Phenological Adaptations in Ficus tikoua Exhibit Convergence with Unrelated Extra-Tropical Fig Trees

Abstract

Flowering phenology is central to the ecology and evolution of most flowering plants. In highly-specific nursery pollination systems, such as that involving fig trees (Ficus species) and fig wasps (Agaonidae), any mismatch in timing has serious consequences because the plants must balance seed production with maintenance of their pollinator populations. Most fig trees are found in tropical or subtropical habitats, but the dioecious Chinese Ficus tikoua has a more northerly distribution. We monitored how its fruiting phenology has adapted in response to a highly seasonal environment. Male trees (where fig wasps reproduce) had one to three crops annually, whereas many seed-producing female trees produced only one fig crop. The timing of release of Ceratosolen fig wasps from male figs in late May and June was synchronized with the presence of receptive figs on female trees, at a time when there were few receptive figs on male trees, thereby ensuring seed set while allowing remnant pollinator populations to persist. F. tikoua phenology has converged with those of other (unrelated) northern Ficus species, but there are differences. Unlike F. carica in Europe, all F. tikoua male figs contain male flowers, and unlike F. pumila in China, but like F. carica, it is the second annual generation of adult wasps that pollinate female figs. The phenologies of all three temperate fig trees generate annual bottlenecks in the size of pollinator populations and for female F. tikoua also a shortage of fig wasps that results in many figs failing to be pollinated.

Full text

RESEARCH ARTICLE
Phenological Adaptations in Ficus tikoua
Exhibit Convergence with Unrelated
Extra-Tropical Fig Trees
Ting-Ting Zhao
1
, Stephen G. Compton
2,3
, Yong-Jiang Yang
1
, Rong Wang
2
,
Yan Chen
1
*
1. Ecological Security and Protection Key laboratory of Sichuan Province, College of Life Science and
Biotechnology, Mianyang Normal University, Mianyang, Sichuan 621000, China, 2. School of Biology,
University of Leeds, Leeds LS2 9JT, United Kingdom, 3. Department of Zoology and Entomology, Rhodes
University, Grahamstown 6140, South Africa
*goose_01@163.com
Abstract
Flowering phenology is central to the ecology and evolution of most flowering
plants. In highly-specific nursery pollination systems, such as that involving fig trees
(Ficus species) and fig wasps (Agaonidae), any mismatch in timing has serious
consequences because the plants must balance seed production with maintenance
of their pollinator populations. Most fig trees are found in tropical or subtropical
habitats, but the dioecious Chinese Ficus tikoua has a more northerly distribution.
We monitored how its fruiting phenology has adapted in response to a highly
seasonal environment. Male trees (where fig wasps reproduce) had one to three
crops annually, whereas many seed-producing female trees produced only one fig
crop. The timing of release of Ceratosolen fig wasps from male figs in late May and
June was synchronized with the presence of receptive figs on female trees, at a
time when there were few receptive figs on male trees, thereby ensuring seed set
while allowing remnant pollinator populations to persist. F. tikoua phenology has
converged with those of other (unrelated) northern Ficus species, but there are
differences. Unlike F. carica in Europe, all F. tikoua male figs contain male flowers,
and unlike F. pumila in China, but like F. carica, it is the second annual generation of
adult wasps that pollinate female figs. The phenologies of all three temperate fig
trees generate annual bottlenecks in the size of pollinator populations and for
female F. tikoua also a shortage of fig wasps that results in many figs failing to be
pollinated.
OPEN ACCESS
Citation: Zhao T-T, Compton SG, Yang Y-J, Wang
R, Chen Y (2014) Phenological Adaptations in
Ficus tikoua Exhibit Convergence with Unrelated
Extra-Tropical Fig Trees. PLoS ONE 9(12):
e114344. doi:10.1371/journal.pone.0114344
Editor: Genlou Sun, Saint Mary’s University,
United States of America
Received: July 24, 2014
Accepted: November 8, 2014
Published: December 4, 2014
Copyright: ß2014 Zhao et al. This is an open-
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and repro-
duction in any medium, provided the original author
and source are credited.
Data Availability: The authors confirm that all data
underlying the findings are fully available without
restriction. All relevant data are within the paper
and its Supporting Information files.
