Evolutionary diversification and geographical isolation in Dubautia laxa
(Asteraceae), a widespread member of the Hawaiian silversword alliance
Mitchell E. McGlaughlin1,2,* and Elizabeth A. Friar1,3
1Rancho Santa Ana Botanic Garden, 1500 N. College Ave., Claremont, CA 91711, USA,2School of Biological Sciences,
University of Northern Colorado, 501 20th St., Greeley, CO 80639, USA and3Division of Environmental Biology, National
Science Foundation, 4201 Wilson Blvd, Suite 655, Arlington, VA 22230, USA
* For correspondence. E-mail email@example.com
Received: 5 July 2010 Returned for revision: 30 September 2010Accepted: 15 November 2010 Published electronically: 30 December 2010
†Background and Aims The Hawaiian silversword alliance (Asteraceae) is one the best examples of a plant adap-
tive radiation, exhibiting extensive morphological and ecological diversity. No research within this group has
addressed the role of geographical isolation, independent of ecological adaptation, in contributing to taxonomic
diversity. The aims of this study were to examine genetic differentiation among subspecies of Dubautia laxa
(Asteraceae) to determine if allopatric or sympatric populations and subspecies form distinct genetic clusters
to understand better the role of geography in diversification within the alliance.
†Methods Dubautia laxa is a widespread member of the Hawaiian silversword alliance, occurring on four of the
five major islands of the Hawaiian archipelago, with four subspecies recognized on the basis of morphological,
ecological and geographical variation. Nuclear microsatellites and plastid DNA sequence data were examined.
Data were analysed using maximum-likelihood and Bayesian phylogenetic methodologies to identify unique
†Key Results Plastid DNA sequence data resolved two highly divergent lineages, recognized as the Laxa and
Hirsuta groups, that are more similar to other members of the Hawaiian silversword alliance than they are to
each other. The Laxa group is basal to the young island species of Dubautia, whereas the Hirsuta group
forms a clade with the old island lineages of Dubautia and with Argyroxiphium. The divergence between the
plastid groups is supported by Bayesian microsatellite clustering analyses, but the degree of nuclear differen-
tiation is not as great. Clear genetic differentiation is only observed between allopatric populations, both
within and among islands.
†Conclusions These results indicate that geographical separation has aided diversification in D. laxa, whereas
ecologically associated morphological differences are not associated with neutral genetic differentiation. This
suggests that, despite the stunning ecological adaptation observed, geography has also played an important
role in the Hawaiian silversword alliance plant adaptive radiation.
Key words: Dubautia laxa, Hawaiian silversword alliance, infraspecific divergence, subspecies, speciation,
geographical divergence, microsatellites, plastid DNA, chloroplast DNA.
In many actively evolving groups, species and infraspecific taxa
(i.e. subspecies and varieties) may represent incipiently diver-
ging lineages (Darwin, 1859; Shaw, 2002; Manier, 2004;
Gamble et al., 2008; Mulcahy, 2008). However, as many organ-
isms exhibit phenotypic plasticity and reproductive isolation is
not absolute, taxa recognized on the basis of geography,
ecology or morphology may not represent genetically distinct
evolutionary units (Zink, 2004; Sotuyo et al., 2007). This
problem is exacerbated in groups that have undergone recent
adaptive radiations, because the degree of genetic divergence
among sister taxa may be low, whereas ecological and morpho-
logical divergence can be large (Schluter, 2000; Hughes and
Eastwood, 2006; Givnish et al., 2009; Meudt et al., 2009).
The Hawaiian silversword alliance (Asteraceae) offers a
prime example of a plant adaptive radiation and the difficulties
associated with untangling the evolutionary patterns of a rapid
diversification. Over the past 5 Myr (Baldwin and Sanderson,
1998), the 33 members of the Hawaiian silversword alliance
have undergone a rapid radiation, colonizing every major
island in the Hawaiian archipelago, exhibiting a diverse
array of morphological characteristics and occupying almost
every available habitat in the archipelago (Carr, 1985;
Baldwin and Robichaux, 1995). Despite this exceptional diver-
sity, phylogenetic and population genetic studies have had dif-
ficulty resolving evolutionary relationships within species
(Friar et al., 2001, 2007, 2008; Lawton-Rauh et al., 2007;
Remington and Robichaux, 2007) and among species (Witter
and Carr, 1988; Baldwin, 1997; Friar et al., 2006). When
examining the relationship of taxa within the group, two
drivers of diversification are apparent: (1) ecologically based
isolation among sister taxa (Robichaux et al., 1990; Baldwin
and Robichaux, 1995; Friar et al., 2006), and (2) geographi-
cally based isolation with sister taxa being isolated between
islands or distinct geographical regions within islands (Carr,
1985; Friar et al., 2001). Previous studies have focused
largely on the ecological drivers of diversification within this
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Annals of Botany 107: 357–370, 2011
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group, but the role of geography has not previously been
Genetic data provide a tool for dissecting the evolutionary
relationships of recently derived taxa, independent of mor-
phology and geography. Studies that employ multiple data
types (Schaal et al., 1998; Schaal and Olsen, 2000; Gamble
et al., 2008; Pineiro et al., 2009) are particularly suited to
understanding the complexities of population-level evolution-
ary processes and relationships that are indicative of both his-
torical and contemporary diversification. However, conflict
among data types, particularly DNA sequence data from
different genomes, has been widely documented (e.g.
Rieseberg and Soltis, 1991; Birky, 2001; Funk and Omland,
2003) raising questions about whether any specific gene tree
(Maddison, 1997; Barraclough and Nee, 2001; Degnan and
In plants, plastid (or chloroplast) capture, in which the plastid
genome has a unique evolutionary history relative to the nuclear
genome, has long been recognized as a potential source of phy-
logenetic error (Rieseberg and Soltis, 1991), but its impact at
the species level remains largely unaddressed (Tsitrone et al.,
2003; Okuyamaet al., 2005;
Additionally, as plastid DNA is maternally inherited in most
flowering plants (Birky, 2001), phylogeographical reconstruc-
tions based solely on plastid DNA generally document the
pattern of seed dispersal, which may be independent of long-
distance gene flow via pollen. Consequently, significant ques-
tions exist as to whether evolutionary groups recognized on
the basis of plastid DNA represent true evolutionary entities
or artefacts of the evolutionary history of a plastid genome
with a relatively small complement of genes.
