Where have all the blue flowers gone: pollinator responses andselection on flower colour in New Zealand Wahlenbergiaalbomarginata

D. R. CAMPBELL*, M. BISCHOFF*, J. M. LORD� & A. W. ROBERTSON�*Department of Ecology and Evolutionary Biology, University of California, Irvine, CA, USA

�Department of Botany, University of Otago, Dunedin, New Zealand

�Institute of Natural Resources, Massey University, Palmerston North, New Zealand

Introduction

Many phenotypic traits of flowers, including the bright

colours of petals, are thought to have evolved in response

to interactions with animal pollinators. This view is a

long-standing one (Darwin, 1862; Faegri & van der Pijl,

1966), and it has greatly influenced evolutionary studies

of flower colour (review in Fenster et al., 2004). There

are, however, few experimental demonstrations that

pollinators cause fitness differences between flowers of

different colours (reviewed by Rausher, 2008). Pollina-

tors may respond to colour for a variety of reasons,

including the ability to perceive and distinguish colours,

innate preference (Raine & Chittka, 2007) and learning

of associations with other traits such as rewards (Menzel,

1979; Melendez-Ackerman et al., 1997). However, flower

pigmentation may also serve other functions besides

visual signalling, suggesting that selective agents besides

pollinators could drive the diversity of petal colours in

nature. For example, flower colour can influence flower

temperature (McKee & Richards, 1998), which in turn

may affect water relations of the plant (Galen, 2006), the

microenvironment for developing seeds (Lacey & Herr,

2005) and ⁄ or attractiveness of the flower to herbivores.

Like other traits in plants and animals (Lande & Arnold,

1983), flower colour may also be selected not because of

its direct effects on fitness components but because of

indirect selection of correlated characters. Some enzymes

used in the synthesis of anthocyanin pigments are also

used to synthesize other flavonoid compounds, which

could generate pleiotropic effects of genes influencing

flower colour (Rausher, 2008). Whereas pigment syn-

thesis can be costly, flower colour mutants not expressing

anthocyanins can be less tolerant of stresses such as

drought and heat (Coberly & Rausher, 2003; Whittall

et al., 2006). Pigmentation in flowers often correlates

with pigmentation in vegetative tissues (Armbruster,

2002) and can affect the level of resistance to herbivores

(reviewed by Strauss & Whittall, 2006).

One common approach to studying selection on floral

traits has been to use phenotypic selection analysis to

Correspondence: Diane R. Campbell, Department of Ecology and

Evolutionary Biology, University of California, Irvine, CA 92697, USA.

Tel.: +1 949 824 2242; fax: +1 949 824 2181;

e-mail: drcampbe@uci.edu

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Keywords:

flower colour;

male fitness;

phenotypic manipulation;

phenotypic selection;

pollination;

solitary bee;

spatial scale;

Wahlenbergia albomarginata

Abstract

Although pollinators are thought to select on flower colour, few studies have

experimentally decoupled effects of colour from correlated traits on pollinator

visitation and pollen transfer. We combined selection analysis and phenotypic

manipulations to measure the effect of petal colour on visitation and pollen

export at two spatial scales in Wahlenbergia albomarginata. This species is

representative of many New Zealand alpine herbs that have secondarily

evolved white or pale flowers. The major pollinators, solitary bees, exerted

phenotypic selection on flower size but not colour, quantified by bee vision.

When presented with manipulated flowers, bees visited flowers painted blue

to resemble a congener over white flowers in large, but not small,

experimental arrays. Pollen export was higher for blue flowers in large arrays.

Pollinator preference does not explain the pale colouration of W. albomargi-

nata, as commonly hypothesized. Absence of bright blue could be driven

instead by indirect selection of correlated characters.

doi: 10.1111/j.1420-9101.2011.02430.x

characterize the relationship of a trait to a fitness

component; a recent review uncovered 56 such studies

on 44 plant species (Harder & Johnson, 2009), although

there were only four estimates included of selection on

petal colour. A limitation of this approach is that it can

only separate direct effects of the trait of interest from

indirect selection of correlated characters if those char-

acters are also included in the analysis (Mitchell-Olds &

Shaw, 1987). Genetic manipulations of flower colour

provide much stronger evidence (Bradshaw & Schemske,

2003), although they do not necessarily eliminate pleio-

tropic effects. An alternative approach is to manipulate a

trait, such as flower colour, phenotypically (Clements &

Long, 1923; Waser & Price, 1981, 1983; Campbell et al.,

1997, 2010; Melendez-Ackerman & Campbell, 1998;

Peter & Johnson, 2008; Dudash et al., 2011). This

approach of phenotypic manipulation, when control

manipulations are included, has the advantage of ascrib-

ing any selection seen directly to flower colour. All

studies referenced above were conducted on small spatial

scales, such that individual plants, inflorescences or

flowers of two experimental colours are intermixed.

Pollinators may, however, show different behavioural

responses depending on the spatial scale, as shown in the

studies of traits other than colour (Campbell et al., 1997;

Leiss & Klinkhamer, 2005). Different responses at differ-

ent scales have been predicted by elements of foraging

theory (Mitchell, 1989) and also observed for animals

foraging at flowers. For example, bumblebees in one

study discriminated between groups of plants with

different nectar rewards only when the groups were

separated by >6 m (Klinkhamer et al., 2001).

Even in those cases where pollinators have been shown

to respond behaviourally to flower colour, the conse-

quences for pollen import and export (female and male

pollination success) are rarely known. A few studies have

examined the net effect of colour manipulation on seed set

(Melendez-Ackerman & Campbell, 1998), and Peter &

Johnson (2008) measured the effect of manipulating UV

reflectance not only on insect visitation but also on

removal and deposition of pollinaria in a deceptive orchid.

But even in that case, the shape of the relationship

between visitation and pollination was not reported.

Given the long history of pollination ecology, there are

remarkably few plant species for which the relationship

between flower visitation and either pollen import or

pollen export has been quantified in the field (Young &

Stanton, 1990; Engel & Irwin, 2003; Price et al., 2005).

In this study, we used both phenotypic selection

analysis and phenotypic manipulations to measure the

effect of flower petal colour on insect visitation and pollen

export (an aspect of male fitness; Campbell, 1989; Galen,

1992) at two different spatial scales. Our study system was

the white- to pale-blue-coloured Wahlenbergia albomargi-

nata Hook. (Campanulaceae) in alpine New Zealand. The

distribution of flower colours in this habitat is unusual.

