Gaidropsarus gallaeciae (Gadiformes: Gaidropsaridae), a New Northeast Atlantic Rockling Fish, with Commentary on the Taxonomy of the Genus

Simple Summary The genus Gaidropsarus is a poorly known group of marine fishes found from the intertidal zone to the deep sea in all three major oceans. The present taxonomic study describes a new deep-sea species of this genus originating from Galicia and Porcupine Banks, two seamount-like structures in the Northeast Atlantic. The results suggest that deep-water coral reefs could be an essential habitat for this species. The existence of this new species was previously flagged by the analysis of mitochondrial DNA sequences of the species of the genus described in the North Atlantic, and has been corroborated by morphological examination of the specimens. Abstract A new species of rockling fish genus Gaidropsarus is described based on six specimens collected in Galicia and Porcupine Banks, in Atlantic European waters. An analysis of morphological characters has confirmed the specific status of specimens of a previously described clade by comparison of DNA sequences. Gaidropsarus gallaeciae sp. nov. it is distinguished from congeners by the following combination of characters: 43–44 vertebrae; 54–60 third dorsal fin rays; 44–52 anal fin rays; 21–23 pectoral fin rays; head length 21.1–25.2% of standard length (SL); length of the pelvic fin 16.2–19% SL; length of the first dorsal fin ray 15.8–27% of head length (%HL); eye diameter 15.8–20.5% HL; and interorbital space 21.7–28% HL. Using the nucleotide sequence of the 5’ end of the mitochondrial COI gene as a molecular marker, the genetic p-distance between the new species and its congeners far exceeds the usual 2%, granting the former the status of an independent taxon, which is in accordance with the morphological identification. A comparison with the other 12 valid species of the genus is presented. The study also highlights the morphological diversity resulting from the meristic and biometric variability of Gaidropsarus species and lays the groundwork for future taxonomic studies on this genus.


Introduction
The genus Gaidropsarus Rafinesque, 1810 shows a remarkable ecological diversity ranging from intertidal and near-shore to deeper areas up to 2000 m depth, and from arctic This study provides a formal description of this set of individuals as a new species explores the current state of knowledge of the genus.

