Reproductive biology of the skates Sympterygia acuta Garman , 1877 and S . bonapartii Müller & Henle , 1841 ( Chondrichthyes : Rajoidei ) in south Brazil

The present study analyzed the sexual development, sizes at maturity and morphometric relationships for both sexes of Sympterygia acuta and S. bonapartii, endemic of south-western Atlantic Ocean. The examined specimens were obtained through research cruises and commercial fishing trips, during 2011 and 2012, along the southern Brazilian coast, in latitudes ranging from 34°28’S to 31°29’S and at depths between 15 and 142 m. Significant differences (p<0.05) in S. bonapartii and in S acuta between sexes for the relationships total length (cm) disc width (cm) and total length total/eviscerated weight (g) respectively, demonstrated sexual dimorphism during the development in both species. The estimated size at maturity for males and females, respectively, were 46.1 and 44.7 cm, for S. acuta, and 58.4 and 59.9 cm, for S. bonapartii. A decrease in size at maturity for both sexes was observed in S. acuta and females of S. bonapartii of the southern coast of Brazil, respect to previous studies carried out over the last 30 years.

Like other elasmobranchs, the rajoids present relatively low fecundity, late sexual maturity and high longevity.These characteristics, together with fish exploitation, have led to population decreases worldwide, including cases of local extinction (Dulvy et al., 2000;Dulvy & Reynolds, 2002;Iglésias et al., 2009).The bignose fanskate S. acuta, is (globally) considered by the IUCN Red List of Threatened species as "Vulnerable".On the other hand, the smallnose fanskate, S. bonapartii, is classified as "Data Deficient" (Massa & Hozbor, 2004;Massa & Lamilla, 2004).However, due to the high fishing pressure to which both species are subject, the mentioned categories may change regionally in a short-term period.
Studies on the reproductive biology, size-at-maturity and abundance of S. bonapartii were carried out in the coasts of Argentina and Uruguay over the last decade, demonstrating an increasing concern for the fisheries effects upon their populations, as well as the need for biological data (especially on reproduction) in order to ensure their sustainable exploitation (Mabragaña et al., 2002;Oddone & Velasco, 2004).
De Queiroz (1986) analyzed the reproductive biology of Sympterygia spp. in the southern Brazilian shelf.Apart from these data being part of an unpublished thesis, they were collected in the period 1981-1984, i.e., almost 30 years ago.Moreover, it is a well-known fact that in the reproductive parameters, such as the size-at-maturity, may change in the rajoids due to the pressure of excessive fishing (Walters & Martell, 2004).
It is, therefore, necessary, to provide an updated study on the reproductive biology of genus Sympterygia in southern Brazil, in order to guide the management decisions needed for their conservation, including measures such as closed areas and minimum landing sizes.The aim of this study was , thus, to provide updated information on the reproductive parameters of S. bonapartii and S. acuta in the southernmost continental shelf of Brazil between Conceição (31° 43'S) and Chui (33°45'S).

