Ecological interactions drive evolutionary outcomes: The first example of extreme and independent host plant shifts mediated by parasitic plants in insects

Ecological interactions drive evolutionary outcomes: The first example of extreme and independent host plant shifts mediated by parasitic plants in insects

Zelvelder B.1,2,*, Kergoat G.J.2, Benoit L.3, Tsuchida T.4, Haran J.3 & Allio R.2,*

1 ISEM, Univ Montpellier, CNRS, IRD, Montpellier, France. ORCID: 0009-0007-8486-2511 (BZ; [e-mail])

2 Centre de Biologie pour la Gestion des Populations, INRAE, IRD, CIRAD, Institut Agro, Univ. Montpellier, Montpellier, France. ORCID: 0000-0002-8284-6215 (GJK; [e-mail]); 000-0003-3885-5410 (RA; [e-mail]) 

3 Centre de Biologie pour la Gestion des Populations, CIRAD, INRAE, IRD, Institut Agro, Univ. Montpellier, Montpellier, France. ORCID: 0000-0003-3740-5346 (LB; [e-mail]); 0000-0001-9458-3785 (JH; [e-mail]) 

4 Faculty of Science, Academic Assembly, University of Toyama, 3190 Gofuku, Toyama, Toyama 930-8555, Japan. ORCID: 0000-0001-6065-4469 (TT; [e-mail])

Running title

Parasitic plants mediated weevil host shifts

Abstract

Phytophagous insects have specialized on virtually every plant lineage. Parasitic plants, however, are uncommon hosts. Among insects, only a single lineage of weevils, the Smicronychini, has successfully radiated on both parasitic and non-parasitic plants in a large panel of distantly related Asterid families. This unusual pattern suggests that major host plant shifts have occurred over the course of their diversification. Through the analysis of a phylogenomic dataset, we reconstruct for the first time their evolutionary history and ancestral host plant associations. Our results show that independent host plant shifts occurred both from parasitic to non-parasitic hosts and between distinct parasitic lineages. These results suggest that host shift mechanisms can be driven by ecological opportunities provided by plant-plant interactions. This first evidence of extreme insect host plant shifts mediated by parasitic plant-plant interactions emphasizes the core importance of ecological interactions as driving forces behind insect host plant shifts.

Figures & Tables

Figure 1: Host repertoire of Smicronychini weevils. From top to bottom: diagrams represent parasitic interactions between Smicronychini weevils and their host plants. Host plant families that make up the host repertoire of Smicronychini are shown below each corresponding diagram, with examples of associated weevils and known galls. Credits: BZ & JH, except for Orobanchaceae (Clémence Massard) and Asteraceae (@sinaloasilvestre, iNaturalist) pictures; diagrams with BioRender ©.

Figure 2: Phylogenomic tree based on the AHE dataset. A. Maximum likelihood tree of the AHE dataset. Nodes poorly supported (SH-aLRT < 80% or UFBS < 95%) are respectively highlighted with gray triangles and circles. For graphical purposes, some branches were shortened to half of their length (//). Pictures of weevils are linked to corresponding species. B. Partitioning scheme of a theoretical AHE loci. Flanking and inserted partitions (f1-3) are highlighted in grey and coding partitions (c1-3) are highlighted in green. The region targeted by the probes (P) is shown in red but does not appear in the final supermatrix. Credits: JH.

Figure 3: Ancestral host plant estimations of Smicronychini weevils and their current ecologies. The phylogenetic tree topology corresponds to one of the ML trees obtained from the extended dataset pruned to one branch per species. Species grafted with COI data are less robust than the AHE backbone and are represented with dashed branches. Gall-inducing species are marked with a “x” between branch tips and species names. Host plants of each species of weevil are colored according to their botanical family (on the right) and parasitic type (in top-right corner) as illustrated in Figure 1, while unknown states are left blank. Pie charts represented on each node correspond to the estimated likelihood of each character state computed by the ace function. Credits: BZ & JH

Appendices

Figure S1: Flowchart of the phylogenomic pipeline developed to process AHE data. Each step, numbered from 1 to 10 as referenced in the main text, is represented, from raw reads to the complete supermatrix. External tools and custom scripts (marked with an *) are given, along with a diagram representing each step of the pipeline.

Figure S2: Complete grafted tree of the extended dataset. ML tree generated with MFP+MERGE model on IQTREE v2.3.2 using the AHE topology as backbone. To increase computation time, -allnni option was not used. Bootstraps show SH-aLRT and UFBS values respectively.

Figure S3: Grafted tree of the extended dataset with one specimen per species. ML tree generated with MFP+MERGE model on IQTREE v2.2 using the AHE topology as backbone. Bootstraps show SH-aLRT and UFBS values respectively.

Table S1: Detailed interactions of species represented in the extended dataset. Informations marked with an asterisk (*) are considered unsure. Parasitic mode (PM): np = non-parasitic, hemip = hemiparasitic, p = 

Table S2: Complete specimen data. Sample IDs highlighted in red were removed from the final dataset, those highlighted in yellow were merged to an AHE sample as mentionned in "COI" column.

Zenodo supplementary files

01_PREPROCESS: Commands and files used to gather AHE probes targeted sequences from Anthonomus grandis genome

see README.sh for a detailed description of files and commands used to obtain them.

