Northeastern Naturalist Vol. 23, No. 2 S.N. White, D.T. Stewart, N.K. Hillier, and R.C. Evans 2016 211 2016 NORTHEASTERN NATURALIST 23(2):211–218 Identification of Mompha capella Busck, a Microlepidopteran Predator of an Endangered Plant, Crocanthemum canadense (L.) Britton, in Nova Scotia Stephanie N. White1, Donald T. Stewart1,*, N. Kirk Hillier1, and Rodger C. Evans1 Abstract – In recent years, a small insect was discovered predating seeds of Crocanthemum canadense (Canada Frostweed or Rockrose), which is an endangered plant with small, localized populations in Nova Scotia. This insect targets primarily chasmogamous flowers (insect-pollinated, open flowers) but not cleistogamous flowers (self-pollinated, closed) of Canada Frostweed. This behavior is of concern because a decrease in the number of seeds produced by outcrossing could cause a decrease in genetic variance within populations at affected sites (e.g., Canadian Forces Base Greenwood, NS). We extracted DNA from larvae collected from chasmogamous flowers and used the mitochondrial cytochrome oxidase subunit I (COI) gene to barcode the DNA. Results from queries showed a 91% match to Mompha (Lepidoptera: Momphidae) species on GenBank, indicating that this insect was a member of the genus Mompha, but that this particular species was not in the database. To further characterize this lepidopteran, we collected and incubated chasmogamous flowers to rear the larvae to adulthood. We identified the reared adults as Mompha capella, a species of Momphidae not previously documented in the Maritime provinces, Canada. Introduction Relationships between plants and insects are often complex, making it difficult to predict potential consequences of their interactions (Dart and Eckert 2015, Emery et al. 2009). To better understand these relationships, it can be useful to use phylogenetic analyses to study the evolutionary history and potential co-evolution of plants and their predators (e.g., Brooks and McLennan 1991). Herein we identify an insect pest of Crocanthemum canadense (L.) Britton (= Helianthemum canadense (L.) Michx. [Reznicek et al. 2011]) (Cistaceae) (Canada Frostweed or Rockrose), a perennial herb distributed along portions of the eastern seaboard of Canada and the US. It was recently estimated that only 5000–5500 mature plants exist in the province of Nova Scotia, primarily in 2 closely associated populations in Kings County, and another population in Queens County (Newell 2007). One of the Kings County populations and the Queens County population are in decline (Newell 2007), an historic population in Halifax County appears to be extirpated, and the species is currently listed as endangered under the Nova Scotia Endangered Species Act (Nova Scotia Department of Natural Resources 2013). The preferred habitat for Canada Frostweed appears to be Corema (crowberry) barrens, a habitat that has been reduced to less than 3% of its historic size in the province (Catling 1Department of Biology, Acadia University, Wolfville, NS, Canada B4P 2R6. *Corresponding author - don.stewart@acadiau.ca. Manuscript Editor: Daniel Pavuk Northeastern Naturalist 212 S.N. White, D.T. Stewart, N.K. Hillier, and R.C. Evans 2016 Vol. 23, No. 2 and Carbyn 2005, Catling et al. 2004). Given that populations in Kings County and Queen’s County, NS appear to be genetically distinct from one another and from the nearest known populations in Quebec and southern Maine (Yorke et al. 2011), the loss of any of the populations in this province will decrease the extent of genetic variability of this species. Canada Frostweed has 2 distinct flower types: chasmogamous (open flower, insect pollinated) and cleistogamous (closed flower, self-pollinated) (Newell 2007). In 2005, Dr. Samuel vander Kloet, a botanist (now deceased) at Acadia University noted predation of seeds of chasmogamous fruits by an unidentified insect. Following up on these observations during the summer of 2013, we observed insect larvae feeding upon developing seeds of chasmogamous fruits pre-dispersal. We found this type of insect predation only at the Canadian Forces Base