310 
Fishery Bulletin 11 7(4) 
were captured at 32.5-33.0°N and depths of 172-251 m, an 
area similar (32.3-33.9°N and depths of 155-302 m) to that 
fished by 3 longline fishermen who captured specimens 
(number of fish sampled [n]=1361) for the earlier study 
(see figure 1 in Wyanski et al., 2000). 
After collection, whole snowy grouper were weighed 
to the nearest gram and the maximum TL (from the 
anterior-most point on the head to the tip of the cau¬ 
dal fin with lobes compressed) and standard length 
were measured to the nearest millimeter. The left sag¬ 
ittal otolith was removed and stored dry prior to pro¬ 
cessing (the right sagitta also was removed when time 
permitted). Samples of gonad tissue were removed and 
fixed in 10-11% seawater-formalin for later histological 
processing. 
Age and growth 
Each otolith was embedded in paraffin (1979-1985) or 
an epoxy resin (1993-2012) and sectioned (thickness: 
0.5-0.7 mm) along a dorsoventral plane through the 
focus with a single high-concentration diamond wheel on 
a Buehler 5 IsoMet Low Speed Saw (Buehler, Lake Bluff, 
IL). Otolith sections were mounted on glass slides with 
thermoplastic or a liquid mounting medium and exam¬ 
ined under a Nikon dissecting microscope (Nikon Corp., 
Tokyo, Japan) (magnification: 7.5-63x) with reflected 
and transmitted light. Sample sections read before 1996 
were covered with cedar wood oil prior to examination. 
Otolith sections were examined by 2 readers inde¬ 
pendently and reexamined jointly when differences in 
age estimation occurred; aging was done without knowl¬ 
edge of specimen length and date or location of capture 
(Harris et al., 2002). If disagreement on age persisted, the 
specimen was eliminated from age and growth analyses. 
Age analyses were based on counts of annual increments 
(one opaque and one translucent zone). We examined 2364 
specimens from 1996 through 2012 and used samples from 
Wyanski et al. (2000) as a historical data set (zz=2263). 
Reproduction 
Histological processing of gonadal tissues followed stan¬ 
dard hematoxylin and eosin techniques. The posterior 
portion of each gonad was fixed for 7-14 d in a 10-11% 
seawater-formalin solution buffered with marble chips 
then transferred to 50% isopropanol for 7-14 d. Repro¬ 
ductive tissue was processed in automated tissue proces¬ 
sors and blocked in paraffin. Three transverse sections 
(thickness: 6-8 pm) were cut from each sample with a 
rotary microtome, mounted on glass slides, stained with 
double-strength Gill’s hematoxylin, and counterstained 
with Eosin Y. Sections were viewed under a Nikon com¬ 
pound microscope (Nikon Corp.) (magnification: 20-400x) 
by 2 readers without knowledge of specimen length or age 
5 Mention of trade names or commercial companies is for identi¬ 
fication purposes only and does not imply endorsement by the 
National Marine Fisheries Service, NOAA. 
and date or location of capture. The readers independently 
determined sex and reproductive phase by using histo¬ 
logical criteria described by Wyanski et al. (2000) and 
Harris et al. (2004); terminology follows a recent review 
by Brown-Peterson et al. (2011). If the assessments 
differed between readers for a section, it was viewed 
jointly by the readers. A specimen was eliminated from 
reproductive analyses when disagreement on its sex or 
reproductive phase persisted. To ensure that inactive 
females were assigned correctly to either the immature 
phase or the regenerating phase, the length-frequency 
histograms of suspected immature and early develop¬ 
ing or regenerating females were compared with the 
histograms for females with evidence of definite matu¬ 
rity (i.e., those that were developing, spawning capable, 
or regressing). The criteria put forward by Sadovy and 
Shapiro (1987) and Sadovy de Mitcheson and Liu (2008) 
were used for the diagnosis of hermaphroditism. 
Specimens with developing, spawning-capable, regress¬ 
ing, or regenerating gonads were considered sexually 
mature. Females of uncertain maturity were excluded 
from all reproductive analyses (Wyanski et al., 2000). 
Juvenile females were excluded from sex composition 
analyses to restrict data to the adult population (Coleman 
et al., 1996), and specimens undergoing sex transition 
(female to male) were considered males, unless noted, 
because these specimens would likely have spawned as 
males in the next spawning season (Sadovy and Shapiro, 
1987). In addition, sex composition analyses were restricted 
to data from gear types that effectively sample the adult 
population (i.e., Kali poles and longlines). Females were 
considered spawners if they possessed maturing oocytes 
(i.e., fusing of yolk globules, germinal vesicle migration 
and breakdown, and hydration), ovulated eggs, or pos¬ 
tovulatory follicle complexes. Spawning indicators were 
based on stages that are thought to last approximately 
60 h—12 h for oocyte maturation and hydration and 
48 h for postovulatory follicle complexes (Hunter and 
Goldberg, 1980; Fitzhugh et al., 1993). We use spawning 
frequency and spawning interval as defined in Lowerre- 
Barbieri et al. (2011). Spawning proportion, called 
spawning fraction by Lowerre-Barbieri et al. (2011), 
is the proportion of adult females that were spawning 
daily. Values of spawning proportion were proportionally 
reduced to a 24-h period. Batches were determined by 
multiplying the proportion of spawners by duration of 
the spawning season. 
Lunar phase analyses were performed to determine if 
the lunar cycle influenced the periodicity of spawning in 
snowy grouper. We combined all samples from all gear 
types that were collected during 1982-2012 and had 
stamps with the exact date and time of their collection. 
The new moon day was defined as day 0, and the full moon 
was defined as day 15. Individual specimens (n=371) were 
given a lunar day based on the new moon date for that 
month and year, their time of capture, and their reproduc¬ 
tive phase. For specimens undergoing oocyte maturation 
(excluding hydration), lunar day was calculated as capture 
date + 1 d because spawning would have most likely taken 
