On the presence of Dipturus nidarosiensis (Storm, 1881) in the Central Mediterranean area

Introduction

Species discrimination is a challenging issue mainly because it is based only on morphological diagnoses. Indeed, species classification errors are frequent because of several factors, such as phenotypic plasticity, the presence of cryptic taxa, morphological changes linked to life stage and the high level of expertise required for this kind of analysis (Hebert et al., 2003).

The misclassification of skates is common (e.g.,  Serena, 2005; Frodella et al., 2016; Iglesias, Toulhoat & Sellos, 2010). It has been estimated that approximately 50 known species are still only partially described or completely undescribed (Coulson et al., 2011). Consequently, in recent decades, DNA-based species discrimination has been applied (Hebert, Ratnasingham & deWaard, 2003) in an increasing number of cases (Spouge, 2016). The use of molecular markers, specifically the cytochrome c oxidase I gene (COI), has proven to be very helpful on several occasions in the identification of ray and skate species in the Mediterranean, Atlantic and other waters worldwide (Ball et al., 2016; Bineesh et al., 2017; Cannas et al., 2010; Cariani et al., 2017; Cerutti-Pereyra et al., 2012; Coulson et al., 2011; Frodella et al., 2016; Griffiths et al., 2013; Iglesias, Toulhoat & Sellos, 2010; Landi et al., 2014; Lynghammar et al., 2014; Mabragana et al., 2011; Ordines et al., 2017; Ramírez-Amaro et al., 2017; Serra-Pereira et al., 2011; Spies et al., 2006; Vargas-Caro et al., 2016; Vella, Vella & Schembri, 2017; Ward et al., 2008; Ward et al., 2005; Martin Dia zDe Astarloa et al., 2008).

In this context, skates (Rajidae) display conservative morphological traits both among and between alive and fossil species (Valsecchi et al., 2005). Hence, for this particular taxonomic group, DNA barcoding has been used to describe new species (e.g., Last & Séret, 2016), extirpation (e.g., Ball et al., 2016) and species’ expansion range (e.g., Ordines et al., 2017). In particular, the misclassification problem is particularly crucial in the genus Dipturus (Griffiths et al., 2010; Iglesias, Toulhoat & Sellos, 2010). In the eastern Atlantic Ocean and in the Mediterranean Basin, such misclassification includes Dipturus batis (Linnaeus, 1758), Dipturus oxyrinchus (Linnaeus, 1758) and Dipturus nidarosiensis (Storm, 1881). There has been a high percentage of misclassification in the fishery data reported for the French coast (Iglesias, Toulhoat & Sellos, 2010), mostly among the following species: D. oxyrinchus, D. batis, D. nidarosiensis and Rostroraja alba (Lacepede, 1803). These findings could have led to the underestimation of the risk of a dramatic decline and collapse of the spawning stock (Griffiths et al., 2010; Iglesias, Toulhoat & Sellos, 2010). Hence, the risk of extinction of these species might be higher than previously calculated (Iglesias, Toulhoat & Sellos, 2010).

D. nidarosiensis is one of the species that is often misclassified in both the Atlantic and the Mediterranean Basin (Cannas et al., 2010; Follesa et al., 2011; Cariani et al., 2017; Ramírez-Amaro et al., 2017). The geographical distribution of the Norwegian skate D. nidarosiensis is known to occur along the northeast Atlantic, from Iceland, the Faroe Islands and northern Norway to Madeira and northern Mauritania (Stehmann, 1990). This species is considered endemic in the northeast Atlantic and the Bay of Biscay (Priede et al., 2010; Stehmann & Bürkel, 1984; Rodríguez-Cabello, Pérez & Sánchez, 2013). However, recently, it has been recorded in the western Mediterranean Sea, along the southern Sardinia coast (Cannas et al., 2010; Follesa et al., 2012; Cariani et al., 2017; Marongiu et al., 2017; Porcu et al., 2017), off Algeria (Cariani et al., 2017), in the Alboran Sea (Ramírez-Amaro et al., 2017) and in the southern Adriatic and Ionian Seas (Cariani et al., 2017). The first catch of Norwegian skate specimens in western Mediterranean waters (2005) was reported to have occurred in Sardinia (Cannas et al., 2010); however, the records of the Alboran Sea are more recent (2012–2016; Ramírez-Amaro et al., 2017). The abovementioned authors hypothesised the ancient presence of the Norwegian skate, which had not been detected due to a misclassification between D. nidarosiensis and D. batis (Cannas et al., 2010).

