Robert K. Robbins

Integration of DNA barcoding into an ongoing inventory of complex tropical biodiversity

Inventory of the caterpillars, their food plants and parasitoids began in 1978 for todays Area de Conservacion Guanacaste (ACG), in northwestern Costa Rica. This complex mosaic of 120 000 ha of conserved and regenerating dry, cloud and rain forest over 0–2000 m elevation contains at least 10 000 species of non‐leaf‐mining caterpillars used by more than 5000 species of parasitoids. Several hundred thousand specimens of ACG‐reared adult Lepidoptera and parasitoids have been intensively and extensively studied morphologically by many taxonomists, including most of the co‐authors. DNA barcoding — the use of a standardized short mitochondrial DNA sequence to identify specimens and flush out undisclosed species — was added to the taxonomic identification process in 2003. Barcoding has been found to be extremely accurate during the identification of about 100 000 specimens of about 3500 morphologically defined species of adult moths, butterflies, tachinid flies, and parasitoid wasps. Less than 1% of the species have such similar barcodes that a molecularly based taxonomic identification is impossible. No specimen with a full barcode was misidentified when its barcode was compared with the barcode library. Also as expected from early trials, barcoding a series from all morphologically defined species, and correlating the morphological, ecological and barcode traits, has revealed many hundreds of overlooked presumptive species. Many but not all of these cryptic species can now be distinguished by subtle morphological and/or ecological traits previously ascribed to ‘variation’ or thought to be insignificant for species‐level recognition. Adding DNA barcoding to the inventory has substantially improved the quality and depth of the inventory, and greatly multiplied the number of situations requiring further taxonomic work for resolution.

Modified Pollard transects for assessing tropical butterfly abundance and diversity

This paper introduces and discusses the consistency and effectiveness of an inexpensive modification of Pollard transects for assessing the diversity and abundance of tropical butterfly communities in two national parks in Rio de Janeiro, Brazil. To measure butterfly abundance, students walked simultaneous timed transects at the interface of forest and cleared areas. They either counted the number of individuals observed or tabulated the number of specimens collected with nets. After a short training period, the number of butterflies observed or collected on simultaneous transects was statistically indistinguishable among student groups, and there was a significant positive correlation between observation counts and collected number of individuals. As a measure of species richness, the number of butterfly species sampled on each simultaneous transect was tabulated and did not differ statistically. To measure diversity, alpha of the logseries model was calculated for each collected sample, and statistical fit to a logseries model was determined. Although virtually all daily samples and the years accumulated sample at one park fit the logseries model, about 35% of the daily samples and the entire years sample did not fit at the other park. Despite these differences between the two parks, values for alpha from daily samples at both parks varied similarly (from 15 to 50 in almost all cases), and values from the entire years samples were statistically indistinguishable. The repeatability of results among novices, such as students, suggests that timed transects have great promise for furthering our understanding of butterfly community demographics.

Extinction and biogeography in the Caribbean: new evidence from a fossil riodinid butterfly in Dominican amber.

We describe a new species of extinct riodinid butterfly, Voltinia dramba, from Oligo–Miocene Dominican amber (15–25 Myr ago). This appears to be the first butterfly to be taxonomically described from amber, and the first adult riodinid fossil. The series of five specimens represents probably the best–preserved fossil record for any lepidopteran. The phenomenon of extant Voltinia females ovipositing on arboreal epiphytes probably explains the discovery of multiple female V. dramba specimens in amber. Voltinia dramba appears to be one of many extinct butterfly species on Hispaniola. The northwestern Mexican distribution of the explicitly hypothesized sister species, the extant V. danforthi, supports the hypothesis that V. dramba reached Hispaniola by the ‘proto–Greater Antillean arc’, dating the divergence of V. dramba and V. danforthi to 40–50 Myr ago. This date is contemporaneous with the oldest known butterfly fossils, and implies a more ancient date of origin for many of the higher–level butterfly taxa than is often conceded.

