Molecular Phylogenetics and Evolution 42 (2007) 38–46
www.elsevier.com/locate/ympev
Phylogeography and demographic history of the deep-sea Wsh
Aphanopus carbo (Lowe, 1839) in the NE Atlantic: Vicariance
followed by secondary contact or speciation?
Sergio Stefanni a,¤, Halvor Knutsen b
a
IMAR/DOP, University of the Azores, Cais Sta Cruz, 9901-862 Horta, Azores, Portugal
b
Institute of Marine Research, Flødevigen, N-4817 His, Norway
Received 30 March 2006; revised 19 May 2006; accepted 23 May 2006
Available online 3 June 2006
Abstract
Comparative phylogeography for the commercially valuable deep-sea Wsh Aphanopus carbo from a large area of the NE Atlantic
revealed remarkable patterns of concordance using two mtDNA markers. Two strongly supported phylogroups were identiWed from
complete sequences of the control region (731–733 bp) and partial sequences of cytochrome b (414 bp) In one of these groups, all
sequences from the Mid-Atlantic Ridge (Faraday seamount), mainland Portugal and Madeira were clustered together. The other group
constituted all the sequences from the southern coast of Pico island (Azores, central group). The remaining sampling localities had
sequences represented in both phylogroups. Although the two clades were strongly diVerentiated (ST D 0.8281 for the CR and
ST D 0.9083 for the cytb) no evidence for any geographical pattern in this structure, was found. Historical demography of the mitochondrial control region was analysed to clarify the phylogenetic signals embedded in each phylogroup. Mismatch distributions for both
clades suggested that both phylogroups were in agreement with sudden expansion models, and both with similar time estimates of expansion ( D 4.30 and D 3.45 for phylogroup one and two, respectively). A molecular clock based on cytb sequences was enforced and dating
of divergence for the two phylogroup was 412.5 KY, a time that coincides with geological events that might have caused a split in the original population of black scabbardWsh. Once climatic conditions and sea level were restored, the two separate populations came into contact again, leaving traces of the historical events in the non-recombinant mtDNA genes. An alternative hypothesis suggested is that two
species of scabbardWsh are present. The outcome from the comparison of the same mtDNA regions of the closely related Aphanopus intermedius from Angola clustered with the ones from phylogroup two (from the southern coast of Pico island, Azores). Therefore, these two
species may have overlapping distribution ranges and are found sympatrically in the Azores.
© 2006 Elsevier Inc. All rights reserved.
Keywords: ScabbardWsh; Phylogeography; Population structure; Speciation; MtDNA; Control region; Cytochrome b; Atlantic
1. Introduction
ScabbardWsh belong to the family Trachiuridae, a group
of deep-sea Wshes with worldwide distribution (Parin,
1986). Black scabbardWsh, Aphanopus carbo (Lowe, 1839),
live in temperate-cold Atlantic waters at depths between
200 and 1800 m (Tucker, 1956; Martins et al., 1987). The
*
Corresponding author. Fax: +351 292200411.
E-mail address: [email protected] (S. Stefanni).
1055-7903/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2006.05.035
more recently described species Aphanopus intermedius
(Parin, 1983; Nakamura and Parin, 1993) is found in tropical and sub-tropical waters at similar range of depths.
Aphanopus carbo is a valuable species in Wsh markets of
the United Kingdom, Ireland, northern France, Spain and
in particular Portugal with annual landings (probably
including A. intermedius oV Madeira) up to 7181 tons
(FAO, 2002). In Madeira, the specialized deep-water longliner Xeet is dedicated to this Wshery (Maul, 1950), with
catches up to 1000 tons per year (FAO, 2002), making up
55% of the total landings.
