Towards a better future for DNA barcoding: Evaluating monophyly- and distance-based species identification using COI gene fragments of Dacini fruit flies.

The utility of a universal DNA 'barcode' fragment (658 base pairs of the Cytochrome C Oxidase I [COI] gene) has been established as a useful tool for species identification, and widely criticized as one for understanding the evolutionary history of a group. Large amounts of COI sequence data have been produced that hold promise for rapid species identification, for example, for biosecurity. The fruit fly tribe Dacini holds about a thousand species, of which 80 are pests of economic concern. We generated a COI reference library for 265 species of Dacini containing 5601 sequences that span most of the COI gene using circular consensus sequencing. We compared distance metrics versus monophyly assessments for species identification and although we found a 'soft' barcode gap around 2% pairwise distance, the exceptions to this rule dictate that a monophyly assessment is the only reliable method for species identification. We found that all fragments regularly used for Dacini fruit fly identification >450 base pairs long provide similar resolution. 11.3% of the species in our dataset were non-monophyletic in a COI tree, which is mostly due to species complexes. We conclude with recommendations for the future generation and use of COI libraries. We revise the generic assignment of Dacus transversus stat. rev. Hardy 1982, and Dacus perpusillus stat. rev. Drew 1971 and we establish Dacus maculipterus White 1998 syn. nov. as a junior synonym of Dacus satanas Liang et al. 1993.

Interspecific gene flow obscures phylogenetic relationships in an important insect pest species complex.

As genomic data proliferates, the prevalence of post-speciation gene flow is making species boundaries and relationships increasingly ambiguous. Although current approaches inferring fully bifurcating phylogenies based on concatenated datasets provide simple and robust answers to many species relationships, they may be inaccurate because the models ignore inter-specific gene flow and incomplete lineage sorting. To examine the potential error resulting from ignoring gene flow, we generated both a RAD-seq and a 500 protein-coding loci highly multiplexed amplicon (HiMAP) dataset for a monophyletic group of 12 species defined as the Bactrocera dorsalis sensu lato clade. With some of the world's worst agricultural pests, the taxonomy of the B. dorsalis s.l. clade is important for trade and quarantines. However, taxonomic confusion confounds resolution due to intra- and interspecific phenotypic variation and convergence, mitochondrial introgression across half of the species, and viable hybrids. We compared the topological convergence of our datasets using concatenated phylogenetic and various multispecies coalescent approaches, some of which account for gene flow. All analyses agreed on species delimitation, but there was incongruence between species relationships. Under concatenation, both datasets suggest identical species relationships with mostly high statistical support. However, multispecies coalescent and multispecies network approaches suggest markedly different hypotheses and detected significant gene flow. We suggest that the network approaches are likely more accurate because gene flow violates the assumptions of the concatenated phylogenetic analyses, but the data-reductive requirements of network approaches resulted in reduced statistical support and could not unambiguously resolve gene flow directions. Our study highlights the importance of testing for gene flow, particularly with phylogenomic datasets, even when concatenated approaches receive high statistical support.

Genomics reveals widespread hybridization across insects with ramifications for species boundaries and invasive species.

As the amount of genomic data for nonmodel taxa grows, it is increasingly clear that gene flow across species barriers in insects is much more common than previously thought. In recent years, the decreased cost and increased accuracy of long-read sequencing has enabled the assembly of high-quality reference genomes and chromosome maps for nonmodel insects. With this long-read data, we can now not only compare variation across the genome among homologous genes between species, which has been the basis of phylogenetics for more than 30 years, but also tease apart evidence of ancient and recent hybridization and gene flow. The implications of hybridization for species adaptation may be more positive than previously considered, explaining its prevalence across many groups of insects. Unfortunately, due to anthropogenic actions, some pest species appear to be benefitting from hybridization and gene flow, facilitating future invasions.

Phylogeny of gracillariid leaf-mining moths: evolution of larval behaviour inferred from phylogenomic and Sanger data.

