Gy ~ ee, ZHBS 20% Newsletter of the Hawatian ace Society Volume 36 Numbers 2 In This Issue Genetic variation among Hawaiian cultivars of ‘uala (Ipomoea batatis) by D N. Adams and C. W. 14% C0} 0 (or 5 Pee a ey) Effects of population fragmentation on genetic variation of Haplostachys haplostachya , an endangered Hawaiian mint by W. Loeffler and Caw. Morden: (o...004 242 42 Identification of Dubautia paleata x D. raillardioides hybrids using RAPD markers by D. A. Carino and C. W. Morden nes ie 47 “Sierra Club Legal Defense Fund” changes its name to “Earthjustice Legal Defense Fund”........ 52 Minutes of the Hawaiian Botanical SOCIELY: noe 53 Inside Next Issue..... 54 July, 1997 Lobelia gloria-montis Genetic Variation Among Hawaiian Cultivars of ‘uala Ipomoea batatis) Dawn N. Adams Department of Botany, University of Hawai‘i at Manoa, Honolulu, HI Clifford W. Morden Department of Botany and C.C.R.T., University of Hawai‘i at Manoa, Honolulu, HI ABSTRACT. Randomly Amplified Polymorphic DNA (RAPD) markers produced by five arbitrary 10-mer primers were used to determine the relationship between 12 cultivars of Hawaiian Ipomoea batatas (L.) Lam. Total DNA were extracted from leaves. Data was analyzed with multivariate principal component and cluster analysis. Leaves with a common shape were closely grouped. Yet within each group there still existed some degree of variability. ©The RAPD procedure provided a good estimate of genetic variation even within species J. batatas (L.) Lam., that can be used as a basis for further studies. There are a number of items, such as linguistics, pottery, fish hooks, and tools, that can link cultures to each other. Plants can also be included as one of these artifacts. Genetic studies on kalo, Colocasia esculenta, presented the idea of two different cultivars based upon the amount of chromosomes. The migration route of one cultivar was traced from mainland Asia to Japan, to Indonesia, and onward to New Caledonia (Abbott 1992). In essence, the study not only arranged a migration pattern for the cultivar but also for the people carrying this kalo. Continued on page 39 38 Newsletter of the Hawaiian Botanical Society Botanical Society which was founded in 1924 to... PRESIDENT Wisteria Loeffler . UH Department of Botan ..advance the science of botany ( P y) in all its applications, encourage VICE-PRECIRERNT research in botany in all its Curtis Daehler phases, promote the welfare of its (UH Department of Botany) members and develop the spirit TREASURER of good fellowship and cooper- Ron Fenstemacher ation among them.” (Ho‘okahe Wai Ho‘oilu ‘aina) SECRETARY Any person interested in the plant a Ce Canvey life of the Hawaiian Islands is ea ar eat eligible for membership. Inform- BS le ' : erald Toyomura aan be obtained from the (Sierra Club) ociety at: Alvin Yoshinaga (Ctr. for Conservation Research & Training) c/o Department of Botany COMMITTEES 3190 Maile Way Appointed by the Executive Committee University of Hawai‘i Ne nena Honolulu, HI 96822 Alvin Yoshinaga UH-CCRT NEWSLETTER Cliff Morden, Editor UH-Botany/CCRT Mindy Wilkinson, Assoc. Ed. UH-Botany CONSERVATION MEMBERSHIP Steve Montgomery UH EARTH DAY The Society year is from December 1 OB ee ty ean el through November 30 UNDERGRADUATE GRANTS Priscilla Millen, Chair Leeward CC : Alvin Chock USDA-APHIS-IS (Ret.) Membership Cost per year Eric Enos Individual $10.00 Greg Koob Lyon Arboretum Lisa Stratton UH-Botan Student $5.00 d Family $12.00 SCIENCE FAIR ; See (ee James Kwon UH-Botany Life (individuals only) $180.00 Winona Char Char and Associates - WERT Tim Motley Char and Associates ee er anes oe NATIVE PLANTS pay Clifford Smith, Chair UH-Botany Ken Nagata USDA, APHIS PPQ Evangelina Funk Botanical Consultants John Obata Bishop Museum Art Medeiros Haleakala Nat’) Park Volume 36 (2) 39 Continued from page 37 Using Randomly Amplified Polymorphic DNA (RAPD), genetic relationships between cultivars can be analyzed and, similar to the information gathered form the kalo experiment, can be used to trace the movement of people and cultivar. The plant of this particular study is Ipomoea batatas (L.) Lam., or sweetpotato of Convolvulaceae. There are three different groups of sweetpotato, the Kumara, Batata, and Comote. All three of these groups are said to have origins in either central or south America. The Comote line was distributed to the Philippines, - Batata was taken from __ the Bahamas to Europe, and the Kumara was believed to enter the Pacific. The sweetpotato, or ‘uala in Hawaiian, was found all throughout the Pacific. Depending on the culture, it was a minor crop such as on the island of Futuna, where it was used as an afternoon snack for women (Yen 1974). In other places such as the eight major Hawaiian islands it was a major crop, second only to kalo. Being a hardy plant, ‘uala was very important in the drier leeward ahupua’as, land divisions that usually stretched from valley ridges to the ocean. Tubers and leaves were cooked in an imu, an underground oven. Tubers were sometimes preserved by drying after cooking and taken on fishing trips, as done by the fishermen of Ka’a (Abbott 1992). At other times the tubers were pounded into poi ‘uala. Some parts of the plant were used medicinally. Old leaves and vines provided padding under floor mats (Wagner et al 1990). The method of propagation was mainly through slips or cuttings. By placing the slips in a damp material such as ti (Cordyline fruticosa) leaves, roots were initiated to grow. After saying prayers to the gods and working the soil, about two or three slips were placed into mounds, or pu'e. This meant a limited number of genetic variability between crops. Corresponding mutation rates that cause changes in morphological expression were estimated to be low (Hernandez et al. 1964). As recorded by Handy, there were more than 230 cultivars in the Hawaiian Islands. The cultivars were classified by Hawaiians according to leaf shape, leaf color (top and bottom), vein color, stem