DNA Barcodes of Microlepidoptera Reared from Native Fruit in Kenya Author(s): Scott E. Miller , Robert S. Copeland , Margaret E. Rosati and Paul D.N. Hebert Source: Proceedings of the Entomological Society of Washington, 116(1):137-142. 2014. Published By: Entomological Society of Washington DOI: http://dx.doi.org/10.4289/0013-8797.116.1.137 URL: http://www.bioone.org/doi/full/10.4289/0013-8797.116.1.137 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. PROC. ENTOMOL. SOC. WASH. 116(1), 2014, pp. 137–142 NOTE DNA barcodes of microlepidoptera reared from native fruit in Kenya DOI: 10.4289/0013-8797.116.1.137 This paper provides metadata for (Copeland et al. 2009). Although the study DNA barcode (COI) data in GenBank was focused on fruit flies (Tephritidae) and for a collection of small moths (micro- their parasitoids, the collections also lepidoptera except Blastobasidae and yielded many Lepidoptera. This is the third Tortricidae) reared from native fruits in a series of papers focused on the Lepi- throughout Kenya. This paper aims to doptera (Adamski et al. 2010, Razowski make DNA barcode data available to and Brown 2012), resulting from ongoing document ongoing research, to contribute collaboration among the International to the International Barcode of Life Centre of Insect Physiology and Ecology (iBOL; www.ibol.org) and Kenya Barcode (ICIPE), the Smithsonian Institution’s of Life projects, and to encourage en- National Museum of Natural History hancement in identifications, in line with (USNM), the National Museums of Kenya the concept of DNA barcode data release (NMK), and International Barcode of Life papers and the Fort Lauderdale principles project based at the University of Guelph. for genetic data (Schindel et al. 2011). This is also a contribution to a series of Many of these records represent un- papers documenting DNA barcodes for described species, and we have purpose- moths from Kenya (Martins et al. 2013, fully refrained from assigning new names Miller et al. 2013). until the relevant taxa can be studied in sufficient detail. Under the Fort Lauder- MATERIALS AND METHODS dale principles, we ask others to refrain The 5-year survey reared insects from from assigning new species names to indigenous fruits in diverse localities these records outside of the context of throughout Kenya. A total of 3838 fruit proper systematic study. Data for 251 se- collections were made representing 910 quences representing 114 barcode clusters distinct plant taxa from 118 families. (putative species) have been released on Lepidoptera were reared from 19% of the GenBank (accession numbers GU695820- samples, representing 349 plant species. GU695823, GU695866, HQ947262, Copeland et al. (2009) provide a sum- JF847884-JF847887, JN284900, KF643008- mary of the program and database of the KF643239, KF808331-KF808337) includ- rearings. Most specimens have a rearing ing the standard fields for the BARCODE lot number for the set of specimens that data standard (Benson et al. 2012) and more emerged from one sample of one species data, including images and host plants, are of fruit (prefaced by A&M or KIP), but available on BOLD (www.boldsystems. some of the later collections were “ad org; Ratnasingham and Hebert 2007, hoc,”madeafterthemainsamplingeffort 2013), in a dataset accessible using a DOI was completed, and thus lack lot numbers. (dx.doi.org/10.5883/DS-KFML1). Three groups of Lepidoptera dominate From 1999 to 2004, Copeland organized the samples in both numbers of species an insect-rearing program from native and individuals; the family Blastobasidae, fruit collected at sites throughout Kenya the family Tortricidae, and the subfamily 138 PROCEEDINGS OF THE ENTOMOLOGICAL SOCIETY OF WASHINGTON Phycitinae (Pyralidae), all of which include and to link to taxonomic names where many species that are specialists in fruit, possible. a niche not utilized by most Lepidoptera We have been able to identify some of (Novotny et al. 2010). All of these, along the microlepidoptera by comparison to with the remaining microlepidoptera re- reference collections, the literature, or ported here, are relatively small moths, matching DNA sequences in BOLD, but mostly with drab colors, and often diffi- because of the limited knowledge of cult to identify without reference to gen- African microlepidoptera (e.g., Mey 