Journal of Biogeography
SUPPORTING INFORMATION
Testing dispersal limits in the sea: range-wide phylogeography of the pronghorn spiny
lobster Panulirus penicillatus
Matthew Iacchei, Michelle R. Gaither, Brian W. Bowen, Robert J. Toonen
Appendix S1. Photographs of Panulirus penicillatus specimens collected from the East
Pacific to the Red Sea. As described in the introduction, there is great variation in color
across the range, even within sites (e.g., Fig. S1.4 are lobsters at the same local market in
Samoa).
M. Iacchei et al.
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Figure S1.1 Photographs of a Panulirus penicillatus specimen collected from the East
Pacific – Revillagigedo Islands. This is the only East Pacific sample we obtained a
photograph for, and this lobster was photographed at night, making it difficult to see the
redder coloration of the body compared to the rest of the range. There are also different
spotting patterns at the base of the tail on the dorsal side near the uropods, not seen in this
photo. Photo credit: Scott Aalbers.
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Figure S1.2 Photographs of two Panulirus penicillatus specimens collected from the
Hawaiian Archipelago. The top lobster has the darker body color and thin, wavy lines
on the legs versus the lighter body and thick, white lines on the legs of the lobster in the
bottom photo. Photo credit: Matthew Iacchei (top) and Joseph O’Malley (bottom).
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Figure S1.3 Photograph of a Panulirus penicillatus specimen collected in Fiji. Photo credit:
Posa Skelton.
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Figure S1.4 Photograph of three Panulirus penicillatus specimens collected at a fish market in
Samoa. Note the variation in body color and the width and color of the leg stripes even within one
sample. Photo credit: John Fitzpatrick.
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Figure S1.5 Photograph of two Panulirus penicillatus specimens collected in the
Western Indian Ocean on Zanzibar. Both of these lobsters were frozen in a local
fisher’s freezer when sampled and photographed. Photo credit: Michelle R. Gaither and
Shelley Jones.
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Figure S1.6 Photograph of Panulirus penicillatus specimens collected at Dream Beach,
Jeddah, Saudi Arabia in the Red Sea. The specimens in the photos correspond to the
following Genbank sequences: Specimen #RS2473: (12S–KT954806; 16S–KT954848;
COI–KT954888). Specimen #RS2474: (12S–KT954807; 16S–KT954849; COI–
KT954889. Photo credit: Tane Sinclair-Taylor.
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Appendix S2 Details of phylogenetic analyses of Panulirus penicillatus including methods
and parameter choices, PARTITIONFINDER results, BEAST results, the specimens from public
databases that were included in all analyses, the specimens used to make the multi-locus
phylogeny, and the condensed maximum likelihood tree for all of the P. penicillatus COI
sequences.
Sample Collection, DNA Extraction, PCR, Sequencing
Tissue samples that were preserved in salt-saturated 20% dimethyl sulfoxide buffer followed
the protocols of Gaither et al. (2011); however, we found no difference in amplfication for P.
penicillatus between EtOH and DMSO-preserved samples.
Three sets of primers were used in this study. Species-specific primers for cytochrome c
oxidase subunit I (COI) (Iacchei et al., 2014) were designed internal to the
LCO1490/HCO2198 primer set of Folmer et al. (1994), and were used to resolve a 460 bp
fragment of COI for all individuals used in the study. For a subset of individuals (Table
S2.4), we also resolved 522bp of 12S mtDNA using the primer set in Podsiadlowski and
Bartolomaeus (2005) and a 440bp region of 16S mtDNA using the Palumbi et al. (1991)
primers (see Table S2.4 for a list of all individuals and which individuals were resolved at
which mtDNA regions).
BEAST
To estimate the time to most recent common ancestor (TMRCA), we used the Bayesian
MCMC approach implemented in BEAST. We kept molecular and mutation models unlinked
across loci and we employed a relaxed uncorrelated lognormal clock for 12S and 16S. We
used a coalescent tree prior assuming expansion growth. Following the results of
PARTITIONFINDER (Table S2.1), we used the TN93 model of mutation (plus gamma for COI
and 12S and invariant sites for 16S) and ran simulations for 20 million generations with
sampling every 1000 generations. Ten independent runs were computed to ensure
convergence. Effective sample sizes (ESS) were calculated for all model parameters to
ensure adequate mixing. Tree files were combined using LOGCOMBINER 1.7.5 after 10%
burn-in and resampled every 4000 trees for a total of 50,000 trees.
Maximum likelihood tree (RAXML)
Following the results of PARTITIONFINDER, the GTR+G model of nucleotide substitution was
implemented for all DNA segments using a random starting tree and four discrete rate
categories. All model parameters were estimated by RAXML and partitions were assigned
according to gene region. We estimated support for individual clades in the tree with 1000
bootstrap replicates.
