Botany
Syconium development in Ficus petiolaris (Ficus, sect. Americanae, Moraceae) and their relationship with pollinator and parasitic wasps.
Journal: Botany
Manuscript ID cjb-2018-0095.R3
Manuscript Type: Article
Date Submitted by the 06-Dec-2018 Author:
Complete List of Authors: Piedra-Malagón, Eva; Universidad Nacional Autónoma de México, NA Hernández-Ramos, Balbina; Universidad del Mar Mirón-Monterrosas,Draft Ana; Universidad Nacional Autónoma de México Cornejo-Tenorio, Guadalupe; Universidad Nacional Autónoma de México, Navarrete-Segueda, Armando; Universidad Nacional Autónoma de México Ibarra-Manríquez, Guillermo; Universidad Nacional Autónoma de México,
Fig wasps, Phenophases, Seed germination, Seedlings, Syconium Keyword: morphology
Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :
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Syconium development in Ficus petiolaris (Ficus, sect. Americanae, Moraceae) and
their relationship with pollinator and parasitic wasps.
Eva María Piedra-Malagón, Balbina Hernández-Ramos, Ana Mirón-Monterrosas,
Guadalupe Cornejo-Tenorio, Armando Navarrete-Segueda and Guillermo Ibarra-
Manríquez
Eva María Piedra-Malagón ([email protected]); Balbina Hernández-Ramos
([email protected]); Ana Mirón-Monterrosas ([email protected]);
Guadalupe Cornejo-Tenorio ([email protected]); Armando Navarrete-Segueda
([email protected]); and Guillermo Ibarra-Manríquez
([email protected]). InstitutoDraft de Investigaciones en Ecosistemas y
Sustentabilidad, Universidad Nacional Autónoma de México, Antigua carretera a
Pátzcuaro 8701. Col. Ex-Hacienda de San José de la Huerta. CP 58190. Morelia,
Michoacán, México.
Balbina Hernández-Ramos. Universidad del Mar, campus Puerto Escondido, Carretera
Oaxaca, Universidad, CP 71980. Puerto Escondido, Oaxaca, México.
Corresponding author: Guillermo Ibarra-Manríquez ([email protected]), Tel.
443-322273; Fax: 443-3222719.
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Abstract
For the first time in a Neotropical Ficus species (F. petiolaris), the external and internal structural changes in its syconia are described together with the development of its fig wasps. Ficus petiolaris is endemic to Mexico and represents the northernmost limit of the geographical distribution of the genus in America. Considering the large variation in syconium morphology, we evaluate whether there are differences between syconium development in F. petiolaris and that described for Paleotropical species. We recorded the development of 428 syconia in two individuals, weekly for a period of five months, from initiation to maturity, as well as that of their associated insects. The time required for completion of syconia development ranged from 91 to 126 days. The external morphology of the syconia (color, ostiole size and thickness of syconium wall) enabled phase recognition. Male and femaleDraft of pollinators (Pegoscapus sp.) and parasitic wasps
(Idarnes sp.) were registered. High seed germination values (50-90 %) tested indirectly the positive role of pollinators. Our results are similar to those reported in Paleotropical
Ficus species and should contribute to our understanding about syconium development as well as to the relationships that they maintain with their Agaonidae wasps.
Key words: Fig wasps, Phenophases, Seed germination, Seedlings, Syconium morphology.
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Introduction The genus Ficus (Moraceae) is characterized by its peculiar inflorescence-infrutescence
with one apical aperture known as ostiole (the fig or syconium), which is commonly
referred to as a “fruit”, while the true fruits (achenes or drupes depending on authors) as
“seeds” (Verkerke 1989). The syconium is an ellipsoid to spherical receptacle, which
encloses structurally and functionally unisexual flowers, as well as the fruits (Berg
1990). Within the monoecious species (Berg 1989), trees produce protogynous syconia
which contain two types of female flowers that differ in style length (short-styled and
long-styled flowers) and male flowers. In contrast, in gynodioecious species, plants
produce either “gall-syconia”, with male and short-styled female flowers (male
individuals) or “seed syconia”, generally only with long-styled female flowers (female individuals) (Berg and Wiebes 1992;Draft Weiblen 2001). Mature syconia represent a food resource for many vertebrate frugivores, particularly birds and mammals (Shanahan et
al. 2001; Ragusa-Netto 2002; Domínguez-Domínguez et al. 2006; Lomáscolo et al.
2010). Members of this genus are therefore considered key components for the
maintenance of tropical forest diversity (Janzen 1979; Shanahan et al. 2001; Harrison
2005).
