Sandalwood is a hemiparasitic tree of the genus Santalum L. (Santalaceae). Two lineages of Hawaiian sandalwoods are believed to have originated from the South Pacific. A red flowered clade comprised of Santalum pyrularium A. Gray, Santalum freycinetianum Gaudich., and Santalum haleakalae Hillebr. This clade is most closely related to species of Santalum L. from French Polynesia and Ogasawara Islands. The white flowered clade composed of Santalum paniculatum Hook. & Arn. and Santalum ellipticum Gaudich. are most closely related to species of Australasian origin. Santalum involutum H. St. John is a federally listed endangered species endemic to Kauai suspected of an ancient hybrid origin. The introduced Indian sandalwood (Santalum album L.) readily hybridizes with the coastal native S. ellipticum Gaudich. These hybrids appear to spread readily and potentially could pose a threat to indigenous members of the genus where their ranges intersect.
As part of his PhD research in the Botany Department at the University of Hawai‘i at Mānoa, Solomon Champion is studying the genetics of native Hawaiian sandalwood. Fifteen putative populations of Santalum paniculatum from around Hawai’i Island have been analyzed here to ascertain relatedness and potential gene flow between them. A total of 66 individuals (n = 66) from wild populations and nurseries were collected for SRAP analysis. Among available individuals, all were collected from populations of Santalum paniculatum without expected introgression from S. ellipticum. Stemmermann (1980), recognized two intergrading varieties of Santalum paniculatum, Santalum paniculatum var. pilgeri (Rock) Stemmerm. and Santalum paniculatum var. paniculatum Hook. & Arn. Otto Degener relates in his Flora Hawaiiensis Plate 100 that the variety paniculatum (as S. paniculatum) is chiefly found in the Kilauea region while variety pilgeri (as Santalum pilgeri) is observed from Kau and Kona. Additional samples of S. paniculatum from the Hawaiian Plant DNA Library (HPDL) were used to supplement this study.
Sequence-Related Amplified Polymorphisms
Purified DNA samples from 66 individual plants were used to investigate genetic diversity among populations using Sequence-related amplified polymorphism (SRAP) marker method. Four species of Santalum from different populations were used to screen over 200 different primer combinations yielding 10 forward and 20 reverse select primers for this study. Eight primer sets producing clear and reproducible bands were selected for further study.
SRAP analyses were conducted using the 20 μl PCR reaction mixture: 5x Green GoTaq PCR Reaction Buffer [50mM Tris-HCL (pH 9.0), 50 mM NaCl, 5mM MgCl2, Promega], 0.25 mg BSA, 0.2 mM dNTPs, 0.5 mM of each forward and reverse primers, 1 unit of Taq GoTaq G2 DNA polymerase, and approximately 15-30 ng of DNA. All reactions were carried out by a MJ Research DNA Thermocycler or Eppendorf thermal Cycler with the following conditions: 5 min of initial denaturation at 94°C, five cycles of three steps: 1 min of denaturation at 94°C, 1 min of annealing at 35°C and 1 min of elongation at 72°C, followed by further 35 cycles with annealing temperature being increased to 50°C, with a final extension on last cycle at 72°C for 5 minutes. Amplified PCR products were mixed with loading dye and separated on 2% agarose gel, stained with EtBr and visualized with a UV light source. Negative control reactions were run without DNA for all PCR amplifications to ensure reaction components were uncontaminated. Each primer combinations PCR quality was carefully examined by the gel bands and repeated if needed with selected samples to confirm the reproducibility of the genetic markers. Size of amplification products was estimated using 100 bp ladder. Final gel products were digitally recorded.
SRAP markers were scored either present (1) or absent (0). The data were entered into a binary matrix and assessed for the level of polymorphism and expected heterozygosity (assumption made that populations were in Hardy-Weinberg equilibrium) across individuals within each population and then averaged across all markers. Expected heterozygosity was calculated for each population in total for each marker as follows: H = 1 – (p² + q²).