Dubautia laxa (Asteraceae) is a widely distributed member
of the Hawaiian silversword alliance, occurring on five of the
six major islands of the Hawaiian archipelago (Carr, 1985).
Partially due to its wide distribution, D. laxa harbours exten-
sive infraspecific variation with four currently recognized sub-
species and as many as 12 varieties being recognized
previously (Carr et al., 2003). Two subspecies have multi-
island distributions, D. laxa subsp. hirsuta occuring on
Kaua‘i, O’ahu and Lana’i and D. laxa subsp. laxa occurring
on O’ahu, Moloka’i and Maui, and two subspecies, D. laxa
subspp. bryanii and pseudoplantaginea, are endemic to the
Ko’olau Range on O’ahu (Table 1; Fig. 1). The apparent
centre of diversity of D. laxa is O’ahu, where all four subspe-
cies occur and populations of subspecies bryanii, laxa and
Sotuyo etal., 2007).
pseudoplantaginea are found in close physical proximity in
the Ko’olau Range. D. laxa subsp. hirsuta is relatively isolated
on O’ahu, where it is restricted to, and is the sole subspecies
occuring in, the Wai’anae Range. Among the subspecies of
D. laxa, there is considerable morphological and habitat vari-
ation with subsp. hirsuta exhibiting the most distinctive
characteristics (Table 1). When considering the biogeographi-
cal relationship of populations distributed on Lana’i, Moloka’i
and Maui, we will use the term Maui Nui, which refers to the
single large island composed of these smaller islands that
began to separate about 0.6 Mya (Price and Elliott-Fisk, 2004).
Despite the current taxonomic treatement of D. laxa (Carr,
1985), it is unclear if the recognized subspecies represent
unique evolutionary entities or if the ecological and morpho-
logical diversity within this group is a product of recurrent
local adaptation or phenotypic plasticity. Further complicating
our understanding of this species is the widespread occurrence
of hybridization among members of the Hawaiian silversword
alliance (Carr and Kyhos, 1981, 1986; Carr, 1985; Carr et al.,
2003). The available data on hybridization within the alliance
indicate that the barriers that do exist to prevent hybridization
among species are ecological in nature rather than genetic
(Friar et al., 2006; Carr, 1985). This finding can be extended
to D. laxa in which subspecies are differentiated according
to morphology and habitat, but are frequently not geographi-
cally separated, allowing potential gene flow among subspe-
cies. This is particularily apparent in the Ko’olau Range on
the island of O’ahu where three of the four subspecies of
D. laxa are sympatric and subspecies are frequently physically
closer to populations of other subspecies than they are to con-
In the present study, we use nuclear microsatellite and
plastid DNA sequence data to investigate genetic differen-
tiation among populations, subspecies and islands to determine
if genetic data support the recognition of divergent infraspeci-
fic taxa and how distinct lineages relate to geographical distri-
bution. Furthermore, the unique distribution of D. laxa allows
us to pose questions about the impact of geography on diver-
sification at multiple spatial scales. This research aimed to
address three questions related to the role of geography in
the diversification of D. laxa. (1) Are sympatrically distributed
subspecies on the island of O’ahu distinct evolutionary units,
suggesting that ecological adaptation is the primary force
maintaining distinct lineages? (2) Is isolation among islands
sufficent to lead to divergence both within and among subspe-
cies with multi-island distributions? (3) What has been the
TABLE 1. Distribution, habitat and morphological characteristics of Dubautia laxa subspecies
D. l. subsp. bryaniiD. l. subsp. hirsutaD. l. subsp. laxaD. l. subsp. pseudoplantaginea
Plant height (m)
Receptacular bract length (mm)
Leaf length (cm)
Leaf width (cm)
Kauai, Oahu, Lanai
Oahu, Molokai, Maui
Open wet forest
Rarely along midvien
Closed wet forest
McGlaughlin & Friar — Diversification in Dubautia laxa358
impact of the pattern of island colonization on diversification
in this group? Together, these questions allow us to address
the role of geography in diversification within the Hawaiian
MATERIALS AND METHODS
The subspecies of Dubautia laxa have been distinguished on
the basis of capitulescence number, receptacular bract length,
flowering time, leaf morphology, leaf pubescence, whole
plant architecture and habitat (Carr, 1985). A matrix of these
characters is provided in Table 1.
D. laxa was sampled from 13 populations on four islands
(Fig. 1; Table 2). Only a single subspecies occurs on each
island except for O’ahu where D. laxa subsp. hirsuta (Dlh)
is restricted to the Wai’anae Range and D. laxa subsp.
bryanii (Dlb), D. laxa subsp. laxa (Dll) and D. laxa subsp.
pseudoplantaginea (Dlp) are found in the Ko’olau Range, fre-
quently in close physical proximity. Thirty individuals were
sampled from each population, except for Dlh Ka’ala and
Dll Moloka’i, which are represented by 29 and 27 individuals,
respectively (Table 2). All leaf tissue samples were shipped on
ice to Rancho Santa Ana Botanic Garden, where they were
deposited in a –80 8C freezer until extraction. DNA extrac-
tions were performed using a modified CTAB protocol
(Friar, 2005) with the addition of 1% (w/v) Caylase (Cayla
Inc., Toulouse, France) and 100 mL Phytopure Nucleon
Resin (Amersham Biotechnologies, Amersham, UK).
For plastid DNA sequencing, three individuals of Dubautia
plantaginea from the island of Lana’i were sampled as an
outgroup. D. plantaginea is not thought to be a close relative of
D. laxa, but it is grouped in the same section of the genus, it
co-occurs with D. laxa on several islands and has been thought
to hybridize with this species in nature. The following members
of the Hawaiian silversword alliance were also included in the
phylogenetic analysis using data collected for other projects in
the E. Friar lab: Argyroxiphium caliginis, A. grayanum,
A. kauense, A. sandwicense, Dubautia arborea, D. ciliolata,
D. latifolia, D. linearis, D. menziesii, D. platyphylla,
D. reticulata, D. scabra and D. waianapanapaensis and Madia
sativa (a California tarweed).
Microsatellites. Five microsatellite loci developed for the
Hawaiian silversword alliance were used as described in
Friar et al. (2000). The microsatellite loci were Ap3MS1,
MKMS2, MKMS3, MKMS4 and 42-2. Each primer popu-
lation pair was optimized for annealing temperature and
MgCl2 concentration. PCR amplifications were carried out
in 15-mL volumes, which included 3 mL genomic DNA
(at 5 ng mL21), 0.6 mL of each primer (at 10 p.p.m.), 0.9 mL
dNTPs (at 2.5 mM), 0.5 U Taq polymerase (Promega,
Madison, WI, USA) and 1.5 mL 10× amplification buffer
(0.1 M Tris-HCl, 0.5 M KCl, 5 %, v/v, glycerine, pH 8.3).
