bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Genome-wide sequence data show no evidence of admixture and introgression among pollinator wasps associated with a community of Panamanian strangler figs

Jordan D. Satler1,*, Edward Allen Herre2, Tracy A. Heath1, Carlos A. Machado3, Adalberto Gómez Zúñiga2, and John D. Nason1

1Department of Ecology, Evolution, and Organismal Biology, Iowa State University, Ames, Iowa 50011 2Smithsonian Tropical Research Institute, Unit 9100 Box 0948, DPO AA 34002-9998, USA 3Department of Biology, University of Maryland, College Park, Maryland, USA 20742 *Corresponding author: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1 Abstract

2 Interactions between plants and their pollinators can shape processes of divergence

3 and gene flow within associated lineages. For example, in the obligate mutualism between figs

4 () and fig pollinator wasps (family ), each wasp typically pollinates a

5 single fig species, potentially reinforcing reproductive isolation among different wasp species.

6 Multiple pollinator species, however, can sometimes reproduce in the same host fig species,

7 potentially enabling hybridization and introgression between wasp species. In a community

8 of Panamanian strangler figs (section Americana), we use genome-wide ultraconserved ele-

9 ment (UCE) loci to estimate phylogenetic relationships and test for hybridization and gene

10 flow among 19 pollinator species associated with 16 host fig species. Previous studies show-

11 ing ongoing pollinator sharing and a history of pollinator host switching are consistent with

12 documented genetic admixture in their host figs. Here we investigate if host sharing and

13 a dynamic evolutionary history including host switching has also resulted in hybridization

14 and gene flow between pollinator species. Phylogenetic analyses recover strong support for

15 well-delimited wasp species coupled with high interspecific divergence. There is no evidence

16 for ongoing hybridization or introgression, even among pairs of pollinator species currently

17 reproducing within the same host. In contrast to work suggesting admixture among Panama-

18 nian host figs, we conclude hybridization and interspecific gene flow have not been important

19 processes shaping the evolutionary history of their pollinating wasps.

20 Keywords: pollination mutualism, introgression, hybridization, phylogeny, Pegoscapus, Fi-

21 cus

1 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

22 Introduction

23 Hybridization produces offspring exhibiting novel genetic combinations derived from di-

24 vergent parental genomes. Introgression results in genetic material moving between species.

25 The influx of genetic material into a lineage introduces new alleles and multilocus combi-

26 nations that, depending on the situation, can be either harmful (Rhymer and Simberloff,

27 1996) or beneficial (Dowling and Secor, 1997). In many lineages, strong pre- and postzy-

28 gotic barriers can limit hybridization and reinforce species boundaries (e.g., Nosil et al.,

29 2005). Evidence from next-generation sequencing, however, has revealed that hybridization

30 and introgression have occurred throughout the evolutionary history of the tree of life (Mallet

31 et al., 2016; Taylor and Larson, 2019). Identifying the ecological processes that either main-

32 tain or undermine species boundaries is useful for generating testable hypotheses concerning

33 factors that affect the evolutionary importance of hybridization and introgression (Twyford

34 and Ennos, 2012).

35 Host specialization in plant-associated generally leads to a reduction in interspe-

36 cific interactions, limiting opportunities for hybridization. This is particularly true for brood

37 pollination mutualisms (e.g., figs and fig wasps, yuccas and yucca moths, globeflowers and

38 globeflower flies) that often exhibit relatively species-specific host–pollinator relationships

39 (Hembry and Althoff, 2016). Figs (Ficus; ca. 800 species) and their obligately-associated

40 pollinating wasps (family Agaonidae) present evolutionarily replicated systems that often

41 vary in their potential for hybridization. While most figs appear to be associated with a

42 single, distinct pollinator fig wasp species, there are many cases where fig species are pol-

43 linated by multiple, co-occurring pollinator species (e.g., Wiebes, 1995; Kerdelhué et al.,

44 1997; Molbo et al., 2004; Machado et al., 2005; Jackson et al., 2008; Wang et al., 2016; Sut-

45 ton et al., 2017). Further, at the species range scale about 30% of figs are pollinated by more

46 than one wasp species (Yang et al., 2015). What is certain is that when wasps share the same

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47 host—especially when different wasp species reproduce in the same individual figs—there are

48 opportunities for hybridization in either the host fig, the pollinator wasp, or both.

49 Fig syconia—urn-shaped, enclosed inflorescences—define the genus Ficus. Chemical, be-

50 havioral, and morphological traits are thought to be important for the female pollinating

51 wasp to identify, locate, and enter a receptive fig (Herre et al., 2008). When a female wasp

52 arrives at a fig tree whose receptivity is signaled by fig floral volatiles (Van Noort et al., 1989;

53 Ware et al., 1993; Grison-Pigé et al., 2002; Hossaert-McKey et al., 2010), she must enter a

54 syconium through a small terminal pore (the ostiole) that excludes other insects. Once inside,

55 the foundress wasp pollinates flowers, oviposits into a subset of them, and dies within the

56 fig (Janzen, 1979). Offspring then develop over several weeks inside the galled, univolulate

57 fig flowers (Galil and Eisikowitch, 1968). Male wasps emerge from their galls, locate galls

58 that contain female wasps, and chew holes in the galls to expose females for mating. After

59 mating, female pollinators emerge from their galls, gather pollen from male flowers in the fig,

60 exit the syconium, and disperse. Female wasps routinely travel many kilometers to encounter

61 another receptive fig inflorescence—on typically a conspecific fig tree—to reproduce (Nason

62 et al., 1998; Ahmed et al., 2009).

63 Given this reproduction biology, if a fig is visited by a single pollinator wasp (foundress),

64 all pollinator wasp offspring will be her direct descendants—haploid sons and diploid daughters—

65 and mating within the fig will be between siblings. In contrast, if two or more foundresses

66 enter and oviposit within the fig, there are opportunities for non-sibling mating. If these

67 foundresses represent different species, then there is the opportunity for hybridization. Since

68 figs vary substantially in their characteristic foundress numbers (Herre, 1989), opportunities

69 for outcrossing and hybridization in pollinator wasps vary among species. Thus, to potentially

70 have hybridization and interspecific gene flow between pollinators, multiple foundresses must

71 occur within the same fig syconium, and those foundresses must represent different species.

72 There are approximately 120 described species of strangler figs (Ficus subgenus Urostigma,

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73 section Americana) in the Neotropics (Berg, 1989). Figs in this group are pollinated by wasps

74 from the genus Pegoscapus (family Agaonidae). Previous studies have emphasized specific

75 associations of particular wasp species with particular host fig species, with its suggestion

76 of strict sense co-speciation of wasp and fig species (e.g., Ramírez, 1970; Bronstein, 1987).

77 Yet, cophylogenetic studies of strangler figs and their pollinators—throughout the Neotropics

78 (e.g., Cruaud et al., 2012) and within Panama (e.g., Jackson et al., 2008)—have identified

79 highly discordant phylogenetic patterns. This indicates that over evolutionary timescales

80 wasps are not particularly host-specific and host switching is an important evolutionary

81 process in this group. In particular, using genome-scale data coupled with a model-based

82 approach, Satler et al. (2019) estimated host switching to be the most important process

83 generating phylogenetic patterns in a community of Panamanian strangler figs and polli-

84 nating wasps. Although contemporary associations in this community are predominantly

85 species-specific, phylogenomic data indicate a dynamic evolutionary history punctuated by

86 host-switching events, with these events creating opportunities for hybridization among both

87 associated pollinator and fig species.

88 Hybrid pollinator wasps have been detected in the Panamanian community of strangler

89 figs. Molbo et al. (2003) sampled emerging pollinators from nine fig species. Using mitochon-

90 drial sequence data and microsatellite genotyping, they identified successful F1 hybrids in 4 of

91 457 (0.9%) broods sampled from one fig species, Ficus obtusifolia. These rare F1s occurred

92 between the two frequently co-occurring sister wasp species, Pegoscapus hoffmeyeri sp. A

93 and Pegoscapus hoffmeyeri sp. B, that locally pollinate Ficus obtusifolia. While occasional

94 wasp hybridization exists, it is unclear whether successful introgression occurs, and raises the

95 question of whether or not hybrids exhibit anything other than negligible fitness. Further,

96 Molbo et al. (2004) recovered two diploid males (out of 18 males sampled from four broods

97 containing hybrid females) among hybrid pollinators associated with F. obtusifolia. They

98 suggest a breakdown in the sex determination mechanism caused by hybridization as a fac-

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99 tor potentially reducing fitness among hybrid pollinators. Molbo et al.(2004) also collected

100 microsatellite data to test for hybridization between two more distantly related wasp species

101 associated with Ficus popenoei. Even though fruits with two foundresses are relatively com-

102 mon for F. popenoei, among 255 sampled wasps they recovered no evidence of hybridization.

103 These studies of co-occurring pairs of pollinator species suggest that while hybrids may be

104 occasionally produced, they are functionally sterile and do not contribute genetic material

105 to the next generation.