Ficus species are also notable for the completely dependent mutualistic
relationship they maintain with their pollinator wasps (Hymenoptera, Chalcidoidea,
Agaonidae). The wasps, in turn, require the syconium in order to grow and reproduce
(Ramírez 1974; Weiblen 2002; Harrison 2005). Originally, this interaction was
considered a model of strict coevolution, where each species of Ficus had its specific
pollinator (Janzen 1979; Jousselin et al. 2003; Ronsted et al. 2005; Herre et al. 2008).
However, there is now evidence of some Ficus species being pollinated by more than
one wasp species and, equally, one species of these insects can act as a pollinator for
more than one species of Ficus (Ramirez 1970; Michaloud et al. 1996; Cook and
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Rasplus 2003; Harrison 2005; Lin et al. 2011). Moreover, there are five subfamilies of non-pollinator wasps (Epichrysomallinae, Otitesellinae, Sycoecinae, Sycophaginae and
Sycoryctinae), with life cycles that are also strictly associated with the syconia and can be parasites of the pollinator wasps or of the ovaries of female flowers (Bouček 1993;
Cruaud et al. 2011).
Diversity in the genus Ficus is estimated to be around 750 species, which are found in the Neo and Paleotropical areas of the world (Berg 1989). Despite the high number of species described in Ficus and the notable variation that exists in the structure, color, shape or number of flowers of their syconia, developmental phases have been characterized only in few species and typically categorized from A (initiation of syconia) to E (mature syconia) phases (Galil and Eisikowitch 1968a; Smith and
Bronstein 1996). A total of 22 speciesDraft of Ficus have been reported in Mexico, of which three are endemic (Ibarra-Manríquez et al. 2012). One of these endemic species is F. petiolaris Kunth, which also represents the northernmost distributional limit of the genus in America. Its monoecious tree grows exclusively on rocky substrates (Fig. 1), mainly in the tropical dry forest (Ibarra-Manríquez et al. 2012). Detailed ecological information about the reproductive aspects of this species (e. g., syconium phase duration, records about their pollinator and parasitic wasps, which animals could disperse their syconia or data about seed germination), are virtually unavailable.
The main objectives of the present study are therefore to: i) describe and illustrate the main changes, both external and internal, in the developmental phases of the F. petiolaris syconia and ii) determine the duration of each phase. Given that the development of the syconium of any species of Ficus cannot be adequately understood without also considering the development of its wasp community (Galil and
Eisikowitch 1968a), another important goal of this study is characterize general aspects
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of the development of the wasps associated with F. petiolaris. We also wish to provide
evidence of the activity of bats, which are likely to be their seed disperser, as additional
indicators of the phase E. Finally, we aim to estimate the seed viability, in order to
indirectly evaluate the effectiveness of the pollinator wasps and further our knowledge
of the seedling morphology. Considering the findings reported for paleotropical species,
we expected to find characters in the syconium external structure (e. g. color and size)
with which to recognize internal developmental changes that occur throughout its
different development phases, particularly in the female (B) and dispersal (E) phases.
Materials and methods
Selection of F. petiolaris trees
Observation of the main external and internal changes in the syconia was conducted in
two individuals of F. petiolaris locatedDraft in Morelia city, Michoacán, Mexico. The first
individual (A) was located in the gardens of the Comisión Forestal del Estado de
Michoacán (19º41’50.15” N, 101º10’53.45” W). The individual (B) is in the Botanic
Garden of the Instituto de Investigaciones en Ecosistemas y Sustentabilidad, Morelia
campus, of the Universidad Nacional Autónoma de México (19º38’56.23” N,
101º13’47.21” W). It had been planted in the spring of 2011 by one of the authors (GI-
M), and originally came from a seasonally dry forest in the La Huacana municipality,
Michoacán. Vouchers of both monitored individuals will be deposited at MEXU and
MO herbaria (Piedra-Malagón 618, 619).
Development of syconia in F. petiolaris
The syconia were measured and monitored in both trees, from initial development
(phase A1) to maturation (phase E). The recording frequency was weekly and covered
the period from July to November 2017. Twenty branches with floral buds were
selected (15 branches in A and 5 in B individuals) and marked once the development of
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buds began and differentiation of the syconium structure became evident. Each syconium was assigned a key for identification and its growth and any external morphological changes recorded. Simultaneously, syconia of equivalent size and color to those of the marked syconia were collected from other non-marked branches. We measured and dissected these syconia in order to record the internal characteristics and accurately identify their developmental phase based on the proposal of Galil and
Eisikowitch (1968a, b), while including the modifications of phases A and C suggested by Smith and Bronstein (1996). In total, eight attributes were recorded in the collected syconia and seven in the marked syconia (Table 1, Fig. 2). The difference in the number of characteristics recorded between both groups was due to the fact that manipulation of the marked syconia had to be minimized in order not to affect their normal development. In total, 48 syconia wereDraft marked (12 IA, 36 IB) and 380 syconia were dissected in different phases. All measurements were recorded using a digital vernier
(Mitutoyo, Absolute model (CD-6” CSX)), accurate to 0.01 mm and with instrumental error margins of ± 0.02 mm (≤ 200 mm) and ± 0.03 mm (> 200 mm). To assess whether the syconia measurement values differed statistically among phases, we applied a repeated measures ANOVA, with a Tukey post hoc test, using SPSS 22 (IBM Corp
2013).
