O R I G I NA L A RT I C L E
doi:10.1111/j.1558-5646.2012.01727.x
POLLINATOR-MEDIATED REPRODUCTIVE
ISOLATION AMONG DIOECIOUS FIG SPECIES
(FICUS, MORACEAE)
Annika M. Moe1,2 and George D. Weiblen3
1
Biology Department, Syracuse University, 114 Life Sciences Complex, 107 College Place, Syracuse, New York 13244
2
3
E-mail: [email protected]
Bell Museum and Department of Plant Biology, University of Minnesota, 1445 Gortner Avenue, Saint Paul, Minnesota
55108
Received January 5, 2012
Accepted June 10, 2012
The extent of isolation among closely related sympatric plant species engaged in obligate pollination mutualisms depends on
the fitness consequences of interspecies floral visitation. In figs (Ficus), interspecific gene flow may occur when pollinating
wasps (Agaonidae) visit species other than their natal fig species. We studied reproductive isolation in a clade of six sympatric
dioecious fig species in New Guinea. Microsatellite genotyping and Bayesian clustering analysis of the fig community indicated
strong reproductive barriers among sympatric species. A total of 1–2% of fig populations consisted of hybrid individuals. A new
experimental method of manipulating fig wasps investigated the reproductive consequences of conspecific and heterospecific
pollinator visitation for both mutualists. Fig wasps introduced to Ficus hispidioides pollinated and oviposited in receptive figs.
Seed development and seedling growth were largely comparable between conspecific and heterospecific crosses. Heterospecific
pollinator fitness, however, was significantly less than that of conspecific pollinators. Heterospecific pollinators induced gall
formation but offspring did not develop to maturity in the new host. Selection on pollinators maintaining host specificity appears
to be an important mechanism of contemporary reproductive isolation among these taxa that could potentially influence their
diversification.
KEY WORDS:
Ceratosolen, coevolution, hybridization, speciation.
The observation that some closely related plants co-occur as distinct species, even in the presence of hybridization, has long motivated the study of reproductive isolating mechanisms (Stebbins
1950). Reproductive isolation in plants can involve prepollination
mechanisms, such as phenological, mechanical, and ethological
barriers, or postpollination mechanisms, such as pollen incompatibility, genetic incompatibility, or hybrid inferiority (Grant 1971).
In animal-pollinated taxa, pollinators may act as agents of reproductive isolation by means of species-specific floral constancy in
foraging behavior (Oyama et al. 2010). Additionally, trait mismatching in heterospecific visitation can prevent heterospecific
C
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pollen deposition or the collection of pollinator rewards (Kephart
and Theiss 2004; Kay 2006).
When reproduction of pollinators and host is interdependent,
selection for specialization may be intense, especially where similar hosts occur in sympatry. Indeed, the evolution of extreme pollinator specialization is observed in cases where plants provide
“nursery” rewards for pollinators (Ollerton 2006). A recent natural
experiment on the obligate pollination mutualism between Joshua
trees and yucca moths (Smith et al. 2009) revealed that phenotype matching plays a role in reproductive isolation of parapatric
host tree varieties. In spite of hybridization between varieties in a
C 2012 The Society for the Study of Evolution.
2012 The Author(s). Evolution Evolution 66-12: 3710–3721
P O L L I NATO R - M E D I AT E D R E P RO D U C T I V E I S O L AT I O N
contact zone, fitness differences between pollinators ovipositing
in the host variety from their native range compared to the host variety from outside their range selects for specificity and serves as
a potential mechanism of reproductive isolation between Joshua
tree varieties.
The obligate pollination mutualism between figs (Ficus,
Moraceae) and fig wasps (Agaonidae, Hymenoptera) is similarly specialized (Janzen 1979; Weiblen 2002; Cook and Rasplus
2003). Life cycles of pollinating fig wasps and their Ficus host
plants are completely interdependent. Volatile signals attract female fig wasps to a receptive fig of a host (Hossaert-McKey et al.
1994; Chen and Song 2008) where they gain access to the unisexual flowers of the enclosed inflorescence through a narrow,
bract-covered opening, called the ostiole. Females oviposit in pistillate flowers and deposit pollen in the process. In most cases,
pollinators do not have a second chance at reproduction as they
cannot leave the fig upon entering (Moore et al. 2003). In monoecious figs, flowers either set seed, form galls that nourish fig wasp
offspring, or produce pollen that eclosing wasps later transport to
other receptive figs. In dioecious species, reproductive functions
are separated between plants bearing either seed-producing inflorescences (female figs) or inflorescences producing wasps and
pollen (male figs).
Ficus is known not only for its obligate pollination mutualism
but also for extreme species richness (>750 species), and diversity
in sympatry. In centers of endemism, such as New Guinea, recent
radiations of closely related species coexist in sympatry (Berg and
Corner 2005). Although the highly specific host associations of
wasps have been implicated in fig speciation, observations of fig
pollinators visiting multiple host species in sympatry (Molbo et al.
