Supporting information
Catarino D, Knutsen H, Veríssimo A, Olsen E, Jorde PE, Menezes G, Sannæs H, Stanković D,
Company JB, Neat F, Danovaro R, Dell’Anno A, Rochowski B, Stefanni S (2015) The Pillars of
Hercules as a bathymetric barrier to gene-flow promoting isolation in a global deep-sea shark
(Centroscymnus coelolepis), Molecular Ecology.
Appendix S1
Material and Methods
PCR amplification
PCR amplifications were based on protocols by Veríssimo et al. (2011a, 2011b) with some
modification as follow:
For mtDNA Control Region, PCR reactions of 20 µL of volume containing 0,5 µL of each
primer (10 µM), 1 µL of DNA template (10-20 ng), 10 µL of PCR Master Mix (Promega
Corporation, USA) and 8 µL of dH2O were conducted under the following thermal cycle
conditions: initial denaturation of 2 min at 94°C, followed by 35 cycles of denaturation for 30 s
at 94°C, annealing for 50 s at 58°C, extension for 1 min 35 s at 72°C, with a final extension of 7
min at 72°C.
Microsatellite amplification was performed in 10 µL reaction volumes, each containing 0.12–
0.30 µL of the forward primer (10 µM) with a fluorescent label (VIC, PET, NED or 6-FAM),
0.12–0.30 µL of the reverse primer (10 µM), 1 µL of the DNA template (10-20 ng), 0.16 µL of
Taq DNA Polymerase (5 U/ µL; Qiagen, Germany), 3 µL of dNTPs (1 mM), 1 µL of 10x PCR
Buffer (Qiagen) and dH2O to reach the final volume. Due to low yield and quality of the DNA
in some of the samples (e.g. Madeira and Ireland samples) 5x Q-Solution (Qiagen) and MgCl2
(25mM; Qiagen) were add to the PCR master mix following the supplier recommendations.
Thermal cycling conditions consisted in an initial denaturation of 4 min at 95°C, followed by
35-45 cycles of denaturation for 45 s at 95°C, 60 s at the corresponding annealing temperature
(Table S2), extension for 60 s at 72°C, with a final extension of 15 min at 72°C.
Statistical analyses
Deviations from the HWE were found initially for locus Ccoe8. This deviation was due to
homozygosity excess at three alleles and affecting seven individuals (three in the IRE/UK and
four in MAD samples). Removing these seven individuals from the dataset removed all
significances (Ccoe8 FIS=0.0596, P=0.13, after FDR correction, Table S2), suggesting that the
departure was due to technical issues rather than a biological phenomenon. Therefore these
seven individuals were eliminated from the locus analyses (coded as missing data) in the final
dataset.
References
Veríssimo A, McDowell JR, Graves JE (2011a) Population structure of a deep-water squaloid
shark, the Portuguese dogfish (Centroscymnus coelolepis). ICES Journal of Marine Science, 68,
555–563.
Veríssimo A, Moura T, McDowell J, Graves J, Gordo L, Hoelzel R (2011b) Isolation and
characterization of ten nuclear microsatellite loci for the Portuguese dogfish Centroscymnus
coelolepis. Conservation Genetic Resources, 3, 299–301.
Table S1. Portuguese dogfish sampled collection sites with averaged coordinates in decimal degrees within sampling area. The average capture depth (range)
in meters was calculated based on the collected sharks in this study for the respective number (N) of individuals. Whenever the capture depth information was
not available for the sharks sampled from one area, the average capture depth for that area was retrieved from the literature in order to provide a value for
average temperature. This table is informative and is only partially linked to the data used for the analyses of the life history traits.