02_SCRIPTS: Scripts used during phylogenomic analysis.

Most scripts are used by the shell pipeline "04-FcC_HMMC_PB_elongated_probes.sh". Other softwares used by the pipeline that are available online are not listed but can be found inside the pipeline script or in figure S1.

bed and gapped_refs folders contain necessary files for the pipeline to run, see 01_PREPROCESS material to see how they were generated.bed - genomic data from Anthonomus grandis reference used by the pipeline steps 7-8gapped_refs - probe consensus sequences targeted on Anthonomus grandis reference genome, split fasta from consensus_probes_gap.fasta

04-FcC_HMMC_PB_elongated_probes.sh - Semi-automatic pipeline routinely used during analysescheck_het.R - estimates genetic distanceclean_duplicated_seq.sh - removes or makes a consensus of sequences from specimens with multiple sequences depending on genetic distancecount_gap_per_site.py - computes number of gaps per sitedrop_short_seq.py - removes sequences shorter than a given fraction of non-gapsHMMcleanNuc.pl - Nucleotide derived HMMCleaner script: Di Franco, A., Poujol, R., Baurain, D. & Philippe, H. (2019). Evaluating the usefulness of alignment filtering methods to reduce the impact of errors on evolutionary inferences. BMC Evol. Biol., 19, 21.improve_parts.R - updates partition files based on Anthonomus grandis reference genome.make_codon_partitions_forProbe_from_charset.py - makes codon partitions from a nexus "charset" filemake_codon_partitions_forProbe.py - makes codon partitions from partition files ".part"make_gapped_parts.py - makes partition files based on the probe reference considering gaps in probe sequencemake_good_fasta.py - converts block fasta to one line formatPaulBlock.pl - removes columns with more than a given threshold of gaps (only for non-coding parts)remove_seq_given_ID.py - removes sequences with a strict given IDremove_seq_given_relaxed_ID.py - removes sequences with a given patternseqCat.pl - cf file headerseqConverter.pl - cf file headertrim_partition_file.R - trims sequences overlapping with each other based on bed formatted reference genome

03_TREES: Intermediate and finales files generated by the phylogenomic pipeline

    AHE: Phylogenomic analyses on AHE dataset

AHE_2024_v8.3_70.phylip - Final sequence file obtained with the phylogenomic pipeline, only keeping loci with >70% speciesAHE_2024_v8.3_70.nex - Partition file with 1 partition per flanking/coding regions (see Fig. 3B in main text)AHE_2024_v8.3.70.MFPMERGE* - Tree generated with MFP+MERGE model

    COI: Phylogenetic analyses on COI dataset

COI_2025.v1.fasta - fasta alignment of all cytochrome oxydase I samples cleaned from bad sequencesCOI_2025.v1.noemptyseq* - COI tree of all samples, cleaned from empty sequences (corresponding to AHE specimen without COI)

    grafting: Phylogenomic analyses on extended dataset

# Phylogenetic analysesconstraintfile.txt - Backbone generated by AHE analysesAHE_COI_2025.v1* - Grafted tree with all samples, launched without --allnni iqtree option to reduce computation timeAHE_COI_2025.v2.monosp.nex | .phylip - Nexus and phylip files resulting from manual removal of all but one representative of each species based on AHE_COI_2025.v1.grafting.noallnni and COI tree (for dentirostris and rubricatus), see tips highlighted in red in AHE_COI_2025.v1.grafting.noallnni.treefile.keepsp.pdf for samples kept in v2AHE_COI_2025.v2* - Grafted tree with one sample per species

# Topology teststopo1.newick - Same topology as AHE_COI_2025.v2bis.grafting.monosp.treefiletopo2* - Alternative topology assuming the monophyly of North American Smicronyx speciestopo3* - Alternative topology assuming the monophyly of North American Smicronyx and Promecotarsus speciestopotest.iqtree - results of the topology test showing no significative difference between the three tested topologies

04_ASE: Ancestral character state estimations are run with ASE_v3.R

topo1 = paraphyletic Asteraceae-feeding speciestopo2 = monophyletic Asteraceae-feeding speciesBoth topologies are equally likely (cf ../03_TREES/grafting/topotest.iqtree)

ACE_HPPM_ER.topo1.pdf - ACE with ace() function on topo1ACE_HPPM_ER.topo2.pdf - ACE with ace() function on topo2ACE_HPPM_Likelihoods.csv - Ancestral state likelihoods at each node of ACE_HPPM_ER.topo2.pdfASE_pruned_topo1_collapsed_HPPM_ER.pdf - ACE with Phytools make.simmap etc. on topo1. NA's were removed and bootstraps below thresholds were left polytomicASE_pruned_topo2_collapsed_HPPM_ER.pdf - same on topo2AHE_COI_2025_70coll.v2.grafting.monosp.contree - topo1 collapsed if bootstraps <70AHE_COI_2025.v2.grafting.monosp.contree - topo1host_plants.txt - table with host repertoire of each species (see Table S1)topo2_70coll.rooted.contree - topo2 collapsed if bootstraps <70topo2.rooted.contree - topo2

Raw data & sequences

Raw target capture data and assembled AHE have been deposited in GenBank (NCBI) under the accession PRJNA1244829.

COI sequences generated for the present study have been deposited on BOLD systems (pending).