Figures 3 and 4 show the standardized dimensions of each morphological measurement as a result of linear regressions against the ML. Differences can clearly distinguish female and male squid according to the geographic subarea. Despite those differences, however, these groups do not form a cline along the latitudinal gradient.

Among the female squid, individuals from the coast of Santa Catarina were the most distinct, with the largest FL, FW, RW, GW, LBRL and the smallest GL amongst all areas. Squid from Mar del Plata were also very distinct, since all measurements (except FL and LBRL) were the smallest among subareas. Squid from Rio de Janeiro, São Paulo and Rio Grande do Sul were similar, but individuals from São Paulo had the largest GL and those from Rio de Janeiro had the smallest LBRL (Fig. 3).

Morphological differences were also evident in males. Except for the FW, differences were noticeable for the remaining measurements. Squid from Rio de Janeiro had the smallest FL and LBRL, whereas those from São Paulo had the largest GL and a relatively large RW. Individuals from Santa Catarina had the smallest GL and RW, whereas Rio Grande do Sul squid had the smallest FL and largest RW, GW and LBRL. Uruguayan squid were fairly alike to Rio de Janeiro individuals, but differed in RW, GW and LBRL. Squid form Mar del Plata had the largest FL and FW (Fig. 4).

Figures 3–4. 

Box-Whisker plots of standardized values of attributes for the soft body parts of Doryteuthis sanpaulensis females (3) and males (4) from each region surveyed. Blue triangles represent mean values. FL: fin length, FW: fin width GL: gill length, RW: raquis width, GW: gladius width, LBRL: lower beak rostral length. RJ: Rio de Janeiro, SP: São Paulo, SC: Santa Catarina, RS: Rio Grande do Sul, Uy: Uruguay, MP: Mar del Plata.

The two species of Doryteuthis presented distinguishable morphological differences along the latitudinal gradient. In Doryteuthis pleii, males and females from the two subareas overlapped >70%, implying that the latitudinal differences were subtle. In contrast, D. sanpaulensis can be separated into at least six distinguishable geographical groups (five in the case of female squid) (Fig. 5).

Doryteuthis sanpaulensis females inhabiting Santa Catarina and Mar del Plata diverged morphologically from their immediate counterparts to the north and south (São Paulo and Rio Grande do Sul, Ω̄ = 60.5–67) and to the north (Rio Grande do Sul coast, Ω̄ = 55.8) respectively. Squid from Mar del Plata also overlapped very little in their morphologies within all remaining subareas. Squid from São Paulo and Rio Grande do Sul were very similar (Ω̄ = 75.2). The smallest overlap between groups were between squids from Santa Catarina and Rio de Janeiro (Ω̄ = 49.3) (Fig. 5).

Doryteuthis sanpaulensis males from Santa Catarina also overlapped very little with their closest neighbours to the north and to the south (Ω̄ = 64.1–68.6) and, again, squid from the São Paulo and Rio Grande do Sul coasts were very similar (Ω̄ = 75.9). The Uruguayan group diverged strongly from their immediate northern neighbour (Rio Grande do Sul, Ω̄ = 53) and was very similar to squid from the northernmost geographical area (Rio de Janeiro, Ω̄ = 72.2) (Fig. 5).

Figure 5. 

Heatmaps depicting the morphological overlap (Ω̄) among geographic areas in Doryteuthis sanpaulensis. RJ: Rio de Janeiro, SP: São Paulo, SC: Santa Catarina, RS: Rio Grande do Sul, Uy: Uruguay, MP: Mar del Plata.

The size structure histograms constructed for D. sanpaulensis were plotted in three figures for better visualization (Figs 6–8). The size of individuals ranged from 14 to 114 mm ML. The size distribution of juvenile squid was unimodal regardless of the geographic area, but squid from the Patos Lagoon estuary were smaller than squid found north of it (which encompassed a similar size range) (Fig. 6).

Figure 6. 

Size structure of Doryteuthis sanpaulensis according to the subareas nested within the SBB (22–29°S). Note de different y-axis.

The largest and smallest female squid were sampled south of 29°S (at Patos Lagoon estuary and off Uruguay, respectively), and in all cases, their size distribution was unimodal (Figs 7, 8). Females from Santa Catarina were larger than their male counterparts (Fig. 6).