Funding: This study was supported by the Natural
Science Foundation of China (31270387) to YC.
The funder had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
PLOS ONE | DOI:10.1371/journal.pone.0114344 December 4, 2014 1/17

Introduction
The times of year when plants flower and set seed are not random, even among
plant species growing in relatively aseasonal tropical environments [1]. Flowering
phenology is subject to selection from a combination of abiotic, biotic and
intrinsic factors linked to life history, and also to the plant’s phylogeny [2,3].
Abiotic factors include constraints imposed by physiological responses to
temperatures, day lengths and other climatic variables, while biological factors
include the availability of pollinators and seed dispersal agents and competition
with other plants flowering at the same time [4,5]. In temperate latitudes, strong
climatic seasonality provides particular constraints, with threshold temperatures
limiting both insect pollinator activity and the length of the period when floral
and seed development can continue. This has led to widespread convergence in
flowering times, most noticeably with a spring-time concentration of flowering
[6,7], despite potential competition for pollinators among animal-pollinated
plants and increased likelihood of receipt of heterospecific pollen from other
species flowering at the same time. Variation in flowering times also has
evolutionary implications, potentially contributing to reproductive isolation and
speciation [8,9,10].
The time of year when flowers are available to be pollinated is especially
important for plants which depend on one or a small number of insect species for
pollination [11]. In the case of nursery pollination systems, where the reward
provided by the plant is a place for the insects to breed, any mismatch in timing
has serious consequences for the population dynamics of both partners in the
mutualism [12]. The more than 800 species of fig trees (Ficus, Moraceae) and
their pollinating fig wasps (Agaonidae) are partners in a largely species-specific
nursery pollination system. Fig trees are often keystone species providing food for
a diverse range of fruit-eating birds, mammals and other vertebrates [13]. The
significance of figs for vertebrates results from their structure, which makes them
easy to eat, their abundance in a variety of habitats and what is often an all-year
round fruiting phenology that makes figs available at times of the year when the
fruits of other plants are absent. The all-year fruiting phenology of many Ficus
species may be linked to their unique pollination system, because it helps
maintain populations of their pollinator fig wasps, with which they have an
obligate association [14]. Highly reciprocal adaptive traits and co-evolutionary
dynamics are ubiquitous between fig hosts and their pollinators, and continue to
stimulate ecological and evolutionary questions [15].
Adult female pollinating wasps do not feed and only survive for one or two days
after emerging from their natal figs [16]. As a consequence, the synchronization of
pollinator release with the production of receptive figs is critical for both the
maintenance of pollinator populations and pollination of the figs [17]. Fig trees
with a monoecious breeding system produce figs that support the development of
both seeds and fig wasp progeny. Individual trees typically produce synchronized
fig crops, each of which has a brief period when they are attractive to wasps and
another when the next generation of fig wasps emerge and disperse. Tree
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populations contain trees that flower at different times, which ensures that the fig
wasps that emerge from each tree have a chance to find suitable figs on other trees
[17]. Dioecious fig tree species have female individuals with mature figs which
only contain seeds and male individuals with figs that produce pollen and support
the development of pollinating fig wasp offspring. The flowering phenologies of
dioecious fig trees are more diverse than those of monoecious species. Figs may or
may not be produced in synchronized crops on individual trees, and the timing of
flowering often differs between the sexes [18,19].
Latitudinal trends in the flowering times of plants are well documented, with
selection for example tending to favour earlier flowering among plants growing at
higher latitudes [20,21]. The vast majority of Ficus species have tropical or sub-
tropical distributions [14], a latitudinal range that appears to be related to global
temperature patterns, because during a warmer period of Earth history they were
also present in northern Europe [22]. A small number of fig tree species currently
extend to higher latitudes [23], where they have evolved atypical fruiting
phenologies in response to the strong seasonality of their environments, in
particular the long winter periods that are too cold for fig wasps to be dispersing
between trees.