S. Stefanni, H. Knutsen / Molecular Phylogenetics and Evolution 42 (2007) 38–46
The information available on the biology, maturity,
spawning and growth of this species (Sanches, 1991; Martins et al., 1994; Figueiredo et al., 2003; Morales-Nin and
Sena-Carvalho, 1996) is scattered and at present, only one
genetic study limited to the eastern Atlantic has been conducted (Quinta et al., 2004). Although spawning areas are
still poorly known, it has been suggested that within the NE
Atlantic, black scabbardWsh from Madeira diVer from the
population oV of mainland Portugal and Hatton Bank
(Quinta et al., 2004).
In an eVort to investigate population history and structure in A. carbo, we have chosen a phylogeographical
approach using two molecular markers displaying diVerent
levels of polymorphism. The sampling localities extend
from the Mid-Atlantic Ridge to the European coast of Portugal, including the Azores, Madeira and several Atlantic
seamounts. We report results obtained with the rapidly
evolving non-coding mtDNA control region and the slower
evolving cytochrome b. In the discussion, sequences from
the same mtDNA regions are compared with the closely
related species A. intermedius.
2. Materials and methods
A total of 103 Wsh were included in the partial sequence of
the cytochrome b (cytb) gene (GenBank Accession Nos.
DQ408777–DQ408879) and 101 were used for the comparison of the control region (CR) (GenBank Accession Nos.
DQ408880–DQ408980). Samples were collected oV mainland
Portugal and Madeira, two north Atlantic seamounts (Sedlo
39
and Seine), one seamount from the Mid-Atlantic Ridge (Faraday), the Azores islands (Flores, Corvo, Graciosa, Fayal,
Pico and Sta Maria islands) and three neighbouring Wshing
banks (Condor, Azores and Princess Alice) (Fig. 1).
In addition, the sequence of Thunnus thynnus (GenBank
Accession No. AB097669) was included as the outgroup for
cytb sequences.
Small portions of white muscle or liver were immediately
Wxed in 95% ethanol before being stored at ¡20 °C.
MtDNA was extracted from white muscle following the
procedure of Sambrook et al. (1989), with slight modiWcations (Stefanni, 2000). Fragments of the mtDNA including
CR were ampliWed using the primers L-Pro1 (Ostellari
et al., 1996) and 1612SAR-H (Palumbi et al., 1991), while
for partial cytb the primers used were cytb-glu-L-cp and
cb2-H (Palumbi et al., 1991). The same pairs of primers
were also used for sequencing.
The thermal cycling proWle started with 94 °C for 2 min followed by 10 cycles of denaturation at 94 °C for 30 s, annealing
at 52 °C (for CR) or 50 °C (for cytb), extension at 72 °C for
1 min 35 s, and followed by an additional 30 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C (for CR) or 52 °C
(for cytb) and extension at 72 °C for 1 min 35 s with an increment of 3 s per cycle, with a Wnal extension at 72 °C for 7 min.
All sequences were aligned using SEAVIEW (Galtier
et al., 1996).
Hierarchical series of likelihood ratio tests (Huelsenbeck
and Rannala, 1997), implemented using Modeltest 3.06
(Posada and Crandall, 1998) were used to identify the
appropriate nucleotide substitution models.
Fig. 1. Map of sampling localities. In the enlargement are shown the location of all islands of the Azores. SD, Sedlo seamount; SN, Seine seamount; FD,
Faraday seamount; PT, mainland Portugal; MD, Madeira <>, Princess Alice Bank; X, Condor Bank; *, Azores Bank.
40
S. Stefanni, H. Knutsen / Molecular Phylogenetics and Evolution 42 (2007) 38–46
For CR, the appropriate model of nucleotide substitution was the HKY (Hasegawa et al., 1985) model with
invariable sites (I) and rate heterogeneity (G). The transition/transversion (ti/tv) ratio, proportion of invariable sites
(i) and gamma shape parameter () were estimated to be ti/
tv D 4.6879, i D 0.8811 and D 1.0342, respectively. The base
frequencies were estimated to be A D 0.3201, C D 0.2069,
T D 0.3056 and G D 0.1536. On the other hand, for cytb, the
appropriate model of nucleotide substitution was HKY
with no invariable sites and equal rate, ti/tv D 2 and base
frequencies were estimated to be A D 0.2456, C D 0.3043,
T D 0.2792 and G D 0.1710.
