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Journal of Biogeography
SUPPORTING INFORMATION
Phylogeography, population structure and evolution of coral-eating butterflyfishes (Family
Chaetodontidae, genus Chaetodon, subgenus Corallochaetodon)
Ellen Waldrop, Jean-Paul A. Hobbs, John E. Randall, Joseph D. DiBattista, Luiz A. Rocha,
Randall K. Kosaki, Michael L. Berumen and Brian W. Bowen
Additional Supporting Information may be found in the online version of this article:
Appendix S1: Additional materials & methods
S1. Supplemental information on PCR amplifications
Mitochondrial DNA
A 605 base pair (bp) segment of mtDNA cytochrome b (cyt b) gene was resolved using primers
Cyb 9 (FOR: 5’-GTGACTTGAAAAACCACCGTTG-3’ Song et al., 1998) and Cyb 7 (REV: 5’AATAGGAAGTATCATTGCGGTTTGATG-3’ Taberlet et al., 1992). Polymerase chain
reaction (PCR) mixes contained 7.5 µl of 2x BioMix Red solution (BioMix Red; Bioline Ltd.,
London, UK), 0.3 µl (10 µM) of each primer and 5 to 50 ng template DNA in 15 µl total volume.
PCRs had an initial denaturing step at 95°C for 3 min, then 35 cycles of amplification (30 s of
denaturing at 94°C, 45 s of annealing at 52°C and 45 s of extension at 72°C), followed by a final
extension at 72°C for 10 min. PCR products were purified with 1.1 units of ExoSAP (USB,
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Cleveland, OH) per 10 µl PCR product at 37°C for 60 min, followed by deactivation at 85°C for
15 min. DNA sequences were resolved with an ABI 3130XL Genetic Analyzer (Applied
Biosystems, Foster City, CA). All specimens were sequenced in the forward direction and
questionable haplotypes were re-sequenced in the reverse direction. The sequences were aligned
and edited to a common length using Geneious Pro 6.0.6 (Drummond et al., 2010). Details of
this protocol are available in Waldrop (2014).
Microsatellite DNA
PCR mixes contained 5 µl of 2x BioMix Red solution (BioMix Red; Bioline Ltd., London, UK),
0.15 µl (10 µM) of reverse primer and dye, 0.035 µl (10 µM) of fluorescently labeled forward
primer and 5 to 50 ng template DNA in 10 µl total volume. PCRs included an initial denaturing
step at 94°C for 5 min, then 35 cycles of amplification (30 s of denaturing at 94°C, 30 s of
primer-specific annealing at temperatures in Table S1.1 and 90 s of extension at 72°C), followed
by a final extension at 72°C for 10 min. All markers reliably amplified in Chaetodon lunulatus
and C. trifasciatus, and product sizes were consistent with expectations (Lawton et al., 2011;
Montanari et al., 2012). PCR products were resolved using an ABI 3130XL Genetic Analyzer
(Applied Biosystems, following DiBattista et al., 2012). Allele sizes were assigned using the
Geneious Pro microsatellite plug in. Additional details are available in Waldrop (2014).
For C. lunulatus the mean number of alleles per locus was 18 (range: 7 to 29 alleles),
allelic richness was 3.56 (range: 1.54 to 5.44) and observed heterozygosity ranged from 0.54
(Lun 14) to 0.95 (Lun 3). Few loci deviated from Hardy-Weinberg equilibrium for within site
comparisons (7 out of 140, P < 0.05). Linkage disequilibrium was detected in 2 out of 45 within
site comparisons after correcting for multiple tests: Mo‘orea Lun 14 and Lun 34, Johnston Atoll
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Lun 8 and Lun 20 (P < 0.011 across all populations), but none of these deviations are consistent
within loci or sites. Microchecker analysis revealed no evidence for scoring error due to
stuttering, and no evidence for large allele dropout. Evidence of null alleles was detected in only
12 out of 140 within site comparisons, although one locus was disproportionately represented,
Lun 20. Therefore analyses were run including and excluding this locus; findings were no
different between datasets and so results from all 10 loci are presented. Overall, there was no
consistent evidence for departure from HWE, linkage disequilibrium or null alleles across all
sampled locations, supporting the decision to retain and examine the entire data set.
