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Colour and odour drive fruit selection
and seed dispersal by mouse lemurs
Kim Valenta
1
, Ryan J. Burke
1
, Sarah A. Styler
2
, Derek A. Jackson
2
, Amanda D. Melin
3
& Shawn M. Lehman
1
1
University of Toronto, Department of Anthropology,
2
University of Toronto, Department of Chemistry,
3
Dartmouth College,
Department of Anthropology.
Animals and fruiting plants are involved in a complex set of interactions, with animals relying on fruiting
trees as food resources, and fruiting trees relying on animals for seed dispersal. This interdependence shapes
fruit signals such as colour and odour, to increase fruit detectability, and animal sensory systems, such as
colour vision and olfaction to facilitate food identification and selection. Despite the ecological and
evolutionary importance of plant-animal interactions for shaping animal sensory adaptations and plant
characteristics, the details of the relationship are poorly understood. Here we examine the role of fruit
chromaticity, luminance and odour on seed dispersal by mouse lemurs. We show that both fruit colour and
odour significantly predict fruit consumption and seed dispersal by
Microcebus ravelobensis
and
M.
murinus
. Our study is the first to quantify and examine the role of bimodal fruit signals on seed dispersal in
light of the sensory abilities of the disperser.
P
lant reproduction often requires animal vectors to provide seed dispersal or pollination services
1
. Numerous
studies have demonstrated that plant signals and cues are critical to fruit selection by animals
2–5
. While ripe
fruit signals refer to traits such as colour and odour that are maintained by natural selection because of their
ability to reliably convey information to other organisms
6
, ripe fruit cues refer to traits that evolved in a context
unrelated to animal signalling (e.g. red anthocyanine pigmentation), that may nonetheless convey reliable
information to dispersers
7
. The question of whether plants have evolved to maximize signal detectability to
potential pollinators and dispersers is contentious
1,8
, as is the question of how much variation in frugivore sensory
phenotypes is driven by fruit signals versus cues
9
.
Plant signals and cues available to animals depend critically on three complex factors: first, the complete signal
being broadcast, second, its arbitration by the local environment
6
, and third, animal sensory phenotypes, which
mediate the detectability of plant signals and cues to potential seed dispersers and pollinators
10
. Plant signals and
cues are highly variable
8
, and comprise visual components - chromaticity (hue, saturation)
11
, brightness or
luminance
12
- and odour components, including individual volatile compounds as well as overall odour plume
13
.
Animal colour vision phenotypes are also highly variable, and for terrestrial vertebrates range from monochro-
macy to tetrachromacy
14
. In the case of odour-detection ability, few studies have sampled olfactory receptor (OR)
gene repertoires
15–17
, although evidence from experimental studies and broad neuroanatomical measures indicate
high variation amongst vertebrates
18,19
. Ideally, to determine the extent of the mutualism between animal sensory
phenotype and fruit signals, models should include quantitative measures of signals as well as the ability of
animals to discern those signals
10
. Recent studies have quantified certain fruit signals and compared them to seed
dispersal
8
, and quantified disperser phenotypes in light of fruit colour
11,20
. For example, recent studies of the role
of plant colour signals and cues in primate foraging decisions indicate that at least some variation in primate
foraging efficiency and preferences results from variation in individual colour vision phenotypes
11,12,21
.
Additionally, a recent study examining the relative sensory reliance in three strepsirrhines emphasized the role
of vision over olfaction for diurnal lemurs
22
. However, to date, no study has yet quantified and qualified fruit
chemistry in combination with quantitatively measuring luminance and chromaticity in light of animal-specific
sensory phenotypes.
Several studies have established the importance of primates as seed dispersers
23,24
. Primates comprise between
25% and 40% of frugivore biomass in tropical forests
25
, and defecate or spit large numbers of viable seeds
26
, which
makes them particularly well suited to be effective dispersers. Seed dispersal by primates is critical to the
maintenance of fruiting tree populations, and has been shown to contribute to the maintenance of biodiversity
in tropical forests
27
. The case of Madagascar is particularly compelling, as primates comprise most of the seed
dispersing species in those forests – indeed, only ten non-primate species in Madagascar have been identified as
seed dispersers
28
, which is in stark contrast to the diverse disperser assemblages of other tropical biomes
29
.
