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Gracillariidae are one of the most diverse families of internally feeding insects, and many species are economically important. Study of this family has been hampered by lack of a robust and comprehensive phylogeny. In the present paper, we sequenced up to 22 genes in 96 gracillariid species, representing all previously recognized subfamilies and genus groups, plus 20 outgroups representing other families and superfamilies. Following objective identification and removal of two rogue taxa, two datasets were constructed: dataset 1, which included 12 loci totalling 9927 bp for 94 taxa, and dataset 2, which supplemented dataset 1 with 10 additional loci for 10 taxa, for a total of 22 loci and 16 167 bp. Maximum likelihood analyses strongly supported the monophyly of Gracillariidae and most previously recognized subfamilies and genus groups. On this basis, we propose a new classification consisting of eight subfamilies, four of which are newly recognized or resurrected: Acrocercopinae Kawahara & Ohshima subfam. n.; Gracillariinae Stainton; Lithocolletinae Stainton; Marmarinae Kawahara & Ohshima subfam. n.; Oecophyllembiinae Réal & Balachowsky; Parornichinae Kawahara & Ohshima subfam. n.; Ornixolinae Kuznetzov & Baryshnikova stat. rev.; and Phyllocnistinae Zeller. The subfamily Gracillariinae is restricted to the monophyletic group comprising Gracillaria Haworth and closely related genera. We also formally transfer Acrocercops scriptulata Meyrick to Ornixolinae and use the name Diphtheroptila Vári, creating Diphtheroptila scriptulata comb. n. An exploratory mapping of larval host‐use traits on the phylogeny shows strong conservation of modes of leaf mining but much higher lability of associations with host plant orders and families, suggesting that host shifts could play a significant role in gracillariid diversification. This published work has been registered in ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:942814A2-DE66-41D4-8AB6-FF0B18C87EDB.

BackgroundResearchers conducting molecular phylogenetic studies are frequently faced with the decision of what to do when weak branch support is obtained for key nodes of importance. As one solution, the researcher may choose to sequence additional orthologous genes of appropriate evolutionary rate for the taxa in the study. However, generating large, complete data matrices can become increasingly difficult as the number of characters increases. A few empirical studies have shown that augmenting genes even for a subset of taxa can improve branch support. However, because each study differs in the number of characters and taxa, there is still a need for additional studies that examine whether incomplete sampling designs are likely to aid at increasing deep node resolution. We target Gracillariidae, a Cretaceous-age (~100 Ma) group of leaf-mining moths to test whether the strategy of adding genes for a subset of taxa can improve branch support for deep nodes. We initially sequenced ten genes (8,418 bp) for 57 taxa that represent the major lineages of Gracillariidae plus outgroups. After finding that many deep divergences remained weakly supported, we sequenced eleven additional genes (6,375 bp) for a 27-taxon subset. We then compared results from different data sets to assess whether one sampling design can be favored over another. The concatenated data set comprising all genes and all taxa and three other data sets of different taxon and gene sub-sampling design were analyzed with maximum likelihood. Each data set was subject to five different models and partitioning schemes of non-synonymous and synonymous changes. Statistical significance of non-monophyly was examined with the Approximately Unbiased (AU) test.ResultsPartial augmentation of genes led to high support for deep divergences, especially when non-synonymous changes were analyzed alone. Increasing the number of taxa without an increase in number of characters led to lower bootstrap support; increasing the number of characters without increasing the number of taxa generally increased bootstrap support. More than three-quarters of nodes were supported with bootstrap values greater than 80% when all taxa and genes were combined. Gracillariidae, Lithocolletinae + Leucanthiza, and Acrocercops and Parectopa groups were strongly supported in nearly every analysis. Gracillaria group was well supported in some analyses, but less so in others. We find strong evidence for the exclusion of Douglasiidae from Gracillarioidea sensu Davis and Robinson (1998). Our results strongly support the monophyly of a G.B.R.Y. clade, a group comprised of Gracillariidae + Bucculatricidae + Roeslerstammiidae + Yponomeutidae, when analyzed with non-synonymous changes only, but this group was frequently split when synonymous and non-synonymous substitutions were analyzed together.Conclusions1) Partially or fully augmenting a data set with more characters increased bootstrap support for particular deep nodes, and this increase was dramatic when non-synonymous changes were analyzed alon...

