Brasil
Zoologia (Curitiba)
Sumário Anterior Atual Seguinte
Sumário
Research Article • Zoologia (Curitiba) 43 • 2026 • https://doi.org/10.1590/S1984-4689.v43.e25054 linkcopy

Open-access A histology-free description of Lineus marcosii (Pilidiophora: Lineidae), a cryptic species resembling Lineus lacteus

AuthorshipSCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

Cryptic biodiversity is one of the main challenges to overcome for an accurate characterization of Earth’s biodiversity. The nemertean species Lineus lacteus has been recorded from Sweden to the Black Sea since its description in the first half of the 19th century. However, molecular data suggest that it actually represents a cryptic species complex formed by at least two species. Here, we reanalyze the molecular information for both lineages and provide new sequences from the Cantabrian Sea. As a result, we describe the Mediterranean and Cantabrian lineage as a new species, Lineus marcosii sp. nov., based on newly collected material. Because no morphological differences were observed between the two species, diagnostic characters were searched for in all available sequences of the cytochrome c oxidase subunit I gene. Moreover, new knowledge on its trophic ecology, parasites, and behavior is provided based on opportunistic observations. There is no known overlap in the geographic distributions of L. lacteus and L. marcosii sp. nov. Thus, these two species are considered an allopatric cryptic species complex, and their entire distributions are inferred from the available molecularly confirmed records.

KEYWORDS:
COI; cytochrome c oxidase subunit I; Heteronemertea; molecular taxonomy; Nemertea; species delimitation

INTRODUCTION

Cryptic species challenge our ability to record Earth’s biodiversity, as it is becoming increasingly common to encounter biological species that resemble other described taxa so closely that they are extremely difficult or even impossible to differentiate based on morphological characters (Knowlton 1993, Kawauchi and Giribet 2010). This situation is more frequent among marine invertebrates, as the marine realm has traditionally been considered devoid of hard barriers to genetic exchange and speciation (Fernández-Álvarez et al. 2020). Thus, many congeneric specimens from distant locations have been assigned to previously described species, or names have been synonymized using over-conservative taxonomic practices (Carrera-Parra et al. 2011, Valdés et al. 2017). However, this view is increasingly challenged by the discovery that many invertebrates with large distribution ranges are actually composed of several cryptic species with smaller distributions (Bickford et al. 2007, Arnold et al. 2025, Church et al. 2025).

Nemerteans are soft-bodied animals with a limited number of external and internal morphological characters, which complicates the appropriate characterization of their diversity (Sundberg et al. 2009). Because external characters, such as coloration or sensory organs, are often difficult or impossible to study in fixed specimens, their morphology has traditionally been examined primarily through anatomical features via histological sections. These histological studies are time-consuming and laborious, potentially increasing the shelf time of species descriptions and discouraging researchers from engaging in nemertean taxonomy (Strand and Sundberg 2011). As a result, histology-free descriptions characterizing the external morphology of nemerteans in combination with molecular characters are becoming the new standard for the characterization of nemertean biodiversity (e.g., Kajihara 2015, Kajihara et al. 2018, Chernyshev et al. 2020, Cherneva et al. 2023, Alfaya et al. 2025).

Within the pilidiophoran nemertean Lineus lacteus (Rathke, 1843), two divergent lineages have been discovered through DNA barcoding (Kang et al. 2015) and population genomics of nuclear markers (Ament-Velásquez et al. 2016). One of these lineages occurs on the Mediterranean and Cantabrian coasts of the Iberian Peninsula and the Mediterranean coast of France, while the other is present on the Atlantic coasts of France and Wales. Ament-Velásquez et al. (2016) designated them as L. lacteus M and L. lacteus A, respectively; this nomenclature is adopted herein. The objective of this study was to resolve the systematics of both lineages through molecular systematics.

MATERIAL AND METHODS

Sampling

On December 27, 2023, 20 specimens of L. lacteus M were collected from gravel under rocks in the upper intertidal zone of Los Chalanos beach, Muros de Nalón, Spain (Cantabrian Sea; 43.560029°N, 6.102786°W). The specimens were transferred to clean Falcon tubes containing a small amount of gravel and seawater. Additional seawater from the sampling site was collected to maintain the animals alive. The specimens were cultured following Zattara and Fernández-Álvarez (2022) until processing on January 25 and February 2, 2024. Although chicken liver was offered as food every three days, no ingestion was observed.