OPEN
SUBJECT AREAS:
ANIMAL PHYSIOLOGY
COEVOLUTION
ANIMAL BEHAVIOUR
BEHAVIOURAL ECOLOGY
Received
14 May 2013
Accepted
25 July 2013
Published
13 August 2013
Correspondence and
requests for materials
should be addressed to
K.V. (kim.valenta@
utoronto.ca.)
SCIENTIFIC REPORTS | 3 : 2424 | DOI: 10.1038/srep02424 1
Understanding the relationship between endemic fruit signals and
seed dispersal by Microcebus spp. is important in the heavily dis-
turbed forests of Madagascar. The potential for Microcebus spp.as
critical seed dispersers in the uniquely depauperate frugivore com-
munities of Madagascar has recently been highlighted
29
. While
numerous studies have demonstrated the importance of fruit mor-
phology, including colour and size, in diurnal primate foraging deci-
sions
21
, data are lacking on morphological characteristics of fruits
consumed by nocturnal primates. This is the first study to compare
quantitative and qualitative measures of fruit odour, as well as quant-
itative measures of luminance and chromaticity on fruit consump-
tion by an animal in light of species-specific sensory phenotypes.
Additionally, this is the first study to quantify fruit chromaticity
and luminance for a nocturnal primate.
Here, we determine whether seeds of endemic plant species are
dispersed by wild-trapped Microcebus ravelobensis and M. murinus
held in short-term captivity in northwestern Madagascar, as evi-
denced by the presence of intact seeds in feces. We then compare
chromaticity, luminance and odour of dispersed and non-dispersed
species. Microcebus is an ideal taxon within which to measure the
interplay between fruit cues and sensory phenotypes because their
capacity for colour discrimination can be accurately modelled based
on known peak cone spectral sensitivities and optical morpho-
logy
30,31
. Additionally, Microcebus spp. have been shown experiment-
ally to be able to reliably distinguish olfactory cues, and retain
extensive neuroanatomical structures associated with enhanced
olfactory discrimination, including moist rhinaria and large olfactory
bulbs
19,32
. Unlike some other nocturnal primates, Microcebus spp.
have retained dichromatic cone function
30
, which may indicate puri-
fying natural selection acting to maintain colour vision
33
. Therefore,
we predict that fruits that are consumed and dispersed will have a
greater chromatic contrast than fruits that are not consumed.
Because dichromatic animals have been shown to respond to lumin-
ance cues
34
, we predict that consumed fruits will have a greater
luminance contrast than unconsumed fruits. Because Microcebus
spp. are strepsirrhines with highly retained OR repertoires and large
olfactory bulbs
19
we predict that dispersed fruits will emit greater
overall volatile organic compounds (VOC) than fruits that are not
consumed or dispersed by Microcebus spp. and that Microcebus-dis-
persed fruits will be characterized by emission of similar chemical
compounds. All of our analyses are one-tailed due to the direction-
ality of our predictions.
