The macroscopic scales used in the present study are based on the female maturity scales from the MEDITS-Handbook Version n. 8 (Anonymous 2016). In particular, one species for each systematic category was chosen:

Mullus barbatus Linnaeus, 1758 (red mullet) for Osteichthyes – iteroparous and batch spawner (Carbonara et al. 2015);
Galeus melastomus Rafinesque, 1810 (blackmouth catshark) for oviparous Elasmobranchii – lecitothrophic with multiple oviparity (Hamlett 2005);
Etmopterus spinax (Linnaeus, 1758) (velvet belly lanternshark) for viviparous Elasmobranchii– lecithotrophic, yolk sac viviparous (Hamlett 2005);
Aristeus antennatus (Risso, 1816) (blue and red shrimp) for Crustacea - iteroparous and total spawner (Carbonell et al. 2006);
Loligo vulgaris Lamarck, 1798 (European squid) for Cephalopoda - semelparous and intermittent terminal spawning (Rocha et al. 2001).

A photo of each gonad was taken according to an established protocol (Follesa and Carbonara 2019). Both the gonad inside the cavity and the unit of measure had to be perfectly visible in order to perceive the ratio between the gonad and cavity dimensions (Follesa and Carbonara 2019). Additionally, the collection of particular secondary maturity structures for Elasmobranchii and Cephalopoda was also included. The microscales proposed in this paper aimed to be as wide-ranging as possible, though the information available was limited to just a few species. Considering the recommendations of the Workshop on Sexual Maturity Staging of Elasmobranchs (WKMSEL2 - ICES 2013), the tables of Elasmobranchii were structured for different reproductive organs by maturity stage: ovaries, oviducal glands and uteri. For Cephalopoda species, the sexual maturity process involves the ovary, the single left oviduct and also glandular structures such as the nidamental, accessory nidamental and oviducal glands.

For each maturity stage, the following sampling biological metrics were reported: Length of specimen; total weight and sampling time. In particular, for fish (Osteichthyes and Elasmobranchii) the total length (TL) with the tail fully extended was recorded to the lowest 0.5 cm. For Crustacea, the cephalo-thoracic length (CL) at the lowest 0.1 mm was measured, while for Cephalopoda, the dorsal mantle length (ML) to the lowest 0.5 cm was obtained.

In order to confirm the assigned macroscopic maturity stages, a histological analysis was conducted following the description and terminology of oocyte stages proposed by Brown-Petersen et al. (2011) for Osteichthyes, by Porcu et al. (2014) for Elasmobranchii, by Cuccu et al. (2013) for Cephalopoda and by ICES (2009b) for Crustacea.

Protocol of the microscopic material collection

The histological protocol used by all involved laboratories for all analysed species followed Mazzi (1977) and Cerri and Sasso-Cerri (2003) for the slide section and the staining respectively, as follows:

fixation in 5% buffered formaldehyde (0.1 M, pH 7.4) for a maximum period of 48 h and storage in ethanol 70%; dehydration through a progressively higher series of alcohol concentrations (70%-100%), embedding in a synthetic resin (GMA, Technovit 7100, Bio-Optica), cutting at 3.5 μm with a rotative microtome (LKB, Historange) and staining with techniques including Gill or Carazzi’s haematoxylin and alcoholic eosin or toluidine blue (to analyse the histological structure of ovaries, and oviducal glands), All sections were then observed using a Zeiss Primo Star optical microscope equipped with a Canon EOS 1100D at different magnifications (40×, 100×, 400×), edited with Adobe Photoshop CS6.

Biological parameters

All reproductive parameters were estimated for 6 GSAs (GSA 9, 10, 11, 16, 18 and 19) considering the years 2015, 2016 and 2017. The sample size for each GSA is showed in Table 1.

The reproductive seasonality of the target species was inferred through an analysis of the seasonal/monthly evolution of the percentage of maturity stages of females.

