Evaluation of spawning frequency in a Mediterranean sardine population (Sardina pilchardus sardina)

Marine Biology (2003) 142: 1169 1179 DOI 10.1007/s00227-003-1028-5 K. Ganias Æ S. Somarakis Æ A. Machias A. J. Theodorou Evaluation of spawning frequency in a Mediterranean sardine population (Sardina pilchardus sardina) Received: 23 January 2002 / Accepted: 3 January 2003 / Published online: 21 March 2003 Ó Springer-Verlag 2003 Abstract The postovulatory follicle method was used to assess the frequency of spawning of sardine (Sardina pilchardus sardina) in the Central Aegean Sea (eastern Mediterranean) in November December 1999 and 2000. A field-based aging key for postovulatory follicles (POFs) was developed, and preliminary evidence is presented of a temperature influence on the degeneration of POFs. Bias in the fraction of day-0 spawners (females that had, were, or would spawn the night of sampling) with respect to sampling time was identified and attributed to spawning behavior. An appraisal of consistency in spawners fractions and sex ratio, based on four sets of sequential trawl hauls, showed a considerably high small-scale variability, which suggested that spawning schools are structures limited in space and/or ephemeral in time. Sampling gear, i.e. commercial purse seines and research pelagic trawls, did not differ significantly with respect to spawners fractions. The fractions of day-1 and day-2 females were the same during both years, and their combination resulted in more precise, composite estimates of spawning frequency. In both years, the Mediterranean sardine spawned every 11 12 days. The incidence of spawning increased significantly with fish size. When compared to all estimates available for other sardine genera, species and subspecies, the spawning frequency of the Mediterranean sardine was lower; this Communicated by R. Cattaneo-Vietti, Genova K. Ganias (&) Æ A. J. Theodorou Laboratory of Oceanography, University of Thessaly, Fytokou Street, 38446 Magnisia, N. Ionia, Greece E-mail: kganias@uth.gr Tel.: +32-421-093053 Fax: +32-421-093054 K. Ganias Æ S. Somarakis Æ A. Machias Institute of Marine Biology of Crete, P.O. Box 2214, 71003 Iraklio, Crete, Greece S. Somarakis Laboratory of Zoology, Department of Biology, University of Patras, 26500 Patras, Rio, Greece was attributed to the synergetic effect of oligotrophy and smaller body sizes in the Mediterranean. Introduction Most clupeoids like anchovies and sardines are indeterminate spawners, producing multiple batches of eggs in a protracted spawning period (Blaxter and Hunter 1982). The spawning frequency or fraction of females spawning per night is an important reproductive variable of indeterminate spawners and a crucial parameter of the daily egg production method (DEPM), a widely used direct method of assessing the spawning biomass of pelagic fishes (Lasker 1985; Hunter and Lo 1997). Besides biomass estimation, series of spawning frequency values for fish stocks can lead to new insights into their reproductive biology, particularly when such values can be compared between species and stocks or habitats and seasons (e.g. Alheit 1993). Since the early 1980s, and based on the pioneer work of Hunter and Goldberg (1980), methods have been developed for the histological estimation of spawning frequency that are based on the state of deterioration of the postovulatory follicles (Hunter and Macewicz 1985). A prerequisite for applying the postovulatory follicle method is validation of the duration that postovulatory follicles (POFs) can be detected in field samples. To develop aging criteria for POFs, fish can be spawned in the laboratory and sampled at known times after spawning (e.g. Hunter and Goldberg 1980). An alternative method is to examine a time-series of field-collected material in relation to peak spawning time (Goldberg et al. 1984; Macewicz et al. 1996). Ideally, the effect of temperature should be taken into account, because the rate of POF resorption is temperature dependent (Fitzhugh and Hettler 1995, and references therein). The estimation of spawning frequency is often subjected to sampling bias, which is usually introduced by the typical behavior of pelagic fish to form spawning