In males, the average ML size and modes tended to decrease from Rio de Janeiro to Santa Catarina, increasing in squid sampled south of 29°S (Rio Grande do Sul) except at the Patos Lagoon estuary, where squid were <40 mm ML (Fig. 7). The largest average ML and modes were found off the Uruguayan coast, decreasing again in squid from Mar del Plata (Fig. 8). The size distribution of male squid from all remaining areas was unimodal, except for exemplars caught off Uruguay (Figs 6–8). Male-biased sex ratios were found in Rio de Janeiro (1 female: 2.2 males), São Paulo (1 female: 1.3 males), Santa Catarina (1 female: 1.9 males) and Mar del Plata (1 female: 1.4 males), and females outnumbered males in Uruguay (1 female: 0.5 males) (χ² = 2.7–19.6, p < 0.05). Regarding bathymetry, the sex ratio was biased toward males in shallow (<31 m) bottoms (1 female: 1.4 males, χ² = 21.67, p = 3.24 × 10^–6) and toward females in deeper (>55 m) bottoms (1.3 females: 1 male, χ² = 4.06, p = 0.04).

Figure 7. 

Size structure of Doryteuthis sanpaulensis according to the subareas nested within the Rio Grande do Sul coast (29–33.7°S). Note de different y-axis.

Figure 8. 

Size structure of Doryteuthis sanpaulensis according to the subareas nested within the Uruguayan-Argentinean coast (33.7–38.2°S).

The proportion of D. sanpaulensis in each maturity stage in relation to the geographic subareas is given in Table 2. Immature females were the most frequent stage in all areas (≥74%), except off Uruguay (Table 2). Mature and spent females were frequent off Santa Catarina (42%) and off Uruguay (89.7%) (Table 2). Spent individuals were mostly recorded off the coast of Santa Catarina (Table 2). Maturing and mature males predominated in all subareas (>67.7%), except at the Patos Lagoon estuary (31.9%) (Table 2). Considering all subareas pooled, implanted spermatophores in the buccal receptacle occurred more frequently in mature females (n = 19), followed by spent (n = 6) and maturing squids (n = 1).

The size structure of Doryteuthis pleii is depicted in Fig. 9. The length of the mantle ranged from 30 to 247 mm. Since the morphological analysis had detected distinct groups (see above), two histograms were plotted separately (Fig. 9). Female squid between Cabo Frio and Paranaguá (23–26°S) had a normal size distribution with a distinct mode around 90–100 mm ML, whereas those off Santa Catarina (26–28°S) had a mode at smaller sizes (80–90 mm ML) (Fig. 9). The size distribution of male squid from both areas was skewed to the left, with a clear mode at 90–100 mm ML, although individuals between Cabo Frio and Paranaguá encompassed a wider size range (Fig. 9). The size range of juvenile squid was similar in both areas, but the mode obtained for squids sampled from Cabo Frio to Paranaguá was greater (Fig. 9).

Figure 9. 

Size structure of Doryteuthis pleii caught off southern Brazil.

In the two areas, the sex ratio of squid was significantly biased toward males (0.78 female: 1 male; χ² = 4.09, p = 0.026). There were, however, no differences regarding bathymetric sex ratio (<30 m: 0.9 female: 1 male, χ² = 1.57, p = 0.209 and >49 m: 0.88 female: 1 male, χ² = 1.10, p = 0.292).

Of the 326 squid sampled for maturity assessment (143 females and 183 males), most (58% of females and 65.6% of males) were in an advanced stage of maturity (i.e. stages B, C and D). Nearly 44.1% of the females were mature, whereas 14% were maturing. Only 13.3% of the females were found spent. Immature squid comprised 34.4% and 42% of all males and females, respectively. No spent males were recorded. Mature females had the highest incidence of implanted spermatophores (n = 31), followed by spent individuals (n = 7) and maturing squid (n = 3).

The size of Lolliguncula brevis varied between 16 and 56 mm ML, females being larger than males, with a wider size interval. Two distinct modes were observed in female squid, 15–20 and 25–30 mm ML, whereas a well-defined mode at 25–30 mm ML (Fig. 10) was obtained for males. Only four females were found with implanted spermatophores, all of which were inside the mantle cavity.

Figure 10. 

Size structure of Lolliguncula brevis caught off southern Brazil (24–26°S).

All mated females were mature (stage C). Maturing and mature females (stages B and C) comprised little more than half of all individuals sampled (53.5%), followed by immature individuals (stage A) (43.6%). Only three spent females were found. Most mature females were caught in September 1979 at depths shallower than 18 m (FAUNEC III survey). In contrast, most male squid were mature (83.1%), and the bulk of them (n = 64) were caught in deeper waters (20–30 m). No spent males were found. The sex ratio was biased towards females (2.24 female: 1 male), although no significant difference was detected (χ² = 3.71, p = 0.053).