Size at first maturity (SFM₅₀= length at which 50% of the individuals are mature) was estimated by fitting maturity ogives to the proportion of mature individuals in each size class (1 cm for Osteichthyes and Elasmobranchii, 0.5 cm for Cephalopoda and 1 mm for Crustacea). The mature portion included stages 2b-4b for Osteichthyes, stages 3a-4b for Elasmobranchii, stages 2b-3b for Cephalopoda and stages 2b-2e for Crustacea. The SFM₅₀ and the maturity range (length range between SFM₇₅ and SFM₂₅; MR=SFM₇₅ - SFM₂₅ where SFM₇₅ and SFM₂₅ are the length at which 75% and 25% of the individuals are mature respectively) were estimated using the following ogive model: M(L)=e(a+bL)/1+e(a+bL), where M(L) is the proportion of adult individuals (ICES 2008) and L the length class. Binomial generalized linear models with logistic link were used to model the proportion of adult individuals on length as an independent variable (ICES 2008). MEDITS data were used for Osteichthyes, Elasmobranchii and Crustacea target species, while for Cephalopoda, when available, commercial data were used.

The maturity stage changes in the gonado-somatic index (GSI) of viviparous elasmobranchs were calculated as follows GSI = (GM/ TW) × 100, where GM was the gonad mass and TW the total weight.

The description of the macroscale of red mullet fits well with what is reported in the MEDITS manual for Osteichthyes (Table 2).

The gonads modify colour with the progression of their maturation development, changing from pinkish/reddish (stages 2a and 2b named respectively virgin developing and recovering) to pinkish orange (stages 2c and 3 named respectively maturing and mature-spawner). Also the dimension switches from being 1/3 - 1/2 of the body cavity in the immature (stage 1) and developing (stage 2a) stages to being from 2/3 to full length of the body cavity in the maturing and mature stages (stages 2c and 3). Post-spawning stages (stages 4a and 4b named respectively spent and resting) are characterized by a reddish progressively shrunken ovary. The eggs are visible to the naked eye in the maturing, mature and spent stages, but they escape freely from the abdomen only in mature condition (Table 2).

The histological criteria used to distinguish the reproductive stages of M. barbatus females are presented in Table 3. The immature stage 1 appears histologically similar to that of other bony fish. This stage can be distinguished by the presence of oogonia and primary growth oocytes (PG), little connective tissue between follicles and a thin ovarian wall. As females move into the developing stage (stage 2a) and maturing stage (stage 2c), they can be histologically distinguished by the initial appearance of cortical alveoli oocytes (CA) and of vitellogenic oocytes (Vtg1 and Vtg2). The appearance of CA oocytes and the physiological initiator for their formation nevertheless provide the definitive marker for entry into the developing stage. Fish enter the developing phase only one time during a reproductive cycle. The mature/spawner stage (stage 3) is defined as the fish is being capable of spawning within the current reproductive cycle. Any fish with Vtg3 oocytes is assigned to the mature stage. Fish undergoing early stages of oocyte maturation (OM) (i.e. germinal vesicle migration) are also considered to be in the mature/spawner stage (stage 3). In M. barbatus, as in other batch spawners, the presence of all stages of oocytes (i.e., PG, CA, Vtg 1, Vtg 2, Vtg 3 and OM) is observed (Carbonara et al. 2015), whereas in total spawners Vtg3 or early OM and PG are the only oocytes present. The evidence that batch spawner fish, in stage 3, have already spawned in a previous event could be detected by the presences of post-ovulatory follicles.

The end of the spawning season is indicated by the capture of numerous females in the spent stage (stage 4a). From the histological point of view this stage is characterized by abundant atresia, POFs, and few (if any) healthy Vtg2 or Vtg3 oocytes. Fish remain at this stage for a relatively short time and then move to the resting stage (stage 4b) distinguished by the presence of a lot of PG, similar to the immature stage, but with a thicker ovarian wall, the presence of more space, interstitial tissue, capillaries around PG oocytes and the presence of atretic oocytes. Fish at this stage are sexually mature but reproductively inactive. Sub-stage 2b represents specimens that have finished a reproductive cycle and are preparing to start another one. At this stage the specimens are inactive mature, the gonad has undergone development but it has taken part in the spawning in the current year. The only fish actually spawning and reproducing in the current year are those in (sub)-stages 2b, 2c, 3 and 4 (both sub-stages 4a and 4b).