Results
The luminance contrast between fruits and background leaves is
similar for both dispersed and non-dispersed species, as indicated
by their large degree of overlap (Fig. 1). Thus, contrary to our pre-
dictions, the effect of luminance on seed dispersal is not significant
(Wald chi-square 5 0.456, df 5 1, p 5 0.245, one-tailed). Our
prediction that Microcebus spp. disperse species with a higher chro-
matic contrast is, however, supported (Wald chi square 5 3.018, df 5
1, p 5 0.041, one-tailed). Fruits of dispersed species have a higher
chromatic (blue-yellow) contrast with background leaves than fruits
of non-dispersed species (Fig. 1). Our prediction that Microcebus-
dispersed fruits are characterized by emission of similar volatile
compounds is not supported: fruit species dispersed by lemurs do
not show a significant difference in VOC compound distributions
(Wald chi-square 5 4.332, df 5 5, p 5 0.252, one-tailed) relative to
non-dispersed species (Wald chi-square 5 2.682, df 5 4, p 5 0.306,
one-tailed). Our prediction that Microcebus-dispersed fruits emit
greater overall VOCs than non-dispersed fruits is supported (F 5
8.001, p 5 0.014, one-tailed). Five of the six species with the lowest
overall VOC emissions (integrated chromatogram area , 15,500)
are non-dispersed. In contrast, eight of the nine species with VOC
emissions with an integrated chromatogram area . 15,500 are dis-
persed (Fig. 2).
Discussion
Our prediction that fruit luminance contrast predicts fruit consump-
tion and seed dispersal was not supported. Conversely, we did find
support for the importance of chromatic contrast. Previous studies
Figure 1
|
Chromatic and luminance contrasts of dispersed and non-dispersed fruits. Scatterplot showing the blue-yellow chromatic contrasts (y axis)
and luminance contrasts (x axis) between ripe fruits and upper leaf surfaces of dispersed and non-dispersed fruits. Reflectance spectra of ripe fruits and
upper leaf surfaces were measured relative to a Spectralon white reflectance standard using a Jaz portable spectrometer and a PX-2 pulsed xenon
lamp emitting a D-65 light source. The chromatic and luminance conspicuity of food items was modeled as a ratio of the quantum catch of photons
incident on the retina by different cone types, using a dichromatic visual model based on the long-wavelength sensitive (L) photopigments (l
max
558 nm)
and short-wavelength sensitive (S) photopigments (l
max
409 nm) possessed by Microcebus spp.
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 3 : 2424 | DOI: 10.1038/srep02424 2
suggest that the persistence of two functional opsin genes in some
nocturnal primate species indicates that dichromatic colour vision is
under purifying natural selection, and confers a foraging advant-
age
33,35
. Our findings that under moonlight conditions - which are
bright enough to support cone function
36
- Microcebus-dispersed
fruits display greater blue-yellow chromatic contrast from leaves
than non-dispersed fruits supports the adaptive function of dichro-
matic colour vision for this genus.
Our prediction that individual VOCs predict fruit consumption
and seed dispersal was not supported. Rather we found no relation-
ship between the top ten most common VOCs and fruit consump-
tion or seed dispersal. While our results are therefore not in
accordance with the role of specific compounds during fruit selec-
tion, previous studies on other mammals support this relation-
ship
13,18
. There are at least two potential explanations for this
discrepancy. First, because olfactory receptor gene repertoires have
not yet been sequenced for this genus, Microcebus’ phenotypic sens-
itivity to specific VOCs is unknown. Ideally, future work will be
aimed at establishing olfactory sensitivity to specific compounds
which would allow for a biologically meaningful approach to the
question of fruit signal VOC specificity. Second, it is possible that
Microcebus, like other olfactory-driven foragers
2
, are responsive to
VOCs that are present even in trace amounts. Thus, rather than
identifying the most common VOCs present in fruits, it would be
beneficial to identify Microcebus’ sensitivity to certain VOCs, in
addition to identifying their presence at critical thresholds in fruits.
Our prediction that overall VOC emission intensity predicts fruit
consumption and seed dispersal is supported, which is consistent
with the expectation that Microcebus spp. rely heavily on olfaction
32
.
Microcebus spp. have been shown experimentally to be able to reli-
ably distinguish olfactory cues, and retain extensive neuroanatomical
structures associated with enhanced olfactory discrimination
19,32
,
which are useful for identifying fruit signals and cues in the wild.