2003; Machado et al. 2005; Marussich and Machado 2007; Su
et al. 2008) and molecular evidence of fig hybridization (Parrish
et al. 2003; Renoult et al. 2009) have called into question the role
of pollinators in historical and contemporary fig reproductive isolation. Recent studies have inferred ancient host switching or host
conservatism from phylogenetic patterns (Lopez-Vaamonde et al.
2002; Weiblen and Bush 2002a; Jousselin et al. 2003; Machado
et al. 2005; Marussich and Machado 2007; Jackson et al. 2008;
Ronsted et al. 2008; Azuma et al. 2010; Moe and Weiblen 2010).
Ecological mechanisms affecting gene flow and reproductive isolation in this system have not been investigated directly. Given
that the nature and extent of isolating mechanisms among sympatric fig species depends on the reproductive consequences of
pollinator host choice, experiments are needed to examine the
fitness of both mutualistic partners. Such experiments can provide direct evidence on the nature of contemporary reproductive
isolation and potential mechanisms for diversification in the past.
This study investigated the extent of hybridization and mechanisms of reproductive isolation among sympatric fig species in
New Guinea. We applied Bayesian clustering methods based on
microsatellite data to identify putative natural hybrids and estimate rates of heterospecific gene flow. We employed a new
method for introducing pollinators to novel hosts that allowed us
to compare fig and pollinator reproduction following conspecific
visitation versus heterospecific visitation and separate host choice
behavior from subsequent pollination and oviposition behaviors.
Methods
STUDY SYSTEM
Ficus bernaysii King, Ficus congesta Roxb., Ficus hahliana Diels,
Ficus hispidioides S. Moore, Ficus morobensis C.C. Berg, and
Ficus pachyrrhachis K. Schum. and Lauterb. are members of
dioecious Ficus subgenus Sycomorus section Sycocarpus. They
comprise the majority of a clade estimated to have originated in
New Guinea at least 15 million years ago and within which precise
phylogenetic relationships are unresolved (Silvieus et al. 2008).
These New Guinea endemic taxa often occur at high density in
patches of secondary regrowth. They have abundant cauliflorous
figs growing along the length of the trunk and are pollinated by
Ceratosolen species (Wiebes 1963). Most fig species in this study
system have uniquely associated pollinator species. For example,
Ceratosolen notus pollinates F. congesta, whereas a distinct but
unnamed Ceratosolen pollinates F. pachyrrhachis (Silvieus et al.
2008).
Landowners at the study site recognized two entities distinguished by the length and density of epidermal hairs on young
shoots and the persistence of stipules that were referred to as
F. bernaysii morphotypes A and B in a previous publication on
pollinator sharing (Moe et al. 2011). Our examination of type
specimens determined that these entities correspond to F. hahliana
Diels and F. bernaysii. The taxon referred to as F. bernaysii in previous work at the study site (Weiblen 2000, 2001, 2004; Novotny
et al. 2002; Weiblen and Bush 2002b; Weiblen et al. 2006, Weiblen
et al. 2010) and subsequently as F. bernaysii “A” (Moe et al. 2011;
Moe and Weiblen 2011) rather corresponds to F. hahliana. Confusion about the identity of F. hahliana dates from at least Wiebes
(1963), where type material for Ceratosolen hooglandii, the pollinator of F. bernaysii, included specimens reared from both F.
bernaysii (Hoogland 4890) and misidentified F. hahliana (NGF
12471). Moe et al. (2011) identified two cryptic clades of C. hooglandii associated with F. hahliana and F. bernaysii, respectively.
No such confusion concerns the other species included in our
study.
SAMPLING
Experimental technique was developed over eight months and
two field seasons in 2007–2008. Experiments were performed
May–August of 2009 at Ohu village in the Madang district of
Madang Province, Papua New Guinea (latitude 5◦ 13 38 S, longitude. 145◦ 40 44 E). Experimental trees were located in a 400 ha
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A . M . M O E A N D G . D. W E I B L E N
patchwork of secondary regrowth and undisturbed forest. Fifty
individuals of each focal species were sampled for microsatellite analyses. Young leaf tissue was collected from each individual and dried over silica gel, and later stored at −80◦ C prior to
DNA extraction. A voucher specimen was also collected from
each individual, alcohol preserved, and later dried for long-term
storage.