In the case of Galeus melastomus, in general the description of the macroscale fitted well with what is reported in the MEDITS manual for oviparous Elasmobranchii. Only some specific integrations are reported in bold in Table 4.

As defined in the WKMSEL2 (ICES 2013), the juvenile females can be assigned to an immature stage (stage 1) when a cut is made in the ovary and no follicles are observed inside. Generally, in this stage, a primordium of the oviducal gland, as a thickening of the uterus, can be identified in some skate species but is less distinguishable in sharks.

The maturing stage (stage 2) is characterized by the presence of ovarian follicles of small-medium size, but no large ones are present. Large yolked follicles, ready to be ovulated, can be observed only in the mature specimens (stage 3a – mature), which will be considered as mature “extruding” only when egg capsules are present (stage 3b mature-extruding). Indeed, the characteristics of the ovary in stage 3a and 3b are the same; the only difference can be found in the presence of egg capsules in 3b (Table 4). In extruding females of G. melastomus, a lecithotrophic species with multiple oviparity, more than one egg capsule in each oviduct can be found, unlike in other members of the Scyliorhinidae or Rajidae family characterized by a single oviparity (Fig. 2).

The resting stage (stage 4a) is characterized by the presence of uteri that have collapsed and reduced in size (compared with the previous stages 3a and 3b). Also, the oviducal gland appears reduced in dimension. The presence of few but large atresic follicles is the key to the assignment of this stage; in the micro sections post-ovulatory follicles (POFs) may also be visible (Tables 4-5). In the regenerating stage (stage 4b), the ovary appears enlarged due to the previous ovarian cycle, showing follicles of small to medium size and some large eggs entering in atresia can be also observed. No pre-ovulatory follicles are present in the ovary in this stage. The duration of a possible resting period may be short for most species, i.e. less than one year.

Table 5

Stage	Photos	Reproductive apparatus description
1 – Immature		Ovaries: primordial and primary follicles (PO), connected to the germinal epithelium and tunica albuginea. Uteri: composed mainly of connective tissue, covered by simple columnar epithelium with some invaginations.
	GSA11 (H&E)
2 – Maturing		Ovaries: late-form of pre-vitellogenic follicles (PVO) of various sizes with visible lipidic drops. Oviducal glands: gland tubules beginning to form (early stage) or completely formed with secreting zones differentiated (late stage). Uteri: epithelium more invaginated and vascularized than in immatures females with some secretory cells.
	GSA11 (H&E)
3A – Mature	GSA11 (H&E)	Ovaries: follicles in all stages of development, vitellogenic follicles (VObeing) the most abundant. Post-ovulatory follicles may be present. Oviducal glands: gland tubules filled with secretions. Uteri: invaginated, longitudinal folds with secretions into the lumen.
3B – Mature/Extruding
4A – Resting		Ovaries: follicles in all stages. Some post-ovulatory and atretic follicles (AO), with collapsed basal lamina.
	GSA11 (H&E)
4B – Regenerating		Ovaries: pre-vitellogenic follicles (PVO) of different sizes are present. Oviducal glands: no secretory material detected. No observations of uteri are detected.
	GSA11 (H&E)

In the immature specimens (stage 1) the ovarian tissue consists of follicles in early pre-vitellogenesis (50-100 μm), connected to the germinal epithelium and to the tunica albuginea. The follicular epithelium surrounding the follicle is single and consists mainly of squamous cells, and lipid inclusions are visible in the cytoplasm (Table 5). In the maturing specimens (stage 2) the ovary contains late-formed pre-vitellogenesis follicles in comparison with the previous stage (~500 μm). In the mature specimens (stage 3a and 3b), the ovary shows follicles in all stages of development, but with a clear predominance of the vitellogenic ones, of high dimensions (up to 3000 μm), showing drops of vitello protein in the cytoplasm (Table 5). Resting females (stage 4a) preside post-ovulatory and atretic follicles in which the basal lamina invading the central lumen appears to be collapsed (Table 5). Finally, in females undergoing regeneration (stage 4b) the ovary shows pre-vitellogenic follicles very similar to those found in maturing stage (stage 2). In the case of the oviparous species, the only specimens actually spawning and reproducing in the current year are those from stages 3 to 4.