1170 groups or schools and synchronize spawning within such aggregations. This might result in time-related changes in sex ratio as well as the fraction of day-0 spawners (females that had, were or would spawn the night of sampling) (e.g. Picquelle and Stauffer 1985). Alheit (1993) speculates on the possibility of biased estimates when collecting samples from commercial fisheries and indicates the importance of collecting independent samples for estimating spawning frequency. However, short-term temporal or spatial variability in spawning fractions has never been assessed. The frequency of spawning might vary seasonally and be affected by the age structure of the population or food abundance (Alheit 1993; Hunter and Lo 1997; Somarakis 1999). Nevertheless, at least for some species or stocks, the spawning frequency can be relatively constant as long as environmental conditions do not change (McEvoy and McEvoy 1992; Hunter and Lo 1997). In the present study we assessed the spawning frequency of the Mediterranean sardine Sardina pilchardus sardina in the eastern Mediterranean (Central Aegean Sea) during November December 1999 and 2000. A field-based aging key for POFs is presented, along with preliminary evidence of a temperature influence on POF degeneration. Sampling bias related to adult spawning behavior was appraised, and spawning frequency estimates were subsequently corrected. Inter-annual, intergear and small-scale variability in spawning fractions were analyzed, and their dependence on fish size was evaluated. Information based on previous applications of the postovulatory follicle method on several stocks of Sardina spp. (Cunha et al. 1992; Garcı a et al. 1992; Lago de Lanzo s et al. 1998; Anonymous 2000; Quintanilla and Pe rez 2000) and Sardinops spp. (Macewicz et al. 1996; Ward et al. 2001) reveals various intricacies of the method that might affect accuracy and precision of the respective spawning frequency estimates. These include: (1) inter-laboratory differences concerning the interpretation of POFs (Anonymous 2000), (2) indication of extended daily spawning periods (Fig. 2 in Quintanilla and Pe rez 2000), which might complicate the correct assignment of females as to the day of spawning, and (3) inconsistency in the incidence of bias with respect to day-0 females, which are occasionally over-represented (García et al. 1992, Galicia), under-represented (Quintanilla and Pe rez 2000), or sampled equally to other daily spawners classes (Cunha et al. 1992). Materials and methods Sample collection and analysis Adult sardines (Sardina pilchardus sardina) were collected at nighttime from coastal locations of the Central Aegean Sea during November December 1999 and 2000 (Tables 1, 2), i.e. the peak of the spawning period (Ganias et al. 2001b; Somarakis et al. 2001). Sampling was carried out on board the commercial purse seine fleet, as well as on the R.V. Philia", by means of a small pelagic trawl (mouth opening: 10 m, cod-end mesh size: 10 mm) towed near the surface (5 40 m) for 45 60 min, in a stepped oblique manner, at 3 4 knots. The placement of samples was attempted to be proportional to the local fish concentrations. A total of 23 purse seine samples and 5 trawl samples were collected in 1999, and 19 purse seine and 11 trawl samples were collected in 2000 (Fig. 1; Tables 1, 2). Each sample consisted of a random collection of 1.5 2 kg of sardines. Fish were fixed on board in 10% neutral buffered formalin. Sex ratio, i.e. the fraction of females by numbers, was determined from a subsample of 50 specimens per sample. Spawning frequency of reproductively active females, i.e. the fraction of females spawning per night, was estimated by the postovulatory follicle method (Hunter and Macewicz 1985). A total of 15 20 females (or all the females, when their number was <15) were randomly selected from each sample, weighed for total weight (0.1 g) and, subsequently subjected to histological analysis of their gonads. Both ovaries were removed, weighed and a piece of tissue (about 0.1 g) was removed from the center of each ovary, dehydrated, cleared in xylol and embedded in paraffin. Sections (4 6 lm) were cut and stained with Mayer s hematoxylin and eosin Y (Clark 1981). Histological scoring included the maturation stage of the most advanced group of oocytes and the presence and histological characteristics of atretic and postovulatory follicles. Ovaries of active females contained a sufficient number of yolked oocytes for a spawning in contrast to ovaries of inactive females, which were in atretic states 2 or 3 (Hunter and Macewicz 1985). Ovaries of immature females contained only oocytes in the primary growth phase. Postovulatory follicle aging The degeneration of postovulatory follicles of the Mediterranean sardine has never been studied in the field, neither has it been calibrated in the laboratory. In order to assign specimens according to the day of spawning, we examined the morphology of postovulatory follicles in detail, in relation to time of collection, the daily spawning period (1900 2300 hours; authors unpublished data on the occurrence of early egg stages in the plankton, see also the Results section) and existing descriptions of POFs for other sardine populations and species (Iberian sardine, S. p. pilchardus: Pe rez et al. 1992; Californian