Optimized amplification temperatures and MgCl2 concen-
trations ranged from 52 to 608C and from 3 to 5 mM, respect-
ively (data not shown). Forty cycles of amplification were
carried out in an MJ Research, Inc. (Waltham, MA, USA)
PTC-100 thermocycler, with 1 min denaturing at 94 8C,
1 min annealing at primer-specific temperatures and 30 s
extension at 72 8C. Amplification products were visualized
on 1 % agarose gels to verify fragment size.
Once amplifications were optimized, microsatellite loci were
amplified using primers labelled with the fluorescent dyes
FIG. 1. Map of sampled populations of Dubautia laxa from the Hawaiian archipelago (the island of Hawaii is not shown). Subspecies of D. laxa, as recognized
by Carr (1985), are identified by three-letter abbreviations and symbols: Dlb ¼ D. laxa subsp. bryanii (triangles), Dlh ¼ D. laxa subsp. hirsuta (squares), Dll ¼
D. laxa subsp. laxa (circles), Dlp ¼ D. laxa subsp. pseudoplantaginea (diamonds). Population names are presented for each locality. Sympatric populations sep-
arated by ,0.5 km were sampled at Poamoho and Manana.
McGlaughlin & Friar — Diversification in Dubautia laxa 359
6-FAM or HEX (Applied Biosystems, Foster City, CA, USA) for
analysis on an Applied Biosystems 3100 genetic analyser. When
possible, fluorescently labeled amplified products were multi-
plexed. All amplification products were co-loaded with a
Plastid DNA sequencing. PCR was employed to amplify the
non-coding plastid psbA-trnH intergenic spacer and rpl16
intron. PCR amplifications were carried out in 20 mL
volumes, which included 2 mL genomic DNA (at 10 ng
mL21), 1 mL of each primer (at 10 pmol), 1 mL dNTPs
(at 2.5 mM), 1.5 U Taq polymerase (Promega) and 2 mL
10× Promega Taq polymerase buffer with MgCl2; 0.2 mL
bovine serum albumin (at 10 mg mL21) was added to proble-
matic samples. Annealing temperatures were varied from 52 to
56 8C. Amplifications were carried out in an MJ Research,
Inc. PTC-100 thermocycler, with initial denaturing at 948C
for 4 min, followed by 35 cycles of 1 min of denaturing at
94 8C, 1 min annealing at primer-specific temperatures and
3 min extension at 72 8C. The annealing temperature was
increased by 0.58C per cycle to a maximum of 728C.
The 35 cycles were followed by a final 10 min extension at
72 8C. Amplification products were visualized on 1%
agarose gels to verify fragment size. Primer pairs used for
the PCR were F71 (5′-GCTATGCTTAGTGTGTGACTCG
TT-3′, Jordan et al., 1996) and R1516 (5′-CCCTTCATTCTT
CCTCTATGTTG-3′, Kelchner and Clark, 1997) for the rpl16
intron and psbAF (5′-GTTATGCATGAACGTAATGCTC-3′)
and trnHR (5′-CGCGCATGGTGGATTCACAATC-3′) for the
psbA–trnH intergenic spacer (modified from Sang et al., 1997).
prior to direct sequencing (Johnson and Soltis, 1995). Cycle
sequencing was performed on an MJ Research PTC-100 thermo-
cycler, using BigDye Terminator v3.1 (Applied Biosystems).
Purified PCR products were subjected to 35 cycles of amplifica-
tion, with 30 s denaturing at 948C, 15 s annealing at 508C and
4 min extension at 608C. The amplification primers for the two
Due to the size of the rpl16 intron, the internal primers F543
(5′-TCAAGAAGCGATGGGAACGATGG-3’; Butterworth and
Wallace, 2004) and rpl16IR (5′-ATTAATGGAGAAGCTA
TGG-3′; Prince and Kress, 2006) were used to ensure complete
coverage of the fragment. Labelled products were purified by
Sephadex column cleaning and then sequenced using an
Applied Biosystems genetic analyser.
Microsatellites. Each population was characterized by the
observed number of alleles (AO), the effective number of
alleles (AE), observed heterozygosity (HO), expected heterozyg-
osity under Hardy–Weinberg expectations (gene diversity; HE)
and the inbreeding coefficient (FIS), calculated as FIS¼ 1 –
HO/HE. One-tailed t-tests and F-tests were performed to deter-
mine significant differences in means and variances between
pairs of populations and subspecies using StatView 5.0.1
(SAS Institute, Cary, NC, USA). Bonferroni corrections were
used to correct for multiple comparisons (Rice, 1989). Tests
for fit to Hardy–Weinberg expectations and pairwise linkage
disequilibrium were performed using Arlequin ver. 2.000
(Schneider et al., 2000). Genetic differentiation between popu-
lations, subspecies and islands was assessed using an analysis of
molecular variance (AMOVA; Excoffier et al., 1992).
The Bayesian analysis STRUCTURE ver. 2.3.1 was used to
assess population structure and ancestry (Pritchard et al., 2000;
Falush et al., 2003). Burn-in and run lengths of 50 000 replicates
lations, the admixture model using correlated allele frequencies
was implemented. The analyses including all sampled individ-
uals used the subspecific designation of each population as a
prior incorporated into the STRUCTURE algorithm (Hubisz
et al., 2009). Subsequent analyses examining subsets of the
sampled individuals did not use any information about subspeci-
fic designation as a prior. The numberof inferred populations, K,
ing values of K, the method presented by Evanno et al. (2005)
was used to determine the ‘true’ number of populations. This
method examines the rate of change in log-likelihood values
between successive K values over 20 replicates to obtain DK.