Phylogenetic trees of the haplotypes were constructed
using PAUP (SwoVord, 1999). The neighbour-joining (NJ)
algorithm (Saitou and Nei, 1987) was implemented to construct a phylogenetic tree from the maximum likelihood
(ML) distances estimated under the selected models. The
support for internal branches within the NJ tree was
assessed using the bootstrap (Felsenstein, 1985) with 1000
replicates. A maximum parsimony (MP) analysis was performed with heuristic search using the TBR options to Wnd
the best MP tree(s). The length (L), consistencies index (CI)
and retention index (RI) of the MP tree(s) were calculated
with parsimony-uninformative sites excluded. For the CR
trees, no outgroup was used for the rooting. The most similar sequences detected using BLAST (Altschul et al., 1990)
were still too distantly related; therefore, trees were rooted
at midpoint.
NJ, MP and ML all reconstruct the evolutionary history
of sequences under the assumption that a tree best represents their phylogenetic relationships. IntraspeciWc data,
however, typically consists of many, very similar sequences,
some of which may be ancestral, and whose phylogenetic
relationships are often more clearly and accurately represented by a network (Posada and Crandall, 2001). Consequently, the Median Joining Network (MJN) method
(Bandelt et al., 1999) was also used for both mtDNA
regions and networks were constructed using NETWORK
4.1.0.9 (Bandelt et al., 1999) based on default parameters.
Levels of genetic diversity and genetic signatures were
estimated using the program ARLEQUIN 2.000 (Schneider
et al., 2000). The number of segregating sites (K), haplotypic diversity (h), nucleotide diversity () and the parameters theta (), including their standard deviations (SD),
were calculated for each of the mtDNA regions. was
estimated, from the average number of pairwise diVerences
(Tajima, 1983) while S was calculated from the number of
segregating sites (Watterson, 1975) for each monophyletic
clade.
Because the HKY model is not implemented in ARLEQUIN the more inclusive Tamura–Nei (TrN) (Tamura and
Nei, 1993) model with the same parameters for ti/tv rate
and was used to calculate the genetic pairwise distances
between haplotypes.
The haplotypic correlation measure (ST) was assessed
considering all samples as belonging to a single population.
Additionally, values of ST were estimated between each
possible sample pair. SigniWcance levels were determined by
a non-permutation procedure 1000 times.
The mean sequence divergence between distinct clades
was corrected for within-group sequence divergence using
the function pAB(corrected) D pAB ¡ (pA + pB)/2, where
pAB is the mean sequence divergence between groups A
and B and pA and pB are the mean sequence divergence
within A and B, respectively (Nei, 1987; Avise and Walker,
1998) implemented in ARLEQUIN.
Molecular clock was enforced for the cytb, locus that has
been universally calibrated in multiple bony Wshes at 2%
(Bowen et al., 2001).
Demographic history was inferred using mismatch distribution analyses implemented in DNASP (Rozas et al., 2003).
Populations that have experienced a rapid expansion in the
recent past show unimodal distributions, while the ones at
demographic equilibrium present multimodal distributions
(Rogers and Harpending, 1992). Therefore, mismatch distribution analyses, under the assumption of selective neutrality,
were also used to evaluate possible historical events of population growth and decline (Rogers and Harpending, 1992;
Rogers, 1995). For each phylogroup, theoretical distributions
under the assumption of constant population size and the
sudden expansion model were compared to the observed
data. While with the former model, the population is
expected to be stable over time, in the latter model, the original population that was at equilibrium (0), generations
ago, suddenly expanded assuming the new size (1). Demographic Parameters (Li, 1977), 0 were estimated from the
data by considering 1 as inWnite (Rogers, 1995). Goodnessof-Wt was determined using estimates of the raggedness statistic (r), Tajima’s D test (Tajima, 1989), Fu’s Fs (Fu, 1996)
and R2 (Ramos-Orsins and Rozas, 2002) tests. These tests
were compared to the empirical distribution expected under
the neutral model as generated by 1000 simulated re-samplings. The null hypothesis of neutrality may be rejected
when a population has experienced demographic expansion,
bottlenecking or heterogeneous mutation rate (Tajima, 1996;
Aris-Brosou and ExcoYer, 1996).