sardine, Sardinops sagax: Goldberg et al. 1984; Japanese sardine, Sardinops melanostictus: Murayama et al. 1994). Inter-annual or regional differences in temperature regimes and their effect on the rate of POF degeneration were addressed by analyzing vertical profiles of temperature taken in the same area during concurrent egg surveys (Somarakis et al. 2001; authors unpublished data). Simple spawning fraction estimates The spawning frequency might be estimated by the fraction of day- 0, day-1, day-2, etc. spawners. Given the daily spawning period (1900 2300 hours) and our sampling times (1800 0500 hours), day-0 spawners were fish that had, were or would spawn the night of sampling (with hydrated oocytes and/or POFs aged <10 h); day-1 spawners were fish that had spawned the previous night (with POFs 23 34 h); day-2 spawners were fish that had spawned two nights before (with POFs 47 58 h); etc. We used the ratio estimator (Picquelle and Stauffer 1985) to estimate the fraction of day-0, day-1, day-2, etc. spawners such that: y ¼ P n P n m i m i y i ð1þ

1171 Table 1 Sardina pilchardus sardina. November December 1999 samples. Fraction of females with gonads classified as immature/ inactive, hydrated, running, or with different kinds of postovulatory follicles (POFs). Samples marked in bold correspond to trawl hauls made sequentially over the same towing route in a single night (EG Evoikos Gulf; SG Saronikos Gulf; PS purse seine; TR pelagic trawl; n number of females examined histologically) Sample Region Gear Date Time (hours) n Sex ratio Fraction of: Immature/ Inactive Hydrated Running POF-0 POF-1 POF-2 No POFs en1 EG PS 13 Nov 1999 0300 15 0.409 0.13 0.13 0.73 en2 EG PS 14 Nov 1999 0400 15 0.600 0.27 0.27 0.07 0.40 en3 EG PS 18 Nov 1999 0300 15 0.567 0.33 0.13 0.07 0.07 0.40 en5 EG PS 19 Nov 1999 0430 15 0.510 0.13 0.07 0.80 sn1 SG PS 24 Nov 1999 0330 20 0.744 0.05 0.05 0.10 0.20 0.60 sn2 SG PS 26 Nov 1999 0300 15 0.540 0.07 0.07 0.07 0.80 sn6 SG PS 27 Nov 1999 0030 15 0.440 0.53 0.07 0.40 Tr01 EG TR 28 Nov 1999 1900 20 0.680 0.05 0.10 0.10 0.75 en7 EG PS 28 Nov 1999 2130 15 0.548 0.13 0.20 0.67 sn5 SG PS 29 Nov 1999 2230 16 0.289 0.06 0.44 0.13 0.38 en8 EG PS 30 Nov 1999 0300 15 0.735 0.07 0.07 0.07 0.13 0.67 sn8 SG PS 30 Nov 1999 0400 15 0.360 0.27 0.07 0.67 sn7 SG PS 30 Nov 1999 2345 15 0.306 0.47 0.53 ed09 EG PS 2 Dec 1999 0300 15 0.429 0.27 0.07 0.07 0.60 Tr03 SG TR 3 Dec 1999 1900 20 0.982 0.15 0.85 Tr04 SG TR 3 Dec 1999 2115 20 0.340 0.35 0.10 0.05 0.50 Tr05 SG TR 4 Dec 1999 0100 17 0.640 0.18 0.18 0.65 ed10 EG PS 5 Dec 1999 2330 15 0.380 0.80 0.13 0.07 ed11 EG PS 8 Dec 1999 0315 15 0.406 0.20 0.33 0.20 0.27 sd2 SG PS 9 Dec 1999 0000 17 0.720 0.06 0.06 0.12 0.76 sd3 SG PS 9 Dec 1999 0430 16 0.583 0.06 0.13 0.06 0.75 sd4 SG PS 9 Dec 1999 0500 16 0.673 0.06 0.25 0.06 0.63 ed12 EG PS 11 Dec 1999 0230 15 0.500 0.07 0.07 0.13 0.73 ed14 EG PS 11 Dec 1999 0230 15 0.620 0.27 0.07 0.13 0.53 ed13 EG PS 11 Dec 1999 2300 5 0.098 0.60 0.40 ed16 EG PS 13 Dec 1999 0300 14 0.424 0.29 0.07 0.64 sd6 SG PS 16 Dec 1999 0100 19 0.940 0.05 0.21 0.16 0.58 Tr10 EG TR 18 Dec 1999 1915 20 0.840 0.10 0.05 0.85 with sample variance: P n m 2 i ðy i yþ 2 VarðyÞ ¼ P n 2 nn ð 1 Þ m i n where y is the estimate of the population mean, n is the number of stations, y i is the value of the ith station and m i is the number of reproductively active females analyzed from the ith catch. Sampling bias For many anchovy and sardine stocks, it has been observed that day-0 females are oversampled during the hours of spawning (Picquelle and Stauffer 1985; Alheit 1993). The effect of time on the proportions of daily spawner classes was quantified using contingency table analysis (Picquelle and Stauffer 1985; Zar 1999). In case of such a bias, day-0 females should be excluded from the analysis and m i in Eq. 1 corrected. This correction can be made by either considering the number of day-0 equal to the number of day-1 spawners (when only day-1 spawners are used in the estimation of spawning frequency, e.g. Picquelle and Stauffer 1985), or, equal to the average number of day-1, day-2, etc. spawners (when females with POFs can be assigned to three or more spawning nights, see e.g. Quintanilla and Pe rez 2000): P n N i N0 ¼ ð3þ n where N0 is the corrected number of day-0 females and N i is the number of day-i spawners. ð2þ Inter-gear and small-scale variability in sex ratio and spawner fractions Fractions of day-0, day-1,..., day-n spawners, sex ratio and mean weight of active females were compared by gear (pelagic trawl, purse seines) using the Mann Whitney test (Zar 1999). The small-scale variability in the same parameters was assessed on four different occasions (nights) by conducting sequential trawl hauls, on approximately the same towing routes (see Fig. 