Detecting and Removing Sample Contamination in Phylogenomic Data: An Example and its Implications for Cicadidae Phylogeny (Insecta: Hemiptera).

Contamination of a genetic sample with DNA from one or more nontarget species is a continuing concern of molecular phylogenetic studies, both Sanger sequencing studies and next-generation sequencing studies. We developed an automated pipeline for identifying and excluding likely cross-contaminated loci based on the detection of bimodal distributions of patristic distances across gene trees. When contamination occurs between samples within a data set, a comparison between a contaminated sample and its contaminant taxon will yield bimodal distributions with one peak close to zero patristic distance. This new method does not rely on a priori knowledge of taxon relatedness nor does it determine the causes(s) of the contamination. Exclusion of putatively contaminated loci from a data set generated for the insect family Cicadidae showed that these sequences were affecting some topological patterns and branch supports, although the effects were sometimes subtle, with some contamination-influenced relationships exhibiting strong bootstrap support. Long tip branches and outlier values for one anchored phylogenomic pipeline statistic (AvgNHomologs) were correlated with the presence of contamination. While the anchored hybrid enrichment markers used here, which target hemipteroid taxa, proved effective in resolving deep and shallow level Cicadidae relationships in aggregate, individual markers contained inadequate phylogenetic signal, in part probably due to short length. The cleaned data set, consisting of 429 loci, from 90 genera representing 44 of 56 current Cicadidae tribes, supported three of the four sampled Cicadidae subfamilies in concatenated-matrix maximum likelihood (ML) and multispecies coalescent-based species tree analyses, with the fourth subfamily weakly supported in the ML trees. No well-supported patterns from previous family-level Sanger sequencing studies of Cicadidae phylogeny were contradicted. One taxon (Aragualna plenalinea) did not fall with its current subfamily in the genetic tree, and this genus and its tribe Aragualnini is reclassified to Tibicininae following morphological re-examination. Only subtle differences were observed in trees after the removal of loci for which divergent base frequencies were detected. Greater success may be achieved by increased taxon sampling and developing a probe set targeting a more recent common ancestor and longer loci. Searches for contamination are an essential step in phylogenomic analyses of all kinds and our pipeline is an effective solution. [Auchenorrhyncha; base-composition bias; Cicadidae; Cicadoidea; Hemiptera; phylogenetic conflict.].

Inflation of Molecular Clock Rates and Dates: Molecular Phylogenetics, Biogeography, and Diversification of a Global Cicada Radiation from Australasia (Hemiptera: Cicadidae: Cicadettini).

Dated phylogenetic trees are important for studying mechanisms of diversification, and molecular clocks are important tools for studies of organisms lacking good fossil records. However, studies have begun to identify problems in molecular clock dates caused by uncertainty of the modeled molecular substitution process. Here we explore Bayesian relaxed-clock molecular dating while studying the biogeography of ca. 200 species from the global cicada tribe Cicadettini. Because the available fossils are few and uninformative, we calibrate our trees in part with a cytochrome oxidase I (COI) clock prior encompassing a range of literature estimates for arthropods. We show that tribe-level analyses calibrated solely with the COI clock recover extremely old dates that conflict with published estimates for two well-studied New Zealand subclades within Cicadettini. Additional subclade analyses suggest that COI relaxed-clock rates and maximum-likelihood branch lengths become inflated relative to EF-1[Formula: see text] intron and exon rates and branch lengths as clade age increases. We present corrected estimates derived from: (i) an extrapolated EF-1[Formula: see text] exon clock derived from COI-calibrated analysis within the largest New Zealand subclade; (ii) post hoc scaling of the tribe-level chronogram using results from subclade analyses; and (iii) exploitation of a geological calibration point associated with New Caledonia. We caution that considerable uncertainty is generated due to dependence of substitution estimates on both the taxon sample and the choice of model, including gamma category number and the choice of empirical versus estimated base frequencies. Our results suggest that diversification of the tribe Cicadettini commenced in the early- to mid-Cenozoic and continued with the development of open, arid habitats in Australia and worldwide. We find that Cicadettini is a rare example of a global terrestrial animal group with an Australasian origin, with all non-Australasian genera belonging to two distal clades. Within Australia, we show that Cicadettini is more widely distributed than any other cicada tribe, diverse in temperate, arid and monsoonal habitats, and nearly absent from rainforests. We comment on the taxonomic implications of our findings for thirteen cicada genera.