3. Results
3.1. Sequence variability
Sequences from the complete mtDNA CR had a length
ranging from 731 to 733 bp. For complete alignment, four
indels were required, giving a total length of 733 bp to all
the sequences. The black scabbarWsh CR contained a total
of 49 polymorphic sites, 38 of which were parsimony informative. From alignment, 58 distinct haplotypes were
deWned among the 101 specimens, giving an overall haplotypic diversity of 0.9659 § 0.0106 and nucleotide diversity
of 0.0191 § 0.0096.
Sequences from the partial cytb had a length of 414 bp
and no indels were necessary for alignment. These fragments contained 12 polymorphic sites, eight of which were
parsimony informative. These sequences deWned 11 diVer-
S. Stefanni, H. Knutsen / Molecular Phylogenetics and Evolution 42 (2007) 38–46
ent haplotypes from the 103 individuals, giving an overall
haplotypic diversity of 0.9453 § 0.0114 and nucleotide
diversity of 0.0071 § 0.0041.
3.2. Phylogenetics
Neighbour-joining (NJ) as well as maximum parsimony
(MP) trees revealed two strongly supported monophyletic
phylogroups (Figs. 2 and 3). In “Group 1” all sequences
from Faraday Seamount, mainland Portugal and Madeira
clustered together. “Group 2” contained all the sequences
from Pico island (south coast). The remaining sampling
localities had sequences represented in both phylogroups
(see tables in Figs. 2 and 3).
The corrected sequence divergence between the two
phylogroups was 39.82% for the CR (5.08% for the
cytb). The sequence divergence within these two phylogroups estimated for the CR was 9.42% for “Group 1”
and 5.80% for “Group 2” (0.64 and 0.30%, respectively
for the cytb).
The maximum parsimony (MP) search for the cytb
sequences retained eight best MP trees (L D 79, CI D 0.9747,
RI D 0.900). The strict component consensus tree of the MP
trees, which displays all and only those groups found in all
the MP trees, resembles the NJ tree (MP trees not shown).
“Group 1” was represented by 39 haplotypes, for the
CR, (seven for cytb) with haplotypic diversity of
1.0000 § 0.0058 (h D 0.5620 § 0.0406 for cytb). This clade
had 28 polymorphic sites and nucleotide diversity of
0.0129 § 0.0067 (k D 6 and D 0.0016 § 0.0014 for cytb).
“Group 2” was smaller and included 19 haplotypes for the
CR (four from the cytb) with haplotipic diversity of
1.0000 § 0.0171 and nucleotide diversity of 0.0079 § 0.0044
(k D 3 and D 0.0008 § 0.0009 for cytb). The average mean
nucleotide distance between the two groups was 0.0084 for
the cytb gene, and enforcing the molecular clock, the divergent time between the two phylogroups was estimated to be
of about 421.5 thousand years ago.
Like the NJ tree, the Median Joining networks (MJ)
with D 0 to reduce to the minimum the number of reticulations, separated the two phylogroups. The number of mutations separating “Group 1” and “Group 2”, between even
the most similar haplotypes, were 17 for the CR and four
for the cytb (Fig. 4).
3.3. Historical demography
Genetic partitioning between Atlantic samples of black
scabbardWsh did not suggest the existence of any geographically structured populations. However, the presence of two distinct phylogroups represented a signature
of some important historical events. High values for ST
were also described for the two groups with highly signiWcant p-values (ST D 0.8281 for the CR and ST D 0.9083
for the cytb).