More than 70% of the flowers are white or near-white,

which is high compared with continental mountain floras

(Godley, 1979). The absence of bright blues and purples

has been suggested to reflect the absence of native social

bees (Wardle, 1978; Godley, 1979; Lloyd, 1985), largely

on the basis of the argument that elsewhere in the world

bright blue is considered part of the pollination syndrome

for plants pollinated by bumblebees (Faegri & van der Pijl,

1966). As many other species of Wahlenbergia in Austral-

asia are much brighter blue, focusing on W. albomarginata

allowed us to examine whether the absence of bright blue

from the New Zealand alpine could be explained by a

preference of native insect pollinators in that habitat for

white flowers. Over four field seasons (2008–2011), we

asked the following specific questions:

1 Do the native pollinators visit flowers of W. albomargi-

nata preferentially on the basis of natural variation in

flower colour and ⁄ or interactions of flower colour

with flower size? We examined visitation rate by

pollinators as a function of petal colour and size using

phenotypic selection analysis (Lande & Arnold, 1983;

Phillips & Arnold, 1989).

2 Do the native pollinators discriminate against flowers

phenotypically manipulated to match the brighter

petal colour of another New Zealand Wahlenbergia?

We used phenotypic manipulations to extend the

range of colour presented, because the absence of

bright blue in the New Zealand alpine might be

explained by insect colour preference even if those

insects do not respond to the intraspecific range of

variation in petal colour.

3 Does success at pollen export to stigmas of surround-

ing flowers, a measure of male pollination success,

depend on flower colour?

4 Do insect visitation rate and pollen export respond to

flower colour in the same way on two different spatial

scales, with equal number of flowers of the two

colours either intermixed on a small scale or presented

in relatively large patches?

5 Is intraspecific variation in flower colour associated

with flower temperature and ⁄ or floral herbivory?

Materials and methods

Study system

Wahlenbergia albomarginata is a small creeping rhizomatous

herb found in tussock grasslands in lowland to alpine areas

of South Island and Stewart Island, New Zealand. The

genus Wahlenbergia originated in South Africa and dis-

persed to Australasia prior to radiating about 3.7 Ma

(Prebble, 2010). Recent molecular evidence suggests two

dispersal events from Australia to New Zealand (Prebble,

2010). One clade, which diversified only about 0.5 Ma,

consists only of lowland species, including the brightly

coloured W. violacea. W. albomarginata belongs to the older

(about 1 Ma) New Zealand clade, as do the other

high-elevation species in New Zealand. The phylogeny of

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Wahlenbergia, along with the observation that the Austra-

lian species are brightly coloured, as are some of the New

Zealand species, suggests that bright pigmentation is

the ancestral condition, with a loss of pigmentation in

W. albomarginata that occurred at or after occupation of

high-elevation habitat in New Zealand.

Eachrametof W. albomarginata producesone flower that

is protandrous. The pollen is shed onto retractable stylar

hairswhile theflower is inbudandthepollenpresented ina

cylindrical layer along the side of the style in a sequential

fashion as the hairs progressively retract (Lloyd & Yates,

1982). This form of secondary pollen presentation is

common in the Campanulaceae (Endress, 2011). After a

variablenumberofdays, thestigmatic lobesexpandandthe

stigma becomes receptive. Flowers last 8 days on average

(D. Campbell & M. Bischoff, unpublished data). Autoga-

mous selfing does not occur, and flowers that are hand-

selfed make <5% as many seeds as flowers that are

outcrossed, suggesting that the species is largely self-

incompatible (Bischoff, 2008). We studied W. albomargi-

nata at elevations of 1650–1750 m in the Rastus Burn

Recreation Area within the Remarkables Range in Otago,

New Zealand, along the trail from the Remarkables Ski

Area to Lake Alta. At this site, W. albomarginata blooms

from early February to mid-March. Solitary bees, Hylaeus

matamoko (Hymenoptera: Colletidae) and Leioproctus spp.

(Hymenoptera: Halictidae), are the major pollinators,

contributing about 20 times as much to pollen deposition

as the other common visitors, syrphid and tachinid flies

(M. Bischoff, D.R. Campbell, A.W. Robertson and J.M.

Lord, unpublished data). In single visits, Hylaeus bees

transfer nearly three times as much germinating pollen to

the stigma as do syrphid flies in the genus Allograpta

(means = 113 vs. 40 grains; M. Bischoff, D.R. Campbell,

A.W. Robertson and J.M. Lord, unpublished data). The

petals are often chewed by grasshoppers (Sigaus australis),

who sometimes consume the entire flower (M. Bischoff

and D. Campbell, personal observation).

Flowers are generally white with bluish markings, but

vary at our site from nearly pure white to much more

intensely coloured (Appendix S1). Although colour can

fade slightly with senescence of the flower, the relative

difference between bluer and paler flowers remains

consistent over time. To characterize the flower colour

of individual W. albomarginata flowers, we measured the

reflectance spectra of the adaxial surface of single petals

over wavelengths from 300 to 700 nm. These spectra

were obtained using an Ocean Optics (Dunedin, FL, USA)

USB 4000 reflectance spectrometer with a 200- to 1000-

nm range and a UV–VIS light source, standardized by a

white reflectance standard. The probe was held at a 45�.

Q1: Do the native pollinators visit preferentially basedon natural variation in flower colour and/or size?

To measure the selection based on natural variation in

colour, we examined insect visitation as a function of

flower colour and morphology in experimental arrays,

each with 16 flowers. These arrays are a subset of those

for which M. Bischoff, D.R. Campbell, A.W. Robertson

and J.M. Lord (unpublished data) reported insect visita-

tion, but not flower traits as analysed here. Arrays were

set up near (usually 5–10 m away), but not in, patches of

natural flowers, and were generally placed into mats of

cushion plants that provided a relatively uniform green

background. Flowers were spaced 10 cm apart in four

rows of four flowers, with each flower in a numbered

1.5-mL microcentrifuge tube filled with water. Flowers

were assigned to tubes at random. We observed each of

10 different flower arrays, totalling 160 flowers (Table 1).