106 Here we estimate phylogenetic relationships and test for hybridization and introgression

107 among 19 species of pollinating wasps associated with a community of 16 species of Pana-

108 manian strangler figs. First, we use genome-wide sequence data representing ultraconserved

109 element loci to estimate a phylogeny of all sampled fig wasps to test if individual wasps

110 sampled from the same host fig species cluster together in phylogenetic space. Next, focusing

111 on five pairs of wasp species known to pollinate, develop, and mate within figs of the same

112 host fig species, we test for evidence of hybridization and gene flow among these co-occurring

113 pollinators. We then test for hybridization and gene flow more broadly among all pollinator

114 species. Finally, we consider the ecological and evolutionary processes shaping diversification

115 dynamics in these pollinator wasps and how they compare with diversification dynamics in

116 their associated host figs.

117 Materials & Methods

118 DNA sampling and sequencing

119 Pollinator wasps were collected from strangler fig species in the vicinity of Barro Col-

120 orado Island Nature Monument in the Canal Zone of central Panama (Table 1). Wasps were

121 allowed to emerge from mature figs in the lab and then stored in 95% ethanol or RNALater

122 for DNA extraction and analysis. A single wasp was selected per fig fruit for sequencing to

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123 ensure independence among samples. We also included four pollinator wasps from an unde-

124 scribed species (Pegoscapus sp.) associated with the Mexican strangler fig Ficus petiolaris to

125 serve as an outgroup; a broader survey has revealed this pollinator to be sister to all other

126 Central American Pegoscapus wasps (J. Satler, unpublished data). Including the outgroup,

127 we generated sequence data from 176 wasp samples representing 20 pollinator species, with

128 an average of 8.8 individuals per species (Table 1). Genomic DNA was extracted with a

129 Qiagen DNeasy Kit (Qiagen Inc., Valencia CA, USA). Illumina libraries were generated with

130 a KAPA Hyper Prep kit. Samples were sheared to an average size of ∼450 bp on a Covaris

131 sonicator. Following library construction described in Glenn et al. (2019), we grouped sam-

132 ples in sets of eight, and hybridized biotinylated RNA probes to capture targeted loci. We

133 used the hymenopteran probe v2 set of Branstetter et al. (2017) to target 2590 ultraconserved

134 element (UCE) loci. After probe hybridization and library amplification, size distributions

135 were checked on a Bioanalyzer and libraries were combined in equimolar concentrations for

136 sequencing. Libraries were sequenced on an Illumina sequencer targeting 150 bp paired-end

137 reads.

138 Data processing

139 We used Phyluce v1.6.7 (Faircloth, 2015) to process raw sequence reads and generate

140 data sets for downstream analysis. Sequence reads were cleaned with illumiprocessor v2.0.9

141 (Faircloth, 2013), a wrapper around Trimmomatic v0.39 (Bolger et al., 2014). Cleaned reads

142 were assembled into contigs with Trinity v2.0.6 (Grabherr et al., 2011). Contigs were then

143 aligned to the hymenopteran v2 UCE locus set to filter non-specific sequences. Loci were

144 subsequently aligned with MAFFT v7.407 (Katoh and Standley, 2013), and ambiguously

145 aligned sites were removed with Gblocks v0.91b (Castresana, 2000) using default parame-

146 ters. All loci sampled for a minimum of 70% of individuals were retained in the final data

147 set. Additionally, we generated a data set of phased alleles for certain downstream analyses

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Pollinator Wasp Host Fig N Foundresses Pegoscapus gemellus sp. A Ficus bullenei & Ficus popenoei 21 1.980 Pegoscapus gemellus sp. B Ficus popenoei 11 2.550 Pegoscapus gemellus sp. C Ficus bullenei 7 1.410 Pegoscapus insularis sp. A Ficus colubrinae & Ficus perforata 16 1.005 Pegoscapus insularis sp. B Ficus perforata 2 1.000 Pegoscapus orozcoi Ficus colubrinae 8 1.010 Pegoscapus hoffmeyeri sp. A Ficus obtusifolia 4 1.050 Pegoscapus hoffmeyeri sp. B Ficus obtusifolia 10 1.050 Pegoscapus sp. Ficus aurea 8 NA Pegoscapus tonduzi Ficus citrifolia 6 1.210 Pegoscapus estherae Ficus costaricana 5 NA Pegoscapus longiceps Ficus dugandii 6 2.160 Pegoscapus sp. Ficus gomezii 8 NA Pegoscapus piceipes Ficus nymphaeifolia 6 2.640 Pegoscapus herrei Ficus paraensis 12 1.050 Pegoscapus silvestrii Ficus pertusa 7 NA Pegoscapus sp. Ficus petiolaris 4 NA Pegoscapus lopesi Ficus near trigonata 10 2.570 Pegoscapus grandii Ficus trigonata 17 4.530 Pegoscapus baschierii Ficus turbinata 8 NA

Table 1: Pollinator wasp sampling. Information includes pollinator species, host fig species, the number of individual wasps sequenced, and the average number of foundresses per host(s). Foundress information, when present, is from Herre (1989). For figs that share a pollinator species, foundress numbers were averaged between the host figs.

148 following the outline provided by Andermann et al. (2018). Taking our aligned loci (before

149 the use of Gblocks), we mapped cleaned sequence reads for each individual to the UCE locus

150 set using BWA-MEM (Li, 2013) in bwa v0.7.17 (Li and Durbin, 2010). We then phased the

151 data with samtools v1.9 (Li et al., 2009) using the phase command, calling two alleles per

152 individual per locus. Loci were then realigned and cleaned as described above, with loci

153 sampled for a minimum of 70% of individuals retained for downstream analysis.

154 Phylogenetics

155 We used both concatenation and coalescent-based species tree methods to estimate phy-

156 logenetic relationships among the fig wasps. We first estimated a concatenated phylogeny

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157 using maximum likelihood (ML) in IQ-TREE v1.6.12 (Nguyen et al., 2015; Chernomor et al.,

158 2016). This approach allows us to test species monophyly as individuals are treated as tips

159 in the tree. The data set was partitioned by UCE locus, with each partition estimated un-

160 der the GTR + γ substitution model. Nodal support values were generated through 1,000

161 repetitions of the ultrafast bootstrap approximation (Hoang et al., 2018).

162 We used two coalescent-based approaches to estimate a species tree. Rather than as-

163 suming all genes evolved under the same tree topology, these methods allow for discordance

164 between the gene histories and species tree by explicitly accounting for the biological process

165 of incomplete lineage sorting. First, we used the program SVDquartets (Chifman and Ku-

166 batko, 2014) as implemented in PAUP* v4.0a166 (Swofford, 2003). SVDquartets uses site

167 patterns in the nucleotide data to estimate a phylogeny under the multi-species coalescent

168 model. We used SVDquartets in two ways. We initially estimated a lineage tree (SVDQLT ),

169 where all individuals are represented as tips in the tree, to confirm species assignment and

170 test species monophyly. We then assigned individuals to species a priori and estimated a

171 species tree (SVDQST ). For both SVDquartets analyses, we evaluated all quartets and used

172 standard bootstrapping to generate nodal support values. Second, we used the program

173 ASTRAL-III v5.6.3 (Zhang et al., 2018) to estimate a species tree. Instead of using site pat-

174 terns in the nucleotide data, ASTRAL-III uses gene trees as input to estimate a species tree.

175 Maximum likelihood gene trees were first estimated in IQ-TREE. For each locus, IQ-TREE

176 selected the substitution model of best fit with ModelFinder (Kalyaanamoorthy et al., 2017)

177 using Bayesian Information Criteria (BIC). We assigned individuals to species a priori (Ra-

178 biee et al., 2019), and then used the ML gene trees as input to estimate a species tree. Nodal

179 support values were quantified with local posterior probability values (Sayyari and Mirarab,

180 2016).

181 Previous work in the Panamanian system has recovered pollinator species as monophyletic

182 and has not brought into question species validity (Machado et al., 2005; Jackson et al., 2008;

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183 Satler et al., 2019). Given the typical pattern of small intraspecific divergence coupled with

184 large interspecific divergence for these wasps, we wanted to ask what proportion of loci

185 recover each species as monophyletic. If the pollinators have been evolving in isolation over

186 evolutionary time, we would expect the species to be monophyletic for all or nearly all sampled

187 loci. In contrast, if interspecific gene flow has been an important process in the evolution of

188 this system, we would expect interacting species to show a lack of monophyly for numerous

189 loci. We used DendroPy v4.4.0 (Sukumaran and Holder, 2010) to count the proportion of

190 gene trees (as estimated above in IQ-TREE) for which a species was monophyletic. We

191 required that at least two individuals were sequenced for a species for a given gene tree to

192 assess monophyly.