In the IUCN Red List, the Norwegian skate D. nidarosiensis is listed as near-threatened (Stehmann et al., 2015); moreover, this species is rare in both the northeast Atlantic and the Mediterranean areas (Williams, Helle & Aschan, 2008; Cannas et al., 2010). Moreover, because the Norwegian skate has biological characteristics that are similar to other elasmobranchs, such as low fecundity, slow growth and high length at first maturity (Follesa et al., 2012), it is particularly vulnerable to environmental and anthropogenic pressures. Until now, information about the life history of the Norwegian skate in the Mediterranean Basin has been limited and based only on specimens caught around Sardinia Island (i.e., the central-western Mediterranean) (Follesa et al., 2012).

The present study is the first, from morphological and genetic points of view, to describe and characterise the Norwegian skate specimens caught in the southern Adriatic Sea and the north-western Ionian Sea (Central Mediterranean Basin), where this species has never been reported (Marano et al., 2002; Sion et al., 2003; Sion et al., 2004; Zupa et al., 2010; Relini et al., 2000; Relini et al., 2010) until very recently (Cariani et al., 2017). This study provides further information about the geographical distribution and the life-history traits of this species. Morphological comparisons with specimens from Sardinian waters and the Alboran Sea are made to provide the correct classification of this species in the Mediterranean Basin (Cannas et al., 2010; Follesa et al., 2012; Ramírez-Amaro et al., 2017).

Methods

Sampling

Samples of D. nidarosiensis were collected on land (e.g., market sampling) along the ports of the Apulia region and, from offshore observers (i.e., discard monitoring) in a fishery sampling programme (Data Collection Framework [DCF]; EU Reg. 1543/2000, 1639/2001, 1581/2004, 199/2008 and 1004/2017) and from fisheries independent scientific surveys (Mediterranean International Trawl Survey - MEDITS) (AAVV, 2017) (Fig. 1). In total, eight specimens were morphologically analysed and preserved in the deep freezer (−20 °C) at the COISPA Institute Laboratory. Morphology and colour analysis as well as morphometric measurements (nearest 1 mm) (Fig. 2), weights (the nearest 1 g) and meristic counts were performed following the previous descriptions of skate species (Ebert & Stehmann, 2013; Serena, 2005). All measurements and meristic counts are shown in Fig. 2 and Table 1.

Sex and maturity were determined macroscopically, and the gonad weights were recorded to the nearest 0.1 g. Gonadal maturity was classified using the MEDITS maturity scale (AAVV, 2017; Follesa & Carbonara, 2019). The following results are shown in Table 2: Stage 1 (virgin immature); Stage 2 (maturing); Stage 3A (mature); Stage 3B (mature/extruding active); Stage 4A (resting); Stage 4B (regenerating).

Ethical compliance

All the Norwegian skate (Dipturus nidarosiensis) analysed in this study, which were drawn from commercial fishing activity (DCF; EU Reg. 1004/2017), did not fit the European Commission recommendations (Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010) or Italian National Law (Decree Law n. 26 of 4 March 2014) regarding the protection of animals used for scientific experiments. Moreover, according to the welfare guidelines under the Decree Law n. 26 of 4 March 2014 (Italian Ministry of Health) and the protocol approved by the Committee on the Ethics of Animal Experiments of COISPA (Italian Ministry of Health 15/2015-UT), the specimens brought alive onboard during the scientific survey or the monitoring on the commercial fishing vessel should be killed using an overdose of anaesthetic. However, all the animals sampled in this study were already dead at the time of sampling: both the specimens sampled at the landing and on board (i.e., discard monitoring and scientific survey when the specimens were brought on board).

Results

The Norwegian skate specimens were captured around the pit of Bari and in the northwest part of the Ionian Sea (Fig. 2) at depths ranging between 320 and 720 m. All measurements, meristic counts, sex and maturity are presented in Table 1. All specimens showed uniformly dark ventral sides, including the ampullary pores (Fig. 3). The disc was wider (WD 64.0–74.0% of TL) than it was long (LD 52.5–59.7% of TL), and it was covered uniformly by a darkish mucous on both sides (Fig. 3). The teeth were pointed in parallel rows in the males and in molar-like sets in pavement in the females (Fig. 4), with the numbers of tooth rows on the upper and lower jaws between 42–45 and 39–43, respectively. It appeared that the number of thorns on the tail increased with the length of the specimens, as shown in Table 1. Moreover, both the dorsal and ventral sides were prickly in the area of the snout (Fig. 5). Morphometric variability between the juveniles and adults was detected in the relationships between the Length Tail (Ltail)/Total Length and Distance interorbital (Dorb)/Total Length (Table 1). In terms of the percentage of TL, Ltail was longer in the juveniles at about half of the TL, and it was one-third of the TL in the sub-adults and adults (Fig. 2). On the contrary, the Dorb increased with the increasing of TL. Moreover, the juveniles did not show spines on the median line of the disc or on both lateral sides of the tail, which were present in the adults (Table 1).

