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: cmorden@hawaii.edu
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). Other assumptions associated with RAPD marker analysis are described in Lynch and
Milligan (1994). A RAPD marker absent in any individual sampled within a population (ie, a
“null”) will have an allelic frequency of 0.17 in Kaua‘ōpu‘u (frequency of null homozygote is
0.03) or 0.14 in Mauna Ku¯wale (frequency of null homozygote is 0.019), exceeding the 0.05 value to be considered polymorphic (Hartl and Clark 1989). Markers present in five or fewer individuals are assumed to be marker/null heterozygotes; a marker must be present in three (in
Kaua‘ōpu‘u) or five (in Mauna Ku¯wale) to have a frequency of 0.05 for the population to be considered polymorphic. Percent polymorphic loci for each population and the species were calculated using MS Excel. Genetic similarity indices were estimated using both Gower (1971) and Nei and Li (1979) similarity coefficients for populations using MVSP Plus ver. 3.1 (Kovach
2007). Expected heterozygosity (H) was calculated across the species as well as separately for each population for each locus as follows:
H = 1 – (p² + q²) where p is the frequency of the present allele and q is the frequency of the null allele. Genetic differentiation among populations was estimated using estimated population and species H based on Wright’s FST where FST = (HT – HS)/HT (Hartl and Clark 1989). Relationships within and among populations and the species were projected from the similarity matrixes using principal coordinate analysis (PCO) and cluster analysis with MVSP Plus ver. 3.1 (Kovach 2007) using
Gower similarity (Gower 1971).
Results
6
RAPD analysis of the eleven primers selected for use with all samples yielded 119 loci
(Table 1). There was an average of 10.8 genetic markers per primer with a range from 5 to 19
markers. Extensive variation was found within the species and in each population (Table 2).
Across both populations, 107 of 120 markers were polymorphic, giving a level of polymorphism
of 89.2%. Within each population, the level of polymorphism was slightly lower in the
Kaua‘ōpu‘u population at 78.3% in comparison to the Mauna Ku¯wale population at 86.7%.
Estimated heterozygosity for both populations combined (HT) was 0.313, and estimated
heterozygosity for each of the populations (HS) was 0.279 at Kaua‘ōpu‘u and 0.302 at Mauna Ku¯ wale (Table 2). FST values calculated from estimates of heterozygosity were 0.072 indicating
genetic structure among the populations.
<< Table 1 near here >>
<< Table 2 near here >>
The two populations were compared for genetic similarity (Gower 1971) where a value
of 0.0 indicates complete dissociation and 1.0 indicates complete genetic identity. Similarity
among individuals within the Kaua‘ōpu‘u population (0.737) and Mauna Ku¯wale population
(0.745) is higher than the similarity among the two populations (0.716). FST and genetic
similarity both reflect some differentiation among the populations. This is visualized in the
Principal Coordinates Analysis (PCO) that shows the populations are largely segregated while
forming a continuum of variation with little overlap (Figure 2). Cluster analyses gave results
consistent with the PCO analyses, and are not presented.
<< Fig. 2 near here >>
Discussion
RAPD data indicate there is a considerable level of variation within and among the two
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populations of E. kuwaleana. The level of polymorphism in each population was about the same
(mean = 82.5%) although the Kaua‘ōpu‘u population is larger in size and expectedly had the
higher level of polymorphism. The total level of polymorphism across both populations was
higher (89.2%) suggesting there is some genetic structure among them.
The amount of variation found within and among populations of E. kuwaleana was
within the range of that found in other closely related Hawaiian native species. The only
congeners for which RAPD marker data are available for comparison are E. skottsbergii and E.
celastroides var. kaenana, taxa also federally listed as endangered. Euphorbia skottsbergii has
the highest levels of variation found for any Hawaiian plant examined, endangered or not, with
99.4% of the loci across all populations polymorphic and 95.9% polymorphic within populations
(Morden and Gregoritza 2005). However, this high level is atypical. Within E. celastroides var.
kaenana, a markedly different pattern is evident where total species polymorphism was 80.2%,
but within population polymorphism ranged from 23.1 to 41.8% (Morden 2011).
