Molecular Phylogeny, Revised Higher Classification, and Implications for Conservation of Endangered Hawaiian Leaf-Mining Moths (Lepidoptera: Gracillariidae: Philodoria)1 Chris A. Johns,2,3,5 Matthew R. Moore,4 and Akito Y. Kawahara2,3,4 Abstract: The leaf-mining moth genus Philodoria Walsingham (Lepidoptera: Gracillariidae) is composed of 30 described species, all of which are endemic to the Hawaiian Islands. Philodoria is known to feed on 10 families of endemic Hawaiian host plants, with several species recorded only from threatened or en- dangered hosts. Beyond their dependence on these plants, little is known of their evolutionary history and conservation status. We constructed a molecular phy- logeny of Philodoria to assess validity of its current subgeneric classification and to help guide future work on this threatened Hawaiian lineage. Mitochondrial and nuclear DNA sequences from three genes (CO1, CAD, EF-1α) combining for a total of 2,041 base pairs, were collected from 11 Philodoria species, incorpo- rating taxa from both currently recognized subgenera. These data were analyzed using both parsimony and model-based phylogenetic approaches. Contrary to the most recent systematic treatment of Philodoria, our results indicate strongly that the two currently recognized Philodoria subgenera are not monophyletic and that morphological characters used to classify them are homoplasious. Based on our robust results, we revised the higher classification ofPhilodoria: the subgenus Eophilodoria Zimmerman, 1978 is established as subjective junior synonym of Philodoria Walsingham, 1907. We also present new host plant and distribution data and discuss host range of Philodoria as it pertains to endangered Hawaiian plants. Philodoria WALSINGHAM, 1907 (Lepidoptera: cromoths that currently includes 30 described Gracillariidae) is a genus of leaf-mining mi- species, all of which are endemic to the Ha- waiian Islands (Zimmerman 1978). The genus is extraordinary in that its larvae mine leaves 1 This research was supported by the National of 10 plant families from seven orders (Swezey Science Foundation (Graduate Research Fellowship to 1954, Zimmerman 1978). Host plant groups C.A.J.; DEB no. 1354585 to A.Y.K.), the National Geo- graphic Society (no. C283-14 to C.A.J.; no. 9686-15 to include iconic and endangered Hawaiian A.Y.K.), the Entomological Society of America (2014 plant taxa such as the silversword alliance SysEB Travel Award to C.A.J.), the University of Flor- (Argyroxiphium DC. and Dubautia Gaudich.) ida’s Tropical Conservation and Development Program and the Hawaiian lobelioids (Clermontia (2014 Field Research Grant to C.A.J.), and the Society for Systematic Biologists (2012 SSB MiniARTS Grant to Gaudich.). Approximately 80% of Philodoria A.Y.K.). Manuscript accepted 29 March 2016. species feed on a single plant host species, and 2 Department of Biology, University of Florida, more than three-quarters of these species are Gainesville, Florida 32611. 3 restricted to a single Hawaiian island (Zim- Florida Museum of Natural History, Gainesville, merman 1978). The genus Philodoria should Florida 32611. 4 Department of Entomology and Nematology, Uni- be considered a conservation priority due to versity of Florida, Gainesville, Florida 32611. the stringent host specificity and limited geo- 5 Corresponding author (e-mail: [email protected]). graphic range of the majority of its species. The taxonomic history of Philodoria has been unstable, and the group’s evolutionary Pacific Science (2016), vol. 70, no. 3:361 – 372 doi:10.2984/70.3.7 relationships remain unknown despite its dis- © 2016 by University of Hawai‘i Press tribution across Hawai‘i and specialization on All rights reserved distantly related plants. Philodoria was origi- 361 362 PACIFIC SCIENCE · July 2016 nally assigned to Tineidae Latreille ( Wal- each subgenus defined by Zimmerman (1978), singham 1907), followed by placement in Philodoria (Eophilodoria) margine strigata and Glyphipterigidae Stainton (Meyrick 1912). Philodoria (Philodoria) succedanea, were cap- Species within Philodoria have also been tured in these collections ( Table 1). Philodoria assigned to various other genera, including collection localities were selected based on Gracillaria Haworth, Elachista Treitschke historical records of Swezey (1954) and Zim- ( Walsingham 1907), and Parectopa Clemens merman (1978). New localities were also (Meyrick 1928). The most recent systematic surveyed based on the presence of known treatment grouped all Hawaiian species previ- Philodoria host plant species. We visually ously assigned to Elachista, Gracillaria, and identified host plants and collected leaves Parectopa into Philodoria (Zimmerman 1978). with signs of leaf miner larval activity. Both Zimmerman