Materials and Methods
Specimens come from two Atlantic seamount-like structures (Figure 1). The G Bank is a non-volcanic seamount located off the northwest of the Iberian Penin between 42°15′ N-43° N and 11°30′ W-12°15′ W, at water depths from 625 to 1800 m at about 125 nautical miles from the coast, and is 50 km long in the E-W direction an km on the N-S axis [8]. The presence of vulnerable species and habitats in this bank, as Lophelia and Madrepora communities and black and bamboo coral aggregations the basis for its inclusion in the Natura 2000 network as a Site of Community Impor [22]. The Porcupine Bank is located in the Northeast Atlantic, from 13° W to 1 longitude and from 51° N to 54° N latitude, 200 km off the west coast of Irelan extends from a depth of 150 m to 4000 m of the abyssal plain, forming a seamoun structure, which is connected by a narrow strip to the continental shelf. The closed w recirculation system in this area favours the retention of nutrients and plankton, cre an area of high productivity. Sampling on the Galicia Bank was conducted aboard the R/V "Thalassa", as pa the INDEMARES project (BanGal0810), an exploratory multi-gear survey, wh sampling on the Porcupine Bank was conducted aboard the R/V "Vizconde de during the annual bottom-trawl surveys (Porcupine 2019), using a Baca-GAV 39/52 a cod-end mesh size of 20 mm. Specimens were taken from the catch and froze Sampling on the Galicia Bank was conducted aboard the R/V "Thalassa", as part of the INDEMARES project (BanGal0810), an exploratory multi-gear survey, whereas sampling on the Porcupine Bank was conducted aboard the R/V "Vizconde de Eza" during the annual bottom-trawl surveys (Porcupine 2019), using a Baca-GAV 39/52 with a cod-end mesh size of 20 mm. Specimens were taken from the catch and frozen on board. In the laboratory, specimens were thawed, examined and photographed. With the exception of total length (TL) and standard length (SL), measurements are distances perpendicular to the length of the fish measured with a digital calliper to the nearest 0.1 mm on the type specimens and to nearest mm on the comparative material. Counts and measurements were recorded following Svetovidov [23,24]. All measurements are expressed as the percentage of standard length (%SL) or head length (%HL). Voucher specimens were deposited in the ichthyological collection of the Museo de Historia Natural, Universidade de Santiago de Compostela (MHNUSC).
To improve the knowledge of the natural variation of the species of the genus, a comprehensive review of the morphological characters, distribution and coloration of valid Gaidropsarus species reported in the ichthyological literature, mainly compiled in Svetovidov [23,24], Barros-García et al. [2] and Biscoito and Saldanha [4], was complemented with measurements and counts of own comparative material, when available.
A single COI sequence representative for each valid Gaidropsarus species available (n = 7) was retrieved from Genbank specimens belonging to the author's project 'Molecular identification of Gaidropsarus fishes' (Code GSRUS) in BOLD systems (https://www. boldsystems.org/, accessed on 27 April 2022), including the sequence of the holotype of Gaidropsarus gallaeciae sp. nov. (KY250297). A sequence of Notacanthus bonaparte Risso, 1840 (KP845234) was used as outgroup.
These sequences were employed to construct a molecular cladogram using the Neighbour-Joining (NJ) method [25] in MEGA 11 [26] with confidence limits tested through a bootstrap procedure [27], after 2000 replicates. board. In the laboratory, specimens were thawed, examined and photographed. With the exception of total length (TL) and standard length (SL), measurements are distances perpendicular to the length of the fish measured with a digital calliper to the nearest 0.1 mm on the type specimens and to nearest mm on the comparative material. Counts and measurements were recorded following Svetovidov [23,24]. All measurements are expressed as the percentage of standard length (%SL) or head length (%HL). Voucher specimens were deposited in the ichthyological collection of the Museo de Historia Natural, Universidade de Santiago de Compostela (MHNUSC).

Gaidropsarus
To improve the knowledge of the natural variation of the species of the genus, a comprehensive review of the morphological characters, distribution and coloration of valid Gaidropsarus species reported in the ichthyological literature, mainly compiled in Svetovidov [23,24], Barros-García et al. [2] and Biscoito and Saldanha [4], was complemented with measurements and counts of own comparative material, when available.
A single COI sequence representative for each valid Gaidropsarus species available (n = 7) was retrieved from Genbank specimens belonging to the author's project 'Molecular identification of Gaidropsarus fishes' (Code GSRUS) in BOLD systems (https://www.boldsystems.org/, accessed on 27 April 2022), including the sequence of the holotype of Gaidropsarus gallaeciae sp. nov. (KY250297). A sequence of Notacanthus bonaparte Risso, 1840 (KP845234) was used as outgroup.
These sequences were employed to construct a molecular cladogram using the Neighbour-Joining (NJ) method [25] in MEGA 11 [26] with confidence limits tested through a bootstrap procedure [27], after 2000 replicates.

Comparative Material Examined
Morphological data of the comparative material examined are shown in Table S1.

Diagnosis
The new species belongs to the genus Gaidropsarus as defined by Iwamoto and Cohen [28] as having three dorsal fins barely separated from each other; the first with a single thickened unsegmented ray, the second with small, unsegmented rays in a fleshy ridge that rises within a groove and the third with segmented rays in an elongate fin, and five prominent individual barbels, four on the snout and one at the tip of the lower jaw. Gaidropsarus gallaeciae sp. nov. is morphologically distinct from all congeners by the following combination of characters: third dorsal-fin rays 54-60, anal-fin rays 44-52, pectoral fin rays 21-23, total vertebrae 43-44; anal fin base short, its length 39.6-48% SL; first dorsal fin ray moderately elongated, its length 15.8-27% HL and a wider interorbital space, 21.7-28% HL.