N with capture
depth info
Sampling area
Code
Azores
AZ
38.657
-29.018
203
South of the Azores
SAZ
30.199
-28.569
27
UK/Ireland
UK/IRE
57.167
-9.417
―
Mauritania
MAU
16.761
-17.130
46
Mediterranean
MED
40.914
3.224
199
Portugal mainland
PT
38.633
-10.167
―
Madeira
MAD
33.718
-14.345
33
Australia
AUS
-42.167
144.283
―
Average Latitude Average Longitude
Average capture depth
Reference for
Average Temperature
(m)
capture depth
(°C)
1364
this study
6.17
(925-2475)
1518
this study
5.4
(1225-1825)
1200
Clarke et al. 2001a,b
6
(500-1900)
1445
this study
4.8
(1215-1598)
2137
this study
13.21
(1500-2853)
1200
Figueredo et al. 2005
10.8
(800-1600)
1519
this study
6
(1175-1975)
1025
Daley et al. 2002
5
(650-1400)
Reference for average Temperature
Azores Oceanographic Data Center,
AZODC
Azores Oceanographic Data Center,
AZODC
New & Smythe-Wright 2001
Colman et al. 2005
Tecchio et al. 2011
World Ocean database, NOAA
Azores Oceanographic Data Center,
AZODC
Trawler database, CSIRO
AZODC, Azores Oceanographic Data Center, IMAR/DOP, University of the Azores, Oceanographic section. http://oceano.horta.uac.pt/azodc/
Clarke MW, Conolly PL, Bracken JJ (2001a) Aspects of the reproduction of the deep water sharks Centroscymnus coelolepis and Centrophorus squamosus
from west of Ireland and Scotland. Journal of the Marine Biological Association of the United Kingdom, 81, 1019–1029.
Clarke MW, Conolly PL, Bracken JJ (2001b) Biology of exploited deep-water sharks West of Ireland and Scotland. North Atlantic Fisheries Organization
Scientific Council Report Doc. 01/108, 18 pp. Available at http://archive.nafo.int/open/sc/2001/scr01-108.pdf [accessed 15 January 2014].
Colman JG, Gordon DM, Lane AP, Forde MJ, Fitzpatrick JJ (2005) Carbonate mounds off Mauritania, Northwest Africa: status of deep-water corals and
implications for management of fishing and oil exploration activities. In: Cold-water Corals and Ecosystems, 1st edn (eds Freiwald A, Roberts M), pp. 417–
441. Springer-Verlag, Berlin Heidelberg.
Daley RK, Stevens JD, Graham K (2002) Catch analysis and productivity of the deepwater dogfish resource in southern Australia. CSIRO Marine Research,
Fisheries Research and Development Corporation (FRDC), and NSW Fisheries, Australia, FRDC Project No. 1998/108.
Figueiredo I, Machado PB, Gordo LS (2005) Deep-water Sharks Fisheries off the Portuguese Continental Coast. Journal of Northwest Atlantic Fisheries
Science, 35, 291–298.
New AL, Smythe-Wright D (2001) Aspects of the circulation in the Rockall Trough. Continental Shelf Research, 21, 777–810.
Tecchio S, Ramírez-Llodra E, Sardà F, Company JB, Palomera I, Mechó A, Pedrosa-Pàmies R, Sanchez-Vidal A (2011) Drivers of deep Mediterranean
megabenthos communities along longitudinal and bathymetric gradients. Marine Ecology Progress Series, 439, 181–192.
Trawler database, CSIRO URL: www.cmar.csiro.au/trawler/
World Ocean database, NOAA URL: www.nodc.noaa.gov/OC5/SELECT/dbsearch/dbsearch.html
Table S2. Nuclear microsatellite loci combined in multiplexes (Set) and annealing temperature
(Ta). Estimated quantities: HT, total heterozygosity; A, number of alleles scored per locus; RS,
mean allelic richness (minimum sample size of 13 individuals); FIS deviations from HardyWeinberg equilibrium; FST, level of genetic differentiation among all sampled locations 1)
without Mediterranean, 2) with Mediterranean. Significant at alpha = ***0.001 after FDR
approach.