Molecular systematics

For DNA barcoding, total genomic DNA was extracted from ethanol-fixed tissue of five specimens from the middle of the body using the Quick-DNA Microprep Plus kit (Zymo Research), following the manufacturer’s protocol, in a total volume of 20 µL. Sequences from the partial mitochondrial cytochrome c oxidase I (COI) gene were amplified using the primer pair LCO1490 and HCO2198 (Folmer et al. 1994). All PCRs were performed in a total volume of 25 μL, containing 2.5 μL of template DNA, 0.5 μM of the primers, 6.25 μL of Supreme NZYTaq 2x Green Master Mix (NZYTech), and ultrapure water up to 25 μL. PCRs consisted of an initial denaturation at 95°C (5 min), followed by 35 cycles of denaturation at 95°C (30 s), annealing at 49°C (30 s), and extension at 72°C (45 s). The amplified fragments (approximately 700 bp) were purified using magnetic beads (MagBind, Omega-Biotek) prior to sequencing both strands on an ABI Prism 3730xl DNA Analyzer (Applied Biosystems, USA), with the same primers as those used in the PCR amplification. DNA sequences were cleaned at the primer ends using Sequencher (Gene Codes Corporation). GenBank accession numbers are provided in Table 1.

Thumbnail
Table 1
Sampling table showing the locality, GenBank accession number and reference of the cytochrome c oxidase subunit I (cox1) sequences used in this work. The outgroups Lineus sanguineus and Lineus longissimus are not included in this table, but the sequences can be accessed through the Supporting FASTA File S1.

All sequences of cytochrome c oxidase subunit I (cox1) allocated to Lineus lacteus were downloaded from GenBank (Table 1). Individuals from the North Atlantic will be designated Lineus lacteus A from now on, and individuals from the Mediterranean and Cantabrian lineage will be referred to as L. lacteus M. All available Lineus sanguineus (Rathke, 1799) and Lineus longissimus (Gunnerus, 1770) sequences were used as sequential outgroups. The matrix was aligned using the MAFFT algorithm in UGENE (Okonechnikov et al. 2012). Recently, Verdes et al. (2025) published the complete genome of Mediterranean L. lacteus. The SRA files from the ENA Accession number ERR13767215 were used to assemble a fragment of cox1 using NOVOPlasty v.3.8.3 (Dierckxsens et al. 2016). The resulting fragment (GenBank accession number PX559724) was shorter than the remaining sequences. Thus, it was not included in the species delimitation analyses, but it was used for allocating the Verdes et al. (2025) genome to one of the previously identified L. lacteus lineages.

A maximum likelihood (ML) analysis was performed on the IQ-TREE server (Nguyen et al. 2015, Hoang et al. 2018). In IQ-TREE, an ultrafast bootstrap with 10,000 iterations was performed to determine the statistical support for the clades. We implemented the ModelFinder tool (Kalyaanamoorthy et al. 2017) to estimate the best-fitting model of substitution following the Bayesian information criterion (BIC), Akaike information criterion (AIC), and the Akaike information criterion corrected (AICc). AIC selected the model K3Pu+F+G4, while the AICc and BIC found that HKY+F+G4 was the best model, which was consequently applied. The ML tree was converted to Newick format using FigTree v.1.4.3 (Rambaut 2018) to run Bayesian Poisson tree processes (bPTP) (Zhang et al. 2013) through the bPTP web portal (http://species.h-its.org/ptp/). The ASAP method (Puillandre et al. 2021) was performed using p-distances through the ASAPy Python tool (Vences et al. 2021). Only the option with the highest likelihood score was considered. The concurrence of molecular species delimitation methods was considered a confirmation of speciation according to the unified species concept (De Queiroz 2007).

Genetic uncorrected p-distances of cox1 within and between species were calculated with MEGA 11 (Tamura et al. 2021). Raw genetic p-distances were visualized as a histogram using ggplot2 (Wickham 2016) in the software RStudio v.1.4.1106, working under R v.4.1.5 (R Core Team 2021). Additionally, the available sequences of the mitochondrial 16S and the nuclear 28S and 18S from both lineages (three of L. lacteus M, and one of L. lacteus A) and a specimen of L. sanguineus were downloaded and aligned via the MAFFT server (https://mafft.cbrc.jp/alignment/server/; Katoh et al. 2009) using the Q-INS-i iterative refinement method. Due to the low number of L. lacteus M and A specimens available for those markers, only p-distance analyses were performed for these three markers (Table 2).

