Acta Oecologica 122 (2024) 103975

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Acta Oecologica
journal homepage: www.elsevier.com/locate/actoec

A new player in the Panamanian fig tree – fig wasp mutualism; a study on
the effect of gall midges on Ficus citrifolia
H. Roberta Hedberg, Lovisa Dück, K. Charlotte Jandér, Lisette van Kolfschoten *Department of Ecology and Genetics, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18D, 752 36, Uppsala, Sweden

A R T I C L E I N F O

A B S T R A C T

Keywords:
Parasite
Ficiomyia
Gall midge
Antagonist
Parasitoid
Physothorax

The mutualism between the fig tree and the pollinating fig wasps is a keystone interaction in tropical forests.
However, many antagonistic interactions also occur in the system, taking advantage of the fig trees and thepollinator. One such example is an antagonistic gall midge (Cecidomyiidae) that develops inside figs. Gall midges
inside figs have been documented in a few Ficus species around the world, but to our knowledge they have notpreviously been observed in Panama. In this study the newly observed Panamanian fig gall midge is documented,
together with its parasitoid wasp. The fig gall midge was only found in Ficus citrifolia figs. We investigated theeffect of fig gall midge presence on the number of seeds and the number of pollinating wasps (Pegoscapus tonduzi)
in a fig and aimed to identify the species based on morphology and barcoding of the COI region. We found thatthe fig gall midge had no, or a negligible effect, on the reproduction of the fig tree - fig wasp mutualism. The fig
gall midge most likely belongs to the genus Ficiomyia, close to Ficiomyia perarticulata. The parasitoid belongs to
the genus Physothorax, close to Physothorax russelli. This study suggests that the potentially newly arrived fig gall
midge currently has no major effect on the fig tree - fig wasp mutualism. However, should infestation rates
increase, it is likely that the fig gall midge would affect the mutualism negatively as it has in other parts of theworld. More studies on the fig gall midge species distributions in this region would be valuable and would
connect these newly observed species to a larger community, adding yet another species to this complex butclassic example of a mutualism.

1. Introduction
Parasitism is one of the most prevalent forms of life, with at least one
parasite species found for each free-living metazoan species (Poulin and
Morand 2004). Parasites have a variety of direct negative impacts on
their hosts and can even indirectly influence ecosystem dynamics andalter biodiversity (Marcogliese 2004; Lafferty and Kuris 2012; Preston
et al. 2016; Frainer et al. 2018). To determine the effect of a parasite on
an ecosystem one must consider (1) the ecological importance of thehost, (2) the parasite’s fitness effect on the host, and (3) the frequency ofinfestation (Lafferty and Kuris 2012). Parasitism is often derived frommutualistic systems, where one or more species alter their mutualistic
interactions and start taking advantage without providing any benefits
back to their partner in the mutualism (Boucher et al. 1982). Mutualisms
are fundamental to the majority of ecosystems (Boucher et al. 1982;
Chomicki et al. 2019; Biedermann and Vega 2020). For instance, mostland plants interact with mycorrhizal fungi that aid plants with nutrient
uptake, and the majority of flowering plants need pollinators to

reproduce (Ollerton et al. 2011; Antoine et al. 2021). A mutualism inwhich fitness effects on both partners can be quantified is the one be­
tween fig trees (Ficus, Moraceae) and their pollinating fig wasps(Agaonidae) (Herre 1989; Herre et al. 2008).
Fig trees are keystone species in tropical ecosystems and are often acrucial food source for frugivores, providing a stable food supply by
producing fruits throughout the year (Shanahan et al. 2001). Bothparties of the fig tree – fig wasp mutualism fully rely on each other for
reproduction, making them highly dependent on each other (Wiebes
1979; Weiblen 2002). The fig (formally syconium, hereafter fig) is an
enclosed infloresence that contains hundreds or thousands of uniovulate
flowers (Cook and Rasplus 2003). Fig trees flower asynchronously
throughout the year and thereby provide the wasps with constantreproductive opportunities. The pollinating female fig wasps are very
short lived: around two days outside the fig (Machado et al. 2005;
Kjellberg et al. 2005; van Kolfschoten et al. 2022) during which they
have to find a receptive fig to lay eggs in within their flying range. The
average flight distance for Panamanian pollinator fig wasps is estimated* Corresponding author.
E-mail addresses: roberta.hedberg@gmail.com (H.R. Hedberg), lisette.vankolfschoten@ebc.uu.se (L. van Kolfschoten).
https://doi.org/10.1016/j.actao.2023.103975
Received 29 January 2023; Received in revised form 26 June 2023; Accepted 11 December 2023
Available online 3 January 2024
1146-609X/© 2023 The Authors.
Published by Elsevier Masson SAS. This is(http://creativecommons.org/licenses/by/4.0/).

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Acta Oecologica 122 (2024) 103975

to be between 5.8 and 14.2 km (Nason et al. 1998). When a receptive fig
is reached, the pollinating female fig wasp enters the fig where shepollinates, oviposits, and dies. The fig wasp offspring develop in galled
flowers for several weeks before hatching. In the Neotropical region,
only the pollinating fig wasps access flowers from inside while othernon-pollinating wasps oviposit from outside the fig (Cook and Rasplus
2003). Fig trees and their pollinators (Agaonidae) are closely co-evolved(Herre 1989; Cruaud et al. 2012). In addition, many antagonistic species
are associated with this mutualism.
The fig can be seen as a microcosm containing many different
mutualistic and antagonistic organisms. The different fig tree species areoften associated with specific compositions of mutualistic and antago­
nistic species (Jackson 2004; Costa and Graciolli 2010; Palmieri and
Pereira 2018). Apart from the pollinator (Agaonidae), a diversity ofantagonistic non-pollinating wasp species (e.g., Torymidae and Pter­
omalidae) can be found, as well as other invertebrates (Bronstein 1991;
West and Herre 1998; Elias et al. 2008, 2012; Farache et al. 2018; Pal­
mieri and Pereira 2018). Some of these organisms have seemingly no
negative effect on the fig host but others definitely do. Host defence
against antagonists is complicated due to the intertwined symbiont
communities within a fig where the mutualistic pollinator might beharmed by drastic actions such as fig abortion. It is therefore argued thatthe fig tree host cannot easily exclude antagonists which consequently
remain (Cook and Rasplus 2003), although antagonist fitness might be
reduced by selective resource allocation to more beneficial figs (Jandérand Herre 2016) or by ant predation (Janzen 1966; Compton andRobertson 1988; Schatz et al. 2006; Bain et al., 2014; Wang et al., 2014;
Jandér 2015). Whereas some antagonists like non-pollinating fig wasp
genera are fairly well documented (Bronstein 1991; West and Herre
1998; West et al. 1996; Elias et al. 2008, 2012; Farache et al. 2018),
much less is known about other invertebrates that develop inside figs
(Bai et al. 2008; Palmieri and Pereira 2018; Piatscheck et al. 2018; vanKolfschoten et al., submitted). One such less studied invertebrate are the
fig-associated gall midges (Diptera: Cecidomyiidae).
Cecidomyiidae can damage cultivated plants and cause economic
losses in forestry and agriculture, but they can also function as biocon­
trol agents against invasive plants and other pests (Kolesik and Gagné2020). Additionally, they can function as pollinators: plant species fromat least seven families (including Moraceae) have Cecidomyiidae polli­nators, but no Cecidomyiidae are known to pollinate Ficus (Gan et al.
2022). Cecidomyiidae induce galls that are usually species-specificgrowths often developing on leaves, vegetative and floral meristems,
flowers, stems, and rarely roots (Kolesik and Gagné 2020). The galls are
induced from phytohormonal changes manipulated by effectors secreted
from Cecidomyiidae larvae (Tanaka et al. 2013; Zhao et al. 2015; Kolesik
and Gagné 2020), similar to other galling insects (Tooker and Helms
2014; Oates et al. 2016; Hearn et al. 2019; Krogaonkar et al. 2021).
Fig-associated Cecidomyiidae are specific to Ficus and have larvae thatdevelop in galls inside figs (Felt 1922; Roskam and Nadel 1990; Bai et al.
2008; Miao et al. 2011). Fig-associated Cecidomyiidae have been
observed since the beginning of the 20th century (Felt 1922), but only
one species has been fully described: Ficiomyia perarticulata in figs from
Ficus citrifolia found in Florida, USA (Felt 1922; Roskam and Nadel 1990;
Gagné 2018). Apart from Ficiomyia perarticulata, other fig-associatedCecidomyiidae have been studied in China on Ficus benjamina andJapan on Ficus macrocarpa, but no formal species descriptions of thoseare yet published (Bai et al. 2008; Miao et al. 2011; Yafuso et al. 2013;
Yukawa and Kim 2021). There have been sporadic reports of
fig-associated Cecidomyiidae in Taiwan, Malaysia, India, Philippines,
Dominica, and Cambodia (Roskam and Nadel 1990; Hsieh et al. 2021;
Yukawa and Kim 2021), but not in Panama and direct neighbouring
countries.
In Panama in November 2021, fig-associated Cecidomyiidae wereobserved in figs of Ficus citrifolia for the first time (field observations by
LD, HRH, LvK, and F. Piatscheck (personal communication)). Figassociated Cecidomyiidae had not previously been observed by fig

