﻿Revalidation of the jumping spider genus Cheliceroides Żabka, 1985 based on molecular and morphological data (Araneae, Salticidae)

﻿Abstract The monotypic genus Cheliceroides Żabka, 1985 is revalidated based on both molecular sequence data (ultra-conserved elements and protein coding genes of mitochondrial genomes) and morphological evidence. Our molecular phylogenetic analyses show that Cheliceroides is not closely related to Colopsus Simon, 1902, not even in the same tribe, and a comparative morphological study also demonstrates significant differences in the genital structures (i.e. in the shape of embolus, and with or without pocket on epigynum) of the two genera. Therefore, we remove Cheliceroides from the synonymy of Colopsus, and its generic status is revalidated.


Introduction
The jumping spider genus Cheliceroides Żabka, 1985 originally contained only the type species, Cheliceroides longipalpis Żabka, 1985, which has been commonly collected from Vietnam and southern China (World Spider Catalog 2024).A second species, Cheliceroides brevipalpis Roy, Saha & Raychaudhuri, 2016, was later reported from India (Roy et al. 2016), but it has been transferred to the genus Bathippus Thorell, 1892 (tribe Euophryini Simon, 1901; see Logunov 2021).Based on the results of a molecular phylogenetic study using Sanger-sequenced data (Maddison et al. 2014), Maddison (2015) included Cheliceroides in the tribe Hasariini Simon, 1903 in the phylogenetic classification of jumping spiders.Later, Logunov (2021) synonymized Cheliceroides with Colopsus Simon, 1902 based on similarities of morphological characters, such as the modified and elongate male chelicerae and male palpal characteristics, and transferred its type species to Colopsus, as Colopsus longipalpis (Żabka, 1985).Colopsus has been placed in the tribe Plexippini Simon, 1901 based on other molecular phylogenetic results (Kanesharatnam and Benjamin 2021).However, in a recent comparative mitogenomic study of jumping spiders, Colopsus longipalpis was not clustered with the other members of the tribe Plexippini on the phylogeny (Zhang et al. 2023a), which challenged Logunov's taxonomic treatment of Cheliceroides.ZooKeys 1196: 243-253 (2024), DOI: 10.3897/zookeys.1196.117921 Long Lin et al.: Revalidation of Cheliceroides Żabka, 1985 Here we thoroughly investigate the phylogenetic placement of Cheliceroides in relation to Colopsus and other putatively related genera using both ultra-conserved element (UCE) and mitochondrial genome datasets.Comparative morphological study on the type species of both Cheliceroides and Colopsus is carried out to further clarify the taxonomic status of Cheliceroides.The implication of phylogenomic results on the classification of salticids is also discussed.

Materials and methods
All specimens are preserved in 85-100% ethanol and stored at −20 °C.The photographs of genitalia were taken under a Leica M205A stereomicroscope.Photographs of palp, epigyne, and spiders were stacked using Helicon Focus v. 7 and retouched in the Adobe Photoshop CC 2022.Specimens were measured by the measuring tool of Leica LAS v. 4.3.Female vulvae were cleared with Pancreatin (BBI Life Sciences) or macerated in clove oil.All specimens studied are deposited in the Museum of Hebei University, Baoding, China (MHBU).Abbreviations used in the study: CD, copulatory duct; CO, copulatory opening; E, embolus; FD, fertilization duct; P, pocket; S, spermatheca; SD, sperm duct; RTA, retrolateral tibial apophysis.
Molecular data were obtained for ultra-conserved elements (UCEs) and mitogenomes to compose the UCE and mitogenomic datasets, each with 46 species (see Table 1 for detailed information).Genomic DNA extraction was performed using the QIAGEN dNeasy Blood & Tissue Kit, and the RNA was removed with 4 μL of rNase A (Solarbio) followed by a 2-minute incubation at room temperature.The library preparation was conducted using the NEXTFLEX Rapid DNA-Seq Kit 2.0 and the NEXTFLEX Unique Dual Index Barcodes (Set C) (Bioo Scientific) following the protocols by Zhang et al. (2023b).UCE enrichment followed the myBaits protocol 5.01 (Daicel Arbor Biosciences) using a modified version of the RTA probes, the "RTA_v3" probe set (42,213 probes targeting 3818 UCE loci) that was proposed by Zhang et al. (2023b).The enriched UCE libraries were then sent to Novogene Co. Ltd for sequencing using the Illumina NovaSeq platform with 150-bp paired-end reads.The UCE loci were extracted from the empirically enriched and sequenced raw reads following the protocols applied in Zhang et al. (2023b) with the PHYLUCE (Faircloth 2016) workflow.For ten species with whole genome sequencing data, the genomes were first assembled using the Phylogenomics from Low-coverage Whole-genome Sequencing (PLWS) pipeline (Zhang et al. 2019), and then the UCEs were harvested using the "RTA_v3" probes and the PHYLUCE workflow (see Zhang et al. 2023b for details).
The UCEs extracted from genomes and target enrichment data were combined and organized by locus, and then aligned using Mafft v. 7.313 (Katoh and Standley 2013) with the L-INS-I strategy.Poorly aligned regions were initially trimmed by the heuristic method "-automated1" in Trimal v. 1.4.1 (Capella-Gutiérrez et al. 2009).We then applied Spruceup v. 2020.2.19 (Borowiec 2019) to convert the remaining obviously misaligned fragments to gaps in each alignment (cutoff as 0.75).The gappy regions in each alignment were later masked using Seqtools (PASTA; Mirarab et al. 2014) with "masksites = 23".Loci with trimmed alignment length less than 200 bp or less than 50% of taxon occupancy were removed, which resulted in 2593 loci in the final dataset for phylogenetic inference.All remaining UCE loci were concatenated by FASconCAT v. 1.0 (Kück and Meusemann 2010).The maximum-likelihood (ML) analyses were conducted in IQ-TREE v. 2.0.6 (Minh et al. 2020) with the best-fitting model and optimized partition scheme inferred using the option "-m MF+MERGE".Ten independent ML tree searches (five with random starting trees and five with parsimonious starting trees) were run with the optimized model and partition scheme, and 5,000 replicates of ultrafast bootstrap analysis was conducted to assess the node supports.Mitochondrial genomes were assembled and annotated using MitoZ v. 3.4 (Meng et al. 2019) and MITOchondrial genome annotation Server (MITOS; Bernt et al. 2013) from the raw reads of UCEs, WGS (whole genome sequencing), or transcriptomes following the protocols described by Ding et al. (2023) and Zhang et al. (2023b).In addition, two mitochondrial genomes were downloaded from the GenBank.Thirteen mitochondrial protein-coding genes (PCGs) were extracted for phylogenetic analysis.Each of the 13 PCGs was aligned using Mafft v. 7.505 (Katoh and Standley 2013) with the L-INS-i strategy, and then the gaps and misaligned sites were trimmed in Trimal v. 1.2rev57 (Capella-Gutiérrez et al. 2009) with the "automated1" mode.The trimmed alignments were concatenated in PhyloSuite v. 1.2.3 (Zhang et al. 2020), and PartitionFinder2 was used to select the best partition and model.ML analyses were performed in IQ-TREE v. 2.2.0 (Minh et al. 2020) using the optimized model and partition scheme, and an ultrafast bootstrap analysis with 1,000 replicates was conducted to assess the node support.

