Genetic diversification among populations of the endangered Hawaiian endemic Euphorbia kuwaleana (Euphorbiaceae) By: Clifford W. Morden*, Troy Hiramoto, and Mitsuko Yorkston Abstract The Hawaiian Euphorbia is an assemblage of 17 species, seven of which are endangered and four others rare. Euphorbia kuwaleana is an endangered species of small shrubs restricted to three small populations in west O‘ahu, Hawai‘i. The species has declined to fewer than 1000 individuals largely due to habitat encroachment by alien plant species and the periodic fires that occur in the vicinity. Genetic variation was assessed among individuals in two populations to determine what impact small population size has had on genetic diversity within the species using RAPD markers. Results demonstrate that polymorphism within these populations is high (mean=82.5%), equal to or exceeding that of many other non-endangered Hawaiian species. Genetic similarities within (0.741) and among (0.716) populations, FST (0.072), and PCO analysis all indicate differentiation among the populations although in close geographical proximity (<1 km apart). Conservation efforts for this species should focus on protection of existing populations from eminent threats and the establishment of new populations in suitable habitats on O‘ahu. *Corresponding Author E-mail: [email protected] Pacific Science, vol. 68, no. 1 July 16, 2013 (Early view) Introduction The genus Euphorbia (Euphorbiaceae) is represented in Hawaii by 17 species from two separate colonizations. The C3 species, Euphorbia haeleeleana, is a tree with succulent stems and is closely related to the Australian succulent species E. plumerioides and E. sarcostemmoides (Zimmerman et al 2010). The other 16 species (previously in the genus Chamaesyce, now recognized as a subgenus of Euphorbia; Yang et al. 2012) all utilize the C4 photosynthesis pathway (Pearcy and Troughton 1975) and are the consequence of a tremendous adaptive radiation from a single colonist whose closest relatives are in section Anisophyllum and are from Mexico or the Southwest United States (Yang and Berry 2011, Yang et al. 2012). These species, commonly referred to as Hawaiian spurge or ‘a¯koko, vary from small shrubs less than 0.1 m tall and stems less than 2 mm in diameter to trees up to 10 m tall with boles up to 30 cm in diameter (Koutnik and Huft 1990). Habitats are similarly diverse with some species occurring along coastal strand or xeric lowland habitats, and others in mountain rainforests or open areas at elevations exceeding 2000 m (Koutnik and Huft 1990). Many of these species are endemic to a single island or region within an island resulting in small and isolated populations. Consequently, the U.S. Fish and Wildlife Service lists seven of the 17 species and three varieties as endangered species and others species and varieties are known to be rare (USFWS, personal communication). Euphorbia kuwaleana is a small shrub normally growing to 0.3 m tall, but it can be as tall as 1.0 m when growing in rocky crevices (Koutnik and Huft 1990; personal observation). It is distinguished from related species by its stalked oval to round leaves with an untoothed margin and curved stalk bearing the cyathea and capsule (Koutnik 1987, Koutnik and Huft 1990). Plants are restricted to coastal arid, rocky exposed volcanic cliffs in a 5 km rim of cinder cone hills that form a ridgeline separating Wai‘anae and Lualualei Valleys in west O‘ahu (USFWS 1991, 1998, 2 2011). The species is named for the type locality, Mauna Ku¯wale, the western-most of the cinder cones and the lowest elevation (190 to 230 m) where plants may be found (Figure 1). Separated by a short valley and approximately 800 m to the northeast is Kaua‘ōpu‘u where plants are found at slightly higher elevations (250 to 380 m) and in subpopulations that extend eastward along the ridgeline. A third population is present at Pu‘u Ka‘i¯lio approximately 2.5 km east of Kaua‘ōpu‘u (Figure 1). It is probable that this species was much more widespread on O‘ahu in the past. Earlier collections were made from Moku Manu, a pair of small islets near Kane‘ohe Bay in east O‘ahu, and other locations have been identified as suitable habitat for E. kuwaleana if alien species can be controlled (USFWS 2003). << Fig. 1 near here >> The habitat where plants occur are primarily dry shrublands dominated by ‘a‘ali‘i (Dodonaea viscosa) and ‘ilima (Sida fallax). However, these are being encroached upon by alien vegetation including koa haole (Leucaena leucocephala), Christmas berry (Schinus terebinthifolius), and invasive African grasses including buffelgrass (Cenchrus ciliaris), guinea grass [Megathyrsus maximus (synonyms = Panicum maximum and Urochloa maxima)], and Natal redtop (Melinis repens) (USFWS 1998, 2003). Precipitation in this region is approximately 750 to 1000 mm that is largely concentrated in the winter months (November to April) with