MICROSATELLITE AMPLIFICATION AND SCORING
DNA was extracted from each individual using a Qiagen DNeasy
Plant Tissue extraction kit. We amplified and genotyped individuals at 14 loci (Table 1). Among the microsatellite loci used, four
primer pairs had been developed for Ficus montana (FM4–15 and
FM3–64) and Ficus septica (FS4–11 and FS3–31) by Zavodna
et al. (2005), four for Ficus racemosa (Frac86) and Ficus rubiginosa (Frub29, Frub38, and Frub436) by Crozier et al. (2007)
and six for F. hahliana (B30, B47, B83) and F. pachyrrhachis
(P164, P211, P215) by Moe and Weiblen (2011). Amplification
of microsatellite loci was performed in an Eppendorf Mastercycler in a total volume of 10 µl using 0.2 mM fluorescent endlabeled forward primer and unlabeled reverse primer, 0.2 mM
buffer solution, 0.2 mM of each dNTP, 0.8 mM BSA, 0.3 units of
TaKaRa Ex Taq polymerase (TAKARA BIO Inc., Shiga, Japan),
and 20–50 ng template DNA. PCR conditions are indicated in
Table 1. Microsatellite alleles were visualized using an ABI 377
Sequencer along with a ROX 500 (Applied Biosystems) size standard and scored by hand using Genotyper 2.5 software (Applied
Biosystems, Foster City, CA).
MICROSATELLITE ANALYSES
We performed kinship analysis using Kinalyzer (Berger-Wolf
et al. 2007; Ashley et al. 2009) to identify siblings among our
samples. One individual from each sibling group was randomly
chosen and included in tests for linkage disequilibrium and deviations from Hardy–Weinberg equilibrium using GENEPOP (Raymond and Rousset 1995; Rousset 2008). We applied Sequential
Bonferroni corrections for multiple tests (Holm 1979). Data were
analyzed in Microchecker (Van Oosterhout et al. 2004) to test for
the presence of null alleles.
In describing genetic differentiation among focal species, we
used all 14 loci to calculate Fst, a measure based on allele identity
(Weir and Cockerham 1984), and Rho (Valdes et al. 1993), a
measure based on allele size and an estimate of Rst (Slatkin 1995).
Bayesian clustering analyses were implemented in
STRUCTURE (Pritchard et al. 2000). This method assigns individuals to one or more ancestral populations based on their
allelic genotypes. Hybrids can be identified by their partial assignment to more than one ancestral population. To test whether
the six named plant species correspond to genetically distinct
clusters, we ran five independent iterations in STRUCTURE with
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ancestral population number (K) set for K = 4, K = 5, K =
6, and K = 7 and without using a priori species identifications.
Each Markov chain included a 100,000-generation burn-in and
ran for 106 additional generations. We used an admixture model
and allowed for correlated allele frequencies among clusters. Using the method described in Evanno et al. (2005), we identified
the most appropriate K value as six, which resulted in consistent cluster assignments over five iterations. These results along
with the observation of six morphological species suggest that the
mostly likely number of ancestral populations is six (Pritchard
et al. 2000). Assuming K = 6, we used prior information on
species assignment, based on morphological identification, and
ran the analysis again at three values of interspecies migration
rate, v = 0.01, 0.05, and 0.10. STRUCTURE estimated the posterior probabilities of each individual being (1) a nonhybrid, but
with an incorrect a priori species assignment, (2) an F1 hybrid,
or (3) an F2 hybrid. Eight individuals with a higher probability of being either misidentified or having hybrid ancestry than
having nonhybrid ancestry were singled out for reexamination.
Leaf vouchers and DNA reextraction and genotyping confirmed
identity for these individuals. Analyses were run again at K = 6
with corrected genotypes and one misidentified individual reassigned to the correct species. The STRUCTURE results reported
are from this second round of analyses.
An additional analysis identifying hybrid individuals was
implemented in BayesAss (Wilson and Rannala 2003). BayesAss
uses Bayesian and Monte Carlo–Markov chain methods to estimate recent migration rates among populations and estimate
each individual’s ancestry. Individuals are classified as an immigrant from a specific population, a nonimmigrant, or the offspring
of an immigrant and a nonimmigrant (hybrid). The program assumes unlinked loci and a relatively low rate of migration among
populations (less than 1/3), but allows for deviations from H-W
equilibrium. A 10,000-iteration burn-in, followed by 3,000,000 iterations and default delta values were used. Individuals assigned
as hybrids were noted. The mean and 95% confidence interval
(95% CI) for estimated pairwise migration rates between focal
species were recorded.
INTERSPECIFIC POLLINATION EXPERIMENTS
Background
Ficus hispidioides served as the pollen recipient for all experiments. A nonreciprocal experimental design, permitting only fitness comparisons associated with conspecific and heterospecific
pollination in one species, was necessitated by field logistics and
natural history. The relatively accessible, abundant, and large figs
(3–4 cm in diameter at receptivity) of F. hispidioides supported
manipulations that were not feasible for other species. Five of
six study species were included as pollen donors due to the limited availability of figs from F. bernaysii during the study period.
P O L L I NATO R - M E D I AT E D R E P RO D U C T I V E I S O L AT I O N
Microsatellite loci analyzed. The total number and length of alleles observed across all species, PCR conditions: annealing
temperature (T a ) and number of cycles, and the original primer note references are shown.
Table 1.
