Griffiths, AM, G. Machado-Schiaffino, E. Dillane, J. Coughlan, JL ...

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1


N
ORTH
A
MERICAN
J
OURNAL OF
A
QUACULTUR
E
(2012)

I
N PRESS
.

C
OMMUNICATION


Population
genetic comparison
s

among

cobia (
Rachycentron canadum
)

from
the
northern Gulf of Mexico
,

U.S.
western
Atl
antic, and Southeast Asia


John R. Gold,
Melissa
M.
Giresi

and

Mark A. Renshaw

Center for Biosystematics and Biodiversity, Texas A&M University, College Station, Texas
77843
-
2258


Jin
-
Chywan Gwo

Department of Aquaculture, Taiwan National Ocean
University, Keelung 20224, Taiwan


Abstract


Nuclear
-
encoded microsatellites and mitochondrial (mt)DNA sequences were assayed from
cobia (
Rachycentron canadum
) sampled from waters offshore of Virginia (U.S. Atlantic),
Mississippi and Louisiana (Gulf of Mex
ico), and Taiwan (Southeast Asia). Global exact tests
and analysis of molecular variance revealed that fish from U.S. waters were homogeneous for
alleles and genotypes at 2
7 nuclear
-
encoded

microsatellites and for a
352

base
-
pair fragment of
mitochondrial

(
mt
)
DNA; fish from Taiwan differed significantly in both genetic markers.
Use of

cobia

brood stock from Southeast Asia in
U.S. aquaculture
facilities
is
not recommended.
Results are compatible with use of cobia brood

stock
from

either the U.S. Atlantic or Gulf of
Mexico
. Caveats to this are discussed.




*Corresponding author: goldfish@tamu.edu



2




3



Cobia,
Rachycentron canadum
,

is a large
, coastal

pelagic fish that is widely distributed in
tropical, subtropical
,

and warm temperate seas except
for

the e
astern Pacific (Shaffer and
Nakamura 1989).


In the western Atlantic, cobia are distributed from Massachusetts and
Bermuda to Argentina (Briggs 1958)
but are mo
st common in the Gulf of Mexico
(
Shaffer and
Nakamura 1
989
).

Cobia is a prized sport fish because of its large size and
food quality,
with

the
majority

of the cobia
caught

in U.S. waters
attributed to recreational fish
ing

(
Shaffer and
Nakamura 1989;
Franks et al. 1999
)
.
A
quaculture
of cobia
worldwide
has been
expanding
rapidly
, especially in Southeast Asia

where roughly 80%
of the world’s cobia production is in
Taiwan and China (FAO 2007). Currently,
facilities for cobia aquaculture

exist in
several

U.S.
Atlantic and Gulf C
oast states (Benetti
et al.

200
8
).

Desirable attributes of cobia for culture
include ease of spawning and larval rearing, rapid growth
,

high survival through the first year,
and low feed
-
conversion ratios (Benetti and Orhun 2002).


A

priorit
y

of the Draft Aquaculture Policy of the
U.S.
National Oceanic and Atmospheric
Administration (NOAA 2011)

is

to ensure protect
ion of

wild species
.

Th
is

concern relates in
part to matching genetic profiles of cultured fish to
local
wild stock
s

of the same species
in order
to mitigate potential ne
gative genetic impacts

of escapees or of hatchery
-
released fish in
restoration programs
(
Triantafyllidis

et al. 2007).
Data

on stock structure of cobia
in U.S waters
is
sparse
.
Franks et al. (1991) and
Hammond (2001)

reported

movement
of cobia
between the
Gulf of Mexico
(hereafter Gulf) and the U.S. Atlantic coast (hereafter U.S. Atlantic)
,

and Biesiot
et al. (1993) could not distinguish between mtDNA haplotypes of cobia sampled from localities
in the Gulf and
U.S. Atlantic.

However, t
here are
differences in growth rate
,

adult size
, and
longevity

between
cobia in the

Gulf and U.S. Atlantic (
Burn
s

et al. 1998
).

4



In this note, we
report tests of genetic homogeneity among cobia sampled from offshore
waters in the Gulf

and

U.S. Atlantic. The study was
designed
in part
to
ask whether cobia in the
Gulf and U.S. Atlantic
are

genetically distinguishable, and if so, to
identify genetic markers that
could

be used to match aquaculture
-
produced fish genetically with wild stocks

in

the same
geographic
region
. The study also was
in response to queries from individuals and private
companies interested in culturing cobia in

the Gulf and
who had asked
whether cobia brood
stock from Taiwan
were compatible genetically with cobia in U.S.
waters.