1b; Tables 1, 2). Parameters were compared by the Kruskal Wallis analysis of variance by ranks (Zar 1999). Parametric tests were not used, because data did not meet the required assumptions. Composite spawning frequency estimates The precision of the spawning frequency estimate could generally be improved, if more than one class of day-i spawners can be identified and, subsequently, combined to produce a composite estimate (e.g. Quintanilla and Pe rez 2000). A prerequisite for such calculations is that different spawner classes have the same statistical distributions (Alheit 1985). The Wilcoxon non-parametric paired sample test was used to compare different classes of day-i spawners. The generalized formula of a composite spawning fraction estimate is: S ¼ 1 n X n ðs i Þ ð4þ

1172 Table 2 Sardina pilchardus sardina. November December 2000 samples. Fraction of females with gonads classified as immature/ inactive, hydrated, running, or with different kinds of postovulatory follicles (POFs). Samples marked in bold correspond to trawl hauls made sequentially over the same towing route in a single night (EG Evoikos Gulf; SG Saronikos Gulf; PS purse seine; TR pelagic trawl; n number of females examined histologically) Sample Region Gear Date Time (hours) n Sex ratio Fraction of: Immature/ Inactive Hydrated Running POF-0 POF-1 POF-2 No POFs sn5-00 SG PS 21 Nov 2000 0300 15 0.734 0.07 0.93 sn6-00 SG PS 22 Nov 2000 0200 15 0.321 0.07 0.20 0.07 0.13 0.53 sn7-00 SG PS 25 Nov 2000 2300 13 0.203 0.69 0.31 en5-00 EG PS 26 Nov 2000 0400 15 0.625 0.20 0.80 en6-00 EG PS 28 Nov 2000 0300 15 0.622 0.13 0.07 0.80 en7-00 EG PS 29 Nov 2000 0300 14 0.556 0.07 0.07 0.86 ed1-00 EG PS 2 Dec 2000 0300 12 0.333 0.17 0.08 0.17 0.58 ed2-00 EG PS 3 Dec 2000 0300 15 0.639 0.07 0.93 ed3-00 EG PS 4 Dec 2000 0300 15 0.444 0.20 0.13 0.13 0.53 ed4-00 EG PS 5 Dec 2000 0300 15 0.429 0.27 0.13 0.60 Tr01-00 SG TR 6 Dec 2000 2030 11 0.338 0.09 0.18 0.09 0.64 Tr02-00 SG TR 6 Dec 2000 2300 15 0.254 0.13 0.47 0.40 Tr03-00 SG TR 7 Dec 2000 0038 13 0.356 0.23 0.77 ed5-00 EG PS 7 Dec 2000 0300 15 0.514 0.07 0.07 0.20 0.67 sn3-00 SG PS 8 Dec 2000 2345 15 0.340 0.40 0.60 Tr06-00 EG TR 10 Dec 2000 2330 15 0.414 0.20 0.13 0.13 0.53 sn1-00 SG PS 11 Dec 2000 2215 15 0.258 0.60 0.07 0.33 ed6-00 EG PS 12 Dec 2000 0300 15 0.667 0.13 0.13 0.13 0.60 sn2-00 SG PS 13 Dec 2000 0500 15 0.804 0.20 0.13 0.07 0.60 Tr07-00 EG TR 13 Dec 2000 2000 15 0.172 0.13 0.13 0.20 0.53 Tr08-00 EG TR 13 Dec 2000 2200 15 0.350 0.13 0.80 0.07 Tr09-00 EG TR 13 Dec 2000 2330 15 0.611 0.47 0.13 0.07 0.33 Tr10-00 EG TR 14 Dec 2000 1930 14 0.736 0.07 0.07 0.86 sd1-00 SG PS 15 Dec 2000 0300 14 0.565 0.21 0.07 0.07 0.64 sd2-00 SG PS 15 Dec 2000 0300 15 0.550 0.27 0.20 0.13 0.40 sd4-00 SG PS 15 Dec 2000 2230 9 0.098 0.67 0.11 0.22 sd3-00 SG PS 15 Dec 2000 2330 10 0.133 0.60 0.20 0.20 Tr11-00 EG TR 15 Dec 2000 1845 15 0.270 0.27 0.25 0.08 0.40 Tr12-00 EG TR 15 Dec 2000 2000 15 0.526 0.20 0.07 0.73 Tr13-00 EG TR 16 Dec 2000 1800 15 0.672 0.13 0.13 0.13 0.60 and variance is given by: " # VarðSÞ ¼ 1 2 X n VarðS i Þþ2 Xn Cov S i ; S j ð5þ n i<j where S is the composite spawning fraction estimate and S i is the fraction of day-i females. Effect of fish size The effect of length on spawning incidence was quantified using contingency table analysis. In Chi-squared analysis of contingency tables, cell frequency values should not be <1.0 and at least 80% of values should be >5.0 (Cohran 1954). Hence, to adequately perform the analysis we used the total fraction of active females having POFs in their ovaries (incidence of recent spawning), corrected for the bias in day-0 fraction, and length was split into three classes (<12.9, 13 15.9 and >16 cm). Results Postovulatory follicle morphology The histological examination and comparative analysis of sardine (Sardina pilcardus sardina) follicles revealed three different classes of POFs with distinct morphological features. POF-0 (0 10 h) In general, oocyte hydration (Fig. 2a) is followed shortly by ovulation, at which time the fully hydrated oocytes are released from their encompassing follicle (Fig. 2b). In the Mediterranean sardine, very new POFs (early POF-0) exhibited a highly convoluted shape, with many folds and a high follicular-tissue/lumen ratio (Fig. 2b, c). Granulosa cells were large, well defined and with a columnar or cuboidal shape, and they were arranged in an orderly manner along the thecal cells. Their nuclei were prominent and displayed a characteristic linear arrangement. The degeneration of POFs-0 seemed to be relatively quick and to depend on ambient temperature. During both years of the study, water temperature was approximately 2 2.5 C higher in Saronikos Gulf than in Evoikos Gulf (Table 3). POFs-0 with signs of degeneration (late POF-0) appeared as early as 2100 hours in Saronikos Gulf and 2300 hours in the N. Evoikos Gulf (Fig. 3). No intact POFs-0 occurred after midnight (0 hours). The degeneration of POF-0 involved the progressive decrease of follicle size and size of folds, the increase in the lumen/follicular-tissue ratio, the loss of linear