1
Primer
No. of alleles
Length (bp)
Ta
No. of cycles
Reference
FM3–64
FM4–15
FS3–31
FS4–11
Frac86
Frub29
Frub38
Frub436
B30
B47
B83
P164
P211
P215
9
18
8
11
10
6
25
15
45
16
16
18
15
18
267–291
232–298
219–243
279–357
141–183
179–199
172–132
97–131
215–347
171–219
165–195
227–288
99–127
212–244
54
53
54
54
50
54
50
53
60
53
53
60
53
53
101 +20
30
101 +20
101 +20
151 +20
101 +20
151 +20
30
101 +20
30
30
101 +20
30
30
Zavodna et al. 2005
Zavodna et al. 2005
Zavodna et al. 2005
Zavodna et al. 2005
Crozier et al. 2007
Crozier et al. 2007
Crozier et al. 2007
Crozier et al. 2007
Moe and Weiblen 2010
Moe and Weiblen 2010
Moe and Weiblen 2010
Moe and Weiblen 2010
Moe and Weiblen 2010
Moe and Weiblen 2010
Indicates number of touchdown cycles starting 10◦ C above the annealing temperature.
We collected phenological and life-history data on F. hispidioides
to appropriately design pollination experiments. We estimated
numbers of foundresses typically encountered in receptive figs
of F. hispidioides, so a comparable number of wasps could be
introduced to experimental figs. A total of 250 receptive figs
yielded an average of five foundresses per fig (SD = 4.06). We
chose to introduce six pollinators per experimental fig to account
for wasp mortality associated with the method of introduction.
We also determined the minimum diameter at which figs are accessible to pollinators to design an effective pollinator exclusion
treatment. We collected 120 figs, measured their diameter, and
classified them as prereceptive (no pollinators inside), receptive
(live pollinators inside), or postreceptive (dead pollinators and/or
developing seed or galls inside). The average diameter of receptive figs was 33. 8 mm (range 25–39.5). Only figs ≤24 mm in
diameter were treated.
Prereceptivity treatment
Seven functionally female and seven functionally male F. hispidioides trees were chosen as experimental trees on the basis of
having large clusters of accessible and unreceptive figs. Figs with
diameters ≤24 mm were tagged loosely around the peduncle. A
ring of Tanglefoot pest barrier was applied around the ostiole
of each fig and organza fabric was adhered to the pest barrier
(Fig. 1A, B). This treatment allowed fluid to escape from the fig
interior and for expansion of the fig during development while
excluding pollinators from entering the ostiole (Fig. 1C). Fig diameters were measured and recorded every two days and the seal
around the ostiole checked for integrity. If a seal was discovered
broken and an ostiole exposed for any period of time, the fig
was considered potentially contaminated and removed from the
experiment. Figs were considered receptive upon reaching a diameter ≥30 mm and when numerous fig wasps had adhered to
the Tanglefoot.
Receptivity treatment
When figs reached receptivity, a cylindrical section of the fig
wall running perpendicular to the shoot apex was cored using
a 3 mm diameter stainless steel borer. A 3 cm section of glass
Pasteur pipet plugged with cotton fiber was inserted into the hole
(Fig. 1D). After two days, pollinators were manually introduced
to treated figs that had not aborted. Figs from all five pollen donor
species were collected and brought to experimental F. hispidioides
trees (Fig. 1E). We first attempted to introduce winged wasps collected from ripe, functionally male figs of the five focal species
that invariably failed to enter experimental figs when deposited
in unplugged Pasteur pipets using a fine-tipped watercolor paintbrush. In 894 trials, no wasp demonstrated taxis along the length
of the pipet toward the fig interior. However, when we removed
foundresses engaged in active pollination from untreated receptive figs, and introduced them to experimental figs through Pasteur
pipets, these wingless wasps, regardless of species, readily entered experimental figs (Fig. 1F, G). In the conspecific pollination
treatment, we introduced six Ceratosolen dentifer wasps collected
from F. hispidioides figs. In the four heterospecific treatments, we
introduced six pollinators from one of four fig species to figs of F.
hispidioides. After wasps had actively entered a fig, the pipet was
replugged with cotton. Local availability of receptive, untreated
figs determined the number of replicates performed. Control figs
received no pollinator introduction. Figs were checked every two
days until all figs aborted or reached maturity (approximately six
weeks per tree).
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A . M . M O E A N D G . D. W E I B L E N
Experimental method. (A–D) Prereceptivity treatment excluded pollinators and provided for controlled introduction. (E–G)
Receptivity treatment. Pollinating wasps collected from receptive figs were introduced to experimental figs through Pasteur pipets.
Figure 1.
Seed and gall formation
Figs were collected, split open, and checked for seed or gall
development either upon abortion or maturity. A 3 mm wide
longitudinal section of each fig was cut and pistillate flowers
were counted under a dissection microscope and categorized as
either undeveloped or setting seed in the case of female trees or
forming galls in the case of male trees.
Seed viability
To determine viability and survivorship of interspecific crosses in
comparison with nonhybrid F. hispidioides, we measured germination, growth, and survival rates for each pollen donor treatment.