The sizes of the three loliginids at maturity (ML₅₀) are given in Table 3. Note that calculations were made according to the availability of maturing/mature squid and, in some cases, not all stages were found in reasonable numbers. Size at maturity in female and male D. sanpaulensis varied according to the latitudinal gradient, with the largest and smallest ML₅₀ found in Rio Grande do Sul and Santa Catarina, respectively, for both sexes (Table 3). Sizes at maturity of 104.66 mm ML and 86.85 mm ML were estimated for females and males of D. pleii, respectively, pooled for the whole study area (Table 3). A ML₅₀ of 36.58 mm ML was estimated for female L. brevis, but in this case, sample size was very small (n = 5), which means that our results should be interpreted with caution. The number of mature males of L. brevis was not sufficient for a similar calculation.

The three loliginids were present on the SBB (22–29°S). Doryteuthis sanpaulensis and D. pleii were also recorded up to the southernmost portion of the study area (~38°S, Mar del Plata). The southernmost occurrence of D. pleii was based on four large male squid (>200 mm ML) sampled off Mar del Plata in 1972 and deposited at the malacological collection of the National Museum. Lolliguncula brevis occurred exclusively nearshore (<30 m) between 24 and 26°S (Fig. 2).

On the wider continental shelf (up to 130 m deep), Doryteuthis spp. had patchy distributions (Fig. 2), with mean catch rates of 21 ± 95.3 (SD) squid haul^–1 for D. sanpaulensis (range 0–812 squid haul^–1) and 6.23 ± 23.2 (SD) squid haul^–1 for D. pleii (range 0–167 squid haul^–1).

An analysis of the catches by depth showed that D. sanpaulensis was disproportionally more abundant in shallower depths (<40 m, 1210 squid caught, catch rate: 39 squid haul^–1) and moderately abundant at the deepest depth strata (100–130 m), although the highest catch rate was found between 70 and 100 m deep (993 squid haul^–1) (Fig. 11). Doryteuthis pleii was abundant on midshelf (395 squid caught, catch rate: 15.8 squid haul^–1), and the highest catch rate was recorded in the shallowest depth strata (<40 m, 369 squid haul^–1) (Fig. 11).

Regarding temporal occurrence, D. sanpaulensis was more abundant during mid-afternoon and in the first third of the night (between 3:00 pm and 9:00 pm). In contrast, the highest abundance of D. pleii was recorded between late night and the first third of the morning (3:00–9:00 am) (Fig. 11).

Figure 11. 

Total number and catch rates of Doryteuthis sanpaulensis and D. pleii collected in different depths and time of day during FAUNEC surveys and within the shared area off southern Brazil. Both metrics were calculated for stations where at least one of the two species was present (FAUNEC surveys) or where both species were present (shared area). Bars: number of squid. Circles: squid per haul. Crosshatched bars and empty circles: D. sanpaulensis. Black bars and dark grey circles: D. pleii.

Doryteuthis sanpaulensis occurred more often in colder waters (T <19.5 °C), a pattern particularly evident for both bottom and surface temperatures. Likewise, the species was frequent in fresher waters (S <34.5), in both surface and the bottom, although it was found in high bottom salinities as well (Fig. 12). Interestingly, a reasonable number of D. sanpaulensis (n = 311) was also caught inside the Patos Lagoon estuary.

The patterns were less clear for Doryteuthis pleii, but apparently the species commonly occurs in warmer surface temperatures (T >21.5 °C), sharing with D. sanpaulensis the same bottom temperature (i.e. T ~19.0 °C). The response of D. pleii to water salinity signatures was also poorly discernible, but the species was apparently more frequent in saltier water (Fig. 12).

Doryteuthis sanpaulensis, D. pleii and L. brevis occurred together in the same haul only in four oceanographic stations conducted at a depth of <15 m. The Fager’s affinity indexes calculated for D. sanpaulensis–L. brevis and D. pleii–L. brevis pairs were 0.28 and 0.31, respectively, indicating a poor association between L. brevis and the two Doryteuthis species.

Figure 12. 

Total number and catch rates of Doryteuthis sanpaulensis and D. pleii collected in different temperatures and salinities during FAUNEC surveys off southern Brazil. Both metrics were calculated for stations where at least one of the two species was present. Bars: number of squid. Circles: squid per haul. Crosshatched bars and empty circles: D. sanpaulensis. Black bars and dark grey circles: D. pleii.

The two species of Doryteuthis co-occurred in only 12 out of the 78 oceanographic stations during the FAUNEC III and IV surveys. Those 12 stations were between 23.5 and 27°S (“shared area”, comprising 32 oceanographic stations, see Fig. 2). A Fager affinity index of 0.7 was calculated for the two co-ocurrying species, suggesting a strong positive relationship between them. However, we believe that it is possible that this index does not reflect a strong affinity, since it was common to collect many exemplars of one species and only a few of the other in the same batch (Fig. 13).

