Supplementary Methods
Primers used for PCR and Sanger sequencing.
Marker
ITS
rpl14-rpl36 spacer
psbB-psbH spacer
atpI-atpH spacer
psbD-trnT spacer
trnH-psbA spacer
rpl16 intron
trnL-F region
(amplified and
sequenced in two
pieces)
LEAFY second
intron
Primers
ITS-I
ITS4
rpL14 and rpL36
psbB and psbH
atpI and atpH
psbD and trnT(GGU)-R
Internal sequencing
primer psbD-trnT F881
Internal sequencing
primer psbD-trnT R1138
trnH (GUG) and psbA
rpl16-71f
rpl16-1516r
trnL intron: trnL-c and
trnL-d
trnL-F spacer: trnL-e and
trnL-f
LFY F2 and LFY R1
LFY1 F629
LFY1 R1415
LFY2 F177
LFY2 R1415
G3pdhC intron
GPDX7F and GPDX9R
GDX7 1F59
GDX7 1R900
GDX7 2F144
GDX7 2R850
Source
Urbatsch et al., 2000
White et al., 1990
Shaw et al., 2007
Shaw et al., 2005
Shaw et al., 2007
Shaw et al., 2007
5’ TTG ATC TTG CGT TCT GGA ATC 3’ (designed in this
study)
5’ CCT AAC CTA TTG CAT GAT GAC 3’ (designed in
this study)
Hamilton, 1999
Jordan et al., 1996
Kelchner & Clark, 1997
Taberlet et al., 1991
Taberlet et al., 1991
Howarth & Baum, 2005
5’ TTC AGA CAC CTT TTG GGT T 3’
(designed in this study)
5’ CTC GAC TTG ATT AGC ATA TTC TTG G 3’
(designed in this study)
5’ GGG TCC ACA GTA TAC CTA CCT AC 3’
(designed in this study)
5’ CCA ACA TGA TTA GCA TAT TCC TGC 3’
(designed in this study)
Strand et al., 1997
5’ TTC ACG CCA TCA CTG GTT AGT C 3’
(designed in this study)
5’ TTA GGT TTC AGC AAG AGA ATC 3’
(designed in this study)
5’ CTC CTT TGA ACT TGT GAT ACT G 3’
(designed in this study)
5’ CAG YAA CAG AAA TGC TAA TGC CC 3’
(designed in this study)
PCR and cloning procedures using copy-specific primers of LEAFY and G3pdhC
Each PCR reaction contained 0.2 μL PfuUltra II Fusion HS DNA Polymerase (Agilent
Technologies, Inc., Santa Clara, California, USA), 1.5 μL 10x buffer, 1.5 μL dNTP mix (2.5
mmol/L), 0.5 μL of each primer (10 μmol/L), 3 μL diluted template DNA, and ddH2O for a final
volume of 15 μL. Cycling conditions were: 94 °C for 4 min; 35 cycles of 95 °C for 20 s, 51°C
for 20 s, 72 °C for 1 min; and a final extension step of 72 °C for 3 min. PCR products were
purified using the QIAquick PCR Purification Kit or the QIAquick Gel Extraction Kit. Purified
PCR products were cloned using Zero Blunt TOPO PCR cloning kit (Invitrogen, Carlsbad,
California, USA)
Processing short inversions in cpDNA
Two short inversions were detected in the rpl16 intron region from a preliminary phylogenetic
analysis using rpl16 intron alone. The first one was a 33-bp inversion found in two outgroup
accessions (E. hirta and E. cinerascens) and in six ingroup individuals (E. remyi var. remyi 5305
and Y356, E. celastroides var. hanapepensis 4169, E. multiformis var. multiformis 4766, E.
celastroides var. tomentella 5597, and E. celastroides var. stokesii 5315). Monophyly of these
eight individuals was strongly rejected by all other cpDNA markers. The second inversion in
rpl16 intron was 38 bp long and was found in E. stictospora, E. velleriflora, and E. mendezii.
Monophyly of these three individuals was strongly supported by all cpDNA regions. A third 23bp long cpDNA inversion was detected in the trnH-psbA spacer and was present in two outgroup
individuals (E. setosa and E. linguiformis) and the ingroup E. celastroides var. amplectens Y396.
Monophyly of these three individuals was again strongly rejected by all other cpDNA markers.
All three short inversions were reversed and complimented in the final matrix for all subsequent
analyses. Bayesian and ML results were congruent when PP≥0.95 and BS≥70 (Fig. 3;
Appendices S5–S7).