Results from mismatch distributions and neutrality tests
were compared between phylogroups and they were esti-
41
mated only for the CR because of the limited number of
haplotypes for the cytb gene.
The estimates of theta derived from the nucleotide diversity () and the number of segregating sites (S) were dissimilar in both phylogroups. “Group 1” had D 5.2038
(SD D 2.8587) and S D 6.1496 (SD D 2.1153), while for
“Group 2”, was 3.8363 (SD D 2.2546) and S D 5.4362
(SD D 2.1866).
Mismatch distribution for both groups appeared to be
unimodal (Fig. 5). Little values for Harpending’s Raggedness index associated to lack of signiWcance were of further
support for unimodal interpretation of the mismatch distribution in both clades (r D 0.019 with p D 0.121 and r D 0.077
with p D 0.637 for group 1 and 2, respectively).
Tajima’s (D D ¡0.930 with p D 0.189 for group 1 and
D D ¡1.400 with p D 0.077 for group 2) as well as Fu’s
(Fs D ¡50.288 with p D 0.000 for group 1 and Fs D ¡22.089
with p D 0.000 for group 2) neutrality tests resulted in negatives, but only the most sensitive of the two tests was statistically signiWcant. In both tests, negative values suggested
that populations experienced demographic expansion. On
the other hand, R2 test resulted in signiWcance only for
“Group 2” (R2 D 0.079, p D 0.130 for “Group 1” and
R2 D 0.070, p D 0.005 for “Group 2”). This may be due to
“Group 1” consisting of two subgroups, as revealed by the
MJ network (Fig. 4).
The tau values (), which indicate the location of the
crests of the mismatch distributions and provide a rough
estimate of when rapid changes in the populations had
occurred, were similar for group 1 and 2 ( D 4.30 and
D 3.45, respectively). However, it should be taken into
account that Rogers’ method of moments (Rogers, 1995),
based on the mean and variance of the observed mismatch
distribution, underestimates the expansion time compared
to other mutation models (Schneider and ExcoYer, 1999).
4. Discussion
This study of two mtDNA regions of black scabbardWsh is the Wrst to compare samples covering a large area
of the NE Atlantic. In both mtDNA fragments, CR and
cytb, phylogenetic analyses presented trees with two
strongly supported clades. Of particular interest is the
distinct phylogroup originating from the waters around
the island of Pico, in the central group of the Azores. Levels of nucleotide diversity from both mtDNA regions
were higher in “Group 1” than in “Group 2”, and this
may be due to either the diVerent number of haplotypes
in the two groups or to an historical event of isolation
followed by secondary contact with “Group 2”. Nevertheless, values of were compatible with estimates from
the same mtDNA regions for other groups of Wshes (Stefanni and Thorley, 2003; Stefanni et al., 2006; Muss et al.,
2001).