In general, the macroscopic scale of the viviparous species fitted well with what is reported in the MEDITS manual, only few integrations are reported in bold in Table 6. The macroscale was also confirmed by histological investigations.

In E. spinax the macro and micro descriptions of the immature (stage 1) and developing (stage 2) females is similar to that reported for the immature and maturing stages of oviparous elasmobranchs (Table 6 cf. Table 5).

The vitellogenesis process consists in the formation of yolk drops, in the pseudo-stratification of the follicular epithelium and in an increase in the peripheral vascularization between the technical layers and the follicular epithelium (Table 6) in the egg cell. Yolk droplets begin to form first in the cortical region of the oocyte (CA >1200 microns); later, their size increases and this allows them to fill the entire cytoplasm in mature females. In some primary and pre-vitellogenic follicles (PG and CA) and at the beginning of the vitellogenesis phase the presence of the “lumpbrush” chromosomes was observed in the nucleus (Table 6).

In general, subjectivity in the macroscopic distinction between maternal stages 3c (mid pregnancy) and 3d (late pregnancy) can influence the attribution of the stage. The capability for noticing this differentiation is only acquired with species-specific experience regarding the state of the yolk sac. In case of difficulties to distinguish between these stages, taking measurements of the embryos is recommended.

It is worth noting that, in the maternal stages the uterus description is not taken into account. However, a distinction among viviparous species between those with synchronicity and those with asynchronicity of uterine and ovarian phases, respectively, should be made. In species such as E. spinax with asynchronicity between the two phases, the ovary after stage 3a begins to regress (Fig. 3). While the uteri were developing more and more, the histological sections of the ovaries in stage 3b highlighted the presence of primary and pre-vitellogenic follicles surrounded by the epigonal organ with the presence, albeit in a much reduced manner, of some vitellogenic follicles and atretic follicles. Instead, in species with synchronicity such as Squalus blainville (Risso, 1827), the ovary continued its development together with the uterus, as shown by the GSI evolution in each maturity stage (Fig. 2).

In the post-natal phase (stage 4a and 4b), the ovarian tissue appeared flaccid and contained only primary and pre-vitellogenic follicles (Table 7). No histological information is present for the late maternal and regression stages (3c-3d-4a-4b) (Table 7). In this case, the only specimens actually spawning and reproducing in the current year are those from 3 to 4.

The macroscale of A. antennatus fitted well with what is reported in the MEDITS manual. No integrations are reported in Table 8. The macroscale was also confirmed by histological investigations (Table 9).

Considerable differences in the colour and shape of the ovaries are observed in A. antennatus. The aspect of the ovaries varies from transparent, small, duct-like, and restricted at the posterodorsal part of the stomach, to violet, greatly swollen, subdivided into distinct lobes, and covering most of the stomach and hepatopancreas. The primary oocytes and early follicular cells (PG) in immature individuals (stage 1) are very small and with no sign of development. Changes in colour and shape of the ovaries match very well with the development of oocytes moving from oogonia and of both early and primary oocytes (PG, stage 1) to early and late vitellogenic oocytes during the maturing and mature stages (stage 2c and 3). The primary oocytes and early follicular cells in stage 1 are very small and with no sign of development. The high number of nucleoli inside the nucleus of developing vitellogenic oocytes on the maturing ovary indicates that these oocytes have become very active. The enlargement of the expanding oocytes in the mature ovary mainly results from expansion of the cytoplasm, which has become acidophilic, probably indicating the presence of yolk droplets, vitellogenic and/or glycoprotein substance. In the resting adult (stage 2e), the presence of atretic oocytes is also detected. Macroscopically, the ovaries appear ivory-coloured with cephalic and lateral lobes small but distinguishable to the naked eye. In these stages the maturative zones are organized into lobuli containing late primary oocytes. The only specimens actually spawning and reproducing in the current year are those from stages 2 to 5.