193 Population genetics

194 In addition to estimating phylogenetic relationships and testing for monophyly, we were

195 interested in understanding the population genetics of the pollinator species. In particular,

196 we wanted to test if genetic diversity within a species varied with average foundress number.

197 If outbreeding is common among species that contain multiple foundresses per fruit, we

198 would expect to see a positive correlation between genetic diversity and average foundress

199 number. We used DendroPy to calculate nucleotide diversity (π), number of segregating

200 sites, and Watterson’s theta (per site) for all pollinator species. For species with foundress

201 number data, we used Pearson’s correlation test in R v3.5.2 (R Core Team, 2018) to test for

202 a correlation between average number of foundresses and summary statistic.

203 Testing for monophyly with mitochondrial DNA

204 To complement analyses and inference with the genome-wide data from the nuclear

205 genome, we used mitochondrial DNA (mtDNA) to test if pollinator species were mono-

206 phyletic in the mitochondrial genome. Even when not targeted, mitochondrial DNA is often

207 collected using sequence capture approaches (do Amaral et al., 2015; Barrow et al., 2017).

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208 We used NOVOPlasty v3.8.3 (Dierckxsens et al., 2017) to identify mitochondrial reads and

209 generate haplotypes from the sequencing files. Since there is no Pegoscapus mitochondrial

210 reference genome available, we aligned reads to a Pegoscapus cytochrome oxidase I (COI)

211 mtDNA sequence (AY148119). We used default settings with an initial kmer value of 39.

212 For samples that either did not generate any matches or produced obvious sequencing errors,

213 we lowered the kmer value to 23. Using a lower kmer value can help with low coverage

214 data, appropriate here since we were only targeting regions of the nuclear genome. Finally,

215 for samples that did not recover any mtDNA, we aligned reads to a longer Pegoscapus COI

216 sequence of a different species (JN103329) using default settings. Mitochondrial haplotypes

217 were aligned with MAFFT and edge trimmed to match the primary seed sequence to reduce

218 missing data. We estimated a maximum likelihood gene tree in IQ-TREE, using ModelFinder

219 to select the substitution model and 1,000 repetitions of the ultrafast bootstrap approxima-

220 tion to generate nodal support. If patterns of monophyly differ between the nuclear and

221 mitochondrial genomes, the cytonuclear discordance would suggest introgression or processes

222 other than genetic drift generating these discordant patterns.

223 Testing for admixture and gene flow

224 Detailed studies of Neotropical strangler figs and their pollinators have revealed excep-

225 tions to the typical 1:1 association between fig and wasp species (Molbo et al., 2003, 2004;

226 Machado et al., 2005). Cases of host sharing, where multiple pollinator species are associated

227 with the same host, are of particular interest because they present clear opportunities for

228 hybridization and introgression. Five cases of host sharing have been described in central

229 Panama: (i and ii) Ficus bullenei and Ficus popenoei share a pollinator, Pegoscapus gemellus

230 sp. A, which co-occurs with Pegoscapus gemellus sp. C in F. bullenei and Pegoscapus gemel-

231 lus sp. B in F. popenoei, resulting in two hosts each with two co-occurring pollinators, (iii

232 and iv) Ficus colubrinae and Ficus perforata share a pollinator, Pegoscapus insularis sp. A,

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233 which co-occurs with Pegoscapus orozcoi in F. colubrinae and Pegoscapus insularis sp B in

234 F. perforata, again resulting in two hosts each with two co-occurring pollinators, and (v)

235 Ficus obtusifolia is pollinated by two co-occurring species, Pegoscapus hoffmeyeri sp. A and

236 Pegoscapus hoffmeyeri sp. B. We refer to these three systems as BP (i and ii), CP (iii and

237 iv), and O (v) (see Figure 1). They include two pollinator species associated with two hosts

238 each, and six pollinator species that are host specific. It is the interaction of wasps within

239 the same fig hosts that provide the greatest opportunities for hybridization and gene flow.

240 The fig species with co-occurring pollinators vary in their average number of foundress

241 wasps per fig (Herre, 1989). Variation in foundress number enables predictions about the pol-

242 linator taxa most likely to contain histories of hybridization and gene flow. Ficus colubrinae

243 and F. perforata are nearly always single foundress (Table 1). Despite two pollinator species

244 associated with each fig species, we expect little opportunity for hybridization since more

245 than one foundress rarely occupies the same syconium. Ficus obtusifolia is mostly single

246 foundress, averaging 1.21 foundresses per fruit, although 16% of sampled fruits had between

247 two and four foundresses (Herre, 1989). We expect intermediate opportunities for hybridiza-

248 tion between the two pollinators associated with this host. Finally, F. bullenei averages 1.41

249 foundresses per fruit and F. popenoei averages 2.55 foundresses per fruit, providing frequent

250 opportunities for pollinator co-occurrence and, potentially, hybridization between their two

251 pairs of wasp species. Together, the five pairs of host-sharing pollinators vary substantially

252 in their expected opportunities for hybridization and interspecific gene flow.

253 We used two approaches to test for hybridization and admixture between co-occurring

254 pollinator species. First, we used principal components analysis (PCA). If sampled individu-

255 als are recent hybrids or backcrosses to parental lineages, we would expect to see individuals

256 intermediate between clusters representing parental lineages. Further, if introgression has

257 been extensive, there should be little separation of clusters in PCA space. PCA analysis was

258 conducted with the dudi.pca function in the R package ade4 (Dray et al., 2007). Missing

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259 data were replaced with mean values of allele frequencies within a given species. We plot-

260 ted the first two axes of variation to visualize genetic clustering of individuals. Second, we

261 used Structure v2.3.4 (Pritchard et al., 2000) to cluster individuals into genetic groupings.

262 Structure clusters individuals by maximizing Hardy-Weinberg equilibrium within clusters and

263 minimizing Hardy-Weinberg equilibrium among clusters. Evidence for recent hybridization

264 and introgression would be reflected by individuals containing multilocus genotypes sampled

265 from multiple clusters. Our choice of the number of clusters (K) was informed by previous

266 work identifying the wasp species within these systems (Molbo et al., 2003; Jackson et al.,

267 2008). For F. bullenei/F. popenoei (BP) and F. colubrinae/F. perforata (CP), we used a K

268 value of 3; for F. obtusifolia (O), we used a K value of 2. We used the admixture model, and

269 allowed allele frequencies to be correlated among populations. Each analysis had a burnin of

270 100,000 steps, followed by 500,000 MCMC reps, and was replicated 10 times. Results were

271 processed with the R package pophelper v2.2.7 (Francis, 2017).

272 For the pollinators in each host system (F. colubrinae and F. perforata, F. bullenei and

273 F. popenoei, F. obtusifolia), genetic data were processed independently to generate phased

274 data sets. By processing each taxon set independently, we retained loci with a minimum

275 of 70% for the taxa of interest only. We followed the same procedure as outlined above for

276 generating the phased data sets, and used unlinked SNPs for both the PCA and Structure

277 analyses. To generate these data sets, we first scanned our phased aligned taxon-specific

278 UCE loci for variable sites. Within a locus, we then selected the SNP that had the highest

279 sample coverage. If a locus had multiple SNPs with equal sample coverage, one of those

280 SNPs was selected at random.

281 Tests of hybridization among co-pollinators provide biologically motivated hypotheses

282 where opportunities for interspecific mating are most likely. If gene flow among these species

283 has been recent or ongoing, we would expect to detect that signal and infer a history of

284 hybridization. This approach, however, is limited to specific sets of taxa based on present

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285 day associations. Given the history of host switching in this system, and accounting for a

286 potential dynamic process of host–pollinator associations through time, it would be useful to

287 test for hybridization among all sampled taxa.

288 To test if historical introgression has been an important process in this community of

289 pollinators, we used TreeMix v1.13 (Pickrell and Pritchard, 2012). TreeMix is a maximum

290 likelihood approach that uses allele frequency data to construct a population graph and places

291 hybridization events on that graph for populations with the least fit to a tree model. Since

292 the number of hybridization events are specified a priori, we can test models varying the

293 number of hybridization events to determine a model of best fit. We used the total variance

294 explained by the model to inform the number of hybridization events that provides a best

295 fit for this community of pollinator species. We used allele frequency data representing

296 unlinked, biallelic SNPs from the phased data set from all pollinator species. To remove

297 effects of missing data, we first identified biallelic SNPs within each locus that were sampled

298 for at least one individual per species. For loci with multiple SNPs, we selected a single SNP

299 with the highest coverage. If a locus had multiple SNPs with equal sample coverage, one

300 SNP was selected at random. We then estimated a maximum likelihood population graph in

301 TreeMix, allowing between zero and five migration events.

302 Although additional approaches are often used to test for hybridization and introgression,

303 they were not applicable for our data set. For example, D-statistics (Green et al., 2010;

304 Durand et al., 2011), often referred to as ABBA/BABA tests, have been widely used to

305 infer a signal of introgression among numerous clades across the tree of life (e.g., Eaton and

306 Ree, 2013; Streicher et al., 2014; Meier et al., 2017; Hughes et al., 2020; Pulido-Santacruz

307 et al., 2020). D-statistics, however, can not estimate gene flow between sister species, and

308 our biologically informed hypotheses do not lend themselves well to the strict phylogenetic

309 pattern (((P1,P2),P3),OG) typically required for testing if one species (P3) has hybridized

310 with another (either species P1 or species P2). In addition, if we use an agnostic approach

13 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

311 and test all possible combinations of taxa, that would require 969 tests (19 choose 3), and

312 would potentially lead to artifacts from unsampled (“ghost”) lineages given the subsampling

313 approach (Pease and Hahn, 2015).

