Is Anatrachyntis another radiation onto cycads? Catherine Hua, Shayla Salzman, Naomi Pierce

Introduction Results All species of cycads are highly toxic and have very few known herbivores. This requires a strategy Discussion for dealing with the host plant’s secondary metabolites, something which must be difficult as very few These new records of the presence of Anatrachyntis on cycads is things feed on them. In fact, there appears to be very few evolutions of the ability to utilize cycads as unconventional, as these host plants are highly toxic and have very few known a food source, often resulting in radiations of genera of onto cycads. Examples include leaf herbivores and suggests that Anatrachyntis may be predisposed to feeding on feeding such as Eumaeus (Contreras-Medina, 2003) and cone feeding coleoptera this uniquely toxic host tissue. Results depict the lack of a one-to-one relationship Rhopalotria (Fawcett and Norstog, 1993). Cycads were once considered strictly wind pollinated, but it between the and cycads. Instead, we identify on Figure 1: Phylogenetic tree. The Zamia integrifolia. is increasingly becoming evident that most, if not all, rely on insect pollination where the host plant three species of moths found to provides a brood site for the pollinator (Terry et al. 2012). The pollinator-plant relationships appear to have emerged from three different populations of cycad were found to Results depict the lack of a one-to-one relationship between the moths and be strictly one-to-one in a classical co-evolution. be three separate species that all belong to the Anatrachyntis cycads. Instead, we identify the generalist Anatrachyntis badia on Zamia An unidentified Anatrachyntis has been reported as a pollinator of on Guam genus. The two moths collected integrifolia. Additionally, the two moths on separate populations of Cycas from Cycas micronesica pollen micronesica were found to be separate species. One of these moths is known to whose larvae develop in the pollen cone (Terry, 2009). Currently, there is no other known Lepidoptera cones on Guam and Yap islands pollinator of cycads (Terry, 2009). However, microlepidoptera morphologically similar to Anatrachyntis both remain unidentified. The pollinate Cycas micronesica (Marler, 2011). The result of this analysis is were found emerging from male cones of Zamia integrifolia in Florida at the time the cones were that emerged from Zamia integrifolia unprecedented because it seems that the Anatrachyntis species found on Guam is identified as Anatrachyntis is more closely related to Anatrachyntis badia than to the other moth found on releasing pollen. badia. Yap Island, which is geographically close as well as shares the same host plant The genus Anatrachyntis consists of about 50 species, but research in this genus has focused primarily on Anatrachyntis badia due to it’s wide geographic range. A. badia is of North American In many cases, herbivores that feed on cycads also specialize in cycad origin (Dawidowicz, 2017). A. badia is known as a scavenger that feeds on a variety of hosts, habitation. This is a niche that is highly beneficial, therefore, once a species or including various fruits and cones of conifers (Bella & Mazzeo, 2006). Upon feeding, it may cause even a whole genus discovers this underlying survival potential, a radiation can damage to the plant, generating tunnels in leaves and causing erosion and yellowing (Bella & shortly follow, meaning that this genus could be diversifying on to cycads. Our Mazzeo, 2006). Despite these damages, an unidentified species of Anatrachyntis is a pollinator of results suggest a potential radiation of this moth genus onto cycads that mirrors the critically endangered cycad species, Cycas micronesica on Guam where it spends its larval stage other known cycad herbivores. To provide further evidence for this hypothesis, in the pollen cone (Marler, 2012). additional genes should be tested. In the same vein, more individuals from across the range of Anatrachyntis should be sampled to further diversify this tree so as Although a moth in this genus feeds on and pollinates Cycas micronesica in the Old World tropics to infer evolutionary history of the association with cycads. (Terry et al., 2009, Marler, 2012), this is the first record of this genera of moth on any other cycad. Zamia integrifolia and Cycas micronesica are threatened and endangered species and knowledge of Additionally, pollen was found on moth specimens that emerged from Zamia another pollinator is beneficial. These one-to-one mutualistic pollinator-plant relationships are fragile; integrifolia during the plant breeding season. Anatrachyntis badia could be a if a pollinator goes extinct, the plant will follow suit. Conservationists are currently working to grow potential pollinator, however, much more groundwork is necessary to confirm. these endangered plants in protected areas before returning them back into the wild. However, after Further exclusion and trapping studies would be needed to tell whether or not this restoration in the wild, these plants will be unable to survive and reproduce if their pollinators are not species of moth pollinates Z. integrifolia. Since the unidentified Anatrachyntis present or have gone extinct. Identification of a generalist pollinator with many host plants like A. Figure 2: Anatrachyntis from Guam is also a known pollinator of C. micronesica (Marler, 2011), the genus badia photographed with has potential pollination ability for Zamiai integrifolia. badia would greatly help conservation efforts for these endangered plants. This moth was DNA the Keyence digital barcoded together with the known Antrachyntis pollinator from Cycas micronesica in Guam and one microscope. Dorsal other unidentified moth that emerged from Yap island (geographically close to Guam). habitus, lateral habitus, ventral habitus. Figure 4: Unidentified Guam Anatrachyntis species Materials and Methods photographed with the Microlepidotera were found emerging from pollen dishing male cones of Zamia integrifolia collected in Florida. Adult moths that Keyence digital microscope. Dorsal habitus, lateral habitus, emerged from pollen cones of Cycas micronesica in Guam and Yap islands were sent from Drs. Irene Terry and William Tang. ventral habitus.