1173 Fig. 1a, b Map of the study area (central-western Aegean Sea), indicating the location of adult sampling stations: a purse seine samples and b pelagic trawl samples (black symbols 1999 samples; open symbols 2000 samples; squares correspond to duplicate or triplicate trawl hauls made sequentially over the same towing route in a single night; EG Evoikos Gulf; SG Saronikos Gulf) Fig. 2a f Sardina pilchardus sardina. Micrographs of histological sections of female gonads: a hydrated female, b running female, c early POF-0, d late POF-0, e POF-1 and f POF-2 (a aperture; gc granulosa cells; H hydrated oocyte; L lumen; n nucleus of granulosa cell; P primary oocyte; t thecal cell layer; YG oocyte in the yolk-granule stage; YV oocyte in the yolk-vesicle stage). Scale bars: 0.1 mm arrangement of granulosa cells and nuclei, the breakdown of cell walls and the appearance of pycnotic nuclei (Fig. 2d). Despite quick degeneration, POFs-0 were always substantially larger and more looped than the next class observed (POFs-1). POF-1 (23 34 h) Because of the large age interval between the latest POF- 0 and the earliest POF-1 in our collections (approximately 13 h, Tables 1, 2), the discrimination of the two classes of follicles was straightforward. At this phase of degeneration, the follicle was greatly shrunken, almost to half the size of the previously described POF-0 follicle. The POFs-1 presented just a few or no loops, and the granulosa cells were indistinct (Fig. 2e). POF-2 (47 58 h) The size of the follicles was greatly reduced (the follicles were often hard to detect at low magnifications), and their occurrence was very scarce. The structure was very compact, with no folds, and generally exhibited a triangular Table 3 Averages for temperature ( C) at 5 m and for the upper water column (0 50 m) in Evoikos Gulf and Saronikos Gulf during November December in 1999 and 2000 1999 2000 5 m 0 40 m 5 m 0 40 m Evoikos Gulf 16.7 16.8 17.1 17.1 Saronikos Gulf 19.2 19.3 18.6 19.2

1174 Fig. 4 Sardina pilchardus sardina. Breakdown of day-0 spawners into ovarian categories by time of sampling Fig. 3 Sardina pilchardus sardina. Evoikos Gulf and Saronikos Gulf. Relative proportions of day-0 females with intact postovulatory follicles (early POF-0) and with first signs of degeneration (late POF-0) by time of sampling shape (Fig. 2f). The follicular lumen was reduced to a small aperture towards the ovarian lumen. The follicles usually contained some residual pycnotic nuclei. Sampling bias In the samples analyzed, hydrated females were found from 1845 to 2000 hours, running females from 2130 to 2200 hours and females with POFs-0 from 1900 to 0500 hours (Fig. 4; Tables 1, 2). The fraction of day-0 spawners was particularly high from 2000 to 2359 hours, and decreased thereafter (Fig. 5). In contrast fractions of day-1 and day-2 spawners did not change with time (Fig. 5). Overall, contingency table analysis revealed a significant effect of sampling time on spawning condition (v 2 =145.21, df=15, P<0.05), which was attributable to day-0 females. There was a strong negative correlation between the fraction of day-0 spawners and sex ratio (fraction of females) during both years (Spearman rank correlation, 1999: r s =)0.586, P<0.01; 2000: r s =)0.698, P<0.01; 1999+2000: r s =)0.714, P<0.01; Fig. 6). Thus, the Fig. 5 Sardina pilchardus sardina. Mean fraction of day-0, day-1 and day-2 spawners by time of sampling larger the fraction of day-0 spawners was, the higher the possibility of a male bias in the sample sex ratio. Inter-gear and small-scale variability in spawners fractions Sex ratio, mean weight of active females and fractions of day-0, day-1 and day-2 females did not differ significantly with respect to sampling gear, i.e. pelagic trawl or purse seine (Mann Whitney test, P>0.05; Table 4). Kruskal Wallis analysis of variance by ranks did not reveal any significant differences among the four sets of sequential trawls with regard to sex ratio (H=2.86, P>0.05), mean female weight (H=1.53, P>0.05), or fractions of day-0 (H=4.225, P>0.05), day-1 (H=4.73, p>0.05) and day-2 females (H=4.199, P>0.05). In each particular set of sequential trawl hauls (Tables 1, 2), the variability in parameters was unexpectedly high. For