Plastid DNA. Primer regions were trimmed and sequences were
edited in Sequencher v. 4.2.1 (Gene Codes Corp., Ann Arbor,
TABLE 2. Population characteristics for all sampled Dubautia laxa populations
No. of individuals
sampled Population sizeElevation (m) Habitat Rainfall (mm)
Open wet forest
Closed wet forest
Open wet forest
Open wet forest
Closed wet forest
McGlaughlin & Friar — Diversification in Dubautia laxa360
MI, USA). Edited sequences were aligned manually using
Se-Al v2.0a11 (Rambaut,
numbers for each dataset are as follows: psbA–trnH IGS:
EU341877–EU341889, GU226075–GU226130; rpl16 intron
The plastid regions were combined into a single concate-
nated dataset for all analyses. Nucleotide diversity (p, Nei,
1987), population mutation parameter (qw, Watterson, 1975),
number of segregating sites (S) and number of haplotypes
(H) were calculated by population and subspecies for the com-
bined plastid DNA dataset using DNASP version 4.0 (Rozas
et al., 2003). The number of haplotypes was also calculated
by hand after coding gaps larger than 1 bp (see below).
Sequence gaps larger than 1 bp were coded using the simple
indel coding method (Simmons and Ochoterena, 2000;
Simmons et al., 2001) as implemented by the program
GapCoder (Young and Healy, 2003). Optimal maximum-
likelihood (ML) settings were determined using likelihood
ratio tests of multiple DNA substitution models as implemented
by Modeltest v. 3.6 (Posada and Crandall, 1998). ML phyloge-
netic trees were constructed using PAUP v. 4.0b10 (Swofford,
2003). All phylogenetic trees are rooted with the California
tarweed Madia sativa. Additionally, a minimum-spanning hap-
lotype network was computed using TCS v. 1.18 (Clement
et al., 2000) with gaps treated as a fifth state and the parsimony
criterion set to 95%.
1996). GenBank accession
A total of 386 individuals representing 13 populations of
D. laxa were analysed for five microsatellite loci. Locus
Ap3MS1 was the most diverse, exhibiting 19 alleles across
all populations, with a maximum of 14 alleles within the
Dlh Kilohana population and a minimum of six alleles
within the Dlh Kaala population (data not shown). Across all
populations, loci MKMS2, MKMS3 and MKMS4 had four,
five and five observed alleles, respectively. Locus 42-2 was
fixed for all individuals except in the Dlb Laie and Dlb
Poamoho populations, which each had one heterozygous indi-
vidual with the same rare allele.
Locus Ap3MS1 showed significant deviation from expected
Hardy–Weinberg frequencies for all populations except Dlp
Manana. Locus MKMS2 also showed significant deviation
from expected Hardy–Weinberg frequencies for all popu-
lations except Dll Moloka’i, Dll Kukui and Dll Hanaula. Dlh
Lana’i showed significant deviations from expected Hardy–
Weinberg frequencies for MKMS3. Both Ap3MS1 and
MKMS2 showed a deficiency of heterozygotes and dimorphic
allele size distributions.
Tests for pairwise linkage disequilibrium among loci
showed significant disequilibrium among loci for most popu-
lations. Only Dlh Kilohana, Dlh Wekiu and Dlh Kaala had
no significant linkage disequilibrium among loci. Significant
linkage was observed between Ap3MS1 and MKMS2 in Dlb
Poamoho, Dlb Manana, Dll Konahuanui and Dlh Lana’i,
between Ap3MS1 and MKMS3 in Dlp Manana, between
Ap3MS1 and MKMS4 in Dlb Laie, Dlb Manana and Dll
Moloka’i, between Ap3MS1 and 42-2 in Dlb Poamoho,
between MKMS2 and MKMS3 in Dll Konahuanui, between
MKMS2 and MKMS4 in Dll Moloka’i, and between
MKMS3 and MKMS4 in Dlb Laie, Dll Poamoho, Dll Kukui
and Dll Hanaula. Due to the apparent randomness associated
with the observed linkage between loci, it is likely that these
results are due to deviations from Hardy–Weinberg equili-
brium within populations (Schneider et al., 2000).
The observed number of alleles (AO), expected number of
alleles (AE), observed heterozygosity (HO), expected hetero-
zygosity (HE) and inbreeding coefficient (FIS) are shown for
all populations in Table 3. Observed heterozygosity ranged
from 0.05 at Dlh Wekiu to 0.37 at Dlb Laie. Grouped by sub-
species, Dlh had significantly lower observed heterozygosity
and a higher inbreeding coefficient than subspecies Dlb and
Dll. No significant differences between heterozygosity values
and variances were found among populations within subspe-
cies. The inbreeding coefficient within populations ranged
widely, but was .0.50 in all Dlh populations. Due to the sig-
nificant differences between Dlh and the other three subspe-
cies, the populations were divided into two groups, the Laxa
group, containing all populations of Dlb, Dll and Dlp, and
the Hirsuta group, containing all populations of Dlh. The div-
ision of D. laxa into two groups is strongly supported by
plastid sequence data reconstructions presented below.
AMOVA was used to assess the distribution of genetic vari-
ation on multiple taxonomic and spatial scales. When the
AMOVA was applied to sympatric members of the Laxa
group on O’ahu, 4.8% of the variation was partitioned
among subspecies, 8.8% among populations within subspecies
and 86.4% within populations (Table 4). When AMOVA was
calculated for Dlh populations, 46.1% of the variation was par-
titioned among islands, 3.2% among populations within islands
and 50.7% within islands (Table 5). When the AMOVA was
applied only to the Laxa group, 14.3% of the genetic variation
was partitioned among islands, 10.5% among populations
within islands and 75.2% within populations (Table 6).
Distributions of mean L(K) and DK values generated from
STRUCTURE are shown in Fig. 2. When all populations are
included, there is moderate support for dividing D. laxa into the
Laxa and Hirsuta groups. The Laxa group exhibited the largest
DKvalueatK ¼ 2(Fig.2F),whereastheHirsutagroupexhibited
the largest DK value at K ¼ 3 (Fig. 2E). Cluster assignment is
shown for D. laxa in its entirety and the Laxa and Hirsuta
groups for the maximum DK value (Fig. 3). Within the Laxa
group, Dlb from the northern Ko’olau Range and the three Dll
populations from Maui Nui are unambiguously assigned to two
distinct clusters. The remaining populations of the Laxa group
exhibit significant admixture. The admixed populations are
located in the central and southern portions of the Ko’olau
range on O’ahu, indicating that gene flow is occurring among
these populations. Within the Hirsuta group, the three inferred
clusters correspond to the island of occurrence. The division of
the Hirsuta group by island indicates that there is limited gene
flow among islands within this group.
bp with an aligned length of 1030 bp. A 133-bp deletion was
McGlaughlin & Friar — Diversification in Dubautia laxa 361
TABLE 4. AMOVA results testing genetic subdivision among sympatric subspecies of Dubautia laxa on the island of Oahu
Source of variation d.f. Sum of squares Variance componentsPercentage of variationFixation indices*P
Among populations/ within subspecies
Fct ¼ 0.0481
Fsc ¼ 0.0927
Fst ¼ 0.1363
* Fixation indices: Fct, the correlation of genotypes within a subspecies relative to the entire data set; Fsc, the correlation of genotypes within a population
relative to subspecific designation; Fst, the correlation of genotypes within a population relative to the entire data set.