researchers working in the area (LD (since 2015), KCJ (since 2003), A.
Gomez (since 1999 (personal communication Jan 2023), E. A. Herre(since 1982 (personal communication Jan 2023)). We therefore believethat the fig-associated Cecidomyiidae could be a recent arrival to the
local fig community or a previously unnoticed endemic species.
Furthermore, a Physothorax (Hymenoptera: Torymidae) presumed to bea parasitoid of the Cecidomyiidae was first observed in March 2022
(HRH).
In this study we describe our findings of a newly observed figassociated Cecidomyiidae and its Physothorax parasitoid and their po­
tential fitness effects on the fig tree – fig wasp mutualism. To determine
the potential fitness effect, the number of seeds and the number ofpollinator offspring were counted in figs with and without the studied
Cecidomyiidae (from now on referred to as gall midge or Panamaniangall midge) and its parasitoid (from now on referred to as Physothorax).
The gall midge and the Physothorax were barcoded, and we present their
Barcode Index Number (BIN) IDs.
2. Materials & methods
2.1. Study species
Ficus citrifolia is a monoecious New World fig species (subgenus
Urostigma, section Americanae) that grows in a wide range from Florida,
USA, to southern Brazil (Dunn et al. 2014). F. citrifolia flowering is
generally asynchronous among trees and synchronous within a tree. In
Panama F. citrifolia is pollinated by Pegoscapus tonduzi (Hymenoptera:
Agaonidae) (Wiebes 1995). The most common invertebrates observed infigs of Ficus citrifolia across its full distribution range are the following:
Pegoscapus spp. (Agaonidae; pollinator), Idarnes group flavicollis spp.
(Hymenoptera: Pteromalidae; non-pollinating gall-inducing wasp),
Idarnes group carme spp. (Hymenoptera: Pteromalidae; non-pollinating
cleptoparasitic wasps), Idarnes group incertus spp. (Hymenoptera: Pter­
omalidae; non-pollinating wasp), caterpillars of Crambidae and Pyr­alidae, nematodes Parasitodiplogazter sp. (Nematoda: Diplogasteridae),mites (Arachnida), and larvae of Charoxus spinifer (Coleoptera: Staph­
ylinidae) and Ceratopus sp. (Coleoptera: Curculionidae) (Herre 1993;
Giblin-Davis et al. 1995; West et al. 1996; West and Herre 1998; Mar­
ussich and Machado 2007; Elias et al. 2012; Frank and Nadel 2012;
Farache et al. 2017; Palmieri and Pereira 2018; van Kolfschoten et al.,submitted). The non-pollinating wasps (Hymenoptera) that are mostcommonly observed for F. citrifolia at BCNM in Panama are Idarnes
group carme and Idarnes group incertus (Marussich and Machado 2007),and rare non-pollinating wasps are: Aepocerus (Agaonidae), Heteran­drium (Agaonidae), Physothorax, (Torymidae) and the subfamily Dor­yctina (Braconidae) (West et al. 1996).
2.2. Fig-associated gall midges
Fig-associated gall midge larvae develop inside figs in individual
galls (Felt 1922; Roskam and Nadel 1990; Bai et al. 2008; Miao et al.
2011; Yafuso et al. 2013). The midge galls in Ficus macrocarpa and Ficus
benjamina figs are induced within female flowers (Bai et al. 2008; Yafuso
et al. 2013), while Ficiomyia perarticulata galls are described as
pocket-shaped outgrowths from the receptacle (fig wall) (Roskam andNadel 1990). All gall midges described in the literature leave emergenceholes on the fig surface from where they exit the fig, but the emergencemechanism and characteristics vary. Sexual dimorphism is present for
all described gall midges. Females are generally larger with apink/red-orange abdomen and with a retractable ovipositor, whilemales are yellowish and grey (Roskam and Nadel 1990; Bai et al. 2008).
2.3. Study site
The field work was conducted in Barro Colorado Nature Monument(BCNM) around the Smithsonian Tropical Research Institute field station
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Figs that contained invertebrates other than pollinators (P. tonduzi), gall
midges (Cecidomyiidae) and the Physothorax were excluded from thestudy (i.e., figs that contained Parasitodiplogazter sp., Lepidoptera larvae,
Coleoptera larvae, Idarnes group incertus, and/or mites). Figs that had
more than one pollinator foundress were also excluded, due to theresulting likely increase in the total number of offspring (Herre 1987,
1989; West and Herre 1998). The number of foundresses per fig wasrecorded directly or estimated from the number of P. tonduzi males in­side a fig, figs with more than 15 males were highly likely to have had
two or more P. tonduzi foundresses and were therefore excluded fromthis study (Herre 1987; West and Herre 1998). However, foundress
numbers could not be assessed for trees 1 & 6 because the P. tonduzi
offspring had already emerged in the field. Sampled figs were stored in afreezer at − 25 ◦ C.
Each fig was divided into quarters and dissected. The following
structures were counted: empty pollinator P. tonduzi galls from which a
P. tonduzi had emerged, galls with P. tonduzi females still inside, gallswith male P. tonduzi still inside, bladders (P. tonduzi galls whose
development failed), undeveloped pistillate (female) flowers, staminate(male) flowers, fig seeds, midge galls, and gall midges inside midge galls(Figure A1). Total number of P. tonduzi offspring was calculated byadding together galls with P. tonduzi still inside and empty P. tonduzi
galls where P. tonduzi had emerged. Number of gall midge emergenceholes on the fig surface were also counted (Fig. 1). An unidentified
parasitoid species, morphologically keyed to Physothorax (Hymenop­
tera: Torymidae) was repeatedly found inside midge galls or emerged
into the sealed petri dishes. Figs that had Physothorax in midge galls or
emerged adults were counted and ordered as a separate group (Gallmidge + Physothorax) and collected for identification.
In 2022, gall midges were found in eight out of the twelve Ficus
citrifolia trees that had figs at the stage when pollinators emerged (Dphase; Galil and Eisikowitch 1968). Two of these eight trees were notused in the analyses because for these trees fewer than five figs remainedin each treatment group (control and gall midge figs) after figs withother invertebrates were excluded. In total, 53 gall midge figs, 55 con­trol figs, and 14 figs with both gall midges and Physothorax were ana­
lysed (Table A1). All collected figs were dissected; the uneven sample
sizes were due to many figs having to be omitted from the study due tothe presence of other invertebrates.

