1. Introduction
Family Ommastrephidae Steenstrup, 1857 includes many economically and ecologically important squid species distributed in all oceans from the sub-Arctic to sub-Antarctic seas [
1]. These abundant, muscular, and fastest-growing squids inhabit shelf, continental slope, and open ocean waters, from the surface to depths of 2000 m, and are one of the most exploited invertebrate fishing resources, representing 50% of the total world cephalopod catch [
1]. In 2021, the total catch of Ommastrephidae in the North Atlantic and Mediterranean waters was 73,590 tons [
2]. Oceanic ommastrephids are considered an abundant underexploited fishery resource, with only
Ommastrephes bartramii and
Dosidicus gigas commercially exploited in the Pacific waters [
1,
3].
Despite ommastrephids’ economic significance, the phylogeny of the
Ommastrephes genus has only just recently been resolved by Fernández-Álvarez et al. [
4].
Ommastrephes d‘Orbigny, 1834 was considered a monotypic genus with only one cosmopolitan species
O. bartramii (Lesueur, 1821) with discontinuous distributions between the southern and northern hemispheres and with three geographically different populations: the North Atlantic, the North Pacific, and the Southern Hemisphere (the South Atlantic, Indian Ocean, and Southwestern) [
1,
5]. However, previously observed morphological (mainly spermatophore morphology) [
6,
7] and metabolic (cholinesterase activities of optical ganglia) [
8,
9] differences between these populations inhabiting different geographic regions brought the monotypy of the
Ommastrephes genus into question. Fernández-Álvarez et al. [
4] performed a molecular analysis of individuals collected throughout the genus distribution area using two mitochondrial markers (COI, subunit I cytochrome c oxidase and 16S rRNA, ribosomal RNA). Their results showed that the genus
Ommastrephes is an allopatric cryptic complex of four species with the following proposed nominal names and distributional ranges:
Ommastrephes caroli (Furtado, 1887) (Northeast Atlantic),
Ommastrephes cylindraceus d‘Orbigny, 1835 (East Tropical and South Atlantic with South Indian),
Ommastrephes brevimanus (Gould, 1852) (South Pacific), and
Ommastrephes bartramii (Lesueur, 1821) (Northwest and Central North Pacific). Furthermore, the authors found that
O. caroli represented a pseudocryptic species, most phylogenetically divergent from others, indicating a longer evolutionary history due to the isolation from the remaining congeneric species.
In the Mediterranean Sea, mesopelagic
O. caroli is still poorly studied, most likely because it is rarely caught by fishing gear and the lack of experimental research in the mid-waters [
10]. In addition to sporadic findings of moribund females drifting in shallow waters or stranded on shore, these squids are occasionally found in the gastric contents of teuthophagous predators [
11,
12]. However, since 2004, the frequency of observations of
O. caroli in the Mediterranean has been increasing [
10], suggesting possible changes in species distribution and abundance. So far, reported records of juvenile individuals [
13,
14,
15] are scarcer than those of larger adults, mostly stranded post-spawning females [
10,
16,
17,
18,
19,
20]. The known range of this species currently covers the entire Mediterranean Sea, i.e., western and central parts, Taranto Bay, Adriatic, Aegean, and Levantine Seas [
4,
10,
15,
20]. According to Lefkaditou et al. [
10], this recent record increase in the Northwest and Northeast Mediterranean could be a consequence of warmer sea surface temperatures that drive the northward expansion of native warm-water species. More recently, Lefkaditou et al. [
10] reported on the body and beak morphometry, diet, and maturity of adult specimens collected from the Aegean and Ionian Sea (Easternmost Mediterranean), while Agus et al. [
20] investigated the morphological, biometric, and reproductive characteristics and assessed the age based on the beak and lens analysis of specimens from the Sardinian waters (Western Mediterranean). Furthermore, Agus et al. [
20] confirmed the findings of Fernández-Álvarez et al. [
4] and additionally suggested potential genetic differentiation between the Mediterranean and Atlantic populations.