site, Greenwood, NS (hereafter CFB Greenwood). We did not observe predation in collections of fruits obtained from a nearby location along Highway 101, Kings County, NS. No other Nova Scotia populations have been examined for evidence of predation (White 2015). The number of seeds present at the dispersal stage has important implications for a plant’s reproductive potential (Crawley 1989). Insect predation on seeds is of particular concern at Nova Scotia’s limited Canada Frostweed populations because it potentially reduces the number of individuals in populations already in decline (Yorke et al. 2011). Insects that target plants pre-dispersal often consume more than 90% of the seeds within the fruit (Rand and Louda 2006). The specific effects of pre-dispersal insect predation on perennial plant populations are understudied (e.g., Lewis and Gripenberg 2008); thus, the future population dynamics of Canada Frostweed in regards to insect predation are uncertain. The first step towards understanding the effects of insect predation on Canada Frostweed was to determine the species of insect feeding on the seeds. DNA barcoding allows researchers to identify insects at any life stage, provided that the unknown species has been previously entered into a barcoding database (Garcia-Robledo et al. 2013). DNA barcoding can be used to help determine if an unidentified insect belongs to a species that could pose a significant threat to a particular plant (e.g., Armstrong and Ball 2005), in this case, the endangered Canada Frostweed population. DNA barcoding uses a standardized DNA region to determine the identity of an organism potentially down to species level (Valentini et al. 2008). For insects, as well as other animals, the mitochondrial cytochromec oxidase subunit 1 (COI) gene is used in barcoding identifications (Hebert et al. 2004, 2009; Jinbo et al. 2011). Recent studies have also integrated morphological identification with DNA barcoding for a more complete analysis (Emery et al. 2009, Garcia-Robledo et al. 2013, Valentini et al. 2008). Morphological identification can be extremely useful because not all species are yet present in GenBank’s databases. In this study, we present morphological and genetic analyses of the unidentified l arval insect predator of Canada Frostweed. Northeastern Naturalist Vol. 23, No. 2 S.N. White, D.T. Stewart, N.K. Hillier, and R.C. Evans 2016 213 Materials and Methods Sample collection and study area To obtain adults in pristine condition for identification, we reared larvae into adults in the Weston Animal Care Facility, Acadia University, Wolfville, NS. During mid-July, we collected 395 post-anthesis (i.e., the period after which a flower opens and becomes functional) chasmogamous flowers from the CFB Greenwood site (44.9804N, 64.9394W), where the Canada Frostweed population was known to be infested. The secondary study site, Highway 101 Exit 17 (44.9911N 64.9653W), showed no evidence of infection in 43 flowers examined. We placed over 100 flowers from the infested CFB Greenwood population on a moistened piece of filter paper (Whatman #8) in petri dishes and incubated them at 20 °C until the larvae within the flowers had fully developed into their adult form. We pinned a total of 40 adult moths and N.K. Hillier identified them through comparison with illustrations of similar Mompha species, previous records of Mompha species feeding on Cistaceae (Hodges 1992), and online resources (Momphidae: Microleps.org, accessed July 2014). We also shipped 11 voucher specimens to the Canadian National Collection of Insects, Arachnids, and Nematodes, Ottawa, ON, Canada, for independent verification of the identification. Lepidoptera infection rates of chasmogamous flowers We collected and placed in vials containing 70% ethanol chasmogamous flowers (n = 395) from CFB Greenwood. We dissected and recorded larval presence for each flower. DNA barcoding and phylogenetic analysis We used 2 samples for DNA barcoding. The first sample was a single larva isolated from a chasmogamous flower. The second sample consisted of all 6 legs from a single adult moth reared at