TABLE 3. Microsatellite DNA diversity statistics (means+s.d.) for Dubautia laxa populations and subspecies
Different letters indicate values that are significantly different at P , 0.05 (unpaired t-test).
* Number of observed alleles.
†Effective number of alleles.
TABLE 5. AMOVA results testing genetic subdivision among islands of D. laxa subsp. hirsuta.
Source of variation d.f.Sum of squaresVariance componentsPercentage of variationFixation indices*P
Among populations/ within islands
Fct ¼ 0.4601
Fsc ¼ 0.0596
Fst ¼ 0.4611
* Fixation indices: Fct, the correlation of genotypes among islands relative to the entire data set; Fsc, the correlation of genotypes within a population
relative to island of occurrence; Fst, the correlation of genotypes within an island relative to the entire data set.
TABLE 6. AMOVA results testing genetic subdivision among islands of members of the Laxa group
Source of variationd.f. Sum of squaresVariance componentsPercentage of variationFixation indices*P
Among populations/ within islands
Fct ¼ 0.1428
Fsc ¼ 0.1223
Fst ¼ 0.2476
* Fixation indices: Fct, the correlation of genotypes among islands relative to the entire data set; Fsc, the correlation of genotypes within a population
relative to island of occurrence; Fst, the correlation of genotypes within an island relative to the entire data set.
McGlaughlin & Friar — Diversification in Dubautia laxa362
Intrapopulation sequence variation for this region was observed
onlyinDllPoamoho. Sequence fragment lengths for thepsbA–
560 bp. Population-specific insertion/deletion events were
observed for Dlh Lana’i, Dll Konahuanui and D. plantaginea
of 6, 15 and 5 bp, respectively. A 20-bp insertion was shared
by all members of the Laxa group. The combined data set
resolves 11 haplotypes with gaps excluded and 15 haplotypes
with gaps coded (Table 7). Only three of the 13 populations
sampled, Dlh Kilohana, Dll Konahuanui and Dll Poamoho,
exhibited any intrapopulation sequence variation for the com-
tide diversity (p ¼ 0.00128) followed by subspecies Dlb (p ¼
0.00078) and the Laxa group (p ¼ 0.00077).
ML analysis was conducted using the General Time
Reversible model plus gamma-distributed rate variation across
sites, as selected by Modeltest. ML reconstructions resolved
two well-supported clades of the D. laxa plastid DNA lineage
(Fig. 4). One group, which will be referred to as the Hirsuta
group, consists of all populations of Dlh and Dll Konahuanui.
The second lineage, which will be referred to as the Laxa
group, includes all populations of subspecies Dlb and Dlp and
all populations of subspecies Dll excluding Dll Konahuanui.
Sequences from D. plantaginea were most similar to the
Hirsuta group. With the inclusion of other members of the
Hawaiian silversword alliance, D. laxa is resolved as polyphy-
letic (Fig. 4). The Hirsuta group is resolved as monophyletic
and groups with old island members of Dubautia, represented
here by D. plantaginea and D. latifolia and all members of
Argyroxiphium. Observed haplotypes within the Hirsuta
group coalesce within islands. The Laxa group is resolved as
Railliardia clade nested within it. There is no clear taxonomic
or phylogeographical resolution within the Laxa group.
The minimum-spanning haplotype network further illus-
trates the divergence between the Laxa and Hirsuta groups
(Fig. 5). The two groups are separated by a minimum of
nine inferred mutational steps, and the Hirsuta group is separ-
ated from D. plantaginea by three inferred mutational steps.
The haplotype network resolves considerable geographical
structure within the Hirsuta group with no haplotypes shared
among populations or islands. The Laxa group exhibits
limited taxonomic or geographical structure with the single
sampled population of Dlp containing a unique haplotype
and one Dll population from Maui Nui containing a unique
haplotype (see legend to Fig. 5).
Speciation is fundamentally a population-level process that
occurs when populations diverge based on morphological, eco-
logical, reproductive and/or genetic characteristics, leading to
the emergence of new evolutionary entities (Coyne and Orr,
2004). Considerable variability exists in the application of
the taxonomic or theoretical concept of species. When exam-
ining genetic data, there is likely to be a continuum below the
rank of species with some populations exhibiting complete
independence, others being only minimally divergent and
many actively exchanging alleles. Investigations that examine
the divergence of infraspecific variants within a species can
provide information about how the speciation process proceeds
and which factors are most important to promote sustained
2 3 4 5 6 7 8 9 101112131415
1 2 3 4 5 6 7123
45678 9 10 11 12
D. laxa – all populationsHirsuta group Laxa group
FIG. 2. (A–C) Mean STRUCTURE likelihood values [L(K)+s.d.] for 20 replicates for each number of K population clusters. (D–F) Second-order rate of
change (DK) of STRUCTURE likelihood values following the method of Evanno et al. (2005).
McGlaughlin & Friar — Diversification in Dubautia laxa363
In the current study, two distinct lineages were resolved
within D. laxa using both plastid DNA and nuclear microsatel-
lites. When the data were analysed independently for the
lineages, significant structure among populations and islands
was observed. Based on these results we feel that D. laxa
should be divided into two groups. The Laxa group is com-
posed of subspecies Dlb, Dll and Dlp. These subspecies
were not significantly genetically distinct, exhibit overlapping
morphological and ecological traits, and have largely sympa-
tric distributions. The Hirsuta group is composed solely of
subspecies Dlh and occurs in a unique habitat, and is geneti-
cally distinct from and has an allopatric distribution relative
to the other members of D. laxa. One population of Dll,
Konahuanui, appears to be an infraspecific hybrid, with the
nuclear genome of the Laxa group and the plastid genome
of the Hirsuta group.