The genetic signature support vicariance followed by
secondary contact, probably from lineages originating allopatrically. In fact, overall values of nucleotide diversity
42
S. Stefanni, H. Knutsen / Molecular Phylogenetics and Evolution 42 (2007) 38–46
Pic
Fco
Gra
Sma
Fa*
SN
SD
BA^
FD
PT
Mad
1
ApcaAc16
58 Az16
ApcaMD04
MD4
ApcaMD03
MD3
ApcaSd6
SD6
ApcaSd4
SD4
ApcaAc17
Az17
ApcaA374
shA374
ApcaP345
shP345
Group 1
ApcaMD10
MD10
ApcaPT06
PT6
ApcaPT07
PT7
shS130
ApcaS130
ApcaAc15
Az15
ApcaA380
shA380
ApcaPT08
PT8
ApcaAc13
Az13
ApcaAc18
Az18
ApcaP190
shP190
ApcaSn7
SN7
ApcaSn9
SN9
ApcaSn11
SN11
ApcaSn2
SN2
ApcaSn10
SN10
ApcaMD08
MD8
ApcaM790
shM790
ApcaSn8
SN8
ApcaSd7
SD7
ApcaPA02
PA2
ApcaFD01
FD1
ApcaAc22
Az22
ApcaFD03
FD3
52
ApcaMD05
MD5
ApcaFD02
FD2
ApcaFD04
FD4
shS109
ApcaS109
ApcaSd2
SD2
APCABA03
BA3
100
ApcaA478
shA478
APCABA04
BA4
ApcaAc20
Az20
ApcaAc01
100
Az1
ApcaAc02
Az2
shA345
ApcaA345
ApcaAc21
Az21
APCABC01
BC1
ApcaB300
shB300
shA334
ApcaA334
ApcaAc28
Az28
ApcaAc24
Az24
Group 2
ApcaAc25
Az25
ApcaAc08
Az8
ApcaA999
shA999
ApcaA129
shA129
ApcaAc75
Az75
ApcaAc06
Az6
ApcaA412
shA412
ApcaAc07
Az7
ApcaAc26
Az26
1
1
1
1
1
2
1
2
1
3
1
1
1
1
2
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
2
1
1
1
3
1
1
1
2
1
1
1
1
8
2
1
1
1
3
1
2
1
1
1
1
1
2
N
1
1
1
1
1
1
4
6
1
1
1
3
1
4
1
1
1
4
1
1
1
1
1
1
3
1
1
1
1
1
1
1
1
1
2
1
1
3
1
1
1
1
3
1
1
2
2
1
1
1
1
16
2
1
1
3
1
1
0.005
Fig. 2. Neighbour-joining tree rooted at midpoint and constructed from sequences of the control region. Numbers above internal branches indicate bootstrap values out of 1000 replicates (only if greater than 50%). The table at the right shows the geographical distribution of the haplotypes. AZ, Azores; BA,
Azores Bank; SN, Seine seamount; FD, Faraday seamount; PT, mainland Portugal; MD, Madeira; Sh, Shared haplotyples; Pic, Pico islands; Fco, Flores
and Corvo islands; Sma, Santa Maria island; Fa*, Fayal island and Condor Bank; BA⵩, Azores and Princess Alice Banks; N, total haplotypes number.
Highlighted in grey are the samples encountered only in one of the two clades.
were very high ( D 0.019 § 0.010). Moreover, when the two
phylogroups were compared, pairwise values of ST were
large and statistically signiWcant for both mtDNA regions
(ST D 0.828 and ST D 0.903 for CR and cytb, respectively).
Isolation by distance is a model that could be considered.
Levels of divergence between phylogroups were extremely
high and some sequences derived from the same sampling
localities were falling into diVerent clades.
S. Stefanni, H. Knutsen / Molecular Phylogenetics and Evolution 42 (2007) 38–46
43
Pic Fco Gra Sma Fa* SN SD BA^ FD PT Mad
52
Group 1
60
1
SH05
1
Group 2
2
3
6
3
1
1
3
1
6
6
1
5
2
1
BA04
1
SN08
SH02
2
SH03
88
67 SH01
65
Az29
18
N
23
1
1
SN03
SH04
100
6
1
Az17
SN01
56
1
2
4
7
36
1
1
3
1
2
2
1
2
2
6
1
2
1
1
33
1
Tthyn
0.02
Fig. 3. Neighbour-joining tree constructed from sequences of the cytochrome b. Numbers above internal branches indicate bootstrap values out of 1000
replicates (only if greater than 50%). Tthyn D Thunnus thynnus sequence used as outgroup. The table at the right shows the geographical distribution of the
haplotypes. AZ, Azores; BA, Azores Bank; SN, Seine seamount; FD, Faraday seamount; PT, mainland Portugal; MD, Madeira; Sh, Shared haplotyples;
Pic, Pico islands; Fco, Flores and Corvo islands; Sma, Santa Maria island; Fa*, Fayal island and Condor Bank; BA⵩, Azores and Princess Alice Banks; N,
total haplotypes number. Highlighted in grey are the samples encountered only in one of the two clades.
