Appendix ES-6 Population genetics of Schiedea Species of Conservation Concern on U.S. Army Lands Oahu Appendix ES-6 Population genetics of Schiedea Species of Conservation Concern on U.S. Army Lands Oahu Cover image: Schiedea obovata (Scherff) W.L.Wagner & Weller Photographer: Lauren A. Weisenberger Appendix ES-6 Population genetics of Schiedea Species of Conservation Concern on U.S. Army Lands Oahu Population genetics of Schiedea species (Caryophyllaceae) of conservation concern on U.S. Army lands, O‘ahu. Final report prepared for: O‘ahu Army Natural Resource Program (OANRP) Schofield Barracks, Hawaii Prepared by: Shelley A. James Hawaii Biological Survey & Pacific Center for Molecular Biodiversity Bishop Museum Honolulu HI 96817, USA September 2012 Contribution No. 2012-019 to the Hawaii Biological Survey Appendix ES-6 Population genetics of Schiedea Species of Conservation Concern on U.S. Army Lands Oahu Population genetics of Schiedea Population genetics of Schiedea species (Caryophyllaceae) of conservation concern on U.S. Army lands, O‘ahu. Introduction Schiedea (Caryophyllaceae) is a morphologically variable monophyletic genus endemic to the Hawaiian Islands. The genus consists of 32 extant species, all of which arose from a single initial colonization of the island chain (Wagner et al. 2005; Willyard et al. 2011). Unfortunately, largely due to land degradation and the impacts of invasive species, 15 species of Schiedea are now federally listed as endangered and two species are now extinct. Six endangered species of Schiedea are currently found on U.S. Army lands on O‘ahu, of which three are the focus of this study: Schiedea kaalae, S. nuttallii, and S. obovata. Phylogenetic analyses using morphological and nrDNA ITS and ETS regions show that S. nuttallii and S. kaalae are closely related members of the S. nuttallii clade (section Mononeura) (Wagner et al. 2005; Willyard et al. 2011). They have fleshy stems, large leaves with a single vein, and attenuate to caudate, strongly reflexed sepals. S. obovata is part of the basal clade of the genus (S. membranacea clade, section Alsinidendron), having broad, multi-nerved leaves with ciliate or toothed margins. Schiedea kaalae Wawra is a long-lived (>10 years) perennial herb with a hermaphroditic breeding system (Wagner et al. 2005). S. kaalae was federally registered as an endangered species in 1991 (Ellshoff et al. 1991, USFWS 2008). The species was once distributed throughout the Wai‘anae and northern Ko‘olau Mountains, but less than 30 mature individuals currently remain within three extant wild populations in the Wai‘anae Mountains, two with only a single individual, and three in the Ko‘olau Mountains, one with only a single individual (Keir and Weisenberger 2011). However, outplanted individuals survive in three populations in the Wai‘anae Mountains and recruitment and vegetative reproduction have been observed, and most founders from extirpated populations are represented in living and seed collections. All plants in the Wai‘anae Mountains are now protected by fencing from habitat destruction caused by ungulates. Invasive plant species, slug, rat and mouse predation, drought, and fire continue to threaten the survival of the species (U.S. Army Garrison 2007, USFWS 2008). Morphological Bishop Museum 1 Hawaii Biological Survey Appendix ES-6 Population genetics of Schiedea Species of Conservation Concern on U.S. Army Lands Oahu Population genetics of Schiedea differences between the Ko‘olau and Wai‘anae Mountain populations have been observed (L. Weisenberger, pers. comm. 2012). Schiedea nuttallii Hook. is an outcrossing hermaphroditic subshrub. Historically, the range of the species extended from O‘ahu, Moloka‘i, to West Maui (Wagner et al. 2005), but it is now restricted only to O‘ahu. At the time of federal listing, only a single population of 25 extant individuals in the Wai‘anae Mountains was known (Russell and Bruegmann 1996), but the discovery of a second population at Kahanahā‘iki in 1999 doubled the number of known individuals (U.S. Army Garrison 2007, USFWS 2007a). As of 2007, 47 wild individuals were known from two populations (Kahanahā‘iki and Kapuna-Keawapilau Ridge), and four outplanting sites were established (U.S. Army Garrison 2007, USFWS 2007a). Currently, only one extant population (Pahole) and one isolated individual (Kahanahā‘iki) occur in mesic habitat in the northern Wai‘anae Range (Keir and Weisenberger 2011). Schiedea nuttallii has previously been characterized as having low isozyme variability, low heterozygosity and percentage of polymorphic loci, and demonstrates inbreeding effects due to small population size (Wagner et al. 2005; Weller et al. 1996). According to the USFWS (2007a, 2009), Schiedea nuttallii is in a phase of quasi-extinction with numbers declining to the point where demographic or environmental events alone could result in its extirpation. Schiedea obovata (Sherff) W.L.Wagner & Weller is a shrub with a hermaphroditic, facultative autogamy breeding system (Wagner et al. 2005). While historically found along the entire Wai‘anae Mountain