Pollination_and_oviposition

Video depicting pollination of and oviposition into flowers of Glochidion lanceolarium by a female Epicephala lanceolaria moth

RAxML_Epicephala

Phylogenetic hypothesis for Epicephala, recovered using maximum likelihood implemented in RAxML. Tip labels represent either specimen numbers and/or host species (in the genera Flueggea, Phyllanthus, Breynia, and Glochidion) from which specimens were reared, and are from the present study as well as Kawakita et al. (2004) Evolution, Kawakita et al. (2009) Proc. R. Soc. B, and Hembry et al. (2013) Proc. Roy. Soc. B. Many tip labels follow the Supplementary Appendix in Hembry et al. (2013) Proc. R. Soc. B. For E. lanceolaria OTUs, numbers refer to particular specimens; two-letter codes indicate collection localities (GB: Gaobangshan, LD: South China Botanical Garden, QA: Qiao Island, WT: Wutongshan); C = pupa with cocoon, NC = pupa with no cocoon. This tree corresponds to Supplementary Figure 2 in the present manuscript

MrBayes_Glochidion.nex.con

Phylogenetic hypothesis recovered using Bayesian inference implemented in MrBayes for Glochidion. Tip labels represent either specimen numbers or specific epithets for Glochidion species and are from the present study as well as Kawakita et al. (2004) Evolution and Hembry et al. (2013) Proc. Roy. Soc. B. Phyllanthus roseus is a very close relative to Glochidion, but is not pollinated by Epicephala (Kawakita and Kato 2009 Proc. R. Soc. B). Many tip labels follow the Supplementary Appendix in Hembry et al. (2013) Proc. R. Soc. B. This tree corresponds to Figure 5 and half of Figure 7 in the present manuscript

Figures_3_4

Data used in generating Figures 3 and 4 in the manuscript (see Excel spreadsheet tab labels). Figure 3 data represent the number of adult Epicephala lanceolaria moths found in individual Glochidion lanceolarium fruit at three sites in Guangdong Province, China. Figure 4 data represent the number of Epicephala lanceolaria pupae with and without cocoons found when dissecting Glochidion lanceolarium fruit at two sites in Guangdong Province, China

MrBayes_Epicephala.nex.con

Phylogenetic hypothesis for Epicephala, recovered using Bayesian inference implemented in MrBayes. Tip labels represent either specimen numbers and/or host species (in the genera Flueggea, Phyllanthus, Breynia, and Glochidion) from which specimens were reared, and are from the present study as well as Kawakita et al. (2004) Evolution, Kawakita et al. (2009) Proc. R. Soc. B, and Hembry et al. (2013) Proc. Roy. Soc. B. Many tip labels follow the Supplementary Appendix in Hembry et al. (2013) Proc. R. Soc. B. For E. lanceolaria OTUs, numbers refer to particular specimens; two-letter codes indicate collection localities (GB: Gaobangshan, LD: South China Botanical Garden, QA: Qiao Island, WT: Wutongshan); C = pupa with cocoon, NC = pupa with no cocoon. This tree corresponds to Figure 6 in the present manuscript

Epicephala_constrained.nex.con

Phylogenetic hypothesis for Epicephala, recovered using Bayesian inference implemented in MrBayes constrained to force monophyly of all Glochidion-associated Epicephala. Tip labels represent either specimen numbers and/or host species (in the genera Flueggea, Phyllanthus, Breynia, and Glochidion) from which specimens were reared, and are from the present study as well as Kawakita et al. (2004) Evolution, Kawakita et al. (2009) Proc. R. Soc. B, and Hembry et al. (2013) Proc. Roy. Soc. B. Many tip labels follow the Supplementary Appendix in Hembry et al. (2013) Proc. R. Soc. B. For E. lanceolaria OTUs, numbers refer to particular specimens; two-letter codes indicate collection localities (GB: Gaobangshan, LD: South China Botanical Garden, QA: Qiao Island, WT: Wutongshan); C = pupa with cocoon, NC = pupa with no cocoon. This tree was used in Shimodaira-Hasegawa tests as described in the present manuscript