Figure 13. 

Catches of Doryteuthis sanpaulensis and D. pleii within the shared area.

Catches by depth strata within the shared area mirrored the general pattern for both species (Fig. 11). Catches by time of day differed between the two species: Doryteuthis pleii peaked between 3:00 am and 9:00 am (361 squid caught, 40 squid haul^–1), whereas D. sanpaulensis took peaked between 3:00 pm and 9:00 pm (915 squid caught, 130 squid haul^–1) (Fig. 11).

Both species were caught in very similar environmental conditions within the shared area, except for the fact that D. sanpaulensis was caught in a slightly lower average salinity (surface: 34.90, bottom: 33.98) than D. pleii (surface: 35.15, bottom: 33.99). The temperature at the time of capture was the same for the two species (surface: 21.6 °C, bottom: 18.5 °C).

The feeding activity of D. pleii was clearly concentrated between late evening and mid-morning (3:00–9:00 am) regardless of the stage of maturity stage and sex of the specimen. There was also an elevated number of empty stomachs found for the same period (Fig. 14).

Figure 14. 

Stomach fullness frequency of Doryteuthis pleii collected during FAUNEC surveys per period of the day. Black bars: empty stomachs, grey bars: 50% full stomachs, white bars: 100% or distended stomachs.

Ideally, studies on morphologic variations in squid populations must be conducted with samples collected simultaneously, to avoid mixed stock effects (Pierce et al. 1994). Even though this recommendation was followed for FAUNEC samples, we were not able to take simultaneous samples from Uruguay and Argentina. Despite the fact that Uruguayan and Argentinean squid were sampled in the same season (austral summer), their samples were obtained on different years, which may have introduced temporal biases to our analysis. Notwithstanding the latter, it was most instructive to keep these samples for comparison purposes, as they expanded our study area southward in nearly six degrees of latitude.

In addition to the limitations evoked above, it is also important to recognize that there were some other issues with our data that deserve mention. Most of our samples were obtained during a very narrow window of time, and since both Doryteuthis species produce at least two cohorts per year (Vidal et al. 2013, Arkhipkin et al. 2015), it is unlikely that we ended up with a representative set of environmentally driven morphological variability.

Cryptic squid species are difficult to separate on the basis of morphological analyses only (Herke and Foltz 2002) because squid display great phenotypic plasticity in response to environmental conditions (Pelc and Jackson 2008). Furthermore, even though it is unlikely that the morphological differences found in the squid from FAUNEC result from an artefact in the selectivity of the fishing gear (e.g. Martins and Perez 2007), since the same net was employed in all surveys, the same may not be the case of the samples collected from Uruguay and Argentina. This certainly must be considered when interpreting the results.

In our data, latitudinal morphological variability was very evident in D. sanpaulensis, as the dimensions of both soft and hard parts diverged significantly among geographically separated groups. This suggest that D. sanpaulensis populations experience different environmental conditions throughout the sampled area. This is in line with the restricted loliginid larval dispersal potential along the SBB (see Martins et al. 2014), which would expose paralarvae and early juveniles to distinct biotic and abiotic conditions. Environmental variability results in different growth patterns, and thus different final dimensions of body parts as squids mature (Pelc and Jackson 2008). In addition, the morphological variability among geographical groups suggests a strong homing behavior in adult squid.

The morphological variability of body parts along with the morphological overlap analysis and size at maturity data found for D. sanpaulensis in the study area suggests that there are at least six distinct groups scattered between Cabo Frio (23°S) and Mar del Plata (37.8°S). On the SBB (23–29°S), squid inhabiting ~26–28°S (Santa Catarina coast) had shorter mantle lengths and matured at smaller sizes than squid to the north and to the south of this region. In addition, males were significantly smaller than females, in contrast with the typical sexual dimorphism described for this species (i.e., males larger than females; Vidal et al. 2013). Actually, the squid from the Santa Catarina coast differed from others in almost all morphological characters investigated.

Doryteuthis sanpaulensis dwelling to the north and to the south of the coast of Santa Catarina were very similar in morphology. This suggests that these geographically divided groups experienced similar environmental conditions, at least during some part of their lifetime. Latitudinal gradients in water temperature may be a major driver of phenotypic divergence in D. sanpaulensis, since temperature has a direct effect in growth-related parameters (Forsythe 2004). However, variability in other environmental conditions, such as dissolved oxygen and food availability, may also play a role (Jackson and Moltschaniwskyj 2001, van der Vyver 2016).

