First Record of Lyconus brachycolus (Gadiformes: Lyconidae) in Spanish Waters: An Update on Taxonomic Knowledge

Abstract

This study reports the first record of Lyconus brachycolus in Spanish waters. A single specimen measuring 216 mm in total length was captured on 13 October 2024 in the Cantabrian Sea (north of Spain, 43.8467 N, −6.2109 W) by bottom trawl at a depth of 412 m. A detailed morphological description, updated characteristics, and illustrations are provided. For comparative purposes, an update of the species’ morphological characteristics was carried out. In an integrative taxonomic approach, the DNA barcode was obtained, which confirmed the morphological identification of the specimen as Lyconus brachycolus. A molecular taxonomic analysis using this marker showed the existence of two clades separated by 5.1%, corresponding to Lyconus brachycolus and Lyconus pinnatus, the two currently valid species of the genus.

1. Introduction

The order Gadiformes is a large and diverse group comprising five suborders, 17 families and 613 species, commonly known as cods, hakes, and grenadiers [1]. Gadiformes inhabit a wide range of marine environments, from shallow tropical and temperate waters to deep waters, including a single freshwater species [2].

The genus Lyconus Günther, 1887 was historically assigned to several families, including Macrouridae, Macruronidae, Merluccidae or as Macruroninae, a subfamily within Merlucciidae [3]. Von der Heyden and Matthee [4] resurrected the family Lyconidae Günther, 1887, and its recognition as a distinct family was subsequently confirmed by a thorough phylogenetic analysis [1]. Members of this genus are distinguished by an elongated, compressed body, with a long, tapering tail and no separate caudal fin; dorsal and anal fins long and continuous; head moderate-sized; mouth terminal, large, and oblique; end of upper jaw extending to the posterior margin of the eye; snout short and rounded; eye large, lying immediately below upper profile; interorbital space rather flat and narrower than eye diameter; both jaws armed with a series of widely set teeth, unequal in size, two canine-like on tip of upper jaw; vomer with a single canine-like tooth on each side; no barbel on chin; opercles very thin; infraorbital bone separating maxilla from eye narrow; origin of dorsal fin situated above base of pectoral fins; anal fin origin located immediately behind anus, its rays considerably shorter than those of dorsal fin; pectoral fins with 12–17 elongated rays, directed obliquely upwards; pelvic fins thoracic, below bases of pectoral fins, composed of 8–10 rays shorter than pectoral fin rays; scales very small, thin, cycloid, and deciduous [5,6].

The genus Lyconus has been revised by Lloris et al. [6], who established two valid species: Lyconus brachycolus, Holt and Byrne, 1906, found in the north-western Atlantic (Canada), north-eastern Atlantic (east of Ireland, southeast of Madeira, and the coast of Sahara), and in the south-eastern Atlantic, and Lyconus pinnatus, Günther, 1887, known from scattered records in the southern Atlantic, Madagascar shelf, southern Australia, and to the east of New Zealand. There is also information about three more undescribed species from near the Java Island, the south of New Zealand, and in the North Pacific Ocean [7].

In the most recent inventory of the marine ichthyofauna of Spain, Báez et al. [8] recorded 1075 species, including 64 (5.9%) gadiforms, but none from the genus Lyconus.

Molecular markers are increasingly being used alongside morphological data in an integrative manner in fish identification. A few molecular markers have been previously used in Lyconus species for taxonomic or phylogenetic purposes: cytochrome c oxidase subunit I (COI) [4,9], cytochrome b [4], and even nuclear loci using a metagenomic approach [1].

Mitochondrial DNA has been successfully used to discriminate species because of its maternal inheritance, rapid evolutionary rate (1–2% per million years), lack of introns, and recombination [10]. Different mitochondrial genes, namely cytochrome b, 12S, and 16S rRNA genes, have been used for fish species identification. However, the availability of universal primers and presence of phylogenetic signals make the COI more appropriate for fish species identification [11].

The aim of this study is to present the first occurrence of L. brachycolus in Spanish waters, looking at on morphometric and meristic data and molecular taxonomy.

2. Materials and Methods

2.1. Sampling Data and Morphological Analysis

The specimen was caught by bottom trawl during “Demersales” surveys (SP-NSGFS) [12], carried out each autumn since 1983 by the Instituto Español de Oceanografía (IEO-CSIC) on soft bottoms along the continental shelves of Galicia and the Cantabrian Sea. These surveys, originally designed to assess commercially important stocks, have followed an ecosystem-based approach since 1992, incorporating biological and ecological data on all faunal groups from the monitoring of demersal communities. After taking a small muscle sample for molecular analysis, the specimen was preserved frozen and deposited in the fish collection of the Museo de Historia Natural, Universidade de Santiago de Compostela, with the reference number MHN USC_25186-3. Species identification was carried out following Lloris et al. [6]. Morphometric measurements were recorded to the nearest mm using a digital caliper by a single observer to reduce bias. Dentition was examined and counted under a binocular microscope. Total length (TL) and standard length (SL) were used throughout. Due to the presence of biases in the morphological data in the literature, formal statistical tests were not appropriate in this case.