The fruiting phenologies of three species of dioecious fig trees with largely
extra-tropical distributions have been described, F. erecta and F. pumila in China
including Taiwan, and F. carica in Europe [24,25,26]. All three species belong to
Ficus subgenus Ficus [27] and are passively pollinated by species of Blastophaga
and Wiebesia belonging to Agaonidae, Subfamily Agaoninae [25,28–31]. The
three species produce relatively synchronized crops, population wide, at set
periods each year. The resulting precise matches between the phenologies of male
and female trees facilitate the pollination of female figs while at the same time
maintaining pollinator populations [25,30]. In Europe, the pollinator of F. carica
has larvae that overwinter in one crop of male figs. They become adults in the
spring, a time when there are many receptive male figs available to lay their eggs
in, often on the same plants. This allows pollinator numbers to increase, but when
the next generation of pollinators emerges in the summer there are very few
receptive male figs available, but many receptive female figs. Seed set is ensured,
but at the expense of an annual bottleneck in pollinator populations [24,32]. Not
all individuals exhibit precisely the same phenology, and an extra generation of
figs is developed in some plants [33]. In most parts of its range, F. erecta has a
flowering phenology that is largely the same as that of F. carica [25]. This changes
when the plant is grown under warmer conditions, outside its native range, where
fig production on male trees becomes asynchronous [34]. The phenology of a
second Asian species, F. pumila, is also similar to those of F. carica and F. erecta,
but with one major difference. Like the other species, male trees produce two
major crops each year, and female trees produce a single major crop. However,
whereas F. carica and F. erecta build up pollinator numbers in the spring by fitting
in a post-winter generation in male figs, this is not the case in F. pumila, where it
is the generation of fig wasps that has overwintered as larvae that emerges at the
same time as female figs are receptive, and so contributes to seed set. Based on the
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phylogenetic relationships of the plants (and also their pollinators), the three
plants are not closely related species [35,36] and their atypical phenologies
represent convergent responses to selection pressures generated by the seasonality
of their environments.
F. tikoua has been assigned to Ficus Subgenus Ficus [27], but molecular
evidence [36] and also its morphology (F. Kjellberg, Pers. Comm.) show that it
should be placed in Ficus Subgenus Sycomorus, which contains both monoecious
and dioecious species. It is pollinated by an undescribed species of Ceratosolen
(Agaonidae, Subfamily Kradibiinae). Both the tree and its pollinator are distantly
related to the other dioecious fig trees with northern distributions. Furthermore,
all described Ceratosolen species are active pollinators [36], in contrast to the
passive pollinators of the other three species. Active pollinators collect pollen into
thoracic pollen pockets before leaving their natal figs. After entry into a receptive
fig the pollen is actively removed and deposited on the stigmas. This behavior is
carried out even if the fig wasp has entered a female fig and is unable to oviposit
there [37]. In passively pollinated fig trees the insect makes no direct effort to
collect or transport pollen, and pollination is dependent on pollen grains that
were transported on the body of the insect. This form of pollen transfer is less
efficient, and requires male plants to produce more pollen. This relative
inefficiency is reflected in the much larger number of male flowers present in figs
of passively pollinated fig trees [28].
Each year, only one of the generations of fig wasps that emerges from male figs
of F. carica, F. erecta and F. pumila has a high probability of entering female rather
than male figs and thereby contribute to the reproductive success of the male
plants from which they emerged. Pollen carried by the pollinators released at
other times of the year represents a metabolic cost for which there is no direct
reward to the plants. Reflecting this, figs on male trees of F. carica and F. pumila
that are produced at other times of the year do not contain functional male
flowers, so the pollinators that emerge from them carry no pollen [24,38]. These
species are passively pollinated, and their pollinators can clearly develop
successfully in male figs that receive no pollen. It is unclear whether F. erecta is
similar.
Unlike the other northern species, F. tikoua has an active pollinator, which may
limit its ability to produce pollen-free male figs if pollen aids pollinator fecundity
[39]. Here, we studied the fruit phenology of F. tikoua within its native range in
China and address the following questions: (1) what is the flowering phenology of
F. tikoua and does it vary between the sexes? (2) what proportion of the figs
produced by F. tikoua are entered by pollinators and does this vary with season?
(3) does this species exhibit convergence in reproductive phenology with F. carica,
F. erecta and F. pumila? And if so (4) are male flowers present in male figs
throughout the year, as in most fig trees, or are female-flower only figs produced
seasonally, as recorded for two of the other extra-tropical dioecious fig tree
species?