The reproductive apparatus changes from its immature condition characterized by small dimension, translucid appearance and absence of oocytes, to a full maturity status (stage 3a) through two distinct phases of developing (stage 2a) and maturing (stage2b) (Table 10). Stage 2b is distinguished from the previous 2a by the larger size of the organs and also by the presence of a lateral brownish strip on the oviducal glands and the appearance of small, slightly red pigmented, accessory nidamental glands. The following fully maturity (stage 3a) is unequivocally defined by enlarged and turgid glandular structures but principally by the presence of a higher percentage of large oocytes in the ovary and ripe oocytes (Ø> = 2 mm) inside the oviduct. Moreover, a markedly lit colour of the oviducal gland lateral strip and of the accessory nidamental gland can be observed.

As shown in Table 10, the ovary in mature phase could also include fewer oocytes and/or a just emptied oviduct. Stage 3a is clearly distinguished from stage 3b (spent) because, in the latter, the organs showed a disorderly and flaccid appearance.

From a histological point of view (Table 11), it is possible to observe the presence of different cells resulting from the oogenesis and their different proportion characterizes the five stages. In particular, the differences between the developing stage (stage 2a) and the maturing stage (stage 2b) are given by the appearance of late primary oocytes and vitellogenic oocytes, respectively. In the mature stage, vitellogenic oocytes are prevalent in the ovary and are displacing their follicular folds towards to the periphery to be ready for ovulation like the smooth ripe oocytes visible inside the oviduct. The only cephalopods actually spawning and reproducing in the current year are those from stages 2b to 3b.

In general, a similar reproductive period of the species is observed between the geographic areas studied (Figs 4-8). Red mullet females showed a reproductive period from April to July with a code in early autumn in all GSAs considered. The peak of the spawning period seems to be concentrated in May-July in GSAs 9-11-18-19, whereas it appears just more extended from April to July in GSA 10 and from April to August in GSA 16 (Fig. 4).

The reproductive season of the viviparous E. spinax and the oviparous G. melastomus occurs all year around in all the investigated GSAs. Specifically, mature (stage 3a) and pregnant individuals (stages 3b, 3c) of E. spinax were found mainly in summer and autumn while mature females (stage 3a) of G. melastomus were found all year around but with low frequencies (Figs 5-6). The spawning time of A. antennatus is comprised between spring and summer period in all analysed GSAs (Fig. 7). Although L. vulgaris is not well represented monthly in all GSAs, its spawning period seems to occur all year in all investigated areas (Fig. 8).

The SFM₅₀ of M. barbatus in GSAs 10, 18 and 19 shows values of about 11 cm TL. The highest SFM₅₀ value was registered in female population of Ligurian Sea (GSA9, 13 cm TL) and the lowest one in Sardinia (GSA11, 10.77 cm TL) (Table 12).

The SFM₅₀ of E. spinax ranges from 32.0 to 36.0 cm TL, with the lowest values in GSAs 9 and 18 and the highest ones in GSAs 11 and 19. G. melastomus females attained the SFM₅₀ at the same length [between 45.16 cm (GSA 10) and 46.59 cm TL (GSA 19)], except for the Ligurian population with the lowest SFM₅₀ observed (41.80 cm TL).

The SFM₅₀ of A. antennatus ranges from a minimum of 21.88 mm CL in GSA 11 to a maximum of 25.67 mm CL in GSA 19. In general, in the whole Tyrrhenian the values are around 23.0-24.0 mm CL, while in the Ionian Sea a higher value was recorded (25.67 mm CL). Finally, L. vulgaris shows values ranging from 14.44 in the southern Adriatic to 17.92 in Sardinia (Table 12).