All observations were made during sunny conditions

between 11:30 and 16:30. Visitation rates were unusu-

ally high during these periods (for dates see Table 1),

probably because both periods corresponded with a spell

of particularly fine weather, so we watched each array

for only 1 h. Only visitors that were foraging for rewards

on the flower were counted. For each insect that entered

the array, we recorded the sequence of flowers visited

until the insect left and either visited a flower outside of

the array or disappeared from sight. Each such sequence

is hereafter called a foraging bout. After observation, we

measured the corolla width and diameter of the entire

flower using callipers. The two morphometric measure-

ments were strongly correlated (r = 0.46, N = 158,

P < 0.0001), and for further analysis, we used only

diameter to represent flower size.

Flowers were also measured using a spectrometer to

characterize petal colour in a way independent of human

vision. For phenotypic selection analysis, we needed a

summary measure of colour. The few previous estimates

of selection gradients on quantitatively varying petal

colour have used optical density of pigment extractions

(Campbell et al., 1997), reflectances at particular wave-

lengths (Frey, 2004) or extracted brightness, chroma and

hue from the spectra (Caruso et al., 2010). As we were

most interested in insect pollinator responses to colour,

we characterized petal colour using measures based on

the properties of insect vision. Most insects have recep-

tors with peak sensitivities near 350 nm (UV receptor),

440 nm (blue receptor) and 530 nm (green receptor;

Briscoe & Chittka, 2001). Flowers of W. albomarginata do

not reflect in the UV (Fig. 1). So, for our first point

measure of colour, we used the ratio between reflectance

in the blue vs. green (R440 ⁄ R530). We refer to this

measure hereafter as the reflectance ratio. Like the

colour measure used by Frey (2004), this reflectance

ratio captures much of the variation between flowers in

shape of the reflectance curve (Fig. 1).

We also used a second point measure of colour, relying

on plots of the reflectance spectra in bee colour space,

following the hexagon model for honeybees (presented

in Chittka & Kevan, 2005). It is likely that the spectral

sensitivities of other bees, including the common visitors

seen here, are very similar (Briscoe & Chittka, 2001). We

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could only plot the flowers observed in 2009, as in 2008

we did not have an available UV spectrometer and only

measured reflectance spectra from 400 to 700 nm. After

determining the x and y coordinates in the hexagon

colour space, which represent excitation values of the

two types of colour opponent neurons (Fig. 2), we used

principal component analysis to find the major axis

explaining the greatest variation in colour as visible to

insects. In this case, the first principal component (PC1)

explained 93% of the variance. We then used the PC1

score as a measure of colour in the phenotypic selection

analysis. Individual flowers at the two ends of this axis

differed by 0.39 Euclidean units in colour space. As bees

can typically learn to discriminate colours, after differ-

ential conditioning, down to about 0.05 units apart (Dyer

& Chittka, 2004), these would have represented distinct

colour stimuli to the insects.

In these arrays of 160 natural flowers (Table 1), 90%

of the flower visits were made by the bees H. matamoko

and Leioproctus spp. (Table 2). These bees collect pollen

from both male-phase and female-phase flowers, the

latter of which generally retain some pollen along the

style. Because these bees are the dominant pollinators

(M. Bischoff, D.R. Campbell, A.W. Robertson and J.M.

Lord, unpublished data), we performed phenotypic

selection analysis based on visitation rate by bees only.

For each flower, we determined the number of visits by

Hylaeus and Leioproctus in the observation period. Because

overall visit rates were highly variable over time, we

standardized these visit rates by finding the residual from

the overall mean for the observation period. We then

regressed residual visit rate (w) on size and colour, after

standardizing each trait to a mean of zero and variance of

1 (represented by X1 and X2):

Table 1 Overview of experimental arrays used to assess pollinator visitation and pollen export.

Experiment

No. flowers

in an array

Independent

variable(s)

Number of

arrays set-up

Array hours of

observation Dates

Q1. Natural arrays 16 Natural petal colour and

Flower diameter

10 10 19–21 February 2008 and

17–19 February 2009

Q2. Small experiment 16 Manipulated colour 7 21 14–17 February 2009

Q3. Pollen export to

surrounding flowers

16 Manipulated donor

colour and Array

8 None 7–18 February 2009

Q4. Large experiment

on pollen export and

visitation

64 Manipulated inner

colour and

Manipulated outer

colour and Array pair

12 38 26 February 2011 to 12

March 2011

Fig. 1 Reflectance spectra for Wahlenbergia albomarginata and

W. violacea. Black line: Mean and 95% CI for 112 W. albomarginata

flowers from the seven arrays observed for phenotypic selection

analysis in 2009. Grey line: Mean and 95% CI for five sample

flowers of W. violacea. Values were binned into the nearest 5 nm

prior to plotting. Numbers on the plot give the ranges for the

reflectance ratio R440 ⁄ R530.

Fig. 2 Petal colours plotted in hexagon colour space. Grey circles:

Individual flowers in the natural arrays used in the phenotypic

selection analysis. Filled blue diamonds: Individual flowers of the

congener W. violacea. Filled black circle: Wahlenbergia albomarginata

painted to resemble W. violacea. Open circle: W. albomarginata

painted white.

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w ¼ aþ b1X1 þ b2X2 þ 0:5c11X21 þ 0:5c22X2

2 þ c12X1X2 þ e;

ð1Þ

This expression contains terms for the strength of

directional selection (b1 and b2) and stabilizing or

disruptive selection on each trait (c11and c22), as well as

the strength of correlational selection (c12). Correlational

selection is here represented by interactions between

effects of traits on a fitness component (Phillips & Arnold,

1989).

Q2: Do the native pollinators discriminate againstflowers phenotypically manipulated to match thebrighter petal colour of other Wahlenbergia?

We set up experimental arrays, resembling the natural

arrays described above, except that half of the 16 flowers

were painted white and half painted a blue-purple to

match the reflectance spectrum of W. violacea. The colour

of W. violacea is most likely similar to the ancestral petal

colour when the ancestor of W. albomarginata migrated to

New Zealand. We matched the average spectrum for five

flowers obtained from wild plants transplanted into a

garden near Wellington (Fig. 1) using a mixture of

approximately half blue and half white and a small

amount of red water-based acrylic paint. The reflectance

ratio (R440 ⁄ R530) for our mixture was 2.26, within the

range of 2.23–2.90 for W. violacea, and flowers painted

were just 0.05 units in hexagon colour space away from

the nearest measured W. violacea (Fig. 2). Flowers

painted white were within the natural range for

W. albomarginata, with a reflectance ratio of 1.01

(Fig. 2). Because the petals are delicate, prior to painting

the flowers we affixed the petals to a plastic support.