Gracillariidae is the most taxonomically diverse cosmopolitan leaf-mining moth family, consisting of nearly 2000 named species in 105 described genera, classified into eight extant subfamilies. The majority of gracillariid species are internal plant feeders as larvae, creating mines and galls in plant tissue. Despite their diversity and ecological adaptations, their phylogenetic relationships, especially among subfamilies, remain uncertain. Genomic data (83 taxa, 589 loci) were integrated with Sanger data (130 taxa, 22 loci), to reconstruct a phylogeny of Gracillariidae. Based on analyses of both datasets combined and analyzed separately, monophyly of Gracillariidae and all its subfamilies, monophyly of the clade "LAMPO" (subfamilies: Lithocolletinae, Acrocercopinae, Marmarinae, Phyllocnistinae, and Oecophyllembiinae) and relationships of its subclade "AMO" (subfamilies: Acrocercopinae, Marmarinae, and Oecophyllembiinae) were strongly supported. A sister-group relationship of Ornixolinae to the remainder of the family, and a monophyletic leaf roller lineage (Callicercops Vári + Parornichinae) + Gracillariinae, as sister to the "LAMPO" clade were supported by the most likely tree. Dating analyses indicate a mid-Cretaceous (105.3 Ma) origin of the family, followed by a rapid diversification into the nine subfamilies predating the Cretaceous-Palaeogene extinction. We hypothesize that advanced larval behaviours, such as making keeled or tentiform blotch mines, rolling leaves and galling, allowed gracillariids to better avoid larval parasitoids allowing them to further diversify. Finally, we stabilize the classification by formally re-establishing the subfamily ranks of Marmarinae stat.rev., Oecophyllembiinae stat.rev. and Parornichinae stat.rev., and erect a new subfamily, Callicercopinae Li, Ohshima and Kawahara to accommodate the enigmatic genus Callicercops.

Novel lures and COI sequences reveal cryptic new species of Bactrocera fruit flies in the Solomon Islands (Diptera, Tephritidae, Dacini).

Results from a snap-shot survey of Dacine fruit flies carried out on three of the Solomon Islands in April 2018 are reported. Using traps baited with the male lures cue-lure, methyl eugenol, and zingerone, 30 of the 48 species previously known to occur in the Solomon Islands were collected. Six species are newly described here: Bactroceraallodistincta sp. nov., B.geminosimulata sp. nov., B.kolombangarae sp. nov., B.quasienochra sp. nov., B.tsatsiai sp. nov., and B.vargasi sp. nov., all authored by Leblanc & Doorenweerd. An illustrated key to the 54 species now known to be present in the country is provided.

Phylogeography of an endemic California silkmoth genus suggests the importance of an unheralded central California province in generating regional endemic biodiversity.

The California Floristic province is a biodiversity hotspot. Understanding the phylogeographic patterns that exist in this diverse region is essential to understanding its evolution and for guiding conservation efforts. Calosaturnia is a charismatic silkmoth genus endemic to large portions of the region with three described species, C. mendocino, C. walterorum, and C. albofasciata. We sampled all three species from across their ranges, sequenced 1463 bp of mitochondrial COI and 1941 bp of nuclear DNA from three genes, and reconstructed phylogenetic relationships and estimated divergence times within the lineages. All three species show pronounced evidence of isolation and, in two cases, secondary reconnection. An unexpected monophyletic mtDNA lineage was found in the Central Coast region, in a region thought to represent an intergrade between C. mendocino and C. walterorum. Our genetic data also significantly revise previous hypotheses as to which species occur in which regions, suggesting that historical ecological changes around four Ma ago isolated some lineages, and a secondary isolation event two Ma ago led to isolation of populations both in the Central Coast region and the southern Sierra Nevada. Our results add to a currently under-appreciated pattern suggesting that coastal Central California is not a transition zone between Northern and Southern California Floristic Province faunas but rather its own unique, periodically isolated, biogeographic region. They also suggest cryptic diversity may be present in many other currently unrecognized groups. Additional research should focus on this central California region because many species may be highly restricted in range and in need of conservation attention.

The Dacini fruit fly fauna of Sulawesi fits Lydekker's line but also supports Wallacea as a biogeographic region (Diptera, Tephritidae).

Although there is scientific consensus on most of the major biogeographic regions in the world, the demarcation of the area connecting Southeast Asia with Australia and Oceania remains debated. Two candidate boundaries potentially explain faunistic diversity patterns in the regions: Lydekker's and Wallace's lines. The islands in between both 'lines' are jointly termed Wallacea, with Sulawesi as the largest landmass. We surveyed Dacini fruit flies (Tephritidae: Dacinae) in Sulawesi between 2016 and 2019 using traps baited with male lures, resulting in 4,517 collected flies. We identified all specimens to species level, which adds 15 new species records to the island, bringing the total number of Dacini species in Sulawesi to 83. The biogeographic affinity of species in the updated checklist reveals a strong connection with former 'Sunda' (41% of species); validating Lydekker's line, but also a high level of endemism (47% of species), confirming the uniqueness of Wallacea as a biogeographic region. We further describe a new species, Bactrocera (Bactrocera) niogreta Doorenweerd, sp. nov. and discuss the taxonomy of several interesting species.