HT
0.825
A
11
Rs
7.2
FIS
0.0244
FST1
0.0007
0.0564***
0.893
28
10.6
0.0211
0.0001
0.1275***
0.933
23
12.6
0.0596
0.0005
0.0762***
0.811
15
6.6
0.0108
-0.0033 0.0941***
0.823
19
8.4
-0.0063
-0.0006 0.0401***
0.954
56
17.4
-0.0042
0.0013
0.0107***
0.843
26
10.2
-0.0054
0.0032
0.0193***
0.819
11
6.4
0.0066
0.0042
0.0699***
0.639
9
3.9
-0.0094
0.0025
0.0538***
0.681
5
3.8
0.0172
0.0055
0.0541***
SacaGA11
0.756
18
6.7
0.0185
0.0047
0.0407***
Overall
0.816
0.0123
0.0016
0.0581***
Locus
Ccoe24
Ccoe75
Ccoe8
Set
1
Ta (°C)
59
Ccoe9
Ccoe13
Ccoe55
2
Ccoe61
57
56
Ccoe16
Ccoe2
Saca3853
3
54
FST2
Table S3. Summary statistics used in the computations in the DIYABC software. Statistics with an observed value calculated were the statistics used to build
the reference table, the model choice analyses and to estimate the parameters. The observed value is the calculated value in the real dataset. For each scenario
the x indicates a summary statistic where the simulated value is similar to the observed value; the x followed by a * or ** indicates summary statistics where
observed and simulated were significantly different at 0.05 or 0.01, respectively. Summary statistics that were not used to build the reference table (i.e.
statistics with no observed value calculated) were then used for model checking analysis.
mtDNA CR
Microsats
mtDNA CR
Microsats
Summary statistics
One Sample
Mean number of alleles
Mean genetic diversity
Mean size variance
Mean Garza-Williamson's M
Number of haplotypes
Number of segregation sites
Mean of pairwise differences
Variance of pairwise differences
Tajima's D
Private segregating sites
Mean of numbers of the rarest
nucleotide at segregating sites
Variance of numbers of the rarest
nucleotide at segregating sites
Two Sample
Mean number of alleles
Mean genetic diversity
Mean size variance
Fst
Classification index
Shared allele distance
2
(dµ) distance
Number of haplotypes
Number of segregation sites
Mean of pairwise differences (W)
Mean of pairwise differences (B)
Fst (Hudson et al ., 1992)
Observe d value
Scenario 1
Sce nario 2
Sce nario 3
Sce nario 4
ATL
MED ATL
MED ATL
MED ATL
MED ATL
MED
19.8182
0.8191
10.0000
0.6568
x
x*
x*
x*
x
x*
x
x
x
x
x
x
x
x
x
x
0.7543
19.0000
14.0000
0.6962 x**
5.0000 x
4.0000 x
x
x
x
x**
x
x
x
x
x
x**
x
x
x**
x
x
x**
x
x
x*
x
x
x**
x
x
x
x
x
-1.6078
-1.6750 x**
x*
8.4615
1.5000
x*
x*
x
x
x
x
x
x*
20.0909
0.8053
x*
x*
0.1037
2.3309
x*
x*
x*
1.6748
x
x
x
x
x*
x*
x
x
x
x*
x
x
x
x
x
21.0000
16.0000
x
x
x
x
x
x
x
x
0.1108
x
x
x
x
Table S4. Pairwise FST results for temporal replicates and combined sampled locations using the 11
nuclear loci. In all pairwise comparisons P>0.05.
Pairwise comparison
FST
AZ2003 vs. AZ2011
-0.0016
IRE vs. UK
-0.0002
MED1 vs. MED2
0.0008
MED1 vs. MED3
0.0011
MED2 vs. MED3
-0.0028
MED2001 vs. MED2009
0.0011
MED2001 vs. MED2012
0.0006
MED2009 vs. MED2012
0.0025
MEDjuveniles vs. MEDadults
0.0006
Table S5. IM model estimates: time since divergence (t), effective population sizes of ancestral and
descended populations (ΘANC, ΘMED, ΘATL) and migration rate (mATL<>MED) with the lower
and upper boundaries of the 95% highest posterior density (HPD) interval. All parameters are scaled
by mutation rate. HiPt is the bin with the highest value in the histogram, while HiSmth is the bin
with the highest value after smoothing. The estimated size of the ancestral population was on the
upper border of the prior range (100), however the slope of the curve starts to come down towards
the upper range of the prior (*). The upper level of 95% confidence interval for migration rate is
outside the upper limit of the prior (1.000), however a single clear peak was still observed within the
range of the priors (**).