range, the species was restricted to three populations in the northern Wai‘anae Mountains of O‘ahu (U.S. Army Garrison 2007, USFWS 2007b) but has subsequently been reduced to one extant wild population (NW Makaleha) of approximately 100 individuals (30 mature) and a few isolated individuals in surrounding areas (Keawapilau and West Makaleha) (L. Weisenberger, pers. comm. 2012). S. obovata was federally listed under the synonym, Alsinidendron obovatum Sherff., as endangered in 1991 (Ellshoff et al. 1991). As for the other two Schiedea species in this study, S. obovata is threatened by small population sizes, and habitat degradation and damage due to invasive plant and animal species (USFWS 2007b). While S. nuttallii and S. obovata occur in sympatry in the Wai‘anae Mountains, differences in Bishop Museum 2 Hawaii Biological Survey Appendix ES-6 Population genetics of Schiedea Species of Conservation Concern on U.S. Army Lands Oahu Population genetics of Schiedea breeding systems have prevented interspecific gene flow between these species and the development of hybrids (Willyard et al. 2011). For all three species, natural recruitment remains poor, and extant populations continue to be threatened by reduced reproductive vigor due to small number individuals and populations (Keir and Weisenberger 2011). Weller et al. (1996), using allozyme analysis, found extremely low levels of polymorphism and heterozygosity in all three species, but particularly for the autogamous S. obovata. Future management actions for Schiedea and determining whether reintroductions or population augmentation of species within their historic range should represent a single population or multiple populations will be based on multiple factors, including inter- population and intra-population genetic variation, and variation in habitats between populations. The goals of this study are to assess the level of genetic variation within and among founder populations of three Schiedea species located on U.S. Army lands, namely S. kaalae, S. obovata, and S. nuttallii, using microsatellite analysis, and compare this variation to the geographic distribution of purported populations to assist with developing management actions for population reintroduction and/or augmentation. Materials and Methods Sampling Immature leaf material of Schiedea kaalae, S. obovata, and S. nuttallii were obtained from shade-house-grown individuals at the Pahole Mid-elevation Nursery in Mokulē‘ia Forest Reserve (656 m elevation) by Oahu Army Natural Resources Staff. Schiedea individuals were derived from distinct wild (founder) population units in the Wai‘anae Mountain Range, O‘ahu, namely ‘Ēkahanui (EKA), Huliwai (HUL), Kalua‘ā (KAL), Makaleha (LEH), Kahanahā‘iki (MMR), Pahole (PAH), and Pālāwai (PAL), Keawapilau (PIL), and four Ko‘olau Mountains populations, Makaua (AKA), Kahana (KNA), Ma‘akua (MAA), and Kaipapa‘u (PAP) (Appendix 1). Leaf tissues were rapidly dehydrated in silica gel and frozen at 80 °C. Genomic DNA was extracted from 6-10 mg dried plant material using a DNeasy Plant Mini Kit (QIAGEN Inc.) following the recommended protocol. Tissues and extracted genomic DNA have been accessioned within the collections of the Pacific Center for Molecular Biodiversity, Bishop Museum (accession numbers 2009.199 and 2011.084) and are stored at 80 °C. Bishop Museum 3 Hawaii Biological Survey Appendix ES-6 Population genetics of Schiedea Species of Conservation Concern on U.S. Army Lands Oahu Population genetics of Schiedea Microsatellite analysis DNA samples were analyzed in multiplexed reactions that incorporated the fluorescent labeling of forward primers during PCR (adapted from Schuelke 2000) following the protocol of Culley et al. (2008). Each microsatellite region was amplified using an unlabelled reverse primer, a forward primer incorporating a unique sequence added to the 5' end, and a third primer composed of this same unique sequence but with a fluorescent label (either WellRED D2, D3 or D4) attached to the 5′ end (Table 1). Primers were developed by Culley et al. (2008) specifically for Schiedea species and had been previously noted to be polymorphic for the three species in this current study. The four unique sequences used were M13(-21) (D4- TGTAAAACGACGGCCAGT), two modifications (M13A: D2-TAGGAGTGCAGCAAGCAT, M13B: D4-CACTGCTTAGAGCGATGC) and T7term (D3-CTAGTTATTGCTCAGCGGT). The forward primer was provided at one-fourth the amount (0.5 μM) of the reverse and fluorescently labeled primers (2 μM). PCR was performed in 10 μL reaction volumes using a QIAGEN Multiplex kit (QIAGEN Inc.) as follows: 5 μL Multiplex PCR Master Mix, 1 μL 10x primer mix, 0.5 μL DNA and 3.5 μL dH2O. Primers were run in one of four sets (Table 1) or individually. Multiplex PCR consisted of 95 °C for 15 min, followed by 30 cycles each of 94 °C for 30 s, 57 °C for 45 s, and 72 °C for 45 s, and then with eight cycles each of 94 °C for 30 s, 53 °C for 45 s, and 72 °C for 45 s. A final extension consisted of 72 °C for 10 min. Product was run on a Beckman Coulter CEQ8000 genetic analysis system with DNA size standard 400.