For C. trifasciatus the mean number of alleles per locus was 17.9 (range: 7 to 29 alleles),
allelic richness was 5.99 (range: 3.04 to 9.92) and observed heterozygosity ranged from 0.41
(Lun 36) to 0.91 (Lun 3). Linkage disequilibrium was detected in only one pair of loci
(Christmas Is., Lun 3 and Lun 9) out of 45 within site comparisons after correcting for multiple
tests (adjusted P < 0.001). The test for Hardy-Weinberg disequilibrium was significant in 7 out
of 40 within site comparisons (adjusted P < 0.005). MICROCHECKER analysis again revealed
no evidence for scoring error due to stuttering or large allele dropout. Evidence of null alleles
was detected in only 9 out of 40 within site comparisons; but again 2 loci were
disproportionately represented, Lun 7 and Lun 20. Analyses were therefore run including and
excluding these loci; findings were no different between datasets and so results from all 10 loci
are presented. To test for the possible impact of null alleles in Lun 7 and Lun 20 on these results,
we also ran FreeNA, which estimates unbiased FST (Weir 1996) following the excluding null
alleles (ENA) method described in Chapuis & Estoup (2007). This test resulted in FST = 0.0038
not using ENA and FST = 0.0044 using ENA correction. Here microsatellite statistics are
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averaged over all loci for each location and species: Number of samples (N), number of alleles
(NA), observed heterozygosities (HO) and expected heterozygosities (HE).
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Table S1.1. Characteristics of microsatellite loci used in this study, developed for Chaetodon
lunulatus by Lawton et al. (2010). Primer sequences, repeat motifs and annealing temperatures
(Ta) are given as per the authors of the original study. Fluorescent dye labels and size ranges are
from the present study.
Locus
Label
Repeat Motif
Primer sequence (5'–3')
Ta (°C)
Size Range
Lun03
PET
(AG)
TGTGTGTCACCACCTGGTCT
58
180-280
55
170-210
58
160-220
55
180-240
58
170–190
58
210-280
58
220-280
55
230-290
58
180–210
58
200-270
ACTCAGTTTTGAGCCGCTTC
Lun05
6FAM
(CAA)
GCAACCCAGTCTCACATCAA
TCTGCTATTTCACAATTTTAGAGCA
Lun07
NED
(TG)
AAGTGCCCTTTAGCAAAGCA
CTCCAGTCGCTTTCTGTGTG
Lun08
NED
(CA)
GGCCTTTGTTTGTGGTCATT
CCTGAAGAGAGAGCTGCTCAA
Lun09
6FAM
(TG)
CCTGTGTTTGTCATCCAACG
CTTTGGGACACACACTTCCA
Lun14
VIC
(TCA)
TACGTTGGACAGTGGCTGTG
TGGCTCTGTGGCATGTATGT
Lun20
6FAM
(CTT)
CAGTGTCGGAGAACAACGAA
TCACTGTGTCACCAATGCAC
Lun29
6FAM
(AC)
CACCCACAGGCAGTGTATTG
GCCAGCCTGTCAAAACTTTA
Lun34
PET
(CA)
CATGCTTGGGTGAGCATGTA
TGTGCGTTTGTGCAAGTGTA
Lun36
VIC
(GT)
GCGTTTGACTTCACGTTTCA
TGCAAAACAACAACCTACGG
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LITERATURE CITED
Chapuis, M.P. & Estoup, A. (2007) Microsatellite null alleles and estimation of population
differentiation. Molecular Biology and Evolution, 24, 621–631.
DiBattista, J.D., Rocha, L.A., Craig, M.T. Feldheim, K.A. & Bowen, B.W. (2012)
Phylogeography of two closely related Indo-Pacific butterflyfishes reveals divergent
evolutionary histories and discordant results from mtDNA and microsatellites. Journal of
Heredity, 103, 617–629.
Drummond, A.J., Ashton, V., Buxton, V., Cheung, M., Cooper, V., Duran, C., Field, M., Heled,
J., Kearse, M., Markowitz, S. Moir, R., Stones-Havas, S., Sturrock, S., Thierer, T. &
Wilson, A. (2010) Geneious version 5.4, Available from http://www.geneious.com.
Lawton, R.J., Bay, L.K. & Pratchett, M.S. (2010) Isolation and characterization of 29
microsatellite loci for studies of population connectivity in the butterflyfishes Chaetodon
trifascialis and Chaetodon lunulatus. Conservation Genetics Resources, 2, 209-213.