Where p is the frequency of the dominant allele and q is the frequency of the null allele. Genetic relationships within and among populations were estimated using the similarity coefficients of Nei and Li (1979) and Principal Coordinates Analysis (PCO) using Gower general similarity coefficients (Gower 1971) were calculated and plotted using MVSP 3.0 (Kovach, 2007). Pairwise similarities were averaged for individuals within and among populations. A Bayesian algorithm, as implemented in STRUCTURE version 2.3.4 (Prichard et al., 2000, Falush et al., 2007), was used to define genetic groups within each species. This algorithm infers genetic discontinuities from individual multilocus genotypes without a priori knowledge of geographic location or taxonomy. The default settings of the program were used, including an admixture model. To determine the most likely number of groups (K) in the data, a series of analyses were performed from K = 1 to 8 (upper limit determined by the number of putative populations plus three following Evanno et al., 2005; 8 selected for Nīhoa, O’ahu, Māui, Moloka’i and Hawai’i plus 3), using a burn-in period and Markov Chain Monte Carlo (MCMC) both set at 100,000 repetitions, with ten iterations per K (Porras-Hurtado et al., 2013). These results were examined using the ∆K method (Pritchard et al., 2000) to identify the most likely number of groups in the data using CLUMPAK (Kopelman et al., 2015).
The principal coordinate analysis of the surveyed populations indicates extensive mixing between Mauna loa and Mauna kea populations of Santalum paniculatum with Kilauea populations appearing intermediate between the two. Hualālai populations from Pu’u Wa’awa’a form a group about Mauna kea samples suggesting a close genetic relationship between the two populations. Santalum paniculatum var. paniculatum as defined by Degener (1934) therefore appears to be intermediate between divergent populations of Mauna loa volcano and the older Mauna kea volcano. Populations sampled from Mauna kea and Hualālai may share a common origin as indicated by close genetic relatedness in the Figure 1.
Bar plots of inferred population assignment when assigned to one of two clusters. Each individual is represented by a single vertical column broken into K=2 (upper) and K=3 (lower)-colored segments, with lengths proportional to each of the K inferred clusters. 1=South Kona, Hawai’i Island; 2=Pōhakuloa, Hawai’i Island; 3=Amy Greenwell Botanical Garden, Hawai’i Island; 4=NW Mauna loa, Hawai’i Island; 5=Hāloa ʻĀina, Hawai’i Island; 6= Kealakekua Mountain Reserve, Hawai’i Island; 7= Queen Emma Estates, Hawai’i Island; 8=Pu’u Wa’awa’a, Hawai’i Island; 9=Undisclosed Location, Hawai’i Island; 10=Kapapala, Hawai’i Island ; 11=Hawai’i Volcanoes National Park, Hawai’i Island; 12=Mauna kea, Hawai’i Island; 13=Wall Ranch, Hawai’i Island; 14=Undisclosed Location, Hawai’i Island; 15=Undisclosed Location, Hawai’i Island; to identify the number of distinct population clusters and the assignment of individuals to those clusters based on SRAP analysis.
Variation clearly exists within all populations of S. paniculatum at both K=2 and K=3 for the STRUCTURE analysis. K=2 suggests that there has been regular gene flow between S. paniculatum var. pilgeri and S. paniculatum var. paniculatum. K=3 may suggest further population subdivision is also taking place within S. paniculatum, perhaps due to hybridization with S. ellipticum. The PCO analysis supports the broad similarity of populations of S. paniculatum across all populations sampled with two Pu’u Wa’awa’a individuals as outliers (removed from Figure 1) to an otherwise clean grouping of individuals from Kona (Mauna loa) populations and a second cluster composed of Mauna kea and Pu’u Wa’awa’a (Hualālai) individuals. Outliers from Pu’u Wa’awa’a may represent relatively recent hybrid individuals with Santalum ellipticum, this relationship will be further resolved with additional sampling and genotype sequencing.