Fig wasps material
Fig wasps were collected from both individuals observed. All wasps were collected from closed syconia in phase D, placed in sealable plastic bags to allow wasps emerge naturally and then, with a fine brush, stored in alcohol 70 %.
Scanning electron microscopy
Floral syconium structures and the associated fig wasps were observed and photographed under a JEOL JSM-IT300 LV scanning electron microscope (SEM). In
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order to avoid charge effects, the samples were observed in low vacuum mode, at
between 20 and 50 Pa depending on the sample. Micrographs obtained by SEM were
pseudocolored in Adobe Photoshop CS6, using the color substitution tool in order to
highlight the floral syconium structures as well as the fig wasps.
Germination of seeds in F. petiolaris
Syconia in phase E were collected from each individual (2 syconia from individual A
and 4 from individual B). We extracted 20 “seeds” of each syconium and placed on
filter paper in Petri dishes, in order to evaluate if seeds were viable. The dishes were
then placed in a germination chamber at 25 °C and the “seeds” watered every third day.
Germination (radicle emergence) was verified daily (n = 120 seeds).
Results
Development of syconia and fig waspsDraft in F. petiolaris
Phase A1 (Differentiation of structures; duration 21–28 days). Like in all other species
of the subgenus Urostigma, syconia are also initiated here in pairs in the axil of a leaf
and begins their development simultaneously as two green ellipsoid buds, each
protected by a pale green bract (Fig. 3). The first structure to differentiate in the interior
of each syconium is the ostiole, which is composed of a series of overlapping bracts,
with no clear differentiation of the syconium cavity. However, there is a slight widening
in its lower part that, in future phases, gives rise to the collar of the syconium and the
peduncle. As the syconium continues its development, the bracts of the buds acquiring a
brown color, separate and drop. Around the 15th day, the syconium is as wide as its
collar and maybe covered with yellow to red macules, depending on the level of
insolation to which it has been exposed (Table 1, Fig. 3).
Phase A2 (female; 7–21 days). In this phase, important growth of the syconium occurs
with respect to the previous phase (Table 1, Fig. 3). The basal bracts of the syconium
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become more visible due to widening of the receptacle and may change in color from green to brown. The macules increase in size and their outline is more defined. The stigmas of the female flowers are observed with a uniform distribution in the cavity of the receptacle; the collar of the receptacle, the peduncle and the ostiole are clearly delimited. In some syconia, it was possible to observe wasps in different phases of development, mainly around the ostiole and in ovaries close to the collar of the syconium, as is the case in phase C1 or C3. All of these emerged wasps belonged to the genus Idarnes sp. (non-pollinators). The females presented red eyes, an ovipositor that exceeded their body length and a metallic green exoskeleton (Fig. 4).
Phase B (pollination; 1–7 days). Given its short duration, this phase is difficult to observe. Externally, a similar morphology to that described in phase A2 is presented in terms of coloration and size (Table Draft1). However, unlike in A2, the syconium has a more defined globular form and the ostiole is slightly sunken (Fig. 3). Strictly, this phase begins with the arrival of the female wasps (Pegoscapus sp., Fig. 4), which enter the syconium through the bracts of the ostiole. When this event has recently taken place, it is possible to observe the wings inserted between the internal bracts of the ostiole or between its most external bracts (Fig. 4). Once within the syconium, the female wasps loaded with a pollen (Figs. 4) pollinate the female flowers and oviposit on some. The flowers have white stigmas (Fig. 5) and their number range from 394 to 562, with an average of 228 (SD ± 63.06) for those with long styles, and of 247.8 (SD ± 37.83) for those with short styles. The overlap of the ostiole bracts causes the wasps to remain trapped in the syconium cavity and eventually die. It is most common during this stage to find only one wasp (Fig. 3). However, in around of 30 % of the observed syconia, up to three were recorded. Finalization of this phase is recognized when the bracts of the ostiole become more constricted (Fig 5).