Each support was made by punching a star-shaped piece

of mylar plastic film (diameter = 24 mm from point to

point) coated with double-sided cellophane tape on each

side and then punching a 5-mm hole through the centre.

The 5-pointed plastic star was mounted on the top of a

1.5-mL microcentrifuge tube after removing the tube’s

lid. We filled each tube with water, placed the flower

stalk through the central hole and with forceps pasted

the five petals down onto the tape on the top of the

plastic mount. Flowers were then painted one of the two

colours, and the tubes were put into wire holders that

held them about 15 cm above the ground. In previous

studies with these same acrylic paints, pollinating insects

at these sites, including flies (Campbell et al., 2010) and

bees (D. Campbell personal observation) did not show

any preference between flowers painted white and

flowers that are naturally white. This indifference to

the paint suggests that it has no unintended effects on

insect visitation that are not driven by colour.

The two colour treatments were assigned at random to

the 16 positions in each 40 cm · 40 cm array. We

observed each of seven different flower arrays for 3 h

each, during the hours of 11:30–16:30 in mid-February

2009 (Table 1), recording insect visitation as described for

the natural arrays. Because our question in this case

centred on insect preference, we analysed the data from

the point of view of the insects by using each foraging

bout as a unit of replication. As individual insects were

not marked, it is possible that some insects made multiple

foraging bouts. However, we frequently observed multi-

ple insects of the same type foraging simultaneously,

reducing the extent of this nonindependence. As the

blue-purple treatment represented a colour not normally

found in the environment, the insects could have

changed behaviour in response to learning over the

course of the experiment.

For each category of insect, we analysed two variables:

the kind of flower (blue or white) first visited by the

insect upon entering the array and the proportion of

visits made to a given flower colour during the foraging

bout (provided the insect made at least two flower visits).

The first variable was compared to the null hypothesis

that both colours were visited equally, using a likelihood

ratio test. The second variable was arcsine-square-root-

transformed to improve normality and compared with

the null hypothesis of 0.5 with a one-sample t test.

Table 2 Distribution of insect visitors at arrays. Values are presented on the basis of per cent of foraging bouts and per cent of flowers visited

for the smaller arrays with 16 flowers. The data for the natural arrays include only those in which we measured flower traits for selection

analysis. For the large colour arrays, individual flower visits were not always recorded for the outer, nontarget, part of the array.

Per cent bouts (Per cent flower visits)

Natural arrays Small colour arrays Large colour arrays

Hylaeus (Colletidae) 81.2 (77.4) 57.9 (58.7) 57.0

Leioproctus (Halictidae) 6.0 (13.4) 0.0 (0.0) 8.7

Allograpta (Syrphidae) 5.8 (3.9) 21.2 (18.2) 13.4

Platycheirus (Syrphidae) 0.2 (0.2) 0.9 (0.4) 1.6

Tachinidae 2.0 (1.4) 11.2 (16.8) 13.7

Other insects 4.0 (3.8) 8.7 (6.2) 1.9

Total N 582 (1453) 321 (499) 321

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Q3: Does success at pollen export to stigmas ofsurrounding flowers depend on flower colour?

To determine the effects on pollen export, in 2009, we

also set up experimental arrays using phenotypic mani-

pulations of flower colour as described earlier for ques-

tion 2, but with all experimental flowers in the male

phase. To estimate pollen export from these flowers, we

used two colours of powdered fluorescent dyes, applied

with a wooden toothpick to the pollen arranged along

the stylar pollen presenter. Although fluorescent dyes do

not perfectly mimic pollen export, previous studies of

plants visited by solitary bees and flies have shown good

correspondence between movement of pollen grains and

movement of dye, used as a pollen analogue (Campbell,

1985; Dudash, 1991). On each of 4 days, we set up two

experimental arrays, one with blue dye applied to the

eight blue-painted flowers and fire orange dye applied to

the eight white-painted flowers and another with the

reverse assignment of dye colours to flower petal treat-

ments. These 40 cm · 40 cm arrays of 16 manipulated

flowers (Table 1) were set up so that they were sur-

rounded by natural flowers of W. albomarginata. These

natural flowers served as potential recipients for dye

transfer. The two arrays were placed sufficiently far apart

(>100 m) that the chance of dye transfer between arrays

was negligible. After 48 h, we collected all female-phase

W. albomarginata flowers within 2 m radius of each of the

experimental arrays. For all flowers, dye particles of each

colour on the stigma were counted under 40· power

using a dissecting microscope. In total, we set up eight

arrays of this type and counted dye particles transferred

to 300 flowers, an average of 18.75 flowers per donor

colour and array.

For each array, we totalled the dye particles success-

fully transferred to stigmas from donor flowers of each

colour and log-transformed the value to improve

normality. Dye transfer was then analysed using a

randomized block ANOVAANOVA, with array as the blocking

factor and donor petal colour as the treatment.

Q4: Do insect visitation rate and pollen exportrespond to flower colour in the same way on twodifferent spatial scales?

To determine how pollinators respond to flower colour

on a larger spatial scale, in 2011 we set up experimental

arrays four times as large as in 2009, each containing 64

phenotypically manipulated flowers in eight rows and

eight columns, separated by 10 cm (Table 1, Fig. 3).

Natural patches of W. albomarginata at this site are often

smaller than this array size, particularly if they are

composed of a single clone, but they can be this large. On

each of six dates, we set out a pair of experimental arrays,

for a total of 768 manipulated flowers in 12 arrays. The

central core of each array contained 16 flowers as before,

eight painted blue and eight painted white, with those

treatments randomized. The two arrays in a pair, how-

ever, differed in the colour of the outer 48 flowers

surrounding that core, with one array having all blue

outer flowers in the female phase and the other array

having all white outer flowers in the female phase. This

arrangement allowed us to test whether the overall

colour context in a larger area would influence visitation,

even if once in the area insects would visit both colours.

