Phylogenomic insights into helmet evolution in Neotropical treehoppers (Hemiptera: Membracidae): Conserved morphological complexity across five subfamilies

We analyzed genomic data from 2,539 nuclear loci (580,110 aligned nucleotides) to investigate phylogenetic relationships among selected treehopper lineages (Hemiptera: Membracidae), with a focus on helmet morphology. Using geometric morphometric and comparative methods, we found that helmet shape—but not size—exhibits strong phylogenetic signal, indicating that shape is highly conserved across evolutionary lineages. Similar morphologies were more common among closely related taxa, particularly within genera and tribes. Our results support the monophyly of major clades (e.g., Centrotinae + Nicomiinae; Membracinae + Darninae + Similiinae), demonstrating the power of UCE-derived nuclear data to resolve relationships within Membracidae. Additionally, patterns of shape variation suggest the evolution of pronotal modularity across lineages.

Methods
Data sequences and processing

Ultraconserved elements (UCEs) are highly conserved regions of organismal genomes shared among evolutionary distant taxa (McCormack et al. 2012; Faircloth et al. 2012). We use a bait designs targeting UCEs for Hemiptera (40,207 baits for 2,731 UCEs) to recover UCE from Membracidae (Treehoppers). We use phyluce and illumiprocessor software to help analyze data (Faircloth, 2013; Faircloth et al. 2012; Faircloth et al., 2015). 

Phylogenetic analysis

Phylogenetic analysis was conducted using RAxML-NG version 0.9.0 (Kozlov et al., 2019) for Maximum Likelihood and MrBayes version 3.2.6 (Ronquist et al., 2012) for Bayesian Inference. Phylogenetic calibration was estimated using a 17 Ma Stegaspidinae fossil and a normal distribution in BEAST 2 (Bouckaert et al., 2019).

Photography and image processing

Photographs were taken with a Rebel XT camera and processed using Adobe Photoshop Lightroom version 24.1.0 (Adobe Systems Software Ireland Ltd. 2023).

Geometric morphometrics

Landmarks were placed using tpsRelw (v1.75) and tpsDig (v2.33) software (SB Morphometrics, NY, USA). Centroid size was estimated using MorphoJ (v1.08.0) (Klingenberg Lab, MC, UK). PCA visualizations were performed using the MorphoJ (v1.08.0) and R Statistical Software (v4.1.2), along with the ape, phytools, and ggplot2 packages (Revell, 2012; Wickham, 2016; R Core Team, 2021). Procrustes ANOVA by taxonomic rank (genus, tribe, and species), using a 1000-permutation test, was also performed in MorphoJ (v1.08.0) (Klingenberg Lab, MC, UK).

References 

Bouckaert R., Vaughan T.G., Barido-Sottani J., Duchêne S., Fourment M., Gavryushkina A., et al. (2019) BEAST 2.5: An advanced software platform for Bayesian evolutionary analysis. PLoS computational biology, 15(4), e1006650.

Faircloth BC, McCormack JE, Crawford NG, Harvey MG, Brumfield RT, Glenn TC. 2012. Ultraconserved Elements Anchor Thousands of Genetic Markers Spanning Multiple Evolutionary Timescales. Syst Biol 61:717-726. pmid: 22232343 doi:10.1093/sysbio/sys004.

Faircloth, B. C., Branstetter, M. G., White, N. D., & Brady, S. G. (2015). Target enrichment of ultraconserved elements from arthropods provides a genomic perspective on relationships among Hymenoptera. Molecular ecology resources, 15(3), 489-501. https://doi.org/10.1111/1755-0998.12328

Faircloth, BC. 2013. illumiprocessor: a trimmomatic wrapper for parallel adapter and quality trimming. http://dx.doi.org/10.6079/J9ILL.

Kozlov, Diego Darriba, Tomáš Flouri, Benoit Morel, and Alexandros Stamatakis (2019) RAxML-NG: A fast, scalable, and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics, 35 (21), 4453-4455 https://doi.org/10.1093/bioinformatics/btz305

McCormack JE, Faircloth BC, Crawford NG, Gowaty PA, Brumfield RT, Glenn TC. 2012. Ultraconserved Elements Are Novel Phylogenomic Markers that Resolve Placental Mammal Phylogeny when Combined with Species Tree Analysis. Genome Res 22: 746–754. pmid: 22207614 doi: 10.1101/gr.125864.111.

R Core Team (2021). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/.

Revell, L. J. (2012). phytools: An R package for phylogenetic comparative biology (and other thing*s). In Methods in Ecology and Evolution *(Vol. 3, pp. 217–223). https://doi.org/10.1111/j.2041-210X.2011.00169.x

Ronquist, F., Teslenko, M., Van Der Mark, P., Ayres, D. L., Darling, A., Höhna, S., ... & Huelsenbeck, J. P. (2012). MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic biology, 61(3), 539-542. https://doi.org/10.1093/sysbio/sys029

Wickham H (2016). ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York. ISBN 978-3-319-24277-4, https://ggplot2.tidyverse.org.