Thumbnail
Table 2
GenBank accession numbers and uncorrected p-distances (%) for the ribosomal markers 16S, 28S and 18S. Alignments of 16S, 28S, and 18S are provided in the Supporting FASTA File S2-S4, respectively.

The cox1 matrix was used to extract molecular synapomorphies for a better diagnosis of the species. Diagnostic molecular characters are nucleotides in a specific position and gene region that are identical in all individuals examined for a single species and consistently distinct from all other known species (autapomorphies). Only homogeneous characters, shared by all members of the taxon under study and not present in the outgroups, were considered. For the presentation of the results and to ensure the reproducibility of this work, the general recommendations of Jörger and Schrödl (2013, 2014) were followed. To ensure transparency in our selection of diagnostic nucleotides, the alignments of cox1, 16S, 28S, and 18S are provided as supporting information (Supplementary materials FASTA Files S1-S4).

Morphology

Six of the specimens showed poor body condition and were not morphologically studied; therefore, all measurements are based on 14 individuals. Specimens were anesthetized in 7.5% magnesium chloride in distilled water, then measured and observed under a stereomicroscope. Afterwards, squash preparations of the anesthetized specimens were examined using a bright-field microscope. Finally, the individuals still under anesthesia were fixed in either 4% buffered seawater formalin (14 specimens) or 96% ethanol (six specimens) and deposited in the Marine Biological Reference Collection of the Marine Sciences Institute (CMBR-ICM, ICM-CSIC, Barcelona; Guerrero et al. 2020). Data obtained during the regeneration experiments of Zattara et al. (2019) were used to complement the descriptions and provide additional data on the ecological interactions of this species. Photographs of specimens observed in the same locality and at La Guarda beach (San Esteban de Pravia, Asturias) between 2009 and 2014 were used to complete the descriptions and provide ecological observations.

RESULTS

All L. lacteus formed a clade with 94% ultrafast bootstrap (ub); sequences of L. lacteus A clustered together with an ub of 99%, while the support for the clade of the Mediterranean and Cantabrian L. lacteus M was 84% ub (Fig. 1). Lineus lacteus and L. sanguineus had 100% ub, and the clades of L. sanguineus and L. longissimus received 98% and 100% support, respectively. The bPTP produced both Bayesian and ML solutions, which in this case were identical, identifying L. lacteus A, L. lacteus M, L. sanguineus, and L. longissimus as distinct species. This result was consistent with the results of ASAP, which also identified each species as distinct.

thumbnail of Figure 1
Figure 1
(A) The inferred distribution of the lineages is represented by shadowed light color (orange, Lineus marcosii sp. nov.; blue, Lineus lacteus), and specific locations with molecularly-identified specimens are signaled with dark color circles, and the type localities with triangles. The type locality of L. lacteus is colored in black as it is not yet molecularly confirmed. Records without a link with molecular information are signaled with two colored concentric circles; records of L. marcosii sp. nov. based on the synonymized record list provided in this work, records of L. lacteus after Gibson (1982). Note that most part of the inferred distribution of both species lacks molecular confirmation at present. (B) Summary of species delimitation analyses from the cox1 database, depicted over the ML analyses obtained through IQTREE (Nguyen et al. 2015, Hoang et al. 2018). Lineus sanguineus was collapsed for visualization due to the large number of sequences. The numbers above the branches refer to the support of the node as the ultrafast bootstrap percentages (%) from the ML analysis. The vertical columns summarize the results from Maximum Likelihood (ML) and Bayesian solutions of the bPTP and ASAP (see Materials and methods for more details). (C) Histogram of percentages of cox1 p-distance obtained in ggplot2 (Wickham 2016) in the software RStudio v. 1.4.1106, working under R v. 4.1.5 (R Core Team 2021).

The uncorrected mean p-distance of cox1 between L. lacteus A and L. lacteus M was 12.4%, while the distance among these two species and L. sanguineus was 14.0% and 13.2%, respectively (Table 3). Lineus longissimus interspecific distances were shorter with L. sanguineus (14.9%) than with L. lacteus A and L. lacteus M (17.2% in both cases). Intraspecific p-distances (Table 4) were 0.5% for L. lacteus A (0.0-1.7, n = 15) and L. lacteus M (0.0-0.9, n = 12), and 0.3% for L. sanguineus (0.0-1.3, n = 170) and L. longissimus (0.0-0.6, n = 11).