Table S2.1 Alignment statistics
a. cpDNA regions
rpl16
trnHpsbA
trnL-F
rpl14rpl36
psbBpsbH
atpIatpH
psbDtrnT
Concatenated
cpDNA
No. terminals
164
164
164
164
164
164
164
164
Aligned length
1432
904
1047
1007
613
993
2282
8278
93
(6.5%)
84
(9.3%)
92
(8.8%)
73
(7.2%)
23
(3.8%)
63
(6.3%)
205
(9.0%)
633 (7.6%)
No. indels coded
41
31
60
17
7
40
94
292
Nucleotide
substitution model
selected by AIC
–
–
–
–
–
–
–
GTR+I+γ
Data set
Variable characters
(proportion)
b. Nuclear regions
Data set
ITS
LFY
G3pdhC copy 1
G3pdhC copy 2-4
G3pdhC copy 5-6
No. terminals
116
90
23
52
5
Aligned length
706
1447
1063
1030
929
177
(25.1%)
405
(29.2%)
126 (11.9%)
171 (16.6%)
148 (15.9%)
–
88
12
19
12
GTR+I+γ
GTR+I+γ
–
–
–
Variable characters
(proportion)
No. indels coded
Nucleotide
substitution model
selected by AIC
Figure S2.1 Best tree from Maximum Likelihood analysis of cpDNA + indel.
Figure S2.2 Maximum likelihood trees recovered from nuclear markers.Numbers above the
branches bootstrap percentages. Following each taxon name is the DNA accession number and
clone number when applicable. (a) ITS. (b) LEAFY. Copy-specific primer pair LFY2 F177 and
LFY2 R1415 amplified copies 1 and 2, and primer pair LFY1 F629 and LFY1 R1415 amplified
copies 3 and 4. (c) G3pdhC. Copy-specific primer pair GDX7 1F59 and GDX7 1R900 amplified
copy 1; primer pair GDX7 2F144 and GDX7 2R850 preferentially amplified copies 2 and 3;
while copies 4, 5 and 6 were obtained from cloning PCR products from the original primer pair
GPDX7F and GPDX9R
a
b
LFY
Island:
H = Hawaii
M = Maui
L = Lanai
Mo = Molokai
O = Oahu
K = Kauai
Habitat type:
1 = coastal strand
2 = scrub
3 = dry forest
4 = mesic forest
5 = wet forest
6 = bog
1.00
100
1.00
100
0.65
celastroides var. kaenana 2934c1, O1
halemanui 4780c2, K4/5
halemanui 4780c1, K4/5
remyi var. remyi Y356c2, K5
0.74
76
celastroides var. kaenana 2934c2, O1
0.76
celastroides var. kaenana 2934c3, O1
halemanui Y357c2, K4/5
0.71
-1.00
rockii 2223c3, O5
0.64
-rockii 2223c4, O5
100
1.00
-degeneri 3656c1, O1
0.86
84
clusiifolia 1353c1, O4/5
-celastroides var. tomentella 4420c1, O4
0.96
herbstii 2796c2, O5
0.71
deppeana Y358c2, O4
56
skottsbergii var. audens 3523c2, Mo1
0.82 -1.00
degeneri 2219c1, O1
-multiformis 4721c3, O3
97
multiformis 4721c2, O3
1.00
skottsbergii var. vaccinioides 3564c1, Mo2
0.64
Copy
celastroides var. hanapepensis 4169c1, K4
93
0.99
olowaluana Y350c3, H3
0.91
-- 50
multiformis var. microphylla 5624c1, H3
1.00
-celastroides var. kaenana 2776c1, O1
olowaluana 5116c1, H3
97
1.00
celastroides var. hanapepensis 4169c2, K4
1.00
celastroides var. kaenana 5840c3, O1
97
1.00
78
skottsbergii var. vaccinioides 3564c2, Mo2
0.63
0.51
-herbstii 2942c2, O5
1.00
skottsbergii var. audens 3523c1, Mo1
--atrococca Y355c1, K3/4
-1.00
herbstii 2787c2, O5
arnottianus var. integrifolia 4422c2, M3
69
0.90
herbstii 2942c1, O5
-olowaluana 5131c1, H3
1.00
olowaluana 5619c3, H3
olowaluana Y352c4, H3
79
1.00
multiformis var. microphylla 5624, H3
olowaluana Y352c2, H3
94
1.00
kuwaleana 5700c1, O2
kuwaleana 5700c2, O2
100
olowaluana Y350c1, H3
1.00
multiformis 4721c1, O3
0.55
deppeana Y358c1, O4
98
herbstii 2796c1, O5
0.97
degeneri 2219c4, O1
-2246multic1
multiformis 2246c2, O3
0.98
herbstii 2787c1, O5
0.97 0.67
arnottianus var. integrifolia 4422c1, M3
99
0.67
-celastroides var. kaenana 5840c2, O1
--remyi var. remyi Y356c1, K5
1.00
skottsbergii var. skottsbergii 3540c1, O1
skottsbergii var. skottsbergii 3540c2, O1
52
multiformis 2246c3, O3
1.00
multiformis 2246c4, O3
0.72
multiformis 4627c3, O3
83
olowaluana 5619, H3
-Copy 2
olowaluana 5116c4, H3