The paradoxical rarity of a fruit fly fungus attacking a broad range of hosts.

Understanding the factors that determine the realized and potential distribution of a species requires knowledge of abiotic, physiological, limitations as well as ecological interactions. Fungi of the order Laboulbeniales specialize on arthropods and are typically thought to be highly specialized on a single species or closely related group of species. Because infections are almost exclusively transmitted through direct contact between the hosts, the host ecology, to a large extent, determines the distribution and occurrence of the fungus. We examined ~20,000 fruit flies (Diptera: Dacinae) collected in Malaysia, Sulawesi, Australia, and the Solomon Islands between 2017 and 2019 for fungal infections and found 197 infected flies across eight different Bactrocera species. Morphology and 1,363 bps of small subunit (18S) DNA sequences both support that the infections are from a single polyphagous fungal species Stigmatomyces dacinus-a known ectoparasite of these fruit flies. This leads to the question: why is S. dacinus rare, when its hosts are widespread and abundant? In addition, the hosts are all Bactrocera, a genus with ~480 species, but 37 Bactrocera species found sympatric with the hosts were never infected. Host-selection does not appear to be phylogenetically correlated. These results suggest a hidden complexity in how different, but closely related, host species vary in their susceptibility, which somehow limits the abundance and dispersal capability of the fungus.

Highly variable COI haplotype diversity between three species of invasive pest fruit fly reflects remarkably incongruent demographic histories.

Distance decay principles predict that species with larger geographic ranges would have greater intraspecific genetic diversity than more restricted species. However, invasive pest species may not follow this prediction, with confounding implications for tracking phenomena including original ranges, invasion pathways and source populations. We sequenced an 815 base-pair section of the COI gene for 441 specimens of Bactrocera correcta, 214 B. zonata and 372 Zeugodacus cucurbitae; three invasive pest fruit fly species with overlapping hostplants. For each species, we explored how many individuals would need to be included in a study to sample the majority of their haplotype diversity. We also tested for phylogeographic signal and used demographic estimators as a proxy for invasion potency. We find contrasting patterns of haplotype diversity amongst the species, where B. zonata has the highest diversity but most haplotypes were represented by singletons; B. correcta has ~7 dominant haplotypes more evenly distributed; Z. cucurbitae has a single dominant haplotype with closely related singletons in a 'star-shape' surrounding it. We discuss how these differing patterns relate to their invasion histories. None of the species showed meaningful phylogeographic patterns, possibly due to gene-flow between areas across their distributions, obscuring or eliminating substructure.

Six years of fruit fly surveys in Bangladesh: a new species, 33 new country records and discovery of the highly invasive Bactrocera carambolae (Diptera, Tephritidae).

We engaged in six years of snap-shot surveys for fruit flies in rural environments and ten protected forest areas of Bangladesh, using traps baited with male lures (cue-lure, methyl eugenol, zingerone). Our work has increased the recorded number of species of Tephritidae in the country from seven to 37. We summarize these surveys and report eight new country occurrence records, and a new species (Zeugodacus madhupuri Leblanc & Doorenweerd, sp. nov.) is described. The highlight among the new records is the discovery, and significant westward range extension, of Bactrocera carambolae Drew & Hancock, a major fruit pest detected in the Chattogram and Sylhet Divisions. We rectify the previously published erroneous record of Bactrocera bogorensis (Hardy), which was based on a misidentification of Zeugodacus diaphorus (Hendel). We also report the occurrence in Bangladesh of nine other Tephritidae, the rearing of three primary fruit fly parasitoids from Zeugodacus, and records of non-target attraction to fruit fly lures.

Descriptions of four new species of Bactrocera and new country records highlight the high biodiversity of fruit flies in Vietnam (Diptera, Tephritidae, Dacinae).

