DOI: 10.5061/dryad.k6djh9wkx
title: Phylogenomic insights into the tribal-level phylogeny of Typhlocybinae

description: 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.
description: # Phylogenomic insights into the tribal-level phylogeny of Typhlocybinae
 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
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