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Phylogeographic analyses
To test for hierarchical population genetic structure, an analysis of molecular variance
(AMOVA) was performed in ARLEQUIN using 100,000 permutations. Pairwise ΦST
comparisons among all locations with N > 5 were also tested for statistical significance using
a permutation analysis with 100,000 permutations. We controlled for false discovery rate as
recommended by Benjamini et al. (2006). Following the PARTITIONFINDER results (Table
S2.1), we used the TrN (Tamura & Nei, 1993) sequence evolution model with a Ti/Tv ratio
of 7.273 and gamma parameter of 1.550 for ARLEQUIN analyses. In accordance with other
crustacean studies, we used Kimura’s two-parameter distance model (K2P; Kimura, 1980) to
calculate genetic distance between mitochondrial lineages to be able to compare our results
with other crustacean studies. Mantel tests were conducted to test for isolation-by-distance
(IBD) among populations in the Western and Central Pacific. Mantel tests were performed
using multiple combinations in full factorial: all sites, all sites except the Sino-Japanese
Province, ΦST including negative values, ΦST with negative values converted to zeroes; log of
ΦST, and log of geographic distance. Each Mantel test was conducted with 10,000 iterations
using the ISOLATION-BY-DISTANCE WEB SERVICE 3.23 (Jensen et al., 2005).
Table S2.1 PARTITIONFINDER results for each of the three sequence regions (Marker) using
the specified number of base pairs (Bp). Results include the recommended model of
sequence evolution for each region and analysis program (BEAST, RAXML, All Models - for
all possible models available in the program).
Marker
Bp
All
Models
COI
460
TrN+G
TrN+G
GTR+G
12S
522
TrN+G
TrN+G
GTR+G
16S
440
TrN+I
TrN+I
GTR+G
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BEAST
RAXML
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Table S2.2 BEAST results for the estimated time to most recent common ancestor (TMRCA)
and 95% highest posterior density (95% HPD) in parentheses for: 1) all samples included in
the tree (TMRCA All); 2) just the P. penicillatus samples with no outgroups (TMRCA All
PP); 3) the Indo Pacific P. penicillatus samples (TMRCA IP); 4) the East Pacific P.
penicillatus samples (TMRCA EP); 5) the Red Sea P. penicillatus samples (TMRCA RS); 6)
the Red Sea P. penicillatus samples combined with the Indo-Pacific P. penicillatus samples
(TMRCA RS+IP). Details on the BEAST runs are provided in the methods section of
Appendix S2. In addition to running the program using a coalescent tree prior assuming
expansion growth, we also ran BEAST using an exponential growth model and it made little
difference.
Parameter
Tree prior
COI clock
TMRCA All
TMRCA All PP
TMRCA IP
TMRCA EP
TMRCA Red Sea
TMRCA RS + IP
M. Iacchei et al.
Prior/Result
(95% HPD)
Expansion growth
1.39%
6.892 (3.48, 10.82)
1.519 (0.890, 2.306)
0.299 (0.172, 0.461)
0.218 (0.091, 0.404)
0.256 (0.103, 0.468)
0.707 (0.416, 1.095)
Testing biogeographic boundaries in the pronghorn spiny lobster
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Table S2.3 Specimens included in this study from Panulirus penicillatus COI sequences previously
catalogued in GenBank. All sequences were trimmed to our COI sequencing region (internal to the
LCO1490 and HCO2198 primers in Folmer et al. (1994)), and checked for ambiguities prior to
analysis. Only sequences from adult individuals with a known sampling location were utilized.
Sequences for population-level analyses are organized by site (Sample Site), and include the number
of individuals used for each site (N), and the GenBank sequence identification number for each
individual (GenBank Sequence ID).
Sample Site
N
Chesterfield Islands
5
Fiji
5
Galapagos Islands
10
India
Indonesia
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4
GenBank
Sequence ID
AB610683
AB610684
AB610685
AB610686
AB610687
AB610678
AB610679
AB610680
AB610681
AB610682
AB610699
AB610700
AB610701
AB610702
AB610703
AB610704
AB610705
AB610706
AB610707
AB576722
JN418940
JQ229881
AB576713
AB576714
AB576715
AB576716
Sample Site
N
Japan
15
New Caledonia
6
Palau
Torres Strait
Tuamotu Archipelago
1
1
6
Testing biogeographic boundaries in the pronghorn spiny lobster
GenBank
Sequence ID
AB576698
AB576699
AB576700
AB576701
AB576702
AB576703
AB576704
AB576705
AB576706
AB576707
AB576708
AB576709
AB576710
AB576711
AB576712
AB610688
AB610689
AB610690
AB610691
AB610692
AB610693
AF339468
Pan1504.1
AB610669
AB610670
AB610671
AB610672
AB610673
AB610674
11
Table S2.4 Specimens used in the Panulirus penicillatus phylogeny. Included are all East Pacific
and Red Sea specimens, and a stratified random subset of specimens from the Indo-Pacific to cover
a broad geographical scope. Listed here are the region, collection location, and GenBank Accession
numbers for each specimen resolved for each of the three different mitochondrial DNA loci
included in the phylogeny: 12S, 16S, and COI.