Cost and evolution of a facultative mutualism between ants and lycaenid larvae (Lepidoptera)

Larvae of Arawacus lincoides (Lycaenidae) reared in the lab with ants took 0.68 d longer to complete development than larvae without ants. Pupal weight was independent of whether a larva had been ant-tended. A 0.68 d delay in reaching age of first reproduction represents an estimated 1.0%-2.2% lowered intrinsic rate of increase for this continuously brooded species. I show that mutualism will be favored when then ratio of larval survival with ants to that without ants is greater than exp (dr), where d is the additional larval development time caused by feeding ants and r is the intrinsic rate of increase. Other things being equal, species with higher intrinsic rates of increase will be less likely to be mutualistic than their relatives with lower rates of increase

Complete genomes of Hairstreak butterflies, their speciation, and nucleo-mitochondrial incongruence

Comparison of complete genomes of closely related species enables research on speciation and how phenotype is determined by genotype. Lepidoptera, an insect order of 150,000 species with diverse phenotypes, is well-suited for such comparative genomics studies if new genomes, which cover additional Lepidoptera families are acquired. We report a 729 Mbp genome assembly of the Calycopis cecrops, the first genome from the family Lycaenidae and the largest available Lepidoptera genome. As detritivore, Calycopis shows expansion in detoxification and digestion enzymes. We further obtained complete genomes of 8 Calycopis specimens: 3 C. cecrops and 5 C. isobeon, including a dry specimen stored in the museum for 30 years. The two species differ subtly in phenotype and cannot be differentiated by mitochondrial DNA. However, nuclear genomes revealed a deep split between them. Genes that can clearly separate the two species (speciation hotspots) mostly pertain to circadian clock, mating behavior, transcription regulation, development and cytoskeleton. The speciation hotspots and their function significantly overlap with those we previously found in Pterourus, suggesting common speciation mechanisms in these butterflies.

The “upside down” systematics of hairstreak butterflies (Lycaenidae) that eat pineapple and other Bromeliaceae

All Lycaenidae larvae that eat Bromeliaceae belong to the Strymon ziba and S. serapio species groups, but confusion with taxonomy has resulted in widespread misidentification of the butterflies in both the ecological and agricultural literature. Published food plant records are assessed, and new rearing records are presented. The species that have been recorded eating Bromeliaceae are Strymon ziba (Hewitson), S. megarus (Godart), S. lucena (Hewitson), S. oreala (Hewitson), S. serapio (Godman & Salvin), S. azuba (Hewitson), and S. gabatha (Hewitson). The first four are recorded from pineapple, with the sympatric S. megarus and S. ziba sometimes being especially destructive in commercial fields. Most published records lump these two species and misidentify them under the names Thecla basilides, Thecla basalides, or Tmolus echion. Strymon ziba has also been reared from other monocotyledon plant families, such as Heliconiaceae and Haemodoraceae. In most cases, caterpillars eat flowers and fruits, but larvae of S. megarus and/or S. ziba may also bore into leaves when flowers and fruits are unavailable. The known bromeliad feeders are illustrated, and distinguishing characters are noted. The female butterflies appear to use reddish color as a visual cue, which could be the basis for a non-insecticidal means of controlling outbreaks in commercial pineapple crops. Todas as larvas de Lycaenidae que se alimentam de Bromeliaceae pertencem aos grupos de espécies de Strymon ziba e S. serapio, porém confusão com sua taxonomia resultou em erros generalizados de identificação destas borboletas tanto na literatura ecológica como agrícola. Registros publicados de plantas alimentares são aqui avaliados, e novos registros com base em criação são apresentados. As espécies que foram registradas se alimentando de Bromeliaceae são Strymon ziba (Hewitson), S. megarus (Godart), S. lucena (Hewitson), S. oreala (Hewitson), S. serapio (Godman & Salvin), S. azuba (Hewitson), e S. gabatha (Hewitson). As quatro primeiras são encontradas em abacaxi, com as espécies simpátricas S. megarus e S. ziba algumas vezes sendo especialmente destrutivas em áreas comerciais. A maioria dos registros publicados considera estas duas espécies como somente uma, identificando-as erroneamente como Thecla basilides, Thecla basalides, ou Tmolus echion. Strymon ziba também foi criada em outras famílias de plantas monocotiledôneas, como Heliconiaceae e Haemodoraceae. Na maioria dos casos, as lagartas comem flores e frutos, mas larvas de S. megarus e / ou S. ziba também podem perfurar as folhas quando as flores e os frutos não estão disponíveis. As espécies conhecidas que se alimentam de bromélias são aqui ilustradas, e seus caracteres distintivos ressaltados. As borboletas fêmeas parecem usar a cor vermelha como um sinal visual, o que poderia ser a base para um meio não-inseticida de controle de focos em lavouras comerciais de abacaxi.