Phenological Convergence of Ficus tikoua with Extra-Tropical Fig Trees
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Materials and Methods
Ethics Statement
Our sampling site was not in a national park or protected area. The studies
species, Ficus tikoua, is not an endangered or protected species, so specific
permission was not required. The specific location of the sampling site is 31.45˚N,
104.60˚E.
Ficus tikoua and its fig wasps
The natural distribution of F. tikoua Bureau covers Southwest and Central China
and montane areas of Northeast India, Laos and North Vietnam, where it is found
in wastelands, grassy banks, rocky areas and open woodland [40]. It is a prostrate
shrub that does not reach a height of more than about 30 centimeters, with figs
located at the leaf axils. The figs are often partially buried in the soil and for this
reason it is called ‘‘di-guo’’ in Chinese, meaning ‘fruit from soil’. The figs are
small, flattened ovoid, reaching 10–20 mm in diameter at maturity. Both male
and female figs remain yellow-brown when ripe, suggesting that terrestrial
mammals may contribute to seed dispersal [13].
Genetic differentiation between adjacent F. tikoua populations suggests that its
Ceratosolen sp. pollinator disperses less widely than the pollinators of most Ficus
species [41]. This may be a consequence of the plant’s small crops and the cryptic
location of its figs, all of which reduce the ‘apparency’ of F. tikoua to pollinators,
and makes long distance detection of suitable figs more difficult. The fig wasp
community associated with F. tikoua is simpler than that of most species of fig
trees. Widespread sampling throughout most of the range of the plant has
detected only the pollinator (Ceratosolen sp. indesc.) and one non-pollinating fig
wasp (NPFW), its presumed parasitoid, a species of Philotrypesis (Pteromalidae,
Sycoryctinae) (Y. Chen et al., unpublished). As in most dioecious fig tree species,
no fig wasp offspring were recorded from female figs.
Locality and methods
Our study population was located on a small hill with sparse deciduous forest
located in Mianyang, Sichuan Province, China (31.45 ˚N, 104.60 ˚
E), which is
towards the northern edge of the natural distribution of F. tikoua. The region has
a subtropical monsoon climate with four distinct seasons. Summers are long, hot
and humid, and winters are relatively short and mild, but with some snow. Mean
minimum temperatures for the coldest month (January) are about 3˚C, and mean
maximum temperatures for the hottest months (July and August) are about 30 ˚C
(http://www.chinaweatherguide.com/sichuan/mianyang-weather.htm).
The creeping growth form of F. tikoua makes it difficult to distinguish between
individuals and to identify which figs are produced by each individual. We
therefore established sampling points with dense F. tikoua foliage that were at least
30 meters apart between each other that were assumed to represent 32 different
plants. One meter square areas were marked at each point. The numbers and
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developmental stages of the figs in each square were generally recorded every
seven to 10 days between 30 November 2012 and 2 March 2014, but recorded
monthly during the winter periods. Fig developmental phases were assigned based
on the scheme of Galil [42]. A phase figs are immature, B phase figs are the stage
when pollinators enter, C phase is when fig wasp offspring and seeds develop, D
phase is when fig wasp offspring vacate male figs and E phase female figs have
become attractive to seed dispersers. To follow the growth of individual figs, we
also marked 20 randomly-chosen figs in each square with small plastic labels. If
less than 20 figs were present the nearest figs outside the squares were also marked.
The maximum diameters of the labelled figs were measured with electronic
Vernier calipers. When one of these marked figs aborted it was replaced by a
nearby fig whenever possible.
To compare the relationship between weight and diameter of male and female
figs, and to relate size to developmental stages, we also sampled 15 to 20 figs at
random away from the marked areas once a month between February and
December 2013. Their maximum diameters and fresh weights were measured.
Late C phase male figs were also collected from outside the marked areas and kept
in netting bags to let the fig wasps emerge naturally. The figs were then dissected
to remove any remaining fig wasps from inside and their contents recorded.
