Sampling, DNA extraction and additional data collection
Larvae, YoY and adult samples of ABFT from the Mediterranean Sea (n=
260), the Gulf of Mexico (n=210) and the Slope Sea (n=49) were obtained
from scientific surveys and commercial fisheries from these three
spawning grounds (Table S1; Figure 1A). From each adult and YoY, a
~1cm3 piece of muscle or fin tissue
sample was excised and immediately stored in RNA-later or 96% molecular
grade ethanol at -20ºC until DNA extraction. Larvae were collected with
a 60 cm diameter bongo net or a 2 x 1-meter frame net and immediately
preserved in 96% molecular grade ethanol. Genomic DNA was extracted
from about 20 mg of tissue or from whole or partial larvae (eyeballs or
tails) using the Wizard® Genomic DNA Purification kit (Promega, WI,
USA), following manufacturer’s instructions for “Isolating Genomic DNA
from Tissue Culture Cells and Animal Tissue”. Extracted DNA was
suspended in Milli-Q water and concentration was determined with
the Quant-iT dsDNA HS assay kit using a Qubit® 2.0 Fluorometer (Life
Technologies). DNA integrity was assessed by electrophoresis, migrating
about 100 ng of GelRed™-stained DNA on a 1.0% agarose gel. For selected
specimens, spawning capability was assessed by histologic inspection
following the criteria described in
Brown-Peterson et al. (2011).
Additionally, for females, ovulation within the past two days was
determined from identification of postovulatory follicle complexes,
which are assumed to degrade within 24-48 h
(McPherson 1991,
Schaefer 1996,
Aranda et al. 2011). For selected adult
specimens, sagittal otoliths were prepared for analysis of stable
isotope signatures of the otolith core (yearling period) according to
the protocol described in Rooker et al.
(2008) and analyzed for δ13C and
δ18O on an automated carbonate preparation device
(KIEL-III Thermo Fisher Scientific, Inc., Waltham, Mass.) coupled to a
gas-ratio mass spectrometer (Finnigan MAT 252 Thermo Fisher Scientific,
Inc.) at the University of Arizona. Stable isotopes of carbon and oxygen
(δ13C and
δ18O) are reported relative to the PeeDee Belemnite
(PDB) scale after comparison to an in-house laboratory standard
calibrated to PDB.