The juveniles (maturity stage 1) were caught in shallow waters at depths between 320 and 377 m, whereas the sub-adult (maturity stage 2) and adult specimens (maturity stage 3A and 3B) were caught in depths ranging between 550 and 720 m.

Furthermore, all samples caught during the MEDITS trawl survey were associated with a biocenosis typical of compact bathyal muds. In particular, the juveniles (specimens 5, 7 and 8; Table 1) caught at upper depths were found to be in association with a biocenosis characterised by tall sea pen Funiculina quadrangularis facies.

Genetic analyses

A 633 bp fragment of the COI gene was obtained from eight individuals from the South Adriatic and Ionian seas. The combination of these data with those available for D. nidarosiensis in other areas in the Mediterranean and the adjacent Atlantic Ocean provided the final alignment of 38 sequences and seven haplotypes (555 bp; Hd: 0.515; k: 0.586; π: 0.00106). The haplotype network (Fig. 6) showed the occurrence of a common haplotype (Hap_1) shared by 26 individuals from all investigated areas except South Africa. It also showed six additional haplotypes, four of which were exclusive to the Mediterranean (Hap_2-Hap_5), and two were exclusive to the Atlantic (Hap_6-Hap_7).

The phylogenetic analyses (Fig. 7) further confirmed the identification of the Ionian–Adriatic specimens as D. nidarosiensis. They were clustered in a strongly supported clade that was clearly distinguishable from the other species of Dipturus found in the Mediterranean and the eastern Atlantic Ocean. The sequences that were the most similar to D. nidarosiensis were those of D. cf flossada (95% base pair similarity), and the least similar were those of R. alba (87.82% base pair similarity).

Discussion

The results of the present study confirmed the presence of the Norwegian skate Dipturus nidarosiensis in the central Mediterranean Sea by identifying morphometric and morphological features and molecular markers. The morphological parameters described are in line with the data collected in Sardinian waters (Cannas et al., 2010) and the Alboran Sea (Ramírez-Amaro et al., 2017). In particular, the relationship between the morphometric measurements and total length was similar to the data reported in the Alboran Sea (Ramírez-Amaro et al., 2017). Specifically, the measurements of the juveniles from the Spanish area (Ramírez-Amaro et al., 2017) were in accordance with those of the Adriatic Basin. Furthermore, the high variability in Length Tail during the different stages of life seemed to be common to other Rajidae species, including R. clavata, D. oxyrinchus and Amblyraja radiata (Mnasri et al., 2009; Capapé et al., 2015; Templeman, 1987).

The genetic analysis of the mitochondrial gene COI made it possible to confirm the morphological identification of the Adriatic–Ionian samples as D. nidarosiensis. In particular, in this study, the molecular data revealed that the Mediterranean specimens of D. nidarosiensis (from Sardinia, Algeria and the Alboran Sea) shared COI mitochondrial haplotypes with the Atlantic specimens (see Table S1 for details). However, additional analyses based on more powerful markers (i.e., microsatellites and SNPs) and larger numbers of individuals are needed to investigate possible intraspecific differences and genetic connectivity among the different areas within the Mediterranean as well as between the latter and the Atlantic populations (Griffiths et al., 2011).

The D. nidarosiensis reported in this paper were captured mainly around the Bari Pit (maximum depth = 1,233 m) in an area between the western Italian coast and the eastern Montenegrin and Albanian coasts. This area is connected with the northern part of the Ionian Sea through the Otranto Channel, and it is part of a system of currents that exchange and mix deep water from the eastern part of the Mediterranean Sea and the northern part of the Adriatic Sea (Vilibic & Orlic, 2002). Although the capture locations of Norwegian skates are dispersed over 200 miles, all these areas are connected by deep-water currents (Artegiani et al., 1997). In the southern Adriatic and Ionian bathyal systems, there is an absence of barriers to gene flow in deep species as well as Aristeus antennatus (Marra et al., 2015). In the case of A. antennatus, it was previously hypothesised that the presence of the population could be structured into two layers: a deeper stock not affected by fishing, which preserves genetic homogeneity; and an upper stock living in the fishing ground. The larval dispersal, adult migration and continuous movements of individuals from deeper grounds to upper areas could explain their genetic homogeneity throughout the Adriatic and Ionian system (Marra et al., 2015). Similarly, the partial overlap between the Norwegian skate’s habitat and fishing grounds in the Adriatic and Ionian Seas suggests that the Adriatic and Ionian D. nidarosiensis specimens are part of the same population.