divided the genus into two sub- inactive and active leaf mines were photo- genera, Philodoria (Eophilodoria) and Philodoria graphed and georeferenced. Leaves with ac- (Philodoria), based on the size of the maxillary tive mines and advanced larval instars were palpus. Under this classification, Zimmerman collected and kept in cool, dry conditions in assigned 16 Philodoria species with the maxil- plastic containers for rearing. Successfully lary palps “fully developed” to the subgenus reared moths were stored in 100% ethanol for Eophilodoria (type species: P. marginestrigata molecular analyses. Larvae that did not suc- Walsingham). Fourteen Philodoria species cessfully pupate and emerge as adults were with this structure “greatly reduced, vestigial, stored in ethanol for future morphological or obsolescent” were assigned to the subgenus and molecular analyses. Moths and the leaves Philodoria (type species: P. succedanea Wal- from which they were reared were kept as singham). In addition, Zimmerman’s treat- voucher material and are deposited at the ment definesPhilodoria species based on scale McGuire Center for Lepidoptera and Biodi- patterns, host plant associations, and distribu- versity (MGCL), Florida Museum of Natural tion. However, no phylogenetic data /analyses History, Gainesville, Florida. Parasitoids reared have evaluated the usefulness of these char- from these collections are also stored at MGCL. acters for defining the subgenera or species. Multiple representatives of two species This study represents the first attempt to (Philodoria auromagnifica, samples CJ-064 and evaluate the usefulness of the maxillary palp CJ-072; Philodoria splendida, samples CJ-049 character (i.e., the monophyly of the sub- and CJ-105) were included in the study to genera) for the subgeneric classification of determine genetic variation between samples Philodoria. collected from different volcanoes or host We constructed the first phylogeny of plants. All adult moths sequenced in this study Philodoria that sampled molecular sequence were reared from active leaf mines as detailed data from one mitochondrial and two nuclear earlier, with the exception of CJ-049, which genes from 11 Philodoria species (see Table 1) was field collected as an adult.Philodoria spe- to test the subgeneric classification of Zim- cies were identified by comparing adult mor- merman (1978). Our results do not support phology with specimens determined by Otto Zimmerman’s subgenera, and we discuss pat- H. Swezey or Elwood C. Zimmerman that terns of host plant associations among our were stored in the Bishop Museum, Honolulu sampled Philodoria species. ( BPBM) or the Smithsonian National Museum, Washington, D.C. (USNM). We also aided our identifications by comparing our locality materials and methods data and larval host plant data with historical Taxon Sampling, Amplification, and Sequencing records. Molecular data were obtained by extract- Thirteen samples representing 11 species of ing the DNA from the entire adult moth. Philodoria were collected during April 2013 at Extraction methods followed manufacturer’s 13 sites on the islands of O‘ahu and Maui protocols for the Qiagen DNEasy kit (Qia- (Figure 1). Specimens of the type species of gen, Inc., Valencia, California). Specimens Molecular Phylogeny of Philodoria Leaf-Mining Moths · Johns et al. 363 Figure 1. Map of the Hawaiian Islands and the collection localities for the taxa sampled in this study. Additional information is available in Table 1. were sequenced for three genes: mitochon- Authors’ Note: Supplemental materials drial Cytochrome c Oxidase subunit 1 [CO1; 603 available only on BioOne ( http://www.bioone base pairs ( bp)], nuclear Carbamoylphosphate .org/ ). Synthase domain of CAD (922 bp), and nuclear Elongation factor 1-alpha (EF-1α) (516 bp); Phylogenetic Analyses the primer sequences for amplification of each fragment are listed in Table 2. We in- Analyses using parsimony (P), maximum like- cluded the same loci for three gracillariids, lihood (ML), and Bayesian inference (BI ) Epicephala relictella, Parectopa robiniella, and were first conducted on individual loci to as- Conopomorpha sp. from the study of Kawahara sess congruence among data sets. Parsimony et al. (2011). These taxa were included as analyses were executed in PAUP* 4.0 (Swof- outgroups because they are known to be ford 2003) using heuristic searches performed close relatives of Philodoria (Kawahara et al. with 1,000 random addition replicates and 2016). Sequences were edited using Ge- tree bisection-reconnection ( TBR) branch neious Pro v5.5.8 (Biomatters 2013), and swapping. For ML and BI, we first partitioned sequence alignments were produced using the the concatenated data set by gene region MUSCLE alignment algorithm