color, tuber color (inside and out), and tuber shape. This study is a preliminary analysis on Hawaiian I. batatas ,and coupled with existing data, such as morphological characteristics and genetic information of other sweetpotato cultivars in Oceania, could result in a better understanding of the cultivar's distributions throughout Polynesia. The step taken here is to provide base-line data on genetic variability and to establish relationships between varieties of Hawalian sweetpotatos. Randomly Amplified Polymorphic DNA (RAPD) procedure was used. By using small, random sequence primers to start the DNA amplification process, Table 1. Ipomoea batatas varieties from the Amy B. H. Greenwell Ethnobotanical Garden. Records of the Garden note that the names may not be the original names. Row 1 is located makai and rows 2 through 4 progressively mauka. Sample Variety Accession Garden Leaf Number Name Number Location Shape 1 ‘uala 95.031 Mound 1 cordate 2 ‘uala hehe nui 95.036 Mound 2 cordate 3 ‘uala hua moa 95.035 Mound 3 cordate 4 ‘uala palaa 95.020 Mound 4 lobed 3) ‘uala kilapaki 95.037 Mound 5 lobed 6 ‘uala papamu 95.019 Mound 6 lobed 7 ‘uala 95.039 Mound 7 lobed 8 ‘uala pulaa Mound 8 lobed 9 ‘uala Row 1 cordate/ lobed 10 ‘uala Row 2 lobed 11 ‘uala Row 3 cordate 12 ‘uala Row 4 lobed 40 Newsletter of the Hawaiian Botanical Society the amplified product may show differences from one individual within a population to the next. This method has an advantage of being able to sample a large number of individuals very rapidly, is relatively inexpensive, and provides a good estimate of the genetic variation among the individuals. The major disadvantage is that statistical approaches to analyzing the data are not straightforward and are still being developed and tested. MATERIALS AND METHODS Leaves of cultivars were obtained from the Amy B.H. Greenwell Ethnobotanical Garden located at Captain Cook, Hawai‘i (Table 1). The material were placed in plastic baggies and stored at 4°C until ready for use. Isolation of total cellular’ DNA and purification was done following the protocol of Morden et al. (1996). Leaves were washed and approximately 1.5 g weighed out. A grinding buffer consisting of 2% CTAB, 100mM Tris- HCL (pH 8), 1.4 NaCl, 20 mM EDTA, 0.2% beta-mercaptoethanol, and dH,O was aliquoted into tubes and placed in a water bath, 60°-65°C. Samples were then ground in a pre-heated mortar with the buffer and some sea sand. Next, specimens were incubated in a water bath at 60°-65°C between 15-60 minutes. The material was then extracted once with SEVAG (24:1, chloroform isoamyl alcohol) and centrifuged at 3000rpm for 10 minutes (Sorvall RT 6000D). The top liquid phase was transferred into anew tube. Using 2/3 volume of 2-propanol, DNA was precipitated. Again the samples were spun at 3000 rpm for 5 minutes to collect the precipitate. The liquid portion was poured off and the precipitate allowed to dry for a few minutes. A wash buffer of 76% ethanol, 10 mM ammonium acetate, and dH,0 was added to the pellet, which was then incubated for 10-60 minutes. Samples were spun, liquid poured off, and pellet dried for 5 minutes. For DNA purification, the pellet was then resuspended in 4 ml of TE and added to 3.9 g of cesium chloride and 50ml of ethidium. bromide. Ultracentrifugation using gradient density allowed DNA bands to be pulled. Ethidium bromide was removed with extractions of water saturated isobutanol and cesium chloride with at least the dialysis in TE. DNA was then stored at 4°C, DNA concentrations were obtained by Shimadzu UV-1201 Spectrophotometer. Table 2. Synthetic deoxyribonucleotides used as primers for amplification of sweetpotato DNA. RAPD Nucleotide # of Amplified Primer Sequence Fragments OPE-16 GGTGACTGTG 5 OPG-05 GTGAGACGGA 10 OPG-08 TCACGTCCAC 5 OPG-09 CTGACGTCAC 5 OPH-03 AGACGTCCAC g Primers were screened, and the best five were used for amplification (Table 2). PCR was carried out using a 25 pl reaction consisting of dH,0, primer in 100 mM stock, 1.25 mM of dNTPs, 10X PCR buffer, 25 mM MgCl, TAQ Polymerase (the latter three supplied by Promega), and approximately 12.5 mg/ml. Reactions were then run on a 1% agarose gel. Along with the DNA samples, pBS markers that had fragments between 2950 and 448 base pairs were run to help approximate the sweetpotato DNA fragment lengths. RESULTS AND DISCUSSION RAPD markers ranged in length from 2100 to 390 base pairs depending on the primer and cultivar (Table 2 and Fig. 1). Markers for each cultivar were scored by either a "0" or a "1" , signifying absence or presence, respectively. A total of 25 bands were used for multivariate principal component and cluster analysis using Minitab for Windows Bit 32. Graphing principal component 4 versus principal component 1, resulted in cultivars being partially arranged according to leaf shape (Fig. 2). Principal component four was mainly responsible for the separation of leaf types. Although similar leaf types were grouped together, there was still variability within each group possibly do to other factors that were not Fig. 1. RAPD markers on 1% agarose gel. pBS=size marker used to determine size of fragments. 