Thumbnail
Table 3
Interspecific mean p-distance percentages (%). The sequences can be accessed through the Supporting FASTA File S1.

Thumbnail
Table 4
Intraspecific p-distance percentages (%). The sequences can be accessed through the Supporting FASTA File S1.

For the remaining molecular markers, uncorrected p-distances are summarized in Table 2. For the mitochondrial 16S, interspecific distances ranged between 4.5% and 5.6%, while intraspecific distances in L. lacteus M were 0.2%. For 28S, the distances among L. sanguineus and the two L. lacteus lineages were 1.3-1.4%, the distance between L. lacteus A and L. lacteus M was 0.7%, and among individuals of L. lacteus M was 0.3%. For 18S, L. lacteus A was the most divergent, with 1.0-1.1% distance with respect to L. sanguineus and L. lacteus M. The distance between L. sanguineus and L. lacteus M was larger (0.3%) than among individuals of L. lacteus M (0.1%).

Molecular diagnostic characters of cox1 are provided in Table 5. Both Lineus lacteus lineages showed 20 synapomorphies each. All this information combined suggests L. lacteus M is an undescribed species, which is described herein.

Thumbnail
Table 5
Molecular diagnostic characters obtained for cytochrome c oxidase subunit I. The sequences can be accessed in Supporting FASTA File S1.

TAXONOMY

Phylum Nemertea Schultze, 1851

Class Pilidiophora Thollesson & Norenburg, 2003

Order Heteronemertea Bürger, 1892

Lineidae McIntosh, 1874

Lineus Sowerby, 1805

Lineus marcosii sp. nov.

Fig. 2

https://zoobank.org/6370EAA1-400A-41C6-9928-9BA57F53AD3B

Diagnosis: Lineus with the mouth some distance posterior to the brain; with 5-21 eyes per row; rhynchocoel extending ~10-15 % of the body and with a slender proboscis; whitish in color with anterior tip of the head reddish or red; cytochrome c oxidase subunit I diagnostic characters: 16, C; 75, A; 99, C; 108, G; 109, C; 165, G; 174, C; 204, A; 225, A; 276, G; 327, A; 388, C; 450, T; 475, T; 477, G; 549, T; 564, G; 570, C; 573, C; 618, G.

Material examined: Holotype: SPAIN. Immature, Los Chalanos beach, Muros de Nalón, Asturias, Spain; coordinates: 43.560029, -6.102786; 27th December 2023; FÁF-Á and JLMV leg.; ethanol fixed ICMCBMR000684. cox1 GenBank Accession number: PX559723. Paratypes: 19 individualized specimens, same collection data as holotype. ICMCBMR000675-ICMCBMR000678 and ICMCBMR000685-ICMCBMR000691, immature unsexed specimens, formalin fixed; ICMCBMR000692-ICMCBMR000694, immature female specimens, formalin fixed; ICMCBMR000679-ICMCBMR000683, immature unsexed specimens, ethanol fixed.

Type locality: Los Chalanos beach, Muros de Nalón, Asturias, Spain; coordinates: 43.560029, -6.102786.

thumbnail of Figure 2
Figure 2
Lineus marcosii sp. nov., all specimens photographed under anesthesia. (A) Paratype specimen ICMCBMR000690, unsexed, the specimen had no colored gut contents. (B) Paratype specimen ICMCBMR000694, maturing female, the specimen had greyish gut contents, so the gonads were easier to spot (black arrowheads). (C) Paratype specimen ICMCBMR000679, unsexed, detail of the dorsal surface of the pre-oral region. (D) Paratype specimen ICMCBMR000677, unsexed, detail of the ventral surface of the pre-oral region; white arrowhead points to the rhynchopore, black arrowhead signals the mouth. (E) Paratype specimen ICMCBMR000676, unsexed, detail of the dorsal surface of the head, a black arrowhead signals the point where the left cephalic slit ends. (F) Paratype specimen ICMCBMR000690, unsexed, detail of the left lateral surface of the head, a black arrowhead signals the point where the left cephalic slit ends. (G) Paratype specimen ICMCBMR000694, maturing female, detail of the two lines of dorsolateral gonopores (black arrowhead) and the transversal constriction of the body (white arrowhead). Scale bars: 1 mm.