Loss of male secondary sexual structures in allopatry in the Neotropical butterfly genus Arcas (Lycaenidae: Theclinae: Eumaeini)

Male secondary sexual characters in Lepidoptera may be present or absent in species that otherwise appear to be closely related, an observation that has led to differences of opinion over the taxonomic usefulness of these structures above the species level. An evolutionary issue raised by this debate is whether male secondary sexual characters (1) can be regained after being lost evolutionarily, (2) are not lost after being evolved, or (3) are ‘switched on and off’ by genes that regulate development. A second evolutionary issue is the conditions under which male secondary sexual characters might be lost or gained evolutionarily. Because these structures are thought to promote species recognition, theory predicts evolutionary losses to be most likely in allopatry; evolutionary gains to be most likely during the process of secondarily establishing sympatry or during sympatric speciation. We updated the species-level taxonomy of the brilliant emerald-winged Neotropical lycaenid butterfly genus Arcas and performed an analysis of phylogenetic relations among species to assess these evolutionary issues. We morphologically detail a scent pouch on the ventral hindwing of Arcas and report that six species possess the pouch with androconia, one possesses the pouch without androconia, and the remaining two species have neither pouch nor androconia. In addition, eight Arcas species have a morphologically species-specific male forewing scent pad, and one lacks a scent pad. This variation appears to be the result of three evolutionary losses and no gains of male secondary sexual organs. The four Arcas species lacking a scent pouch or a scent pad are allopatric with their closest phylogenetic relatives while four of five with both of these structures are sympatric. Although Arcas is a small genus, these results are significantly more extreme than predicted by chance. For taxonomy, this study provides a rationale for the evolutionary loss of male secondary sexual structures and suggests that their absence, but itself, does not indicate a lack of relationship above the species level.

Nomenclatural Changes in the Neotropical Eumaeini (Lepidoptera, Lycaenidae, Theclinae)

Nomenclatural actions are taken in the Neotropical Eumaeini in advance of publication of the Atlas of Neotropical Lepidoptera Checklist. Lectotypes are designated for eleven species group names: Lycaena astiocha Prittwitz, 1865; Thecla azia Hewitson, 1873; Thecla beroea Hewitson, 1868; Thecla cupa Druce, 1907; Thecla daraba Hewitson, 1867; Thecla duma Hewitson, 1878; Thecla erenea Hewitson, 1867; Thecla galliena Hewitson, 1867; Thecla guacanagari Wallengren, 1860; Thecla stagira Hewitson, 1867; and Thecla thoria Hewitson, 1867. Thecla duma Hewitson, 1878 and Thecla columbinia Strand, 1916 are transferred from Eumaeini to Deudorigini (Theclinae). Lycaena vanessoides Prittwitz, 1865 is transferred from Polyommatinae to Theclinae (Eumaeini). Six type localities are changed: Colombia to Africa for Thecla columbinia Strand, 1916; Amazon to Guayaquil for Thecla daraba Hewitson, 1867; Colombia to Southeast Asia for Thecla duma Hewitson, 1878; Bolivia to Westem North America for Ignata illepida K. Johnson, 1992; Argentina to the United States for Strymon nivnix K. Johnson, Eisele & MacPherson, 1990; and Dominican Republic to mainland Central and South America for Tmolus victoria K. Johnson & Matusik, 1989. Seven new synonyms are: Lycaena vanessoides Prittwitz, 1865 = Thecla hygela Hewitson, 1868 syn. nov.; Thecla saepium Boisduval, 1852 = Ignata illepida K. Johnson, 1992 syn. nov.; Thecla tyriam H.H. Druce, 1907 = Zigirina minutia K. Johnson & Adams, 1997 syn. nov.; Thecla halciones Butler & H. Druce, 1872 = Decussata colombiana K. Johnson, Austin, Le Crom & Sal azar, 1997 syn. nov.; Papilio celmus Cramer, 1775 = Tmolus victoria K. Johnson & Matusik, 1989 syn. nov.; Thecla daraba Hewitson, 1867 = Thecla tyleri Dyar, 1913 syn. nov.; and Thecla galliena Hewitson, 1877 = Thecla iopas Godman & Salvin, 1887 syn. nov. The generic name Decussata K. Johnson, Austin, Le Crom & Salazar, 1997 is a new junior synonym of Ostrinotes K. Johnson, Austin, Le Crom & Salazar, 1997. The unavailable infra-subspecific name Thecla orobiana forma ♀ cosmophila Tessmann, 1928 is available as Thecla cosmophila Bridges, 1988. Seven new combinations are: Hypokopelates columbinia (Strand, 1916) comb. nov.; Mithras cosmophila Bridges, 1988 comb. nov.; Nicolaea cupa (Druce, 1907) comb. nov.; Salazaria elizabetha (Salazar, Velez & K. Johnson, 1997) comb. nov.; Ostrinotes halciones (Butler & H. Druce, 1872) comb. nov.; Strephonota tyriam (H.H. Druce, 1907) comb. nov.; and Aubergina vanessoides (Prittwitz, 1865) comb. nov. The holotypes of seven eumaeines are composed of parts belonging to different species: Strymon andrewi K. Johnson & Matusik, 1988; Decussata colombiana K. Johnson, Austin, Le Crom & Salazar, 1997; Trochusinus elizabetha Salazar, Velez & K. Johnson, 1997; lgnata illepida K. Johnson, 1992; Zigirina minutia K. Johnson & Adams, 1997; Strymon nivnix K. Johnson, Eisele & MacPherson, 1990; and Tmolus victoria K. Johnson & Matusik, 1989. Some parts of these holotypes are excluded to clarify the identity of these names. Three chronic misspellings are corrected: Papilio ganimedes Crarner, 1775, for Papilio ganymedes [sic] Fabricius, 1776;Thecla atnius Herrich-Schaffer, [1853], for Thecla atrius [sic] Herrich-Schaffer, [1858]; and Rusticus minyas Hubner, [1809], for Rusticus minijas [sic] Poey, 1832. The name Electrostrymon minikyanos K. Johnson & Matusik, 1988, is treated as a nomen dubium. The subjective synonymy of Thecla guacanagari Wallengren, 1860, and Thecla azia Hewitson, 1873, is referred to the International Commission on Zoological Nomenclature for conditional suppression of the first name. Precedence for the names Papilio dion Schaller, 1788, and Hesperia columella Fabricius, 1793, is reversed under Article 23.9.1 of the International Code of Zoological Nomenclature.