12.3 4 5 6 7 8 9 10 11 12 pBS 932 2.96 KIO Volume 36 (2) 4] considered in this project, such as_ leaf pubescence, stem color, tuber color, etc. These other factors might have been considered in principal component one. Cordate and lobed leaves were again placed together when doing a cluster analysis (Fig. 3). The oddly shaped leaf of ‘uala sample 9 also appears to be the furthest related to everyone, this was also seen in the principal component graph. ‘Uala hua moa, or sample 3, in the cluster analysis is not grouped with the cordate leaves but rather more closely with sample 9. This could also be in part to other characteristics. Two ways in which this analysis could be more complete is to use more primers to acquire more RAPD markers and to including other morphological features of the cultivars. Despite those two drawbacks, RAPD analysis proved to be useful even to find variability within species. Fig. 2 and 3. Genetic relationships among varieties of ‘uala. Fig. 2: Multivariate Principal Component Analysis. Fig. 3. Multivariate Cluster Analysis. Principal Cormponent 4 vs. Principal Component 1 (37% variabi tty) $5 45 45 -25 15 05 05 pe Obs ervatio ns ACKNOWLEDGMENTS Special thanks to Alice Mendoza for her guidance; the Botany 662, Vickie Caraway and James Kwon for their helpful insights; Dr. Abbott for instructional support; and The Amy B.H. Greenwell Ethnobotanical Garden for providing the cultivars for this project; and The Waimea Arboretum & Botanical Garden for information and photographs of cultivars. This research was conducted as a course project for Advanced Systematics at in the Dept. of Botany, UH Manoa. LITERATURE CITED Abbott, LA. 1992. La‘au Hawai‘i, traditional Hawaiian uses of plants. Bishop Museum Press, Honolulu, HI. Hernandez, T.P., T. Hernandez, and J.C. Miller. 1964 Frequency of somatic mutation in several Sweet potato varieties. Proc. Amer. Soc. Hort. Sci. 85:430-433. Jarret, R.L., N. Gawel, and A. Whittemore. 1992. Phylogenetic relationships of the Sweetpotato, Ipomoea batatas (L.)Lam. J. Amer. Soc. Hort. Sci. 117(4):633-637. Morden, C., V. Caraway, and T. Motley. 1996. Development of a DNA Library for Native Hawaiian Plants. Pacific Science, 50:324-335. Wagner, W., D. Herbst, and S.H. Sohmer. 1990. Manual of the flowering plants of Hawai'i. Bishop Museum Press, Honolulu, HI. p.555 Yen, D.E. 1974. The sweet potato and oceania, an essay in ethnobotany. B.P. Bishop Museum Bulletin 236. Bishop Museum Press, Honolulu, HI. Ipomoea indica (J. Burm. ) Merr. (from Manual of the Flowering Plants of Hawai‘i) 42 Newsletter of the Hawaiian Botanical Society Effects of Population Fragmentation on Genetic Variation of Haplostachys haplostachya, an endangered Hawaiian Mint Wisteria Loeffler Department of Botany, University of Hawai‘i at Manoa, Honolulu, HI Clifford W. Morden Department of Botany and C.C.R.T., University of Hawai‘i at Manoa, Honolulu, HI ra ABSTRACT. Randomly Amplified Polymorphic DNA (RAPD) are used to examine genetic variability within three sub-populations of the endangered mint H. haplostachya (Gray) St. John on the island of Hawai’i. Number of fixed genetic markers within the sub-populations were analyzed and show a genetic bottleneck within the small, disturbed sub-population at Pu’u_ Leilani. This study gives recommendations for future restoration efforts of H. haplostachya and illustrates the importance of genetic analysis prior to endangered species restoration. Until a recent rediscovery on the Pohakuloa Plateau of Hawai‘i, the endangered mint Haplostachys haplostachya (Gray) St. John (honohono), was thought to be extinct (USFWS). At one time a prevalent species in the mid-elevation (2000-3000 m) dry-forest between Mauna Loa and Mauna Kea on Hawai‘i (Wagner et al. 1990), grazing pressure by feral ungulates, loss of habitat from military activity and conversion of forest to pastures have fragmented its range to small subpopulations. These remaining subpop- ulations are located within the military Pohakuloa Training Area (PTA) on several cinder cones and in the forested region of Kipuka Kalawamauna (USFWS). An endemic genus within the Lamiaceae, Haplostachys once contained five species all thought to share a common ancestor with the genus Phyllostegia (Wagner et al. 1990). Four out of the five documented species are now extinct with the only extant representative, H. haplostachya, federally listed as endangered. Historically known from scattered collections at a low elevation sites on Kaua‘i and Maui, H. haplostachya are now restricted to a single Historical Pre nt D population on the island of Hawai‘i (Fig. 1). = Ca se ay They appear as erect perennial herbs with white, typically lamiaceaeous flowers found in Fig. 1. Historical and Present Day Range of Haplostachys haplostachya. Volume 36 (2) 43 dense terminal racemes (Wagner et al. 1990). Due to the historical rarity of H. haplostachya very little information on its biology is known (Wagner et al. 1990). This study focuses on three distinct subpopulations: Pu‘u Leilani, Pu‘u Ka Pele, and Kipuka Kalawamauna. Until 1991, there had been no efforts to protect any of these subpopulations. At this time, the Army and the State Department of Land and _ Natural Resources (DLNR) came to an agreement that resulted in a four-foot fence being erected around the recently discovered population at the cinder cone Pu‘u Ka Pele. Estimates of the number of individuals found at this cinder cone prior to fencing were about 3000 individuals; the population now has over 10,000 individuals, including numerous juveniles. The sub-population of Kipuka Kalawamauna also contains thousands of individuals and shows much reproductive activity. In contrast, Pu‘u Leilani, a non-fenced cinder cone less than 2 km away, has 14 individuals remaining with no visible evidence of reproduction (either flowering or juveniles). However, even after the erection of fences, there are still several threats to the population. Feral ungulates (primarily goats, sheep and pigs) are commonly found at PTA, and these have been observed browsing the inflorescenses of H. haplostachya (Applet et al 1991a, 1991b), or digging up the soil. Military training poses threats to the plants from trampling during maneuvers as well as dust raised by passing vehicular traffic on the unimproved roads throughout the area. Fires also occur frequently at PTA as a result of various military activities; a 1992 fire destroyed several acres with numerous endangered species in different locations at PTA. As a result of the intense habitat frag- mentation and destruction of H. haplostachya, a once large, continuous population is now broken into small potentially inbreeding subpopulations. Inbreeding depression may be one of the most important genetic consequences of small population size (Lande 