1175 Table 5 Sardina pilchardus sardina. Spawning frequency estimates using different classes of female spawners. Bias correction was made using Eq. 3. Coefficients of variation in parentheses Year Female spawners Uncorrected estimates Bias corrected estimates Fig. 6 Sardina pilchardus sardina. Scatterplot of the fraction of day- 0 spawners on sex ratio (fraction of females) (r s Spearman rank correlation coefficient) example, the sex ratio in trawls Tr03, Tr04 and Tr05 (Table 1), which were made sequentially from 1900 to 0100 hours, over approximately the same towing route (Saronikos Gulf 1999), varied from 0.982 to 0.340. Spawning frequency estimates For both years, estimates of spawning fraction, based on females spawning the night of capture (day-0 spawners), were higher than those based on day-1 or day-2 females (Table 5). Correction for oversampling the day-0 females resulted in significant differences between the noncorrected and the corrected estimates of fractions of both day-1 and day-2 females in 2000 (Wilcoxon test, P<0.05), but did not significantly change the fractions in 1999 (Wilcoxon test, P>0.05). Estimates based on day-1 and day-2 were similar (Table 5), and the paired sample tests showed no statistically significant differences (Wilcoxon test, P>0.05). Thus, day-1 and day-2 females could be combined, and the resulting composite estimates were more precise, especially in 2000 (Table 5). Effect of fish length In general, the bias-corrected fraction of females with incidence of recent spawning (females with POFs/total Table 4 Sardina pilchardus sardina. Mean fraction of day-0, day-1, day-2 females, sex ratio (fraction of females) and average weight of active females by sampling gear Variable Sampling gear Pelagic trawl Purse seine Fraction of day-0 females 0.210 0.230 Fraction of day-1 females 0.100 0.070 Fraction of day-2 females 0.070 0.070 Sex ratio 0.511 0.492 Average weight of active females 20.41 21.18 1999 Day-0 0.190 (0.222) Day-1 0.089 0.100 (0.160) (0.159) Day-2 0.077 0.080 (0.172) (0.133) (Day-1+Day-2)/2 0.083 0.093 (0.126) (0.104) 2000 Day-0 0.258 (0.171) Day-1 0.079 0.096 (0.179) (0.162) Day-2 0.077 0.093 (0.168) (0.152) (Day-1+Day-2)/2 0.078 0.095 (0.105) (0.048) active females) increased with fish length (Table 6). Contingency table analysis revealed a significant effect of length on the fraction of females with POFs (v 2 =7.93, df=2, P<0.05). Discussion The degeneration of postovulatory follicles is a continuous process. Hence, when sampling is carried out on a 24-h basis, it is often difficult to delimit discrete classes of POFs corresponding to different spawning nights. In the present study, samples were collected only during night-time (1800 0500 hours), and, consequently, the observed microscopic differences between the three identified classes of POFs were large enough to assure correct daily age assignment. The postovulatory follicle method has been applied to several sardine stocks (Table 7), and the number of POF classes used ranged from two to four. The latter can be attributed to inter-laboratory methodological differences concerning the interpretation of POF classes (Anonymous 2000), to differences in sampling time (Table 7) or to the particular populations involved and their preferred spawning temperature (Hunter and Macewicz 1985; Fitzhugh and Hettler 1995). For example, in the Central Aegean Sea, different temperature regimes between Saronikos Gulf and Evoikos Gulf resulted in different rates of POF deterioration; this was particularly noticeable for the early stages of deterioration (Fig. 3; Table 3). However, differences in water temperature were not large enough to generate perceptible difference in the duration of full follicle resorption. For both years, in the warmer waters of Saronikos Gulf, females with POF-2 were not significantly fewer than females with POF-1 (P>0.05), indicating that full follicle resorption lasted at least 58 h, as in the colder Evoikos Gulf. In the Atlantic, Spanish

1176 Table 6 Sardina pilchardus sardina. Reproductively active females. Bias-corrected fractions per length class of females with postovulatory follicles in their ovaries (females with incidence of recent spawning). Percent length frequency distribution is also given Length class Females with incidence of recent spawning Frequency distribution (%) <13 0.176 23.56 13 16 0.245 71.17 >16 0.384 5.17 workers identified an additional class of POFs for the Iberian sardine (POF-3, Table 7), which might be due to a temperature effect, i.e. slow-down of the rate of POF resorption in the colder Atlantic waters (12.5 15 C, Sola et al. 1992). The correct classification of female spawners to discrete spawning days is facilitated when spawning is synchronous with respect to time of day, i.e. fish accomplish spawning in a narrow time interval during the 24-h cycle. For example, in Scomber scombrus spawning occurs throughout the 24-h cycle, which precludes the use of the postovulatory follicle method and