For four of the six sets, we also applied two colours of

fluorescent dye (blue and pink) to the flowers in the

central core to estimate pollen export to flowers in the

outer areas. For a given pair of arrays, we used the same

assignment of dye colours to the paint treatments, but

these assignments were reversed for the next pair of

arrays, so that each dye assignment was used equally

often.

The two large arrays in a pair were observed simulta-

neously for insect visitation, for 3–4 h each, between

11:30 and 17:30. One pair of arrays was not included in

the final data set for visitation, as on both days that we

(a)

(b)

Fig. 3 Schematic of a sample pair of large experimental arrays. (a)

Large blue array. (b) Large white array. A pair of arrays was always

observed simultaneously by two observers. Filled symbols: Painted

blue. Open symbols: Painted white. The inner box surrounds the 16

inner male-phase flowers (triangles), which were also the pollen

donors. Two colours of dye were used to track pollen from these

flowers to the outer 48 female-phase flowers (circles) in the array.

6 D. R. CAMPBELL ET AL.

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attempted visitation we saw no insect visitation after

45 min. For each array, we noted the number of insects

of each type that entered the array and foraged on at least

one of the 64 flowers. We also determined the number of

bee (Hylaeus + Leioproctus) visits per hour and the num-

ber of fly (Syrphidae + Tachinidae) visits per hour to the

inner blue flowers and to the inner white flowers. These

visitation rates were analysed with split-plot ANOVAANOVA

(implemented in Proc GLM of SAS v9.1.3; SAS Institute

Inc., Cary, NC, USA), with array pair as the blocking

factor, outer colour of the array as the whole-plot factor

and colour of the inner flowers as the split-plot factor.

Flowers were collected after 48 h of exposure to count

dye particles transferred to stigmas of the outer flowers.

In total, we counted dye particles on 384 flowers from

eight arrays. Dye transfer from inner flowers of a

particular colour was log-transformed (after adding 1)

to improve the normality of residuals and then also

analysed using split-plot ANOVAANOVA. To examine the rela-

tionship between insect visitation and overall dye export,

we calculated correlation coefficients between the total

dye moved in an array (log-transformed) and the

numbers of bees and flies observed entering those large

arrays and visiting at least one flower.

Q5: Is intraspecific variation in flower colourassociated with flower temperature and/or floralherbivory?

We examined two other processes besides visual responses

of pollinators hypothesized to be influenced by flower

colour. First, flowers of different colours might differ in

temperature, which could affect attractiveness to insect

visitors and ⁄ or the rate at which stigmas become receptive

or pollen tubes grow. Over three afternoons between 26

February to 9 March 2010, we chose a total of 20 pairs of

natural flowers (usually in male phase) growing within

5 m of each other in similar light conditions, one of which

was unusually white and one of which was unusually

blue. Using a dual thermocouple (Omega model

HH2002A; Stamford, CT, USA), we took simultaneous

readings of the temperature inside the petals and ambient

air temperature and found the discrepancy between these.

For each flower, we averaged measures of discrepancy

after 60 and 90 s of stabilization. We then immediately

measured the other colour flower in the pair, with the

order chosen at random, the same way. Using a random-

ized block ANOVAANOVA with pair of flowers as the blocking

factor, we compared the temperature discrepancies

between the two flower colours. Mean ambient air

temperature during these measurements was 19.7 �C.

After recordings were finished, flowers were collected and

their reflectance spectra measured.

To examine floral herbivory, we located pairs of

unusually blue and unusually white male-phase flowers

growing within about 5 m of each other. We marked 25

pairs with metal rings and a small numbered paper tag on

27 February 2010, 24 pairs on 4 March 2010 and 23 pairs

on 7 March 2011. These pairs were spread out over sites

spanning an elevational range from approximately

1600 m (along a stream below the ski parking lot) to

1750 m (approximately 50 m below Lake Alta). After

4 days (except 5 days for those marked on 4 March

2010), we collected the flowers. Herbivory was assessed

by number of petals (0–5) that were damaged, with a

score of 6 used for flowers that were completely eaten.

For analysis, we collapsed damage scores into three

classes: none (0), partial (1–4) or complete (5–6). If the

entire ramet was gone, we scored the result as missing

data. Reflectance spectra were characterized for a

random sample of those blocks in which both flowers

still retained at least one petal. Herbivory itself did not

change the petal colour. We used contingency analysis to

examine whether damage depended on colour class of

the flower. To supplement this analysis, we also used

logistic regression to model damage (yes or no) as a

function of the reflectance ratio R440 ⁄ R530. The analysis

was implemented in Proc Genmod of SAS v9.13.

Results

Q.1: Do the native pollinators visit preferentiallybased on the natural variation in flower colour and/orsize?

At the 10 arrays of natural, unmanipulated flowers, we

observed 1453 flower visits in 582 foraging bouts by

insects. On average, flowers were 15.9 mm in diameter

with a reflectance ratio of 1.08 (N = 158). Larger flowers

received many more bee visits, with an additional 1.6

visits per hour for every 1 standard deviation (=2.9 mm)

increase in diameter (P < 0.001; Table 3). Selection on

this trait appeared directional, with no evidence for

curvature in the effect on fitness (Table 3). We detected

no selection on flower colour within the range of natural

variation, regardless of whether it was measured as the

reflectance ratio (Table 3A) or as the PC1 in bee colour

space (Table 3B). We also saw no interaction between

the effect of flower size and colour on visitation rate by

bees. Given the absence of nonlinear selection, we also

used multiple regression to analyse bee visitation rate as a

function of three traits: diameter and the two axes in

hexagon colour space. This model also yielded no

significant phenotypic selection on colour (P > 0.50 for

both colour axes).

Q2: Do the native pollinators discriminate againstflowers phenotypically manipulated to match thebrighter petal colour of other Wahlenbergia?