Differential Diagnosis
A detailed comparison between the Gaidropsarus gallaeciae sp. nov. and the other 12 valid congeners is provided in Tables 2 and 3. According to our current knowledge of the genus, only three species, G. macrophthalmus, G. capensis and G. granti, share the low number of vertebrae found in the new species, while all other species have more than 45 vertebrae. It differs from G. capensis by having more third dorsal fin rays (54-60 vs.

Etymology
The name gallaeciae derives from the latin Gallaecia, an ancient Roman Iberian province, now called Galicia, the westernmost region of Spain, in reference to the name of the Galicia Bank where the holotype was collected.

Description
Counts and measurements of type specimens are shown in Table 1. Body elongate and relatively slender, maximum body depth is contained from 5 to 6.4 times in SL; moderate and round eyes, horizontal eye diameter 1 to 1.5 times in snout length; snout short and rounded, its length 4.1 to 4.8 times in head length; mouth large ( Figure 4A), slightly oblique, reaching a vertical through the posterior margin of orbit; upper jaw slightly protruding beyond lower jaw; first dorsal fin short, contained 3.7 to 6.3 times in HL; a small anterior nostril placed near de base of the barbel; posterior nostril oval, large, close to orbit; barbel present on chin, its length approximately equal to eye diameter and 1 to 1.4 times in snout lenght, and one barbel at each anterior nostril on the snout; first dorsal fin ray elongated, followed by a second dorsal fin of short fleshy filaments. The dentition consists of densely packed bands of small conical elements in both jaws ( Figure 4B,D); the outermost row in the upper jaw and the innermost row in the lower jaw are larger; fang-like teeth absent in both jaws; conical teeth on the vomer boomerang-formed ( Figure 4C); palatine teeth absent; gillrakers in the form of dentated tubercles ( Figure 4F), 1 + 6 -8 on the outer side of the first arch and 1+5-8 on the inner side. The colouration of fresh specimens is pinkish-reddish on the head, body and fins, and greyish on the ventral visceral part (Figures 2 and 3). Figure 5 shows a molecular cladogram of valid COI Gaidropsarus species sequences publicly available. In this figure, Gaidropsarus gallaeciae sp. nov. is located at an independent and well differentiated branch. The genetic distance of the sequence of the new species from those of its congeners far exceeds 2%, which is the cut-off value for species delimitation in teleost marine fishes [29]. This figure partially illustrates previously obtained results, in which this species is reported as Gaidropsarus sp. 1 [2] or as Gaidropsarus sp. [3]. publicly available. In this figure, Gaidropsarus gallaeciae sp. nov. is located at an independent and well differentiated branch. The genetic distance of the sequence of the new species from those of its congeners far exceeds 2%, which is the cut-off value for species delimitation in teleost marine fishes [29]. This figure partially illustrates previously obtained results, in which this species is reported as Gaidropsarus sp. 1 [2] or as Gaidropsarus sp. [3].

Habitat and Distribution
Known specimens were collected from two seamount-like structures in the Northeast Atlantic, the Galicia and Porcupine Banks, at 788 and 751 m depth, respectively ( Figure 1). All specimens were caught together with a large amount of live and dead cold-water coral of D. pertusum, Desmophyllum dianthus (Esper, 1794) and Madeprora oculata Linnaeus, 1758, a fact that support this being the preferred habitat of the species. In the Galicia Bank,this habitat was named as "Summit Sands with CW coral reef patches", corresponding with A6.611 Deep-sea D. pertusum reefs in the EUNIS classification [22]. Considering the presence of cold-water coral communities around the