Acadia University. We employed the “DNA purification from tissues” protocol from the QIAamp DNA mini kit handbook (Qiagen, Mississauga, ON, Canada) to extract DNA from the samples, with 3 modifications. In step 3, we pulse-vortexed the samples an additional 4 times before overnight incubation at 56 °C. The following day, we added an additional 10 μl of proteinase K solution and incubated the samples for 2 h. Instead of following Step 5 of the procedure, we centrifuged the sample tubes at 20,000 rpm for 5 min. We used a nanospectrophotometer (Implen, Munich, Germany) to measure the DNA concentration and absorbance ratios of each sample. We employed polymerase chain reaction (PCR) using the primers LepF 5'-ATTCAACCAATCATAAAGATATTGG-3' and LepR 5'-TAAACTTCTTCTGGATGTCCAAAAAATCA- 3' to amplify the mitochondrial COI region (Hebert et al. 2004). The reaction mixture consisted of 22 μl Platinum® Blue PCR Supermix (ThermoFisher Scientific, Pittsburgh, PA), 1 μl LepF primer (10μm), 1 μl LepR primer (10μm), and 1 μm DNA sample (5 ng/μl). We used a third tube without DNA as a negative control. We placed the tubes in a thermo-cycler (MJ Research, GMI, Ramsey, MN) and employed the following protocol: an initial denaturation run of Northeastern Naturalist 214 S.N. White, D.T. Stewart, N.K. Hillier, and R.C. Evans 2016 Vol. 23, No. 2 94 °C for 3 min; 5 cycles of 94 °C for 30 s, 45 °C for 90 s, and 72 °C for 60 s; 35 cycles of 94 °C for 30 s, 51 °C for 30 s, and 72 °C for 60 s; and a final step of 72 °C for 5 min. We electrophoresed PCR products on a 1% agarose gel in 1x TAE buffer. A 100-bp DNA ladder confirmed the presence of an ~650-bp product from each DNA sample. No bands appeared in the negative control, indicating that our samples were not contaminated. We sent PCR products to the McGill University and Génome Québec Innovation Centre (Montréal, QC, Canada) for sequencing. We used the Clustal Omega program (Goujon et al. 2010) to align the resulting sequences. After alignment, we removed the primer sequences and queried the sequence with the “nucleotide BLAST” search tool on the NCBI website (http://blast. ncbi.nlm.nih.gov/Blast.cgi). We employed the computer program MEGA6 (Tamura et al. 2013) to calculate pairwise p-distances for 658 bp of COI sequence data and construct a simple phylogenetic tree with the neighbor-joining algorithm for the Mompha species shown in Table 1. Taxa in the tree included Mompha capella Busck (Lepidoptera: Momphidae; GenBank Acc. No. KP123434), M. miscella (Denis and Schiffermüller) (Acc. No. JF818770), M. cephalonthiella (Chambers) (Acc. No. KF492439), M. idaei (Zeller) (Acc. No. GU097014), M. sexstrigella (Braun) (Acc. No. HM863601), M. conturbatella (Hübner) (Acc. No. HM865878), M. epilobiella (Denis and Schiffermüller) (Acc. No. JF859807), M. unifasciella (Chambers) (Acc. No. GU096503), and an outgroup taxon, Urodeta hibernella (Staudinger) (Lepidoptera: Elachistidae; Acc. No. KF644393). Results and Discussion We identified all 40 adult moths reared from larvae feeding on the chasmogamous Canada Frostweed flowers as Mompha capella. No other species of Mompha or any other insects emerged from the incubated material. Results from the BLAST search of the COI DNA barcode data indicated that our sample was most-closely Table 1. Sequence-divergence values (p-distances) among COI sequences for 8 species in the genus Mompha and 1 outgroup taxon, Urodeta hibernella. Species 1 2 3 4 5 6 7 8 9 1. U. hibernella (Staudinger) 2. M. conturbatella (Hübner) 0.138 3. M. epilobiella (Denis & 0.128 0.07 Schiffermüller) 4. M. unifasciella (Chambers) 0.131 0.065 0.053 5. M. sexstrigella (Braun) 0.164 0.134 0.122 0.131 6. M. miscella (Denis & 0.151 0.102 0.105 0.096 0.102 Schiffermüller) 7. M. idaei (Zeller) 0.153 0.112 0.108 0.109 0.102 0.073 8. M. capella Brown, Adamski, 0.164 0.117 0.119 0.128 0.105 0.09 0.093 Hodges & Bahr 9. M. cephalonthiella Chambers 0.147 0.114 0.116 0.116 0.111 0.093 0.094 0.085 Northeastern Naturalist Vol. 23, No. 2 S.N. White, D.T. Stewart, N.K. Hillier, and R.C. Evans 2016 215 related (91% sequence similarity) to Mompha miscella among all