Phylogenomic insights into the tribal-level phylogeny of Typhlocybinae
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Jan 28, 2026 version files 127.58 MB
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Abstract
Typhlocybinae is one of the most diverse groups of leafhoppers, constituting an important component of phytophagous insect diversity. The traditional tribal-level phylogenetic relationships within this subfamily remain contentious, with differing hypotheses implying distinct evolutionary histories. This study contributes to resolving these controversies using phylogenomics. We newly sequenced low-coverage whole genomes for 54 species spanning the six classic tribes of Typhlocybinae. From these data, we extracted thousands of universal single-copy orthologs (USCOs) and ultraconserved elements (UCEs). Robust tribal-level phylogenies were reconstructed using multiple dataset matrices (USCO50, USCO70, USCO90, USCO_fna, ClipKIT_USCO70, UCE_fna) and tree-building strategies, including partitioned maximum likelihood with homogeneous models, unpartitioned heterogeneous mixture models, and the multi-species coalescent model. A particular focus was placed on elucidating the complex taxonomic status between Zyginellini and Typhlocybini, integrating molecular results with morphological evidence. Our findings indicate that the choice of molecular marker type and modeling methods can influence the inferred tribal-level relationships. Data filtering improves tribal-level support. The final analyses reveal that Zyginellini is not monophyletic but is intermixed with Typhlocybini, although some Zyginellini lineages appear to have originated earlier than the Old World Typhlocybini. The other four tribes of Typhlocybinae are all monophyletic. The tribal-level phylogenetic relationship is: ((Zyginellini_Typhlocybini) + (Dikraneurini + Erythroneurini)) + (Alebrini + Empoascini). Furthermore, integrating wing venation morphology with previous molecular evidence, we propose the Eualebrina subtribe nov. of Typhlocybini (sensu lato). This study provides unprecedented genomic-scale data for Typhlocybinae and offers a framework to address similar phylogenetic challenges in other organisms.
This README.txt file was generated on 2026-01-27 by Weiwei Ran
File overview
To ensure universal readability, we have provided both .docx and .pdf formats for the file containing the same content (i.e., Supplementary_Material). This Supplementary_Material file includes all the information necessary to support the research findings, including the underlying data (Table S1) used to substantiate the conclusions. Additionally, we have provided separate charts for the Supplementary_Material.
File List:
Table_S1.csv Data statistics for phylogenomic studies
Definition for column headers:
Subfamily: A taxonomic rank above the level of Tribe.
Tribe: A taxonomic rank above the level of Genus.
Species: Taxonomic units at the species level.
Type of data: Indicates whether genomic or transcriptomic data were used.
Source: The provider of the data.
Sampling site: Geographic location where the species was collected.
NCBI/SRA number: Accession number(s) corresponding to the species in the NCBI.
Table_S2.csv Assembly information statistics
Definition for column headers:
Species: Taxonomic units at the species level.
Raw base (G): The size of raw sequencing data (in gigabases) for newly sequenced genomes in this study, or the size of raw transcriptomic data downloaded from NCBI.
Average read depth (×): Raw base (Gb) / Assembly size (bp).
Assembly size (bp): The size of the assembly at the scaffold level for the corresponding species, generated using SPAdes v4.0.0.
SPAdes time (hour): Time (in hours) required for genome or transcriptome assembly using SPAdes v4.0.0.
Redundans (minute): Time (in minutes) taken for heterozygous redundancy removal from the assembled scaffolds.fasta using Redundans v2020.01.28.
BUSCO (minute): Time (in minutes) required for Benchmarking Universal Single-Copy Ortholog (BUSCO) extraction using BUSCO v5.4.7.
Number of Scaffolds: The total count of scaffolds generated from genome or transcriptome assembly using SPAdes v4.0.0.
Max scaffold length (bp): The length (in base pairs) of the longest scaffold in the assembly generated using SPAdes v4.0.0.
Avg_len of Scaffold: The average length (in base pairs) of scaffolds in the assembly generated using SPAdes v4.0.0.
N50 length (bp): The N50 statistic (in base pairs) of the assembly generated using SPAdes v4.0.0.
Scaffold Contig Coverage: The overall completeness of scaffold coverage by contigs in the assembly results from SPAdes v4.0.0, expressed as a percentage.
GC (%): The proportion (as a percentage) of guanine (G) and cytosine (C) bases in the assembled data.
Table_S3.csv Statistics of BUSCO extraction results
Definition for column headers:
Species: represents taxonomic units at the species level.
BUSCO extraction: the extraction results of Benchmarking Universal Single-Copy Orthologs (BUSCOs) for each species.