Considering comparisons using RAPD markers among Hawaiian species from other
families, E. kuwaleana also exhibits a high level of variation. Haplostachys haplostachya, an
endangered mint (Lamiaceae) on Hawaii Island, had only 49% polymorphism across all populations sampled (Morden and Loeffler 1999). Similarly, the two species of kauila
(Alphitonia ponderosa and Colubrina oppositifolia; Rhamnaceae) had 41% and 47% polymorphism at the species level and 27% and 29% within populations, respectively (Kwon and
Morden 2002). Non-endangered species typically have higher levels of polymorphism as demonstrated by Dubautia ciliolata and D. scabra (91% and 87%, respectively; Asteraceae;
Caraway et al. 2001) and Touchardia latifolia (77.6%; Urticaceae; Loeffler and Morden 2003).
Thus, the variation in E. kuwaleana at the species level, although geographically limited, is greater than other endangered species that have been examined and similar to that found among
8 other non-endangered species in Hawai‘i.
Genetic differentiation of the two populations of E. kuwaleana was surprising given the close proximity of the locations less than 1 km apart. There were no diagnostic markers that distinguished either of the populations from one another yet changes in the frequency of markers indicates these populations are undergoing natural selection or genetic drift resulting in their separation. Populations in such close proximity that are genetically differentiated are unique. The two congeners of E. kuwaleana, E. skottsbergii and E. celastroides var. kaenana, both have populations much more widely dispersed yet have values of genetic differentiation similar to that found here. Of these Euphorbia species, E. celastroides var. kaenana had the highest levels of differentiation among its populations with an average FST of 0.107 (ranging from zero to 0.284).
These populations are widely separated around west O‘ahu from Wai‘anae to Ka‘ena Point and extending eastward. In contrast, the most variable of the three species, E. skottsbergii, has the lowest FST values ranging from 0.040 among the two populations of var. audens on Molokai to
0.087 among populations of var. skottsbergii from O‘ahu and var. vaccinioides from Molokai.
Genetic differentiation among the two E. kuwaleana populations, separated by less than 1 km, exceeded that found among population of these other species that were in several cases greater than 10 km apart and approached that of levels among different varieties on separate islands.
This seemingly unlikely scenario is a function of the total levels of variation found within and among the populations of each species (highest in E. skottsbergii and lowest in E. celastroides var. kaenana) and is not to suggest that the populations should be recognized at any subspecific level. However, the degree of differentiation among the populations is noteworthy.
Conservation Implications
Genetic variation within these populations of E. kuwaleana was sufficient that species
9
decline due to depauperate genetic diversity is unlikely. It is probable that the population
formerly known from Moku Manu had allelic diversity distinct from the Wai‘anae populations,
but testing for this is no longer possible given that only a few plants representing the population
are deposited in herbaria. However, recovery efforts for this species within existing populations
can continue with little concern for maintaining genetic diversity unless population size should
continue to decline. USFWS (2011) has designated critical habitat for this species in 19 locations
including coastal and dry cliff habitats where plants would be re-established or new populations established based on potentially suitable habitat. Coastal locations include off-shore islets of windward O‘ahu and locations on Southern O‘ahu. Dry cliff locations include locations along ridges and mountains of the Waianae coast region including the vicinity of the extant populations. Plants used as founders in these locations should be from seed or cuttings collected widely in the existing populations to maximize the initial variation introduced there.
The greatest threat to populations of E. kuwaleana is the periodic fires that occur in the vicinity of their habitat. A secondary impact known to be a consequence of fires in dry, lowland ecosystems is rapid establishment of invasive grasses such as the prevalent buffelgrass
(Cenchrus ciliaris; Smith and Tunison 1992). This western region of O‘ahu gets little rain during the year (Giambelluca et al 2011) and the soils along the ridge tops where these plants occur are shallow, which minimizes establishment of invasive woody vegetation that might displace native plants. Plants of E. kuwaleana were present in all size classes at both populations (personal observations) suggesting that there is active regeneration and recruitment occurring. However, fires do occur in this region frequently and fuel load on the ridge slopes is sufficient to carry the fire up and over the ridge crest as occurred in June 2012 when a significant portion of the
Kaua‘ōpu‘u population was affected (Sailer 2012). It is unknown at this time if the 2012 fire was fast-burning enough to impact only the above ground stems, and it is hoped that plants in the
10
affected areas may re-sprout with the onset of winter rains. However, future efforts to protect this
species must incorporate a strategy to protect the remaining populations from brush fires. The
only reasonable cost-effective mechanism to do this is by the control of alien species, primarily invasive grasses, to create a buffer zone around these populations. Periodic fires will continue in
this area and it is likely that this species will eventually be extirpated unless steps are taken to
protect these populations.