samples of Mompha on that database. Given that there was >3% sequence divergence between M. miscella and our Mompha sample, which is a generally accepted threshold for interspecific divergence for COI sequences (Hebert et al. 2003, 2004), we concluded that our sample was distinct, and that this species had not yet been entered into the GenBank database. Our COI sequence data for this sample of Mompha capella have been entered into GenBank (Acc. No. KP123434). Mompha capella belongs to the family Momphidae, superfamily Gelechioidea, a lesser-studied group of Lepidoptera (Kaila et al. 2011). A primary reason for the lack of definitive research on members of this family is the difficulty in identifying these moths in the field. For morphological identification, the moths must be dissected to examine the genitalia or, alternatively, genetic barcoding must be conducted (Kaila et al. 2011). However, due to the lack of research on this family, relatively little is known about its phylogeny. The genus Mompha has over 100 species with only 16 registered in GenBank (Emery et al. 2009). It is estimated that only 10–40% of the genus has been classified (Emery et al. 2009) . Some members of the genus Mompha are suspected to be host-specialists targeting plants from the family Onagraceae and the family Cistaceae, to which Canada Frostweed belongs (Emery et al. 2009). Larvae of a close relative, M. miscella (Fig. 1), as well as M. passerella (Busck), feed upon seed capsules of other members of the genus Helianthemum (= Crocanthemum) (Momphidae: Microleps.org, accessed July 2014), and may also be leaf miners (Pitkin et al. 2015). It has been suggested that members of the family Momphidae feed on plants from the families Lythraceae and Rubiaceae (Momphidae: Microleps.org, accessed July 2014). The phylogenetic tree based on a 658-bp COI sequence from 8 Mompha species and 1 outgroup is presented in Figure 1. Two deeper clades in the tree had high bootstrapping values (>95%), which may be indicative of subgeneric divisions within the genus Mompha (e.g., Bernasconi et al. 2000). The clade containing Figure 1. A neighbor-joining tree presenting the relationship between M. capella and other members of the genus Mompha based on 658 bp of cytochrome oxidase I sequence. Bootstrap values are shown for each node. The number of substitutions per nucleotide between samples is shown relative to the scale bar. Northeastern Naturalist 216 S.N. White, D.T. Stewart, N.K. Hillier, and R.C. Evans 2016 Vol. 23, No. 2 M. capella, M. cephalonthiella, M. sextrigella, M. miscella, and M. idaei had an average pairwise COI divergence value of 0.095 ± 0.011. The group containing M. conturbatella, M. epilobiella, and M. unifasciella had an average pairwise divergence value of 0.063 ± 0.007. In contrast, the average inter-group divergence value was 0.115 ± 0.009. The bootstrap values were lower towards the tips of the tree and, accordingly, we have lower confidence in the precise sister-group relationships at the species level. Mompha miscella and M. passerella are the only other members of this genus known to feed specifically upon species of Crocanthemum (Momphidae: Microleps.org, access July 2014). Although not sister species, M. capella and M. miscella are in the same sub-clade (Fig. 1). Feeding on Crocanthemum (or their close relatives) may be a synapomorphy for this sub-group; future work on M. cephalonthiella, M. sextrigella, M. idaei, and other members of this sub-clade should examine whether these species also parasitize Crocanthemum. Mompha passerella is a North American species (Harrison 2011), but it has not been barcoded, and its phylogenetic relationship to other Mompha species has yet to be examined. Implications of Mompha capella predation on Canada Frostweed The M. capella infection of the CFB Greenwood population of Canada Frostweed could be devastating for this provincially endangered species. A direct impact upon genetic variation could be possible because White (2015) determined that M. capella had infested 59% of developing chasmogamous fruits in 1 population in 2014. The effect of host