As the focal species is a pioneer of primary succession in forest
gaps, growth rate could be an important aspect of fitness. Seed
from experimental figs was germinated in a light chamber with
40–100 seeds from each fig in separate petri dishes. The proportion of seeds germinating per fig was recorded. Germinated
seeds were randomly assigned to planters in 12 × 6 arrays in a
chamber at 26.6◦ C, grown with 12 h of light per day and fertilized biweekly. Growth was monitored by length of the longest
leaf, measured once a week, and seedling height, measured once
a month for 139 days.
Seedling genotyping
We extracted DNA from one seedling grown from each experimental fig that produced viable seed. These DNAs were screened
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EVOLUTION DECEMBER 2012
for microsatellites and subjected to Bayesian clustering analysis
to confirm that seedlings were in fact hybrids resulting from the
experimental treatments.
Analyses
Data from functionally male and female trees were analyzed separately because gall development in male figs contributes to wasp
fitness whereas seed development in female figs is directly associated with plant fitness. Control treatments assessed the effectiveness of pollinator exclusion. The development of a few control
figs indicated rare events where naturally occurring pollinators
circumvented our exclusion treatment. Although genotyping of
seedlings distinguished experimental introductions from contamination in the case of seed figs, it was not possible to genotype
wasps in galls. Therefore, to test whether gall formation in treated
figs was significantly more frequent than in control gall figs, we
performed pairwise Fisher’s exact tests on counts of developed
and undeveloped figs. Pairwise Fisher’s exact tests also compared
the proportion of figs that initiated gall and seed development in
each heterospecific treatment against development in the conspecific treatment where F. hispidioides served as the pollen donor.
We performed ANOVA to determine the effects of pollen
donor species and maternal tree on seed germination rates and
survivorship after 139 days with the fig as the unit of replication.
We then performed 2 × 2 contingency (Fisher’s exact) tests to
compare each heterospecific treatment against the conspecific
P O L L I NATO R - M E D I AT E D R E P RO D U C T I V E I S O L AT I O N
treatment. We also performed ANOVA to determine the effect
of pollen donor and maternal tree on plant height and maximum
leaf length as measures of growth with the seedling as the unit of
replication.
Results
Posterior probabilities of assignment of seven individuals identified as putative hybrids from Bayesian clustering analysis
Table 2.
(K = 6) using a priori species identity information with migration
rate priors of 0.01, 0.05, and 0.10 are shown. Bold type values
indicate the ancestry assignment with the highest posterior probability. Bold type individuals are most likely of hybrid ancestry at
all tested migration rates.
MICROSATELLITE ANALYSES
Kinship analysis revealed a large number of siblings in our samples that reduced the effective sample size of each species from
50 to 17–23 individuals (Table S1) for the calculations of genetic
differentiation, tests of Hardy–Weinberg equilibrium, and linkage
disequilibrium. After sequential Bonferroni multiple test corrections, linkage disequilibrium was not detected among the loci we
sampled overall. However, in four of six species (F. hahliana,
F. congesta, F. hispidioides, F. pachyrrhachis), a different locus was found to be significantly heterozygote deficient in each
species after multiple test corrections (Tables S1 and S2), suggesting the presence of null alleles (Table S1). Mean Fst values for
each species ranged 0.1875–0.2346 and mean Rho values for each
species ranged 0.2349–0.4227 (Table S1). Genotypes are archived
in the Dryad Digital Repository (doi: 10.5061/dryad.c3h3v).
Hybrid species
assignment
Migration Non
F1
F2
rate
hybrid hybrid hybrid
F. hahliana×
bernaysii F2
0.01
0.931 0.006 0.063
0.05
0.10
0.01
0.668 0.027 0.305
0.410 0.051 0.536
0.507 0.004 0.489
0.05
0.10
0.01
0.145 0.007 0.848
0.066 0.007 0.927
0.027 0.899 0.074
0.05
0.10
0.01
0.003 0.926 0.071
0.001 0.930 0.069
0.920 0.008 0.071
0.05
0.10
0.01
0.658 0.034 0.307
0.470 0.051 0.521
0.933 0.011 0.054
0.05
0.10
0.01
0.676 0.057 0.267
0.433 0.105 0.462
0.784 0.000 0.216
0.05
0.10
0.01
0.386 0.001 0.612
0.222 0.002 0.776
0.452 0.003 0.545
0.05
0.10
0.128 0.005 0.867
0.062 0.005 0.933
F. hahliana×
pachyrrhachis F2
F. morobensis×
pachyrrhachis F11
F. morobensis×
pachyrrhachis F21
BAYESIAN ANALYSES
Seven out of 300 individuals were identified with high posterior probability of hybrid ancestry using the highest migration
rate prior (m = 0.10; Table 2, Fig. 2). Two of these also exhibited hybrid ancestry at the lowest migration rate (m = 0.01).