complicates the estimation of the spawning fraction (Priede and Watson 1993). For the eastern Cantabrian coast, Quintanilla and Pe rez (2000) have reported the incidence of POFs aged 0 6 h in sardine (Sardina pilchardus sardina) throughout the 24-h cycle. In the case of the Mediterranean sardine, running females were observed from 2130 to 2200 hours, and females that had already accomplished spawning were observed by 1900 hours (Fig. 4; Tables 1, 2). Analysis of plankton samples from concurrent egg surveys (Somarakis et al. 2001; authors unpublished data) revealed the incidence of unfertilized sardine eggs (stage I, Gamulin and Hure 1955) from 1900 to 2300 hours. Thus, S. p. sardina spawns in a relatively limited daily period. Assuming that spawning is normally distributed around the midpoint of the daily spawning period (Smith and Hewitt 1985), mean spawning hour is 2100 hours. During and a few hours after spawning, a bias was observed with respect to sex ratio and day-0 females. Such a bias is a common feature in anchovies and sardines, and is attributed to spawning behavior (Alheit 1993). Alheit has suggested that males and day-0 females segregate themselves from other females during the peak hours of spawning to a depth (or an area) where they are more vulnerable to fishing gears. Correction for over-sampling the day-0 females did not significantly change the fractions of either day-1 or day-2 spawners in 1999. However, samples in 2000 contained more day-0 females (because of the increased pelagic trawl sampling within the daily spawning period), and bias correction resulted in significant differences between corrected and non-corrected estimates of day-1 and day-2 females. Despite bias, fractions of day-1 and day-2 females did not differ significantly from each other (in both 1999 and 2000), and these spawner classes were combined to produce a more precise composite estimate of the spawning frequency (Table 5). An important element of the spawning frequency parameter is that it encompasses a population rather than an individual estimate. Parrish et al. (1986) found that the daily spawning incidence and total annual fecundity were heavily age dependent in northern anchovy. This dependency of spawning frequency on fish age/ size has rarely been studied for pelagic schooling fishes. In the present study, length was found to significantly affect spawning incidence in the Mediterranean sardine. When age/size dependency is present, samples used for the estimation of spawning frequency should have the same age/size structure as the assessed population. In comparing all available spawning frequency estimates for several sardine and anchovy stocks around the world, Alheit (1993) noticed higher values of spawning frequency for stocks sampled from the professional fishery and suggested that the commercial catches might be biased if fish form spawning aggregations that are attractive to the commercial fishery. Alternatively, when size has a significant effect on the fish inter-spawning interval, higher values of population spawning frequency might be due to commercial fishery selecting large fish. The coastal area of the Central Aegean Sea consists of a series of semi-enclosed gulfs (Fig. 1), where the distribution of local school clusters and corresponding egg patches largely overlap, being highly localized and small (Anonymous 2001; Ganias et al. 2001a; Giannoulaki et al. 2001; Somarakis et al. 2001). These clusters consist of closely spaced schools of different-sized fish and are targeted by the commercial purse seine fishery (Anonymous 2001). It was thus expected that the purse seine catches would not be selective with regard to fish size or spawning condition. Indeed, no statistical differences were detected between samples from the commercial fishery and the research trawls for spawner fractions, average weight of active females or sex ratio (Table 4). Sequential trawl hauls made over the same towing routes during four different nights revealed an unexpectedly high small-scale variability in both spawner fractions and sex ratio. Hauls in each of the four sets were practically independent of each other, and the within-set variability was as large as the between-set variability, despite the fact that the sets consisted of different locations, nights or years. This is to our knowledge the first attempt to assess variability in spawner fractions on such a small scale, and points out our limited knowledge of the spawning behavior of pelagic fish. According to our results, spawning aggregations must be structures that are limited in space and/ or ephemeral in time. Indeed, underwater camera observations on the Japanese sardine (Shiraishi et al. 1996) indicate that the length of the spawning school measures several meters.