Lawton, R.J., Messmer, V., Pratchett, M.S. & Bay, L.K. (2011) High gene flow across large
geographic scales reduces extinction risk for a highly specialised coral feeding
butterflyfish. Molecular Ecology, 20, 3584-3598.
Montanari, S.R., van Herwerden, L., Pratchett, M.S., Hobbs, J.P.A & Fugedi, A. (2012) Reef fish
hybridization: lessons learnt from butterflyfishes (genus Chaetodon). Ecology and
Evolution, 2, 310-328.
Song, C.B., Near, T. J. & Page, L.M. (1998) Phylogenetic relations among percid fishes as
inferred from mitochondrial cytochrome b DNA sequence data. Molecular Phylogenetics
and Evolution, 10, 343–353.
Taberlet, P., Meyer, A. & Bouvert, J. (1992) Unusually large mitochondrial variation in
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populations of the blue tit, Parus caeruleus. Molecular Ecology, 1, 27–36.
Waldrop, E. 2014. Phylogeography and Evolution of Butterflyfishes in the Subgenus
Corallochaetodon: Chaetodon lunulatus, Chaaetodon trifasciatus, Chaetodon austriacus,
Chaetodon melapterus. Thesis, University of Hawai‘i, Honolulu
Weir, B.S. (1996) Genetic Data Analysis II. Sinauer Associates, Sunderland, Mass.
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Appendix S2: Supporting tables & figures
Table S2.1. Pairwise comparisons within Hawaiian sampling locations: mtDNA ST values are
above the diagonal and microsatellite FST values are below the diagonal. Significant P values are
highlighted in bold (P < 0.05). All negative ST and FST values were adjusted to 0.
Location
Hawai‘i
Island
Oahu
French Frigate
Maro Reef
Lisianski
Pearl &
Hermes
Midway
Kure
Hawai‘i Island
─
0.056
0.016
0.378
0.305
0.332
0.498
0.493
Oahu
0.010
─
0.264
0.768
0.750
0.712
0.916
0.913
French Frigate
0.034
0.037
─
0.196
0.144
0.150
0.327
0.322
Maro Reef
0.047
0.054
0.020
─
0.000
0
0.024
0.023
Lisianski
0.086
0.090
0.060
0.020
─
0
0
0
Pearl Hermes
0.070
0.085
0.052
0.003
0.023
─
0.051
0.050
Midway
0.066
0.076
0.039
0.000
0.016
-0.007
─
0
Kure
0.048
0.058
0.020
0.005
0.047
0.015
0.011
─
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Table S2.2. MsatDNA statistics for Chaetodon lunulatus and C. trifasciatus.
N
NA
HO
HE
Christmas Island
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6.3
0.883
0.824
Indonesia
27
11.1
0.802
0.795
American Samoa
18
8.5
0.744
0.764
Fiji
37
12.2
0.784
0.79
Kanton Island
17
9.8
0.831
0.79
Marshall Islands
54
13.6
0.785
0.795
Mo‘orea
32
9.5
0.776
0.729
Okinawa
14
8.7
0.752
0.808
Pohnpei
30
10.7
0.793
0.787
Kiribati
40
12.7
0.779
0.783
Palau
33
12.3
0.788
0.795
Johnston Atoll
42
8.6
0.731
0.695
MHI
50
9
0.782
0.747
NWHI
203
13.3
0.688
0.671
Diego Garcia
29
11.6
0.686
0.775
Seychelles
34
14.3
0.764
0.800
Christmas Island
58
15.0
0.757
0.811
Indonesia
17
9.9
0.765
0.800
Location
C. lunulatus
C. trifasciatus
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Table S2.3. Estimation of divergence between species using a mitochondrial DNA cytochrome b
mutation rate of 2% per Myr. Mean genetic distance between species is below the diagonal (d),
and divergence time in years is above the diagonal.
Species
C. lunulatus
C. trifasciatus
C. melapterus
C. austriacus
─
3,000,000
2,900,000
2,900,000
C. trifasciatus
0.060
─
750,000
750,000
C. melapterus
0.058
0.015
─
50,000
C. austriacus
0.058
0.015
0.001
─
C. lunulatus
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Figure S2.1. STRUCTURE HARVESTER analysis used to determine that the most likely value
of K was 3 for Chaetodon lunulatus.
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Figure S2.2. STRUCTURE HARVESTER analysis used to determine that the most likely value
of K was 2 for Chaetodon trifasciatus.
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