Material and Methods
Samples of Sympterygia spp.were obtained from two sources: one-day-long research cruises aboard the research vessel "Larus" (Instituto de Oceanografia, Universidade Federal de Rio Grande), done in May, June, July and August 2011; and two commercial fishing trips carried out along the coast of Rio Grande do Sul State (southern Brazil), from September 22 nd to 30 th 2011 and from January 30 th to February 10 th 2012.The studied area was located between the latitudes of 34°28'Sand 31°29'S at depths between 15 and 142 m (Fig. 1).Voucher specimens of S. acuta (SA001C; SA002C) and S. bonapartii (SB003C; SB004C) were stored in the fish collection of the Laboratory of Morphology and Histology, ICB / FURG, Brazil.A total of 92 males and 125 females of S. bonapartii and 17 males and 28 females of S. acuta were obtained and analyzed.Of each specimen, the following data were registered: total length (TL) from the snout to the tip of the tail, the width of the disc (DW) measured as the length between opposite pectoral fin tips, total weight (TW), eviscerated weight (EW), liver weight (LW) and gonad weight (GW) (measured values in cm and g, respectively) (sensu Oddone et al. 2007a).
For males, the clasper and clasper gland length, the number of alar thorns and rows of alar thorns were recorded.Alar thorns considered as ''developing'' were registered as a circular light mark on a darker epidermis, being evident laterally on the dorsal region of both pectoral fins (sensu Oddone & Vooren, 2005) The calcification of the clasper was manually assessed and classified as "flexible" or "rigid".From females, the height of the oviducal glands, according to Serra Pereira et al. (2011), the number of vitellogenic follicles larger than 1 cm (Mabragaña et al., 2002) and smaller than 1 cm (Díaz Andrade et al., 2011), the uteri width, diameter and the colour of the largest ovarian follicle, as well as the presence or absence of egg capsules in the uteri and in the cloacal region were registered (Oddone & Vooren, 2005).According to the development of the above-mentioned reproductive structures in relation to TL, the individuals were classified in three maturity categories: immature, adolescent and mature (Oddone et al., 2007a).
Morphometric measurements of both species and sexes were compared.Total length-width disc, total length-total weight and total length-eviscerated weight were logtransformed to become linear and the F-test was used to establish comparisons (Souza, 1998).In this respect, a covariance analysis was applied, where the variable "sex" was a factor.We verified if the curves fitting separately substantially reduced the sum of the squared residuals.The logistic equation was used to estimate the size of sexual maturity, as PTL=1/ (1-e (a+bTL) ) where, PTL is the fraction of mature individuals for each class of TL, a and b are the model parameters.The TL 50 , or the size at which 50% of the population is mature, is given by the ratio a/b (Restrepo & Watson, 1991).As the relationships between clasper length and TL and clasper gland length and TL followed a sigmoid-shaped curve, the logistic equation was also fitted in order to estimate the inflexion point of these relationships, and therefore assess the adolescent phase through these variables.In using parametric/non-parametric tests, normality and homogeneity of variance of the variables were tested by Lilliefors' and Levene's tests, respectively.Parametric comparisons were performed using the Student ttest, which was applied to compare the mean number of alar thorns and the mean number of rows of alar thorns and to assess the statistical significance of the differences between the number of ovarian follicles in mature females and eggbearing females (Sokal & Rohlf, 2012).Reproductive parameters were expressed in terms of mean and standard deviation as mean ± S.D. In all cases "n" is the size of the sample.A level of significance of 0.05 was considered for all the applied tests.
According to F-test results, significant differences were detected between sexes for to the relationship TL-DW, TL-TW and TL-EW for S. bonapartii (Table 1, Fig. 2a-c).
Immature individuals with developing thorns had TLs smaller than 62.5 cm.An adolescent individual with TL of 50.0 cm showed developing thorns, which were also recorded in two mature specimens with TLs of 59.6 and 61.3 cm respectively (Fig. 3e).
The size at which 50% of the population is mature, TL 50 was estimated at 58.4 cm (r=0.98;n=92) for male S. bonapartii (Fig. 4).
Individuals with TL between 43.8 and 52.5 cm, had values of clasper gland length between 2.3 and 5.2 cm.(Fig. 5b).
The relationship TL-DW was sexually dimorphic in S. bonapartii, similarly to the congeneric species, S. lima, endemic of the coast of Chile, Southeast Pacific Ocean (Lamilla et al., 1984).
The TL-DW relationship in S. acuta indicated that immature males had higher size when compared with females in this first stage of development.However, in adolescents and mature individuals just the opposite was observed.Such morphometrics differences throughout the ontogeny were also recorded in Atlantoraja cyclophora (Regan, 1903) and Rioraja agassizi (Muller & Henle, 1841) in southern Brazil (Oddone & Vooren, 2004;Oddone et al., 2007b).On the other hand, the growth pattern in S. bonapartii demonstrated that, in this species, females had larger DW than males for any TL considered.Minimum size at first maturity was similar (or close) for both sexes of S. bonapartii.Mabragaña et al. (2002) observed similar situation in the same species off Argentina.However, according to Mabragaña et al. (2002) different populations might have distinct size at maturity, as it seems to be the case for individuals captured off Argentina (63.5 cm for females and 65.0 cm for males) when compared with those obtained in the present study, off southern Brazil.
The number of rows of alar thorns in males of S. bonapartii varied between 1 and 6, in agreement with data reported by Mabragaña et al. (2002), and also similar to the pattern observed in A. cyclophora (Oddone & Vooren, 2005) and Bathyraja albomaculata (Ruocco et al., 2006).This sexual character present in males can vary among species, being an important parameter used for maturity staging.
Sympterygia bonapartii showed a high level of overlap among the three stages of sexual development, regarding the following variables: testicle weight, diameter of lobule, clasper gland length, number of rows of thorns and alar thorns.However, mean values of these variables indicate differences between each stages of sexual development.