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Phase C1 (early interfloral; 14–21 days). At this stage of development, the syconium
continues increasing in size (Table 1), while the “seeds” and wasps begin to develop
(Table 1). The external bracts of the ostiole are slightly flattened. New macules are
observed, although of smaller diameter than those observed previously. Inside of the
syconium, the cavity is well defined and no difference is detectable between the ovaries
that contain the immature seeds and those that contain the wasps. However, due to their
loss of receptivity, the stigmas of all flowers acquire a pale brown color (Fig. 3). In
some cases, a hyaline and viscous liquid covers the syconium cavity.
Phase C2 (mid interfloral; 14–21 days). The syconium continues growing, particularly
the ostiole, and the peduncle reaches its maximum size (Table 1, Fig. 3). However, all
of the external changes during phase C are barely perceptible and the change in size is
particularly gradual, which complicatesDraft differentiation among the C phases. The
changes are more evident in the interior. The wasp larvae and “seeds” continue
developing, such that the syconium cavity becomes smaller. The flowers that contain
the wasps and the “seeds” differ in that the latter are smaller and flatter. Besides, both
female flower types also differ in terms of the tonality of the yellow color of the ovaries.
The exoskeleton of the progenitor wasp is compressed, dehydrated and can be found in
different degrees of decomposition. It is also frequently possible to observe nematodes
feeding on the exoskeleton. In other cases, the wasps and the syconium cavity are
covered by fungal hyphae and a dark brown hyaline liquid (Fig. 3) partially or totally
covers the syconium cavity.
Phase C3 (late interfloral; 14–21 days). The ostiole in this phase appears flattened,
becoming a small apical protuberance (Fig. 3). In the interior of the syconium, the
wasps and “seeds” have almost completed their development, such that the syconium
cavity is reduced markedly. The bodies of the new generation of wasps become visible
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through the ovary walls, which acquire a dark coloration, while those that contain the
“seeds” are yellowish. The anthers begin to emerge among the remains of the female flowers and the interfloral bracts.
Phase D (male; 1–7 days). The syconia increase notably in size and change in color from green to light green and their walls become slightly softer, with pale external macules (Table 1, Fig. 3). Inside, 12 to 45 (mean 26.9, SD ± 9.07) male flowers reach anthesis, and exert their stamens between and above the gall and “seeds” flowers (Fig.
5). The male wasps of the genus Pegoscapus sp. are the first to emerge (Fig. 4), breaking the walls of the ovary with their mandibles, copulating with the females and facilitating their exit from this structure. Most of the males remain within the syconium but some excavate a tunnel through the bracts of the ostiole (Fig. 5), which is used by the fecundated, pollen carrying femalesDraft to leave the syconium. The males non-pollinator wasps of the genus Idarnes sp. can be also observed (Fig. 4).
Phase E (post-floral; 1-7 days). In this final phase, the syconium decreases faintly in size (Table 1) and acquires a pale green to yellow brown color, with opaque macules
(Fig. 3). The mature syconium is completely soft to the touch and a sweet smell can be perceived. The ostiole is almost flattened and compact (Fig. 5). Internally, the structures within the syconium cavity acquire an overall dark brown color. In the ovaries of the female flowers that had been occupied by wasps, the holes through which the wasps emerged can be observed (Fig. 5). The stamens begin to lose turgidity and the “seeds” are now mature (122–214 per syconium; mean 155, SD ± 41.43; n=4) and surrounded by a mucilaginous and hyaline tissue layer (Fig. 5).
Seed germination in F. petiolaris
Seed germination in the studied species, from radicle emergence (started at ninth day) until the presence of foliaceous cotyledons (Figure 1), occurs at between 25 and 30
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days, with 67.5 % of the seeds germinating by day 25. Seed germination percentage
registered in individual A were 55 and 65 % in two syconia, respectively, whereas in the
four compared for individual B they were 50, 60, 85 and 90 %.
Discussion
Syconia development phases in F. petiolaris
The time required of phases A1 to E in the syconia of F. petiolaris ranged from 91 to
126 days. This result agrees with that reported by Verkerke (1989), who reported very
wide variation in this trait (between 40 and 181 days). Similarly, Zhang et al. (2006)
reported a wide range (around 44–111 days), but it varied among the seasons of the year
and was longer during the foggy-cool season. Other studies have indicated a shorter
development of between 28 and 55 days (Galil and Eisikowitch 1968b; Baijnath and
Naicker 1989; Smith and BronsteinDraft 1996; Zhang et al. 2006). Smith and Bronstein
(1996) mentioned that theirs findings may be explained by the fact that the species were
found on the boreal limit of their geographic distribution, which could affect different
aspects of their reproductive cycles and those of their Agaonidae pollinators.
Phases A1 and A2 of F. petiolaris also coincide with that described in previous
studies (Verkerke 1986, 1988; Zhang et al. 2006) in that the most notable development
was the increased size of the syconium, which almost doubled in our study (Table 1).