2011), italic dissections or DNA sequences. We this process will take considerable time. have approached analysis of these groups Thus, we are making these data avail- in different ways, depending on the state of able, while identifications are in progress. taxonomic knowledge and the tools avail- Where taxonomic names are not readily able to us. We approached Blastobasidae available from existing literature, the from a traditional morphological taxo- DNA cluster-based morphospecies can nomic perspective, dissecting most of the be used as species hypotheses that can be available specimens, comparing them to confirmed by future taxonomic studies the type specimens of the named species, in broader context of the African fauna and providing a complete taxonomic (Schindel and Miller 2010, Ratnasingham framework (Adamski et al. 2010). During and Hebert 2013). the process, high throughput DNA bar- Field methods and host plants: Rearing coding became available, so we super- methods and sampling strategy for the imposed barcode results onto the already native fruit-rearing program are provided finished morphological study, providing by Copeland et al. (2006, 2009). All fruits confirmation of identifications, and most were collected from native plant species importantly, the ability to match males except for Passiflora mollissima (Kunth) and females (Adamski et al. 2010). Spec- L. H. Bailey (Passifloraceae). Ripe and imens of Tortricidae were examined by unripe fruits were collected from plants two taxonomic specialists on the family, and occasionally from the ground; sam- John Brown and Jozef Razowski, the ples showing noticeable rotting were dis- latter of whom had previously worked carded. Because of the rearing conditions extensively on the African fauna. Thus, and multiple techniques involved, the they were able to analyze the specimens quality of adult moths varied, and some in a morphological taxonomic frame- specimens were in poor condition. Host work, describing 13 new species in the plants were identified by Quentin Luke process (Razowski and Brown 2012). and are stored at ICIPE. For Phycitinae, we took a pragmatic ap- proach and sequenced DNA from one to Lepidoptera methods several specimens from each rearing lot (a collection of a single species of fruit This study is based on the analysis of from a single locality), depending on their approximately 675 reared specimens, over morphological variability. We dissected 30 genitalic dissections, and 251 cyto- the genitalia of at least one specimen from chrome c oxidase I (COI) sequences. We each cluster of DNA sequences, as puta- attempted to analyze at least one sequence tive morphospecies based on experi- from each morphologically distinctive ence in other studies (Adamski et al. species from each rearing lot, but up to 2010, Craft et al. 2010, Ratnasingham 5 specimens from some rearing lots. and Hebert 2013), to test species concepts Genitalic dissections follow Robinson VOLUME 116, NUMBER 1 139 (1976). Morphological comparisons were Cosmopterigidae made to literature and collections of the We appear to have up to six species National Museum of Natural History tentatively placed in the genus Stilbosis, (USNM), Natural History Museum, reared from several species of Garcinia London (BMNH), National Museums of (Clusiaceae) – lots A&M 953, 1076, Kenya (NMK), and Kenya Agriculture 1192, 1513, 1612, 1687, 1832, 2116, Research Institute, Nairobi (KARI). Ad- 2378 and KIP 269. They are similar to ditional context was provided by inten- the species reared by Ghesquie`re (1940: sive samples of moths from light at Mpala 76) from Garcinia in the Democratic Research Centre in central Kenya, made Republic of Congo, and published as from 1998 to 2011 by Scott Miller and Stilbosis firma (Meyrick), a species de- Tina Kuklenski (Adamski et al. 2010). scribed from the Seychelles. Unfortu- Vouchers are retained by USNM, NMK nately the type of Stilbosis firma in and ICIPE. General field and laboratory BMNH is missing its abdomen, but Le- methods for Lepidoptera are described in grand (1965: 63) published a figure of Adamski et al. (2010) and Copeland et al. the genitalia of the species (as Pyroderces (2009). DNA sequencing (COI barcode) firma), which is similar to, but not the followed standard methods at the Bio- same as, our genitalia slides USNM