Species
Region
Collection Location
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
East Pacific
East Pacific
East Pacific
East Pacific
East Pacific
East Pacific
East Pacific
East Pacific
East Pacific
East Pacific
East Pacific
East Pacific
Indo-Pacific
Indo-Pacific
Indo-Pacific
Indo-Pacific
Indo-Pacific
Indo-Pacific
Indo-Pacific
Indo-Pacific
Indo-Pacific
Indo-Pacific
Indo-Pacific
Indo-Pacific
Indo-Pacific
Indo-Pacific
Indo-Pacific
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
Indo-Pacific
Indo-Pacific
Indo-Pacific
Indo-Pacific
Indo-Pacific
Clarion Island
Clarion Island
Clarion Island
Clarion Island
Clarion Island
Clarion Island
Clarion Island
Clarion Island
Clarion Island
Clarion Island
Clarion Island
Galapagos Islands
Hawai'i Island
Hawai'i Island
Hawai'i Island
Fiji
Fiji
French Frigate Shoals
Lisianski Island
Maro Reef
Marquesas
Marquesas
Marquesas
Palau
Palau
Taiwan
Taiwan
Torres Straight,
Australia
Wake Island
Wake Island
Wake Island
Western Samoa
M. Iacchei et al.
GenBank Accession Numbers
12S
16S
COI
KT954773
KT954812
KT954855
KT954774
KT954813
KT954856
KT954775
KT954814
KT954857
KT954776
KT954815
KT954858
KT954777
KT954816
KT954859
KT954778
KT954817
KT954860
KT954779
KT954818
KT954861
KT954780
KT954819
KT954862
KT954781
KT954820
KT954863
KT954782
KT954821
KT954864
KT954783
KT954822
KT954865
KT954823
KT954866
KT954784
KT954824
KT954867
KT954785
KT954825
KT954868
KT954786
KT954826
KT954869
KT954787
KT954827
KT954870
KT954788
KT954828
KT954871
KT954829
KT954872
KT954830
KT954873
KT954789
KT954831
KT954874
KT954790
KT954832
KT954875
KT954791
KT954833
KT954876
KT954792
KT954834
KT954877
KT954793
KT954835
KT954794
KT954836
KT954795
KT954837
KT954796
KT954838
KT954878
KT954797
KT954798
KT954799
KT954800
KT954801
KT954839
KT954840
KT954841
KT954842
KT954843
Testing biogeographic boundaries in the pronghorn spiny lobster
KT954879
KT954880
KT954881
KT954882
KT954883
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Species
Region
Collection Location
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
P. penicillatus
Indo-Pacific
Indo-Pacific
Indo-Pacific
Indo-Pacific
Red Sea
Red Sea
Red Sea
Red Sea
Red Sea
Red Sea
Red Sea
Western Samoa
Western Samoa
Zanzibar
Zanzibar
Saudi Arabia
Saudi Arabia
Saudi Arabia
Saudi Arabia
Saudi Arabia
Saudi Arabia
Saudi Arabia
Outgroups
P. echinatus
P. echinatus
P. echinatus
P. echinatus
M. Iacchei et al.
GenBank Accession Numbers
12S
16S
COI
KT954802
KT954844
KT954884
KT954803
KT954845
KT954885
KT954804
KT954846
KT954886
KT954805
KT954847
KT954887
KT954806
KT954848
KT954888
KT954807
KT954849
KT954889
KT954808
KT954850
KT954890
KT954851
KT954891
KT954809
KT954852
KT954892
KT954810
KT954853
KT954893
KT954811
KT954854
KT954894
AF339454
AF337965
JQ412159
AB248090
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Figure S2.7 Condensed Maximum Likelihood tree for 829 COI sequences of Panulirus
penicillatus. Panulirus echinatus is included as an outgroup. Data were analysed using MEGA
6 (Tamura et al., 2013). Based on the PARTITIONFINDER results, the Tamura and Nei model
(gamma distributed) was employed, with bootstrap (BS) values calculated based on 100
replicates. Only nodes with BS values of >75 are labelled.