specialists such as members of the genus Mompha on perennial plant populations seems to be dependent on the frequency and intensity of the infection (Doak 1992, Emery et al. 2009). However, these factors will likely be highly variable depending on the species of Mompha and the species of plant that the moth is targeting (Dickerson and Weiss 1920, Doak 1992, Emery et al. 2009). In a previous study of cryptic insect-pest predation, Emery et al. (2009) used DNA barcoding to identify several species of Mompha that were feeding on Camissoniopsis cheiranthifolia (Hornem. ex Spreng.) W.L. Wagner & Hoch (Beach Evening Primrose, Onagraceae). Like Canada Frostweed, Beach Evening Primrose grows in sandy habitats, but the latter species is found on the west rather than the east coast of North America. Emery et al. (2009) also found that Mompha species tend to target outcrossing flowers rather than self-fertilizing flowers, possibly because the outcrossed flowers are larger and provide more resources for the larvae (Dart and Eckert 2015). These authors also speculate that long-term exposure to the moth could play a role in the evolution of floral traits (Dart and Eckert 2015, Emery et al. 2009). Further studies should be conducted on the effect of M. capella on the CFB Greenwood population of Canada Frostweed to quantify the intensity and frequency of Lepidoptera infection rates of chasmogamous flowers. It is not known if this is a new infestation or if this Canada Frostweed population has co-existed with this moth for some time. In the long-term, Mompha species have been found to cause a decline in some perennial populations (e.g., Chamerion [= Epilobium] latifolium [L.] Holub [Dwraf Fireweed]; Doak 1992). Our findings raise concerns Northeastern Naturalist Vol. 23, No. 2 S.N. White, D.T. Stewart, N.K. Hillier, and R.C. Evans 2016 217 that management actions may be required to limit the spread of M. capella to other populations of Canada Frostweed in Nova Scotia. Acknowledgments We would not have undertaken this study without the key insights and keen observations of the late Dr. Sam vander Kloet. This project would also not have been possible without the cooperation of staff at CFB Greenwood. Funding was provided by Acadia University 25.55 Fund, E.C. Smith Herbarium, and the Strategic Co-op Education Incentive. We also thank the following: J.-F. Landry, Canadian National Collection of Insects, Arachnids and Nematodes, Ottawa, Canada, for independent identification of the moth as Mompha capella; M. Elderkin, Nova Scotia Department of Natural Resources, for helpful information on Corema barrens in Nova Scotia; C. Little and L. Thomas for rearing of larval samples; and B. Robicheau and N. LeBlanc for assistance in the lab. Literature Cited Armstrong, K.F., and S.L. Ball. 2005. DNA barcodes for biosecurity: Invasive species identification. Philosophical Transactions of the Royal Society, Series B 360:1813–1823. Bernasconi, M., J. Pawlowski, C. Valsangiacomo, J.-C. Piffaretti, and P. Ward. 2000. Phylogeny of the Scathophagidae (Diptera, Calyptratae) based on mitochondrial DNA sequences. Molecular Phylogenetics and Evolution 16:308–315. Brooks, D.R., and D.A. McLennan. 1991. Phylogeny, Ecology, and Behaviour: A Research Program in Comparative Biology. University of Chicago Press, Chicago, IL. 441 pp. Catling, P.M., and S. Carbyn. 2005. Invasive Scots Pine, Pinus sylvestris, replacing Corema, Corema conradii, heathland in the Annapolis Valley, Nova Scotia. Canadian Field-Naturalist 119:237–244. Catling, P.M., S. Carbyn, S.P. vander Kloet, K. MacKenzie, S. Javorek, and M. Grant. 2004. Saving Annapolis heathlands. The Canadian Botanical Association Bulletin 37:12–14. Crawley, M.J. 1989. Insect herbivores and plant-population dynamics. Annual Review of Entomology 34:531–564. Dart, S., and C.G. Eckert. 2015. Variation in pollen limitation and floral parasitism across a mating system transition in a Pacific coastal dune plant: Evolutionary causes or ecological consequences? Annals of Botany 115:315–326. Dickerson, E., and H. Weiss. 