Figure_S1.jpg (A): Statistical trend of nRCFV values for all genes based on trimmed USCOs amino acid sequences. (B): Statistical trend of nRCFV values for all genes based on trimmed USCOs nucleotide sequences. (C): Statistical trend of evolutionary rate values for all genes based on nRCFV-filtered USCOs nucleotide sequences. (D): Statistical trend of treeness values for all genes based on nRCFV and evolutionary rate-filtered USCOs nucleotide sequences.
Figure_S2.jpg Extraction results of universal single-copy orthologs (USCOs) from tribe Zyginellini
Figure_S3.jpg Extraction results of universal single-copy orthologs (USCOs) from tribe Typhlocybini
Figure_S4.jpg Extraction results of universal single-copy orthologs (USCOs) from tribe Alebrini (species starting with "Al_"), Dikraneurini (species starting with "Di_"), Empoascini (species starting with "Em_") and Erythroneurini (species starting with "Er_")
Figure_S5.jpg Extraction results of universal single-copy orthologs (USCOs) from the outgroups: subfamilies Cicadellinae (species starting with "Ci_"), Deltocephalinae (species starting with "De_"), and Evacanthinae (species starting with "Ev_")
Figure_S6.jpg The average length of the species in each ultraconserved element loci
Figure_S7.jpg Phylogenetic tree of the subfamily Typhlocybinae based on matrix USCO50 using the partitioning model in IQ-TREE. The values at the branch nodes are the support values (%) of SH-aLRT and UFBoot, respectively
Figure_S8.jpg Phylogenetic tree of the subfamily Typhlocybinae based on matrix USCO50 using the wASTRAL coalescent model. The values at the branch nodes are the support rates
Figure_S9.jpg Phylogenetic tree of the subfamily Typhlocybinae based on matrix USCO70 using the partitioning model in IQ-TREE. The values at the branch nodes are the support values (%) of SH-aLRT and UFBoot, respectively
Figure_S10_.jpg Phylogenetic tree of the subfamily Typhlocybinae based on matrix USCO70 using the heterogeneous mixture model PMSF (with the initial guide tree being the partitioned ML tree of USCO70) in IQ-TREE. The values at the branch nodes are the support values (%) of SH-aLRT and UFBoot, respectively
Figure_S11.jpg Phylogenetic tree of the subfamily Typhlocybinae based on matrix USCO90 using the partitioning model in IQ-TREE. The values at the branch nodes are the support values (%) of SH-aLRT and UFBoot, respectively
Figure_S12.jpg Phylogenetic tree of the subfamily Typhlocybinae based on matrix USCO90 using the wASTRAL coalescent model. The values at the branch nodes are the support rates
Figure_S13.jpg Phylogenetic tree of the subfamily Typhlocybinae based on matrix USCO90 using the heterogeneous mixture model PMSF (with the initial guide tree being the partitioned ML tree of USCO50) in IQ-TREE. The values at the branch nodes are the support values (%) of SH-aLRT and UFBoot, respectively
Figure_S14.jpg Phylogenetic tree of the subfamily Typhlocybinae based on matrix USCO90 using the heterogeneous mixture model PMSF (with the initial guide tree being the partitioned ML tree of USCO70) in IQ-TREE. The values at the branch nodes are the support values (%) of SH-aLRT and UFBoot, respectively
Figure_S15.jpg Phylogenetic tree of the subfamily Typhlocybinae based on matrix USCO90 using the heterogeneous mixture model PMSF (with the initial guide tree being the partitioned ML tree of USCO90) in IQ-TREE. The values at the branch nodes are the support values (%) of SH-aLRT and UFBoot, respectively
Figure_S16.jpg Phylogenetic tree of the subfamily Typhlocybinae based on matrix ClipKIT_USCO70 using the wASTRAL coalescent model. The values at the branch nodes are the support rates
Figure_S17.jpg Phylogenetic tree of the subfamily Typhlocybinae based on USCOs that have been filtered by treeshrink using the wASTRAL coalescent model. The values at the branch nodes are the support rates
Figure_S18.jpg Phylogenetic tree of the subfamily Typhlocybinae based on matrix USCO_fna using the partitioning model in IQ-TREE. The values at the branch nodes are the support values (%) of SH-aLRT and UFBoot, respectively
Figure_S19.jpg Phylogenetic tree of the subfamily Typhlocybinae based on matrix USCO_fna using the wASTRAL coalescent model. The values at the branch nodes are the support rates
