp ResponsesTest (1a + R)3 MinPhase 2
#Wasp ResponsesCtrl (1a + 2a)1st MinPhase 2
# Wasp ResponsesCtrl (1a + 2a)3 MinPhase 2
Tests at 1:1 ratio
2b
0
0
0
2
10
2c
0
0
0
0
5
2d
0
0
0
3
7
2e
0
0
0
3
10
2f
0
0
0
4
14
2g
0
0
0
8
14
2h
0
0
0
1
9
2i
0
0
0
2
10
2j
0
0
0
3
11
Tests at 1:4 ratio
2b
0
0
0
3
13
2c
0
0
0
4
6
2d
0
0
0
6
25
2e
0
0
NA
6
NA
2f
0
0
NA
6
NA
2g
0
0
NA
9
NA
2h
0
0
NA
5
NA
2i
0
0
NA
4
NA
2j
0
0
NA
6
NA
3
0
0
NA
10
NA
NA = not assessed
Table 1. Outcomes of sequential bioassays comparing the
pollinator wasp response to (S)-β-citronellol (1a) with each of the
other nine regioisomers (2b-2j or 3 individually, indicated by R in
the table header) against the control blend of 1a and 2a. Results
are shown for tests at 1:1 and 1:4 blend ratios. Phase 1 consisted
of the presentation of test blend for 3 min. This was followed by
phase 2, where both the initial test blend and the control blend
was presented for up to 3 min. The total wasp responses over two
trials, after 1 min and 3 min respectively (where applicable), are
shown for each phase.
1
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
1b+2a (1:4) 1b+2a (1:4) 1a+2a (1:4)
Phase 1 Phase 2
Pro
port
ion
A L C
0
30
60
90
120
1b+2a (1:4) 1a+2a (1:4)
Phase 2
Cou
nt
G=146.6 P<0.0001
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
1b+2a (1:4) 1b+2a (1:4) 1a+2a (1:4)
Phase 1 Phase 2
P
r
o
p
o
r
t
i
o
n
A L C
0
30
60
90
120
1b+2a (1:4) 1a+2a (1:4)
Phase 2
C
o
u
n
t
G=146.6
P<0.0001