2.2. Molecular Analysis

Molecular identification of the Lyconus brachycolus specimen was performed using the fish DNA barcoding mitochondrial marker COI-5P, consisting of approximately 650 nucleotides from the 5′ region of the gene coding for subunit I of the mitochondrial respiratory chain enzyme cytochrome oxidase. For this purpose, DNA was extracted from a muscle tissue sample (with the E.Z.N.A. Tissue DNA Kit) and amplified by PCR, using the Phire Green Hot Start II PCR Master Mix and the universal primer cocktail C_FishF1t1-C_FishR1t1, according to the authors’ instructions [13]. Sequencing reactions of the amplicon were performed in the forward and reverse directions to subsequently obtain a consensus sequence of 652 nucleotides in length. This sequence, along with a photograph of the specimen, its geographical location, and associated metadata, has been deposited in the BOLD database (http://boldsystems.org, accessed on 28 May 2025), under the project ‘Unusual fish from the northeaster Atlantic Ocean and the Mediterranean Sea’ (code UNAFI) with code UNAFI032-25. The sequence can also be found in GenBank (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 28 May 2025), with accession number PV697336.

To construct a molecular taxonomic cladogram of the genus Lyconus, all publicly available COI-5P sequences were downloaded from the BOLD and GenBank databases for alignment. Nucleotide differences between sequences were calculated using the uncorrected percentage distance (p-distance) [14], and represented in the form of tree using the Neighbor-Joining algorithm [15]. These procedures were performed using MEGA12 Version 12 software [16].

3. Results

On 13 October 2024, a single specimen of Lyconus brachycolus was caught with bottom trawl gear on the Cantabrian Sea, 43.8467 N, –6.2109 W, at a depth of 412 m (Figure 1). The specimen measured 216 mm TL and weighed 31.2 g (Figure 2). The bottom substrate was fine sand, the bottom temperature was 11.1 °C, and the salinity was 35.57.

The body is elongated and depressed posteriorly, tapering towards the caudal fin (Figure 2a), maximum body depth 7.2 times in SL; head moderately long (Figure 2b), 5.6 times in SL; snout short and blunt, 3.6 times in head; eye large, its diameter 4 times in head; interorbital space 2.9 times in head; mouth large, terminal and oblique, the jaws extend to the posterior edge of the eye; teeth in a single row on jaws and vomer; premaxilla with one fang anteriorly followed by 18 smaller sharp teeth in a single diminishing series on the left side and two fangs anteriorly, followed by 22 smaller sharp teeth on the right side; dentaries with four fangs in the left side and seven on the right side (Figure 2c); three smaller vomer fangs on the left side and four on the right; ventral fins jugular; barbels absent. The main meristic and morphometric characters are summarized in

Table 1. Morphometric and meristic data of Lyconus brachycolus MHN USC 25186-3 from Spanish waters compared with the holotype [17] and previous records [9,18,19,20,21]. The last column shows the mean and standard deviation of all reported specimens, including the one described here.

	Lyconusbrachycolus
	MHN USC 25186-3	Holotype	Previous Studies Range	Mean ± SE (n)
Total length (mm)	216	237
Standard length (mm)	210	232	180–349
As % SL
Head length	17.9	17.9	17.1–20.6	18.1 ± 1.6 (9)
Preorbital length	5	5.2	4.8–6.1	5.1 ± 0.5 (7)
Postorbital length	8.1		7.8–10
Eye diameter	4.5	4.5	3.2–5.1	4.4 ± 0.6 (7)
Interorbital width	6.2	5.6	5–6.6	5.8 ± 0.5 (7)
Upper jaw length	8.9
Lower jaw length	8.4
Predorsal1 fin length	21.1	19.4	18.3–23.9	21 ± 2 (9)
Preanal fin length	42.1	40.5	35.5–50.6	43 ± 4.3 (9)
Prepectoral fin length	20.9		17.8–21.1	19.9 ± 1.8 (3)
Prepelvic fin length	20		18.5–22.5	20.3 ± 2 (3)
Dorsal1 fin base	5.7		4.3–5	5 ± 0.7 (3)
Dorsal2 fin base	72
Anal fin base	58.1
Pectoral fin length	13.8	11.2	11–24.4	14.7 ± 4.9 (7)
Pelvic fin length	5	7.3	4.6–8.6	6.7 ± 1.6 (6)
Body depth at pectoral fin	13.8	14.2	12.9–15	14.1 ± 0.9 (9)
Body width	5.8	7.8	5.9–7.2	6.7± 0.9
Meristic
Dorsal1 fin rays	8		9–10
Dorsal2 fin rays	107		105–111
Anal fin rays	87		86–96
Pectoral fin rays	13	13	13–14
Pelvic fin rays	8	9	8–9
Branchiostegal rays	7		7
Gill rakers	3 + 10		3–5 + 9–12
Vomer teeth	3 + 4	4 + 4	2–4 + 2–4

. A total of 12 individuals were previously studied.