New genera and new species of Western Australian cicadas (Hemiptera: Cicadidae).

The cicada fauna of Western Australia is briefly reviewed. Six genera and 14 species are recorded from the State for the first time bringing the total of known species and subspecies to 105 and a list of all 105 is provided. Among the taxa here recorded are five new genera and 13 new species belonging to the tribes Macrotristriini (Illyria viridis sp. n.), Pictilini (Chrysocicada trophis sp. n.), and Cicadettini (Calipsalta gen. n., Calipsalta brunnea sp. n., C. fumosa sp. n., C. viridans sp. n., Kalarko gen. n., Kalarko ferruginosus sp. n., Ewartia adusta sp. n., Parvopsalta gen. n., Parvopsalta victoriae sp. n., Pedana gen. n., Pedana hesperia sp. n., Pegapsaltria gen. n., Pegapsaltria lutea sp. n., Pyropsalta amnica sp. n., Py. patula sp. n., and Py. rhythmica sp. n). In addition, Erempsalta hermannsburgensis (Distant, 1907) is redescribed and its presence in Western Australia (and four other States) documented for the first time. Songs are analysed for all species except two species of Pyropsalta where recordings were unavailable.

A molecular phylogeny of the cicadas (Hemiptera: Cicadidae) with a review of tribe and subfamily classification.

A molecular phylogeny and a review of family-group classification are presented for 137 species (ca. 125 genera) of the insect family Cicadidae, the true cicadas, plus two species of hairy cicadas (Tettigarctidae) and two outgroup species from Cercopidae. Five genes, two of them mitochondrial, comprise the 4992 base-pair molecular dataset. Maximum-likelihood and Bayesian phylogenetic results are shown, including analyses to address potential base composition bias. Tettigarcta is confirmed as the sister-clade of the Cicadidae and support is found for three subfamilies identified in an earlier morphological cladistic analysis. A set of paraphyletic deep-level clades formed by African genera are together named as Tettigomyiinae n. stat. Taxonomic reassignments of genera and tribes are made where morphological examination confirms incorrect placements suggested by the molecular tree, and 11 new tribes are defined (Arenopsaltriini n. tribe, Durangonini n. tribe, Katoini n. tribe, Lacetasini n. tribe, Macrotristriini n. tribe, Malagasiini n. tribe, Nelcyndanini n. tribe, Pagiphorini n. tribe, Pictilini n. tribe, Psaltodini n. tribe, and Selymbriini n. tribe). Tribe Tacuini n. syn. is synonymized with Cryptotympanini, and Tryellina n. syn. is synonymized with an expanded Tribe Lamotialnini. Tribe Hyantiini n. syn. is synonymized with Fidicinini. Tribe Sinosenini is transferred to Cicadinae from Cicadettinae, Cicadatrini is moved to Cicadettinae from Cicadinae, and Ydiellini and Tettigomyiini are transferred to Tettigomyiinae n. stat from Cicadettinae. While the subfamily Cicadinae, historically defined by the presence of timbal covers, is weakly supported in the molecular tree, high taxonomic rank is not supported for several earlier clades based on unique morphology associated with sound production.

A new genus and species of tettigarctid cicada from the early Miocene of New Zealand: Paratettigarctazealandica (Hemiptera, Auchenorrhyncha, Tettigarctidae).

A new genus and species of primitive cicada (Hemiptera: Tettigarctidae) is described from the early Miocene of southern New Zealand. Paratettigarctazealandica gen. et sp. n. is the first cicada (Cicadoidea) fossil from New Zealand and exhibits wing venation patterns typical for the subfamily Tettigarctinae. It differs from other fossil taxa and the extant genus Tettigarcta in the early divergence of CuA2 from the nodal line in the forewing, its parallel-sided subcostal cell, the early bifurcation of vein M and long apical cells of the hindwing, and in wing pigmentation patterns.