Description: Body 95.9 ± 31.6 (44-150) mm long and up to 2 mm wide, dorsoventrally compressed (Fig. 2A-B). The head is well demarcated from the body with two long and deep lateral cephalic slits (Fig. 2C-D). The number of eyes in each row is variable (10.6 ± 3.8, 5-21) and typically asymmetrical, and they expand from the anterior dorsal tip of the head to approximately the mid-dorsal point of the cephalic slits (Fig. 2E-F). The size of the eyes is variable and sometimes they do not form a perfect line. The first two eyes are often separated from the remaining ones. The brain can be seen through transparency as two large reddish masses ending approximately at the posterior end of the lateral cephalic slits (Fig. 2C). The rhynchopore is frontal, situated at the anteriormost point of the head (Fig. 2D). The mouth is located 6.8 ± 1.4 (4-10) mm apart from the anterior tip of the body. The body is slender anteriorly to the mouth, up to 1.5 mm wide. The rhynchocoel expands approximately until the foregut-midgut region of the body (~10-15 % of the body). The width of the proboscis is approximately ~5-10 % of the body. The color is white, but the gut content can give it brownish or greyish tones, especially in the intestinal region (Fig. 2B). The anterior part of the body is from reddish to bright red, at least extending until the mouth level (Fig. 2A-B, D), but sometimes reaching the anterior part of the foregut region (Fig. 2C). It typically reaches its maximum color intensity at the level of the brain and fades progressively towards the posterior end of the red area. Some individuals have brighter red areas than others. Shallow transversal constrictions along the body, especially anteriorly to the mouth. Gonads are whitish and difficult to spot unless the gut is full of greyish or brownish material. Gonopores are dorsolateral (Fig. 2G).

Distribution: This species was molecularly recorded in four Mediterranean locations: Fréjus and Banyuls-sur-Mer in France, and Los Genoveses beach in Cabo de Gata and Isla de Tabarca in Spain; and the type locality in the Cantabrian Sea. Until further molecular confirmation is obtained, we propose here that the Mediterranean and Black Sea records of L. lacteus should be allocated to L. marcosii sp. nov. (Fig. 1).

Etymology: This species is named after Marcos Fernández Iglesias (1964-2022), friend of the two co-authors of this manuscript. Marcos was an amateur zoologist who collected, filmed, and curated a personal collection of thousands of marine animals, mostly marine invertebrates. He was skilled in identifications of complicated taxa such as cnidarians, polychaetes, nudibranchs, platyhelminths, and arthropods. He contributed 15 regional records of various species, including several first records for the Iberian Peninsula, and demonstrated an exceptional ability to document every specimen he encountered. His meticulously kept field notebooks remain a valuable legacy. He also shared photographic evidence of his findings through the website http://www.asturnatura.com, and was always generous in offering his photographs, videos, and knowledge to professional researchers.

Remarks: During the regeneration experiments of Zattara et al. (2019), 15 specimens were studied. All individuals were sexually mature and their total length was recorded. Males were 168 ± 74.1 mm long (70-300 mm, n = 9), and females were 131.7 ± 38.2 mm long (80-180 mm, n = 6). It must be pointed out that the 20 specimens studied in this work were measured under anesthesia, while the specimens from Zattara et al. (2019) were measured awake, when the animal stopped moving after being removed from the culture chamber. Thus, it is likely that the measurements taken from awake specimens might be underestimated or overestimated due to different body contractions.

Habitat and ecology: Among gravel under rocks in the upper and mid-intertidal zones. A single specimen was observed eating the snail Phorcus lineatus (da Costa, 1778) (Fig. 3A), and most of the specimens collected on December 27, 2023, were feasting on rotten barnacles, Pollicipes pollicipes (Gmelin, 1789), likely placed under a rock by an illegal fisherman (Fig. 3B). Although photographs were not taken, L. marcosii sp. nov. was sometimes found in close association with large specimens of L. sanguineus, although the latter species was less abundant in those habitats. The intestinal region of L. marcosii sp. nov. is frequently infested by unidentified nematodes of ~10 mm length (Fig. 3C) and microscopic gregarines (Fig. 3D). During regeneration experiments, some amputated individuals developed mucous cocoons (Fig. 3E), which were also observed in L. sanguineus specimens.

