Phylogenomics illuminates the phylogeny of flower weevils (Curculioninae) and reveals ten independent origins of brood-site pollination mutualism in true weevils

Phylogenomics illuminates the phylogeny of flower weevils (Curculioninae) and reveals ten independent origins of brood-site pollination mutualism in true weevils (142 /150 characters) Haran J.1*, Li X.2,3,4*, Allio R.5*, Shin S.3,4,6, Benoit L.1, Oberprieler R.G.7, Farrell B.D.8, Brown S.D.J.9, Leschen R.A.B.10, Kergoat G.J.5 & McKenna D.D.3,4 * Equal contribution Affiliations 1 CBGP, CIRAD, INRAE, IRD, Institut Agro, Univ. Montpellier, Montpellier, France. ORCID: 0000-0001-9458-3785 (JH); 0000-0003-3740-5346 (LB) 2 Department of Entomology, College of Plant Protection, China Agricultural University, Beijing 100193, China. ORCID: 0000-0002-0622-2064 (XL) 3 Department of Biological Sciences, University of Memphis, Memphis, TN 38152 ORCID: 0000-0002-7823-8727 (DDM) 4 Center for Biodiversity Research, University of Memphis, Memphis, TN 38152 5 CBGP, INRAE, IRD, CIRAD, Institut Agro, Univ. Montpellier, Montpellier, France. ORCID: 000-0003-3885-5410 (RA); 0000-0002-8284-6215 (GJK) 6 School of Biological Sciences, Seoul National University, Seoul 08826, Republic of Korea. ORCID: 0000-0002-4258-8661 (SS) 7 CSIRO, Australian National Insect Collection, GPO Box 1700, Canberra, ACT 2601, Australia. ORCID: 0000-0002-1837-580X (RGO) 8 Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, USA. ORCID: 0000-0002-6843-0539 (BDF) 9 Bio-Protection Research Centre, P.O. Box 85084, Lincoln University, Lincoln 7647, New Zealand. Current address: The New Zealand Institute for Plant and Food Research, Mount Albert Research Centre, Private Bag 92169, Auckland 1142, New Zealand. ORCID: 0000-0001-7112-421X (SDJB) 10 Manaaki Whenua - Landcare Research, PB 92170, Auckland, New Zealand. ORCID: 0000-0001-8549-8933 (RABL) Abstract Weevils are an unusually species-rich group of phytophagous insects, for which there is increasing evidence of frequent involvement in brood-site pollination. This study examines phylogenetic patterns in the emergence of brood-site pollination mutualism among one of the most speciose beetle groups, the flower weevils (subfamily Curculioninae). We analyzed a novel phylogenomic dataset consisting of 214 nuclear loci for 202 weevil species, with a sampling that mainly includes flower weevils as well as representatives of all major lineages of true weevils (Curculionidae). Our phylogenomic analyses establish a uniquely comprehensive phylogenetic framework for Curculioninae and provide new insights into the relationships among lineages of true weevils. Based on this phylogeny, statistical reconstruction of ancestral character states revealed at least ten independent origins of brood-site pollination in higher weevils through transitions from ancestral associations with reproductive structures in the larval stage. Broadly, our results illuminate the unexpected frequency with which true weevils — typically specialized phytophages and hence antagonists of plants — have evolved mutualistic interactions of ecological significance that are key to both weevil and plant evolutionary fitness and thus a component of their deeply intertwined macroevolutionary success. Figures Figure 1 (part I). Maximum-likelihood tree resulting from analyses of 214 nuclear protein-coding genes (focus on the CEGH clade and outgroups). Support at node refers to SH-aLRT values ≥ 80% and uBV ≥ 95% (**). Single * refer to SH-aLRT values ≥ 80% only. Clades with black branches and highlighted in blue are classified in Curculioninae sensu Caldara et al. (2014). Taxa displayed on the left: 1 - Hypsomus sp. (Styphlini); 2 - Myllorhinus sp. (Storeini s. lat.); 3 - Encosmia sp. (Storeini s. lat.). Figure 1 (part II). Maximum-likelihood tree resulting from analyses of 214 nuclear protein-coding genes (focus on the CCCMS clade). Node support values refer to SH-aLRT values ≥ 80% and uBV ≥ 95% (**). Single * refer to SH-aLRT values ≥ 80% only. Clades with black branches and highlighted in blue are classified in Curculioninae sensu Caldara et al., (2014). Clades highlighted in darker blue contain genera engaged in brood-site pollination mutualism and the corresponding genera are highlighted in orange (higher taxonomic rank when specific genera are not included in the tree). Other lineages of the CCCMS clade are in bold font. Taxa displayed on the right: 1 - Tychius sp. (Tychiini); 2 - Anthonomus sp. (Athonomini); 3 - Tachyerges sp. (Rhamphini); 4 - Derelomus sp. (Derelomini); 5 - Cionus sp. (Cionini); 6 - Daeneus sp. (Ochyromerini); 7 - Meriphus sp. (Eugnomini); 8 - Archarius sp. (Curculionini); 9 - Dorytomus sp. (Ellescini); 10 - Cleopomiarus sp. (Mecinini). Figure 2. Results of the ASE analysis of larval tissue specialization carried out on the CCCMS clade, with an ER model and using a continuous-time reversible Markov model with 1000 simulations. In addition, red arrows are used to underline the independent origins of brood-site mutualism inferred in another ASE analysis (see Fig. S4). Two clades including brood-site pollinator genera that were not sampled in our study are also highlighted using red rectangles. Additional files Figure S1. Full ML tree with support values. Figure S2. Support for ML analyses. Figure S3. Results of the ASE analysis of the evolution of the tissue specialization by weevil larvae in the CCCMS clade, with an ER model and using a continuous time-reversible Markov model with 1000 simulations. Figure S4. Results of the ASE analysis on the evolution of brood-site pollination in the CCCMS clade, with an ER model and using a continuous time-reversible Markov model with 1000 simulations. Zenodo supplementary files AHE_pipeline.txt shows the detailed step-by-step script used to generate the phylogeny obtained in this study from raw sequencing data. ASE Analyses.zip contains the script and the associated raw results of the ASE analyses. Cole_tcas_probes.fasta contains the Coleopteran probes used. IBA results.zip contains IBA results. IQ-TREE files.zip contains input and output files of the IQ-TREE analysis. Scripts.zip contains the scripts associated with the file AHE_pipeline.txt