1988). The eventual effect of an increase in inbreeding within a population is reduced fitness and potential extinction due to increases in homozygosity, and therefore effectiveness of selection against recessive detrimental alleles (Barrett and Charlesworth 1991; Lande 1994; Lynch et al. 1995). It is the purpose of this study to investigate the genetic diversity that remains among sub- populations of H. haplostachya, and determine what affect population fragmentation has had on its genetic structure. The results of this study could have significant impact on recovery efforts being planned for this and other species with small fragmented populations. A method known as Randomly Amplified Polymorphic DNA (RAPD) was chosen in order to determine the relative amount of genetic variation within and among the subpopulations of H. haplostachya. This method, introduced by Williams et al. (1990), uses single primers of arbitrary nucleotide sequence (8-10 base pairs in length) to amplify a set of fragments of nuclear DNA. Amplification products (usually 2-10 fragments per primer) are then visualized by electrophoresis in ethidium bromide-stained agarose gels. Polymorphisms among _ the amplification products are common and can be useful as dominant genetic markers inherited in a mendelian fashion (Williams et al. 1990; Howland and Arnau 1991; Hadrys et al. 1992; Roderick 1996). In the few years since the potential of this procedure in population’ studies was recognized, the RAPDs method has become a popular method of genetic mapping in plant and animal breeding studies (Dunemann et al. 1994, Rieseberg et al. 1994), discerning hybrid origin (Arnold et al. 1991; Rieseberg and Gerber 1994), determining rates of outcrossing in plant populations (Fritsch and Rieseberg 1992), and detection of gene introgression (Amold et al. 1991; Waugh et al. 1992; Orozco- Castillo et al. MATERIALS AND METHODS Plant Collection. Under _ section 10(a)(1)(A) of the Endangered Species Act, 16 U.S.C. 1531 et seq. a collecting permit (#PRT- 811049) was issued by the U.S. Department of the Interior and leaf tissue from plants of H. haplostachya was collected from _ three subpopulations at PTA. A single leaf was removed from plants spaced every 10 m along a vertical transect from the top to bottom of Pu‘u Ka Pele (total of 16 plants sampled). 1000 Abundant Flowering, Abundant Flowering, Many Juveniles Many Juveniles 29 13 79 93 “Number of individuals estimated at time cindercone was fenced. Subpopulation now estimated at ca 10,000 individuals. 46 Newsletter of the Hawaiian Botanical Society ACKNOWLEDMENTS This research was conducted as a course project for Advanced Systematics in the Dept. of Botany, UH Manoa. LITERATURE CITED Applet, G. H., R. D. Laven, and R. B. Shaw. 1991. A preliminary status report on Haplostachys haplostachya (A. Gray) St. John. an endangered mint restricted to the U. S. Army's Pohakuloa Training Area, Hawaii. Colorado State University, Fort Collins, Colorado. 12 pp. Arnold, M. L., C. M. Buckner, and J. J. Robinson. 1991. Pollen-mediated intro- gression and hybrid speciation in Lousiana irises. Proc. Natl. Acad. Sci. 88: 1398-1402. Barrett, S.C.H. and D. Charlesworth. Effects of a Change in the Level of Inbreeding on the Genetic Load. Nature. 352: 522-524. Dunemann, F., Kahnau, R. and H. Schmidt. 1994. Genetic Relationships in Malus Evaluated by RAPD ‘Fingerprinting’ of Cultivars and Wild Species. Plant Breeding. 113: 150-159. Fritsch, P. and L. H. Rieseberg. 1992. High outcrossing rates maintain male and herma- phrodite individuals in populations of the flowering plant Datisca glomerata. Nature. 359: 633-636. Hadrys, H., M. Balick, and B. Schierwater. 1992. Applications of random amplified poly- morphic DNA (RAPD) in molecular ecology. Molecular Ecology. 1: 55-63. Howland, D. and J. Arnau. 1991. RAPDs: Random Amplified Polymophic DNAs. Genes and Ecology. Blackwell Sci. Pub. Oxford. Lande, R. 1988. Genetics and demography in biological conservation. Science 242:1455- 1460. Lande, R. 1994. Risk of Population Extinction from Fixation of New Deleterious Mutations. Evolution. 48(5): 1460-1469. Lynch, M., J. Conery, and R. Burger. 1995. Mutation Accumulation and the Extinction of Small Populations. The American Naturalist. 146(4): 489-518. Morden, C. W., V. Caraway, and T. J. Motley. 1996. Development of a DNA Library for Native Hawaiian Plants. Pac. Sci. 50(3): 324- 335. Nei, M. and Li, W. H. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA 76: 5269-5273. Orozco-Castillo, C., K.J. Chalmers, R. Waugh, and W. Powell. 1994. Detection of genetic diversity and selective gene introgression in coffee using RAPD Markers. Theor. Appl. Genet. 87: 934-940. Rieseberg, L. H. and D. Gerber. 1995. Hybridization in the Catalina Island Mountain Mahogany (Cercocarpus traskiae): | RAPD Evidence. Conservation Biology. 9(1): 199- 203. Rieseberg, L. H., G. J. Allan, Y.Bui, J. Greene, P. L. Morrell, S. C. Spencer, and B. O’Brien. 1994. Genetic Fingerprinting of Various Native California Cultivars. Madrono. 41(1): 30-38. Roderick, G. K. 1996. Geographic Structure of Insect Populations: Gene Flow, Phylo- geography, and Their Uses. Annu. Rev. Entomol. 41: 325-52. United States Fish and Wildlife Service. Recovery Plan for Haplostachys haplostachya and Phyllostegia sp. Wagner, W. L.; Herbst, D. R.; Sohmer, S. H. 1990. Manual of the Flowering Plants of Hawai‘i. B. P. Bish. Mus. Spec. Pub. 83, University of Hawaii Press and Bish. Mus. Press. Honolulu. Waugh, R., E. Baird, and W. Powell. 1992. The use Of RAPD markers for the detection of gene introgression in potato. Plant Cell Reports. 11: 466-469. Williams, C. E. and D. A. St. Clair. 1993. Phenetic relationships and levels of variability detected by restriction fragment length polymorphisms and randomly amplified polymorphic DNA analysis of cultivated and wild accessions of Lycopersicon esculentum. Genome. 