Habitat and Distribution
Known specimens were collected from two seamount-like structures in the Northeast Atlantic, the Galicia and Porcupine Banks, at 788 and 751 m depth, respectively ( Figure 1). All specimens were caught together with a large amount of live and dead cold-water coral of D. pertusum, Desmophyllum dianthus (Esper, 1794) and Madeprora oculata Linnaeus, 1758, a fact that support this being the preferred habitat of the species. In the Galicia Bank, this habitat was named as "Summit Sands with CW coral reef patches", corresponding with A6.611 Deep-sea D. pertusum reefs in the EUNIS classification [22]. Considering the presence of cold-water coral communities around the world, the new species it is likely to be widely distributed, but most probably throughout the eastern Atlantic and Mediterranean areas.
The four specimens of the Porcupine Bank were collected along with one another rockling species, G. granti [9], and 33 other fish species, including also several gadiform fishes such as Trachyrincus scabrus (Rafinesque, 1810), L. lepidion, Phycis blennoides (Brünnich, 1768) or H. johnsonii among others. The list of bottom living invertebrates collected from the same site included 38 species of crustaceans, molluscs, echinoderms and cnidarians (dead coral, D. pertusum, D. dianthus).

Discussion
The morphology of the genus Gaidropsarus is conservative, making it difficult to find diagnostic characters and to establish an identification key for all known species. Gaidropsarus gallaeciae sp. nov. shares many morphological characters with the other congeneric species. A combination of meristic, biometric, colouration, geographical distribution and depth characters is therefore needed to differentiate the new species from all congeneric species.
The number of vertebrae is an important diagnostic character in distinguishing species of Gaidropsarus. On this basis, the species of this genus can be divided into two groups, either those that may have 45 vertebrae or less or those with more than 45 vertebrae. Our newly described species, Gaidropsarus gallaeciae sp. nov. is included in the first group together with G. macrophthalmus, G. capensis and G. granti.
Traditional taxonomy is descriptive, but the diagnostic characters of many hitherto unrevised fishes come from early manuscripts, which often refer to the examination of only a few specimens, and these results have come down to the present day with minimal changes. However, the magnitude of the variation of morphological characters in fishes, mainly biometrics and meristics, is not properly known and they are often underestimated, being the cause of erroneous denominations and the emergence of synonymies [30]. This seems to be the case for Gaidropsarus species. For example, G. gutattus, now considered a synonym of G. mediterraneus [2,3], was originally described by Collett [31] based on two specimens and only three more were subsequently analysed [23]. Therefore, the morphology of this species has only been based on the examination of five specimens, which is clearly insufficient to know the natural morphological variation of a species. The number of specimens examined was also low for the rest of the species. The specific distinction of G. insularum, G. novaezealandiae and G. parini carried out by Svetovidov [23] is based on only three, ten and two specimens, respectively, so it is not surprising that the valid status of these species has been questioned by Andrew et al. [32].
The main distinctive characters found in Gaidropsarus gallaeciae sp. nov. were the aforementioned number of vertebrae, the interorbital space, the length of the anal fin base, the length of the first dorsal fin ray and the length of the chin barbel. However, given the small number of specimens of Gaidropsaurus species examined, the number of distinctive characters may be reduced in the future.
Among the descriptive characters, the meristic ones have traditionally been used in the identification keys of Gaidropsarus. In fact, the number of fin rays is an important feature for taxonomic discrimination between species of this genus [23,24]. Short, nonoverlapping ranges of morphological characters will favour the detection of distinctive characters, while wide, overlapping ranges make it difficult. The main diagnostic characters of the genus were compiled by Svetovidov [23,24] and successive updates [2,4]. In their revision, Barros-García et al. [2] show a large interspecific overlap of the meristic features, resulting in a set of conservative morphological traits. On the other hand, only a few other characters were used as diagnostics. The length of the first dorsal fin ray is longer in the boreal G. ensis and G. argentatus with respect to other species, whereas G. macrophthalmus is distinguished by the presence of enlarged canine teeth on the upper jaw [23,24,28]. Small eyes, contained five or more times in the head length separate G. vulgaris, G. granti and G. mediterraneus from G. macrophthalmus, with large eyes, contained less than five times in the head length [28]. However, measurements of comparative material of G. macrophthalmus show the eye diameter is contained 4.9 to 6.3 times in the head length, which refutes this character as diagnostic. Further sampling effort would be needed to gain knowledge of the true morphological variation of Gaidropsarus species in order to create more reliable identification keys.
The coloration pattern is also a diagnostic character in the genus Gaidropsarus [4,23,24,28]. The coloration of G. granti, with a whitish sinuous longitudinal band is a quick and useful diagnostic character for the species [9], and the presence of dark spots on the dorsal parts of head and body, and on the second dorsal and caudal fins is also a diagnostic character for G. vulgaris [28]. The colour pattern of this genus varies with depth, from a polymorphic colour in shallower species to a uniform coloration in the deeper ones, most likely tied to how light penetrate the ocean water and camouflaging is needed. Thus, G. mediterraneus, the shallower species of the genus, shows a cryptic and variable coloration, which has probably led to the consideration of two different species, G. mediterraneus from the continental area and G. gutattus from the insular one, when, in fact, they are one and the same [2,3]. Whereas G. mediterraneus has a more or less uniform brown colouration, G. gutattus exhibits a whitish mottled one. This synonymy was already pointed out by Orsi Relini & Relini [33], who reported that G. mediterraneus sometimes showed the typical coloration of G. gutattus, with irregular light spots on its dorsal and lateral dark brown surfaces. This is also the most probable cause of the erroneous record of G. guttatus in continental area [34].