of Barro Colorado Island (BCI) in the Panama Canal in Panama. Data
were collected 17 Feb – Apr 13, 2022 and 25 Jan – Feb 19, 2023. The
data collected 2022 was used to estimate the gall midge effect on the fig
tree-fig wasp mutualism. Data was also collected for gall midge obser­
vation and identification, Physothorax observation and identification,gall midge prevalence, and gall midge infestation rates. Data collected2023 was used to study the gall midge ability to prevent abortion of
unpollinated figs, and to estimate gall midge prevalence and infestation
rates.
Ficus citrifolia trees that were approaching the flowering stage werevisited and their flowering status, together with any presence of gall
midges, were observed. Trees were revisited and surveyed for gall midge
presence if they would get figs with emerging pollinators (D phase figs;
Galil and Eisikowitch 1968) within the study period. A total of twelve
F. citrifolia trees were at the correct fig maturation stage and could be
included in this study (field season 2022). In 2023, we continued
monitoring gall midge prevalence together with fig abortion: a total ofeight F. citrifolia trees were screened in 2023.
2.4. Field collection
Figs were collected in the field if there were signs indicative of gall
midge presence on the fig surface (Fig. 1). As soon as gall midge preemergence spots occurred, 30 figs without pre-emergence spots (con­trol) and 30 figs with pre-emergence spots (gall midge figs) werehaphazardly collected from each tree. If there were fewer than 30 gall
midge figs available on a tree, all gall midge figs that could be reachedwere collected. In total, we collected figs for dissection from eight trees
in 2022.
The ability of gall midges to prevent abortion of unpollinated figs
(Jandér and Herre 2010) was estimated in 2023 on one tree. We placedmesh bags over twigs with pre-receptive figs (B phase figs; Galil andEisikowitch 1968) to prevent pollinator foundresses from entering figs.
The tree was re-visited after 16 days when pollinator wasps were soon to
emerge from figs that had not been covered by mesh bags. All figs in themesh bags were collected and the number of midge galls inside aborted
and un-aborted figs was counted.
In 2023 we also surveyed eight F. citrifolia trees in the area with figs
at the correct maturation stage (late C-phase: early D-phase) for pres­ence of gall midges. From each tree we haphazardly collected fifty figs
from accessible parts, then counted the number of midge galls insideeach fig.

2.6. Barcoding
For the barcoding, in 2022 we collected seven midge pupae and three
Physothorax pupae from their galls and stored them at − 20 ◦ C in abso­lute ethanol. For the complete DNA extraction, an adapted high salt
method was used, based on the original method from Aljanabi andMartinez (1997). The region cytochrome oxidase subunit I (COI) gene of
mtDNA was amplified in a Polymerase chain reaction with 35 cycles of

2.5. Laboratory procedure
Each fig was cut in half and sealed in a Petri dish at room tempera­ture to allow all pollinators (P. tonduzi) and gall midges to emerge before
being frozen. Presence of other invertebrates in the fig was recorded.

Fig. 1. Figs with gall midge emergence holes. A: three gall midge pre-emergence spots (white colour) where the gall midge has not yet emerged and five emergenceholes with some protruding pupal exuviae (brown colour). All emergences took place within 1–2 days (n = 99). B: close up of gall midge emergence hole where pupal
exuviae from a gall midge can be seen (photo taken the same day as emergence occurred). C: fig with gall midge emergence hole (after emergence).
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Acta Oecologica 122 (2024) 103975

30 s 95 ◦ C, 30 s of 50 ◦ C and 90 s of 72 ◦ C, using the primer pairs COIS
forward(5′-GGATCACCTGATATAGCATTCCC-3′)
COIA
reverse(300-402bp)
and(5′-CCCGGTAAAATTAAAATATAAACTTC-3′)
LCO1490
forward(5′-GGTCAACAAATCATAAAGATATTGG-3′)
HCO2198
reverse(5′-TAAACTTCAGGGTGACCAAAAAATCA-3′)(645bp) for both the gall midge and the parasitoid wasp. The productwas cleaned with EXO1 clean-up (Thermo Fisher Scientific). Samples
were sent to Eurofins for sanger DNA sequencing and the obtained se­quences were uploaded to BOLD in the dataset “DS-KOLFMI” (DOI
requested) to get Barcoding Index Numbers (BINs) assigned.

figs containing midge galls, unpollinated figs that remained on the tree
contained a higher number of gall midge galls (4,7,7,7,7,7,8,8,10) than
did unpollinated figs that aborted (1,1,1,4), (t-test t = − 5.992, df =6.066, p = 0.000933).
3.1. Description of the gall midge in Panama
The gall midge larvae developed inside the fig in individual galls.
The midge galls varied in size but were 2–3 times larger (n = 177,
Fig. 2E) than P. tonduzi galls. The midge galls were hard and thickpocket-shaped broad-based outgrowths from the receptacle (fig wall),often with flower remnants on its exterior (Fig. 2). Unlike Ficiomyia
perarticulata (Roskam and Nadel 1990) there was no observabledimorphism between male and female midge galls (n = 36). Midge gallcolouration varied from orange/yellow to pale yellow (n = 177). Colour
dimorphism was observed between adult male and female gall midges(n = 36). The abdomen of adult females was pink/red and the thorax andovipositor were yellow (n = 21, Fig. 3E). Adult males were pale yellow
with spots of grey (n = 14, Fig. 3E). Both females and males had grey
hair sporadically covering their whole body (n = 36). The abdominal
hair grew in a spotted pattern. Males had fewer antenna flagellomeres(32–33 stalked flagellomere) than females (42–47 stalked flagellomere)(n = 5). The general shape of the antennae can be seen in Fig. 3. The gall
midge most likely belongs to the Ficiomyia family, close to Ficiomyia
perarticulata (Felt 1922; Roskam and Nadel 1990).
When the gall midge reached the adult stage, it emerged from its gall
directly through the receptacle, without passing the fig lumen. White
pre-emergence spots were seen developing prior to emergence and the
spots remained after emergence on the fig surface (n = 99, Fig. 1). The
white tissue appears to be derived from plant tissue, but further analysis
is needed. The gall midge pre-emergence spots started to appear within aweek before P. tonduzi emerged. The earliest signs noticed of gall midge
emergence was 22 days after the receptive stage, i.e. just a few days
before pollinator emergence. Pupal exuviae often remained protrudingfrom the emergence hole after adult emergence (Fig. 1: A & B). Eachmidge gall had an individual emergence hole (n = 99).

2.7. Statistical analysis
To test if the different treatment groups differed in their number ofseeds, number of offspring, total number of flowers, undeveloped
pistillate flowers, or bladders, multiple Linear Mixed-Effect Models(LMER) were built. ANOVA (Analysis of variance) were used for groupcomparisons for the three treatment groups: control, gall midge figs, andfigs with both gall midges and Physothorax. Group category was treatedas the independent variable and Tree ID as a random factor to avoid
pseudoreplication.
LMER models were also used to test if the degree of gall midge
infestation influenced the number of seeds, number of offspring, total
number of flowers, or number of undeveloped pistillate flowers. Degreeof infestation rate was recorded as the number of midge galls inside figs
(independent variable) and tested against the different response vari­ables measured. Tree ID was treated as a random factor and presence ofPhysothorax as a fixed effect. Models with both normal and poisson
distributions and without the interaction factor were compared using
AIC (Akaike information criterion). In all cases the LMER with a normal
distribution had the lowest AIC value. The statistical analyses were
performed in RStudio (RStudio Team, 2020) using the lme4 package(v1.1-29, Bates et al. 2015). Significance for all statistical analyses was
set at p < 0.05.
3. Results
The gall midge was only found in figs of Ficus citrifolia. Other Ficus
species in the study area had no visible signs of gall midges on figs; Ficus
americana subsp. americana eugeniifolia-form (Berg 2007; also referred toin publications from the study site as Ficus perforata (Croat 1978)), Ficus
bullenei, Ficus aff. crocata (undescribed; also referred to in publications
from the study site as Ficus triangle or Ficus near trigonata), Ficus
costaricana, Ficus insipida, Ficus maxima, Ficus nymphaeifolia, Ficus obtu­
sifolia, and Ficus popenoei. Eight out of the twelve F. citrifolia trees that
had D phase figs (Galil and Eisikowitch 1968) showed presence of gall
midges in 2022, and five out of eight trees showed presence of gall
midges in 2023. Based on this, a rough estimation of the gall midge
infestation rate in the study area around the time of the study would bethat two thirds of the F. citrifolia trees were affected, keeping in mind
that infestation could be higher because not all parts of tree canopies
could be properly examined due to restrictions in tree and branchaccessibility. The mean number of midge galls in figs with gall midges
was 2.64 ± 2.24 galls (Mean ± SD, n = 67) in 2022 and 3.35 ± 3.15
galls (Mean ± SD, n = 156) in 2023. In 2023, 35% (156 of 452) ofcollected figs contained midge galls. On the five trees with gall midges
present, 56% (156 of 277) of figs were infested with gall midges. The
highest number of midge galls we observed in a single fig was 12 in 2022
and 26 in 2023. In the dissected figs of 2022 the Physothorax was foundin 19.4 percent of the figs with gall midge galls (n = 67), and seven out of
the eight trees with gall midges also harboured Physothorax. No morethan two Physothorax was found in a single fig (n = 67).
The presence of gall midge galls could prevent abortion of unpolli­
nated figs (unentered by pollinator foundresses) if the number of midge
galls reached a certain threshold, seemingly around four midge galls. All
unpollinated figs that did not contain any gall midge galls aborted. Of