These same individuals and a third were identified as putative
hybrids through BayesAss analysis (Table 2). Pairwise migration rates among species estimated in BayesAss were very low
(0.11–0.64%). The highest estimated migration rates were from
F. pachyrrhachis to F. morobensis at 0.41% (95% CI ≤ 0. 01–
2.26%), and vice versa at 0.64% (95% CI ≤ 0. 01–2.50%). All
other pairwise estimates were < 0.3% with upper bounds of 95%
CIs < 1.6%.
F. morobensis× hahliana
F2
F. morobensis×
pachyrrhachis F2
F. morobensis×
pachyrrhachis F21
1
Hybrids identified by BayesAss.
POLLINATION EXPERIMENTS
Data from five male and six female trees were collected whereas
data from three additional experimental trees were lost to foraging fruit bats. Among 563 gall figs and 345 seed figs of
F. hispidioides receiving the pre-receptivity treatment, we observed abortion rates of 17.9% and 31.3%, respectively. A total of
410 gall figs and 216 seed figs that survived the coring treatments
either received experimental pollinator introductions or served as
controls. Heterospecific pollinators of functionally male figs induced gall formation in a significantly lower proportion of figs
than conspecific pollinators (Fisher’s exact test P < 0.01), but
only figs that received the conspecific treatment produced mature
galls and adult wasp offspring (Table 3). One of 62 control figs
developed galls, indicating effective but not absolute exclusion
of pollinators. Only figs treated with pollinators from F. hispidioides, F. congesta, and F. pachyrrhachis initiated gall formation
in a significantly greater number of figs than controls (Fisher’s
exact P < 0.01).
In functionally female figs, seed set was observed in all treatments except F. hahliana (Table 4), including one of 33 control
figs (Table 4). Microsatellite genotyping of seedlings allowed the
identification and exclusion of nonexperimental pollinations from
further analyses (see “Seedling genotyping” in Methods).
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Figure 2.
Barplots of ancestral population assignments for 300 individuals from Bayesian clustering analysis assuming six, color-coded
ancestral populations (K = 6). Priors were used on population information based on morphological identification and an assumed
migration rate of (A) 0.10, (B) 0.05, and (C) 0.01. Asterisks indicate individuals identified with the highest probability as either first or
second generation hybrids.
Experimental gall fig treatments. Pollinator species,
pollen donor species, and numbers of Ficus hispidioides figs
treated, figs that initiated gall development, and mean (± SE)%
Table 3.
flowers galled per fig.
1
Introduced
pollinator
Pollen
donor
C. dentifer
C. notus
C. hooglandii
C. sp. ex Ficus
morobensis
C. sp. ex Ficus
pachyrrhachis
Control
F. hispidioides
F. congesta
F. hahliana
F. morobensis
n
n
n
treated initiated matured
90
81
36
17
511
121,2
22
22
251
0
0
0
F. pachyrrhachis 125
411,2
0
1
0
None
62
Denote a significant difference from control figs (pairwise contingency
test, Fisher’s exact P < 0.01).
2
Denotes a significant difference from F. hispidioides (pairwise contingency
test, Fisher’s exact P < 0.01).
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GERMINATION AND GROWTH
More than 50% of seed resulting from all successful crosses
germinated (Table 5) and ANOVA showed no significant effect
of pollen donor or maternal tree (P = 0.138 and P = 0.189,
respectively). However, pairwise 2 × 2 contingency tests showed
that F. congesta × hispidioides seed germinated at a significantly
lower rate than F. hispidioides (Fisher’s exact P < 0.01). Nonhybrid F. hispidioides had the highest survivorship among germinated seeds (Table 5) and ANOVA did not detect a significant effect of pollen donor treatment (P = 0.284) or maternal
tree (P = 0.307). However, 2 × 2 contingency tests showed that
F. morobensis × hispidioides and F. pachyrrhachis × hispidioides
seed had significantly lower survivorship than nonhybrid F. hispidioides (Fisher’s exact P < 0.01). Survivorship of seedlings was
generally low across all seedling types due to fungal infections,
followed by overly dry conditions in the growth chamber.
Seedlings of all types grew at comparable rates (Fig. 3,
growth data archived in the Dryad Digital Depository
P O L L I NATO R - M E D I AT E D R E P RO D U C T I V E I S O L AT I O N
Experimental seed fig treatments. Pollinator species,
pollen donor species, and numbers of Ficus hispidioides figs
Table 4.
treated, figs that initiated seed development, figs that matured
seed, and mean (± SE) seed set.
1
Introduced
pollinator
Pollen
donor
n
n
n
treated initiated matured
C. dentifer
C. notus
C. hooglandii
C. sp. ex Ficus
morobensis
C. sp. ex Ficus
pachyrrhachis
Control
F. hispidioides
F. congesta
F. hahliana
F. morobensis
54
45
23
16
11
4
0
1
F. pachyrrhachis 52
7
6
None
1
1
33
1
10
4
01
1
Significant difference from F. hispidioides (pairwise contingency test,
Fisher’s exact P < 0.05).