The level of plastid DNA divergence between the Laxa and
Hirsuta groups is striking. Based on inferred mutational steps
among plastid DNA haplotypes (Fig. 5), the two groups are
separated by a minimum of nine mutational steps with no
observed intermediate haplotypes. Furthermore, phylogenetic
analysesincludingspecies throughout the Hawaiian
silversword alliance showed that the plastid genome of both
groups are more similar to other members of the Hawaiian sil-
versword alliance than they are to each other (Fig. 4). The
most comprehensive phylogenetic studies of the alliance
have been conducted by Baldwin and collaborators using
both nuclear ribosomal DNA (Baldwin and Robichaux,
1995; Baldwin and Sanderson, 1998) and plastid DNA
(Baldwin et al., 1990, 1991), but they did not sample widely
within species. All phylogenetic studies using nuclear DNA
have found D. laxa to represent a cohesive evolutionary unit,
sharing a common ancestor (Baldwin and Robichaux, 1995;
Baldwin and Sanderson, 1998; M. E. McGlaughlin, unpubl.
res.), which contrasts sharply with the current plastid DNA
reconstructions in which D. laxa is polyphyletic. There are
clear similarities to Baldwin’s plastid DNA phylogeny
(Baldwin et al., 1991) in both of the major clades resolved
in the current study, but the substantial divergence between
the Hirsuta and Laxa groups is an unexpected result.
Although not as distinct as the plastid DNA data, microsatel-
lite analyses show consistent divergence between the Laxa and
Hirsuta groups. When all populations of D. laxa were used in a
single analysis, using the Bayesian assignment procedure
implemented in STRUCTURE, there was a clear clustering of
Wekiu Kaala Lanai
FIG. 3. Bar plots of inferred population assignment using STRUCTURE. (A) All sampled individuals assigned to two clusters corresponding to the Laxa and
Hirsuta groups. (B) All members of the Laxa group (subspecies bryanii, laxa and psuedoplantaginea) assigned to two clusters. (C) All members of the Hirsuta
group (subspecies hirsuta) assigned to three clusters.
McGlaughlin & Friar — Diversification in Dubautia laxa364
populations belonging to the Laxa or the Hirsuta groups, with
very limited admixture between those groups (Fig. 3). The
unity of members of the Hirsuta group is striking because popu-
lations span .300 km on three islands and Dlh populations on
O’ahu and Lana’i are geographically isolated from other Dlh
populations and are physically much closer to populations of
the other three subspecies. When the Hirsuta group was ana-
lysed singly, three clusters that conformed to the island of
occurrence were resolved. The two Kaua’i Dlh populations,
Kilohana and Wekiu, were not substantially differentiated
from each other, but the Kilohana population was moderately
admixed with Dlh Kaala (on O’ahu) and slightly admixed
with Dlh Lana’i. Within the Laxa group, two population clus-
ters were resolved that were not admixed representing the geo-
graphical extremes of its distribution, Dll populations from
Maui and Moloka’i and Dlb populations from the northern
Ko’olau Range. The remaining Laxa group populations exhib-
ited significant admixture between the two pure cluster types.
Despite significant morphological and ecological diversity,
sympatrically distributed populations of the Laxa group on
the island of O’ahu do not show any clear patterns of
genetic differentiation. This is supported by AMOVA, which
found ,5% of the microsatellite variation was distributed
among subspecies (Table 4), the extensive admixture observed
in the STRUCTURE analysis (Fig. 3) and the sharing of
Together, these results indicate that sympatrically distributed
subspecies have not diverged genetically, at least not detecta-
bly. The lack of resolution among subspecies could be due to
the maintenance of ancestral polymorphisms, continuing gene
flow or recent divergence. Sympatric speciation remains an
actively debated topic in speciation research (Sites and
Marshall, 2003; Fitzpatrick et al., 2008; Hendry et al.,
2009), with most examples being constrained to uncommon
Savolainen et al., 2006; Nosil, 2008). Our data do not
support the suggestion that infraspecific taxa in D. laxa rep-
resent incipient lineages diverging based on localized ecologi-
cal adaptation. Rather, these data support the generally held
idea that the disruption of gene flow among sympatric popu-
lations is difficult to achieve.
Considerable genetic divergence was observed between
populations occurring on different islands within both the
Laxa and Hirsuta groups. Although Dll is distributed on
both O’ahu and Maui Nui, a greater level of divergence is
observed between the two than among subspecies within the
Laxa group. However, the dominant plastid DNA haplotype
found on O’ahu (haplotype A) is found in every Dll population
on Maui Nui, indicating that the dispersal between O’ahu and
Maui Nui occurred relatively recently and there has not been
substantial divergence between the two. Within the Hirsuta
group, almost 50 % of the microsatellite variation is distributed
among islands and no plastid DNA haplotypes are shared
among islands. This high level of divergence is probably due
TABLE 7. Plastid DNA diversity of the combined rpl16/psbA–trnH data set for Dubautia laxa populations and subspecies
All D. laxa
11 15 15
1Subspecies abbreviations: Dlh ¼ D. laxa subsp. hirsuta; Dlb ¼ D. laxa subsp. bryanii; Dlp ¼ D. laxa subsp. pseudoplantaginea; Dll ¼ D. laxa subsp.
2Population abbreviations: WK ¼ Wekiu; KH ¼ Kilohana; KB ¼ Kaala; PK ¼ Konahuanui; LN ¼ Lanai; LA ¼ Laie; PO ¼ Poamoho; MN ¼ Manana;
MO ¼ Molokai; HN ¼ Hanaula; KU ¼ Kukui.
3Number of individuals sampled.
4Number of haplotypes identified excluding gaps.
5Number of haplotypes identified with gaps coded.
6Nucleotide diversity (Nei, 1987).
7Watterson’s population mutation parameter (Watterson, 1975).
8Number of positions analysed.
9Number of segregating sites.
McGlaughlin & Friar — Diversification in Dubautia laxa365
to the age of the Hirsuta group and the limited amount of
inter-island gene flow. Furthermore, these data indicate that
geographical isolation among islands is a significant impedi-
ment to gene flow, particularly in the form of seed dispersal.
The level of genetic variation contained within populations
can also offer insight into evolutionary history and how
populations are interacting genetically. D. laxa, like most
members of the Hawaiian silversword alliance, is believed to
be self-incompatible (Carr et al., 1986), thereby increasing
the importance of the level of genetic variability contained
within populations. The genetic diversity data further support
Microsatellite variability in the Laxa group was high in all
sampled populations. Populations of the Hirsuta group had
significantly lower observed heterozygosity and exhibited sig-
nificantly higher levels of inbreeding than those of the other
three subspecies. In relation to the other subspecies, Dlh has
the smallest mean population size and the greatest distance
among populations and it is found in a rare habitat, bog
Laxa and Hirsutagroups.