sporadic summer showers resulting from orographic condensation associated with the trade winds blowing over the Wai‘anae Mountains and carrying moisture to these habitats. The summer precipitation seems vital to the survival of these plants as plants are most often positioned on the east-facing slopes along the ridge to best capture this infrequent precipitation and few or none are found on west-facing slopes (personal observation). Populations of E. kuwaleana have been in decline over the past 10 years of observation. Approximately 2000 individuals were observed at the time of the species listing (USFWS 1991, 3 1998) and when critical habitat was designated (USFWS 2003) with about 1000 individuals present at Kaua‘ōpu‘u, 500 at Mauna Ku¯wale and “several hundred” at Pu‘u Ka‘i¯lio (USFWS 1998, 2003). By 2007, the total number of individuals had declined and was estimated to be 1200 plants in only two remaining locations (USFWS 2011). A 2012 survey of plants on Kaua‘ōpu‘u found that this population had further declined to approximately 300 individuals (Sailer 2012). Fire, promoted by alien species invasion, is a continual threat to this species (USFWS 1998, 2003). Periodic fires of unknown causes occur in this region on a regular basis, and fires have occurred near these populations as recently as 2003 and 2012, the latter known to have resulted in the loss of 57 plants and damage to several others (Sailer 2012). The purpose of this study was to investigate the genetic variation within and among populations at Mauna Ku¯wale and Kaua‘ōpu‘u to assist in management decisions to improve the recovery of these species. The population at Pu‘u Ka‘i¯lio was not included in this study because this land is in a high security military zone and access was not sought. Random amplified polymorphic DNA (RAPD) markers were used to assess genetic variation in the populations. Previous studies examining genetic variation with RAPD markers had shown that other endangered Euphorbia species have high levels of variation within populations (Morden and Gregoritza 2005, Morden 2011). It was expected that total variation within E. kuwaleana would be similarly high. Given the limited spatial distance between these two populations (ca. 800 m), it was expected that these populations would not be genetically differentiated and that population structure will be absent. Materials and methods Leaf tissue from populations of E. kuwaleana was collected from Kaua‘ōpu‘u (33 plants) and Mauna Ku¯wale (52 plants), two cindercones that form a ridgeline separating Lualualei and 4 Wai‘anae Valleys in western O‘ahu, in January and April, 2008, respectively (Table 1). DNA was extracted from approximately 1.0 g of fresh leaf tissue using the CTAB method of Doyle and Doyle (1987) with minor modification (Morden et al. 1996) and accessioned into the Hawaiian Plant DNA Library [HPDL no. 5700-5752 (Kaua‘ōpu‘u; 19 samples possibly contaminated and omitted from analysis) and HPDL no. 5773-5824 (Mauna Ku¯wale); Randell and Morden, 1999]. Voucher material was not collected because of their endangered status; representative specimens of each population are deposited at BISH (Appendix A). DNA samples were diluted to approximately 25 ng/µl to create working solutions for amplification using the polymerase chain reaction (PCR). DNA (25 ng) was amplified in 15 µl reactions under the following conditions: 0.2 µM random 10-mer oligonucleotide primers (Primer kits OPA through OPD; QIAGEN Operon, Alameda, California, USA), 0.2 mM each of dATP, dCTP, dGTP, and dTTP (Promega, Madison, Wisconsin), 1X Taq polymerase PCR buffer, 2.0 mM MgCl2, 0.1% bovine serum albumin, and ca. 1 unit Taq polymerase (Promega, Madison, Wisconsin, USA). Amplification was performed in a MJ Research PTC-200 Peltier Thermal Cycler with an initial denaturation of 94°C for 2 minutes followed by 45 cycles of 94°C for 45 seconds, 35°C for 45 seconds, and 72°C for 2 minutes, and an extended elongation of 5 minutes on the final cycle. Amplified products were combined with 3 µl loading dye (20 mM EDTA, 10% glycerol, 1% sarcosyl, bromophenol blue, 1% xylene cyanol) and separated in a 1.5% agarose gel in a 0.5X tris-borate-EDTA buffer with 125 ng ethidium bromide per liter. DNA marker size was estimated by comparison to a plasmid (pBS KS+; Stratagene, La Jolla, California) digested with restriction enzymes to produce fragments between 0.448-2.96 kb. Final gel products were viewed with the UVP BioImaging Systems Gel HR Camera-6100 series and recorded on the UVP GelDoc-It TS software. Five individuals were screened with RAPD primers for consistent amplification and 5 brightly staining markers. For those primers selected for further analysis, amplifications were performed on all samples. Bands from reproducible amplification phenotypes (determined from replicated analyses) were scored for either presence (1) or absence (0) at each locus (Rieseberg 1996).