1920. The insects of the evening primroses in New Jersey. Journal of the New York Entomological Society 28:32–74. Doak, D.F. 1992. Lifetime impacts of herbivory for a perennial plant. Ecology 73:2086– 2099. Emery, V.J., J.-F. Landry, and C.G. Eckert. 2009. Combining DNA barcoding and morphological analysis to identify specialist floral parasites (Lepidoptera: Coleophoridae: Momphinae: Mompha). Molecular Ecology Resources 9:217–223. Garcia-Robledo, C., E. Kuprewicz, C. Staines, W. Kress, and T. Erwin. 2013. Using a comprehensive DNA barcode library to detect novel egg- and larval-host plant associations in a Cephaloleia rolled-leaf beetle (Coleoptera: Chrysomelidae). Biological Journal of the Linnean Society 110:189–198. Goujon, M., H. McWilliam, W. Li, F. Valentin, S. Squizzato, J. Paern, and R. Lopez. 2010. A new bioinformatics analysis-tools framework at EMBL-EBI. Nucleic Acids Research 38 (Suppl. 2):W695–W699. Northeastern Naturalist 218 S.N. White, D.T. Stewart, N.K. Hillier, and R.C. Evans 2016 Vol. 23, No. 2 Harrison, T.L. 2011. Microlepidoptera of Illinois Hill Prairies. Ph.D. Dissertation. University of Illinois at Urbana-Champaign, Urbana, IL. 91 pp. Hebert, P.D., A. Cywinska, S.L. Ball, and J.R. deWaard. 2003. Biological identifications through DNA barcodes. Proceedings of the Royal Society of London, Series B 270:313–321. Hebert, P.D., E. Penton, J. Burns, D. Janzen, and W. Hallwachs. 2004. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proceedings of the National Academy of Sciences of the United States of America 101:14,812–14,817. Hebert, P.D.N., J. R. deWaard, and J.F. Landry. 2009. DNA barcodes for 1/1000 of the animal kingdom. Biology Letters 6:359–362. Hodges, R.W. 1992. Two new species of Mompha from California (Lepidoptera: Momphidae). Journal of the New York Entomological Society 100: 203–208. Jinbo, U., K. Toshihide, and I. Motomi. 2011. Current progress in DNA barcoding and future implications for entomology. Entomological Science 14:107–124. Kaila, L., M. Mutanen, and T. Nyman. 2011. Phylogeny of the mega-diverse Gelechioidea (Lepidoptera): Adaptations and determinants of success. Molecular Phylogenetics and Evolution 61:801–809. Lewis, O., and S. Gripenberg. 2008. Insect seed-predators and environmental change. Journal of Applied Ecology 45:1593–1599. Momphidae: Microleps.org. Available online at http://www.microleps.org/Guide/Momphidae/ index.html. Accessed 21 July 2014. Newell, R. 2007. Nova Scotia provincial status report on Rockrose (Canada Frostweed) Helianthemum canadense (L.) Michx. Nova Scotia Species at Risk Working Group, Nova Scotia Department of Natural Resources, Kentville, NS, Canada. 33 pp. Nova Scotia Department of Natural Resources. 2013. Nova Scotia endangered species act: Legally listed species. Available online at http://novascotia.ca/natr/wildlife/biodiversity/ species-list.asp. Accessed 31 May 2015. Pitkin, B., W. Ellis, C. Plant, and R. Edmunds. 2015. The leaf and stem mines of British flies and other insects. Available online at http://www.ukflymines.co.uk/Moths/Mompha_ miscella.php. Accessed 3 November 2015. Rand, T., and S. Louda. 2006. Invasive insect abundance varies across the biogeographic distribution of a native host plant. Ecological Applications 16:877–890. Reznicek, A.A., E.G. Voss, and B.S. Walters. Michigan flora online. 2011. University of Michigan. Available online at http://michiganflora.net/genus.aspx?id=Crocanthemum. Accessed 30 October 2015. Tamura, K., G. Stecher, D. Peterson, A. Filipski, and S. Kumar. 2013. MEGA6: Molecular evolutionary genetics analysis, version 6.0. Molecular Biology and Evolution 30:2725–2729. Valentini, A., F. Pompanon, and P. Taberlet. 2008. DNA barcoding for ecologists. Trends in Ecology and Evolution 24:110–117. White, S.N. 2015. A tale of two flowers: A comparative analysis of chasmogamous and cleistogamous flower development in Helianthemum canadense (L.) Michx. B.Sc. (Hons.). Acadia University, Wolfville, NS. 46 pp. Yorke, A., S. Mockford, and R.C. Evans. 2011. Canada Frostweed (Helianthemum canadense (L.) Michx.; Cistaceae) at the northeastern limit of its range: Implications for conservation. Botany 89:83–89.