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

Interactions between plants and their animal pollinators can shape processes of divergence and gene flow within associated lineages. For example, in the obligate mutualism between figs (Ficus) and fig pollinator wasps (family Agaonidae), each wasp species typically pollinates a single fig species, potentially reinforcing reproductive isolation among different wasp species. Multiple pollinator species, however, can sometimes reproduce in the same host fig species, potentially enabling hybridization and introgression between wasp species. In a community of Panamanian strangler figs (section Americana), we use genome-wide ultraconserved element (UCE) loci to estimate phylogenetic relationships and test for hybridization and gene flow among 19 pollinator species associated with 16 host fig species. Previous studies showing ongoing pollinator sharing and a history of pollinator host switching are consistent with documented genetic admixture in their host figs. Here we investigate if host sharing and a dynamic evolutionary history including host switching has also resulted in hybridization and gene flow between pollinator species. Phylogenetic analyses recover strong support for well-delimited wasp species coupled with high interspecific divergence. There is no evidence for ongoing hybridization or introgression, even among pairs of pollinator species currently reproducing within the same host. In contrast to work suggesting admixture among Panamanian host figs, we conclude hybridization and interspecific gene flow have not been important processes shaping the evolutionary history of their pollinating wasps.


Introduction
Hybridization produces offspring exhibiting novel genetic combinations derived from di-23 vergent parental genomes. Introgression results in genetic material moving between species. 24 The influx of genetic material into a lineage introduces new alleles and multilocus combi-25 nations that, depending on the situation, can be either harmful (Rhymer and Simberloff, 26 1996) or beneficial (Dowling and Secor, 1997). In many lineages, strong pre-and postzy-27 gotic barriers can limit hybridization and reinforce species boundaries (e.g., Nosil et al., 28 2005). Evidence from next-generation sequencing, however, has revealed that hybridization 29 and introgression have occurred throughout the evolutionary history of the tree of life (Mallet 30 et al., 2016;Taylor and Larson, 2019). Identifying the ecological processes that either main-31 tain or undermine species boundaries is useful for generating testable hypotheses concerning 32 factors that affect the evolutionary importance of hybridization and introgression (Twyford 33 and Ennos, 2012). 34 Host specialization in plant-associated insects generally leads to a reduction in interspe-   (1989). For figs that share a pollinator species, foundress numbers were averaged between the host figs.
following the outline provided by Andermann et al. (2018). Taking our aligned loci (before using maximum likelihood (ML) in IQ-TREE v1.6.12 (Nguyen et al., 2015;Chernomor et al., selected  Previous work in the Panamanian system has recovered pollinator species as monophyletic 181 and has not brought into question species validity (Machado et al., 2005;Jackson et al., 2008;182  Pegoscapus hoffmeyeri sp. B. We refer to these three systems as BP (i and ii), CP (iii and 236 iv), and O (v) (see Figure 1). They include two pollinator species associated with two hosts 237 each, and six pollinator species that are host specific. It is the interaction of wasps within  SNP was selected at random. We then estimated a maximum likelihood population graph in 300 TreeMix, allowing between zero and five migration events.

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Although additional approaches are often used to test for hybridization and introgression,  Figure S1). To further investigate, we tested species monophyly for each sampled 328 UCE locus. All species were monophyletic for either all or nearly all loci (         . 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.

Testing for monophyly with mitochondrial DNA
We were able to capture mitochondrial DNA of the COI region from 164 individuals (out 374 of 176). Following alignment and edge trimming, these data comprised 816 base pairs of 375 the COI mtDNA gene. All wasp species are recovered as monophyletic with strong support, 376 with all but one species having a bootstrap value of 100 ( Figure S3). There is, however,   TreeMix estimated a population graph consistent with the other phylogenetic approaches 398 ( Figure S4). Some differences were seen, mainly at places with short internal branches 399 and low support along the backbone of the tree. Adding migration events to the tree only 400 incrementally improved the proportion of variance explained by the model (Table 3). For 401 example, the population graph with no admixture events explained 98.45% of the variation. have been detected in this community (Molbo et al., 2003(Molbo et al., , 2004. We found that all 19  (Figures 1-2, S1-S2), and these are the only two species with evidence supporting rare 466 F1 hybrid events (Molbo et al., 2003(Molbo et al., , 2004. The other co-occurring pollinators span the 467 phylogenetic breadth of our sampled community, with wasps associated with F. bullenei and 468 F. popenoei being relatively closely related (but not sister taxa) while wasps associated with F. colubrinae and F. perforata are distantly related (Figures 1-2 wasp population structure which in turn will affect many aspects of these wasp species (e.g.,

569
Consistent with the findings that pollinator species are often well-delimited molecularly,     Figure S1: Lineage tree analysis with SVDquartets (SVDQ LT ) 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 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.    (Table 3). The undescribed pollinator associated with Ficus petiolaris was used to root the tree.