Recent snap-shot surveys for fruit flies in Vietnam in 2015 and 2017 using traps baited with the male Dacinae fruit fly lures methyl eugenol, cue-lure and zingerone, collected 56 species, including 11 new country records and another 11 undescribed species, four of which are described in this paper. This increases the number of described species known to occur in Vietnam from 78 to 93. Species accumulation curves, based on the Chao 2 mean estimate, suggest that we collected 60-85 % of the local fauna at the sites sampled, and that species diversity decreases with increasing latitude. The four new species are named: Bactrocera (Tetradacus) ernesti Leblanc & Doorenweerd sp. n., B. (Asiadacus) connecta Leblanc & Doorenweerd sp. n., B. (Parazeugodacus) clarifemur Leblanc & Doorenweerd sp. n., and B. (Bactrocera) adamantea Leblanc & Doorenweerd sp. n. In addition to morphological data COI DNA sequence data of both the COI-5P and COI-3P mitochondrial DNA gene regions is provided. Three of the four newly described species are morphologically and genetically easily distinguished from all other members of Dacini. Bactroceraclarifemur sp. n. is superficially similar to B.pendleburyi (Perkins) based on morphology, but there are several apomorphic characters to distinguish the two. Both COI and a segment of the nuclear gene Elongation Factor 1 alpha separate the two species as well.

Description of a new species of Dacus from Sri Lanka, and new country distribution records (Diptera, Tephritidae, Dacinae).

A fruit fly survey in the Sinharaja and Knuckles National Parks in Sri Lanka (2016), using traps baited with the male lures methyl eugenol, cue-lure, and zingerone, yielded 21 species of Dacini fruit flies. Of these, three species, viz. Bactroceraamarambalensis Drew, B.dongnaiae Drew & Romig, and B.rubigina (Wang & Zhao), are new country occurrence records, and Dacus (Mellesis) ancoralis Leblanc & Doorenweerd, sp. n. is described as a new species. The Sri Lankan Dacini fruit fly fauna is now comprised of 39 species.

Tracking the Origins of Fly Invasions; Using Mitochondrial Haplotype Diversity to Identify Potential Source Populations in Two Genetically Intertwined Fruit Fly Species (Bactrocera carambolae and Bactrocera dorsalis [Diptera: Tephritidae]).

Bactrocera carambolae Drew and Hancock and Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) are important pests of many fruits. These flies have been spread across the world through global travel and trade, and new areas are at risk of invasion. Whenever new invasive populations are discovered, quick and accurate identification is needed to mitigate the damage they can cause. Determining invasive pathways can prevent further spread of pests as well as subsequent reinvasions through the same pathway. Molecular markers can be used for both species identification and pathway analysis. We analyzed 1,601 individuals from 19 populations using 765 base pairs of the mitochondrial cytochrome oxidase I (COI) gene to infer the haplotype diversity and population structure within these flies from across their native and invasive ranges. We analyzed these samples by either grouping by species or geographic populations due to the genetic similarity in the mitochondrial genome. We found no genetic structure between B. dorsalis and B. carambolae and our findings suggest recent and most likely ongoing, genetic exchange between these two species in the wild. Hyper-diverse mitochondrial genetic diversity in the native range suggests large population sizes and relatively high mutation rates. Only 52% of the haplotypes found in the trap captures from California are shared with haplotypes from flies found in our global survey, indicating significant genetic diversity in the native range that is missing from our samples. However, these results provide a foundation for the accurate determination of the provenance of invasive populations around the world.

Transpacific coalescent pathways of coconut rhinoceros beetle biotypes: Resistance to biological control catalyses resurgence of an old pest.

Biological control agents have several advantages over chemical control for pest management, including the capability to restore ecosystem balance with minimal non-target effects and a lower propensity for targets to develop resistance. These factors are particularly important for invasive species control. The coconut rhinoceros beetle (Oryctes rhinoceros Linnaeus) is a major palm pest that invaded many Pacific islands in the early 20th century through human-mediated dispersal. Application of the Oryctes nudivirus in the 1960s successfully halted the beetle's first invasion wave and made it a textbook example of successful biological control. However, a recently discovered O. rhinoceros biotype that is resistant to the nudivirus appears to be correlated with a new invasion wave. We performed a population genomics analysis of 172 O. rhinoceros from seven regions, including native and invasive populations, to reconstruct invasion pathways and explore correlation between recent invasions and biotypes. With ddRAD sequencing, we generated data sets ranging from 4,000 to 209,000 loci using stacks and ipyrad software pipelines and compared genetic signal in downstream clustering and phylogenetic analyses. Analysis suggests that the O. rhinoceros resurgence is mediated by the nudivirus-resistant biotype. Genomic data have been proven essential to understanding the new O. rhinoceros biotype's invasion patterns and interactions with the original biotype. Such information is crucial to optimization of strategies for quarantine and control of resurgent pests. Our results demonstrate that while invasions are relatively rare events, new introductions can have significant ecological consequences, and quarantine vigilance is required even in previously invaded areas.