The most distinct geographic group of D. pleii was also found off the coast of Santa Catarina. Nevertheless, the species had just two distinguishable groups, and these were much less distinct than those of the co-generic species. Thus, it is suggested that the phenotype of D. pleii in Santa Catarina also results from local environmental conditions. However, due to the subtle morphological differences between those two groups, we hypothesise that D. pleii individuals move more between latitudes than D. sanpaulensis, perhaps “homogenizing” the environmental conditions experienced during the squid’s lifetime, since they actively track suitable temperature fields (see Haimovici and Perez 1991b). In addition, these latitudinal movements may promote strong gene flow among different stocks at the SBB. This hypothetical scenario is consistent with a recent genetic analysis that failed to find any relevant genotypic differences between squid from this area and their counterparts on the rest of the South American Atlantic coast (Sales et al. 2017).

In our data, Doryteuthis spp. have specific thermal preferences. Doryteuthis sanpaulensis was found more often in colder waters, whereas D. pleii was associated with tropical (warmer) waters. The two species occurred together only when the water was thermally stratified (when both cold and warm water were present in the same location but in different strata) in the shared area. This ability to actively select water strata that are suitable for them has allowed these two species of squid to exploit locations where they would not normally occur.

We found small and mostly immature representatives of D. sanpaulensis at the Patos Lagoon estuary in January, February and March 1979. The presence of these squid inside the estuary during three consecutive summer months does not seem to be by chance. It is worthy of note that D. sanpaulensis had not been recorded in estuaries (at least in Brazilian waters), and to the best of our knowledge, this is the first record of a loliginid squid other than L. brevis (which has well-described physiologic mechanisms to tolerate low salinities, see Hendrix et al. 1981) inside an estuary.

These small D. sanpaulensis may enter the estuary during events of physiologically tolerable environmental conditions to exploit the food-rich, productive estuarine areas. This could be related to landward intrusion of the salt edge into the upper estuary, a near-bottom, high-salinity tongue of seawater, which is particularly strong during austral summer months, when the freshwater discharge is less (Möller et al. 2001). Off the coast Rio de Janeiro, juvenile D. sanpaulensis were found entering the warm Guanabara Bay (22°43’S), during flood tide, tracking bottom landward intrusions of cold SACW. In this case, squid seems to use the physiologically suitable near-bottom temperature field to exploit the abundant food resources inside this eutrophized tropical coastal bay (Moraes and Lavrado 2017).

When this study was conducted, our data extended the southernmost distribution range of D. pleii (29°S, Palacio 1977) by at least nine degrees of latitude, owing to some specimens obtained at ~38°S (Mar del Plata, Argentina). In fact, latter studies found that D. pleii occurs during spring and summer to the south of its normal range over the shelf break between 29 and 34°S, tracking the warm, fast flowing superficial Brazil Current (BC) (Haimovici and Perez 1991b). Recently, D. pleii was recorded as far as Península Valdez, Argentina (42°S) (Augusto Crespi-Abril 2017, personal communication). Thus, it is clear that stronger poleward BC penetration in some years may carry vagrant individuals toward higher latitudes during austral warmer seasons.

According to our results, the two species of Doryteuthis, which are morphologically similar, employ several mechanisms to avoid direct competition in the wider continental shelf. For instance, the diel activity pattern was inverse, with D. pleii being more active during daylight, whereas D. sanpaulensis was more active at night. Furthermore, both species had clearly different thermal preferences: D. sanpaulensis was found more often in colder temperatures than D. pleii. In addition, the most common depths where each species was found were different. The feeding schedule in D. pleii peaks between late night and early morning, whereas D. sanpaulensis feeds more intensively between mid-morning and late afternoon (Andriguetto and Haimovici 1997). This temporal mismatch between feeding activity schedules may be regarded as an additional mechanism that prevents direct competition.

This study revisits data from a time when climate change had not yet been perceived as an agent of significant environmental variability in the ocean (Abran et al. 2016). Thus, the information reported here may be regarded as a “snapshot” on the ecology of sympatric squids in a colder and less acidic marine environment (see Nagelkerke and Connell 2015). It seems that the abundance of the commonest loliginid squids off the southern Brazilian coast has shifted in the last few decades. Doryteuthis pleii apparently replaced D. sanpaulensis as the most abundant squid in some areas where the latter species used to be dominant, such as off the coast of northern Rio de Janeiro (~23°S) (e.g., Costa et al. 2017). Whether this apparent shift was caused by climate change or not is currently unknown. Accordingly, our results may be particularly important for a better understanding of more recent studies on the biology and ecology of loliginid squids in the southwestern Atlantic Ocean and elsewhere.