In the Adriatic Sea, the first record of
O. caroli dates back to March 1910, when an individual of 165 mm DML was caught near Vodice (Central -Eastern Adriatic) [
21]. In March 1986, in the Central Western Adriatic (20 mls off Falconara Marittima), a female of 560 mm DML was caught by midwater trawl [
22]. More recently, Franjević et al. [
18] reported a large female caught with a spear at a depth of 1.5 m in February 2013 in the waters of the island of Šipan (Central Eastern Adriatic), weighing 9 kg and measuring 1.3 m in length (without tentacles). Franjević et al. [
18] identified this specimen using the mitochondrial COI gene.
Apart from the three above-mentioned findings, of which only one was molecularly identified, knowledge about O. caroli in the Adriatic is lacking. Therefore, when we collected a larger sample of this species, it was a rare opportunity to investigate and complement the scarce existing knowledge about this oceanic squid. The aim of this study was to describe the morphological features of the body and beak, estimate the age using statolith microstructure analysis, and genetically identify and characterize the collected individuals using two mitochondrial genes (COI and 16S rRNA). The results of the present study will greatly contribute to future taxonomic and ecological research of the Ommastrephes genus.
4. Discussion
This is the first comprehensive study on the morphometry, age, and genetic structure of
Ommastrephes caroli in the Adriatic Sea. All twenty-three investigated individuals were caught in September 2020, providing the most abundant sample of this species in the Mediterranean waters. Up to date, only three individuals were seen in the Adriatic, one juvenile in 1910 of 165 mm DML [
21] and two adult females in 1986 [
22] and 2013 [
18] of 560 mm DML and 1300 mm in length without tentacles, respectively. Interestingly, these mature females were reported in February and March, and the juveniles in this study were caught in September. Similarly, in the Eastern Mediterranean (Aegean Sea), Lefkaditou et al. [
10] reported juveniles (DML < 143 mm) mainly in August and September, while the largest specimens (560–660 mm DML) were caught from April to June, coinciding with the significant warming of the Tyrrhenian Sea surface. In the Western Mediterranean (Sardinian waters), Agus et al. [
20] also recorded large mature
O. caroli specimens in the spring–summer period (March–July). These observations reveal the species’ reproductive activity in the Mediterranean, with spawning occurring during the summer months, which is congruent with our aging results showing that the squids hatched during July and August. According to Lefkaditou et al. [
10], the spawning ground of
O. caroli could be in the warmer Eastern basin due to the more frequent presence of large moribund females in the Sea of Crete, while the findings of predominately smaller squid from the coast of western Italy indicate the colder Western basin as an important feeding ground. The
O. bartramii migrations between the spawning and feeding grounds in the North Pacific have been connected with a suitable surface sea temperature (SST) and chlorophyll a concentration (chl. a) [
54,
55], with its distribution affected by the Kuroshio Current [
27]. The sea surface temperature is significantly correlated with the monthly latitudinal gravity center of CPUE (catch per unit effort), strongly suggesting that
O. bartramii distribution is controlled by an optimal thermal habitat [
3]. From the above-mentioned, it can be assumed that the Adriatic Sea could also represent a feeding ground for Mediterranean
O. caroli individuals.
The length–weight relationship of
O. caroli juveniles inferred in this study showed an isometric growth, proportional to the weight and length, which is a general pattern for oceanic
Ommastrephes species that in comparison with coastal squid, have an isometric to positively allometric growth [
55,
56,
57]. Considering our small sample size, this should be taken cautiously, especially since short-lived squids, characterized by high plasticity in growth and maturation, can easily adapt their morphology to environmental or ecosystem change, leading to interannual differences in size and growth rates [
58]. Available data regarding the
O. caroli length–weight relationship are provided by Lefkaditou et al.’s [
10] study on 30 mature individuals using an exponential model, which showed a better fit than the traditional power model. Of course, there is more available information on length–weight relationships and the growth of
Ommastrephes species inhabiting the Pacific [
54,
57,
59,
60,
61,
62] and South Indian waters [
56] reporting on higher growth rates in females than males and significant interannual variation in length–weight relationships from isometric to positive allometric growth. Unfortunately, due to our small sample size, we could not test any variation between the sexes or seasons; therefore, future studies with a larger number of individuals are very much needed to describe the growth pattern of Mediterranean
O. caroli as this information is critical to understand the species’ life-history traits and population dynamic, and essential for assessment, management, and conservation [
63].