Estimated rates with 95% confidence intervals
t
HiSmth (95%Lo–95%Hi)
0.495 (0.195 – 1.125)
ΘATL
HiPt (95%Lo–95%Hi)
15.55 (10.05 – 27.15)
ΘMED
HiPt (95%Lo–95%Hi)
4.05 (2.35 – 6.45)
ΘANC
HiPt (95%Lo–95%Hi)
> 100 (86.15 - *)
mATL<>MED
HiPt (95%Lo–95%Hi)
0.756 (0.394 – **)
Table S6. Parameter estimates, with standard error (SE) and P-values, of the generalized linear
models used for describing Portuguese dogfish maturity ogives. Explanatory variables are area of
capture (A), sex (S), body length (L) and body weight (W). Females and Atlantic were coded as zero
(reference level) in the models.
Model
Length ogive
Weight ogive
Model term
Parameter estimate
SE
P-value
Intercept
−23.78
3.10
<0.001
APacific
−0.32
0.58
0.58
AMediterranean
11.53
1.50
<0.001
SMale
−2.15
1.21
0.07
L
0.22
0.03
<0.001
SMale×L
0.10
0.02
<0.001
Intercept
−7.15
1.06
<0.001
APacific
−0.69
0.62
0.27
AMediterranean
5.60
0.98
<0.001
SMale
0.65
0.37
0.07
W
0.70
0.11
<0.001
SMale×W
1.03
0.16
<0.001
Fig. S1DIYABC schematic representation of the four alternative scenarios used for Approximate
Bayesian computation simulations. All scenarios assume that at present time t(0), there are two
populations Atlantic (Pop 1) and Mediterranean (Pop 2) and that they diverged from a single
population in the past at t generations. N1b and N2b indicate periods where population size was
allowed to change. Scenario 1: constant population size over time; scenario 2: MED population size
is allowed to change after divergence; scenario 3: ATL population size is allowed to change after
divergence; scenario 4: both populations sizes (ATL and MED) are allowed to change after
divergence.
Fig. S2 Genetic diversity patterns across all localities, based on 11 microsatellite loci. Sampled sites
abbreviations are given in Table 1.
Fig. S3 Graphical representation of the allele frequencies and haplotype frequencies for the 11
microsatellites markers and mtDNA CR for the three main regions analysed. All the Atlantic
sampling sites were merged within one Atlantic main graph in order to simplify interpretation of the
results. To simplify the graphs only alleles names and alleles frequencies equal or higher to 10% are
shown.
Fig. S4 Relative posterior probability of the demographic scenarios estimated using the logistic
regression approach. This approach was based on the 1% simulated datasets closest to the observed
dataset. Scenario 1: green; scenario 2: red; scenario 3: blue; scenario 4: purple. Scenario 1, 2 and 3
are overlapping at the bottom of the graph (see Table 3 for probability values and 95% CI).
Fig. S5 DIYABC posterior probability distributions (green lines) for the six parameters
estimated: Atlantic Ne (N1), Mediterranean Ne (N2), Atlantic Ne after divergence (N1b),
Mediterranean Ne after divergence (N2b), time since divergence (t), and t1 under the best
supported scenarios (S4). See Fig. S1 for scenarios schematic representation.
Fig. S6 DIYABC analyses with estimated distributions for the genetic markers and control quality of each scenario. The posterior probability distributions
(green lines) and modal values (95% Credible Interval) for the microsatellites and mtDNA mean mutation rates. Principal component analyses (PCA) using
10000 simulations with the fitting of the models to the dataset, using a different set of summary statistics from those used to estimate the parameters.