There is also agreement in the differentiation of the ostiole and of the female flowers.
However, the duration of these phases in F. petiolaris (4–7 weeks) was greater than the
1.5-4 weeks indicated by both Verkerke (1989) and Zhang et al. (2006). In contrast, the
relatively brief duration (1–7 days) of phase B recorded in F. petiolaris coincides with
that indicated by these authors.
Phase B of the syconia is easily recognized when remains of the wings of the
foundress wasps are observed in the ostiole (Fig. 4). In this sense, the record of one, and
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rarely up to three, wasp(s) per syconium in the studied species is comparable to the 1–4 foundress wasps reported by Gibernau et al. (1996) and Hernández-Sosa and Saralegui-
Boza (2001), while in other species (Galil and Eisikowitch 1968a; Compton and Nefdt
1990), the range is wider (between 1 and 10 wasps). This variable influences the number of wasps of next generation and “seeds” produces that can be found in the syconium and can thus affect the population dynamics of both partners in this mutualist interaction. The development of this phase differs from that reported previously by
Smith and Bronstein (1996) and Zhang et al. (2006). In the former study, it was hypothesized that the syconium stops growing during this phase, with entry of the female wasps to the interior of the syconium. However, we observed gradual and constant growth in the diameter and length of the syconium in phases subsequent to phase B (Table 1, Fig. 3). ThroughoutDraft the growth of the syconium, we also observed a clear thickening of the syconium wall in phases AD; this structure showed significant differences between phases A1 and D (Table 1).
On the other hand, based on a phenological study conducted by Morrison on the
Isla de Barro Colorado, Panamá, Janzen (1979) inferred that the separation between phases B and D was four weeks. The records for F. petiolaris present a longer period (6 to 9 weeks), which matches exactly the range indicated for F. racemosa (Zhang et al.
2006). This result can be explained by the proposal of Verkerke (1989), who left open the possibility of a longer duration of phase B, depending on the arrival of the female wasps to the syconium. This situation was later corroborated in other subsequent studies
(Bronstein 1988; Khadari et al. 1995), in which an extension of this phase of up to four weeks was recorded when there was no pollination of the female flowers. These flowers then rapidly lose their receptivity once pollination occurs.
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For phase C, Verkerke (1989) suggested that the development of the syconia
could present marked variations as also demonstrated for F. sycomorus (Galil and
Eisikowitch 1968a, b) and F. racemosa (Zhang et al. 2006). While it was also detected
in F. petiolaris that it is these phases (C1–C3) together that require more time in the
overall development of the syconium, only gradual external changes were detected in its
size (Table 1). In contrast, important modifications are observed inside, especially the
growth, color and texture of the ovaries of the female flowers. All of these variations
eventually derive in a reduction of the syconium cavity, although this does not
completely disappear. This latter finding differs from that described by Smith and
Bronstein (1996), who indicated that the syconium cavity closes completely in phase
C2. On the other hand, there is currently no certainty regarding the function of dark
brown hyaline liquid detected in theDraft syconium cavity of F. petiolaris in C1 and C2,
which was also described during phase C in F. ottoniifolia (subgen. Urostigma) by
Verkerke (1986), in the subgenus Sycomorus (Berg and Wiebes 1992) and in F.
racemosa (Zhang et al. 2006).
Phase D of the syconium of the studied species presented a loss of turgidity as
the most prominent external trait of the syconium, although it also began to become
slightly paler (Fig. 3). Comparatively, the syconium presented greater dimensions than
those recorded in the previous phases (Table 1), enabling clear differentiation of the
female flowers with apical holes, as a result of the emergence of the new generation of
wasps Pegoscapus sp., as well as the ovaries that contained “seeds” and the male
flowers (Figs. 3 and 5). All of this is in general agreement with previous descriptions
(Verkerke 1989, Smith and Bronstein 1996 but see Zhang et al. 2006). In contrast to
that reported in F. aurea with respect to the fact that the exit hole for the female P.
jimenezi wasps is excavated by the males through the lateral walls of the syconium
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(Bronstein and Hassaert-McKey 1996, Hernández-Sosa and Saralegui-Boza 2001), in F. petiolaris, this hole is always found in the ostiole, which possibly represents an easier alternative when attempting to escape the syconium, since the ostiolar bracts are probably less compact than the wall. However, validation of this hypothesis would require future anatomical studies. We found only one exit hole in the ostiole but it was possible to observe two or three such holes on rare occasions (Fig. 5). Other authors reported the same observation in F. sycomorus (Galil and Eisikowitch 1968a) and F. aurea (Hernández-Sosa and Saralegui-Boza 2001).