DIh Wekiu (3,K)
DIh Kilohana (K)
DIh Kilohana (2,K)
DIh Kaala (2,O)
DIh Kaala (O)
DIl Konahuanui (O)
DIl Konahuanui (2,O)
DIh Lanai (3,MN)
D. plantaginea (3, MN)
D. latifolia (K)
Old island Dubautia clade
A. caliginis (MN)
A. caliginis (MN)
A. grayanum (MN)
A. grayanum (MN)
A. kauense (2,H)
A. sandwicense (H)
A. kauense (H)
A. sandwicense (MH)
Madia sativa (California)
Dll Kukui (2,MN)
Dll Kukui (MN)
Dll Hanaula (3,MN)
Dll Poamoho (O)
Dll Molokai (3,MN)
Dlb Laie (3,O)
Dlp Manana (3,O)
Dlb Manana (3,O)
Dlb Poamoho (3,O)
Dll Poamoho (O)
Dll Poamoho (O)
D. waianapanapaensis (MN)
D. reticulata (MN)
D. platyphylla (MN)
D. menziesii (MN)
D. linearis (MN)
D. arborea (H)
D. ciliolata (H)
D. linearis (H)
D. linearis (H)
D. arborea (H)
D. scabra (2,MN,H)
D. ciliolata (H)
D. scabra (H)
D. linearis (MN)
D. linearis (MN)
D. ciliolata (H)
FIG. 4. ML phylogram of Dubautia laxa and other members the genus Dubautia, Argyroxiphium and Madia sativa as an outgroup. Subspecies of D. laxa are
identified by three-letter abbreviations: Dlb ¼ D. laxa subspecies bryanii, Dlh ¼ D. laxa subspecies hirsuta, Dll ¼ D. laxa subspecies laxa, Dlp ¼ D. laxa sub-
species pseudoplantaginea. Shown in parentheses are the number of haplotype copies observed and island of occurrence: K ¼ Kauai, O ¼ Oahu, MN ¼ Maui
Nui, H ¼ Hawaii. Clades within the phylogram are grouped into evolutionary units (vertical bars), discussed in the text.
McGlaughlin & Friar — Diversification in Dubautia laxa366
margins. At the other end of the spectrum is subsp. bryanii,
which exhibited the highest observed levels of genetic diver-
sity, is found in large populations, in close physical proximity
to consubspecific populations and populations of other subspe-
cies (,5 km apart) and on exposed ridges affording the great-
est potential gene flow by generalist pollinators.
The observed microsatellite heterozygosities for Dlb, Dll
and Dlp are within the range of values reported from natural
populations of the closely related genus Argyroxiphium
(mean HO¼ 0.24; Friar et al., 2000, 2001) and generally
higher than values reported for species in Dubautia section
Railliardia (mean HO¼ 0.17; Friar et al., 2006, 2007;
E. A. Friar, unpubl. res.). The differences in genetic variation
between D. laxa and members of section Railliardia are most
likely due to the younger age of the Railliardia taxa, which
occur on islands with a maximum age of 1.76 Myr, although
some populations do exhibit similar levels of variation. For
all subspecies, the expected heterozygosity is higher than
values reported from allozyme data for D. laxa (HE¼ 0.07)
and other members of the silversword alliance (mean HE¼
0.07; Witter and Carr, 1988).
The plastid DNA sequence data provide information about
genetic variability on a different time scale relative to the
nuclear microsatellites due to the slower mutation rate of the
plastid genome. The difference between the Laxa and
Hirsuta groups was particularly pronounced for the plastid
DNA data. The number of haplotypes resolved for the two
groups are comparable despite a larger sampling of individuals
and populations belonging to the Laxa group (24 vs. 15;
Table 7). The Laxa group is characterized by low levels of
nucleotide diversity and a dominant haplotype (haplotype A)
occurring in 62 % of the populations. The low levels of
plastid DNA diversity suggest that the Laxa group has under-
gone a historical population bottleneck, possibly when the
group originated, and has a more recent origin. By contrast,
the Hirsuta group has a much higher level of nucleotide diver-
sity and no haplotypes are shared among populations.
Furthermore, two of the four populations of the Hirsuta
group exhibit multiple plastid DNA haplotypes when gaps
The combination of nuclear microsatellite and plastid DNA
sequence data provides extensive data to untangle the pattern
of dispersal of D. laxa through the Hawaiian Archipelago.
Based on the plastid DNA diversities and the phylogenetic pla-
cement of the Hirsuta group, it appears that D. laxa originated
on the island of Kaua’i. At a later date members of the Hirsuta
group dispersed from Kaua’i to O’ahu, establishing the popu-
lations currently found in the Waianae Range. The Hirsuta
group then dispersed again to Lana’i. The subdivision of the
Hirsuta group by islands is supported by both data sets and
AMOVA analyses. The lack of shared plastid DNA haplotypes
among islands within the Hirsuta group suggests that there was
a single seed dispersal event to colonize each island.
Furthermore, the fact that the Dll Konahuinui population con-
tains a Hirsuta group plastid DNA haplotype indicates that
there has been seed dispersal between the Waianea and
Ko’olau mountain ranges on O’ahu. Denser sampling in the
vicinity of the Konahuinui population would further clarify
the frequency of seed dispersal between the two mountain
The plastid genome of the Laxa group appears to have a
complex history with two possible evolutionary scenarios.
First, the plastid DNA of the Laxa group could have originated
from the Hirsuta group on O’ahu. This scenario would require
substantial divergence of the Hirsuta group plastid DNA
leading to the origin of the Laxa group. The shared ancestry
and recurrent gene flow via pollen between the two groups
could have maintained the relative cohesion of the nuclear
genome. A second scenario is that the plastid DNA of the
Laxa group represents a plastid capture event from an
unsampled Dubautia species. A plastid capture scenario has
also been suggested to explain evolutionary patterns observed
in old island members of Dubautia (Baldwin, 1997) and in
D. scabra (Baldwin, 1997; Baldwin et al., 1990; Friar et al.,
2008), a member of the Railliardia clade. Determining the
likely scenario for the origin of the Laxa group could be
aided by additional sampling of Dubautia species from
Kaua’i and the inclusion of other regions from the plastid
Hirsuta groupLaxa group
D. laxa subsp. bryanii
FIG. 5. Haplotype network for the combined cpDNA data set. Relative sizes of circles, squares and triangles indicate haplotype frequency, and the shaded slices
represent the proportion of the haplotype in each species. The number of inferred steps between haplotypes is indicated by small open circles. The subspecies and
population belonging to each letter haplotype are as follows: A, Dlb Laie, Dll Poamoho, Dll Moloka’i, Dll Hanaula, Dll Kukui; B, Dlb Poamoho; C, Dll
Poamoho; D, Dlb Manana; E, Dll Poamoho; F, Dlp Manana; G, Dll Kukui; H, Dlh Lana’i; I, Dlh Wekiu; K and J, Dlh Kilohana; L and M, Dlh Kaala; O
and N, Dll Konahuinui; P, D. plantaginea Lanai.