A global checklist of the 932 fruit fly species in the tribe Dacini (Diptera, Tephritidae).

The correct application of the scientific names of species is neither easy nor trivial. Mistakes can lead to the wrong interpretation of research results or, when pest species are involved, inappropriate regulations and limits on trade, and possibly quarantine failures that permit the invasion of new pest species. Names are particularly challenging to manage when groups of organisms encompass a large number of species, when different workers employ different philosophical views, or when species are in a state of taxonomic flux. The fruit fly tribe Dacini is a species-rich taxon within Tephritidae and contains around a fifth of all known species in the family. About 10% of the 932 currently recognized species are pests of commercial fruits and vegetables, precipitating quarantines and trade embargos. Authoritative species lists consist largely of scattered regional treatments and outdated online resources. The checklist presented here is the first global overview of valid species names for the Dacini in almost two decades, and includes new lure records. By publishing this list both in paper and digitally, we aim to provide a resource for those studying fruit flies as well as researchers studying components of their impact on agriculture. The list is largely a consolidation of previous works, but following the results from recent phylogenetic work, we transfer one subgenus and eight species to different genera: members of the Bactrocera subgenus Javadacus Hardy, considered to belong to the Zeugodacus group of subgenera, are transferred to genus Zeugodacus; Bactrocera pseudocucurbitae White, 1999, stat. rev., is transferred back to Bactrocera from Zeugodacus; Zeugodacus arisanicus Shiraki, 1933, stat. rev., is transferred back to Zeugodacus from Bactrocera; and Z. brevipunctatus (David & Hancock, 2017), comb. n.; Z. javanensis (Perkins, 1938), comb. n.; Z. montanus (Hardy, 1983), comb. n.; Z. papuaensis (Malloch, 1939), comb. n.; Z. scutellarius (Bezzi, 1916), comb. n.; Z. semisurstyli (Drew & Romig, 2013), comb. n.; and Z. trilineatus (Hardy, 1955), comb. n. are transferred from Bactrocera to Zeugodacus.

Systematics of Phyllocnistis leaf-mining moths (Lepidoptera, Gracillariidae) feeding on dogwood (Cornus spp.) in Northeast Asia, with the description of three new species.

During an ongoing DNA-barcoding campaign of the leaf-mining moths that feed on woody plants in Northeast Asia, four lineages of the genus Phyllocnistis (Gracillariidae, Phyllocnistinae) were discovered on dogwood (Cornus spp): P. cornella Ermolaev, 1987 on C. controversa Hemsl. (Japan: Hokkaido) and three new species - one feeding on C. controversa, C. florida L. and C. macrophylla Wall. in Japan (Honshu, Shikoku, Kyushu), a second species on C. macrophylla in China (Yunnan) and a third on Siberian dogwood Cornus alba L. in Russia (Siberia). All these species showed differences in morphology, in the barcode region of the cytochrome c oxidase I gene and in two nuclear genes (histone H3 and 28S ribosomal RNA). No correlation was found between the deep mitochondrial splits observed and the Wolbachia infection pattern. Based on both morphological and molecular evidence, the three recently discovered lineages are described here as new species: P. indistincta Kobayashi & Triberti, sp. n. (Japan), P. saepta Kirichenko, Ohshima & Huang, sp. n. (China) and P. verae Kirichenko, Triberti & Lopez-Vaamonde, sp. n. (Russia). In addition, the authors re-describe the adult morphology of P. cornella, provide the first record of this species from Japan and highlight the diagnostic characters that allow these Cornus-feeding Phyllocnistis species to be distinguished.

Incongruence between molecules and morphology: A seven-gene phylogeny of Dacini fruit flies paves the way for reclassification (Diptera: Tephritidae).