Phase E in F. petiolaris, in which the syconium and the “seeds” are mature, is externally distinguishable by a very soft texture, green yellow to green brown color
(Fig. 3) and slightly sweet odor. This phase is very brief (1–7 days) and is similar to that found by Zhang et al. (2006) for F. Draftracemosa. In this phase, the syconia of the studied species were rapidly removed by frugivores that, given the characteristics of the bite
(Fig. 5), were probably bats. The night watchmen at the site in which individual A is located reported visits of these mammals around the crown. In addition, it should be noted that the syconia of F. petiolaris present the typical bat-syndrome traits proposed by Lomáscolo et al. (2010).
Syconia flowers and fig-wasp community
The number of flowers of the syconium of F. petiolaris (406–607) is within the very broad range of 50 to 7000 flowers reported for the genus (Verkerke 1989). However, a recently reported pattern is that the number of female flowers is always higher than that of the male flowers (Verkerke 1989; Ibarra-Manríquez and Wendt 1992; Compton
1993, Hernández-Sosa and Saralegui-Boza 2001), presenting a proportion of 10:1 (Berg and Wiebes 1992). In the subgenera Pharmacosycea and Sycidium, the percentage of male flowers can vary by between 10 and 30 % (Berg 1989; Ibarra-Manríquez and
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Wendt 1992). In F. petiolaris, this percentage was lower (3–7.4 %), a finding that is
similar to that reported by Compton (1993) for F. burtt-davyi Hutch. (1.4 to 4.8 %).
While the number of flowers in the syconia depends to a large extent on their size
(Verkerke 1989, Ibarra-Manríquez and Wendt 1992), other more complex relationships
can arise with variation in these proportions. For example, the numbers of wasps and
“seeds” increase directly with the number of flowers recorded in a syconium (Bronstein
and Hossaert-McKey 1996).
Under laboratory conditions, the female fig wasps found in F. petiolaris can
survive outside of the syconium for three to eight days; a similar period is reported by
Compton (1993) and Janzen (1979). However, it was observed that the lifespan of
females of Pegoscapus sp. is shorter than that of Idarnes sp. Another notable finding
was the presence of wasps of the genusDraft Idarnes sp. in phase A2, with an absence of
wasps of Pegoscapus sp. Wasps of the former genus are characterized by being smaller
or of the same size of the pollinator wasps but presenting an ovipositor that exceeds
their body length (Fig. 4). These characteristics enable them to oviposit on the female
flowers from outside of the syconium, days before the presence of pollinators is
detected (Elias et al. 2008). Cruaud et al. (2011) also suggest that these wasps can
oviposit on the female flowers through the stigmas and style, depositing their eggs
between the interior of the integument and the nucellus. In this sense, the oviposition of
these non-pollinators is similar to that of the pollinators, but in this case, no pollination
will occur and there is current uncertainty regarding the possibility of development of
viable syconia in the absence of pollinators, or if they simply abort. It should be noted
that, in the syconia of individual A in phase C1, females of Idarnes sp. were observed
ovipositing through the walls of syconia (Fig. 4) that already had pollinator wasps in the
interior, as inferred by the presence of their wings in the ostiole.
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Seed germination in F. petiolaris
The seeds of F. petiolaris presented high germination percentage values (5090 %).
Previous studies report similar germination values of between 57 and 100 % in other
Ficus species (Verkerke 1986, 1988; Utzurrum and Heideman 1991; Ibarra-Manríquez
1992). The seeds required around nine days for the radicle to emerge and ca. 25 days to exhibit green and foliaceous cotyledons (Fig. 1). This development closely comparable with the findings of Ibarra-Manríquez (1992), who germinated five species of the section Pharmacosycea. Based on the classification proposed by Garwood (1996), F. petiolaris has the phanerocotylar epigeal seedling type with foliaceous cotyledons, which were associated with small seeds in tree seedlings in a tropical rain forest (Ibarra-
Manríquez et al. 2001). The high germination percentage recorded in F. petiolaris also can be considered an indirect measureDraft of the positive role of female wasps Pegoscapus sp. as pollinators, despite the apparently isolated condition of the monitored individuals.
The known natural populations of F. petiolaris are located at distances of ca. 59
(Tacámbaro, Michoacán) and 79 (Tuxpan, Michoacán) km from Morelia. These locations could serve as sources from which the wasps can migrate towards Morelia.
Future genetic studies are required in order to corroborate the validity of this hypothesis.