DNA extraction and Barcoding DNA was extracted from the unknown Microlepidotera with the DNeasy® Blood & Tissue kit and procedure. PCR of the CO1 gene was done using Omega 2x master mix (Bio-Tek, Norcross, Georgia)(with LCO and HCO primers (citation). The Figure 5: Pollen found adhered to Anatrachyntis badia body at magnification 4x, 40x and 40x. This PCR protocol consisted of a 2 min initial denaturing step at 95°C, 37 cycles of 30 sec at 95°C, 1 min at 50°C, and 1 min 30 sec at moth pupates in the pollen cone peduncle and emerges as the cone is dispersing pollen. 72°C, and a final 5 min 72°C extension. The PCR product was sequenced by Eton Bioscience Inc using sanger sequencing methods. Sequence reads were assembled and edited using Geneious (version 10.2.3). Use of the NCBI BLAST database determined that the Citations moths that emerged from Zamia integrifolia had 100% identity to Anatrachyntis badia and identified the other two moths as belonging to the Anatrachyntis genus. Bella, S., & Mazzeo, G. (2006). First record of Anatrachyntis badia (Hodges, 1962) (Lepidoptera ) in Italy. Bollettino di Zoologia agraria e di Bachicoltura, Milano, Ser. II, 38(3), 255-260. Phylogenetic Analysis Contreras-Medina, R., Ruiz-Jiménez, C. A., & Luna Vega, I. (2003). Caterpillars of Eumaeus childrenae (Lepidoptera: Lycaenidae) feeding on two species of cycads (Zamiaceae) in the Huasteca region, Mexico. Revista de All 109 Anatrachyntis CO1 sequences available on the Barcode of Life Database (BOLD) were included in phylogenetic analysis. All biología tropical, 51(1), 201-204. 113 sequences were aligned using the Geneious alignment algorithm and manually edited. The model of evolution for the Co1 gene Dawidowicz, L. (2017). Anatrachyntis badia (Hodges, 1962)(Lepidoptera: Cosmopterigidae): the first report from alignment was determined using jModelTest Both Bayesian information criterion and Akaike information criterion determined that the Turkey and a case of importation to Poland. Turkish Journal of Zoology, 41(1), 60-63. model of evolution was Generalised time-reversible + Gamma (GTR+G)(negative log likelihood 1998.0423). A phylogenetic tree was Fawcett, P and K Norstog. 1993. Zamia pumila in South Florida: A preliminary report on its pollinators R. Slossoni, created using PhyML as implemented in Geneious and the GTR+G model using the best of nearest neighbor interchange and subtree and snout weevil and P. zamiae, a clavicorn beetle. In: The biology, structure and systematics of the cycadales. DW Stevenson and KJ Norstog Editors. aPalm & Cycad Societies of Ltd. pp 109-119. pruning-regrafting tree topology searches. The resulting maximum likelihood tree had a negative log likelihood of -2020.75868. Figure 3: Unidentified Yap Island Anatrachyntis species photographed Marler, T. E., & Niklas, K. J. (2011). Reproductive effort and success of Cycas micronesica KD Hill are affected by habitat. International Journal of Plant Sciences, 172(5), 700-706. Microscope Analysis with the Keyence digital microscope. Dorsal habitus, lateral habitus, ventral We used light microscopy to explore the life history of the Anatrachyntis badia-Zamia integrifolia relationship. A dissecting scope was habitus. Terry, I. R. E. N. E., Roe, M. A. R. E. N., Tang, W. I. L. L. I. A. M., & Marler, T. E. (2009). used to to look for pupal casings and identify where these moths live in the pollen cones of the host plant. Slides were made of adult Cone insects and putative pollen vectors of the endangered cycad, Cycas micronesica. Micronesica, 41(1), 83-99. moths and examined under 4x, 10x, and 40x power to look for the presence of pollen on the insect bodies.