3.2. Description of the gall midge parasitoid in Panama
We believe that the Physothorax repeatedly found inside midge galls
is a parasitoid of the gall midge. Physothorax larvae developed inside thefig in individual galls (n = 64). The Physothorax galls did not differ
conspicuously from galls with midges inside, and Physothorax occur­
rence was only established after opening a supposed midge gall or by
examining emergence holes. The emergence hole of the Physothorax
lacked both exuviae and the white tissue found around a gall midge
emergence hole (n = 64, Figs. 1, Figure 4: A-D). Instead, the Physothorax
emergence hole was similar in appearance (but different in location) tothe chewed emergence hole created by P. tonduzi males. However, anemergence hole connected to a Physothorax gall cannot be chewed by
P. tonduzi because the gall’s walls prevent P. tonduzi in the fig lumen
from entering the gall (n = 64, Fig. 4: E − G). It is therefore likely that
the Physothorax chews itself out with their mandibles, instead ofmanipulating plant tissue as does the gall midge. A mixed emergencehole was occasionally found on figs where a gall midge pre-emergencespot could be seen together with a chewed Physothorax emergencehole (Fig. 4: C & D). We interpret the mixed emergence hole as a
Physothorax emergence, where the Physothorax incapacitated its host
only after the gall midge had started to induce its emergence process.
Based on morphology, we identified the parasitoid up to the genus
Physothorax (Hymenoptera: Torymidae), close to Physothorax russelli
Crawford (Boucek 1993). Jean-Yves Rasplus kindly confirmed thisidentification using photos (personal communication June 2023). The
Physothorax was bright metallic green with large red compound eyes andwas 3 mm from head capsule to the abdomen and 6 mm in total fromantennae to ovipositor (n = 16, Fig. 4: G-H, Fig. 5). The ovipositor was
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Fig. 2. Photos of gall midge pupae and midge galls. A: a midge gall separated from a fig, B: a midge gall cut open showing the internal walls, C: a midge pupa in a cut
open gall, D: gall midge pupae from different perspectives, E: fig cut in half where a midge gall can be seen, inside the white circle, as an outgrowth from thereceptacle (fig wall).

Fig. 3. Photos of the gall midge. A–B: male gall midge, C: newly emerged adult female gall midge resting on a fig, D: head and antenna of male gall midge, E: four
females in the upper row and two males in the lower row, F: gall midge wing, G–H: female gall midge. The terminal antennal segments are broken in H.

approximately 3 mm long and protruded from the abdomen at an almost
90-degree angle (Fig. 5B). The red eye colour appeared early on in the
pupal stage while the rest of the pupal body was transparent in colour(Fig. 5C).

offspring (F2,120 = 1.85, p = 0.16; Fig. 6B). Furthermore, the treatmentgroups did not significantly differ in total number of flowers (F2,120 =2.39, p = 0.097), undeveloped pistillate flowers (F2,120 = 1.09, p =0.34), or bladders (F2,120 = 2.44, p = 0.092).
We then tested whether the number of midge galls in a fig had aneffect on the reproduction of the tree or the pollinators. The number ofmidge galls did not significantly affect the number of seeds in a fig (t121= − 0.440, p = 0.66; Fig. 7A), the number of fig wasp (P. tonduzi)
offspring (t121 = − 0.446, p = 0.66; Fig. 7B), total number of flowers(t121 = − 1.48, p = 0.14), or undeveloped pistillate flowers (t121 =− 1.55, p = 0.12) (Table A2).

3.3. Barcoding
Seven gall midges and three Physothorax individuals were barcoded;sequences are deposited in BOLD, dataset “DS-KOLFMI” (dx.doi.org/
10.5883/DS-KOLFMI). BOLD assigned the gall midge a unique BIN(AEZ0215), the Physothorax has not yet been assigned a BIN. Within
BOLD, the gall midge had a distance to the nearest neighbour of 14.3%;
there was a 86% overlap with non-fig gall midges of the family Ceci­
domyiidae like Asteromyia carbonifera and Asteromyia modesta, both
known from North America (Gagné 1989). The Physothorax in our study
had 88% overlap with the known gall midge parasitoid Physothorax
bidentulus from Florida (U.S.A.), associated with Ficus citrifolia (Burks
1969; Roskam and Nadel 1990). The relatively low percentage ofoverlap suggests that there are no previous records of the two species ofour study within the BOLD system. Blasting the sequences in the NCBI
system did not give different results.
The gall midge effect on the fig tree - fig wasp mutualism.
We first tested whether the presence of midge galls in a fig had any
effect on the reproduction of the tree or the pollinator (P. tonduzi). The
treatment groups (control figs, gall midge figs, & gall midge + Phys­
othorax figs) did not significantly differ from each other in the number ofseeds (F2,120 = 0.169, p = 0.85; Fig. 6A) or the number of P. tonduzi

4. Discussion
The observed gall midge in this study is either an endemic species
previously unnoticed, an infrequent visitor, or a new arrival to central
Panama. The level of gall midges inside figs was low during the study
period, and the gall midge had no significant effect on the reproductionof the pollinator (P. tonduzi) and fig tree. The gall midge most likely
belongs to the Ficiomyia genus due to its close resemblance to Ficiomyia
perarticulata.
We only found the gall midge in figs of Ficus citrifolia. Other sym­
patric fig species that produced figs during the study showed no signs ofgall midges. This suggests that the gall midge specialises on F. citrifolia
which is a very common fig species in the area. Similarly, Ficiomyia
perarticulata was only found in Ficus citrifolia figs in Florida and not in
the more abundant species Ficus aurea (Roskam and Nadel 1990). Both5

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Acta Oecologica 122 (2024) 103975

Fig. 4. Photos of the gall midge parasitoid (Physothorax). A: Physothorax emergence hole, B: side view of fig quarter showing Physothorax emergence hole and gall,
C–D: mixed emergence holes where a gall midge pre-emergence spot can be seen together with a Physothorax emergence hole, E: fig half displaying Physothorax in its
gall, F–G: Physothorax in midge gall before and after dissection, H: close up of Physothorax in midge gall.