Direct methods for assessing the age and growth of cephalopods use hard body parts, i.e., gladius, cuttlebone, beak, lens, and statolith because these structures remember ontogenetic events by forming periodic growth increments. For squid, the analysis of the statolith microstructure is the most widespread method of investigating age and growth. Size-at-age data, based on statolith readings, have been shown as a very useful tool in the identification of seasonal cohorts and growth rate estimation for commercially exploited ommastrephids [
24,
64,
65,
66,
67,
68]. For
O. bartramii in the North Pacific waters, this method has been used to determine growth rates and lifespan [
69], examine the somatic and statolith growth of wild and artificially reared paralarvae and wild juveniles of the autumn cohort [
59], analyze the growth parameters of two allopatric stocks, and propose a tempo-spatial migration model [
60]. By reading statolith growth increments on 237 individuals, Yatsu et al. [
69] concluded a 1-year lifespan of
O. bartramii reaching maturity at the age of about 7–10 months, with almost year-round spawning activity. In the present study, the estimated age of the caught
O. caroli juveniles (65 to 152 mm DML) ranged from 36 to 64 days, with a mean of 47.6 days. Considering that our sample consisted of only 23 individuals, and that squids can exhibit considerable plasticity in respect to environmental conditions, our data were insufficient to develop an appropriate growth model representative of the population. Based on the beaks and eye lenses, Agus et al. [
20] estimated the mean lifespan of Mediterranean
O. caroli at around 12 to 13 months.
The beak represents a resistant chitinous structure in squids used for age determination, species and population identification, or investigation of trophic ecology [
70,
71,
72]. Cephalopods are key players in oceanic food webs and understanding their trophic interactions is essential for understanding marine ecosystems. In this study, we have inferred relationships between the dorsal mantle length and various beak measures of juvenile
O. caroli in the Adriatic. Four measures showed a linear relationship with the DML: LHL, UHL, ULWa, and UWL, of which the LHL and UHL seem to represent the best fit to the data. Due to the small sample size, all the tested models (linear, power, exponential, and logarithmic) showed a relatively equal fit to the data. Lefkaditou et al. [
10] described the fit of the DML to LRL with the power equation, which was second-ranked in our study after the logarithmic with just a small increase in the AICc value. A larger sample size and long-term studies are needed to provide more reliable estimates, especially as it has recently been demonstrated that Pacific
O. bartramii changed the body size and growth pattern of the beak shape in response to environmental change [
61]. Nevertheless, the models provide a useful starting point in understanding the relationship between beak morphology and body size in the Adriatic Sea.
In line with recent molecular phylogenetic findings and the resurrection of four nominal species within the genus
Ommastrephes, the Adriatic samples clustered with other COI and 16S sequences from the North Atlantic [
4], the Western Mediterranean [
20], and the Eastern Adriatic [
18] into a separate clade specific for
O. caroli, and separately from other
Ommastrephes species. The species identification was further corroborated by a range of intra- and interspecific
p-distances: the intraspecific distances did not exceed 1.75% for COI and 3.77% for 16S, while the interspecific distances ranged from 2–9.40% for COI and 0.29–5.22% for 16S. The barcoding gap was evident in the genetic distances for COI [
73], and overlap in 16S distances, as observed by Fernández-Álvarez et al. [
4]. Haplotype analyses and differentiation indexes point to the existence of genetic structuring between the Atlantic and Mediterranean/Adriatic samples, as first suggested by Agus et al. [
20]. This analysis is, however, hampered by a small number of COI sequences available for
O. caroli of Atlantic origin in public databases. A better sample is available for 16S, which further supports the separation between the Atlantic and Mediterranean/Adriatic basins. However, a much smaller fragment of 16S was analyzed in this study, and its taxonomic resolution for this species as well as suitability for population studies need to be further investigated [
4,
20]. The distributional range of the
Ommastrephes species is largely shaped by oceanic gyres, currents, and surface water temperatures affecting the dispersion of paralarvae [
1]. Population structuring between the Atlantic and the Mediterranean has been observed in other marine species, cephalopods included, and attributed to the existence of historical and contemporary hydrographic barriers to the gene flow: the Gibraltar Strait and the Almería–Oran front, in addition to isolation by distance [
74,
75,
76,
77]. This might also be a significant contributing factor shaping
O. caroli structure. For further research, a larger number of individuals should be sampled throughout the Mediterranean and Atlantic using additional genetic markers to clarify these processes.