Seasonal differences in male and female flower numbers and fig wasp contents
in male figs were tested using Generalized Linear Models (GLMs) assuming
quasiPoisson distribution of residuals. Pair-wise comparisons were carried out
using multiple tests with Bonferroni correction. All analyses were carried out
using R [43].
Results
No sampling squares were recorded to produce both male and female figs. Among
32 sampling squares, 20 produced male and 8 produced female figs, so they were
regarded as male and female plants respectively. The rest four sampling squares/
plants produced no figs within our observation period.
The phenology of F. tikoua
Peak numbers of figs in the 1 m
2
areas were noticeably higher on the male plants,
with maximum densities recorded on the 20 males ranging from 26 to 223,
compared with 15 to 28 figs on female plants. Figs were present on some of the
male plants throughout the year, but were only at very low densities during the
winter months (Figure 1,Figure S1 in File S1). A spring burst of fig production
resulted in the highest average density of male figs being recorded in March–April,
followed by a gradual decline through to the end of the year, interrupted by a
second, smaller burst of fig production in early Autumn (Figure 1). Female figs
were not recorded during the winter. They were mainly produced during the
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summer months, but small numbers were recorded on some plants through to the
start of the winter (Figure 1).
Figs were also absent from most of the demarcated areas on male plants during
the winter months, and pollinated figs (that contained fig wasp offspring) were
recorded from just one of the plants during the first winter (Figure 2). In the
following spring, all 20 male plants had young (phase AB) figs present and the first
of these were entered by fig wasps (phase C) in late March and early April. Just
one fig from within the 20 demarcated squares released fig wasps at that time, so
most of the pollinators that entered the B phase figs in spring will have emerged
from figs outside our sampling squares. The next generation of adult pollinators
emerged in early summer (late May and June), when very few AB phase figs were
present on the male plants (Figure 2). Fig production resumed on some of the
male plants in mid-late summer, and these supported a generation of pollinators
that emerged in autumn (Figure 2). Immature figs were present in a few of the
sampling areas at that time, providing oviposition sites for the autumn fig wasps
and allowing the fig wasp population to persist on five of the plants through the
second winter. In summary, across the sampling period as a whole, the
demarcated areas of all the male plants released pollinators in early summer and
about half also released smaller numbers of pollinators once or twice more later in
the year (Figure 2), some of which had the offspring that overwintered.
Figure 1. Seasonal variation in the densities of figs from 28 demarcated areas of Ficus tikoua plants in Mianyang.
doi:10.1371/journal.pone.0114344.g001
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Fig production by female F. tikoua was concentrated in the summer months,
when all eight demarcated squares contained figs (Figure 2). Several of the plants
also produced a second, but much smaller, crop in the autumn, but none of these
figs survived the winter (Figure 2,Table S1 in File S1). The receptive (phase B)
periods of both crops of female figs corresponded closely with the periods when
fig wasps were being released from male figs (phase D figs, Figures 2 &3). The
much larger early summer female crop also corresponded with the larger numbers
of male figs releasing wasps at that time, and also will have benefitted from a
virtual absence of competition for pollinators from B phase male figs (Figure 3).
This contrasts with the smaller supplementary second crops of female figs, which
was produced at a time when many of the pollinators emerging from male figs had
the opportunity to enter receptive figs on the same plants.
The development, pollination and abortion of male and female figs
Relationships between fig size and weight were recorded for 201 male and 100
female figs from outside the demarcated areas. A power-function relationship was
present between their fresh weights and diameters (R
2
50.91 and 0.99 for male and
female figs respectively, Figure S2 in File S1). The female figs were a little heavier
than male figs of similar diameters, especially after they had been pollinated (from
Figure 2. The fruiting phenologies of 20 male (M) and 8 female (F) Ficus tikoua in Mianyang. Phases A and B are hard to distinguish and are combined
here. Grey and black bars indicate Phases AB and C, respectively. The D phase of male individuals and E phase of female individuals is shown by dotted
bars.
doi:10.1371/journal.pone.0114344.g002
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the beginning of C-phase). However, no significant differences were found
between the regression equations of each sex (likelihood ratio test, x
2
50.298,
p50.585). The smallest female figs that had been entered by pollinators had a
diameter of 6.09 mm, compared with 7.11 mm for male figs (Figure S2 in File
S1), but mature female figs (E phase) were considerably larger than male figs at
the time that they released pollinators (D phase) (Female figs have no equivalent
to D phase and pass directly from C to E phase).