Fig. 5. Photos of the gall midge parasitoid (Physothorax). A: Physothorax adult ready for emergence and Physothorax pupa in midge galls, B: adult Physothorax, D:
Physothorax pupa.

the gall midge and the pollinator (P. tonduzi) are dependent on receptive
figs within flying range. The gall midge is therefore thought to repro­
duce throughout the year similar to P. tonduzi.
The mean number of midge galls in figs with gall midges was 2.64 ±2.24 galls (Mean ± SD, n = 67) in 2022 and 3.35 ± 3.15 galls (Mean ±SD, n = 156) in 2023. The highest amount of midge galls in a fig was
twelve in 2022 and 26 in 2023. Higher levels of gall midge infestation
have been observed in China on Ficus benjamina where over 250 Ceci­
domyiidae were found in one fig (Bai et al. 2008). However, local con­ditions might alter the level of infestation. It should be added that Ficus
benjamina has a much higher number of female flowers per fig (1128 ±42.6 (Mean ± SE, n = 30) (Bai et al. 2008);) compared to Ficus citrifolia(325 ± 38 (Mean ± SD, n = 55) (Herre 1989)), which potentially could
facilitate increased Cecidomyiidae infestation. However, we expect thatthe number of flowers does not limit the Panamanian gall midge:

because the Panamanian gall midge induce galls from the receptacle,
whereas the Ficus benjamina Cecidomyiidae induce galls from female
flowers. For receptacle gallers, fig size could instead be a limiting factor.
However, these two fig species are fairly similar in size (Ficus benjamina:
mature Fi gs. 12–25 mm in diameter (Bai et al. 2008), Ficus citrifolia:
14.1–16.5 mm in diameter (Korine et al. 2000)). The differences ininfestation rate might instead be explained by the different Cecido­
myiidae taxonomic groups, that the Panamanian gall midge population
is likely still increasing, and/or Physothorax abundance. Presence of ants
in trees might also reduce the infestation of Cecidomyiidae like it doesfor parasitic wasps (Jandér 2015) and fig consuming moth larvae (vanKolfschoten et al., submitted).
In our data set about two thirds of F. citrifolia trees were infested withgall midges both in 2022 and 2023. On trees with gall midges, over 50%
of figs were infested in 2023. This relatively high infestation rate could
6

H.R. Hedberg et al.

Acta Oecologica 122 (2024) 103975

Fig. 6. Boxplots comparing the (A) number of seeds and (B) number of fig wasp (P. tonduzi) offspring between three different treatment groups. There was no
significant difference between the groups when tree ID was accounted for as a random factor (A: F2,120 = 0.169, p = 0.85; B: F2,120 = 1.85, p = 0.16).

Fig. 7. Regression graphs showing no significant relation between the number of midge galls in a fig and (A) the number of seeds and (B) the number of fig wasp
(P. tonduzi) offspring (A: t121 = − 0.440, p = 0.66; B: t121 = − 0.446, p = 0.66). The blue dots represent figs in which the Physothorax was present.

hypothetically have negative impact on the F. citrifolia population if thenumber of midge galls per fig increases. We would like to point out thatthe infestation rates mentioned here are estimates based on the branches
that were accessible for inspection, but there could be considerable
variation among branches even within the same tree. Some trees were
uniformly infested by gall midges while other trees had some brancheswith no visible signs of gall midge emergence spots and other brancheswith clusters of gall midge figs.
The gall midge had no significant effect on seed number or number ofpollinator (P. tonduzi) offspring in this study (Figs. 6 and 7, Table A2).
However, if the gall midge effect on P. tonduzi offspring number was
significant, the effect we found was so small that we suggest it could be
neglected with this level of infestation, especially compared to othersources of variation in reproductive success (Herre 1989; Herre 1993,
West et al. 1996, Jandér and Herre 2010; van Kolfschoten et al., sub­mitted). Although we expect that the gall midges might have a larger

impact on the fig tree - fig wasp mutualism if the infestation rate in­creases. High levels of fig-associated Cecidomyiidae infestations havesevere negative effects on the fig tree - fig wasp mutualism on Ficus
benjamina in China due to fig abortion, lower number of seeds (20–50%
decrease in Cecidomyiidae infested figs) and pollinator offspring(90–100% decrease in Cecidomyiidae infested figs) (Bai et al. 2008;
Miao et al. 2011). Another reason why seed number and P. tonduzi
offspring were not significantly affected in our study might be because
we only studied single foundress figs. One foundress might not becapable of saturating all available flowers in a fig and the fig might
therefore have sufficient resources for both the developing flowers andthe gall midge development.
The gall midge might also have a negative effect on the fig tree by
preventing fig abortion. Fig trees can save resources by aborting
unpollinated figs; F. citrifolia is known to do this (Jandér & Herre 2010,2016). If abortion is hindered the fig tree will continue to allocate
7H.R. Hedberg et al.

Acta Oecologica 122 (2024) 103975

resources to unpollinated figs without gaining any reproductive bene­fits. We found that fig abortion of unpollinated figs could be prevented
when a threshold of four or more gall midges inside one fig was reached,
presumably by the midge galls acting as a strong nutrient sink (Jandérand Herre 2016). Our observation also suggests that gall midge ovipo­
sition can occur before the pollinator P. tonduzi arrives.
The effect of the Ficus benjamina Cecidomyiidae in Bai et al.’s (2008)
study was more severe for pollinator offspring than for seeds. Bai et al.
hypothesized that the negative effect from Cecidomyiidae is caused bydiversion of nutrient resources from other contents of the fig to Ceci­
domyiidae. Pollinators appeared to be more susceptible to nutrient
diversion in comparison to fig seeds. However, Miao et al. (2011)
showed that seed weight and quality, measured by germination successand root growth rates, was greatly reduced whenever Cecidomyiidae
were present, while seed number was only affected at high Cecido­
myiidae densities. The germination success of Ficus benjamina in Miaoet al.‘s study was close to 100% for seeds from control figs, but only 23%
for seeds from figs containing Cecidomyiidae. A future study of nutrient
diversion and germination success in Panamanian gall midge figs would
be of importance to further understand how the gall midge affects the fig
tree - fig wasp mutualism. A first step might be to check if seed size andweight are altered in figs with gall midges.
Because Physothorax use the same gall as a gall midge, we expect theeffect of Physothorax on the fig tree - fig wasp mutualism to be similar toa gall midge’s effect. However, some other genera of non-pollinating fig
wasps (Idarnes, Critogaster, and Aepocerus) have negative effects on thepollinator and seed production (West et al. 1996). Physothorax as agenus are parasitoids of fig gallers with multiple hosts. Apart from
fig-associated Cecidomyiidae (this study, Roskam and Nadel 1990),
Physothorax have also been found consuming both Aepocerus (West et al.
1996; Conchou et al. 2014) and possibly Ficicola sp. (Conchou et al.
2014). Physothorax may depress populations of their Aepocerus hosts in
Ficus dugandi (West et al. 1996). Lower infestation rates of Physothorax
hosts, for example Aepocerus or Cecidomyiidae, may benefit the fig tree –fig wasp mutualism.
The gall midge of this study shares more similarities in bothmorphology and other aspects of its biology with Ficiomyia perarticulata(Roskam and Nadel 1990) than with the old-world Cecidomyiidae of
Ficus macrocarpa (Yafuso et al. 2013) and Ficus benjamina (Bai et al.
2008; Miao et al. 2011). Our specimens appear morphologically related
to Ficiomyia perarticulata (Roskam and Nadel 1990), but we have not yet
been able to send specimens to an expert for identification. However, we
believe the gall midge in this study to be in the Ficiomyia genus because
it has more than 25 antennal flagellomeres and the females had moreantennal flagellomeres than did the males (Gagné 2018). The only
species described so far in the Ficiomyia genus is Ficiomyia perarticulata
which have ♂ 29–31 and ♀ 37–39 stalked flagellomeres. Although the
Panamanian gall midge has more antennal flagellomeres for both sexes(♂ 32–33 and ♀ 42–47), the number of flagellomeres are known to be avariable trait in the Lasiopteridi, the supertribe of Ficiomyia (R. J. Gagné(personal communication 2022). The infestation rate of the gall midgeof this study (x = 2.64) is also more similar to F. perarticulata (a couple toseveral midge galls per fig (Roskam and Nadel 1990)), compared to theconsiderably higher rates of the Ficus macrocarpa Cecidomyiidae (x =25.5 (Yafuso et al. 2013)) and Ficus benjamina (x = 67.8 (Bai et al.
2008)). The gall midge of this study induces galls from the receptacle
like F. perarticulata (Roskam and Nadel 1990) while the Cecidomyiidae
of Ficus benjamina (Miao et al. 2011) and Ficus macrocarpa (Yafuso et al.
2013) develop inside galled female flowers. Furthermore, the emergenceholes of the Panamanian gall midge (Fig. 1) and F. perarticulata sharesimilarities in their crown-like appearance, pupae exuviae, and neither
emerges from host plant tissue projections like Ficus macrocarpa Ceci­
domyiidae (Yafuso et al. 2013). However, F. perarticulata galls have clear
sexual dimorphism and shared emergence holes (Roskam and Nadel
1990). Gall dimorphism and shared emergence holes were not observed
for the Panamanian gall midge.