Our finding is consistent with results for other nocturnal mammals
that have been found to distinguish between ripe and unripe fruit
based solely on olfactory cues
3
. For example, one study of fruit bats
found that they were able to reliably select ripe fruit based on the
presence and intensity of VOCs
13
. Another study compared VOC
emissions of bird- and bat-dispersed fig fruits, and found that figs
dispersed primarily by olfactorily-driven bats emitted higher overall
VOCs than figs dispersed primarily by visually-oriented birds
8
. That
olfactory cues may be tightly linked to the foraging effectiveness of a
nocturnal primate makes evolutionary sense - animals functioning in
low ambient light environments can be expected to rely on non-
visual signals and cues during foraging.
Microcebus spp. are active under nocturnal conditions that are
sub-optimal for colour vision, yet our study reveals that fruit chro-
maticity still informs some foraging decisions. Therefore the physical
properties of their diet may directly contribute to the maintenance of
dichromacy in Microcebus spp. while it has been lost in other noc-
turnal primates
30,37
. Though the activity patterns and resulting sens-
ory adaptations of early primates is contested
38
, olfaction and colour
vision have traditionally been portrayed as antagonistic, such that the
advent of enhanced visual specializations is expected to co-vary with
a decrease in reliance on olfaction
17
. Yet the variable patterns of loss
and retention of vision and olfaction in different lineages under
similar ecological pressures (i.e. nocturnality) reveals the details are
more nuanced than this and need to be considered more carefully to
identify relationships between diet, activity patterns and sensory
systems.
Microcebus potentially represent their own seed disperser niche in
the frugivore communities of Madagascar, as they are the only small
bodied nocturnal frugivores that do not hibernate for most of the
year. Because of their small body size they are restricted to dispersing
seeds normally available to birds with opposite foraging patterns and
sensory phenotypes. While avian seed dispersers are tetrachromatic
and diurnal, and rely heavily on visual cues during foraging,
Microcebus spp. are capable of fewer chromatic distinctions but have
improved olfactory capabilities than sympatric, frugivorous birds.
The difference in plant signals implied by these two sets of conflicting
Figure 2
|
Volatile organic compound emission intensity of dispersed and non-dispersed fruits. Frequency distribution of surface-area-scaled
volatile organic compound (VOC) emissions of dispersed and non-dispersed fruits. Surface-area-scaled VOC emission intensity is determined by
integrating areas under gas chromatography-mass spectrometry (GC-MS) chromatograms, and scaling GC-MS chromatograms by the total surface area
of all fruits sampled.
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 3 : 2424 | DOI: 10.1038/srep02424 3
adaptations predicts functional separation in small fruit morpho-
logy. The co-occurrence of two small-seeded disperser guilds with
differing sensory abilities is likely to result in different selective pres-
sures on small fruit morphology, favoring both chromatic conspi-
cuity that attracts highly visual diurnal birds, and VOC emissions
and blue-yellow chromatic contrasts that attract olfactory-driven,
dichromatic primates. Future research on the role of sensory pheno-
types of Malagasy avian dispersers during foraging will help to illus-
trate the degree to which these adaptations either integrate or
conflict.
Methods
Data were collected adjacent to Ampijoroa forestry station in the tropical dry forest in
Ankarafantsika National Park, northwestern Madagascar (ANP - 15059’–16u22S,
470569–47012E). Over a three month period (May–July, 2012), we opportunistically
collected ripe fruits of 20 species found growing within the study area. We offered a
minimum of 40 individual ripe fruits (N 5 676) of 20 plant species to wild-trapped
Microcebus (N 5 99) held in short-term captivity (,12 hrs) and identified and
counted all seeds contained in feces collected from traps (N 5 1324). Only fruit
species that contain seeds where the mean size is equal to or lesser than 11 mm in
maximum diameter, which is the largest maximum diameter found in Microcebus
fecal samples, were included in the analysis (N 5 16). A fruit species was considered
to be dispersed when seeds of that species were present in feces. A fruit species was
considered to be non-dispersed when a minimum of 40 fruits of that species were
offered to captive Microcebus, and neither consumed nor discovered in Microcebus
feces. In all cases but one (Monanthotaxis valida, Annonaceae), all fruit species were
dispersed by both species of Microcebus. This research adhered to the Laws of
Madagascar governing primate research, the American Society of Primatologists
principles for the ethical treatment of primates, and the University of Toronto
(Animal Care Protocol #20009283).