Sequence_data.fasta

Novel DNA sequence data generated for this study from the species Glochidion lanceolarium (Phyllanthaceae) and Epicephala lanceolaria (Insecta: Lepidoptera: Gracillariidae). These data represent five loci: for Epicephala, mitochondrial COI, and nuclear EF1-α and ArgK; for Glochidion, nuclear ribosomal ITS and ETS. Sequences are the same as GenBank numbers KY078233-KY078291; however, GenBank numbers are not included in this file. For correspondences between sample numbers and GenBank sequences for Epicephala, please see Table 2 in the manuscript. Glochidion lanceolarium ITS sequence corresponds to GenBank number KY078253 and ETS sequence corresponds to GenBank number KY078252

Table S4. Midge sequences included in this study with their GenBank accession numbers from The largest early-diverging angiosperm family is mostly pollinated by ovipositing insects and so are most surviving lineages of early angiosperms

Insect pollination in basal angiosperms is assumed to mostly involve ‘generalized' insects looking for food, but direct observation of ANITA grade (283 species) pollinators are sparse. We present new data for numerous Schisandraceae, the largest ANITA family, from fieldwork, nocturnal filming, electron microscopy, barcoding and molecular clocks to infer pollinator/plant interactions over multiple years at sites throughout China to test the extent of pollinator specificity. Schisandraceae are pollinated by nocturnal gall midges that lay eggs in the flowers and whose larvae then feed on floral exudates. At least three Schisandraceae have shifted to beetle pollination. Pollination by a single midge species predominates, but one species was pollinated by different species at three locations and one by two at the same location. Based on molecular clocks, gall midges and Schisandraceae may have interacted since at least the Early Miocene. Combining these data with a review of all published ANITA pollination data shows that ovipositing flies are the most common pollinators of living representatives of the ANITA grade. Compared to food reward-based pollination, oviposition-based systems are less wasteful of plant gametes because (i) none are eaten and (ii) female insects with herbivorous larvae reliably visit conspecific flowers

Table S5. Midge eggs and larvae found in the flowers of representative Schisandraceae from The largest early-diverging angiosperm family is mostly pollinated by ovipositing insects and so are most surviving lineages of early angiosperms

Insect pollination in basal angiosperms is assumed to mostly involve ‘generalized' insects looking for food, but direct observation of ANITA grade (283 species) pollinators are sparse. We present new data for numerous Schisandraceae, the largest ANITA family, from fieldwork, nocturnal filming, electron microscopy, barcoding and molecular clocks to infer pollinator/plant interactions over multiple years at sites throughout China to test the extent of pollinator specificity. Schisandraceae are pollinated by nocturnal gall midges that lay eggs in the flowers and whose larvae then feed on floral exudates. At least three Schisandraceae have shifted to beetle pollination. Pollination by a single midge species predominates, but one species was pollinated by different species at three locations and one by two at the same location. Based on molecular clocks, gall midges and Schisandraceae may have interacted since at least the Early Miocene. Combining these data with a review of all published ANITA pollination data shows that ovipositing flies are the most common pollinators of living representatives of the ANITA grade. Compared to food reward-based pollination, oviposition-based systems are less wasteful of plant gametes because (i) none are eaten and (ii) female insects with herbivorous larvae reliably visit conspecific flowers

Table S2. Study sites, observation times, and herbarium vouchers for studied plants from The largest early-diverging angiosperm family is mostly pollinated by ovipositing insects and so are most surviving lineages of early angiosperms

Insect pollination in basal angiosperms is assumed to mostly involve ‘generalized' insects looking for food, but direct observation of ANITA grade (283 species) pollinators are sparse. We present new data for numerous Schisandraceae, the largest ANITA family, from fieldwork, nocturnal filming, electron microscopy, barcoding and molecular clocks to infer pollinator/plant interactions over multiple years at sites throughout China to test the extent of pollinator specificity. Schisandraceae are pollinated by nocturnal gall midges that lay eggs in the flowers and whose larvae then feed on floral exudates. At least three Schisandraceae have shifted to beetle pollination. Pollination by a single midge species predominates, but one species was pollinated by different species at three locations and one by two at the same location. Based on molecular clocks, gall midges and Schisandraceae may have interacted since at least the Early Miocene. Combining these data with a review of all published ANITA pollination data shows that ovipositing flies are the most common pollinators of living representatives of the ANITA grade. Compared to food reward-based pollination, oviposition-based systems are less wasteful of plant gametes because (i) none are eaten and (ii) female insects with herbivorous larvae reliably visit conspecific flowers