314 Results

315 DNA sampling and sequencing

316 We generated 468,733,333 total sequence reads from the 176 fig wasp samples, resulting

317 in an average of 2,663,258 (±1, 273, 010) reads per individual. Assembly in Trinity resulted

318 in an average of 139,295 (±80, 671) contigs per individual. After filtering and retaining

319 contigs that matched UCEs in the probe set, we retained an average of 1,590 (±179) UCE

320 loci per individual. Filtering loci for those that contained at least 70% taxon sampling,

321 we retained 1,504 loci for the complete taxonomic data set. In the three systems (processed

322 independently) with shared and unique pollinators, at 70% sequencing threshold, we retained

323 1,438 (BP), 1,388 (CP), and 1,390 (O) loci for analysis.

324 Phylogenetics

325 When individuals are treated as tips in the tree, phylogenetic analyses recover each pol-

326 linator species as monophyletic with strong support. This result is consistent with both

327 concatenation (Figure1) and in the coalescent-based lineage tree analysis with SVDquartets

328 (SVDQLT , Figure S1). To further investigate, we tested species monophyly for each sampled

329 UCE locus. All species were monophyletic for either all or nearly all loci (Table 2). 58% of

330 species were monophyletic in at least 95% of loci, while 84% of species were monophyletic

331 in at least 90% of loci. Only three species were monophyletic at less than 90% of their loci,

332 with the lowest proportion being 82% (P. gemellus sp. A (hosts: F. bullenei/F. popenoei).

333 For species with foundress data (see Table 1), there is no significant correlation between

334 average foundress number and proportion of monophyletic loci (Pearson’s correlation test,

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100 P. sp. (F. petiolaris) 100 P. sp. (F. aurea) 100 100 100 P. silvestrii (F. pertusa) 100 P. orozcoi (F. colubrinae, 1.01) 100 100 (F. costaricana) 81 P. estherae 100 P. tonduzi (F. citrifolia, 1.21) 100 P. piceipes (F. nymphaeifolia, 2.64) 100 100 P. longiceps (F. dugandii, 2.16) 100 95 P. lopesi (F. near trigonata, 2.57) 100

100 100 P. grandii (F. trigonata, 4.53)

100 P. bashierii (F. turbinata) 100 100 100 100 P. hoffmeyeri sp. A (F. obtusifolia, 1.05) 100 P. hoffmeyeri sp. B (F. obtusifolia, 1.05)

100 P. insularis sp. B (F. perforata, 1.00) 100 100 100 P. herrei (F. paraensis, 1.05) 0.009 100

100 P. sp. (F. gomezii) 100 100 100 P. gemellus sp. B (F. popenoei, 2.55)

100 P. gemellus sp. C (F. bullenei, 1.41)

Host Sharing Systems 100 100 (F. colubrinae, 1.01 & P. insularis sp. A F. perforata, 1.00) Hosts: F. colubrinae, F. perforata 100 100 Hosts: F. bullenei, F. popenoei * (F. bullenei, 1.41 & P. gemellus sp. A Host: F. obtusifolia * F. popenoei, 2.55)

Figure 1: Maximum likelihood phylogeny representing relationships among Pegoscapus wasps. Host fig species are displayed next to their associated wasp species, with average foundress number data included when available. See Herre (1989) for details. Node labels represent bootstrap support values. The insert shows the three systems with co-occurring pollinators, denoted by a circle (green), square (red), or triangle (blue). Two individuals (rep- resented by asterisks) of Pegoscapus gemellus sp. A (hosts: Ficus bullenei/Ficus popenoei) were sampled from Ficus dugandii, which is not its normal host. The undescribed pollinator associated with Ficus petiolaris was used to root the tree.

15 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

335 r = 0.212, P = 0.466). Although we do not distinguish the causes of non-monophyly (e.g.,

336 deep coalescence, introgression, gene tree estimation error), these conservative estimates are

337 considerably high and provide strong support for the validity of the wasp species and their

338 isolation over evolutionary time.

339 Phylogenetic relationships are congruent across all three methods. For the concatenated

340 tree, all nodes are strongly supported (most have a bootstrap value of 100), with a general

341 pattern of small intraspecific divergence and large interspecific divergence (Figure1). Two

342 P. gemellus sp. A (hosts: F. bullenei/F. popenoei) individuals were sampled from Ficus

343 dugandii, indicating an example of non-host specificity. In the species tree analyses SVDQST

344 (Figure2) and ASTRAL-III (Figure S2), there is strong support for most relationships among

345 species, although a few nodes in both trees are weakly supported. The minor differences in

346 the phylogenetic analyses occur at nodes with low support or at short internal branches. For

347 example, IQ-TREE supports Pegoscapus herrei (host: Ficus paraensis) sister to (Pegoscapus

348 sp. (host: Ficus gomezii), P. gemellus sp. B (host: F. popenoei)), with P. insularis sp. B

349 (host: F. perforata) sister to this clade, while SVDQST and ASTRAL-III places P. herrei

350 (host: F. paraensis) as sister to a clade of the other three species. In addition, IQ-TREE

351 and SVDQST place Pegoscapus tonduzi (host: Ficus citrifolia) sister to Pegoscapus estherae

352 (host: Ficus costaricana) with weak support, while ASTRAL-III places P. estherae (host:

353 F. costaricana sister to a large clade of fig wasps, with P. tonduzi (host: F. citrifolia) sister

354 to them.

355 Phylogenetic relatedness varies for the three systems containing co-occurring (i.e., shared

356 and host-specific) pollinators. The two pollinators (P. hoffmeyeri sp. A and P. hoffmeyeri

357 sp. B) associated with F. obtusifolia are sister taxa in the phylogenies, while the three pol-

358 linator species associated with F. colubrinae and F. perforata are phylogenetically distantly

359 related (Figures 1–2, S1–S2). Pollinator species associated with F. bullenei and F. pope-

360 noei show phylogenetic relatedness intermediate to these two systems, where they are less

16 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

P. sp. (F. petiolaris)

P. sp. (F. aurea) 100 P. orozcoi (F. colubrinae) 100 P. silvestrii (F. pertusa)

P. tonduzi (F. citrifolia) 51 P. estherae (F. costaricana) 100 P. piceipes (F. nymphaeifolia)

(F. dugandii) 3 1 P. longiceps P. lopesi (F. near trigonata) 100 100 P. grandii (F. trigonata)

P. bashierii (F. turbinata) 9 7 100 P. hoffmeyeri sp. A (F. obtusifolia) 100 P. hoffmeyeri sp. B (F. obtusifolia)

100 P. gemellus sp. C (F. bullenei) 100 P. gemellus sp. A (F. bullenei, F. popenoei) 100

100 P. insularis sp. A (F. colubrinae, F. perforata)

P. herrei (F. paraensis) 100 P. insularis sp. B (F. perforata) 5 8 P. sp. (F. gomezii) 100 P. gemellus sp. B (F. popenoei)

Figure 2: Species tree analysis with SVDquartets (SVDQST ). Nodal support is denoted with bootstrap support values. Host fig species are displayed next to their respective wasp species. Co-occurring pollinators are denoted by a circle (green), square (red), or triangle (blue). The undescribed pollinator associated with Ficus petiolaris was used to root the tree.

361 divergent from one another than the pollinators associated with F. colubrinae and F. per-

362 forata yet are not sister taxa within the tree. Results for these phylogenetic patterns are

363 consistent with both concatenation (Figure1) and species tree approaches (Figures 2, S2),

364 and highlight a range of evolutionary relatedness among the systems containing co-occurring

365 pollinator species.