M
ATERIALS AND
M
ETHODS


Fin clips were taken from
131

wild
-
caught
cobia sampled
during the summers of
2010 and
2011

from localities offshore of
Virginia (35), Mississippi (46),
Louisiana

(14), and Taiwan (36).
Fin clips were

fixed in
2
0% DMSO
buffer (Seutin 1991).
Whole genomic DNA was extr
acted
using
a

C
helex resin (Bio
-
rad®) extraction protocol
(
Estoup et al. 1996
) and all individuals

were
genotyped
at

28

nuclear
-
encoded microsatellites. Polymerase chain reaction (PCR) primers,
repeat motif
s, and annealing
conditions

may be found in Renshaw et al
.
(
2005
)
.
A
mplification
products were electrophoresed
using

an ABI 377 automated sequencer (Applied Biosystems Inc.,
Foster City, CA)
. G
el images
were analyzed in
G
ENESCAN

v 3.1.2

(
Applied
Biosystems)

and
al
leles were scored with
G
ENOTYPER

v 2.5
(
Applied Biosystems).

Genotypes at the 2
8

microsatellites for each individual assayed are available at
http://agrilife.org/wfsc/doc/

under the
file name

‘Cobia microsatellite genotypes.’
A total of
352

bases from the
cytochrome
b

(cyt
b
)

protein
-
coding mitochondrial gene were acquired from five individuals from
each of the four
sample localities
. PCR primers used were

H15497 and L15080 (Finnerty

and Block 1995)
.

The
PCR protocol was
as follows:
initial denaturation at 95
o
C for 2 min; 38 cycles of denaturation at
95
o
C for 30 sec, annealing at 53
o
C
for 45 sec
, elongation at 72
o
C for 90 sec; and final elongation
5


at 72
o
C for 20 min.

PCR amplifications were electrophoresed on 2% agarose gel
s
. S
uccessful
amplifications were band
-
cut and cleaned with QIAquick Gel Extraction Kits (Qiagen).
Fragments were sequenced
(
both directions
)

using
the amplification primers

a
nd ABI BigDye
T
ERMINAT
OR

v

1.1 (Applied Biosystems).


P
roducts were cleaned with Sephadex columns and
electrophoresed on an ABI

3100 automated DNA sequencer (Applied Biosystems).
Sequences
were edited and aligned with
S
EQUENCHER

v
3.0 (Gene Codes Corporation
).


Tests of
conformance of genotypes at each microsatellite to Hardy
-
Weinberg expectations
and tests of genotypic equilibrium between pair
s

of microsatellites were executed utilizing
G
ENEPOP

v 4.0.10
(Raymond and Rousset 1995; Rousset

2008). Exact probability tests
employed a Markov chain approach (Guo and Thompson 1992) with
10
,
000 dememorizations,
1,0
00 batches and
10
,
000 ite
rations per batch. Sequential B
onferroni
correction

(Rice 1989) was
used to adjust
for multiple tests
carrie
d out simultaneously. Occurrence of null alleles, stuttering
,

and large allele dropout was evaluated utilizing
M
ICROCHECKER

v 2.2.3
(va
n Oosterhout et al.
2004)
.

Estimates of allelic richness
,

gene diversity, and F
IS

were generated using F
-
STAT

v
2.9.3.2

(Goudet 1995).
Homogeneity among samples in allelic richness and gene diversity was
tested using Friedman rank tests as implemented in
S
YSTAT

v 13 (Systat Inc., Evanston, IL);

tests
between pairs of sample localities employed Wilcoxon signed
-
rank tests
(
also

implemented in
S
YSTAT
)
.
Global tests of h
omogeneity of
allele

and genotype distributions (microsatellites) and
haplotype distribution

(mtDNA)

employed

exact tests
,

as implemented in
G
ENEPOP
,

and analysis
of molecular variance (
A
MOVA
), as implemented in
A
RLEQUIN

v 3.5.1.3
(
Excoffier and Lischer
2010).
Exact probabilities were estimated
using the same

Markov chain approach
as above
;

s
ignificance of
F
ST

(from
A
MOVA
) was assessed by permutation (
10,000

replicates)
for
both
microsatellites

and mtDNA.
Exact tests also were used to test homogeneity
of
allele and
6


genotype distributions (microsatellites) and haplotype distribution (mtDNA) between pairs of
samples, using the same Ma
rkov chain approach as above.
The degree of div
ergence in
microsatellites and mtDNA between pairs of samples was estimated as
F
ST

and
Ф
ST
, respectively,
using
A
RLEQUIN
.