The majority of visitors at the small arrays where we

manipulated flower colour were bees of the species

H. matamoko (Table 2). These native bees did not dis-

criminate against bright blue flowers. Instead, they made

Pollinators and selection on Wahlenbergia colour 7

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their first visit to blue and white flowers approximately

equally often (45% vs. 55%, N = 186, likelihood ratio

test, P = 0.1425), and in bouts of two or more visits made

a proportion indistinguishable from 0.5 to blue flowers

(N = 73, t72 = 1.37, P = 0.1750, Fig. 4). The syrphid fly

visitors were primarily Allograpta (Table 2), and these

flies did discriminate against blue. Only 18% of first visits

were made to blue flowers (N = 68, P < 0.0001). In

longer foraging bouts of two or more flower visits, on

average they visited blue 0.27 of the time (N = 16,

P < 0.05, Fig. 4). Tachinid flies also discriminated against

bright blue, making only 6% of first visits to blue flowers

(N = 36, P < 0.0001) and visiting blue an average pro-

portion of 0.22 in longer foraging bouts (N = 20,

P < 0.0001; Fig. 4).

Q3: Does success at pollen export to stigmas ofsurrounding flowers depend on flower colour?

In our experiments with eight small arrays of 16 flowers

each, we saw no difference between donor flowers

painted blue and those painted white in the amount of

dye transferred to stigmas of surrounding flowers (ran-

domized block ANOVAANOVA, F1,7 = 0.01, P = 0.9295; Fig. 5).

For a given donor colour, across the eight arrays, 10–88%

of the potential recipient flowers had at least one particle

of dye on the stigma.

Q4: Do insect visitation rate and pollen exportrespond to flower colour in the same way on twodifferent spatial scales?

The large experimental arrays attracted a similar mix of

insects as had the smaller arrays 2 years previous

(Table 2). Visitation rates by the bees varied strongly

across pairs of arrays (Table 4). At the small spatial scale,

inner blue flowers received significantly more bee visits

than did inner white flowers (effect of inner colour in

split-plot ANOVAANOVA, P < 0.05, Table 4, Fig. 6). Visitation

rates by flies were much more consistent, with no

significant variation among pairs and no effects of flower

colour detected (Table 4). Pollen export (estimated with

dyes) differed with colour on the large spatial scale, but

not detectably so on the small spatial scale. Large arrays

with all of the outer flowers painted blue actually had

greater amounts of dye transfer (effect of outer colour in

split-plot ANOVAANOVA, P < 0.05, Table 4, Fig. 6). Dye export

Table 3 Estimates of phenotypic selection on standardized flower size (diameter) and standardized colour in arrays of natural flowers.

The fitness component examined was the number of visits in an hour by bees, as a residual from the mean value for the array. Analysis

employed multiple regression based on eqn 1 in the text.

(A) Colour measured as the reflectance ratio (R440 ⁄ R530). Measurements were made for all but two of the 160 flowers in the 10 arrays

Parameter Estimate Standard error t152 P

Intercept )0.1866 0.4364 )0.43 0.6697

Size 1.6115 0.3263 4.94 < 0.0001

Colour (R440 ⁄ R530) 0.1788 0.4464 0.40 0.6893

Size2 0.3493 0.4772 0.73 0.4654

Colour2 )0.0909 0.4182 )0.22 0.8282

Size · Colour 0.3204 0.3488 0.92 0.3598

(B) Colour measured as principal component 1 for flowers plotted in hexagon colour space. This analysis used only the flowers from 2009

Parameter Estimate Standard error t105 P

Intercept )0.1632 0.5678 )0.29 0.7743

Size 1.5573 0.4261 3.66 0.0004

Colour (PC1) )0.0600 0.4417 )0.14 0.8921

Size2 0.4564 0.5828 0.78 0.4353

Colour2 )0.5633 0.4529 )1.24 0.2164

Size · Colour 0.0127 0.4872 0.03 0.9793

Fig. 4 Proportion of visits made to blue-painted rather than white-

painted flowers in small experimental arrays of Wahlenbergia

albomarginata.

8 D. R. CAMPBELL ET AL.

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from a given inner colour in an array correlated with bee

visitation (r = 0.58, N = 12, P = 0.0481) but not with fly

visitation (r = )0.30, N = 12, P = 0.3508). After dye

export was summed across the two inner flower colours

to yield a single value for an array, total dye export in an

array also correlated highly with the total number of bees

observed visiting the array during four daytime hours of

those 48 h (r = 0.81, N = 8, P = 0.0140). Number of bees

seen at an array in 4 h ranged from 0 to a high of 24,

with more seen on warmer days. Because visitation rates

by bees were higher to blue flowers, blue flowers in

mostly blue arrays received 92% of their visits from bees,

the more effective pollinators, whereas white flowers in

mostly white arrays received only 68% of their visits

from bees (Fig. 6). Weighting these values by the relative

effectiveness of these two types of insects in single visits

(113 vs. 40 pollen grains) predicts that 97% of pollen

transfer to blue flowers in blue arrays was by bees and

86% to white flowers in white arrays was by bees.

Q5: Is intraspecific variation in flower colourassociated with flower temperature and/or floralherbivory?

The flowers assigned to the blue vs. white categories

differed in colour, as judged by the reflectance ratio

R440 ⁄ R530 (mean ± SE = 1.19 ± 0.4 and 0.96 ± 0.02 for

blue and white, respectively, randomized block ANOVAANOVA,

P < 0.0001). Flowers in both colour groups had internal

temperatures <1� warmer on average than ambient

(mean ± SE = 0.65 ± 0.27 and 0.66 ± 0.32), and there

was no detectable difference between blue and white

flowers in their temperature anomalies from ambient

(randomized block ANOVAANOVA, P = 0.99).

We were able to record herbivore damage for 116 of the

original 144 flowers that we marked. The others had

completely senesced prior to re-checking or were entirely

missing, including the flower stem, for unknown reasons.

As in the temperature study, the flowers assigned to the

blue vs. white categories again differed in the reflectance

ratio (randomized block ANOVAANOVA, P < 0.0001). Petal dam-

age was similar between colour classes, regardless of

whether the analysis used all damage levels individually or

lumped them into three categories of no damage, partial

damage or complete damage (likelihood ratio v2 = 2.5245,

d.f. = 2, P = 0.2830). Logistic regression also yielded no

Fig. 5 Dye exported from painted donor flowers in small experi-

mental arrays in 2009 as a function of colour.

Table 4 Split-plot analysis of variance for (A) insect visitation and (B) dye transfer in the large experimental arrays. The effects of

pair and outer colour were tested over the pair · outer colour interaction, which is equivalent to the whole-plot error.