The COI molecular sequence obtained in this study was compared with reference libraries for positive identification. The molecular taxonomic cladogram (Figure 3) is the result of aligning the 16 publicly available sequences of the COI-5P marker of the genus Lyconus in BOLD and GenBank databases. Twelve of them correspond to the southern species L. pinnatus and four to the northern species L. brachycolus. Among the latter is the sequence of the specimen caught in Spanish waters in the Cantabrian Sea. The molecular marker commonly used in fish barcoding perfectly separates both species. The 120 comparisons made between all pairs of sequences reveal a conspicuous barcoding gap, with an upper limit for intraspecific distances of 1.7% and a lower limit for interspecific distances of 5.1%. The genetic distances within each group are 0.2% in L. pinnatus and 1.1% in L. brachycolus. This value may be biased due to the small number of nucleotide sequences compared.

4. Discussion

This is the first documented occurrence of L. brachycolus in Spanish waters, representing both a new genus and a new species for the area. Despite long-standing efforts to catalog Spain’s marine fish fauna, the discovery of new unreported species is not uncommon. The main sources of these new records include the arrival of new southern species due to rising ocean temperatures and climate change [22], the presence of introduced species [23], or the detection of unreported deep-sea species [24]. The most likely reason that L. brachicolus has not yet been recorded to date is that it is a rare, deep-water species that is difficult to sample. This is also reflected in the small number of specimens examined worldwide, totaling about 12 individuals.

Regarding the morphological description, the main characteristics of the specimen examined are consistent with those previously documented in the literature. The number of teeth shows considerable variability in L. brachycolus [20]. The premaxilla typically has one or two anterior fangs, followed by more than 10 smaller articulated teeth [6], whereas the holotype had 15 sharp teeth [17]. The dentaries have seven separated teeth that are unequal in size [20], and the vomer generally presents one to three teeth on each side. Our specimen showed a higher number of teeth than previously reported, thereby extending the known range number for this trait in the species. This confirms the great variability in the number of teeth of this species, but also probably the small number of specimens examined so far.

Deep-sea ecosystems represent the largest biomes in the global biosphere, but knowledge of their biodiversity is still scant [25]. Environmental DNA (eDNA) metabarcoding has great potential to improve our understanding of deep-water fishes, although the deepest samples (>1400 m depth) had significantly lower DNA concentrations than surface or mid-depth samples [26].

Lyconus brachycolus is a rare bathythermal fish found at depths of 150–700 m, for which only isolated references exist [6]. The Spanish record is within the known distribution range of the species in the eastern Atlantic, from about 60° N off Scotland to about 10° N, and also off Namibia [27]. Therefore, the present Spanish record fills a gap in the known distribution range of the species.

The barcoding gap supports species-level identification. The Barcode of Life Data System (BOLD) [28] contains information on roughly 9500 fish species. In most marine fishes, a DNA barcoding gap is evident between different genera with a lower value of average intra-specific genetic distance (0.18–0.39%) than the inter-specific distance value (3.75–11%) [29].

There are only four COI sequences for L. brachycolus available in publicly accessible databases, but only three of these meet the length and quality requirements for molecular analysis. DNA barcoding for species identification typically requires sequences of 500–650 base pairs (bp) to ensure sufficient genetic variation for accurate discrimination. This length balances technical feasibility with the need to capture enough interspecific divergence while minimizing intraspecific variation [30].

The limited number of available sequences is a common feature of many deep-water fishes due to their rarity and the difficulty of sampling them, but also due to the limited effort expended on sequencing. The number of sequences available would need to be increased to allow for the more accurate identification of deep-sea fishes. A small sample size in DNA barcoding may underestimate intraspecific genetic diversity, which can have a considerable impact on biodiversity studies and species identification. Consequences may include the underestimation of haplotypic and nucleotide diversity, the appearance of an exaggerated barcoding gap, and the impossibility of detecting or insufficient perception of existing hidden diversity. Depending on the taxon and geographical distribution, studies recommend sample sizes well above 5 or 10 individuals per species, with recommended sample sizes in some cases reaching 25 or more in the case of widely distributed species [31].

The standard DNA barcoding marker for fish confirmed the identification of the specimen caught in Spanish waters, as its sequence matched those already existing under the same name, with a similarity of 99.4% with MW908004 from Ireland. Furthermore, the marker made it possible to differentiate, without any doubt, between the two existing nominal species of this genus, in line with traditional taxonomy. Considering the set of pairwise distances, the maximum intraspecific value was 1.7%, while the minimum interspecific value between the two species was 5.1%. These data align with those commonly observed in fish, where intraspecific distances exceeding 2% result in a congeneric probability of greater than 95% [32].

Despite the existence of undescribed Lyconus species [7], this is not yet reflected in the present molecular analyses, probably due to the limited number of sequences and geographical areas sampled. The morphological review reflects the insufficient knowledge of the morphology of L. brachycolus due to the rarity of catches and the limited number of descriptions. Since its original description [6] only a few specimens have been analyzed [9,18,19,20,21]. While this report provides new insights into the molecular, morphological and distributional aspects of L. brachicolus, the knowledge of this genus remains incomplete and further research is clearly needed.