366 Population genetics

367 Population genetic statistics were variable across pollinator species, with generally wide

368 ranges and large variances (Table 2). There is a significant positive correlation between

369 average number of foundress per species and genetic diversity (Figure 3). Pearson’s corre-

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A) B) C) 8 0.005 0.0030 r = 0.694 | P = 0.006 r = 0.867 | P < 0.001 r = 0.833 | P < 0.001 0.0025 0.004 6 0.0020 0.003 4 0.0015 s Theta (per site) 0.002 0.0010 Segregating Sites 2 atterson ' Nucleotide Diversity (π) 0.001 W 0.0005 0 0.000 0.0000 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 Foundress Number Foundress Number Foundress Number

P. insularis sp. A (F. colubrinae, F. perforata) P. gemellus sp. A (F. bullenei, F. popenoei) P. insularis sp. B (F. perforata) P. gemellus sp. B (F. popenoei) P. orozcoi (F. colubrinae) P. gemellus sp. C (F. bullenei) P. hoffmeyeri sp. A (F. obtusifolia) P. longiceps (F. dugandii) P. hoffmeyeri sp. B (F. obtusifolia) P. lopesi (F. near trigonata) P. herrei (F. paraensis) P. piceipes (F. nymphaeifolia) P. tonduzi (F. citrifolia) P. grandii (F. trigonata)

Figure 3: Correlation between population genetic summary statistics and average foundress number per pollinator species. Summary statistics include (A) nucleotide diversity, (B) number of segregating sites, and (C) Watterson’s theta (per site). The Pearson’s product- moment correlation was used to test significance. Fourteen wasp species were included for which we had average number of foundress information (see Herre, 1989). If a wasp had multiple hosts, we used the average for the number of foundresses from the shared hosts.

370 lation test recovered a statistically significant positive correlation for nucleotide diversity (r

371 = 0.694, P = 0.006), number of segregating sites (r = 0.867, P < 0.001), and Watterson’s

372 theta (r = 0.833, P < 0.001) versus number of foundresses.

18 (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376

Pollinator Wasp Host Fig π SS θw Monophyly Pegoscapus gemellus sp. A Ficus bullenei, F. popenoei 0.0005 (±0.0014) 2.0410 (±2.9494) 0.0012 (±0.0018) 0.82 (1502) Pegoscapus gemellus sp. B Ficus popenoei 0.0013 (±0.0021) 2.4979 (±3.4233) 0.0016 (±0.0022) 0.89 (1496) Pegoscapus gemellus sp. C Ficus bullenei 0.0005 (±0.0025) 0.5179 (±1.4514) 0.0005 (±0.0018) 0.92 (1482) Pegoscapus insularis sp. A Ficus colubrinae, F. perforata 0.0004 (±0.0018) 0.8855 (±2.4233) 0.0007 (±0.0020) 0.91 (1499)

Pegoscapus insularis sp. B Ficus perforata 0.0003 (±0.0032) 0.1600 (±1.5342) 0.0003 (±0.0026) 0.98 (1337) available undera Pegoscapus orozcoi Ficus colubrinae 0.0024 (±0.0135) 2.3022 (±2.6740) 0.0021 (±0.0025) 0.94 (1484) Pegoscapus hoffmeyeri sp. A Ficus obtusifolia 0.0004 (±0.0022) 0.3750 (±1.7908) 0.0004 (±0.0021) 0.93 (1479) Pegoscapus hoffmeyeri sp. B Ficus obtusifolia 0.0004 (±0.0026) 0.6432 (±1.7933) 0.0005 (±0.0018) 0.87 (1493) Pegoscapus sp. Ficus aurea 0.0005 (±0.0015) 0.7237 (±2.0046) 0.0006 (±0.0019) 0.99 (1490) Pegoscapus tonduzi Ficus citrifolia 0.0006 (±0.0025) 0.7403 (±2.2139) 0.0006 (±0.0019) 0.99 (1467) CC-BY 4.0Internationallicense ; Pegoscapus estherae Ficus costaricana 0.0007 (±0.0022) 0.8545 (±2.5590) 0.0008 (±0.0022) 0.99 (1467) this versionpostedDecember9,2020.

19 Pegoscapus longiceps Ficus dugandii 0.0009 (±0.0026) 1.0470 (±2.3325) 0.0009 (±0.0023) 1.00 (1472) Pegoscapus sp. Ficus gomezii 0.0003 (±0.0013) 0.4135 (±1.4957) 0.0004 (±0.0015) 0.96 (1491) Pegoscapus piceipes Ficus nymphaeifolia 0.0015 (±0.0030) 1.8930 (±3.1201) 0.0015 (±0.0026) 0.99 (1476) Pegoscapus herrei Ficus paraensis 0.0008 (±0.0025) 1.4675 (±2.4902) 0.0011 (±0.0025) 0.91 (1499) Pegoscapus silvestrii Ficus pertusa 0.0004 (±0.0016) 0.4931 (±1.7470) 0.0004 (±0.0017) 0.97 (1488) Pegoscapus sp. Ficus petiolaris 0.0004 (±0.0013) 0.4183 (±0.9953) 0.0004 (±0.0011) 1.00 (1431) Pegoscapus lopesi Ficus near trigonata 0.0013 (±0.0027) 2.1351 (±3.5987) 0.0015 (±0.0024) 0.99 (1484) Pegoscapus grandii Ficus trigonata 0.0029 (±0.0029) 8.1135 (±7.9945) 0.0047 (±0.0040) 0.96 (1501) Pegoscapus baschierii Ficus turbinata 0.0005 (±0.0024) 0.6733 (±1.6915) 0.0006 (±0.0015) 0.95 (1484) .

Table 2: Population genetic summary statistics for the pollinator wasp species. Summary statistics include nucleotide The copyrightholderforthispreprint diversity (π), number of segregating sites (SS), and Watterson’s theta per site (θw). Monophyly shows the proportion of gene trees for which a pollinator species is monophyletic. A gene tree was tested for monophyly when two or more sequences for a given species were sampled, with total trees tested per species in parentheses. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

373 Testing for monophyly with mitochondrial DNA

374 We were able to capture mitochondrial DNA of the COI region from 164 individuals (out

375 of 176). Following alignment and edge trimming, these data comprised 816 base pairs of

376 the COI mtDNA gene. All wasp species are recovered as monophyletic with strong support,

377 with all but one species having a bootstrap value of 100 (Figure S3). There is, however,

378 little support for phylogenetic relationships among species, as most nodal support values are

379 low. Consistent with how individuals cluster within species with the nuclear data, the same

380 clustering of individuals within species is recovered with data from the mitochondrial genome,

381 providing strong support for what we consider a pollinator species in this community. Given

382 the low support values for interspecific relationships, we make no comparisons between the

383 structure of the nuclear genome phylogeny and the mitochondrial gene tree. Comparing how

384 individuals cluster in the nuclear phylogeny and mitochondrial gene tree, however, these data

385 provide no evidence of recent cytonuclear discordance.

386 Testing for admixture and gene flow

387 There was no evidence of admixture or introgression in the three systems (BP, CP, O)

388 containing co-occurring pollinator species. Distinct clusters corresponding to species are

389 recovered in the PCA analyses, with no signal for hybridization (Figure4). For both BP and

390 CP, wasp species plot in distinct PCA space with little intraspecific variance in comparison

391 to the much larger interspecific variance. The two wasp species in O are well differentiated

392 in PCA axis 1, but show some intraspecific spread in PCA axis 2, although this axis only

393 comprises 0.35% of the variance. These results were further supported in Structure, as

394 individuals were assigned to their respective species with no evidence of admixture (Figure

395 4). In contrast to seeing shared ancestry indicative of admixture and introgression, each

396 individual maps unambiguously to its respective species. Thus, in systems where opportunity

397 for hybridization is present, we detect none.

20 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A)

25 P. gemellus sp. C 20 15 10 5 Axis 2 (35.16%) 0 P. gemellus sp. B P. gemellus sp. A −5

−10 0 10 20 Axis 1 (62.69%)

B)

25 P. insularis sp. B 20 15 10 Axis 2 (13.47%) 5

P. orozcoi 0 P. insularis sp. A

−30 −20 −10 0 10 Axis 1 (85.43%)

C) 3 2

1 P. hoffmeyeri sp. A 0

Axis 2 (0.35%) P. hoffmeyeri sp. B −1 −2

−30 −20 −10 0 10 Axis 1 (97.52%)

Figure 4: Principal components analysis (PCA) and Structure plots for the three systems where multiple pollinators interact with one or two fig species. Panel A represents three pollinator species interacting with Ficus bullenei and Ficus popenoei. Panel B represents three pollinator species interacting with Ficus colubrinae and Ficus perforata. Panel C represents two pollinator species interacting with Ficus obtusifolia 21. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Model Admixture Events Percent Variation m0 0 98.45% m1 1 98.62% m2 2 98.76% m3 3 98.81% m4 4 98.90% m5 5 98.96%

Table 3: Proportion of variation explained by the different models in TreeMix.

398 TreeMix estimated a population graph consistent with the other phylogenetic approaches

399 (Figure S4). Some differences were seen, mainly at places with short internal branches

400 and low support along the backbone of the tree. Adding migration events to the tree only

401 incrementally improved the proportion of variance explained by the model (Table3). For

402 example, the population graph with no admixture events explained 98.45% of the variation.

403 Adding one hybridization event to the graph increased the proportion of variation explained

404 by the model to 98.62%. The minimal improvement in models including migration events

405 suggests hybridization is not an important process in this pollinator community.

