DeMartini et al.: Body sizes at maturation and at sex change, and spawning seasonality and sex ratio of Hyporthodus quernus 
125 
(Fig. 1) — the leeward low island region of the Hawai- 
ian Archipelago — individuals are protogynous sequen- 
tial hermaphrodites, like most epinephelids elsewhere 
(Shapiro, 1987; Sadovy and Domeier, 2005; Sadovy de 
Mitcheson and Liu, 2008). To date, however, data on 
body size at first sexual maturity, spawning seasonality, 
and adult sex ratio of Hawaiian grouper have not been 
adequately quantified, and information on its possible 
size-at-sex-change and sex and gonadal allocation pat- 
terns are lacking. 
Our primary goal is to provide a comprehensive case 
study of the reproductive life history of Hawaiian grou- 
per in the NWHI in which all of the aforementioned 
metrics are estimated (one of the few such studies so 
for any grouper). All of these metrics in combination 
are key elements necessary for conducting comprehen- 
sive reproductive studies of groupers elsewhere. We 
also evaluate the gonad indices of Hawaiian grouper 
in detail to explore possible relations among gonadal 
allocation pattern, male type, adult body size, and ag- 
gregation spawning. 
Materials and methods 
Fish collections and measurements 
Hawaiian grouper were collected during three (“early,” 
“mid-term,” “recent”) capture series. All captures were 
made with similar (hydraulic-powered) handline gear. 
Early specimens were caught by fisheries-independent 
research vessels during the period from May 1978 to 
August 1981. Mid-term specimens were obtained by 
fisheries-independent research cruises during August 
and December 1992 and June and September 1993. 
Most recent (October 2005- June 2008) specimens were 
purchased from contracted Hawaii-based commercial 
bottomfishing vessels and were fishery-dependent. All 
fish were collected from the NWHI, except 26 speci- 
mens (3.5% of all fish) collected from Niihau-Kauai at 
the northwestern edge of the MHI (Fig. 1). The limited 
availability of fish of a sufficient range of sizes from 
elsewhere in the MHI precluded separate analyses for 
fish from this region of relatively high fishing effort. For 
our analyses, we used a total 745 fish collected from the 
NWHI and Niihau-Kauai only (Fig. 1). 
Body length (total length, TL, from tip of snout to 
posterior margin of caudal fin) was measured (mm) for 
each fish either aboard ship (research cruises) or at a 
fisheries laboratory ashore (for commercial specimens). 
The error of length measurements was about 0.5 cm. 
Fish were weighed (total round weight, RW, including 
all viscera, to 10 g) by using a bench scale. Additional 
details of specimen and shipboard data collection are 
provided elsewhere (Nichols and DeMartini 2 ; Ever- 
son 3 ) The Kolmogorov-Smirnov two-sample test (Zar, 
1984) was used to compare body-length distributions 
and numbers of fish by month of collection between 
the total sample and a subsample used for histological 
examination. 
Gonad extraction and processing 
Both left and right lobes of gonads were either dissected 
from freshly caught specimens aboard the research 
ship or from iced fresh fish (commercial specimens) at 
a shore laboratory in Honolulu. Before further process- 
ing, gonads were weighed (to the nearest g). The gonads 
of 611 fish were examined microscopically. Histologi- 
cal slides were prepared for 604 fish including early 
and recent specimens (100 and 504 fish, respectively); 
no gonads from mid-term specimens were examined 
microscopically. In preparation for histological exami- 
nation, gonads were first fixed in 10% (seawater buff- 
ered) formalin for a minimum of 2 months. A segment 
from the mid-region of one lobe (either left or right, 
random choice, including lumen and gonad wall) was 
then cut and placed in a histology cassette and sent 
to a contracted laboratory for further processing. The 
contractor then dehydrated tissues in an alcohol series, 
embedded them in paraffin wax, sectioned them at 
6-7 microns, stained them with Harris’s hematoxy- 
lin, and counter-stained them with eosin (Hunter and 
Macewicz, 1985). One slide of 2-6 (mode and median 
of 4) successive sections was prepared for each gonad 
specimen. The gonads of several ripe female specimens 
from the early series were prepared and examined for 
reproducibility of reproductive scores based on sec- 
tions from the anterior, middle, and posterior regions 
of ovaries. 
Sexual identity and reproductive status 
Gonad appearance based on conventional macroscopic 
criteria (West, 1990) was insufficient to distinguish 
either sexual identity or maturation, and all determi- 
nations of sexual identity and maturation for Hawai- 
ian grouper presented herein were based on standard 
microscopic criteria (West, 1990). After sexual identity 
was determined, each specimen was scored for reproduc- 
tive stage by following a protocol (Table 1) similar to 
those used by others (Pears et al., 2007, and references 
therein) to evaluate gonadal stages of protogynous epi- 
nephelids. Key criteria included the presence of oogonia 
and primitive testicular tissue in the gonads of bisexual 
juveniles, the presence of brown body atretic structures 
in testes of mature sex-changed males as evidence of 
prior female function, and the occurrence of transitional 
individuals whose gonads contained both developing 
male and regressing female tissue types (Sadovy de 
Mitcheson and Liu, 2008). Fish were classified as mature 
females if ovaries were categorized at stages 3 through 
6 (Table 1). The staging of oocytes was complemented by 
estimation of the median diameter of the largest mode 
of viable oocytes present in ovarian tissues by using a 
technique developed for Hawaiian bottomfishes (Lau 
and DeMartini, 1994). Size distributions of all yolked 
oocytes were described for a random subsample of ripe 
stage-5 fish and evaluated for multimodality by using 
the Kolmogorov-Smirnov one-sample test (Zar, 1984). 
Males were considered mature if at stage 7 or greater 
