Molecular Phylogeny, Revised Higher Classification, and Implications for Conservation of Endangered Hawaiian Leaf-Mining Moths (Lepidoptera: Gracillariidae: Philodoria)

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 endangered hosts. Beyond their dependence on these plants, little is known of their evolutionary history and conservation status. We constructed a molecular phylogeny 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, incorporating 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 of Philodoria: 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. 1 This research was supported by the National Science Foundation (Graduate Research Fellowship to C.A.J.; DEB no. 1354585 to A.Y.K.), the National Geographic Society (no. C283-14 to C.A.J.; no. 9686-15 to A.Y.K.), the Entomological Society of America (2014 SysEB Travel Award to C.A.J.), the University of Florida’s Tropical Conservation and Development Program (2014 Field Research Grant to C.A.J.), and the Society for Systematic Biologists (2012 SSB MiniARTS Grant to A.Y.K.). Manuscript accepted 29 March 2016. 2 Department of Biology, University of Florida, Gainesville, Florida 32611. 3 Florida Museum of Natural History, Gainesville, Florida 32611. 4 Department of Entomology and Nematology, University of Florida, Gainesville, Florida 32611. 5 Corresponding author (e-mail: [email protected]). 362 PACIFIC SCIENCE · July 2016 nally assigned to Tineidae Latreille ( Walsingham 1907), followed by placement in Glyphipterigidae Stainton (Meyrick 1912). Species within Philodoria have also been assigned to various other genera, including Gracillaria Haworth, Elachista Treitschke ( Walsingham 1907), and Parectopa Clemens (Meyrick 1928). The most recent systematic treatment grouped all Hawaiian species previously assigned to Elachista, Gracillaria, and Parectopa into Philodoria (Zimmerman 1978). Zimmerman divided the genus into two subgenera, Philodoria (Eophilodoria) and Philodoria (Philodoria), based on the size of the maxillary palpus. Under this classification, Zimmerman assigned 16 Philodoria species with the maxillary palps “fully developed” to the subgenus Eophilodoria (type species: P. marginestrigata Walsingham). Fourteen Philodoria species with this structure “greatly reduced, vestigial, or obsolescent” were assigned to the subgenus Philodoria (type species: P. succedanea Walsingham). In addition, Zimmerman’s treatment defines Philodoria species based on scale patterns, host plant associations, and distribution. However, no phylogenetic data /analyses have evaluated the usefulness of these characters for defining the subgenera or species. This study represents the first attempt to evaluate the usefulness of the maxillary palp character (i.e., the monophyly of the subgenera) for the subgeneric classification of Philodoria. We constructed the first phylogeny of Philodoria that sampled molecular sequence data from one mitochondrial and two nuclear genes from 11 Philodoria species (see Table 1) to test the subgeneric classification of Zimmerman (1978). Our results do not support Zimmerman’s subgenera, and we discuss patterns of host plant associations among our sampled Philodoria species. materials and methods Taxon Sampling, Amplification, and Sequencing Thirteen samples representing 11 species of Philodoria were collected during April 2013 at 13 sites on the islands of O‘ahu and Maui (Figure 1). Specimens of the type species of each subgenus defined by Zimmerman (1978), Philodoria (Eophilodoria) margine strigata and Philodoria (Philodoria) succedanea, were captured in these collections ( Table 1). Philodoria collection localities were selected based on historical records of Swezey (1954) and Zimmerman (1978). New localities were also surveyed based on the presence of known Philodoria host plant species. We visually identified host plants and collected leaves with signs of leaf miner larval activity. Both inactive and active leaf mines were photographed and georeferenced. Leaves with active mines and advanced larval instars were collected and kept in cool, dry conditions in plastic containers for rearing. Successfully reared moths were stored in 100% ethanol for molecular analyses. Larvae that did not successfully pupate and emerge as adults were stored in ethanol for future morphological and molecular analyses. Moths and the leaves from which they were reared were kept as voucher material and are deposited at the McGuire Center for Lepidoptera and Biodiversity (mgcl), Florida Museum of Natural History, Gainesville, Florida. Parasitoids reared from these collections are also stored at mgcl. Multiple representatives of two species (Philodoria auromagnifica, samples CJ-064 and CJ-072; Philodoria splendida, samples CJ-049 and CJ-105) were included in the study to determine genetic variation between samples collected from different volcanoes or host plants. All adult moths sequenced in this study were reared from active leaf mines as detailed earlier, with the exception of CJ-049, which was field collected as an adult. Philodoria species were identified by comparing adult morphology with specimens determined by Otto H. Swezey or Elwood C. Zimmerman that were stored in the Bishop Museum, Honolulu ( bpbm) or the Smithsonian National Museum, Washington, D.C. (usnm). We also aided our identifications by comparing our locality data and larval host plant data with historical records. Molecular data were obtained by extracting the DNA from the entire adult moth. Extraction methods followed manufacturer’s protocols for the Qiagen DNEasy kit (Qiagen, Inc., Valencia, California). Specimens Molecular Phylogeny of Philodoria Leaf-Mining Moths · Johns et al. 363 were sequenced for three genes: mitochondrial Cytochrome c Oxidase subunit 1 [CO1; 603 base pairs ( bp)], nuclear Carbamoylphosphate Synthase domain of CAD (922 bp), and nuclear Elongation factor 1-alpha (EF-1α) (516 bp); the primer sequences for amplification of each fragment are listed in Table 2. We included the same loci for three gracillariids, Epicephala relictella, Parectopa robiniella, and Conopomorpha sp. from the study of Kawahara et al. (2011). These taxa were included as outgroups because they are known to be close relatives of Philodoria (Kawahara et al. 2016). Sequences were edited using Geneious Pro v5.5.8 (Biomatters 2013), and sequence alignments were produced using the MUSCLE alignment algorithm with default parameters (Edgar 2004). Each gene alignment was manually concatenated together into a single alignment that totaled 2,041 bp. Supplemental Table S1 lists GenBank accession numbers; the single gene trees, concatenated data set, and photos of sequenced tissue are available from the Dryad data depository ( http://datadryad.org). Authors’ Note: Supplemental materials available only on BioOne ( http://www.bioone .org/ ). Phylogenetic Analyses Analyses using parsimony (P), maximum likelihood (ML), and Bayesian inference (BI ) were first conducted on individual loci to assess congruence among data sets. Parsimony analyses were executed in PAUP* 4.0 (Swofford 2003) using heuristic searches performed with 1,000 random addition replicates and tree bisection-reconnection ( TBR) branch swapping. For ML and BI, we first partitioned the concatenated data set by gene region and codon position, and determined the bestfitting models of sequence evolution for each partition in PartitionFinder 1.0.1 (Lanfear et al. 2012) using the Akaike Information Criterion. The models for each partition were used in the following analyses and are listed in Supplemental Table S2. ML analyses were implemented in RAxML 8.1.12 (Stamatakis 2014), with 1,000 bootstrap replicates. BayesFigure 1. Map of the Hawaiian Islands and the collection localities for the taxa sampled in this study. Additional information is available in Table 1.