thumbnail of Figure 3
Figure 3
Ecological interactions of Lineus marcosii sp. nov. (a) Specimen ingesting a specimen of the snail Phorcus lineatus. (b) Several specimens (white arrowheads) scavenging rotting barnacles Pollicipes pollicipes (black arrowheads), likely placed under a rock by a person. (c) Posterior fragment of a specimen from the regeneration experiments of Zattara et al. (2019) showing two large parasitic nematodes expelled through the anus. (d) Squash prep of the posterior intestinal region of a mature male showing a large parasitic nematode in the intestinal lumen and gregarines attached to the intestinal wall. (e) Squash prep of the intestinal region of a mature female showing one parasitic nematode. (f) Squash prep of the anal region of a specimen showing a nematode near the anus (white arrowhead) and many gregarines attached to the rectum wall (black arrowhead). (g) Squash prep of the anal region of a specimen showing a detailed view of three gregarines. (h) Specimen packed inside a mucous cocoon, likely responding to an unidentified stress signal during regeneration experiments. Scale bars are not available, but the estimation of the width of the nemerteans is 1.5-2 mm.

Synonymized records

Lineus lacteus - Bürger (1892, 1895, 1904): partial, those records referring to Mediterranean waters (Banyuls, Villafranche-sur-mer, [?] Naples); Anadón (1980a, 1980b, 1981); Anadón and Lastra (1986); Vernet and Bierne (1983, 1988); Vernet and Anadón (1991a, 1991b, 1994); Martins et al. (2018); Zattara et al. (2019): partial, those used for the regeneration experiments and molecular systematics; Verdes et al. (2025).

Lineus (Ramphogordius) lacteus lacteus_M - Ament-Velasquez et al. (2016).

[?] Nemertes lactea - Grube (1855); Czerniavsky (1881)

Ramphogordius lacteus - Fernández-Álvarez and Machordom (2014); Herrera-Bachiller and Junoy (2014); Kvist et al. (2014); Herrera-Bachiller et al. (2015).

Ramphogordius lacteus network 9 - Kang et al. (2015).

DISCUSSION

The heteronemertean L. lacteus was described more than 180 years ago in Norway (Rathke 1843) and has been extensively cited across Europe, including Naples (Bürger 1895), the British Isles, and the Black Sea (Czerniavsky 1881, Gibson 1982). Wijnhoff (1912) questioned its presence in Sweden; however, L. lacteus is listed in the Swedish nemertean catalogue of Sundberg et al. (2007). It has been widely used as a laboratory model in experiments on neuroendocrinology (Vernet and Bierne 1988), histocompatibility (Vernet and Bierne 1983), reproduction (Vernet and Anadón 1994), and regeneration of this species and its sister taxa (Gontcharoff 1951, Ament-Velásquez et al. 2016, Zattara et al. 2019). Despite how long this species has been known and the fact that its biology is well studied compared to other nemerteans, it was not until the 21st century that research suggested cryptic biodiversity existed under this name (Kang et al. 2015, Ament-Velásquez et al. 2016). Ament-Velásquez et al. (2016) characterized two lineages-one occurring in North Atlantic waters and the other in the Mediterranean and Cantabrian Seas-the latter of which is described herein as a new species: L. marcosii sp. nov. As the type locality of L. lacteus is Molde, Norway (Rathke 1843), the North Atlantic lineage should retain this name. Before erecting a new name, the synonym list of L. lacteus was consulted (Gibson 1995). All synonyms of L. lacteus were variations of the same name due to misspellings or ascriptions to different genera, with the single exception of Gordius (minor) albusDalyell, 1853, from the British Isles. As L. lacteus as defined in this work is also present in Wales (Fig. 1), G. albus is considered herein a synonym of this species, and no available name for resurrection exists in the area where L. marcosii sp. nov. occurs.

While the most frequently sequenced marker for this species (and many other nemerteans) is the mitochondrial marker cox1, it is important to note that the mitochondrial 16S and the nuclear 28S and 18S also showed differences between L. lacteus and L. marcosii sp. nov. (Table 2), and distances within conspecific L. marcosii sp. nov. were consistently lower than between species. Moreover, the transcriptomic data from Ament-Velásquez et al. (2016) showed 1,014 and 386 private SNPs in the Atlantic (L. lacteus) and Mediterranean (L. marcosii sp. nov.) lineages, respectively, while only 37 were shared between them. This number of shared SNPs is within the range of other closely related lineids (12-49, if Lineus pseudolacteusGontcharoff, 1951 is excluded due to its hybrid origin; see Ament-Velásquez et al. 2016). Thus, there is robust mitochondrial and nuclear evidence that L. lacteus and L. marcosii sp. nov. are distinct species.