Within the BOLD database our submitted sequence of the gall midge
was assigned a unique BIN, which can be seen as a proxy taxonomic unit
(Ratnasingham & Hebert 2007, 2013), and had a distance to the nearest
neighbour of 14.3%. This suggests that this is the first sequence of thisspecies to be uploaded to the BOLD system. The closest match that was
found in the BOLD database was an 86% overlap with a
non-fig-associated Cecidomyiidae. Physothorax has, to date, been
assigned a BIN. This might be because the uploaded sequences for the
Physothorax, were no longer than 400 bp. The nearest neighbour of the
Physothorax in the BOLD database had an 88% overlap, suggesting it not
to be the same species. Future taxonomic work and molecular phylo­
genetic analyses will hopefully clarify the species identities of both the
Panamanian gall midge and the Physothorax and might link Physothorax
to a future BIN.
We do not currently know if the gall midge has recently migrated toPanama or if it previously existed in the study area without being
noticed. Gall midges are generally difficult to notice by non-specialists
due to their small size. Cecidomyiidae presently contains less than7000 species (Gagné and Jaschof 2021), but recent estimates suggestthat there could be 1.8 million taxa (Hebert et al. 2016). The fig gall
midge could therefore be an undescribed native species. However, we
believe that the gall midge might be a recent arrival to the local fig
community because the Panama Canal area is intensely studied by fig
researchers, and fig gall midges have not been observed previously in­side figs (LD, KCJ, A. Gomez (personal communication), E. A. Herre(personal communication)). Further argument for a recent arrival is the
low infestation rate of the gall midge in 2022, followed by a seeming
increase in infestation rate in 2023. If the gall midge has recently
arrived, and if it can continue to survive and reproduce in Panama, wemight in the future see a further increased population with higher
infestation rates. The increase would continue until it reaches carrying
capacity or gets further stabilised by parasitoids. The Physothorax foundinside midge galls could have arrived simultaneously with the gall
midge, or later. Roskam and Nadel (1990) mentions Physothorax para­
sitoids for Ficiomyia perarticulata in Florida. They observed galls para­
sitised by Physothorax bidentulus (Burks 1969) and Physothorax russelli inthe same samples examined with Ficiomyia perarticulata. No
fig-associated Cecidomyiidae parasitoids are mentioned for Ficus
microcarpa nor Ficus benjamina (Bai et al. 2008; Miao et al. 2011; Yafuso
et al. 2013). Another alternative is that Physothorax could be an
opportunistic generalist that existed in the area before the gall midge.
Physothorax species are present in the study area as parasitoids of
non-pollinating fig wasps that produce large galls within figs (West et al.
1996; Marussich and Machado 2007; Conchou et al. 2014; Jandér 2015).
We would like to see further taxonomic and molecular phylogenetic
studies for the entire Neotropics to better understand prevalence andmovement of the gall midge and Physothorax.
5. Conclusion
The gall midge found inside figs of Ficus citrifolia in central Panama ispresumably either a recent arrival to the area, an infrequent visitor, oran endemic species previously unnoticed. Our species identification andbarcoding suggest that (1) the gall midge most likely belongs to thegenus Ficiomyia, close to Ficiomyia perarticulata, and (2) the parasitoid
wasp belongs to the genus Physothorax, close to Physothorax russelli. In
this study infestation rates were low, and the gall midge had no signif­
icant effect on the reproduction of the fig tree - fig wasp mutualism.
However, should infestation rates increase, it is likely that the gall midge
would affect the mutualism negatively.
Data accessibility
Data and R code is available on FigShare (DOI:10.6084/m9.
figshare.24910983)

8

H.R. Hedberg et al.

Acta Oecologica 122 (2024) 103975

Funding

the work reported in this paper.

Sederholm Stipends (LvK), the B. Lundman’s Foundation for Botan­
ical Studies (LvK and HRH), and the Swedish Research Council (KCJ).

Data availability
Data will be made available on request.

Authors contributions

Acknowledgements

Lovisa Dück: Conceptualization (lead), Methodology (supporting),
Investigation (supporting), Writing – review and editing (supporting),supervision (supporting). K. Charlotte Jandér: Conceptualization (sup­porting), Methodology (supporting), Writing – original draft (support­ing); Writing – review and editing (supporting), Supervision(supporting). H. Roberta Hedberg: Conceptualization (lead), Method­
ology (lead), Formal analysis (equal), Investigation (lead), Writing –original draft (lead); Writing – review and editing (equal). Lisette vanKolfschoten: Conceptualization (supporting), Methodology (support­ing), Formal analysis (equal), Investigation (supporting), Writing –original draft (supporting); Writing – review and editing (equal), Su­
pervision (lead).

We want to thank Finn Piatscheck and Adalberto Gomez for valuable
field information, Raymond J. Gagné for advice on species identificationof the gall midge, Jean-Yves Rasplus for identification of the Phys­
othorax, Martin Lind for statistical advice, Simon Segar and Yvonne
Meyer-Lucht for advice on the barcoding and understanding the BOLD
system, Finn Kjellberg for helpful comments on the manuscript, and two
anonymous reviewers for comments that helped imporve the manu­
script. We also want to thank Jon Ågren for his contribution as a su­
pervisor of the master project of HRH and in helping to develop theresearch question, as well as his help with the project’s practicalities. We
are thankful for financial support from Sederholm Stipends (LvK), the B.
Lundman’s Foundation for Botanical Studies (LvK and HRH), and theSwedish Research Council (KCJ). We want to thank the Smithsonian
Tropical Research Institute for maintaining the facilities that made thisstudy possible.

Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence

Appendix

Fig. A1. Structures of dissected figs, here split into quarters and pinned for dissection. Control fig (left) and Gall midge fig (right). Fig structures are describedby arrows.
Table A1
Number of dissected figs (2022) included in the study per sampled tree.
Tree ID

Control figs

Gall midge figs

Gall midge þ Physothorax

collection Date

1
2
3
4
5
6
7
8

10
16
14
–
–
5
5
5

10
16
14
–
–
5
3
5

5
–
1
–
–
5
3
–

17 Feb 22
22 Feb 22
24 Feb 22
25 Feb 22
08 Mar 22
08 Mar 22
16 Mar 22
09 Apr 22

9

H.R. Hedberg et al.

Acta Oecologica 122 (2024) 103975

Table A2
The results of the regressions (LMER). Degree of syconia gall midge infestation (independent variable) against the different measured response variables. Presence of
the Physothorax parasitoid was set as a fixed effect and tree ID as random factor.
Response variables

Fixed effect

EstimateSEdft-value

Pr (>|t|)

Fig seeds

Intercept
Gall midge
Physothorax
Gall midge + Physothorax
Intercept
Gall midge
Physothorax
Intercept
Gall midge
Physothorax
Intercept
Gall midge
Physothorax
Intercept
Gall midge
Physothorax

119− 0.696− 8.003.22
171− 0.98010.1
155− 0.427
5.56
418− 3.2812.0
84.6− 2.80− 2.45

4.00
1.58
10.1
2.23
10.2
2.20
8.73
14.0
2.35
9.40
15.176
2.22
8.88
15.4
1.80
7.25

3.19
118
117
115
6.61
117
119
5.93
116
119
5.64
115
118
5.50
115
117

29.7
− 0.440− 0.791
1.45
16.7− 0.4461.15
11.1− 0.1810.591
27.5− 1.48
1.35
5.48− 1.55− 0.339<0.001
0.661
0.430
0.151
<0.0010.657
0.252<0.0010.856
0.556<0.0010.141
0.180
0.00204
0.123
0.736

Total NO. of fig wasp offspring
Empty fig wasp galls
Total no. of flowers
Undeveloped flowers