Few figs could be recovered after they became detached from the plant, but
repeated measurements of the same figs allowed us to decide which figs had
aborted without being pollinated, based on their diameters on the last occasion
when they were still attached to the plants. Figs with smaller diameters than the
observed maximum diameter of un-pollinated figs were assumed to have aborted
without being entered by fig wasps. Abortions were frequent among both male
and female figs and ‘replacement’ figs often themselves had to be replaced after
they also aborted (Table S1,S2). Overall, 62% of the initially-marked male figs
(436/700) and 33% of the female figs (67/205) aborted before they matured
(reached D phase if male figs, or E phase if female figs). Most of these abortions
(including those from some replaced figs) took place before the figs reached the
size when pollinators entered (Figure 4) and an estimated 86% (589/683) of the
Figure 3. Seasonal variation of the densities of figs at different developmental stages in demarcated areas of Ficus tikoua plants in Mianyang. The
times when pollinators were emerging (D-phase figs) of spring and early-summer crops of male plants are highlighted by dash-dot and dash frames
respectively. The arrow indicates an additional isolated occurrence of D-phase male figs.
doi:10.1371/journal.pone.0114344.g003
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aborted male figs and 81% (162/200) of the aborted female figs had fallen from
the plants without being entered by pollinators.
Abortion rates varied considerably between crops. Just 32% (126/400) of the
initially-marked male figs in the spring crops completed their development and
released fig wasp offspring, compared with 72% (130/180) of the summer crop
and only 6.7% (8/120) of the autumn crop (Table S1 in File S1). Substantially
different abortion rates were also found among female crops, with most spring
crop figs reaching maturity (86%, 138/160), whereas no summer crop figs reached
maturity (Table S2 in File S1). There were also large between-crop differences in
the sizes of the figs when they aborted. Most spring and summer crop abortions
among male figs occurred when the figs were small, indicating a shortage of
pollinators, whereas the high abortion rates among autumn male crop figs
resulted from a combination of early abortions among un-entered small figs and
losses of larger, pollinated figs, through the winter period (Figure 4). Female figs
showed a similar pattern (Figure 4).
Contents of the figs
Mature figs from outside the marked areas were collected and dissected in 2013.
Spring (main) crop female figs, collected in August, contained 782.00¡111.55
Figure 4. The last recorded diameters of figs of Ficus tikoua that became detached from the plants
before they had reached maturity. The observed maximum diameter of un-pollinated figs is indicated by the
horizontal dotted line. Figs which became detached before reaching these diameters had aborted after failing
to attract pollinators.
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seeds (mean ¡SD, n530 figs). No summer crop female figs could be found. Male
figs could support far fewer pollinator offspring because they contained a much
smaller number of female flowers. Male flowers made up only a small proportion
of the total flowers, reflecting the active pollination exhibited by Ceratosolen
species. Pollinator sex ratios were strongly female-biased. The putative parasitoid
Philotrypesis was always rare (Table 1).
Only flower numbers were recorded from the overwintered fig crop initiated in
autumn 2012 that matured in spring 2013 because the wasps had already emerged
when the figs were sampled at the end of March. Numbers of male flowers in male
figs did not vary among seasons (GLM: LR56.10, df52, p50.110), but female
flower numbers were significantly higher in autumn than spring with intermediate
values in summer figs (GLM: LR5301.81, df52, p,0.01; pair-wise comparisons:
autumn vs. spring: t54.57, p,0.001; autumn vs. summer: t52.00, p50.140;
spring vs. summer: t522.189, p50.0903) (Figure S3 in File S1).