406 Discussion

407 We collected genome-wide ultraconserved element (UCE) loci from 19 pollinator wasp

408 species associated with all 16 host species in a strangler fig community from the vicinity

409 of the Panama Canal. We used these data to estimate phylogenetic relationships and test

410 for hybridization and introgression among the pollinators. This group of congeneric pollina-

411 tors (Pegoscapus) has exhibited frequent host switching throughout their shared evolutionary

412 history with their associated fig hosts (Satler et al., 2019), providing opportunities for hy-

413 bridization in both host fig and pollinator wasp species. Further, rare F1 pollinator hybrids

414 have been detected in this community (Molbo et al., 2003, 2004). We found that all 19

415 pollinator species are well-delimited genetically and show high interspecific divergence. Even

416 among co-occurring pollinator species in which heterospecific contact is most likely (repro-

22 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

417 duction within the same fruits of the same host fig species), we detected no evidence for

418 hybridization that led to successful introgression. Our results suggest that successful hy-

419 bridization and gene flow across species does not contribute meaningfully to the evolutionary

420 history of these Panamanian pollinators, and plays a negligible role in their co-evolution with

421 their host figs.

422 Because the reproduction of both host and pollinator occur in the same general structure

423 (the fig inflorescence = syconium = fig), there is considerable overlap in the conditions that

424 facilitate hybridization in both partners in this mutualism. In both the host fig and the

425 pollinator wasp, opportunities for hybridization and introgression depend on physical access

426 of heterospecific gametes (e.g., pollen and ovaries for the figs, sperm and eggs for the wasps).

427 The conditions for successful hybridization, however, are less restrictive in the host figs. Here,

428 although foundress wasps must be attracted to the floral volatiles of a different host species

429 and enter and pollinate receptive fig inflorescences, only a single wasp from a heterospecific

430 host need enter a fig to unite heterospecific pollen and ovules. The resulting hybrid seeds

431 must be viable, and introgression is only possible if the hybrid seeds produce viable seedlings

432 that survive to reproduce and backcross, most likely with one of the parental species. The

433 conditions for wasp hybridization are more stringent. Here, two or more foundress wasps

434 from different species must enter the same individual receptive fig inflorescence, with the

435 opportunity for foundress co-occurrence greatly restricted in host species typically producing

436 single-foundress figs (Table 1). After pollination and oviposition, heterospecific male and

437 female offspring must mate, the mated females must successfully disperse to a new receptive

438 fig, and the resulting hybrid offspring must survive, develop, disperse, and reproduce. Intro-

439 gression requires that these hybrid wasps contribute to successful backcrosses that also must

440 take place within the same fig where other foundresses have successfully oviposited, which

441 will again be restricted in host species with single-foundress figs.

442 In contrast to our findings in the Panamanian fig wasps, hybridization followed by intro-

23 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

443 gression is strongly suggested in their Panamanian fig hosts (Machado et al., 2005; Jackson

444 et al., 2008). For example, analyses that account for shared ancestral polymorphism show

445 that an isolation-only model provides a poor fit to the data from three Panamanian fig species

446 (Machado et al., 2005). Although based on limited sequence data (three nuclear loci), these

447 authors suggest this poor fit is best explained by hybridization and introgression among the

448 Panamanian figs. Further, Jackson et al. (2008) presented detailed haplotype analyses that

449 were most consistent with widespread hybridization and subsequent introgression across most

450 of the Panamanian figs they studied, which included 14 of the 16 host fig species represented

451 in our study. These findings are consistent with expectations based on the demonstration

452 of pollinator sharing and an evolutionary history of host switching by the pollinators as-

453 sociated the Panamanian figs (Molbo et al., 2003, 2004; Satler et al., 2019). Further, both

454 direct and indirect evidence suggest that hybridization occurs in figs more generally, and that

455 introgression is a potentially important process in the evolutionary history of Ficus (Comp-

456 ton, 1990; Machado et al., 2005; Jackson et al., 2008; Compton et al., 2009; Renoult et al.,

457 2009; Cornille et al., 2012; Van Noort et al., 2013; Bruun-Lund et al., 2017). Specifically,

458 cytonuclear discordance between plastid and nuclear genomes suggest ancient hybridization

459 and introgression have played an important role in shaping diversification patterns in Ficus

460 (Renoult et al., 2009; Bruun-Lund et al., 2017). Accumulated evidence suggests that host

461 switching and mixed plant–pollinator associations affect the likelihood of hybridization and

462 introgression in each mutualist lineage differently.

463 Among the pairs of Panamanian wasp species that share the same host fig species, there

464 appears to be no clear pattern to their degree of phylogenetic relatedness. Only P. hoffmeyeri

465 sp. A and sp. B associated with F. obtusifolia are recovered as sister species in this commu-

466 nity (Figures 1–2, S1–S2), and these are the only two species with evidence supporting rare

467 F1 hybrid events (Molbo et al., 2003, 2004). The other co-occurring pollinators span the

468 phylogenetic breadth of our sampled community, with wasps associated with F. bullenei and

24 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

469 F. popenoei being relatively closely related (but not sister taxa) while wasps associated with

470 F. colubrinae and F. perforata are distantly related (Figures 1–2, S1–S2). The Panamanian

471 observations are consistent with previously reported estimates that 32.1% of co-occurring

472 pollinators of monoecious fig species are sister species (Yang et al., 2015). Importantly, de-

473 spite the evidence for rare F1 hybridization events between the two F. obtusifolia pollinators

474 (Molbo et al., 2003, 2004), we found no evidence for any backcrosses or introgression in any

475 of the species we studied. Even if our current sampling missed F1 hybrid individuals, if

476 hybridization is important then we would expect to have detected some backcross genotypes

477 or genetic signal of introgression. Given our observations, how might different processes that

478 affect prezygotic (e.g., host recognition, likelihood of sharing individual fig inflorescences) and

479 postzygotic (e.g., developmental incompatibility with the host fig, reproductive incompati-

480 bility between wasp species) barriers limit hybridization and introgression among pollinator

481 species?

482 Host choice by the pollinator fundamentally influences the potential for hybridization and

483 gene flow for both the fig and the wasp. Receptive fig inflorescences produce complex volatile

484 chemical blends recognized by potential pollinators. Generally, the blends produced by dif-

485 ferent figs are sufficiently distinct for wasps to distinguish among potential hosts (Van Noort

486 et al., 1989; Ware et al., 1993; Grison-Pigé et al., 2002; Hossaert-McKey et al., 2010; Cornille

487 et al., 2012). The degree of affinity for particular wasp species towards particular host blends

488 appears to be an important prezygotic barrier limiting encounter rates of heterospecific wasps.

489 In 17 of the 19 wasp species sampled in the Panamanian community, wasps are predominately

490 attracted to a single host fig species, suggesting high host specificity likely based in large part

491 on recognition of host volatile chemical signals (Table 1)(Molbo et al., 2003, Oldenbeuvin-

492 gen, Florez, and Herre, unpublished data). Wasp species that share fig hosts, however, are

493 often not sister species, both in this community and more broadly across Ficus (Yang et al.,

494 2015). The observation of multiple, phylogenetically divergent pollinator species attracted

25 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

495 to the same host fig raises important questions: What processes have shaped the genetics

496 and biochemical pathways of host chemical signal production, of wasp chemosensory detec-

497 tion, and of wasp volatile preference? Addressing this complex series of questions will require

498 studies that explicitly connect host volatile chemical composition with wasp preferences (e.g.,

499 antennogram and gene expression studies) within the contexts of detailed phylogenetic and

500 ecological studies, preferably across multiple sites and fig–wasp taxa.

501 Individual fig fruit crops can exhibit tens of thousands of figs, and effective population

502 sizes of figs consist of hundreds of individuals (Nason et al., 1998; Korine et al., 2000). Even

503 if heterospecific wasps recognize the same host fig species, a precondition for hybridization is

504 that two (or more) heterospecific foundress wasps oviposit successfully in the same individual

505 fig. In the Panamanian strangler figs we studied, the average foundress numbers per fruit

506 ranges between one to four and a half (Herre, 1989). For species pollinated almost exclusively

507 by a single foundress (e.g., Panamanian populations of F. perforata and F. colubrinae, Table

508 1), the opportunities for wasp hybridization are limited. In contrast, in fig species commonly

509 having multiple foundresses per fruit, the offspring of pollinators have greater opportunities to

510 encounter heterospecifics in the same fig. Higher mean foundress numbers will also determine

511 wasp population structure which in turn will affect many aspects of these wasp species (e.g.,

512 sexual competition and sex ratios, heterozygosity) (Herre, 1985, 1989, 1993; Molbo et al.,

513 2003, 2004). In particular, we found a significant positive correlation between the average

514 number of foundresses and genetic diversity of UCE loci across pollinator species (Figure

515 3). As was the case with species that show occasional F1 hybrids, however, we detected no

516 evidence of hybridization and introgression in pollinator species that exhibited higher average

517 foundress numbers, including Ficus nymphaefolia (2.6 foundresses) and Ficus trigonata (4.5

518 foundresses). Thus, when multiple foundresses are present, outbreeding occurs within species,

519 but outbreeding—and subsequent introgression—does not occur between species.