References

Felt, E.P., 1922. A new and remarkable fig midge. Fla. Entomol. 6, 5–6.
Frainer, A., McKie, B.G., Amundsen, P.-A., Knudsen, R., Lafferty, K.D., 2018. Parasitism
and the biodiversity-functioning relationship. Trends Ecol. Evol. 33 (4), 260–268.
Frank, J.H., Nadel, H., 2012. Life cycle and behaviour of Charoxus spinifer and Charoxus
major (Coleoptera: Staphylinidae: Aleocharinae), predators of fig wasps(Hymenoptera: Agaonidae). J. Nat. Hist. 46 (9–10), 621–635.
Gagné, R.J., 1989. The Plant-Feeding Gall Midges of North America. Comstock Pub.
Associates.
Gagné, R.J., 2018. Key to adults of North American genera of the subfamily
cecidomyiinae (Diptera: Cecidomyiidae). Zootaxa 4392 (3), 401–457.
Gagné, R.J., Jaschof, M., 2021. A Catalog of the Cecidomyiidae (Diptera) of the World,
fifth ed. Digital. U.S. Department of Agriculture, c/o Smithsonian Institution,
Washington, D.C.
Galil, J., Eisikowitch, D., 1968. Flowering cycles and fruit types of FICUS sycomorus in
Israel. New Phytol. 67, 745–758.
Gan, S.-R., Du, W., Wang, X.-F., 2022. Functional differentiation of floral color and scentin gall midge pollination: a study of a schisandraceae plant. Plants 11, 974.
Giblin-Davis, R.M., Center, B.J., Nadel, H., Frank, J.H., Ramirez, W., 1995. Nematodes
associated with fig wasps, Pegoscapus spp. (Agaonidae), and syconia of native
Floridian figs (Ficus spp.). J. Nematol. 27 (1), 1.
Hebert, P.D.N., Ratnasingham, S., Zakharov, E.V., Telfer, A.C., Levesque-Beaudin, V.,
Milton, M.A., Pedersen, S., Jannetta, P., deWaard, J.R., 2016. Counting animal
species with DNA barcodes: Canadian insects. Philisophical Transac. Royal Soc. B
371, 20150333.
Hearn, J., Blaxter, M., Schönrogge, K., Nieves-Aldrey, J.-L., Pujade-Villar, J., Huguet, E.,
et al., 2019. Genomic dissection of an extended phenotype: oak galling by a cynipid
gall wasp. PLoS Genet. 15 (11), e1008398.
Herre, E.A., 1987. Optimality, plasticity and selective regime in fig wasp sex ratios.
Nature 329, 627–629.
Herre, E.A., 1989. Coevolution of reproductive characteristics in 12 species of New
World figs and their pollinator wasps. Experientia 45, 637–647.
Herre, E.A., 1993. Population structure and the evolution of virulence in nematodeparasites of fig wasps. Science 259 (5100), 1442–1445.
Herre, E.A., Jandér, K.C., Machado, C.A., 2008. Evolutionary ecology of figs and theirassociates: recent progress and outstanding puzzles. Annu. Rev. Ecol. Evol. Syst. 39,
430–458.
Hsieh, Y.-T., Lin, S.-F., Liao, S.-F., 2021. First report of insect galls on Ficus macrocarpa
in penghu islands, taiwan. Taiwanese J. Entomol. Stud. 6 (4), 30–38.
Jackson, A.P., 2004. Cophylogeny of the Ficus microcosm. Biol. Rev. 79, 751–768.
Jandér, K.C., Herre, E.A., 2010. Host sanctions and pollinator cheating in the fig tree – fig
waspmutualism. Proc. Royal Soc. B 277, 1481–1488.
Jandér, K.C., Herre, E.A., 2016. Host sanctions in Panamanian Ficus are likely based on
selective resource allocation. Am. J. Bot. 103 (10), 1753–1762.
Jandér, K.C., 2015. Indirect mutualism: ants protect fig seeds and pollen dispersers form
prasites. Ecol. Entomol. 40, 500–510.
Janzen, D.H., 1966. Coevolution of mutualism between ants and acacias in Central
America. Evolution 20, 249–275.
Kjellberg, F., Jousselin, E., Hossaert-McKey, M., Rasplus, J., 2005. Biology, ecology, andevolution of fig-pollinating wasps (chalcidoidea, Agaonidae). In: Raman, A.,
Schaefer, C.W., Withers, T.M. (Eds.), Biology, Ecology and Evolution of GallInducing Arthropods. Science Publishers Inc, NY, USA, pp. 539–571.
Kolesik, P., Gagné, R.J., 2020. A review of the gall midges (Diptera: Cecidomyiidae) of
Indonesia: taxonomy, biology and adult key to genera. Zootaxa 4847 (1), 1–82.
Korine, C., Kalko, E.K., Herre, E.A., 2000. Fruit characteristics and factors affecting fruit
removal in a Panamanian community of strangler figs. Oecologia 123 (4), 560–568.
Krogaonkar, A., Han, C., Lemire, A.L., Siwanowicz, I., Bennouna, D., Kopec, R.E.,
Andolfatto, P., Shigenobu, S., Stern, D.L., 2021. A novel family of secreted insect
proteins linked to plant gall development. Curr. Biol. 31, 1836–1849.

Aljanabi, S.M., Martinez, I., 1997. Universal and rapid salt-extraction of high quality
genomic DNA for PCR-based techniques. Nucleic Acids Res. 25, 4692–4693.
Antoine, S., Hériché, M., Boussageon, R., Pierre-Antoine, N., van Tuinen, D., Wipf, D.,
Courty, P.E., 2021. A Historical Perspective on Mycorrhizal Mutualism Emphasizing
Arbuscular Mycorrhizas and Their Emerging Challenges, vol. 31. Springer,
Mycorrhiza, pp. 637–653.
Bai, L., Yang, D., Compton, S.G., 2008. A gall midge inhabiting the figs of Ficus benjamina
in Xishuangbanna, south-western China. Symbiosis 45, 149–152.
Bain, A., Harrison, R.D., Schatz, B., 2014. How to be an ant on figs. Acta Oecol. 57,
97–108.
Bates, D., Machler, M., Bolker, B., Walker, S., 2015. Fitting linear mixed-effects models
using lme4. J. Stat. Software 67 (1), 1–48.
Berg, C.C., 2007. Proposal for treating four species complexes in Ficus subgenus
Urostigma section Americanae (Moraceae). Blumea 52, 295–312.
Biedermann, P.H.W., Vega, F.E., 2020. Ecology and evolution of insect-fungus
mutualisms. Annu. Rev. Entomol. 65, 431–455.
Boucher, D.H., James, S., Keeler, K.H., 1982. The ecology of mutualism. Annu. Rev. Ecol.
Systemat. 13, 315–347.
Boucek, Z., 1993. The genera of chalcidoid wasps from Ficus fruit in the New World.
Journal. Hist. 27 (1), 173–217.
Bronstein, J.L., 1991. The nonpollinating wasp fauna of Ficus pertusa: exploitation of amutualism? Oikos 61, 175–186.
Burks, B.D., 1969. Redefinition of two genera of chalcidoids from figs, with new Florida
species (hymenoptera). Fla. Entomol. 52, 115–122.
Conchou, L., Ciminera, M., Hossaert-McKey, M., Kjellberg, F., 2014. The non-pollinating
fig wasps associated with Ficus guianensis: community structure and impact of thelarge species on the fig/pollinator mutualism. Acta Oecol. 57, 28–37.
Costa, P.C., Graciolli, G., 2010. Insects associated with syconia of Ficus citrifolia(Moraceae) in central Brazil. Rev. Bras. Entomol. 54 (4), 707–709.
Chomicki, G., Weber, M., Antonelli, A., Bascompte, J., Kiers, E.T., 2019. The impact ofmutualisms on species richness. Trends Ecol. Evol. 34 (8), 698–711.
Cook, J.M., Rasplus, J., 2003. Mutualists with attitude: coevolving fig wasps and figs.
Trends Ecol. Evol. 18 (5), 241–248.
Croat, T.B., 1978. Flora of Barro Colorado Island. California. Stanford University Press,
Stanford.
Compton, S.G., Robertson, H.G., 1988. Complex interactions between mutualisms: ants
tending Homopterans protect fig seeds and pollinators. Ecology 69, 1302–1305.
Cruaud, A., Rønsted, N., Chantarasuwan, B., Chou, L.S., Clement, W.L., Couloux, A.,et al., 2012. An extreme case of plant-insect codiversification: figs and fig-pollinating
wasps. Syst. Biol. 61 (6), 1029–1047.
Dunn, D.W., Jandér, K.C., Lamas, A.G., Pereira, R.A.S., 2014. Mortal combat andcompetition for oviposition sites in female pollinating fig wasps. Behav. Ecol. 26 (1),
262–268.
Elias, L.G., Menezes Jr., A.O., Pereira, R.A.S., 2008. Colonization sequence of nonpollinating fig wasps associated with Ficus citrifolia in Brazil. Symbiosis 45, 107–111.
Elias, L.G., Teixeira, S.P., Kjellberg, F., Pereira, R.A.S., 2012. Diversification in the use ofresources by Idarnes species: bypassing functional constraints in the fig-fig wasp
interaction. Biol. J. Linn. Soc. 106, 114–122.
Farache, F.H.A., Cruaud, A., Genson, G., Rasplus, J., Pereira, R.A.S., 2017. Taxonomic
revision and molecular phylogenetics of the Idarnes incertus species-group(Hymenoptera, Agaonidae, Sycophaginae). PeerJ 5, e2842.
Farache, F.H.A., Cruaud, A., Rasplus, J., Cerezini, M.T., Rattis, L., Kjellberg, F.,
Pereira, R.A.S., 2018. Insights into the structure of plant-insect communities:
specialism and generalism in a regional set of non-pollinating fig wasp communities.
Acta Oecologica, Elsevier 90, 49–59.