Males S. bonapartii were found to mature with a TL of 58.4 cm.De Queiroz (1986), however recorded a value of 52 cm for this parameter for southern Brazil, indicating that TL 50 may have increased in 3.3 % (6.4 cm) over a ~30-year period.However, the differences in the estimated sizes at maturity may also be due to methodological differences (Oddone & Velasco, 2004).De Queiroz (1986) methodology for recording clasper length (based on Hubbs & Ishiyama, 1968) differed from the one used in the present paper, though he used virtually the same reproductive variables for male sexual maturity staging.For male S. bonapartii, TL 50 (58.4cm) also differed from those calculated from other south western Atlantic areas, such as Argentina (65 cm) and Uruguay (57 cm) (Mabragaña et al., 2002;Oddone & Velasco 2004), though the value reported by Oddone & Velasco (2004) off Uruguay (50.0-57.0cm) was very close to the one obtained in the present study.This similarity may be a consequence of the proximity of the area of the sampled specimens (in the extreme south of Brazil), with the border area between the coasts of Brazil and Uruguay.
In male S. acuta, the inflection point of the curve fit for the relationship between clasper length and TL coincided with the value of minimum TL for the clasper calcification.Therefore, the size at maturity based solely on the TL-clasper length relationship could be underestimated.A similar situation was also observed in S. bonapartii, demonstrating the importance of the determination record of the clasper calcification degree used for assessing maturity in males of Chondrichthyes.
Adult males of S. acuta bore a maximum of 6 rows of alar thorns, similarly to S. bonapartii.Other studies have also demonstrated a similarity in this number of maximum of rows of alar thorns, for other species of the same family (Mabragaña et al., 2002;Oddone & Vooren, 2005;Ruocco et al., 2006).
Contrarily to what was observed for males of S. bonapartii, a decrease in TL 50 corresponding to 3.0 % (5.9 cm) was observed for males of S. acuta, when the present data are compared with the value obtained by De Queiroz (1986) for the same region.
Likewise, the ovary weight of mature females of S. bonapartii analysed by De Queiroz (1986) varied from 25 to 170 g.For mature specimens of S. acuta the same author recorded ovaries weight of 10 to 50 g.When compared to the data found in this study, ovary weights for mature females seem to have decreased since the work done by De Queiroz (1986).Females of both species may have developed at least some degree of reproductive plasticity, reflected in this case in a decrease of gonadal size of mature individuals, which could be a result of fishing mortality in the region during the past decades.
Ovarian follicles larger than 1.0 cm were considered vitellogenic by Mabragaña et al. (2002) for S. bonapartii.Agreeing with these authors, De Queiroz (1986) stated that vitelogenesis started at 1.0 and 1.5 cm in S. bonapartii and S. acuta, respectively.However, Díaz Andrade et al. (2011) examined ovarian follicles smaller than 1.0 cm in mature females of S. bonapartii through the histological analysis of the ovary and concluded that the traditional macroscopic assessment of the follicular vitellogenesis may turn into a subjective parameter of analysis, turning the classification of the development stage difficult.
They demonstrated that vitellogenic follicles with diameters larger than 0.3 cm are characteristic of mature females of S. bonapartii, a size that is very close to the diameter of vitellogenic follicles found in the present study (0.4 cm).The analysis performed by Díaz Andrade et al. (2009) in females of S. acuta, in turn, revealed that, at histological level, vitellogenesis would start at 0.55 cm, a size again similar to the obtained in the present study, where vitellogenesis was macroscopically detected from follicles with diameters of 0.6 cm onward.
Serra Pereira et al. (2011) analyzed the relationship of various measures of oviducal gland, including height, width and thickness, in Raja clavata (Linnaeus, 1758), in relation to maturity stages (i.e., "in development"; "spawning capable"; "actively spawning"), finding differences among stages.Differences in the OGH were also detected in S. bonapartii and S. acuta, whit their values increasing during sexual development.
The size of maturity obtained for females of S. bonapartii and S. acuta, when compared with those values reported by De Queiroz (1986) in southern Brazil, indicated a considerable decrease in the size of maturity over the last 30 years, corresponding to 7.0 and 4.4 % (or 10 and 8.3 cm) for female S. bonapartii and S. acuta, respectively.Such a reduction may be caused by fishing mortality (as well as fishing gear selectivity), which is very intense in the southern region of Brazil.This was demonstrated in other areas by Walters & Martell, 2004;Paesch & Oddone, 2008.The TL 50 estimates obtained for both sexes of S. bonapartii demonstrated differences at regional level.The restricted distribution of the species could indicate the existence of two different populations of S. bonapartii, the first occupying the higher latitudes of the south western Atlantic Ocean, in the coast of Argentina, and a second one composed by individuals that transit between the coastal zones of Uruguay and southern Brazil.This could be attributed to the transport of water masses from the Brazil Current and the Malvinas Current, accentuated mainly during summer and winter, respectively (Miloslavich et al., 2011).
The genus Sympterygia from the south-western Atlantic may have suffered a decline as a consequence of strong fishing pressure upon their populations over the decades.Characteristics related to reproduction such as the maturity size may change in oviparous skates that are affected by fishing (Ebert et al., 2008;Paesch & Oddone, 2008;Orlando et al., 2011).As it is the case of Sympterygia spp., it is essential to have current data on reproduction.Furthermore, future research should also be focused on the reproductive cycle both temporally and spatially.Significant decrease in the size at maturity as a result of fishing pressure was documented for Dipturus chilensis (Paesch & Oddone, 2008) in the southwestern Atlantic Ocean.
Data on the reproductive biology of the genus Sympterygia endemic to the south-western Atlantic Ocean presented in this study, may provide tools for the evaluation of the stocks, in order to properly conserve and manage the populations of Sympterygia spp. that are used for human consumption and exploited unrestrictedly as fishery resources.

Fig. 1 .
Fig. 1.Map of the study area.Left map represents South America.The square is a detail of the study area.The right map represents south of Brazil indicating the trawling stations where the specimens of Sympterygia were captured; research cruises of the research vessel "Larus" (triangles) and commercial bottom trawl fishing trips (circles).

Fig. 4 .
Fig. 4. Percentage of mature individuals by total length class for males and females Sympterygia acuta (black line) and S. bonapartii (grey line).Empty triangles represent male Sympterygia bonapartii and full triangles, male S. acuta.Empty circles represent female S. bonapartii and full circles, female S. acuta.