To quantify fruit odour, fruits were collected in the field, and measured in the
laboratory in three dimensions (height, width and depth) using sliding calipers, and
placed inside plastic sampling bags (Reynold’s large oven bags). The atmosphere
within each bag was sampled using a vacuum pump (Gilian 5000, Sensidyne), which
pulled air through the sample bag (1 L/min, 240 min) and into two odourant-
absorbent filters (Amberlite XAD-2, 400–200 mg, Sigma-Aldrich). Contamination of
the sampling enclosure with ambient VOCs was minimized by passing incoming air
through a container of activated carbon.
In order to analyz e the trapped VOCs, XAD resin beds were removed from their
cartridges and shaken manually in 4 mL hexane (Sigma Aldrich) for 5 min. Main and
breakthrough XAD beds were extracted separately. Extracts were analyzed using an
Agilent 7890A gas chromatograph interfaced with an Agilent 5975 inert mass
spectrometer operating in electron ionization (EI) mode. All injection volumes were
1 mL and performed in the splitless mode with an inlet temperature of 250uC.
Separation was achieved using an Agilent DB-5 column (30 m 3 0.25 mm 3
0.25 mm) at a constant helium flow rate of 1 mL/min. The oven program consisted of
an initial hold at 50uC for 2 min followed by a 10uC/min ramp to 150uC and a 30uC/
min ramp to 300uC. The transfer line temperature was held at 300uC. Analytes were
monitored in full scan mode using a selected mass range of 40–300 Da.
In order to control for variation in fruit number and fruit surface area, we scaled the
GC-MS chromatograms by the total surface area of all fruits in the sample bag. We
determined total VOC emission intensity for each fruit species by summing the area
under the surface-scaled GC-MS chromatograms, and compared values for dispersed
and non-dispersed species with a one-way analysis of variance (ANOVA). Two fruit
species (Elaeocarpus subserratus, Elaeocarpaceae, and Psorospermum crassifolia,
Hypericaceae) were excluded from the analysis because samples were run using a
different GC-MS, and thus were not quantitatively comparable. We determined the
ten largest compound peaks for each fruit species and tentatively identified the five
compounds that appear to be driving variation using MassLy nx software (V4.1). To
determine the effect of individual VOCs on fruit choice and seed dispersal, we ana-
lysed the largest ten VOC values in both dispersed and non-dispersed fruits using a
one-tailed generalized linear model (GLM) with a log link function. We computed the
Wald Likelihood statistic (SPSS V20), for both dispersed and non-dispersed cat-
egories, to test whether the shape of the distribution is significantly different from a
Poisson distribution.
Reflectance spectra of ripe fruits (targe ts) and upper leaf surfaces (backgrounds)
were measured rel ative to a Spectralon white reflectance standard (Labsphere) on-
site in Ma dagascar using a Jaz portable spectrometer and a PX -2 pulsed xenon
lamp (Oc ean Optics Inc.) emitting a D-65 light source. The fruit scanning angle
was fixed at 45u, and external light was blocked using thick bl ack fabric. The
chromatic and luminance conspicuity of food items was modeled as a ratio of the
quantum catch of photons incident on the re tina by different cone types following
established methods
12,39,40
, using a dichromatic visual model based on th e long-
wavelength sensitive (L) photopigments (l
max
558 nm) and short-wavelength
sensitive (S) photopigments (l
max
409 nm) possessed by Microcebus
30
.The
quantum catches of each photoreceptor (cone) type were calculated according to
the formula:
Qi ~
ð
max
min
R(l)I(l)Si(l)dl
where Q
i
represents the quantum catch of a photoreceptor i over the range of the
primate visual spectrum, from 400 nm (min) to 700 nm (max), R(l)represents
the reflectance sp ectrum, I(l) repre sents the irradiance spectrum, and S
i
(l)isthe
spectral sensitivity function of the i-th photoreceptor (containing S or L photo-
pigments). For the representative irradiance spectrum, we used down-welling
moonlight in a large forest gap
36
. The spectral sensitivity function for each
photoreceptor type was calculated as per Hiramatsu et al.