Table 5. Germination and survival of hybrid and nonhybrid
seeds. Totals are pooled over all experimental figs/replicates that
developed seed. Ficus hispidioides was the maternal parent for all
crosses. As such, all rows correspond to hybrid seed except that of
F. hispidioides.
Pollen
donor
Total n
Total n Total n
germinated Total n
seeds
seeds
seeds
seedlings
collected germinated planted
survived
F. congesta
F. hahliana
F. hispidioides
F. morobensis
F. pachyrrhachis
177
0
371
61
278
115
0
327
52
245
82
0
80
45
136
32
0
36
2
31
doi: 10.5061/dryad.c3h3v). Pollen donor species had no significant effect on the height (P = 0.958) or length of the longest leaf
(P = 0.569) after 139 days. The maternal tree also had no effect
on the height (P = 0.975) or length of the longest leaf (P = 0.918)
after 139 days.
Discussion
As in other studies on hybridization in dioecious figs (Ramirez
1994; Parrish et al. 2003), we found little evidence of gene flow
among sympatric fig species. Bayesian clustering analysis of microsatellite genotypes demonstrated little admixture among sympatric species and that naturally occurring hybrids are rare in
the study area. Approximately 1–2% of the sampled genotypes
could be regarded as putative hybrids assuming a liberal migration rate of 10%. With a more conservative migration rate of 1%,
which aligns more closely with Bayesian estimates, less than 1%
of individuals are considered hybrids. Given that the majority of
individuals were assigned to only one ancestral population with
high probability and estimated migration rates were low, the pattern suggests limited backcrossing and introgression that might
be explained by the results of our pollination experiment.
Our experimental method of bypassing host recognition and
passage through the ostiole resulted in hybridization in three
of four heterospecific crosses. Comparable seed development in
F. hispidioides from conspecific and heterospecific crosses involving several close relatives argues against postpollination barriers
to heterospecific fertilization or embryogenesis. Growth rates of
hybrid and nonhybrid seed appeared to be comparable although
we admit that statistical power to separate the effects of individual pollen donors and maternal trees was weak. Germination of
F. congesta × hispidioides and survivorship of F. morobensis ×
hispidioides and F. pachyrrhachis × F. hispidioides was in fact
lower than nonhybrids in the greenhouse. Selection against hybrids might indeed act as a mechanism of reproductive isolation
in nature but our experiments on functionally male figs suggest a
more immediate reproductive isolating mechanism.
Cross-pollinating wasps failed to achieve fitness in a nonnatal
host species. Wasps induced gall development in a novel host but
their offspring did not reach maturity. That gall formation was initiated in some proportion of all heterospecific treatments suggests
that cross-pollinating wasps were at least capable of oviposition
in the novel host. Reduced rates of gall formation in F. hispidioides by species other than C. dentifer could be attributed to
morphological mismatches between ovipositors and style lengths
(Weiblen 2004), whereas physiological mechanisms might explain why galls initiated in heterospecific treatments failed to
reach maturity. Such mechanisms could include the failure of larvae in a novel host to induce the proliferation of the nucellus or
failure to feed upon it, as required by the diet. Host sanctions have
also been detected in other fig lineages (Jander and Herre 2010)
and there is recent speculation that figs may selectively abort figs
that have been self-pollinated (Gates and Nason 2012).
In any event, the failure of pollinators to colonize a novel
host suggests that species-specific recognition of suitable hosts
may be strongly selected. Even newly emerged female resident
pollinators of F. hispidioides refused to enter experimental figs
through a Pasteur pipet, whereas females removed from receptive
figs while in the act of pollination readily entered experimental
figs. This suggests two aspects of wasp behavior. First, attraction
to flowers and subsequent oviposition and pollination behaviors
are conditioned upon having identified a suitable host and passed
through an ostiole. Second, after a wasp has passed through, it
does not discriminate among host species. The chemosensory
attraction and discriminatory behavior of wasps, therefore, only
EVOLUTION DECEMBER 2012
3717
A . M . M O E A N D G . D. W E I B L E N
A
Figure 3.
B
Seedling growth. (A) Average seedling height after 139 days. (B) Average length of the longest leaf after 139 days. Error bars
are 1 SD.
occurs prior to oviposition and indeed prior to entering a fig. As
antennal segments bearing sensillae are torn from wasps’ heads
in the process of entering the fig, we might not expect to find
discriminatory behavior beyond this life-history stage. This idea
is also supported by the low number of heterospecific wasps found
in naturally pollinated syconia of these sympatric dioecious figs
(Weiblen et al. 2001; Moe et al. 2011).
Volatile chemical cues are known to play a role in pollinator
host choice. Studies have shown that fig volatile cues at receptivity
are species-specific (Ware et al. 1993; Grison-Pige et al. 2002b;
Proffit et al. 2009) and wasps are attracted to the volatile bouquet
of particular hosts (Bronstein 1987; Ware and Compton 1994;
Grison-Pige et al. 2002a; Chen et al. 2009). Recently, Lu et al.