36: 619-629. Williams, J.G.K., A.R. Kubelik, K. J. Livak, J. A. Rafalski, and S.V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18(22): 6531-6535. Volume 36 (2) 47 Identification of Dubautia paleata x raillardioides Hybrids Using RAPD Markers Debbie Ann M. Carino Department of Botany, University of Hawai‘i at Manoa, Honolulu, HI Clifford W. Morden Department of Botany and C.C.R.T., University of Hawai‘i at Manoa, Honolulu, HI ABSTRACT... Hybridization appears to be a common phenomenon in the Hawaiian silversword alliance, where 35 naturally occurring hybrid combinations have been reported between species in the genera Agyroxiphium, Wilkesia, and Dubautia. MHybridization has been suspected to occur between Dubautia paleata and D. raillardioides based on _ the occurrence of morphological intermediates between the two taxa; however, no research has yet been done to document hybridization between them. Random amplified polymorphic DNA (RAPD) markers were used to determine if 6 of these morphologically intermediate plants are, in fact, hybrids. Five primers were used to generate 64 markers, 5 of which were specific for D. raillardioides, and 4 of which were specific to D. paleata. All 4 bands specific to D. paleata and 2 bands specific to D. raillardioides were present in the hybrids. In PCA and cluster analysis, the hybrids cluster between D. paleata and D. raillardioides, although the hybrids appear more closely aligned to D. paleata. This data suggest that the putative hybrids are hybrids between D. paleata and D. raillardioides, and show introgression with D. paleata. The evolutionary importance of through recombination, and to form new hybridization is illustrated by the estimation that species through stabilization of the hybrid perhaps 70% of all flowering plants species population (Rieseberg 1995; Carr 1995). have arisen through hybridization (Whitham et Hybrids may be able to exploit new habitats al. 1991). Recent studies are addressing new unoccupied by either parent species (Lewontin ideas and approaches concerning its and Birch 1966; Arnold 1994), mechanisms and consequences (Rieseberg In geologically unstable and geographically 1995). Perhaps the most well-studied hybrid isolated floras such as in Hawaii, hybridization plant system is the Louisiana iris complex. may be critical in the maintenance of colonizing Amold and his colleagues have found the F, species, and in the exploitation of new habitats hybrid formation is Louisiana irises is rare (Carr 1995). Founding populations have a (Armold 1993); intraspecific pollen is a superior limited gene pool, and recombination through competitor to interspecific pollen and has a_ hybridization represents a way to maximize greater capacity to produce mature seeds genetic variation in small populations (Arnold et al. 1993), and the hybrid population (Rieseberg 1995; Carr 1995). Many species in represents advanced generations that have a_ the Hawaiian flora are, in fact, noted for their large degree of introgression (Amold et al. lack in internal barriers to hybridization (Gillet 1990; Cruzan and Amold 1993). Studies 1972; Carr 1995). The greater degree of which assess key characteristics of hybrid genetic variation that hybridization affords populations will lead to a better understanding populations may also facilitate a more rapid of evolutionary consequences of hybridization. advance into new habitats (Gillet 1972; Cruzan Probable roles of hybridization may be to and Amold 1993). Because varying habitats increase the genetic variability in a population are closely juxtaposed in Hawaii, the 47 48 probability that a hybrid may reach a habitat where it might become stabilized is increased (Carr 1995). The occurrence of hybridization is well- documented in Hawai‘i (Smith et al. 1962; Gillet 1972; Carr 1995). Notable examples of hybridization in the Hawaiian flora include Scaevola, Cyrtandra, and the silversword alliance. For example, 67 natural hybrid combinations have been found between Cyrtandra species (Smith et al. 1962). Within the silversword alliance, 35 naturally occurring hybrid combinations have been documented (Carr 1985). Furthermore, intergeneric hybridization between Argyroxiphium sandwincense ssp. macrocephalum and Dubautia menziesii occurs (Carr 1995). The morphological and ecological diversity of the silversword alliance stem from a single introduction (Carr and Kyhos 1981), and have resulted in a radiation comprising 28 endemic species in the genera Argyroxiphium, Wilkesia, and Dubautia (Carr 1985). Species in the silversword alliance are found in habitats as diverse as bogs, wet forests, and alpine deserts (Carr 1985). Dubautia is the most speciose genus in the alliance, with 21 _ species distributed across 6 of the main islands, the majority of species being single island endemics (Baldwin and Robichaux 1995). In Dubautia, hybridization is a frequent occurrence (Carr and Kyhos 1981). Carr and Kyhos (1981) studied 7 different Dubautia hybrid crosses for cytogenetic analysis. Carr (1985) lists a remarkable number of collected hybrids for several Dubautia species. For example, D. paleata is known to hybridize with D. imbricata spp. acrononaea, D. laxa spp. laxa, and D. walalealae (Carr 1985). D. paleata, a Kauai endemic, occurs in open boggy areas of the Alakai Swamp at elevations ranging from 1100 to 1550 m (Wagner et al. 1990). ). D. paleata is a small dense shrub, 0.3 to 2.5 cm in length. Its leaves are pubescent, opposite or ternate, lanceolate, and 3 to 20 cm in length (Wagner et al. 1990). D. raillardioides, a closely related species (Baldwin and Robichaux 1995), and is also present in the Alakai Swamp. However, its habitat is in wet forests at the margins of bogs, at elevations from 600 to 1375 m (Wagner et al. 1990). D. raillardioides is an understory shrub, up to 3 m tall, with a lax, partly decumbent branching shape. Its leaves are ternate, glabrous, lanceolate, 9-25 cm in length, with margins 48 Newsletter of the Hawaiian Botanical Society conspicuously toothed form the apex to halfway to the base (Wagner et al. 1990). In marginal areas between bogs and wet forests in the Alakai Swamp region, large leafed plants with fewer trichomes than typical for D. paleata are observed (Wagner et al. 1990; Roderick, pers. comm.). Because morphology of these plants seem intermediate between D. paleata and D. raillardioides, these individuals appear to be hybrids between these two species. Hybrids between Dubautia species are commonly intermediate in the foliar characters (Carr and Kyhos 1981; Kim 1987), although this is not necessarily true of hybrids in general (Rieseberg 1995). Also, this marginal habitat appears to be intermediate between the habitats of D. paleata and D. raillardioides; thus, intuitively it seems possible that hybrids may occur here. Here, we attempt to clarify the identity of these individuals using RAPD markers. These ‘morphological intermediates’ may either be of hybrid origin, or represent larger morphologies of D. paleata. This determination is an important first step in assessing the dynamics between these two Dubautia species. Genetic analysis using RAPD markers have been useful in determining hybridization in several taxa, including Louisiana irises (Cruzan and Armold 1993), Catalina Island mountain mahogany (Rieseberg and Gerber 1995), and xMargyrancaena skottsbergii (Crawford et al. 1993). The use of RAPD markers is effective because many markers can be generated, and little DNA is required (Crawford et al. 1993). MATERIALS AND METHODS Plant material. Plant samples were collected from the Alakai Swamp on the summit plateau of the island of Kauai, Hawaii, at approximately 1250 m in elevation. Leaves of 10 plants suspected to be of hybrid origin were collected from a putative hybrid zone located close to the end of the boardwalk trail, where the trail begins to loop northward. This area is a transition zone between the bog and wet forest. These tentative hybrids appeared to have vegetative parts that are of intermediate size and pubescence between D. paleata and D. raillardioides. 7 samples of D. paleata were collected along the boardwalk trail in the flat, boggy areas of the swamp, at least 10 m away from the putative hybrid zone. 9 samples of Volume 36 (2) 49 Table 1. Species-specific bands (in kb) present D. paleata and D. raillardioides, and their occurrence in putative hybrid plants. Primer D. pal. __D. pal. xrail. _D. rail. OPG-6 = ef 1.55 = 1.40 1.40 0.56 0.56 = OPG-9 0.96 0.96 2 OPG-11 as = a4 1.4 1.4 = OPG12 2.6 2.6 - _ 1.4 1.4 OPI-13 nes es 0.55 D. raillardioides were collected at the beginning of the boardwalk trail at the margin of wet forest. DNA _ Extraction and Amplification. Total nuclear DNA was extracted using a method based on Doyle and Doyle’s (1987) modification of the CTAB isolation procedure and is described in Morden et al. (1996). 10-mer random primers (Operon) were used for amplification of Dubautia DNA. RAPD amplifications were carried out in 25 yl reaction mixtures containing 1x Fisher buffer, 2.0 mM MgCl,, 0.1 mM each of dATP, dTTP, dCTP, and dGTP, 8uM primer, 0.75 units of Taq DNA polymerase, and approximately 25 ng of DNA. The reaction mixture was overlaid with approximately 25 wl of mineral oil. Amplification was performed in a Hybaid OmniGene thermocycler with the following conditions: one cycle of 94 ‘C for 3 min., 35°C for 30 sec., 72°C for 2 min.; 43 cycles of 94°C for 45 sec., 35°C for 30 sec., 72°C for 2 min.; a final cycle of 94°C for 45 sec., 35C for 30 sec. 72C for 6 min., for one cycle. Amplification products were separated on 1.5% agarose gels. Ethidium bromide was included in the gel and electrophoresis buffer (0.5x TBE), and products were visualized and photographed under ultraviolet light. Data analysis. For each individual, bands from all markers were scored as either absent (QO) or present(1). The amplification products 49 were inspected for bands which were restricted to either D. paleata or D. raillardioides and also found in the putative hybrids. Multivariate Statistics of principal components analysis (PCA) and cluster analysis were performed for further data analysis. Minitab _ statistical software was used for multivariate analyses. RESULTS Seven individuals of D. paleata, six individuals of D. raillardioides, and six putative hybrids consistently amplified with 5 primers: G6, G9, G11, G12, and 113. The five primers produced a total of 64 bands, of which 9 were relatively species-specific (Table 1). 5 of these bands were restricted to D. raillardioides, and the other 4 were restricted to D. paleata. The putative hybrids exhibited all four markers restricted to D. paleata, and 2 markers restricted to D. raillardioides. PCA shows that D. raillardioides is a distinct population from D. paleata and the putative hybrids (figs. 1 and 2). Individuals of D. paleata appear to be highly variable in relation to each other; thus, loosely clustered. The putative hybrids appear to be somewhat clustered together and interspersed in the D. paleata,; however, a weak segregation between the putative hybrids and a majority of D. paleata individuals is evident along the x-axis (PCA 1). Also, the putative hybrids segregate between D. raillardioides and the edge of D._ paleata, indicating that D. raillardioides is more similar to the putative hybrids than it is to D. paleata. For the cluster analysis, 28 out of the 64 markers that accounted for most of the variation as determined from PCA were used. These results also indicate that D. raillardioides form a distinct cluster from D. paleata and the putative hybrids (fig. 3). Individuals of D. paleata and the putative hybrids are more closely aligned. However, it is clearly evident that the putative hybrids form a distinct group from D. paleata, and that the putative hybrids again cluster in between D. raillardioides and D._paleata, indicating that the putative hybrids are genetically intermediate between these two species. DISCUSSION Based on this preliminary data, the putative hybrids identified