(A) Insect visits to flowers per hour

Bees Flies

Source of variation d.f. MS F P MS F P

Pair 4 22.15 41.19 0.0017 0.45 0.81 0.5764

Outer colour 1 0.77 1.43 0.2984 0.43 0.77 0.4290

Pair · Outer colour 4 0.54 1.50 0.2884 0.55 1.91 0.2015

Inner colour 1 2.17 6.06 0.0392 0.08 0.27 0.6163

Outer colour · Inner colour 1 0.64 1.80 0.2171 0.90 3.14 0.1143

Residual error 8 0.36 0.29

(B) log(Dye export + 1)

Source of variation d.f. MS F P

Pair 3 26.44 242.94 0.0004

Outer colour 1 1.36 12.53 0.0384

Pair · Outer colour 3 0.11 0.15 0.9262

Inner colour 1 0.09 0.12 0.7405

Outer colour · Inner colour 1 0.02 0.02 0.8888

Residual error 6 0.73

Pollinators and selection on Wahlenbergia colour 9

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effect of the reflectance ratio (measured for both flowers in

18 blocks) on the occurrence of petal damage (v2 = 0.16,

P = 0.6905). Overall, 39% of the flowers in the blue colour

class and 44% of the flowers in the white colour class

received partial or complete petal damage over the 4- to

5-day period.

Discussion

Pollinators as a source of selection on flower colour

The distribution of flower colours in alpine New Zealand

is unusual in the high percentage of white- to pale-

coloured flowers (Mark & Adams, 1993). Like some other

plants in this habitat (e.g. species of Gentiana and Hebe;

Lloyd, 1985), flowers of W. albomarginata are presumed

to have lost the high level of pigmentation that was present

in ancestral relatives. The traditional explanation offered,

including on New Zealand Department of Conservation

(Te Papa Atawhai) signs at the field site, is that the insect

visitors lack colour selectivity entirely (Wardle, 1978) or

that they discriminate against bright blues, purples and

reds, as those colours are largely missing from the habitat

(Heine, 1938). Here, we found that the major pollinators of

W. albomarginata, small solitary bees such as H. matamoko,

do not discriminate against blue petals painted to resemble

those of a congener, W. violacea, and in some circum-

stances even prefer that bright blue. The progenitors of

Hylaeus bees in New Zealand originated from Australia

(Donovan, 2007), just as did the progenitors of Wahlen-

bergia herbs, so this may be a case where ancestral

responses to blue petals have been retained. This behav-

iour, in combination with the higher pollen transfer

observed in mostly blue-flowered arrays, indicates that

pollinator discrimination cannot explain the absence of

bright blue petals in W. albomarginata. Although some flies

did show a preference for white over blue W. albomarginata

flowers, they were only minor contributors to pollination.

In our large arrays, we estimated that 86–97% of pollen

transfer (depending on flower colour) was by bees rather

than flies. Furthermore, pollen export (as estimated with

dyes) correlated highly with bee visitation, but not with fly

visitation. We have shown elsewhere that the Allograpta

flies preferred yellow- over white-painted petals for three

of four plant species on which they were tested (Campbell

et al., 2010). In combination with those earlier results, the

studies reported here also counter the idea that insect

visitors to New Zealand alpine flowers lack colour selec-

tivity entirely.

Responses to flower colour by the bee pollinators

depended on the range of colour variation offered and on

the spatial scale of the manipulation. When presented with

arrays containing natural variation in petal colour, we

detected no colour preference. This apparent absence of

preference was not due to low statistical power, as we

easily detected preference for visiting larger flowers. In

small experimental arrays with a choice between eight

white flowers and eight flowers painted bright blue to

resemble W. violacea, the bees again showed no preference.

Again, we had sufficient statistical power to detect strong

preferences, as we witnessed significant overvisitation of

white flowers by both syrphid flies and tachinid flies in this

experiment. In contrast to the results in small-scale

manipulations, when flower colour was manipulated over

a larger spatial scale, the bees showed a preference for

flowers painted blue to resemble W. violacea. Pollen trans-

fer (as estimated with dyes) was higher overall in the large

arrays with mostly blue flowers than in the large arrays

with mostly white flowers, although it was not higher

specifically from the blue donor flowers in those arrays.

These findings suggest that bee responses to colour are

dependent on the spatial context. At this field site, the bees

would have had no natural experience with that bright

blue and may only have developed a search image for it

when presented with the large patches of 64 flowers,

presumably offering a rich concentration of rewards. This

(a)

(b)

Fig. 6 Insect visitation rates and dye export in large experimental

arrays of painted flowers in 2011. (a) Visits to inner flowers per hour

for bees and flies visiting the four colour combinations. (b) Dye

export on a log-transformed scale. The scale bars on the left indicate

the square root of the mean square error in the split-plot ANOVAANOVA.

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dependence on spatial scale indicates the need to consider

the larger spatial context of available flowers when

measuring pollinator preferences (see also Klinkhamer

et al., 2001).

If the major pollinators at this site do not prefer the pale-

coloured petals that are typical of W. albomarginata, then

why does this species not have brighter blue petals? It is

conceivable that flies provide a much greater proportion of

the pollination in some other parts of the species range,

although this is unlikely given their low effectiveness, and

that there is likely gene flow from those other sites. It is also

theoretically possible that even in the absence of innate

preference for white flowers, many of the herb species

have converged on white as a common floral signal.

However, in that case, we could have expected that

pollinators would visit the occasional brightly coloured

flower in an otherwise white patch, but avoid larger

patches of bright blue flowers. Yet, in our experimental

manipulations on the larger spatial scale, neither bees nor

flies preferred the large white arrays over the large blue

ones. We cannot rule out the possibility entirely that

flowers in this habitat have converged on white as a signal,

despite localized preference of H. matamoko for blue- over

white-painted W. albomarginata, Leioproctus bees for yel-

low- over white-painted Brachyglottis bellidioides and

Allograpta flies for yellow- over white-painted Ourisia

glandulosa (Campbell et al., 2010), but, if so, the process

would have to work on an even larger spatial scale than we

were able to investigate here.