Molecular and morphological research often suggest conflicting results. Selective pressure on certain morphologies can confound understanding of evolutionary relationships. Dacini is one of the most diverse tribes of tephritid flies and contains many economically important pest species. Their black and yellow patterned body markings are presumed to act as wasp mimicry, and the characters separating species and groups are limited and in some cases phenotypically plastic. The traditional taxonomy of the tribe is controversial because groupings are based on unique combinations of morphological characters without the use of cladistic methods, though recent phylogenetic and taxonomic analyses have resulted in significant changes to their taxonomy. The monophyly of the three largest genera in the tribe has been tested with only small numbers of representatives per genus and a limited number of genes. To further understand the taxonomy and evolution of Dacini we sequenced seven genes from 167 Dacini species and five dipteran outgroups to construct a robust phylogeny and test phylogenetic relationships between genera, subgenera, and species complexes. Our phylogeny confirms the monophyly of Dacus, Bactrocera, and Zeugodacus. However, most groups below the genus level are not monophyletic, and only through further revision will we be able to understand their evolution and clarify the taxonomy within this tribe.

A preliminary molecular phylogeny of shield-bearer moths (Lepidoptera: Adeloidea: Heliozelidae) highlights rich undescribed diversity.

Heliozelidae are a widespread, evolutionarily early diverging family of small, day-flying monotrysian moths, for which a comprehensive phylogeny is lacking. We generated the first molecular phylogeny of the family using DNA sequences of two mitochondrial genes (COI and COII) and two nuclear genes (H3 and 28S) from 130 Heliozelidae specimens, including eight of the twelve known genera: Antispila, Antispilina, Coptodisca, Heliozela, Holocacista, Hoplophanes, Pseliastis, and Tyriozela. Our results provide strong support for five major Heliozelidae clades: (i) a large widespread clade containing the leaf-mining genera Antispilina, Coptodisca and Holocacista and some species of Antispila, (ii) a clade containing most of the described Antispila, (iii) a clade containing the leaf-mining genus Heliozela and the monotypic genus Tyriozela, (iv) an Australian clade containing Pseliastis and (v) an Australian clade containing Hoplophanes. Each clade includes several new species and potentially new genera. Collectively, our data uncover a rich and undescribed diversity that appears to be especially prevalent in Australia. Our work highlights the need for a major taxonomic revision of the family and for generating a robust molecular phylogeny using multi-gene approaches in order to resolve the relationships among clades.

New taxa, including three new genera show uniqueness of Neotropical Nepticulidae (Lepidoptera).

After finding distinct clades in a molecular phylogeny for Nepticulidae that could not be placed in any known genera and discovering clear apomorphic characters that define these clades, as well as a number of Neotropical species that could be placed in known genera but were undescribed, three new genera and nine new species are here described from the Neotropics: Stigmella gallicola van Nieukerken & Nishida, sp. n. reared from galls on Hampea appendiculata (Malvaceae) in Costa Rica, representing the first example of a gall making Stigmella; Stigmella schinivora van Nieukerken, sp. n. reared from leafmines on Schinus terebinthifolia (Anacardiaceae) in Argentina, Misiones; Stigmella costaricensis van Nieukerken & Nishida, sp. n. and Stigmella intronia van Nieukerken & Nishida, sp. n. each from a single specimen collected the same night in Costa Rica, Parque Nacional Chirripó; Stigmella molinensis van Nieukerken & Snyers, sp. n. reared from leafmines on Salix humboldtiana, Peru, Lima, the first Neotropical species of the Stigmella salicis group sensu stricto; Ozadelpha van Nieukerken, gen. n. with type species Ozadelpha conostegiae van Nieukerken & Nishida, sp. n., reared from leafmines on Conostegia oerstediana (Melastomataceae) from Costa Rica; Neotrifurcula van Nieukerken, gen. n. with type species Neotrifurcula gielisorum van Nieukerken, sp. n. from Chile; Hesperolyra van Nieukerken, gen. n.. with type species Fomoria diskusi Puplesis & Robinson, 2000; Hesperolyra saopaulensis van Nieukerken, sp. n., reared from an unidentified Myrtaceae, Sao Paulo, Brasil; and Acalyptris janzeni van Nieukerken & Nishida, sp. n. from Costa Rica, Guanacaste. Five new combinations are made: Ozadelpha ovata (Puplesis & Robinson, 2000), comb. n. and Ozadelpha guajavae (Puplesis & Diskus, 2002), comb. n., Hesperolyra diskusi (Puplesis & Robinson, 2000), comb. n., Hesperolyra molybditis (Zeller, 1877), comb. n. and Hesperolyra repanda (Puplesis & Diskus, 2002), comb. n. Three specimens are briefly described, but left unnamed: Ozadelpha specimen EvN4680, Neotrifurcula specimen EvN4504 and Neotrifurcula specimen RH2.