using morphological characteristics appear to be of hybrid origin. The putative hybrids contain bands which were specific to both parental species, indicating that 50 Newsletter of the Hawaiian Botanical Society the hybrids contain genetic material from both species. PCA and cluster analysis also confirm that the pututive hybrids are intermediate between D. paleata and D. raillardioides. Because the hybrids display more markers specific to that of D. paleata, and that in PCA and cluster analysis, the hybrids are more closely aligned to D. paleata, the individual hybrids sampled may represent advanced generations which show introgression into D. paleata. The greater degree of separation between D. raillardioides and the hybrids, than between D. paleata and the hybrids shown in the PCA and cluster analysis is not surprising given the spatial distribution of the populations sampled. The populations of D. paleata and the hybrids which were sampled were within 10 m to 50 m of each other. The population of D. raillardiocides that was sampled = was approximately 1.6 km from the hybrid zone. Thus, one would expect to see a greater degree of similarity between the samples of D. paleata Principal Component 2 5 3 4 1 3 5 Principal Component 1 Principal Component 3 bgbobpe bt osenvoauna Principal Component 1 Figs. 1 and 2. Principal components analysis of D. paleata (0), D. raillardioides (x), and hybrids (+). 1. PCA 1 and PCA 2 accounting for 40.3% of the total variation. 2. PCA 1 and PCA 3 accounting for 33.5% of the total variation. and the hybrids than between D. raillardioides and the hybrids. If samples of D. raillardioides were taken from closer to the hybrid zone, then perhaps the data would show a greater degree of similarity between D. raillardioides and the hybrids. A population of D. raillardioides within 10 m of the hybrid zone was not found in a cursory search (C. Ewing, per. comm.). However, this search was not thorough, and the intertwining habit of D. raillardioides, along with the topography of the region would have made any individuals hard to find. Because of the small number of hybrids and parental species sampled, and the small number of markers generated, it is not possible to make any detailed assessment of the population dynamics found within this group in this study. However, the great degree of similarity between the hybrids and D. paleata does suggest that the hybrids are not F, hybrids, and that this population is introgressing with D. paleata. In order to more fully assess the characteristics of this hybrid zone, several studies may be undertaken to find a) the population of D. raillardioides that contribute to the hybrid zone b) the frequency of F, hybrid formation and c) the degree of recombination that occurs between D. paleata and D. raillardioides due to hybridization. Given the phylogenetic closeness of D. paleata and D. raillardioides, the close juxtaposition of their habitats in the Alakai Swamp, and D. paleata’s propensity to hybridize, it is not surprising that these two taxa have hybridized in at least one region of the Alakai Swamp. This study offers another example of the lack of genetic barriers within the Hawaiian flora. % Similarity Wh 100 — PP PP UP UP CE bee P Be oe aR Re Re RR Fig. 3. Cluster analysis of D. paleata (P), D. raillardioides (R), and putative hybrids (H). 50 Volume 36 (2) 51 ACKNOWLEDGMENTS Our thanks to Jessica Garb and Curtis Ewing for the field teamwork in the EECB field class which lead to the collection of the samples used in this project; Wisteria Loeffler for guidance in the lab and data analysis; Vickie Caraway for help selecting primers, and the Ecology, Evolution, and Conservation Biology (EECB) program for travel funds to Kaua‘i. This research was conducted as a course project for Advanced Systematics in the Dept. of Botany, UH Manoa. LITERATURE CITED Arnold ML (1993) Rarity of hybrid formation and introgression in Louisiana Iris. Plant Genetics Newsletter 9: 14-17. Arnold ML (1994) Natural hybridization and Louisiana Irises: Defining a major factor in plant evolution. BioScience 44: 141-147. Amold ML, Hamrick JL, Bennett BD (1990) Allozyme variation in Louisiana irises: a test for introgression and hybrid _ speciation. Heredity 65: 297-306. Amold ML, Hamrick JL, Bennett BD (1993) Interspecific pollen competition and reproductive isolation in Iris. J. Heredity 84: 13-16. Baldwin BG, Robichaux RH (1995) Historical biogeography and ecology of the Hawaiian Silversword alliance (Asteraceae): New molecular phylogenetic perspectives. In Hawaiian Biogeography: Evolution on a hot spot archipelago, WL Wagner and VA Funk (eds.). Smithsonian Institution Press, Washington. pp. 259-287. Carr GD (1985) Monograph of the Hawaiian Madiinae (Asteraceae): Argyroxiphium, Dubautia, and Wilkesia. Allertonia 4: 1-123. Carr GD (1995) A fully fertile intergeneric hybrid derivative from Argyroxiphium sandwincense spp. macrocephalum x Dubautia menziesii (Asteraceae) and _ its relevance to plant evolution in the Hawaiian Islands. Amer. J. Bot. 82: 1574-1581. Carr GD, Kyhos DW (1981) Adaptive radiation in the Hawaiian silversword alliance (Compositae-Madiinae): Cytogenetics of spontaneous hybrids. Evolution 35: 543- 550. Crawford DJ, Brauner S, Cosner MB, Stuessy TF (1993) Use of RAPD markers to document the origin of the intergeneric hybrid xMargyrancaena skottsbergii (Rosaceae) on the Juan Fernandez Islands. Amer. J. Bot. 80: 89-92. Cruzan MB, Arnold ML (1993) Ecological and genetic associations in an iris hybridzone. Evolution 47: 1432-1445. Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Photochem. Bull. 19: 11-15. Gillet GW (1972) The role of hybridization in the evolution of the Hawaiian flora: Jn Taxonomy, Phytogeography and Evolution, DH Valentine (ed.). Academic Press, New York. pp. 205-219. Kim I (1987) Comparative anatomy of some parents and hybrids of the HawaiianMadiinae (Asteraceae). Amer. J. Bot. 74: 1224-1238. Lewontin RC, Birch LC (1966) Hybridization as a source of variation for adaptation to new environments. Evolution 20:315-336. Morden CW, Caraway V, Motley TJ (1996) Development of a DNA library for native Hawaiian plants. Pac. Sci. 50: 324-335. Rieseberg LH (1995) The role of hybridization in evolution: old wine in new skins. Amer. J. Bot. 82: 944-953. Rieseberg LH, Gerber D (1995) Hybridization in the Catalina Island mountain mahogany (Cercocarpus traskiae): RAPD evidence. Cons. Biol. 9: 199-203. Wagner WL, Herbst DR, Sohmer SH (1990) Manual of the flowering plants of Hawaii. University of Hawaii Press. Honolulu, HI. Whitham TG, Morrow PA, Potts BM (1991) Conservation of hybrid plants. Science 254: 779-780. a e Dubautia raillardioides Hillebr. (From Manual of the Flowering Plants of Hawai‘i) y a 52 Newsletter of the Hawaiian Botanical Society ‘Sierra Club Legal Defense Fund” Changes Name to “Earthjustice Legal Defense Fund” On June 14, 1997, the Sierra Club Legal Defense Fund announced that it is changing its name to Earthjustice Legal Defense Fund, effective immediately. The decision was made by unanimous vote of the organization’s Board of Trustees meeting in Tiburon, California, on June 14, Earthjustice Legal Defense Fund is a non- profit, public interest, environmental law firm that employs approximately 50 attorneys in nine offices across the country. While its name is changing, the Legal Defense Fund’s mission of protecting people and resources by enforcing and strengthening our environmental laws continues as strong as ever. The group has its headquarters in San Francisco and field offices in Juneau, Honolulu, Seattle, Denver, New Orleans, Tallahassee, Washington, D.C., and Bozeman, Montana. The Mid-Pacific office, located in Honolulu, opened in 1988 with a grant from the MacArthur Foundation. The Fund has successfully litigated matters under state and federal laws, including the Endangered Species Act, Clean Water Act, National Environmental Policy Act, State Water Code, and Hawai'i's “Sunshine” Law. In addition to legal services to the public, which are provided free of charge, the Legal Defense Fund assists its clients in community organizing, lobbying, and media relations. Since 1995 the Mid-Pacific office has also sponsored the Ahupua‘a Action Alliance, a statewide coalition of over 65 environmental and Hawaiian organizations, which was created as part of the Legal Defense Fund’s Marine Biodiversity Project. The Legal Defense Fund represented the Hawaiian Botanical Society in litigation that resulted in the listing of over 180 candidate Hawaiian plants as threatened or endangered. Currently, the Legal Defense Fund represents the Hawaiian Botanical Society, Conservation Council for Hawai‘i, and Sierra Club in a legal action to compel the U.S. Fish and Wildlife Service to designate critical habitat for these plants, as required by federal law. “We are proud to continue to represent grass-root efforts to protect Hawai‘i’s special environment and culture.” said Paul Achitoff, managing attorney for the Mid-Pacific of fice. In addition to Achitoff, the current staff in Hawai‘i include associate attorney David Henkin, resource analyst Marjorie Ziegler, Ahupua‘a Action Alliance coordinator Lynette Cruz, part- time resource analyst/Alliance staff Kat Brady, office manager Kim Ramos, litigation secretary Everett Ching, secretary/receptionist Linda Shapin, and office assistant Karen Miles. Capparis sandwichiana DC (From Manual of the Flowering Plants of Hawai‘i) Volume 36 (2) 53 Minutes of the Hawaiian Botanical Society April Meeting e The April 7 meeting of the Hawaiian Botanical Society was called to order by Wisteria Loeffler, President. e The minutes were approved as read. Ron Fenstamacher ¢ Treasurer’s Report. read the treasurer’s report. ¢ Membership Report. Alvin Yoshinaga, membership. chair, announced five new members. ¢ Old Business ¢ Botanical Society’s garage sale netting $430 and the announcement of the Science Fair winners. ¢ New Business e The Earth Day chair, Orlo Steele, asked = 7 GELS NYE RSD PR. 47k for volunteers to help staff the society’s exhibit. e The annual plant raffle for the May meeting was announced. ¢ The April hike is combined with a Miconia search with Pat Conant as trip leader. ¢ The Plant of the Month talk was Tahitian Flora Endangered by Miconia calvescens, given by Jean-Yves Meyer, Minister of Health and Research at Tahiti. ¢ Guest Speaker. The speaker for April was Kenneth Nagata of US Department of Agriculture, talking about the USDA International Pre-Clearance Program: Dutch Flower Bulb Inspections. Rubus macraei A. Gray 54 Newsletter of the Hawaiian Botanical Society Inside Next Issue Volume 36 (3) ¢ A Preliminary Study on the Origin of the Hawaiian Endemic Genus Hibiscadelphus (Malvaceae). D. Weniger and C. W. Morden ¢ Common Native Hawaiian Plants Worthy of Cultivation. J. K. Obata ¢ Growing Native Hawaiian Plants. J. K. Obata Do you have something you would like to contribute to the Newsletter? All contributions are welcome. They may be technical articles related to on-going research, comments about current events, or field observations you may have made from a recent expedition. Contributions may be sent to the Newsletter Editor via manuscript, disc (please provide hard-copy also), or E-mail at: Cliff Morden Department of Botany, 3190 Maile Way University of Hawai‘i at Manoa Honolulu, HI 96822 E-mail: cmorden@hawaii.edu fax: 808-956-3923 Abutilon menziesii Seem. and Abutilon sandwicense (Degener) Christoph. (From Manual of the Flowering Plants of Hawai‘i) 54 Volume 36 (2) ff) Ny i Sh ay y Ni Sy 1 Y WN ; PN SV r/ iY Fal) —> SS \) >. \— ss i NU Wa \), Nj Asplenium contiguum Kaulf. 2D 55 SMITHSONIAN INSTITUTION LIBRARIES 7349 OWSLETTER OF THE HAWAIIAN BOTANICAL SOCIETY +) DEPARTMENT OF BOTANY OIVERSITY OF HAWAI'I AT MANOA ©)0 MAILE WAY eo NOLULU, HI 96822 3 90 a inn Inst. Libraries Acquisitions Srvcs, NHB 25 10th St x Constitution Ave NW Washington, DC 20560 S46 Jom PN ti a5 8 9 \ »\A ¥ ys Reg