R
ESULTS

AND DISCUSSION


Summary statistics for microsatellite
s
at all four sample localities
are given in Appendix
Table 1
.
Significant departures from Hardy
-
Weinberg equilibrium expectations before
Bonferroni correction were detected at 11 microsatellites;
only
two (
Rca
1BD10


Virginia;

Rca
1H01


Taiwan
) remained sig
nificant following correction
. F
IS

values at both microsatellites
were positive, indicating a deficit of heterozygotes. Subsequent tests of homogeneity in allelic
richness, gene diversity, and allele and genotype distributions across sample localities were run
with and without these two m
icrosatellites; results in
all

cases remained essentially unchanged,
hence results reported
(below)
for these tests include
d

Rca
1BD10 and
Rca
1H01
.
Analysis with
M
ICROCHECKER

indicated possible occurrence of null alleles at
Rca
1BF06 (Louisiana and
Taiwan),

Rca
1BD10 (Virginia), and
Rca
1E05 and
Rca
1H01 (Taiwan).

A total of
42

of 1
,
51
2

pairwise tests of genotypic equilibrium were significant before sequential Bonferroni correction;
excluding comparisons of
Rca
1
BE08A and
Rca
1BE08B
, which
w
ere

expected
to be
significant
a
s
these

microsatellites
were

isolated from the same clone (Renshaw et al.
2005)
,
no

tests
remained significant after correction. Because of the expected (and confirmed) tight linkage
between
Rca
1BE08A and
Rca
1BE08B,
Rca
1BE08B

was omitted from

all
subsequent analyses
.


A
verage allelic richness (± SE) and average (unbiased) gene diversity

(± SE)
, respectively,

across all microsatellites
were
4.69 ±
0.68

an
d 0.445 ± 0.
066

(Virginia), 4.66

±
0.70

and 0.436 ±
0.
067

(
Mississippi), 4.6
5

±
0.68

and 0.
462

± 0.
066

(Louisiana), and 7.01 ±
0.69

and 0.69
1

±
7


0.0
47

(Taiwan).
Both allelic richness and gene diversity differed significantly (Friedman’s rank
test) among the four sample localities:
Q
[3]

=
21.189
,
P
=
0.000 (allelic richness) and
Q
[3]

=
21.320
,

P

= 0.000 (gene diversity).
W
ilcoxon’s

signed
-
rank tests revealed significant differences
(
P

= 0.000
)
in
both parameters only in pairwise comparisons involving the sample from Taiwan.


Because of the small sample size (
n

= 14) from Louisiana, exact tests o
f allele and genotype
distributions
(microsatellites)
were carried out to determine if the two samples from the Gulf
(Louisiana and Mississippi) could be pooled into a single (‘Gulf’) locality.
Exact tests for allele
s

and genotypes were non
-
significant (
P

=
0.
530

and
P

= 0.636
, respectively
)
; consequently, all
remaining tests involving microsatellites utilized three localities


U.S. Atlantic (Virginia), Gulf
(Mississippi + Louisiana), and Taiwan
.

Global e
xact tests of homogeneity of allele and genotype
d
istributions among
the three
localities were significant (
P

= 0.000 for both allele and genotype
distribu
tions)
. Results from A
MOVA

also indicated significant genetic heterogeneity

(
F
ST

=
0.29
2
,
P

=
0.000). Exact tests of p
air
wise comparison
s

(T
able
1
) indicated that only
comparisons between fish from Taiwan and fish
from
U.S. localities different significantly both
before and after sequential Bonferroni correction.
The p
airwise comparison between fish from
the Gulf of Mexico and Virginia was not sign
ificant
.


The spatial distribution of recovered mtDNA haplotypes
and GenBa
nk
numbers

are

given in
Appendix Table 2
.
Haploptype #1

was the most common in U.S. waters; all five fish assayed
from Taiwan possessed Haplotype #
4

(not found in U.S. waters).
Sig
nificant
heterogeneity in
haplotype distribution was indicated by a global exact test (
P

= 0.
000
) and
A
MOVA

(
Ф
ST

= 0.
623
,
P

= 0.
000
).
Exact tests of pairwise comparisons
of

haplotype distributions

indicated that only
comparisons between fish from Taiwan and fish from U.S. localities different significantly both
before and after sequential Bonferroni correction

(data
not shown
).