The occurrence of the Norwegian skate in the southern Adriatic Sea and northern Ionian Sea sheds new light on the dispersion of this species in the Mediterranean Basin. In interviews with fishermen in the southern Adriatic, it emerged that the occurrence of this species, locally called “black monk”, is not unusual.

However, the presence of this species in the Mediterranean was unnoticed until 2010 (Cannas et al., 2010), which could be attributed to two main reasons. Firstly, it had been misclassified with others species of the Dipturus genus (Cannas et al., 2010) as well as for the French coast (Iglesias, Toulhoat & Sellos, 2010). Indeed, the presence of the Dipturus species is not recent (Griffiths et al., 2011). In the southern Adriatic, catches of D. oxyrinchus were reported in 1994–1995 (Relini et al., 2000; Marano et al., 2002), and in the western Ionian, catches of D. batis and D. oxyrinchus were recorded in 1992 and 2000–2001, respectively (Relini et al., 2000; Sion et al., 2003). In the central-western part of the basin, D. oxyrinchus has been caught since the first surveys that were funded by the DCF in mid-1980 (Relini et al., 2000; Relini et al., 2010). Secondly, in the southern Adriatic, the bottom trawl fleet fishing grounds are concentrated on the shelf and the upper part of slope (Mannini & Sabatella, 2015). In the western Ionian, as in the other part of the basin, they are located mainly on the slope to a depth of 800 m (Russo et al., 2017). Hence, the catches of D. nidarosiensis characterised by a wider depth distribution (between 300 and 1,700 m) (Cannas et al., 2010; Follesa et al., 2012; Ramírez-Amaro et al., 2017) could be unrecorded. In the studied areas, the depth of the fishing activity only partially overlaps the deep distribution of the Norwegian skate. In addition, in the Mediterranean sites where D. nidarosiensis specimens were found, the seabed presents similar characteristics (e.g., canyons and slopes of high depths), which are favourable to this species but unfavourable to the trawling activity of both commercial (Mannini & Sabatella, 2015) and research vessels (AAVV, 2017).

The presence of maturing and mature males and females in early autumn (end of September/beginning of October) and summer, respectively, could support previous preliminary observations on the reproductive biology of the species in Sardinian waters (Follesa et al., 2012). Furthermore, the collection of empty egg cases on the sea floor as well as individuals in the different life stages (embryos within egg case, juveniles, sub-adults and adults) of both sexes suggests the presence of a species with a permanent reproductive allocation in the Mediterranean Sea (Cannas et al., 2010; Follesa et al., 2012; Cariani et al., 2017; Porcu et al., 2017; Ramírez-Amaro et al., 2017).

Based on the data recorded, despite the few specimens analysed, the Norwegian skate showed that a bathymetric segregation of juveniles occurred only in the upper part of the slope, and sub-adults and adults occurred at lower depths, which was previously reported in Sardinian waters (Follesa et al., 2012). This deep segregation by the ontogenic phase was also observed in other species of skates, including Raja clavata, R. asterias and Rhinoptera bonasus (Rousset, 1990; Ferrá et al., 2016; Fisher, Call & Grubbs, 2013; Ellis et al., 2005). In addition, the finding of Ionian and Adriatic D. nidarosiensis specimens on compact mud bottoms confirmed that the species prefers this type of sea bed, which was also observed by Follesa et al. (2012) in Sardinian waters. Moreover, the present study is the first to record the presence of juveniles in association with a biocenosis characterised by bathyal muds with tall sea pen Funiculina quadrangularis facies.

Nevertheless, several aspects of D. nidarosiensis remain poorly investigated. For this reason, it would be useful to continue the monitoring and sampling of this species in order to collect additional data to clarify aspects of its biology (e.g., reproduction and growth) and ecology (e.g., trophic relationship and geographical distribution).

Conclusion

The results obtained in this study confirm the presence of Dipturus nidarosiensis in the central Mediterranean Sea (southern Adriatic and northwest Ionian seas) and provide information about its geographical distribution, life-history and morphological traits in the Mediterranean Basin. Indeed, misclassification of the Mediterranean skate species has occurred frequently (Iglesias, Toulhoat & Sellos, 2010), resulting in the misinterpretation of several biological characteristics. The morphological data were similar to those recorded in Sardinia and Alboran Sea (Cannas et al., 2010; Ramírez-Amaro et al., 2017). Moreover, the morphological differences between juveniles and adults were highlighted in terms of morphological and meristic characteristics. The genetic analysis conducted through the mitochondrial gene COI (Spouge, 2016) made it possible to confirm the morphological identification of the Adriatic–Ionian samples as D. nidarosiensis.

Supplemental Information