Discussion
Like other dioecious fig trees, seed production in F. tikoua depends on pollen-
carrying adult female fig wasps being available at the times when receptive figs are
present on female trees. Over longer time periods, the stability of the mutualism
also depends on male plants being able to support populations of their specific fig
wasp pollinators. Genetic evidence [41] suggests that F. tikoua’s Ceratosolen
pollinators are relatively sedentary and do not fly the long distances seen in some
congeneric species [44], so populations of F. tikoua cannot rely on the services of
fig wasps that developed elsewhere, and must maintain their own local pollinator
populations. Fig wasps develop only in the figs of male plants, so it is only their
flowering phenology that is constrained by the need to support the development
of the pollinators, whereas the phenology of female plants is subject to the same
selection pressures, from the environment and mutualists, that influence
flowering times among plants in general. In Mianyang, F. tikoua male trees
produced one to three crops annually (similar intraspecific variation in the
number of fig generations has also been reported in European populations of F.
carica [33]). This ensured that some male figs containing pollinator offspring were
present locally throughout the year, though whether or not the overwintering
stages entered a true diapause is unclear. The fruiting phenology of F. tikoua
results in pollinator populations going through two bottlenecks each year, once
during the winter and another when many of the adult pollinators become
trapped in female figs. These bottlenecks are temporary, because male trees
produced additional crops with sufficient figs to allow subsequent generations of
pollinators to recover. Female plants produced a single annual major crop of figs
that was pollinated in late spring. Pollination was facilitated by synchrony with the
release of pollinators from the main crop of male trees that bear few receptive figs
at that time. Smaller numbers of receptive figs were present on female trees later in
the year, but at that time they were competing with male trees to attract
Phenological Convergence of Ficus tikoua with Extra-Tropical Fig Trees
PLOS ONE | DOI:10.1371/journal.pone.0114344 December 4, 2014 11 / 1 7

pollinators and it was not confirmed whether any of this later crop set seed
successfully. Figs on male plants contain male flowers throughout the year, even
though it is only those figs that release fig wasps in late spring that are likely to
contribute to their reproductive success. At other times, pollen carried into male
figs can nonetheless indirectly benefit pollen donor trees if it increases the
fecundity of pollinators, but only if these fig wasps have entered receptive figs on
the same plant [45]. If they enter figs on other male plants then there is no benefit
accruing from pollen production.
The evolution of a dioecious breeding system provided many potential
advantages for fig trees relative to their ancestral monoecious breeding system,
including avoidance of self-pollination, the potential to chemically defend ovules
from non-pollinating fig wasps and the partial decoupling of flowering times of
male and female individuals [46,47]. Tropical dioecious fig trees exhibit a wide
range of phenologies, including seasonal concentrations of flowering that allow
peaks in pollinator release from male plants to coincide with peaks in the numbers
of female figs waiting to be pollinated [14,23,48,49]. The flowering phenologies
of extra-tropical dioecious species can be seen as extensions of these to cope with a
cold winter period.
The flowering phenology of F. tikoua in Sichuan Province, China shows striking
convergence with that of F. pumila and F. erecta elsewhere in China and F. carica
in Europe, despite being unrelated to these other temperate dioecious fig trees.
The independently-evolved similarities in fruiting patterns that they exhibit
illustrate the likely constraints acting on fig trees growing under strongly seasonal
conditions, combined with vicariant selection operating reciprocally on the two
sexes of the plants to flower at appropriate times [39]. Low temperatures limit the
rate of pollinator larval development, when adult male fig wasps can chew exit
holes to allow emergence of their females and the times of year when females can
disperse and carry pollen between male and female fig trees. Temperatures also
Table 1. The contents of mature male figs of Ficus tikoua collected in Mianyang from outside the marked areas.
Crops
Sample
Date
N figs
Flowers/
Wasps Flowers per fig (mean ¡SD)
Fig Wasps per fig
(mean ¡SD)
NPFW %
(mean ¡SD)
Sex ratio of
pollinators
(mean ¡
SD)
Male Female Total Pollinators Total wasps
Autumn
2012
March
2013
42/0 24.31¡6.11 260.44¡65.89 284.74¡68.60 ////
Spring
2013
June
2013
80/80 25.26¡5.15 209.39¡44.20 234.65¡45.68 186.43¡40.91 205.80¡44.38 0.095¡0.028 0.063¡0.029
Summer
2013
September
2013
39/24 22.90¡6.62 233.33¡72.93 256.23¡72.22 174.88¡49.36 192.58¡54.40 0.092¡0.020 0.098¡0.040
Total 161/104 25.06¡5.67 218.40¡63.45 243.46¡63.80 183.76¡43.03 202.75¡46.93 0.094¡0.026 0.071¡0.035
The only NPFW recorded was a species of Philotrypesis.