McGlaughlin & Friar — Diversification in Dubautia laxa367
genome. However, given the recently documented case of
plastid capture in D. scabra (Friar et al., 2008) and the
degree of divergence among the D. laxa plastid lineages, we
feel that a plastid capture event involving an unsampled
Dubautia species is the most likely scenario.
Therefore, the Laxa group most likely originated on O’ahu,
diverging from existing Dlh populations. This finding is sup-
ported by the high level of diversity for both plastid DNA
and microsatellites on O’ahu, plastid DNA haplotypes that
are shared between O’ahu and Maui Nui populations, and
nuclear phylogenetic reconstructions that find members of
the Laxa and Hirsuta groups as sister taxa (Baldwin and
Robichaux, 1995;M. E.
However, as the exact origin of the Laxa group is unclear, it
is possible that it originated on Kaua’i and then dispersed to
O’ahu. Once completely diverged, the Laxa group dispersed
from O’ahu to Maui Nui. The current distribution could have
resulted from a single seed dispersal event from O’ahu to
Maui Nui followedbyshort-range
Moloka’i and West Maui, or two or more dispersals from
O’ahu. All populations on Maui Nui share the dominant
plastid DNA haplotype found on O’ahu, but the Dll Kukui
population also contains a rare plastid DNA haplotype that is
not observed in any other population. The tight clustering of
the Maui Nui Dll in the Bayesian analyses could be interpreted
to indicate a single colonization from O’ahu, or high levels of
gene flow that have homogenized the nuclear genome of the
Maui Nui populations. The dispersal scenarios presented
here follow the basic progression rule that has been suggested
to occur with other Hawaiian taxa (Funk and Wagner, 1995),
under which species gradually move from older to younger
islands. Furthermore, a single collection of Dll has been
made from the youngest mountain range on Maui, Haleakala
(Carr, 1985), but this population could not be located when
the region was surveyed for this project.
The phylogenetic reconstructions presented here also
provide valuable information about how the plastid genome
has been transferred among Hawaiian silversword species
colonizing younger islands. It is clear that the Laxa group
plastid DNA is the progenitor of the Railliardia clade.
Within the combined lineage of the Laxa group and the
Railliardia clade, there is a clear pattern of plastid DNA dis-
persal from older to younger islands, with the most derived
members of the Railliardia clade occurring on Hawaii, the
youngest island, as concluded from previous analyses of
plastid DNA restriction site variation (Baldwin et al., 1990).
The rarity of inter-island seed dispersal inferred for both the
Hirsuta and the Laxa groups is therefore consistent with
interpretations for the Railliardia clade and for the silversword
alliance in general (Baldwin and Robichaux, 1995). But what
do these data tell us about species formation in the Hawaiian
silversword alliance? It is clear from nuclear DNA data for
D. laxa and members of the Railliardia clade (Friar et al.,
2006, 2007, 2008; E. A. Friar, unpubl. res.) that most
members of Dubautia lack barriers to introgression. This
potential for introgression among species leads to the con-
clusion that plastid DNA may not be as valuable for indicating
boundaries between species as for providing a source of bio-
geographical evidence. It follows that extending the plastid
DNA data set to include all members of the Hawaiian
silversword alliance could lead to a detailed understanding
of the plastid coalescent processes in endemic island taxa.
The data presented here clearly illustrate the need to incor-
porate multiple data types to best understand the evolutionary
history of species. Although the nuclear and plastid data sets
are largely congruent, the degree of divergence among the
Laxa and Hirsuta groups within the two data sets is consider-
able. Members of the Laxa group, subspecies Dlb, Dll and Dlp,
were not clearly distinguishable based on the plastid DNA
data. However, the microsatellite data suggest that there are
pure populations of subspecies Dlb in the western portion of
the Ko’olau range (Laie and Poamoho) and of subspecies
Dll on Maui Nui (Kukui, Hanaula and Moloka’i), but that
all other populations are admixed between these genetic
types. This leads us to suggest that Dlh should be elevated
to the rank of species, and D. laxa should be recognized as
containing two subspecies, Dlb and Dll, that intergrade
throughout most of the Ko’olau Range on O’ahu.
Our data shed light on the importance of geographical sep-
aration to reinforce local adaptation and population divergence
within the Hawaiian silversword alliance. Consistent genetic
differentiation was only observed among allopatric popu-
lations, whether it was among Hirsuta or Laxa group popu-
lations on different islands. This indicates that although there
is considerable ecological and morphological
among D. laxa populations, the available genetic data do not
support the suggestion that sympatric populations are under-
going incipient genetic divergence. It should be noted that
divergent selection for particular traits could be occurring,
but this was not resolved here due to our usage of neutral
We thank L. M. Prince, J. T. Columbus, R. Robichaux,
V. Soza, K. Wood and T. Anderson for laboratory and field
assistance. Collection permits, access to preserves and logisti-
calsupport was generously
Department of Forestry and Wildlife, Hawaii Natural Area
Reserve System, Hawaii State Parks, Maui Land and
Pineapple Company, The Nature Conservancy of Hawaii,
Moloka’i and Maui Programs, Schofield Barracks Army
Environmental Office, O’ahu National Wildlife Preserve,
Alexander Baldwin Company, East Maui Irrigation and the
Bishop Museum. This research was funded by the following
awards to M.E.M.: the Cynthia Lee Smith Botany Award,
the Andrew W. Mellon Foundation, the Howard and Phoebe
Graduate Program Award, the Goldhamer Scholarship Award
and the Rancho Santa Ana Botanic Garden Alumni Award.
Sampling of outgroup taxa for the phylogenetic analyses was
supported by a grant to E.A.F. from the W. M. Keck
Foundation and Rancho Santa Ana Botanic Garden. Any
opinion, finding,and conclusions
expressed in this material, are those of the authors and do
not necessarily reflect the views of the National Science
provided bythe Hawaii
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