Higher taxa often show increasing species richness towards tropical low latitudes, a pattern known as the latitudinal biodiversity gradient (LBG). A rare reverse LBG (with greater richness towards temperate high latitudes) is exhibited by Gracillariidae leaf-mining moths, in which most described species occur in northern temperate areas. We carried out the first assessment of gracillariid species diversity in two Neotropical regions to test whether the relatively low tropical species diversity of this family is genuine or caused by insufficient sampling and a strong taxonomic impediment. Field surveys in six French Guianan and one Ecuadorian site produced 516 gracillariid specimens that were DNA barcoded to facilitate identification and to match larvae inside leaf mines with adults. Species delineation from sequence data was approximated using Automatic Barcode Gap Discovery and Refined Single Linkage Analysis through the Barcode Index Number system, and the proportion of described/undescribed species was estimated after comparison with types of 83% of described species. Locally, alpha-diversity far exceeds that of any known temperate fauna, with as many as 108 candidate species (59.3% as singletons) collected at one site, and with an estimated species richness lower bound of 240 species. Strikingly, at least 85% of the species collected as adults were found to be undescribed. Our sampling represents the most thorough survey of gracillariid species diversity in the Neotropics to date and the results from both our molecular and morphological analyses indicate that the current reverse LBG seen in this group is an artefact of insufficient sampling and a strong description deficit in the Neotropics.

Afrotropical Lithocolletinae are known from 26 described species, mainly from southern Africa. In the present study we describe 41 new species, bring to 66 the number of species from this geographical region. The new species include: Hyloconis luki De Prins, n. sp., Neolithocolletis mayumbe De Prins, n. sp., N. nsengai De Prins, n. sp., Cameraria fara De Prins, n. sp., C. landryi De Prins, n. sp., C. perodeaui De Prins, n. sp., C. sokoke De Prins, n. sp., C. torridella De Prins, n. sp., C. varii De Prins, n. sp., C. zaira De Prins, n. sp., Phyllonorycter aarviki De Prins, n. sp., P. achilleus De Prins, n. sp., P. acutulus De Prins, n. sp., P. adderis De Prins, n. sp., P. agassizi De Prins, n. sp., P. albertinus De Prins, n. sp., P. dombeyae De Prins n. sp., P. fletcheri De Prins, n. sp., P. gato De Prins, n. sp., P. grewiaephilos De Prins, n. sp., P. hibiscola De Prins, n. sp., P. ipomoellus De Prins, n. sp., P. jabalshamsi De Prins, n. sp., P. kazuri De Prins, n. sp., P. maererei De Prins, n. sp., P. mida De Prins, n. sp., P. mwatawalai De Prins, n. sp., P. ocimellus De Prins, n. sp., P. ololua De Prins, n. sp., P. rongai De Prins, n. sp., P. ruizivorus De Prins, n. sp., P. ruwenzori De Prins, n. sp., P. silvicola De Prins, n. sp., P. trochetellus De Prins, n. sp., P. tsavensis De Prins, n. sp., P. turensis De Prins, n. sp., P. umukarus De Prins, n. sp., Cremastobombycia morogorene De Prins, n. sp., C. kipepeo De Prins, n. sp., Porphyrosela desmodivora De Prins, n. sp., P. gautengi De Prins, n. sp. Furthermore, Cameraria hexalobina (Vári, 1961), n. comb. is transferred from Phyllonorycter to Cameraria, and a neotype for Porphyrosela homotropha Vári, 1963 is designated. Lithocolletis aurifascia Walker, 1875, previously placed in Phyllonorycter, is excluded from Lithocolletinae. We designate lectotypes where necessary and provide morphological descriptions of males and females, information on host plants, and detailed distribution data. We also include dichotomous keys to species groups and species, accompanied by DNA barcode sequences of 19 species. We also discuss generic relationships within Lithocolletinae based on both morphology and molecules with a special emphasis on Afrotropical taxa.