a
Group 1
Group 2
A999
b
Group 1
Group 2
SH01
SH04
SH05
Fig. 4. Median Joining Network from: (a) complete CR sequences, and (b) partial cytb. Circles represent haplotypes and size is proportional to the relative
frequencies. Numbers of substitutions are indicated with bars when more than one. X D 10 substitutions and V D 5 substitutions. Dashed lines are showing
the two phylogroups, as in Figs. 2 and 3, while dot lines are indicating the two subgroups in “Group 1”. Only large shared haplotypes were coded following the criteria adopted in Figs. 2 and 3.
Date of divergence from cytb sequences suggested about
400 KY, timing that coincide with geological events that
might have created a vicariant barrier between two populations of black scabbardWsh. The late Pleistocene sees the
origins of the youngest islands of the central group and
aging by K/Ar isotope suggests 0.73 MY for Fayal,
0.55 MY for São Jorge and 0.25 MY for Pico (Fig. 4 in
França et al., 2003). During the same period of time, a series
of glacial events took place causing considerable changes in
water temperature and sea level (Droxler et al., 2003).
Approximately 400 KY ago, Atlantic organisms were experiencing an unusual warm period with higher sea level,
44
S. Stefanni, H. Knutsen / Molecular Phylogenetics and Evolution 42 (2007) 38–46
b
a
0.25
Tau=4.30
Fs= -50.288 (p<0.001)
0.20
Tau=3.45
Fs= -50.288 (p<0.001)
0.15
0.10
0.05
0.00
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21
Group 1
Group 2
Fig. 5. Mismatch distributions from CR sequences, represented as solid bars for the observed pairwise diVerences, continuous line the expected distribution for having passed through a demographic expansion and dashed line for expected distribution under constant population size model. (a) for group 1
and (b) for group 2, as represented in Fig. 2. The past demographic parameter Tau () is indicated for each group along with the Fu’s Fs test for selective
neutrality and its p value.
when compared to the present (Droxler et al., 2003). Subsequently, climatic conditions changed cooling the water and
dropping the sea level approximately 150 m. The joint
eVects of which, in proximity to the central group of the
Azores islands may have strengthened the physical barrier
between the two populations. Subsequently, once climatic
conditions were restored, contact between the two diVerentiated populations was resumed, leaving a genetic signature
in the non-recombinant genes of the mtDNA.
Mismatch distributions were unimodal for both phylogroups with approximate similar values. However, it is
known that values estimated using Rogers’ method of
moments (Rogers, 1995) underestimate up to 50% of the
expansion time () for fast mutating sequences (Schneider
and ExcoYer, 1999). Introducing the appropriate corrections, the mutation rate of the CR was estimated to be 3.8–
4.2%. Data on generation time were extrapolated from data
on reproductive cycle (Figueiredo et al., 2003) and otoliths
reading (Morales-Nin and Sena-Carvalho, 1996) for A.
carbo.
These mutation rate estimates for CR are acceptable and
similar values have been encountered in other Wsh species
(Zhu et al., 1994; McMillian and Palumbi, 1997; Donaldson
and Wilson, 1999; Sato et al., 2003; Stefanni et al., 2006).
Date of divergence from CR sequences suggested about
1 MY, early Pleistocene. This is an earlier period from the
one suggested by cytb estimates and it was characterised by
inter-glacial conditions with high global temperature and
little ice locked in polar caps (Lambeck et al., 2002).