520 Postzygotic barriers to successful wasp hybridization include developmental incompati-

26 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

521 bility with the host fig during the process of gall formation and the development of wasp

522 offspring (Compton, 1990; Compton et al., 2009; Van Noort et al., 2013; Martinson et al.,

523 2014). For example, Moe and Weiblen (2012) experimentally introduced pollinator wasps

524 into figs of a species that are not their normal hosts. Although individuals of the non-normal

525 wasp species were able to oviposit and induce gall development, the galls and the wasp off-

526 spring were unable to develop to maturity, suggesting selective constraints on heterospecific

527 wasp development. It appears that when heterospecific pollinator mating does occur, the

528 hybrid offspring exhibit moderately to extremely reduced fitness compared to non-hybrid

529 individuals (Molbo et al., 2003, 2004).

530 Another potential postzygotic barrier to hybridization and introgression in the wasps

531 is Wolbachia. These maternally inherited cytoplasmic bacteria are known to cause dras-

532 tic reductions of hybrid formation in Nasonia, another chalcidoid wasp (Bordenstein et al.,

533 2001). Wolbachia are commonly found across species (Werren, 1997), and are partic-

534 ularly common in fig wasps (e.g., Haine and Cook, 2005; Sun et al., 2011). Pollinator and

535 non-pollinator wasps associated with the fig community in central Panama show Wolbachia

536 occurrence in 59% of wasp species (Shoemaker et al., 2002), including many of the species

537 sampled in this study. In all fig species in which different wasp pollinators co-occur, at

538 least one of the pollinator species shows partial or complete Wolbachia infection (Shoemaker

539 et al., 2002), raising the possibility that these bacteria influence the likelihood of successful

540 hybridization.

541 Many studies of the associations of pollinating wasps with their host figs tend to focus

542 on one or a few species interactions at one or a few locales. The coupling of geographic

543 sampling with detailed genetic characterization of figs and their pollinators, however, often

544 reveals that multiple pollinators are associated with a single host fig across the range of

545 the fig species (e.g., Michaloud et al., 1996; Haine et al., 2006; Peng et al., 2008; Darwell

546 et al., 2014; Bain et al., 2016). For example, Yu et al. (2019) recovered nine parapatrically

27 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

547 distributed pollinator species associated with Ficus hirta in South-East Asia. Multiple pol-

548 linator species in allopatric and parapatric distributions and associated with the same host

549 suggests pollinator wasps speciate in allopatry before potentially coming back into sympatry.

550 Souto-Vilarós et al.(2019) examined six species pairs of figs and wasps found in New Guinea,

551 and found an increased rate of population genetic divergence with geographic distance among

552 the wasps relative to their host figs. They suggest pollinators have an increased speciation

553 rate, with populations of wasps diverging in isolation faster than their hosts, effectively de-

554 coupling cospeciation processes in the two mutualist taxa (Machado et al., 2005; Peng et al.,

555 2008; Compton et al., 2009; Van Noort et al., 2013; Satler et al., 2019).

556 Despite the ability of pollinating wasps to disperse many kilometers (Nason et al., 1998;

557 Ahmed et al., 2009), broad geographic sampling suggests that multiple distinct pollinator

558 species—in allopatric and parapatric distributions—characterize host fig species across their

559 ranges. Molecular data suggests a previously underappreciated prevalence of host switching

560 in Neotropical strangler figs, as well as figs in general (Jackson et al., 2008; Cruaud et al.,

561 2012; Yang et al., 2015; Satler et al., 2019). We suspect that combinations of pre- and

562 postzygotic barriers contribute to the reproductive isolation of pollinator wasps (Molbo et al.,

563 2003, 2004; Satler et al., 2019). Specifically, the relative inability of hybrids to detect, locate,

564 and enter a receptive fig, or to their inability to successfully pollinate and oviposit and

565 have offspring successfully develop in a receptive fig, or the effects of hybrid developmental

566 incompatibilities (e.g., hybrid diploid male offspring) would all select against hybrids and for

567 stronger reproductive isolation. When pollinator species experience secondary contact, we

568 suspect these barriers contribute to less fit hybrids and reinforces species boundaries (Nosil

569 et al., 2003).

570 Consistent with the findings that pollinator species are often well-delimited molecularly,

571 and that F1 hybrids have only rarely been found in the most closely related Panamanian

572 fig wasp species, we expect this to be true among relatively distantly related species (Molbo

28 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

573 et al., 2003, 2004; Satler et al., 2019). We suggest that the still poorly understood processes

574 of pollinator speciation and extinction play a more important role than hybridization and

575 introgression in shaping evolutionary dynamics of fig pollinating wasps and their host as-

576 sociations in space and time. Future biogeographical, phylogenetic, and population genetic

577 studies coupled with targeted experimental studies are needed to determine the relative im-

578 portance of different mechanisms in preventing interspecific mating, hybrid production, and

579 introgression in pollinating fig wasps.

580 Conclusions

581 Given the reproductive biology of fig wasps, multiple events are important for maintain-

582 ing species boundaries. Before there can be opportunity for hybridization, foundresses from

583 different fig pollinator species need to locate, enter, and oviposit in the same fruit, with suc-

584 cessful development of offspring to reproductive age. Although we focused on a Panamanian

585 fig community with an evolutionary history of host switching, and sampled pollinators from

586 hosts that averaged more than one foundress per fruit, we detect no signal of hybridization

587 and introgression among the wasps. The lack of hybridization and introgression indicate that

588 the Panamanian fig pollinators are cohesive evolutionary units. More generally, our results

589 suggest that processes other than interspecific gene flow have been important to the evolution

590 of these fig pollinating wasps.

29 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

591 Acknowledgements

592 We thank Brant Faircloth and the Faircloth lab for generously providing time, space, and

593 resources for learning sequence capture approaches for generating UCE data. We thank mem-

594 bers of the Heath lab and Nason lab for discussion and comments regarding the manuscript.

595 Funding was provided by the National Science Foundation (DEB-1556853) to JDN, TAH,

596 and EAH, and by the Smithsonian Scholarly Studies Grant to EAH. Computational re-

597 sources were provided by ResearchIT and the College of Liberal Arts and Sciences at Iowa

598 State University.

599 Data Accessibility

600 Raw sequence data will be available from the NCBI Sequence Read Archive (SRA) under

601 BioProject ID: ### (###). All data sets and custom scripts will be made available on

602 Dryad.

603 Author Contributions

604 JDS, TAH, EAH, and JDN designed the study, AGZ, EAH, and CAM collected the fig

605 wasp samples, JDS generated and processed the sequence data, JDS conducted all analyses,

606 JDS, EAH, and JDN wrote the paper, and all authors contributed to revised versions of the

607 manuscript and approved of the final version.

30 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

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Supplemental Information for:

Genome-wide sequence data show no evidence of admixture and introgression among pollinator wasps associated with a community of Panamanian strangler figs

Jordan D. Satler, Edward Allen Herre, Tracy A. Heath, Carlos A. Machado, Adalberto Gómez Zúñiga, and John D. Nason

39 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

100 P. sp. (F. petiolaris) 100 P. sp. (F. aurea) 100 100 P. silvestrii (F. pertusa) 100 100 100 P. orozcoi (F. colubrinae)

100 P. tonduzi (F. citrifolia)

100 100 P. piceipes (F. nymphaeifolia) 100 (F. costaricana) 29 P. estherae 100 P. longiceps (F. dugandii) 3 4 100

100 P. lopesi (F. near trigonata)

100 100

P. grandii (F. trigonata)

100 9 7 P. bashierii (F. turbinata) 100 100 100 P. hoffmeyeri sp. A (F. obtusifolia) 100 P. hoffmeyeri sp. B (F. obtusifolia)

100 100 P. herrei (F. paraensis) 100

93 P. insularis sp. B (F. perforata) 54 100 100 P. sp. (F. gomezii) 100 100 P. gemellus sp. B (F. popenoei)

100 P. gemellus sp. C (F. bullenei)

100 100

(F. colubrinae, P. insularis sp. A 100 F. perforata)

100 * (F. bullenei, P. gemellus sp. A F. popenoei) *

Figure S1: Lineage tree analysis with SVDquartets (SVDQLT ) where each individual is treated as a tip in the tree. Nodal support reflecting bootstrap values are shown for species and interspecific nodes. Host fig species are displayed next to their associated wasp species. Co-occurring pollinators are denoted by a circle (green), square (red), or triangle (blue). Two individuals (represented by asterisks) of Pegoscapus gemellus sp. A (hosts: Ficus bul- lenei/Ficus popenoei) were sampled from Ficus dugandii, which is not its normal host. The undescribed pollinator associated with Ficus petiolaris was used to root the tree.