10H.R. Hedberg et al.

Acta Oecologica 122 (2024) 103975
Shanahan, M., So, S., Compton, S.G., Corlett, R., 2001. Fig-eating by vertebrate
frugivores: a global review. Biol. Rev. 76, 529–572.
Tanaka, Y., Okada, K., Asami, T., Suzuki, Y., 2013. Phytohormones in Japanese mugwort
gall induction by a gall-inducing gall midge. Biosci. Biotechnol. Biochem. 77 (9),1942–1948.
Tooker, J.F., Helms, A.M., 2014. Phytohormone dynamics associ- ated with gall insects,
and their potential role in the evolution of the gall-inducing habit. J. Chem. Ecol. 40,
742–753.
van Kolfschoten, L., Dück, L., Lind, M.I., Jandér, K.C., 2022. Rising temperatures
threaten pollinators of fig trees—keystone resources of tropical forests. Ecol. Evol. 12(9), e9311.
van Kolfschoten, L., Adu Asantewaa, M., Dück, L., Segar, S., & Jandér, K. C. submitted.
Fitness Losses Inflicted by Specialist Fig-Consuming Moths Can Be Mitigated by Ant
Bodyguards.
Wang, B., Geng, X.Z., Bin Ma, L., Cook, J.M., Wang, R.W., 2014. A trophic cascade
induced by predatory ants in a fig-fig wasp mutualism. J. Anim. Ecol. 83,
1149–1157.
Weiblen, G.D., 2002. How to be a fig wasp. Annu. Rev. Entomol. 47, 299–330.
West, S.A., Herre, E.A., Windsor, D.M., Green, P.R.S., 1996. The ecology and evolution of
the New World non-pollinating fig wasp communities. J. Biogeogr. 23 (4), 447–458.
West, S.A., Herre, E.A., 1998. Partial local mate competition and the sex ratio: a study on
non-pollinating fig wasps. J. Evol. Biol. 11 (5), 531–548.
Wiebes, J.T., 1979. Co-evolution of figs and their insect pollinators. Annu. Rev. Ecol.
Systemat. 10 (1), 1–12.
Wiebes, J.T., 1995. Agaonidae (hymenoptera Chalcidoidea) and Ficus (Moraceae): fig
wasps and their figs, xv (Meso-American Pegoscapus). Proc. R. Neth. Acad. Arts Sci.
98 (2), 167–183.
Yafuso, M., Adaniya, S., Yukawa, J., 2013. Host plant manipulation by fig gall midges(Diptera: Cecidomyiidae) which induce emergence projections on flower galls in thesyconia of Ficus microcarpa (Moraceae). Entomol. Sci. 16 (4), 413–420.
Yukawa, J., Kim, W., 2021. Behavior. In: Yukawa, J., Tokuda, M. (Eds.), Biology of Gall
Midges. Entomology Monographs, pp. 151–166.
Zhao, C., Escalante, L.N., Chen, H., Benatti, T.R., Qu, J., Chellapilla, S., Waterhouse, R.
M., Wheeler, D., Andersson, M.N., Bao, R., et al., 2015. A massive expansion ofeffector genes underlies gall-formation in the wheat pest Mayetiola destructor. Curr.
Biol. 25, 613–620.

Lafferty, K.D., Kuris, A.M., 2012. Ecological consequences of manipulative parasites:
chapter 9. In: Hughes, D.P., Brodeur, J., Thomas, F. (Eds.), Host Manipulation by
Parasites. Oxford Universal Press, Oxford, pp. 158–168.
Machado, C.A., Robbins, N., Gilbert, M.T., Herre, E.A., 2005. Critical review of hostspecificity and its coevolutionary implications in the fig/fig-wasp mutualism. Proc.
Natl. Acad. Sci. USA 102, 6558–6565.
Marcogliese, D.J., 2004. Parasites: small players with crucial roles in the ecologicaltheater. EcoHealth 1 (2), 151–164.
Marussich, W.A., Machado, C.A., 2007. Host-specificity and coevolution among
pollinating and nonpollinating New World fig wasps. Mol. Ecol. 16 (9), 1925–1946.
Miao, B., Yang, D., Liu, C., Peng, Y., Compton, S.G., 2011. The impact of a gall midge on
the reproductive success of Ficus benjamina, a potentially invasive fig tree. Biol.
Control 59 (2), 228–233.
Nason, J.D., Herre, E.A., Hamrick, J.L., 1998. The breeding structure of a tropical
keystone plant resource. Nature 391, 685–687.
Oates, C.N., Denby, K.J., Myburg, A.A., Slippers, B., Naidoo, S., 2016. Insect gallers andtheir plant hosts: from omics data to systems biology. Int. J. Mol. Sci. 17 (11), 1891.
Ollerton, J., Winfree, R., Tarrant, S., 2011. How many flowering plants are pollinated byanimals? Oikos 120 (3), 321–480.
Palmieri, L., Pereira, R.A.S., 2018. The role of non-fig-wasp insects on fig tree biology,
with a proposal of the F phase (Fallen figs). Acta Oecol. 90, 132–139.
Piatscheck, F., Van Goor, J., Houston, D.D., Nason, J.D., 2018. Ecological factors
associated with pre-dispersal predation of fig seeds and wasps by fig-specialist
lepidopteran larvae. Acta Oecol. 90, 151–159.
Poulin, R., Morand, S., 2004. Parasite Biodiversity. Smithsonian Books, Washington.
Preston, D.L., Mischler, J.A., Townsend, A.R., Johnson, P.T.J., 2016. Disease ecology
meets ecosystem science. Ecosystems 19, 737–748.
Ratnasingham, S., Hebert, P.D.N., 2007. BOLD: the Barcode of life data system. Mol.
Ecol. Notes 7, 355–364. www.barcodinglife.org.
Ratnasingham, S., Hebert, P.D.N., 2013. A DNA-based registry for all animal species: theBarcode Index number (BIN) system. PLoS One 8.
Roskam, J.C., Nadel, H., 1990. Redescription and immature stages of Ficiomyia
perarticulata (Diptera: Cecidomyiidae), a gall midge inhabiting syconia of Ficus
citrifolia. Proc. Entomol. Soc. Wash. 92 (4), 778–792.
RStudio Team, 2020. RStudio. Integrated Development Environment for R. RStudio, PBC,
Boston, MA. URL: http://www.rstudio.com/. (Accessed 22 May 2022).
Schatz, B., Proffit, M., Rakhi, B.V., Borges, R.M., Hossaert-Mckey, M., 2006. Complex
interactions on fig trees: ants capturing parasitic wasps as possible indirect
mutualists of the fig-fig wasp interaction. Oikos 113, 344–352.

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