0.76
0.58
multiformis 4627c1, O3
--multiformis 4627c2, O3
0.53
0.52
olowaluana 5116c2, H3
--olowaluana 5116c3, H3
celastroides var. kaenana 2776c2, O1
0.94
remyi 482c2, K5
halemanui Y357c1, K4/5
0.77 54
remyi 482c1, K5
skottsbergii var. vaccinioides 3587c1, Mo2
-sparsiflora Y360c1, K6
1.00
rockii 2223c1, O5
97
rockii 2223c2, O5
skottsbergii var. audens 3512c1, Mo1
skottsbergii var. vaccinioides 3587c2, Mo2
1.00
degeneri 2219c2, O1
0.95
89
degeneri 2219c5
0.99
olowaluana 5619, H3
0.93
Copy 3
-multiformis var. microphylla 5624c4, H3
64
54
kuwaleana 5700c4, O2
degeneri
2219c3
1.00
olowaluana 5619, H3
0.78
96
multiformis var. microphylla 5624c2, H3
Copy 4
0.81
55
rockii 2223c5, O5
82
kuwaleana 5700c3, O2
1.00
clusiifolia 1353c2, O4/5
celastroides var. kaenana 5840c1, O1
79
0.95
velleriflora c1
0.87
velleriflora c2
86
83
stictospora
0.94
cinerascens c2
1.00
cinerascens c3
74
cinerascens c1
100
0.02
1.00
1.00
1.00
59
98
89
1
c
91
56
G3pdhC
degeneri 4708, O1
degeneri 4751, Mo1
multiformis var. multiformis 4766, O3
degeneri 3656, O1
multiformis 4627, O3
kuwaleana 5700, O2
arnottianus var. integrifolia 4422, M3
68
skottsbergii var. vaccinioides 3564, Mo2
skottsbergii var. vaccinioides 3587, Mo2
72
Copy 1
96
olowaluana 5619, H3
olowaluana 5131, H3
87
olowaluana 5116, H3
100
Copy 2
herbstii 2796, O5
multiformis 2246, O3
100
celastroides var. hanapepensis 4169, K4
celastroides var. kaenana 2776, O1
herbstii 2942, O5
celastroides var. kaenana 2934, O1
skottsbergii var. audens 3512, Mo1
celastroides var. kaenana 5840, O1
multiformis var. microphylla 5624c1, H3
multiformis var. microphylla 5624c2, H3
100
stictospora
61
64
olowaluana 5619, H3
68
100
Copy 5
velleriflora c2
velleriflora c1
Copy 6
kuwaleana
5700, O2
Copy 3
10 changes
stictospora
Copy 4
51
100
skottsbergii var. audens 3523c1, Mo1
kuwaleana 5700c1, O2
degeneri 4708c2, O1
degeneri 4708c1, O1
skottsbergii var. audens 3512c1, Mo1
skottsbergii var. vaccinioides 3564c1, Mo2
multiformis var. multiformis 4766c1, O3
degeneri 3656c2, O1
herbstii 2942c1, O5
herbstii 2942c2, O5
herbstii 2787c1, O5
herbstii 2787c2, O5
multiformis 2246c2, O3
kuwaleana 5700c2, O2
celastroides var. kaenana 2776c3, O1
celastroides var. kaenana 2776c4, O1
multiformis var. microphylla 5624c3, H3
celastroides var. kaenana 5840c2, O1
olowaluana 5619c3, H3
multiformis var. multiformis 4766c1, O3
multiformis 4721c1, O3
olowaluana 5619c1, H3
skottsbergii var. vaccinioides 3564c1, Mo2
herbstii 2796c1, O5
celastroides var. kaenana 5840c1, O1
multiformis 2246c1, O3
herbstii 2787c3, O5
herbstii 2796c3, O5
degeneri 3656c3, O1
degeneri 4708c3, O1
skottsbergii var. audens 3523c2, Mo1
skottsbergii var. vaccinioides 3564c2, Mo2
multiformis var. multiformis 4766c2, O3
multiformis var. microphylla 5624c4, H3
multiformis 2246c4, O3
celastroides var. kaenana 2776c1, O1
skottsbergii var. audens 3523c3, Mo1
skottsbergii var. audens 3512c3, Mo1
skottsbergii var. vaccinioides 3564c2, Mo2
degeneri 3656c1, O1
multiformis 2246c3, O3
skottsbergii var. audens 3512c2, Mo1
olowaluana 5619c2, H3
celastroides var. kaenana 2776c2, O1
multiformis 4721c2, O3
herbstii 2942c3, O5
celastroides var. hanapepensis 4169c1, K4
multiformis var. microphylla 5624c5, H3
multiformis var. microphylla 5624c6, H3
celastroides var. kaenana 5840c3, O1
celastroides var. kaenana 5840c4, O1
celastroides var. kaenana 5840c5, O1
Figure S2.3 Maximum clade credibility tree recovered from molecular dating analysis. Node
labels are median age, and node bars are 95% highest posterior density interval.
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