1177 Table 7 Sardina spp., Sardinops spp. Spawning frequency estimates (S), and corresponding coefficients of variation (CV), and average female weights (W) for Sardina and Sardinops stocks (BT bottom trawls; PT pelagic trawls; PS purse-seines; GN gill nets; L lines). Age ranges (hours) of the classes of postovulatory follicles (POF) used in each specific study are also given; subspecies are according to Parrish et al. (1989) Taxon Country Region Year Month Sampling times (hours) Gear Mean spawning time (hours) POF-0 POF-1 POF-2 POF-3 S CV W Source Sardina pilchardus pilchardus Sardina pilchardus sardina Sardinops melanostictus Sardinops neopilchardus Sardinops sagax musica Sardinops sagax caerulea Portugal 1988 Mar 1000 0330 PT, BT and PS 1900 0.126 0.18 40.94 Cunha et al. (1992) 1997 0.130 0.19 41.28 Anonymous (2000) 1999 Jan Feb 0700 0130 BT and PS 0 24 24 48 >48 0.101 0.15 44.42 Anonymous (2000) Spain E. Cantabria 1988 Apr May 0900 0130 PT 1900 0 6 18 30 42 54 >66 0.210 0.13 86.31 García et al. (1992) W. Cantabria Apr May 1400 2300 PT 1900 0 6 18 30 42 54 >66 0.130 0.11 79.34 Galicia Apr May 1700 0100 PT 1900 0 6 18 30 42 54 >66 0.080 0.20 64.93 E. Cantabria 1990 0.230 0.30 Lago de Lanzo s et al. W. Cantabria 0.090 1.04 (1998) Galicia 0.120 0.19 E. Cantabria 1997 Mar 0600 2000 PT 1900 0 6 18 30 42 54 >60 0.180 0.45 70.05 Lago de Lanzo s et al. (1998) Hellas C. Aegean 1999 Nov Dec 1900 0500 PT and PS 2100 0 10 23 34 47 58 0.093 0.10 18.73 Present study C. Aegean 2000 Dec 1800 0500 PT and PS 2100 0 10 23 34 47 58 0.095 0.05 19.01 Present study C. Ionian 2001 Jan Feb 1000 2345 PT and BT 2100 0 10 23 34 47 58 0.087 0.12 15.87 Somarakis et al. (2001) Japan 1991 Jan Mar 1800 1230 GN and L 0000 0 24 24 48 0.117 Aoki and Murayama (1993) Australia 1998 Jan Mar GN 0200 0 6 7 30 31 53 0.140 0.10 45.18 Ward et al. (2001) 1999 Jan Mar GN 0400 0 6 7 30 31 53 0.180 0.13 52.28 Ward et al. (2001) Peru 1982 Sep Oct 0100 0 6 7 30 31 53 Goldberg et al. (1984) Chile 1988 Aug Dec Day 0 24 24 48 >48 0.152 Claramunt and Herrera (1994) 1989 Aug 0 24 24 48 >48 0.153 Claramunt and Herrera (1994) California 1986 Aug PT and PS 0.127 Macewicz et al. (1996) 1987 July PS 0.121 Macewicz et al. (1996) 1988 May PS and L 0.200 Macewicz et al. (1996) 1994 Apr May 1930 0830 PT and PS 2100 0 5 20 30 0.121 82.50 Macewicz et al. (1996)

1178 Spawning frequency estimates for the Mediterranean sardine are relatively consistent. The overall estimates in 1999 and 2000 were very close, and another estimate made for the Ionian Sea sardine stock during January February 2001 (Somarakis et al. 2001) was also similar (Table 7). In all these cases, active females seemed to spawn every 11 12 days. Similar inter-annual estimates have also been reported for the Pacific sardine (Sardinops sagax, Macewicz et al. 1996) and the Bay of Biscay anchovy (Engraulis encrasicolus, Motos et al. 1996). In order to explain consistency, Hunter and Lo (1997) introduced the biorhythm hypothesis, which suggests that the frequency of spawning is relatively constant for the females of the population that are actively spawning as soon as the habitat conditions remain about the same. Besides habitat conditions (e.g. prey fields, temperature), spawning incidence was shown in the present study to be significantly affected by the size of the spawner. Thus, consistency between the yearly estimates could also be attributed to similar between-year mean body sizes of the assessed populations (Table 7). Hence, the biorhythm hypothesis could be expanded to include a body-size effect such that: the frequency of spawning is relatively constant for the equally sized active females as soon as the habitat conditions remain about the same. Compared to other worldwide estimates, the spawning frequency of the Mediterranean sardine seems to be generally low (Table 7). A factor reported to regulate spawning frequency and, therefore, reproductive potential of a stock is variability in adult prey fields and energetic reserves. Somarakis (1999) observed higher values of spawning frequency for the N. Aegean anchovy stock during years of increased mesozooplankton productivity and higher somatic condition. Kawaguchi et al. (1990) found that laboratory-starved Japanese anchovy regulate spawning frequency according to the decrease in somatic condition following starvation. Indeed, the Mediterranean sardine is the only sardine that does not profit on the productive food web of a coastal upwelling system. Lower spawning frequency in the Mediterranean might be attributed to the oligotrophy of this sea, especially in its eastern basin (Stergiou et al. 1997). Lower spawning frequency could additionally or alternatively be explained in terms of the considerably lower average female weight of the Hellenic populations (Table 7), which is related to both lower sizes-at-age and fewer age classes in the Aegean and Ionian Seas (Tserpes and Tsimenides 1991; Anonymous 2001) than, for example, in the N. Atlantic Ocean (Quintanilla and Pe rez 2000). Acknowledgements We thank Dr C. Papaconstantinou, E. Caragitsou and A. Siapatis from the National Centre for Marine Research in Athens for their collaboration in the collection of the purse seine samples. Dr G. Koumoundouros and Dr. N. Papandroulakis are thanked for their assistance in microscopic photography and N. Papaconstantinou for her help in laboratory analysis. This research was funded by an EU research project, DG XIV, contract no. 98/0039. References Alheit J (1985) Spawning frequency of Peruvian anchovies taken with a purse seine. In: Lasker R (ed) An egg production method for estimating spawning biomass of pelagic fish: application to the northern anchovy, Engraulis mordax. NOAA Tech Rep NMFS 36:99 101 Alheit J (1993) Use of the daily egg production method for estimating biomass of clupeoid fishes: a review and evaluation. Bull Mar Sci 53:750 767 Anonymous (2000) Report of the workshop on the estimation of spawning stock biomass of sardine. 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