Molecular phylogeny
The newly sequenced raw reads and assembled mitogenomes were submitted to the GenBank with accession numbers provided in Table 1.The phylogenies resulted from the UCE and 13-mitochondrial-PCG datasets are presented in Figs 1, 2. Both results show that Cheliceroides longipalpis (JXZ584) is distantly related to Plexippini, including a potential species of Colopsus (JXZ795).In the UCE phylogeny, Cheliceroides longipalpis is recovered as sister to the clade with Hasariini (excluding Bristowia Reimoser, 1934), Agoriini, and Chrysillini (Fig. 1), whereas in the mitogenomic phylogeny it is clustered as sister to other Hasariini (excluding Bristowia; Fig. 2).Therefore, the molecular phylogenetic results support removing Cheliceroides from the synonymy of Colopsus.Other implications of the molecular phylogenetic results are addressed in the discussion.Diagnosis.Cheliceroides longipalpis differs from members of Hasariini by the presence of iridescent scales on the body (Figs 4,6,7,12), the elongate male chelicera, the male palp with long and whip-like embolus originating at 2 o'clock (left palp) and coiling around the rounded tegulum 16), and the female epigynum with anterior window-like structure and relatively long and coiled copulatory ducts (Figs 14,15,17,18).It is similar to Colopsus species in having modified and elongate male chelicera and a relatively long male palpal tibia (equal to or longer than the cymbium) (Żabka 1985: 210;Logunov 2021Logunov : 1023Logunov -1024;;Kanesharatnam and Benjamin 2021: 54;Fig. 3)   Cheliceroides was considered to be a member of Hasariini in the phylogenetic classification of jumping spiders (Maddison 2015), which was supported by the mitogenomic phylogeny but with poor support (bootstrap = 77%; Fig. 2).The UCE phylogeny recovered Cheliceroides as sister to the clade containing Hasariini (excluding Bristowia), Agoriini, and Chrysillini with strong support (bootstrap = 100%; Fig. 1).This indicates the placement of Cheliceroides within Hasariini is questionable.Another interesting finding from our study is the phylogenetic placement of Bristowia, which was also earlier included in the tribe Hasariini (Maddison 2015).We included the type species, Bristowia heterospinosa Reimoser, 1934 (JXZ743 and DRR297628), in our phylogenetic analyses, and the results show that it is not closely related to other Hasariini.The UCE phylogeny suggests it is sister to the clade containing Euophryini, Leptorchestini, Aelurillini, Salticini, and Plexippini (bootstrap = 100%; Fig. 1), and the mitogenomic phylogeny recovered it as sister to the clade composed of Agoriini and Chrysillini, but with low support (bootstrap = 44%; Fig. 2).Further phylogenetic study with an extended taxon sampling of major lineages of jumping spiders is needed to further clarify their phylogenetic placement.

Table 1 .
Information of the representative taxa used in the phylogenetic analyses.Accession numbers with an asterisk (*) indicate newly obtained sequences in this study.
, but it can be distinguished by the S-shaped trajectory of the sperm duct on the tegulum of the male palp (vs C-shaped in Colopsus; compare Figs 16, 19), the longer embolus coiling in a circle around the tegulum (vs shorter and coiling in half a circle at most in Colopsus; compare Figs 16, 19), the absence of epigynal coupling pocket on epigynum (vs with two pockets in Colopsus; compare Figs 17, 20), and the long, coiled copulatory ducts (vs short and not obviously coiled in Colopsus; compare Figs 18, 21).