One final hypothesis that could involve pollinators is

that they would be preferentially attracted to white

flowers because of stronger reflectance of long wave-

lengths, heating the interior of the flower and the insect

and possibly increasing pollen germination (McKee &

Richards, 1998). For example, the interior temperature is

higher in the white morph compared with the blue morph

of an alpine species of Gentiana on the Qinghai-Tibetan

Plateau (Mu et al., 2010), although in other cases darker

coloured flowers are more efficient at raising internal

flower temperature (McKee & Richards, 1998). We found

no detectable difference in floral temperature associated

with the natural range of flower colour and no preference

of the major pollinators for white flowers anyway. Thus,

this hypothesis is not supported for W. albomarginata,

perhaps not surprisingly given its relatively open flower.

One remaining potential agent of direct selection on flower

colour would be floral herbivores, but again we detected

no association of florivory with flower colour.

Indirect selection vs. pollinator-mediated selection

Ratherthanbeingmaintainedbyinsectpollinators, it seems

likely that the white or pale petals in W. albomarginata

are maintained by indirect selection on correlated

characters. Although our study, with its reliance on

phenotypic manipulations, was designed to examine

the direct selection on flower colour, and primarily the

hypothesis that pollinators select on flower colour,

there are other hypotheses involving indirect selection

mediated by pleiotropic effects of colour genes on other

traits. At this point, we do not know the extent to which

the intraspecific variation in flower colour is genetic vs.

environmental; however, the species difference between

W. albomarginata and W. violacea is presumably genetic,

suggesting that a loss-of-function mutation or downre-

gulation has occurred in W. albomarginata. The pigment

that produces the petal colour is an anthocyanin with

cyanidin indicated by thin-layer chromatography as

the core anthocyanidin (J. Lord, personal observation).

The anthocyanin biosynthesis pathway is controlled by

the expression of six major structural loci (Holton &

Cornish, 1995), so the potential cost savings for other

functions of downregulating pigment production might

depend on how early in the pathway a loss-of-function

mutation has occurred. In many plant species, there are

pleiotropic effects on flower pigmentation and pigmen-

tation in the vegetative stem or leaves (Armbruster,

2002); however, expression levels of a key gene in the

pathway, chalcone synthase, were decoupled in the

petals and leaves of Parrya nudicaulis (Dick et al., 2011).

In several cases reviewed by Strauss & Whittall (2006),

morphs that produced more anthocyanins or were more

brightly coloured had leaves that were better defended

against vegetative herbivores.

In addition to affecting leaf herbivores, the production

of anthocyanins can affect physiological properties of

the plant. Anthocynanins are involved in multiple

stress-related processes, including drought and heat

tolerance, and protection from photoinhibition (Gould,

2004). In other species, anthocyanin production has

increased fitness under drought conditions (Schemske &

Bierzychudek, 2001), sometimes trading off with lower

fitness under well-watered conditions (Warren &

Mackenzie, 2001), suggesting that there is a cost under

wet conditions of producing the floral pigment. Antho-

cyanin pigments have also been reported to increase the

tolerance to both heat and cold stress. White mutants in

Ipomoea purpurea produced by a mutation at the first step of

the anthocyanin biosynthesis pathway showed poor

tolerance to heat stress (Coberly & Rausher, 2003),

whereas white mutants in an arctic mustard were more

common in cold climates (Dick et al., 2011). In the case of

W. albomarginata and other high-elevation New Zealand

species, the radiation of a clade into a novel environment

might have altered the selective advantage of anthocyanin

production. If tolerance to the heat stress that would be

more common at low elevations is lost in white-flowered

individuals, or if there is a greater cost to producing the

pigment under relatively mesic conditions in the alpine,

such physiological responses could provide hypotheses for

why Australian species and lowland species in New

Zealand can be more brightly coloured than those in the

New Zealand alpine. These potential scenarios for indi-

rect selection mediated by environmental stress or by

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vegetative herbivores warrant further investigation in

New Zealand Wahlenbergia as well as other plants.

One way to test for the effect of a particular agent of

selection is to manipulate both the environmental source

of selection and the trait (in this case flower colour; Wade &

Kalisz, 1990). We are now completing such an experiment

with pollination in W. albomarginata, using two extremes

of intraspecific variation in colour to produce two colour

classes crossed with three experimental levels of pollen

supply. To explain the absence of bright blue in species at

high elevation in comparison with low elevation, it would

be valuable to repeat such manipulative studies at a variety

of elevations. Such studies would allow investigating

whether selection by pollinators in favour of blue is even

stronger at lower elevation in the genus Wahlenbergia and

whether there is indirect selection through effects on

herbivores, heat tolerance or other physiological aspects

that may be advantageous at low elevation but not at high

elevation. Our results support the need to consider

hypotheses other than pollinator behaviour (Strauss &

Whittall, 2006) as drivers of flower colour evolution in the

New Zealand alpine, particularly those involving indirect

selection of other characters.

Acknowledgments

This research was partly supported by grant 8621-09

from the Committee for Research and Exploration of

the National Geographic Society and contract

CO9X0503 from the New Zealand Public Good Science

Fund. We are grateful for the field assistance of Mary

Price, Maria Minor, and Nickolas Waser and the

postgraduate students who helped during a field class

held while the first author was a Fulbright Specialist in

Environmental Sciences at Otago University. Mary

Anne Miller and Vickey Tomlinson assisted with

arrangements for supplies. NZ Ski Ltd. provided space

for housing and laboratory work on the mountain. Two

anonymous reviewers provided helpful comments on

the manuscript.

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Supporting information

Additional Supporting Information may be found in the

online version of this article:

Appendix S1 Colour plate showing (A) Wahlenbergia

albomarginata growing in the field, (B) a tachinid fly

visiting a manipulated flower within an experimental

array, (C) an unusually white flower of W. albomarginata,

(D) an unusually blue flower of W. albomarginata, (E)

close up of the colletid bee Hylaeus matamoko on a petal of

W. albomarginata painted to resemble W. violacea, and (F)

a flower of W. violacea.

As a service to our authors and readers, this journal

provides supporting information supplied by the authors.

Such materials are peer-reviewed and may be re-

organized for online delivery, but are not copy-edited

or typeset. Technical support issues arising from support-

ing information (other than missing files) should be

addressed to the authors.

Received 23 August 2011; revised 21 October 2011; accepted 26 October

2011

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