Figure_S20.jpg Phylogenetic tree of the subfamily Typhlocybinae based on the nucleotide sequences of USCOs that have been trimmed by ClipKIT using the wASTRAL coalescent model. The values at the branch nodes are the support rates
Figure_S21.jpg Phylogenetic tree of the subfamily Typhlocybinae based on matrix UCE_fna using the partitioning model in IQ-TREE. The values at the branch nodes are the support values (%) of SH-aLRT and UFBoot, respectively
Figure_S22.jpg Phylogenetic tree of the subfamily Typhlocybinae based on matrix UCE_fna using the wASTRAL coalescent model. The values at the branch nodes are the support rates
Figure_S23.jpg Phylogenetic tree of the subfamily Typhlocybinae based on the UCEs that have been trimmed by ClipKIT using the wASTRAL coalescent model. The values at the branch nodes are the support rates
Figure_S24.jpg (A) is the strict consensus of eight equally parsimonious total evidence trees (Balme, 2007); (B) is the maximum likelihood bootstrap analysis based on the concatenated anchored hybrid enrichment nucleotide sequence dataset, and only the low support rates are shown (Dietrich et al., 2017)
Figure_S25.jpg (A): the maximum likelihood phylogenetic tree constructed based on multiple genes including H3, H2A, 28S rDNA D2, 16S rDNA, and COI. The numbers beside the branches represent the maximum likelihood bootstrap scores and Bayesian posterior probabilities (a dash “-” indicates that the branch was not recovered in the Bayesian analysis) (Lu et al., 2021). (B): the results of the maximum likelihood and Bayesian analyses based on mitochondrial protein-coding genes, with only low support rates shown (Lin et al., 2021)
Figure_S26.jpg (A): the phylogenetic tree inferred from the mitochondrial cox1 gene using the maximum likelihood method and Bayesian inference. The first number at each node represents the bootstrap support rate of the maximum likelihood analysis, and the second number represents the Bayesian posterior probability (Chen et al., 2021). (B): the phylogenetic tree constructed using the Bayesian method based on the site-heterogeneous model CAT+GTR for the amino acids of mitochondrial protein-coding genes (Yan et al., 2022)
Figure_S27.jpg (A): the maximum likelihood phylogenetic tree based on 665 anchored hybrid loci. Only low support rates are shown, and the remaining support rates are 100 (Cao et al., 2023). (B): the phylogenetic tree constructed by the Bayesian and maximum likelihood methods based on mitochondrial protein-coding genes and ribosomal RNA genes. Only low support rates are presented, and the remaining support rates are 1/100 (Zhou et al., 2023)
Supplemental Files References
Balme G R. 2007. Phylogeny and systematics of the leafhopper subfamily typhlocybinae (insecta: Hemiptera: cicadellidae).[D]. North Carolina State University.
Cao Y, Dietrich C H, Kits J H, et al. 2023. Phylogenomics of microleafhoppers (Hemiptera: Cicadellidae: Typhlocybinae): morphological evolution, divergence times, and biogeography[J]. Insect Systematics and Diversity, 7(4): 1.
Chen X, Li C, Song Y. 2021. The complete mitochondrial genomes of two erythroneurine leafhoppers (hemiptera, cicadellidae, typhlocybinae, erythroneurini) with assessment of the phylogenetic status and relationships of tribes of typhlocybinae[J]. ZooKeys, 1037: 137-159.
Dietrich C H, Allen J M, Lemmon A R, et al. 2017. Anchored Hybrid Enrichment-Based Phylogenomics of Leafhoppers and Treehoppers (Hemiptera: Cicadomorpha: Membracoidea)[J]. Insect Systematics and Diversity, 1(1): 57-72.
Lin S, Huang M, Zhang Y. 2021. Structural features and phylogenetic implications of 11 new mitogenomes of typhlocybinae (hemiptera: Cicadellidae)[J]. Insects, 12(8): 678.
Lu L, Dietrich C H, Cao Y, et al. 2021. A multi-gene phylogenetic analysis of the leafhopper subfamily Typhlocybinae (Hemiptera: Cicadellidae) challenges the traditional view of the evolution of wing venation[J]. Molecular Phylogenetics and Evolution, 165: 107299.
Yan B, Dietrich C H, Yu X, et al. 2022. Mitogenomic phylogeny of Typhlocybinae (Hemiptera: Cicadellidae) reveals homoplasy in tribal diagnostic morphological traits[J]. Ecology and Evolution, 12(6): e8982.
Zhou X, Lei Y, Dietrich C H, et al. 2023. Investigating Monophyly of Typhlocybini Based on Complete Mitochondrial Genomes with Characterization and Comparative Analysis of 19 Species (Hemiptera: Cicadellidae: Typhlocybinae)[J]. Insects, 14(11): 842.