Acknowledgements
We thank Julie Rivers and Cory Campora (Lualualei Navmag Natural Resource Managers) and
Joel Lau for assistance in collecting samples and access to the field site, Ya Yang (Univ.
Michigan) for helpful discussions, Sarah Kuioka and Elbereth Walker for laboratory assistance,
Dan Sailer for information regarding fires affecting populations, and Maggie Sporck and two
anonymous reviewers for helpful comments on the manuscript. Funding provided by the Pacific
Cooperative Studies Unit.
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Table 1
Primers used from kits OPA through OPD with nucleotide sequence and number of scored
markers in the genetic analysis of E. kuwaleana.
Primer Sequence (5’ to 3’) Scorable Markers
OPA-14 TCTGTGCTGG 8
OPA-16 AGCCAGCGAA 14
OPA-18 AGGTGACCGT 14
OPB-08 GTCCACACGG 19
OPB-16 TTTGCCCGGA 7
OPB-19 ACCCCCGAAG 12
OPC-12 TGTCATCCCC 9
OPC-14 TGCGTGCTTG 10
OPC-18 TGAGTGGGTG 10
OPD-05 TGAGCGGACA 11
OPD-13 GGGGTGACGA 5
12
Table 2
Variation in within and among populations of E. kuwaleana.
Species/Population N a %P b H c
Kaua‘ōpu‘u 33 78.3% 0.279
Mauna Ku¯wale 52 86.7% 0.302
Total 85 89.2% 0.313
a Number of individual samples. b Percent polymorphism. c Estimated heterozygosity.
13
FIGURE 1. Geographic locations of the three extant populations of E. kuwaleana in west O‘ahu.
Topographic lines represent 200 feet (61 meter) intervals.
14
FIGURE 2. PCO based on RAPD markers for individuals of E. kuwaleana at Kaua‘ōpu‘u (closed
circles) and Mauna Ku¯wale (open circles). The first two principal coordinates accounted for
41.9% of the total variation, the first axis 33.5% and the second axis 8.4%.
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Appendix A
Voucher specimens and collection label information of E. kuwaleana on deposit at B. P. Bishop
Museum Herbarium (BISH).
Mauna Ku¯wale
O. Degener 19613 & T. Murashige. Mauna Kuwale, Waianae, Isl. Oahu on arid volcanic cliffs, alt. 800 ft. June 12, 1949. HOLOTYPE (photo) and ISOTYPE.
Moku Manu (south islet) Kaneohe
F. R Fosberg 14092 & F. E. Egler. Thin guano soil on basaltic rock. Prostrate shrub, summit, west end of island. One plant only. Alt: 60 m. June 18, 1937.
Kaua‘ōpu‘u
J. Obata 400. Along Ridge top, exposed, rocky, dry, on two peaks at 321 and 275 m. alt., over
100 plants, Aug 19, 1978.
Steve Perlman 6822 & John Obata. 1050 ft. el., end of ridge on mauka (east) side, below Pu‘u
Kalena, in Dodonaea viscosa lowland dry shrubland with Sida fallax, Artemisia australis,
Opuntia, Leucaena leucocephala, several hundred plants, shrubs 2-4 ft. ht. with flowers and fruit. Nov. 8, 1987.
J. Obata 87-681, S. Perlman, D. Palmer. Along dry, open, rock faces; mostly north facing side; population stable, over three hundred on and around both peaks. 320 m (1050 ft). Nov. 8, 1987.
16
Pu‘u Ka‘i¯lio
K. R. Wood 3171 & J. Lau. West aspect in mixed native & alien assoc. Panicum beecheyi,
Eragrostis variabilis, Dodonaea viscosa, Heteropogon contortus, Sida falax, Kalanchoe pinnata,
Cenchrus ciliaris, Leucaena leucocephala, Chamaesyce kuwaleana, Artemisia, Carex meyenii,
Acacia confusa, Lantana camera, Opuntia, Grevillea robusta, Bidens torta, Doryopteris,
Ageratina riparia. 1000+ plants, variable in size, prostrate to 2 meter tall with
Schiedea ligustrina, Lobelia niihauensis. Weeds, Goats. Elev: 1470 ft = 448 m.
08 May 1994
17
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