Conclusions
For the first time in a Neotropical Ficus species, the external and internal structural changes in its syconia are described, and these events related to the development of its fig wasp communities. Our study shows that the different developmental phases of the syconium in F. petiolaris conform, in general terms, to those described previously for others species in the genus. However, since this is the only species in America to be characterized in this respect, certain characteristics emerge that should be evaluated in
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other species in order to determine the breadth of similarity that exists among the
congeners. For example, the frequency at which the emergence of female wasps occurs
in phase D through a tunnel in the ostiole, or the role of the hyaline substance found
within the syconium cavity in phases C2–C3. One important consideration is that the
development of the syconia and their fig wasps is, in general, similar to that observed in
natural communities throughout the geographical range of F. petiolaris in Mexico (Fig.
6). Nevertheless, it is recommended to examine and observe the most critical syconium
developmental stages (B, D and E) more frequently (e.g. each week), and it is very
important to determine how many animal species interact as “seed” dispersal vectors in
the natural communities where F. petiolaris are found.
Another particular aspect is the record of new species of wasps of the genera
Pegoscapus and Idarnes. The recordDraft of wasps of the genus Heterandrium is of note
since there is little information available about this genus due to the fact that it is only
occasionally recorded, and there is no certainty regarding the type of interactions it has
with the syconium and with other groups of wasps. Our study confirms that priority
studies in Ficus should integrate the morphology of the syconia and their interactions
with the Agaonidae wasps. These types of studies will be more valuable among species
that belong to different sections and subgenera, particularly during phases B and D.
Finally, we expect to generate interest in conducting similar studies in other species of
the genus, especially in American species, which would advance our understanding of
the complex relationships maintained by species of Ficus with their pollinators and
dispersers.
Acknowledgements
This study is part of the activities carried out by EMPM during her postdoctoral
research (scholarship DGAPA–Universidad Nacional Autónoma de México, UNAM),
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and BHR as part of an investigation conducted within the Summer of Science Program.
We thank the authorities of the Comisión Forestal del Estado de Michoacán for providing facilities for this study, particularly Sr. Daniel Cuamva, who provided us with information about the activity of bats around individual A, and Dr. Orlando Hernández
Cristóbal, head of the Microscopy Laboratory of the ENES Morelia (UNAM), who helped us to obtain the SEM photographs. We greatly appreciate the support of Dr.
Jean-Yves Rasplus, who provided us with valuable taxonomic data about wasps found in our study (all of them are undescribed species). Finally, we thank Dr. Christian
Lacroix, Dr. Julien Bachelier and two anonymous reviewers for their valuable comments that have improved the manuscript.
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Draft
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TABLE 1. Traits measurement for syconium development phases of Ficus petiolaris.
Except for syconium wall thickness, for each structure, we indicate width and length in mm (± DS). Means with the same superscript do not differ statistically (Repeated measures ANOVA with Tukey post hoc test < 0.05) from other means among phases.
Phase Syconium Peduncle Ostiole Syconium wall
Thickness A1 6.83 (2.12)a 2.22 (0.23)a 1.62 (0.29)a 0.71 (0.35)a A2 11.31 (1.07)b 2.12 (0.19)b 2.06 (0.24)b 0.92 (0.18)b B 12.96 (1.71)b 2.32 (0.27)b 2.10 (2.28)b 1.15 (0.27)b C1 13.82 (1.44)c 2.35 (0.25)c 2.23 (0.27)c 1.25 (0.24)c C2 14.3 (1.27)c 2.37 (0.25)c 2.38 (0.29)c 1.50 (80.27)c C3 17.89 (1.9)c 2.47Draft (0.36)c 2.54 (3.77)c 1.88 (0.36)c D 20.54 (2.01)d 2.38 (0.36)d 2.42 (0.32)d 2.28 (0.56)d E 19.55 (2.5)c 2.69 (0.41)c 2.34 (0.34)c 1.79 (0.34)c Length A1 6.42 (2.06)a 8.26 (3.11)a 1.32 (0.24)a A2 10.12 (1.11)b 13.29 (3.96)b 1.47 (0.25)b B 11.64 (1.28)b 15.54 (6.93)b 1.57 (0.30)b C1 12.76 (1.34)c 21.93 (9.86)c 1.81 (0.44)c C2 13.14 (1.3)c 22.51 (6.85)c 1.86 (0.37)c C3 16.49 (2.64)c 20.51 (4.47)c 1.78 (0.41)c D 17.76 (2.26)d 20.86 (6.93)d 1.54 (0.33)d E 15.26 (1.90)c 19.15 (4.42)c 1.33 (0.24)c
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FIGURES
FIGURE 1. Habit and germination aspects of Ficus petiolaris. A) Rupicole habit and
trunk with yellow bark. B) Rupicole young plant with cordiform leaves. C) Germination
of the seed showing a white radicle. D) Seedling with radicle, hypocotil and foliaceous
cotyledons.