When coi barcodes deceive: Complete genomes reveal introgression in hairstreaks

Two species of hairstreak butterflies from the genus Calycopis are known in the United States: C. cecrops and C. isobeon. Analysis of mitochondrial COI barcodes of Calycopis revealed cecrops-like specimens from the eastern US with atypical barcodes that were 2.6% different from either USA species, but similar to Central American Calycopis species. To address the possibility that the specimens with atypical barcodes represent an undescribed cryptic species, we sequenced complete genomes of 27 Calycopis specimens of four species: C. cecrops, C. isobeon, C. quintana and C. bactra. Some of these specimens were collected up to 60 years ago and preserved dry in museum collections, but nonetheless produced genomes as complete as fresh samples. Phylogenetic trees reconstructed using the whole mitochondrial and nuclear genomes were incongruent. While USA Calycopis with atypical barcodes grouped with Central American species C. quintana by mitochondria, nuclear genome trees placed them within typical USA C. cecrops in agreement with morphology, suggesting mitochondrial introgression. Nuclear genomes also show introgression, especially between C. cecrops and C. isobeon. About 2.3% of each C. cecrops genome has probably (p-value < 0.01, FDR < 0.1) introgressed from C. isobeon and about 3.4% of each C. isobeon genome may have come from C. cecrops. The introgressed regions are enriched in genes encoding transmembrane proteins, mitochondria-targeting proteins and components of the larval cuticle. This study provides the first example of mitochondrial introgression in Lepidoptera supported by complete genome sequencing. Our results caution about relying solely on COI barcodes and mitochondrial DNA for species identification or discovery.

Review of the species level taxonomy of the neotropical butterfly genus Oenomaus (Lycaenidae, Theclinae, Eumaeini)

Abstract Seven new species of the Neotropical hairstreak genus Oenomaus are described: Oenomaus mancha Busby & Faynel, sp. n. (type locality Ecuador); Oenomaus gwenish Robbins & Faynel, sp. n. (type locality Panama); Oenomaus lea Faynel & Robbins, sp. n. (type locality Ecuador); Oenomaus myrteana Busby, Robbins & Faynel, sp. n. (type locality Ecuador); Oenomaus mentirosa Faynel & Robbins, sp. n. (type locality Peru); Oenomaus andi Busby & Faynel, sp. n. (type locality Ecuador) and Oenomaus moseri Robbins & Faynel, sp. n. (type locality Brazil, Santa Catarina). For each new Oenomaus species, we present diagnostic characters and notes on its habitat and biology. We illustrate adults, genitalia, and distribution. New distributional and biological data are presented for 21 previously described Oenomaus species. Oenomaus melleus guyanensis Faynel, 2008 is treated as a new synonym of Oenomaus melleus melleus (Druce, 1907). Females are described and associated with males for ten species using a variety of factors, including mitochondrial COI DNA “barcode” sequences. We summarize the reasons why the number of recognized Oenomaus species has grown in the past decade from one species to 28 species. Finally, we overview the habitats that Oenomaus species occupy and note that the agricultural pest on Annonaceae, Oenomaus ortygnus, is the only Oenomaus species that regularly occurs in greatly disturbed habitats.