8



Based on the foregoing, cobia in U.S. waters differ markedly in both nuclear
-
encoded
micros
atellite genotypes and mitochondrial DNA sequences from cobia in waters off Taiwan.
The degree of genetic
divergence

indicates virtually no gene exchange between cobia in the
western Pacific and western Atlantic, despite the species’ pelagic life style and broad
distribution. This
result
is not surprising, given that genetic differences between ocean basins
have been r
eported for several conspecific pelagic fish species (Díaz
-
Jaimes 2010).
Cobia from
waters off Taiwan also were more genetically variable than cobia from U.S. waters, having
significantly greater allelic richness and gene diversity.
R
easons for this are
not known.
Regardless, to the extent that (presumed) selectively neutral microsatellite alleles and variable
mtDNA sequences serve as surrogates for alleles at genes impacting adaptively important life
-
history and production traits, usage of brood fish fr
om Southeast Asia in aquaculture facilities in
U.S.
waters

would appear to be precluded.


Cobia sampled from waters off Virginia, Mississippi, and Louisiana were genetically
homogeneous based on assays of both microsatellite genotypes and mtDNA haplotypes.

This
finding is consistent with observed migration patterns and with tag
-
and
-
release studies. Briefly,
adult
cobia
appear to overwinter primarily off the Florida Keys and then undergo seasonal
migrations during the spring both to the north along the U.S
. Atlantic Coast and to the north and
west into the Gulf of Mexico (Shaffer and Nakamura 1989; Franks et al. 1991)
,
while

limited
tagging studies (Franks et al. 1991; Hammond 2001) indicate fairly regular mixing between the
Gulf and
U.S. Atlantic. Interes
tingly, there are reports of cobia overwintering in deep waters in
the Gulf (Franks et al. 1991
)
and of tagged fish in both the
Gulf and U.S.
Atlantic that were
recaptured near the release locality over one year later (Franks et al. 1991; Hammond 2001).
R
egardless, the range of sample localities in this study
approximate

the range where facilities for
9


cobia aquaculture occur in U.S. waters (
Benetti and Orhun 2002
), suggesting that brood stock
from either the Gulf or U.S. Atlantic could be
used in cobia aqu
aculture
in either region.


There are two caveats

to the above. First, microsatellites generally are presumed to be
selectively neutral and not necessarily indicative of
geographic patterns at
selectively adaptive
genes

that
affect quantitative traits
important to
life
-
history or
aquaculture prod
uction (McKay
and Latta 2002).
This means simply that there could be adaptively useful alleles at coding genes
in cobia that differ between the
Gulf and
U.S. Atlantic
, and it has been reported that cobia from
t
he
Gulf and
U.S. Atlantic
di
ffer
in growth rate, adult size, and longevity (Burns et al. 1998).
Second,
even though a dataset of 2
7

microsatellites
is rather large
compared to

most genetic
studies of stock structure in marine fishes (e.g.,
Carson et al.
2009; Griffiths et al. 2010
; Saillant
et al. 2012
),
cobia possess 24 haploid chromosomes (Jacobina et al. 2011)
, meaning that there is
relatively little genome coverage even with 2
7

markers. Future studies interested in this issue
will need to
utilize nex
t
-
generation sequencing technology (Mardis 2008; Stapley et al. 2010) to
achieve wider genome coverage.
A final note is that cobia
are

being raised
to market
elsewhere
in the western Atlantic, including
Martinique, Mexico, Belize,
Panama, and
Brazil (Bene
tti et al.
200
8
).

Generating

more complete
genetic profiles
of cobia from
additional localities

in the
western Atlantic could be useful relative to permitting
decisions regarding

brood stock
selection
for use
in U.S. facilities.

ACKNOWLEDGMENTS


We thank
B. Falterman
,
J. Franks
, and

J. Graves for assistance in procuring specimens from
U.S. waters
, and K. Burns

and

J
. Franks for comments on a
draft of the manuscript
. Work was
supported by TexasAgriLife under Project H
-
6703

and by the National Science Counc
il, Taiwan
.

10


This paper is number
XX

in the series ‘Genetics Studies in Marine Fishes’ and Contribution No.
XXX

of the Center for Biosystematics and Biodiversity at Texas A&M University


.


11


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14


Table 1. Pairwise F
ST
values (upper diagonal) and exact probabilities
that F
ST

=

0 (lower
diagonal) for pairwise comparisons of three samples of cobia (
Rachycentron canadum
). Boldface
indicates significance before and following sequential Bonferroni correction. Samples are U.S.
Atlantic (Virginia), Gulf of Mexico (Mississip
pi and Louisiana, pooled), and Taiwan.

________________________________________________________________________
___


Sample




U.S. Atlantic Gulf of Mexico Taiwan

U.S. Atlantic




---







0.
003






0
.
373

Gulf of

Mexico



0.
0
96






---






0.
387

Taiwan





<0.001






<0.001






---

___________________________________________________
________________________