Another equally plausible possibility that might support
the outcome of the study is that we are dealing with two
separate species. Fairly recently, a new species has been
described, A. intermedius (Parin, 1983), distinguishable
from A. carbo by smaller adult size (100 cm vs 110 cm),
higher number of vertebrae (102–108 vs 97–100) and species distribution (Froese and Pauly, 2005). No distinguishable diVerences in meristic characters and coloration exist
between the two species. Therefore, we suggest here that the
two species may live sympatrically in the Azores. Unfortunately, all the specimens collected for this study were not
preserved for taxon investigation; it was originally taken
for granted that all Wsh were from the same species. However, we were able to sequence the same fragments from the
mtDNA from three specimens of A. intermedius collected
oV Angola (GenBank Accession Nos. DQ408981–
DQ408985) and the outcome indicated that these sequences
were extremely similar, when not identical, to those in
“Group 2”. For cytb, specimen one and two showed identical sequence to the shared haplotypes SH01 while specimen
three was identical to SH02. Sequences from CR were available only for specimens one and two. The Wrst was shared
with haplotypes A999 while the second was unique and it
diVered from haplotypes A129 for one substitution in position 255 (an A replaces a C) from the complete alignment.
Geological events described above might have been the
driving force to induce allopatric speciation and high values for sequence divergence between the two phylogroups
(39.82% for the CR and 5.08% for the cytb) are of further
support to this hypothesis.
Focusing the attention on “Group 1”, the phylogroup
including all sequences from Madeira, mainland Portugal
and Faraday Seamount, we did not Wnd any evidence of the
population structure reported by Quinta et al. (2004). A
possible explanation is that in the collection of scabbardWsh
from Madeira used by Quinta et al. (2004), included not
only A. carbo, but also the closely related A. intermedius,
which can be found in Madeiran waters too (FAO, 2002).
Further study is necessary to clarify the taxonomy of these
two species. The level of interaction between these two sympatric species is also in need of investigation. There is limited knowledge regarding hybridization events and its
consequences in marine Wshes (but see Nielsen et al., 2003),
and special attention is necessary when dealing with species
of commercial interest.
S. Stefanni, H. Knutsen / Molecular Phylogenetics and Evolution 42 (2007) 38–46
The presence of sympatric mtDNA clades within species
has been interpreted as evidence for vicariance followed by
reinvasion (Avise, 2000) and examples can be found for
highly migratory species such as the Atlantic mackerel
(Nesbo et al., 2000; Zardoya et al., 2004), swordWsh (Bremer
et al., 1995, 2005; Buonnacorsi et al., 2001; Graves and
McDowell, 2003), blue marlin (Buonnacorsi et al., 2001),
sailWsh (Graves and McDowell, 2003), Atlantic bonito
(Viñas et al., 2004) and Atlantic big eye (Martínez et al.,
2006). Our present knowledge of the biology of this species
is too limited to consider the scabbardWsh a highly migratory species.
Acknowledgments
We thank Dr. G. Menezes (DOP), A. Canha (DOP), Dr.
R. Castilho (University of Algarve, Portugal) and J. Maniscalco (SUNY, Stony Brook, USA) for help, discussion and
comments. A great thank goes to Dr. R. Hanel (IFM-Geomar, Germany) for donating samples of A. intermedius. We
are also extremely grateful to captains, crew and technicians
onboard the R/V Arquipelago and R/V G.O. Sars for their
excellent contribution while at sea. All molecular work was
supported by the research project OASIS funded by the
European Commission (Contract No. EVK3-CT-200200073-OASIS). Samples from the Azores were donated from
the research project PESCPROF, project co-Wnanced by EU
Interreg III B program. Samples from Faraday Seamount
were donated from the research project MAR-ECO, a pilot
project within the Census of Marine Life. Part of the contribution comes from MarBEF (Network of Excellence:
“Marine Biodiversity and Ecosystem Functioning”—Contract No. GOCE-CT-2003-505446). S.S. is a postdoctoral fellow funded by FCT (Foundation for Science and
Technology, Portugal) ref IMAR/FCT–PDOC/002/2001
and SFRH/BPD/14981/2004. IMAR/DOP is funded
through the pluri-annual and programmatic funding scheme
as research unit #531 and associate laboratory #9.
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