(2009) found molecular evidence of selection on a gene influencing olfactory reception in Ceratosolen solmsi, the pollinator of
dioecious, Ficus hispida. Species-specific chemical signals might
serve to reinforce reproductive isolation among sympatric species
whose hybrids are less fit. Alternatively, pollinator behavior in
response to variation in fig chemistry may be one of few isolating mechanisms among genetically compatible sympatric species,
which implies that pollinator specificity could potentially play a
role in diversification, as modeled by Kiester et al. (1984). If the
reproductive consequence of selecting an unsuitable host is as
dire as it appears for Ceratosolen in F. hispidioides, selection on
3718
EVOLUTION DECEMBER 2012
wasp behavior alone may be sufficient to reproductively isolate
host figs. This may be counted among few specific examples of
the often-speculated potential for pollinator adaptation to affect
plant diversification in general (Grant 1971; Moe et al., in press).
A previous study on pollinator sharing rates (Moe et al.
2011) did not detect pollinator sharing between F. morobensis and
F. pachyrrhachis, but the only two hybrids identified in the field
shared these parental species. Artificial hybrids of F. hispidioides
and F. congesta grew and survived at comparable rates to nonhybrids and yet pollinator sharing between these parental species is
rare (Moe et al. 2011). This suggests that behavioral barriers could
be stronger than postreproductive barriers. However, lifetime fitness estimates of hybrids are needed to evaluate the potential for
reinforcement to shape pollinator behavior.
Critical insights on host specificity in the fig pollination mutualism (Machado et al. 2005) has motivated the reinterpretation of
examination of phylogenetic and cophylogenetic patterns (Haine
et al. 2006; Marussich and Machado 2007; Jackson et al. 2008;
Jousselin et al. 2008; Su et al. 2008; Renoult et al. 2009; Azuma
et al. 2010; Moe et al. 2011). These patterns invite simple explanation, but it is possible that multiple processes yield similar patterns
and a particular process may result in diverse patterns (Irwin 2002;
Revell et al. 2008; Cavender-Bares et al. 2009; Crisp and Cook
2009). Working hypotheses on host specificity and hybridization
P O L L I NATO R - M E D I AT E D R E P RO D U C T I V E I S O L AT I O N
developed from phylogeny require independent testing through
other lines of inquiry, experimentation, and analysis. For example, evidence of gene flow among Ficus species has been interpreted as evidence of host switching and low pollinator specificity
(Renoult et al. 2009). Our cross-pollination experiment, although
unidirectional by necessity, offers an alternative explanation. We
found that gene flow could occur among sympatric species in at
least one direction without requiring the successful colonization
of a new host. This finding highlights the importance of examining ecological context and the reproductive consequences for
mutualistic partners in concert with patterns of cophylogeny and
contemporary host-pollinator associations.
Our results invite the further speculation that patterns of host
specificity and codiversification in a system often touted as a textbook example of coevolution need not be maintained by reciprocal
selection. Extreme species specificity (Weiblen et al. 2001; Moe
et al. 2011) and patterns of codivergence in New Guinea Sycomorus figs (Weiblen and Bush 2002b; Silvieus et al. 2008) can
be explained by selection on pollinating wasps imposed by host
figs, without comparable selection on host figs imposed by pollinating wasps, similar to adaptive deme formation (Mopper 1996).
Highly specific wasp behavior could be an effective reproductive
isolating mechanism without significantly reduced fig hybrid fitness. Additional fitness differences may exist which this study
was not able to detect, such as infertility of hybrids, or reduced
attractiveness of hybrids to pollinators. However, in theory, these
differences need not exist to explain the high specificity of pollinators, the rarity of hybrids, or congruent phylogenetic patterns.
The development of evolutionary models of unidirectional selection imposed by one organism on another, where lifecycles of the
interacting organisms are interdependent, could evaluate whether
reciprocal selection is actually necessary to explain patterns long
regarded as products of coevolution.
ACKNOWLEDGMENTS
This work was supported by The American Society of Plant Taxonomists,
The American Philosophical Society’s Lewis and Clark Exploration Fund,
The Garden Club of America’s Award in Tropical Biology, a Carolyn
Crosby Fellowship, a Bernard and Jean Phinney Graduate Fellowship, a
Myrna G. Smith International Fellowship, and The Bell Museum of Natural History’s Dayton Research Fellowship. Thanks to the New Guinea
Binatang Research Center and the Ohu Bush Laboratory for logistical
support, to B. Isua, M. Brus, D. Sau, F. Pius, and R. Fafen for assistance
in field experimentation, to E. Treiber for molecular work, to C. Berg for
consultation on Ficus identification, to R. Shaw, G. Heimpel, P. Tiffin,
D. Althoff, K. Segraves, J. Nason, and two anonymous reviewers for
comments and suggestions.
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Associate Editor: C. A. Buerkle
Supporting Information
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Table S1. Species differentiation.
Table S2. Number of alleles/expected heterozygosity/observed heterozygosity for each locus and species (n = 50).
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