In contrast, the North Atlantic deeper species such as G. granti, G. argentatus, G. mauli and Gairopsarus gallaeciae sp. nov. show a similar homogeneous pink-reddish coloration which could difficult their correct identification, particularly in juvenile stages.
The analyses of Barros-García et al. [2,3] seems to point to the real composition of species of this genus, with a reduction of valid shallower species, due to synonymy recognition, suggesting, furthermore, the existence of a greater diversity hidden in the deep. The recent record of G. mauli [4] and Gaidropsarus gallaeciae sp. nov. themselves would confirm this hypothesis. Moreover, according to the molecular results, other deep-sea species remain yet undescribed [2]. Considering that most of the deep-sea areas are unexplored and that the occurrence of Gaidropsarus species reported by in vivo sampling techniques (ROVs, AUVs, BCs) is not unusual, an increased number of undiscovered deep-sea species of this genus is predictable.
Eggs, larvae and juveniles of Gaidropsarus species are pelagic [6]. Pelagic early stages and the absence of physical barriers in the ocean should prevent rapid speciation events, but this statement contrasts with the fact of finding greater diversity at depth. However, depth-related ecological niche axis along which divergence occurs is due to local adaptation to diverse feeding habitats, light conditions, spawning sites, or other ecological factors [35]. For instance, speciation in the Pacific rockfish genus Sebastes is associated with divergence in habitat depth and a depth-associated morphology, in the absence of geographic barriers [36].
Therefore, diversification processes according to deep-sea environments is proposed for the genus Gaidropsarus. Ecological speciation occurs when adaptation to different environments or resources causes reproductive isolation [37]. Changing habitats with depth can create ecological opportunities for colonisation processes, promoting species diversification. The recently discovered species G. mauli was first found in a hydrothermal vent site, in crevices along rocky walls in the vicinity of the venting fluids [4]. Given the abundant presence of live and dead coral in the catches of all of the specimens, the occurrence of Gaidropsarus gallaeciae sp. nov. could be associated with the presence of cold-water coral reefs, as also occurs with G. granti [9]. In fact, both species were caught in the same haul in the Porcupine Bank, and both were also found in samples with coral in the Galicia Bank [38] which reinforces this likely coexistence and niche overlap. Several other fish species caught alongside the new species such as M. moro, L. lepidion and G. latifrons are also associated with the occurrence of cold-water corals [39], reinforcing the above. Association between Gaidropsarus species and cold-water corals has been often observed in live specimens on and between live and dead coral thickets [12,17,39,40]. Underwater observations have also shown much imagery evidence of a strict territorial behaviour of Gaidropsarus sp. in D. pertusum or M. oculata colonies [41]. The size of Gaidropsarus gallaeciae sp. nov. appears to be small, up to 11 cm TL, which could also be a morphological adaptation to shelter among the branches of corals as protection against predators. Hydrothermal vent fields and cold-water corals are two of the varied habitats responsible for the high biodiversity found in the deep ocean [42].
DNA barcoding greatly facilitates the grouping of individuals into putative species, which must then be validated through morphological scrutiny by taxonomic experts. Though morphology is the traditional technique used in alpha taxonomy, genetic tools are becoming increasingly common in studies describing new species, especially when morphological data are ambiguous [43]. In this aspect, Renner et al. [44] recommended DNA-based diagnoses of new species in all taxonomic groups, not just bacterial. A DNA barcoding analysis including the calculation of COI genetic distances [2] clearly differentiated Gaidropsarus gallaeciae sp. nov. (then reported as Gaidropsarus sp. 1) from six valid and two unidentified species from the North Atlantic. Considering that this previous study was based only on mitochondrial DNA sequences, a multilocus species delimitation analysis was carried out, including both mitochondrial (COI, CytB, ND2) and nuclear (Rho, ZIC1) genetic markers, that finally confirmed this previous finding [3].
Although no southern hemisphere species sequences are available, a BOLD search of Gaidropsarus gallaeciae sp. nov. returns a difference of 4.11%, a typical species differentiation distance, with a private COI sequence assigned to southern species G. novaezealandiae. Interestingly, this would be the smallest genetic distance found between the new species and all other congeneric species, as this species is more distantly related to all of the North Atlantic species examined, ranging from 13.21 to 17.36% [2].
A revision of this genus based on extensive collections of specimens and DNA sequencing is needed [4]. Therefore, it is necessary to complete the sequence database with specimens from southern hemisphere to better understand the interspecific relationships of this genus. Without the slightest doubt, the integrative study of traditional and molecular taxonomy can highlight identification mistakes and incongruities between the two disciplines, helping to reveal cryptic species, to identify immature specimens, and to clarify synonymies [45].