40 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

P. sp. (F. petiolaris)

P. sp. (F. aurea)

1.0 P. orozcoi (F. colubrinae) 1.0

P. silvestrii (F. pertusa)

P. tonduzi (F. citrifolia)

P. estherae (F. costaricana) 1.0

P. piceipes (F. nymphaeifolia) 0.81

P. longiceps (F. dugandii)

0.67 P. grandii (F. trigonata) 1.0 1.0 P. lopesi (F. near trigonata)

P. bashierii (F. turbinata)

1.0 1.0 P. hoffmeyeri sp. B (F. obtusifolia) 1.0

P. hoffmeyeri sp. A (F. obtusifolia)

1.0 P. gemellus sp. C (F. bullenei)

1.0 (F. bullenei, P. gemellus sp. A F. popenoei) 1.0

(F. colubrinae, P. insularis sp. A 1.0 F. perforata)

P. herrei (F. paraensis)

2.0 1.0 P. insularis sp. B (F. perforata)

0.86 P. gemellus sp. B (F. popenoei)

1.0 P. sp. (F. gomezii)

Figure S2: Species tree analysis with ASTRAL-III. Nodal support values represent local posterior probabilities. Host fig species are displayed next to their associated wasp species. Branch lengths are in coalescent units. Co-occurring pollinators are denoted by a circle (green), square (red), or triangle (blue). The undescribed pollinator associated with Ficus petiolaris was used to root the tree.

41 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

100 P. sp. (F. petiolaris) 100 P. sp. (F. aurea) 100 P. longiceps (F. dugandii) 100 100 87 P. hoffmeyeri sp. A (F. obtusifolia)

100 P. hoffmeyeri sp. B (F. obtusifolia) 62 99 P. lopesi (F. near trigonata) 46 100 P. estherae (F. costaricana) 46 69 100 P. bashierii (F. turbinata) 94

100 P. grandii (F. trigonata)

44 100 70 P. silvestrii (F. pertusa) 100 P. tonduzi (F. citrifolia) 100 P. orozcoi (F. colubrinae) 27 40 100 (F. nymphaeifolia) 70 P. piceipes 100 P. gemellus sp. B (F. popenoei) 48 100 P. herrei (F. paraensis)

100 69

(F. bullenei & P. gemellus sp. A F. popenoei)

34 * 100 0.02 * (F. colubrinae & P. insularis sp. A F. perforata) 40

100 87 P. gemellus sp. C (F. bullenei) (F. perforata) 72 100 P. insularis sp. B 100 P. sp. (F. gomezii)

Figure S3: Maximum likelihood gene tree representing relationships among mitochondrial haplotypes. Nodal support reflecting bootstrap values are shown for species and interspecific nodes. Host fig species are displayed next to their associated wasp species. Co-occurring pollinators are denoted by a circle (green), square (red), or triangle (blue). Two individuals of Pegoscapus gemellus sp. A (hosts: Ficus bullenei/Ficus popenoei) sampled from Ficus dugandii are denoted by asterisks. The undescribed pollinator associated with Ficus petiolaris was used to root the tree.

42 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.418376; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A) m = 0 B) m = 1

P. sp. (F. petiolaris) P. sp. (F. petiolaris) P. silvestrii (F. pertusa) P. silvestrii (F. pertusa) P. orozcoi (F. colubrinae) P. orozcoi (F. colubrinae) P. sp. (F. aurea) P. sp. (F. aurea) P. tonduzi (F. citrifolia) P. longiceps (F. dugandii) P. longiceps (F. dugandii) P. insularis sp. B (F. perforata) P. baschierii (F. turbinata) P. gemellus sp. B (F. popenoei) P. hoffmeyeri sp. A (F. obtusifolia) P. sp. (F. gomezii) P. hoffmeyeri sp. B (F. obtusifolia) P. gemellus sp. C (F. bullenei) Migration P. insularis sp. B (F. perforata) Migration P. insularis sp. A (F. colubrinae, F. perforata) weight P. gemellus sp. B (F. popenoei) weight P. gemellus sp. A (F. bullenei, F. popenoei) 0.5 P. sp. (F. gomezii) 0.5 P. herrei (F. paraensis) P. gemellus sp. C (F. bullenei) P. baschierii (F. turbinata) P. insularis sp. A (F. colubrinae, F. perforata) P. hoffmeyeri sp. A (F. obtusifolia) P. gemellus sp. A (F. bullenei, F. popenoei) P. hoffmeyeri sp. B (F. obtusifolia) 0 P. herrei (F. paraensis) 0 P. piceipes (F. nymphaeifolia) P. piceipes (F. nymphaeifolia) P. lopesi (F. near trigonata) P. estherae (F. costaricana) P. grandii (F. trigonata) P. grandii (F. trigonata) P. estherae (F. costaricana) P. lopesi (F. near trigonata) P. tonduzi (F. citrifolia)

0.00 0.05 0.10 0.15 0.00 0.05 0.10 0.15

Drift parameter Drift parameter

C) m = 2 D) m = 3

P. sp. (F. petiolaris) P. longiceps (F. dugandii) P. silvestrii (F. pertusa) P. lopesi (F. near trigonata) P. orozcoi (F. colubrinae) P. grandii (F. trigonata) P. sp. (F. aurea) P. estherae (F. costaricana) P. longiceps (F. dugandii) P. herrei (F. paraensis) P. insularis sp. B (F. perforata) P. gemellus sp. C (F. bullenei) P. gemellus sp. B (F. popenoei) P. insularis sp. A (F. colubrinae, F. perforata) P. sp. (F. gomezii) P. gemellus sp. A (F. bullenei, F. popenoei) P. gemellus sp. C (F. bullenei) P. insularis sp. B (F. perforata) Migration P. insularis sp. A (F. colubrinae, F. perforata) Migration P. sp. (F. gomezii) weight P. gemellus sp. A (F. bullenei, F. popenoei) weight P. gemellus sp. B (F. popenoei) 0.5 0.5 P. herrei (F. paraensis) P. baschierii (F. turbinata) P. baschierii (F. turbinata) P. hoffmeyeri sp. A (F. obtusifolia) P. hoffmeyeri sp. A (F. obtusifolia) P. hoffmeyeri sp. B (F. obtusifolia) P. hoffmeyeri sp. B (F. obtusifolia) P. piceipes (F. nymphaeifolia) 0 0 P. piceipes (F. nymphaeifolia) P. tonduzi (F. citrifolia) P. lopesi (F. near trigonata) P. sp. (F. aurea) P. grandii (F. trigonata) P. orozcoi (F. colubrinae) P. estherae (F. costaricana) P. silvestrii (F. pertusa) P. tonduzi (F. citrifolia) P. sp. (F. petiolaris)

0.00 0.05 0.10 0.15 0.00 0.05 0.10 0.15

Drift parameter Drift parameter

E) m = 4 F) m = 5 P. orozcoi (F. colubrinae) P. tonduzi (F. citrifolia) P. silvestrii (F. pertusa) P. herrei (F. paraensis) P. sp. (F. aurea) P. gemellus sp. C (F. bullenei) P. longiceps (F. dugandii) P. insularis sp. A (F. colubrinae, F. perforata) P. estherae (F. costaricana) P. gemellus sp. A (F. bullenei, F. popenoei) P. lopesi (F. near trigonata) P. gemellus sp. B (F. popenoei) Migration P. grandii (F. trigonata) Migration P. sp. (F. gomezii) weight weight 0.5 P. herrei (F. paraensis) 0.5 P. insularis sp. B (F. perforata) P. gemellus sp. C (F. bullenei) P. baschierii (F. turbinata) P. insularis sp. A (F. colubrinae, F. perforata) P. hoffmeyeri sp. A (F. obtusifolia) P. gemellus sp. A (F. bullenei, F. popenoei) P. hoffmeyeri sp. B (F. obtusifolia) 0 P. insularis sp. B (F. perforata) 0 P. piceipes (F. nymphaeifolia) P. sp. (F. gomezii) P. estherae (F. costaricana) P. gemellus sp. B (F. popenoei) P. lopesi (F. near trigonata) P. baschierii (F. turbinata) P. grandii (F. trigonata) P. hoffmeyeri sp. A (F. obtusifolia) P. longiceps (F. dugandii) P. hoffmeyeri sp. B (F. obtusifolia) P. orozcoi (F. colubrinae) P. piceipes (F. nymphaeifolia) P. silvestrii (F. pertusa) P. tonduzi (F. citrifolia) P. sp. (F. aurea) P. sp. (F. petiolaris) P. sp. (F. petiolaris)

0.00 0.05 0.10 0.15 0.00 0.05 0.10 0.15

Drift parameter Drift parameter

Figure S4: TreeMix results. Results show population graphs without admixture (A), and with between 1 and 5 admixture (m) events (B–F). A model without hybridization (A) is best supported by the data (Table 3). The undescribed pollinator associated with Ficus petiolaris was used to root the tree.

43