12
, w ith one alteration.
Because lemurs do not possess a macula lutea, our pre -receptoral filter included
only the effects of the lens, as opposed to the combined effects of the lens and
macular pigment. Although the rods may contribute to colour perception at dim
light levels, the perceptual effects of this are not well understood, and we omit the
contribution of rods here for simplicity.
The blue-yellow chromaticities of target and background objects can be repre-
sented and plotted as the relative quantum catches of the S cones to the L cones, S/L.
Because the S cones do not contribute meaningfully to perception of luminance
contrast, the relative luminance value of each object was estimated by dividing the
quantum catch of the L cones by that of a hypothetical white surface that reflects 100%
of the illuminant. To estimate the blue-yellow chromatic contrast (BY) and the
luminance contrast (LUM) between each target fruit and its respective leaf back-
ground, we calculated a contrast value for each channel: BY 5 jln(Q
L
f
) 2 ln (Q
L
b
)j 2
j
ln(Q
S
f
) 2 ln (Q
S
b
)
j
;LUM5
j
ln(Q
L
f
) 2 ln (Q
L
b
)
j
, where Q denotes the quantum
catch of the L cones (L) or S cones (S) for fruits (f) or backgrounds (b)
12
.
To determine the effect of luminance and chromaticity on fruit choice and seed
dispersal, we analyzed the differences between the leaves and ripe fruits using a one-
tailed GLM for binomial distribution with a logit link function and Wald Likelihood
statistic (SPSS V20).
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Acknowledgements
We thank MICET and Madagascar National Parks, for permission to conduct this research
in Madagascar. We thank Dr. Scott Mabury for the loan of instrumentation. We are grateful
to Mr. Paul Tsiveraza, Tantely, Jhonny, Radoniaina Rafaliarison and Jean de-la-Dieu for
contributions in the field. We thank Sharon Kessler, Cindy Canale, Ute Radespiel,
Blanchard Randrianambinina and Sylvia Lomascolo for their help in the initial phases of
this project. We appreciate assistance from Dr. Chihiro Hiramatsu and Dr. James Higham
with aspects of the colour modelling. For helpful commentary, we thank C.J. Toborowsky
and Paul R. Duffy. We are grateful to Dr. Michael Huffman, Dr. James Higham, and an
anonymous reviewer for valuable comments on this manuscript. This research adhered to
the Laws of Madagasc ar governing primate research, the American Society of
Primatologists principles for the ethical treatment of primates, and the University of
Toronto (Animal Care Protocol #20009283). For funding we thank Sigma Xi, GM Women
in Science (K.V.), the University of Toronto (K.V. and R.J.B.) and Natural Sciences and
Engineering Research Council of Canada (K.V., R.J.B., A.D.M., S.M.L.).
Author contributions
K.V., R.J.B. and A.D.M. designed the study. K.V. and R.J.B. carried out data collection. K.V.,
S.A.S. and D.A.J. designed the VOC collection system, and extracted and analyzed chemical
data. A.D.M. completed colour modeling and analysis. R.J.B. and S.M.L. carried out all
statistical analyses. All authors contributed to the writing and editing of the manuscript.
Additional information
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Valenta, K. et al. Colour and odour drive fruit selection and seed
dispersal by mouse lemurs. Sci. Rep. 3, 2424; DOI:10.1038/srep02424 (2013).
This work is licensed under a Creative Commons Attribution-
NonCommercial-NoDerivs 3.0 Unported license. To view a copy of this license,
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SCIENTIFIC REPORTS | 3 : 2424 | DOI: 10.1038/srep02424 5
