Hyposmocoma, FROM

Doorenweerd, Camiel, Austin, Kyhl A., Rubinoff, Daniel, n, sp., n, sp., n, sp., n, sp. & n, sp., 2017, Small survivors: unexpected endemic diversity of Hyposmocoma (Lepidoptera: Cosmopterigidae) moths on Kaho olawe, a degraded Hawaiian island, Zoological Journal of the Linnean Society (Zool. J. Linn. Soc.) 180 (3), pp. 570-592 : 571

NEW HYPOSMOCOMA FROM KAHOʻOLAWE ISLAND 571

paired with over a century of overgrazing by goats,War and that are considered ‘lost causes’ might still hold proits use as a bombing range for the US military Reserve from mise, such as small patches of degraded habitat frag- World War II until the early 1990.hawaii s (Kahoʻolawe.gov/history.shtml Island). mented by development in urban areas. Reserve Commission, http://kahoolawe.hawaii.gov/his- clitory.shtml). The combination of its very small impoversize and MATERIAL AND METHODS arid climate would suggest that it supported an impov- and erished native fauna prior to human settlement remain, and Adult moth specimens were collected from sites that little or no endemic biodiversity may remain disturafter around Kahoʻolawe ( Fig. 1 ) using UV lights (unless its history of extensive anthropogenic disturbance the. otherwise noted) over several nights in October 2008 Understanding the level of diversity and the phylo- would (Medeiros), July 2011 (Medeiros), and March 2013 genetic relationships on such an island would inform gen- (Haines & Rubinoff). We also searched intensively not only evolutionists concerned with the generation apparently of for case-making larvae in March 2013, and endemic diversity on smaller, apparently lower diver- seek attempted to rear them in the laboratory. Legs of sity landmasses, but also those that seek to estimate land- adult moths were preserved in 95% ethanol for DNA the conservation value of degraded landscapes. The of extraction. Genitalia were prepared and mounted on extended military occupation and use of Kahoʻolawe many slides using the following protocol: abdomens were as a bombing range left behind many unexploded haz- soaked in simmering 10% KOH solution for 1 h, genordinances making it an extremely hazardous virtually envi- italia were removed, stained with lignin pink and ronment; the island has been virtually unavailable when to chlorazol black, soaked in a sequence of 30% ethyl research until the past decade when ownership was the alcohol, 90% ethyl alcohol, 100% isopropyl alcohol, transferred State of Hawai from i. the military to the State of Hawai`i. and Euparal essence (Bioquip, Rancho Dominguez, The endemic Hawaiian moth genus Hyposmocoma CA , USA), then spread on microscope slides and Butler, 1881, is one of the most widespread and mounted in Euparal. Digital photographs were taken diverse lineages in the archipelago, with species using an Olympus Q-Color3 camera mounted on an occurring from sea level to over 3000 m elevation, Olympus SZX10 light microscope. and essentially 100% of these species being single- DNA was extracted from the legs of field-caught island endemics. Precious few island-endemic ani- and loaned specimens using the standard protocol mals or plants have been found on Kahoʻolawe [but described in Qiagen’s (Valencia, CA, USA) DNeasy see Lorence & Wood (1994) for a new genus within kits. Using PCR, we amplified fragments of four the plant family Fabaceae and Medeiros & Adamski nuclear genes (elongation factor 1-a, carbamoyl- (2012) for three new species of Lepidoptera ]; thus, phosphate synthetase 2, aspartate transcarbamylase, our recent surveys of the diversity of Hyposmocoma and dihydroorotase, malate dehydrogenase, and on Kahoʻolawe might provide important information ribosomal protein S5) and one mitochondrial gene concerning the potential for such a small island to (cytochrome oxidase 1). Primers and their annealing generate and maintain endemic diversity in the face temperatures are listed in Appendix 1. Thermal proof wholesale ecological destruction. As we know the files for PCR began with denaturation at 95 ° C ecological roles of Hyposmocoma species based on (2 min), followed by 35 cycles of denaturation at their larval case types, we can use the presence of 95 ° C (1 min), an annealing step (1 min), and elongaspecies on Kahoʻolawe as an indicator of which kinds tion at 72 ° C (1 min). Cycles were followed by a final of microhabitats still occur on the island – even when extension step at 72 ° C (12 min). Amplified PCR collecting adult moths. This information may be used products were purified using the QIAquick PCR to guide future surveys to locate those microhabitats Purification Kit (Qiagen) and run on an ABI Prism that may harbour additional cryptic taxa that are 377 XL automated DNA sequencer. The final data not as well known as Hyposmocoma . Knowledge of set consisted of a total of 3993 bp, including the the evolution of endemic Hyposmocoma species on mitochondrial gene CO1 (1422 bp) and the nuclear Kahoʻolawe will not only be important in under- genes EF-1 a (771 bp), CAD (705 bp), MDH (498 bp), standing the evolution of this ultra-diverse lineage and RpS5 (597 bp). across the archipelago (e.g. Haines et al., 2014), but New sequences from Kahoʻolawe Hyposmocoma also in assessing whether Kahoʻolawe might still were added to many additional sequences of Hyposharbour endemic species in other cryptic arthropod mocoma representing a diversity of larval case types groups. Perhaps more importantly, such data could and islands that had previously been published in guide conservation efforts by indicating how and Haines et al. (2014). The concatenated data matrix where endemic species might have survived cen- had 164 taxa and 3993 characters (see Appendix 2 turies of severe habitat alteration. Results from such for GenBank accession codes and for specimens with research on a small, heavily impacted island could incomplete coverage in terms of the five gene regions

Figures

Figure 1. Map of collection sites on Kahoʻolawe: (1) Kealaikahiki, (2) Kaukaukapapa, (3) drainage near Honokanaiʻa, (4) Honokanaiʻa base camp, (5) Kealialalo, (6) Kaukamoku gulch, (7) base of Puʻu o Moaʻula Iki, (8) old Erythrina tree at Puʻu o Moaʻula Nui, (9) planting site Puʻu o Moaʻula Nui, and (10) planting area at Hakioawa. The Hawaiian Islands are shown in grey in the upper right (boxed area contains Kahoʻolawe).