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Appendix S3 Acknowledgements
We greatly appreciate the assistance of the following individuals in collecting specimens, as
without their assistance, the breadth of this project would never have been possible: Jesse
Hapdei, Tammy Mae Summers, Mark Michael, Scott Aalbers, Kacy Lafferty, Chugey
Sepulveda, Shelley Jones, Joseph DiBattista, Mark Priest, Gustav Paulay, J.P. Hobbs,
Michael Berumen, Tin Yam Chan, Posa Skelton, John Fitzpatrick, Eric Tong, Zoltán Szabó
, Cecile Fauvelot and colleagues, Hal Koike, Paolo Usseglio, Calvin Gerry, Maria, and
Danny at the Seychelles Fishing Authority (SFA), Steve Karl, Jill Zamzow, Sam Kahn,
Meaghan Sundberg, Palmyra Crew: Kydd “Captain Chaos” Pollock, Dan “Danimal”
Wagner, Zachary “ Bohar” Caldwell, Ginny Kim, Captain Steve, Will, Wendy, Jen, and
Bobbi; Robert Moffitt, Kona Division of Aquatic Resources (especially Brent Carman and
Kosta Stamoulis), Derek Skillings, Jon Puritz, Carl Meyer, Woody Woodward, Krista
Woodward and Captain Woody’s Snorkel Charters, Terry Lilley, Jon Barretto, Geoff Walker,
Mac Poepoe, Joseph O’Malley, Jeff Eble, James Ashe, George Thompson and Fathom Five
Divers, Terry Buholm, Patrick Conley, Lawrie Provost, Tanya Beirne and Big Island
Spearguns, Kevin Schneider, Lisa Nelson, Megan Lamson, Bob Carrol, Skippy Hau, Victoria
Martocci, Eric Stein and the crew of Extended Horizons, Greg Concepcion, Nyssa Silbiger,
Megan Donahue, Toby Daly-Engel, Illiana Baums, Michael Stat, Megan Huggett, Jen
Salerno, Jon Dale, Melanie Hutchinson, Elizabeth Keenan, Molly Timmers, Scott Godwin,
Kelvin Gorospe, Ben Wainwright, Kevin Flanagan, Kim Tice, Miguel Castrance, Tim Clark,
Kim Weersing, Matt Craig, Anela Choy, Matt Ross, Megan Ross, Jon Yeh, Heather Leba,
Dana Crompton, Mike Musyl, Derek Smith, Anne Mooney, Kelly Gleason, Will Love, Josh
Copus, Iria Fernández-Silva, and Chris Bird. We thank the University of Hawai‘i Dive
Safety Program (Dave Pence, Kevin Flanagan, Keoki Stender, Tina Tsubota, Maria
Laamang), the scientists and crew of the NOAA ship Hi‘ialakai, NWHI Monument Staff, the
Hawai‘i Institute of Marine Biology office and fiscal staff for all of their logistical support
and facilitation of this project. We thank the Papahānaumokuākea Marine National
Monument, US Fish and Wildlife Services, and Hawai‘i Division of Aquatic Resources
(DAR) for coordinating research activities and permitting. We are grateful to Erik Conklin at
TNC and Amanda Meyer at the US Fish and Wildlife Service Palmyra for making collections
there possible. Collections in Saudi Arabia were conducted under the general auspices of the
King Abdullah University of Science and Technology (KAUST) with the assistance of the
KAUST Reef Ecology Lab. The Red Sea collections were funded by the KAUST Office of
Competitive Research Funds (OCRF) under Award No. CRG-1-2012-BER-002 and baseline
research funds to Michael Berumen.
Many thanks to Alexander Shiarella, Kaleonani Hurley, and Richard Coleman who
assisted with lab work. Also, thanks to Shaobin Hou and the University of Hawaii’s
Advanced Studies of Genomics, Proteomics, and Bioinformatics staff for their assistance
with DNA sequencing, as well as Sarah Daley, Rajesh Shrestha, Mindy Mizobe, and Amy
Eggers and the HIMB EPSCoR Core Genetics Facility. Many thanks to the ToBo Lab for
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Testing biogeographic boundaries in the pronghorn spiny lobster
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assistance both in the lab and for thoughtful contributions to this paper, especially Chris Bird,
Kimberly Conklin, Richard Coleman, Joseph DiBattista, Iria Fernandez-Silva, Zac Forsman,
Ingrid Knapp, Derek Skillings, Marieke Sudek, and Jon Whitney. This paper was greatly
improved thanks to comments from Jonatha Giddens, Charles Birkeland, Megan Donahue,
and Alison Rieser. The views expressed herein are those of the authors, and may not reflect
the views of the EPA, NOAA, or their sub-agencies.
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