FIGURE 2. Attributes recorded in the marked and collected syconia. A) Length. B)
Width. C) Basal neck width. D) Internal wall width (this character was only recorded in
collected syconia), E) Peduncle length. F) Peduncle width. G) Ostiole length. H) Ostiole
width. Draft
FIGURE 3. Internal and external characteristics of syconia development phases in
Ficus petiolaris. The photographs on the right hand side present details of different
structures of the syconia and fig wasps. A) Floral bud cut showing the beginning of
differentiation of the syconium structure (ostiolar bracts and basal neck). B)
Differentiation between ostiole (OS), syconium cavity (SC) and basal neck (BN). C)
Development of female flowers (PF) and gall flowers (GF) of parasitic wasp Idarnes sp.
D) Syconium cavity widening with a female foundress wasp (FW) Pegoscapus sp.,
ovipositing on female flowers (PF). E) Initiation of the constriction of the ostiole
internal bracts with no differences detectable in the cavity between the ovaries of female
and gall flowers (GF and PF, respectively); the body of the foundress wasp (FW) is
visible. F) The wasp larvae and “seeds” continue developing, such that the syconium
cavity constricts even further; the flowers that contain the wasps (GF) and “seeds” (SF)
differ mainly in their color (yellow blackish and brown yellowish, respectively) and the
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body of the foundress wasp (FW) is still visible. G) In the interior of the syconium, the wasps (GF) and “seeds” (SF) have almost completed their development, such that the syconium cavity is reduced almost totally. H) Syconium increases in proportion to its dimensions, male flowers (SF) are visible among the gall flowers (GF) and “seeds”
(SD). The fig wasps emerge. I) Syconium mature size increase and ostiole is almost flattened and compact; internally, the cavity of the fig presents a dark brown color. The holes in the ovaries of the gall flowers (GF) through which wasps emerged and “seeds”
(SD) can be observed.
FIGURE 4. Fig wasps associated with Ficus petiolaris. A) Female Idarnes sp. B)
Females Idarnes sp. ovipositing externally through the walls of syconia. C) Female
Pegoscapus sp. attempting to enter Draftthe syconium cavity through the ostiole. D) Female
Pegoscapus sp. E) Remains of pollinator wasp wings (WW) in the ostiole bracts (OB).
F) Female wasp corbicle of Pegoscapus sp. showing the pollen load. G) Male
Pegoscapus sp. H) Male Heterandrium sp. I) Male Idarnes sp.
FIGURE 5. Details of syconium morphology of Ficus petiolaris. A) Ostiole in early phases of syconium development. B) Female flowers. C) Stamen of the male flower
(SF) and gall flower (OG). D) Broken ovary gall. E) Three exit holes excavated by males through the bracts of the ostiole. F) Detail of exit hole. G) Ostiole flattened, characteristic in phases D-E of syconium development. H) “Seeds” (SD) and ovary galls
(OG). I) Syconia, probably bitten by bats.
FIGURE 6. Syconium development phases in different populations across natural distribution of F. petiolaris. Locations of the populations in Mexico (locality, state) are
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showed with a number in superscript. A) Floral bud 1; B) Syconia 1; C) Syconium cavity
2; D) Syconia 2; E) and F) Female wasps of Pegoscapus sp., 1,3; G) Syconia with
remains of pollinator wasp wings in the ostiole bracts and parasitic wasps Idarnes sp. 1;
H) Syconium 4; I) Phase C2 observed in dissected syconia 3; J) Syconia 5; K) Syconium
6; L) Big and small syconia in phases D and, respectively 7; M) Syconia with ant
coming to the cavity by the exit hole in the ostiole bracts 4; N) Syconium open showing
“seeds”, empty galls and some male wasps 8; O) Syconium open showing “seeds” and
empty flower galls 9; P) Syconium probably bitten by bats 1. Number of locations: 1
Todos Santos, Baja California Sur; 2 Nacapule, Sonora; 3 Chilpancingo, Guerrero; 4
Juncalito, Loreto, Baja California Sur; 5 Nizanda, Oaxaca; 6 Izúcar de Matamoros,
Puebla; 7 Tacámbaro, Michoacán; 8 San José del Cabo, Baja California Sur; 9 Tequila,
Jalisco. Draft
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A B
Draft
C D
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B H
G
A
E Draft
C F
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B OS Phase A1
1cm CS BN
C
Phase A2 PF GF
D PF Phase B
1cm FW
E FW GF Phase C1
1cm PF Draft F GF
Phase C2 FW
SF
1cm
G
Phase C3 SF
1cm GF
H GF
Phase D SF
SD 1cm
I
SD Phase E GF
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Draft OB
WW
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A B C SF
500 um
500 um 500 um OG
g h D E F
500 um
500 um 1 cm500 u m 200 um Draft G I
500 um H OG
500 um
SD 1 cm
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Draft
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