doi:10.1371/journal.pone.0114344.t001
Phenological Convergence of Ficus tikoua with Extra-Tropical Fig Trees
PLOS ONE | DOI:10.1371/journal.pone.0114344 December 4, 2014 12 / 17

influence rates of seed development and germination, and the ability of seedlings
to establish. The timing of mature seed production in F. tikoua may simply be as
early in the year as can be achieved given pollination constraints, but their fruiting
phenology has a clear benefit in that it avoids the need for figs containing
developing seeds to be retained on female plants through the winter period.
F. tikoua is the only one of the four temperate dioecious fig trees to benefit from
active pollination of its flowers. Inflorescence structure in figs from different crops
of male F. erecta has not been compared, but in F. carica and F. pumila only one
crop of male figs each year contains male flowers - the crop that is synchronized
with the availability of female figs to pollinate. Pollen production is more costly
for these passively-pollinated species than in the actively-pollinated F. tikoua,
because they need to produce more pollen to achieve adequate fertilization.
Passive pollinators haphazardly distribute pollen within the male and female figs
they enter, but fertilization of female flowers in male figs is inhibited [50]. Because
F. tikoua is actively pollinated, its male figs contain fewer male flowers than would
be required for passive pollination [28], so the cost of retaining male flowers is
lower and selection for their loss in figs produced at times of year when no female
figs are available is likely to be less than in passively-pollinated species.
Furthermore, if some second crop female figs do manage to survive to maturity,
even at very low frequencies, then benefits would accrue to male plants that are
releasing pollen-carrying pollinators in late summer.
Results from other fig tree species where pollination is active provide an
alternative or additional explanation for the retention of male flowers in all crops
of F. tikoua. The behavior of adult female fig wasps that actively pollinate fig
flowers increases the likelihood that their larvae will develop in pollinated flowers
[50]. Experiments where pollinator foundresses that lack pollen are introduced
into figs suggest that actively-pollinating species often suffer reduced reproductive
success, due to increased larval mortalities, whereas passively pollinating species
appear not to benefit from pollination [51,52]. The presence of male flowers in
male figs of F. tikoua throughout the year may therefore also reflect selection on
the trees acting via pollinator fecundity.
Reproduction by fig trees is often limited by the number of figs entered by fig
wasps [53,54], but abortion rates in the autumn and spring crops of male and late
summer crop of female F. tikoua seem particularly high. This resulted from a
combination of a lack of pollinators and over-wintering losses among autumn-
crop figs and a shortage of pollinators that had survived the winter and became
available to pollinate the spring male crop. In contrast, abortion rates among the
main late-spring crops of female figs were much lower, and emphasize that the
plant’s phenology delivers effective pollination and seed production despite the
seasonal lows in pollinator populations that it generates.
F. tikoua is a short creeping plant with rather small crops of small,
inconspicuous figs. As such, the plant and its figs have a low ‘apparency’ to insects
and they are likely to be hard to find from long distances [55,56]. Perhaps
reflecting this, its pollinators rarely disperse far [41]. Our study was carried out in
an area with a large, dense population of F. tikoua that was clearly able to
Phenological Convergence of Ficus tikoua with Extra-Tropical Fig Trees
PLOS ONE | DOI:10.1371/journal.pone.0114344 December 4, 2014 13 / 17

maintain a resident population of pollinators. Small founder populations may not
be able to do so, and vegetative reproduction may prove to be a significant
component of the plant’s overall reproductive strategy.
Supporting Information
Acknowledgments
We would like to show our appreciation to Xing Tong and Rui Zhao for their
suggestions on our studies, to Jun-Ying Deng and Lu-Shui Zhang for their
assistance with the observations, and to Finn Kjellberg for his constructive
comments.
Author Contributions
Conceived and designed the experiments: SGC YC. Performed the experiments:
TTZ YJY. Analyzed the data: RW YC. Contributed reagents/materials/analysis
tools: RW. Contributed to the writing of the manuscript: SGC YC TTZ.
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