Conclusions
The occurrence of a new fish species Gaidropsarus gallaeciae sp. nov. is well supported by morphological and molecular analyses that clearly differentiate it from other known species. The taxonomy of the genus Gaidropsarus remains poorly understood. The relatively large number of species in this genus, the scattered distribution of many of them, and the small number of specimens examined and/or found are probably the main reasons for this insufficient knowledge. Although morphological characters are conservative, overlapping to a large extent between species, some diagnostic characters can be identified. These, together with colouration, geographical distribution and depth range can currently be applied for species identification. Molecular taxonomy of North Atlantic species has helped to resolve some taxonomic inaccuracies, but also flags the presence of undescribed species. Examination of more specimens and obtaining DNA sequences of southern hemisphere species should be the next objective to clarify the taxonomy of this difficult group.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biology11060860/s1, Table S1. Morphological data of the material comparative examined.  Institutional Review Board Statement: Ethical review and approval was waived for this study because all specimens examined were from fishery catches and were already dead.

Informed Consent Statement: Not applicable.
Data Availability Statement: The sequences employed in the current study are available in the BOLD systems (https://www.boldsystems.org/, accessed on 27 April 2022) and GenBank (https: //www.ncbi.nlm.nih.gov/genbank/, accessed on 27 April 2022) repositories. All specimens used in this study for taxonomical purposes are deposited in the fish collection of the Museo de Historia Natural, Universidade de Santiago de Compostela (MHNUSC) in Santiago de Compostela, Spain (see methods). All other data are included in this article.