One of the observed specimens was eating a Ph. lineatus snail under a rock. Even though the predation event was not directly observed, it is assumed the snail was killed by the nemertean, as the snail and L. marcosii sp. nov. co-occur in intertidal microhabitats. This is consistent with the ecology of the related lineid Riseriellus occultusRogers et al., 1993, which predates on snails and other mollusks (Beckers et al. 2015). In both cases, the nemertean introduces its head inside the snail shell to consume the tissue. Many specimens were found eating detached rotting Po. pollicipes barnacles, below a large rock in the upper intertidal zone. As Po. pollicipes inhabits lower intertidal and subtidal environments, it is extremely unlikely that those specimens were naturally detached from their habitat and washed into the gravel under a heavy rock. It is quite common to see people illegally harvesting barnacles on that beach (Fernández-Álvarez, pers. obs.); therefore, our best assumption is that they were hidden there by a poacher to avoid a fine if law enforcement was nearby. Regardless of the reason why these barnacles ended up there, it is evident that L. marcosii sp. nov. exhibits opportunistic scavenging behavior. Vernet and Anadón (1991a) pointed out that this species can be gregarious, so we speculate that a feeding concentration is possible, though other possibilities, such as reproductive aggregations, cannot be ruled out. Most studied specimens showed high parasitic loads of nematodes and gregarines in their digestive tracts. Trophic interactions are crucial for the transmission of parasites (e.g., Santoro et al. 2023), as early ontogenetic phases of the parasite are ingested with prey tissues. A better understanding of L. marcosii sp. nov. trophic ecology might clarify the transmission of these parasites toward the nemertean predator. Lineus marcosii sp. nov. was encountered in close association with the sister species of the clade formed by this species and L. lacteus: the facultative fissiparous L. sanguineus. Gontcharoff (1951) hypothesized that the strictly fissiparous L. pseudolacteus was a product of hybridization between L. sanguineus and L. lacteus. Ament-Velásquez et al. (2016) proved this hypothesis through population genomics, demonstrating it occurred from an unreduced oocyte of L. sanguineus and a spermatozoon of L. lacteus. The close association of these nemerteans living in the same environments might have facilitated this unlikely hybridization phenomenon. Beckers et al. (2015) also showed that R. occultus lives in close association with L. lacteus.

In recent decades, the increased use of molecular markers has accelerated the species discovery rate of nemerteans (e.g., Chen et al. 2010, Hiebert and Maslakova 2015, Krämer et al. 2017), including cryptic species complexes (e.g., Verdes et al. 2021). As there was no overlap in the distribution of molecularly confirmed records of L. lacteus and L. marcosii sp. nov., they are considered an allopatric cryptic species complex; however, it remains unclear what the biogeographic break between the two species is, as no major biogeographic barriers have been identified between them. It is possible that detailed histological studies might reveal morphological differences between them, but the collection, fixing, and histological work would delay the species description for years (Strand and Sundberg 2011). Thus, we decided to adopt the modern approach of basing nemertean taxonomic descriptions on external morphological characters and DNA sequences, as currently practiced by many researchers (e.g., Kajihara 2015, Kajihara et al. 2018, Chernyshev et al. 2020, Cherneva et al. 2023, Alfaya et al. 2025). According to Gibson (1982), L. lacteus reaches 30-60 cm in length, while the L. marcosii sp. nov. individuals measured in this work were up to 30 cm long. Vernet and Anadón (1991a) reported a size of up to 25 cm in length and 1.5 mm in width for individuals identified as L. lacteus in Vigo (NW Iberian Peninsula), which are interpreted here as L. marcosii sp. nov. and whose size is consistent with our data. Nemertean length can be heavily influenced by body contraction, so our measurements would only be comparable to those of Gibson (1982) and Vernet and Anadón (1991a) if those specimens were also examined under anesthesia. Excluding this apparent larger size of the British L. lacteus, there are no known external morphological differences between L. lacteus and L. marcosii sp. nov.; however, the use of molecular autapomorphies allowed us to find diagnostic characters for the new species (Table 5). Molecular characters are highly useful for the characterization of cryptic biodiversity (Jörger and Schrödl 2013, 2014, Fernández-Álvarez et al. 2020). Based on the molecularly confirmed records (Fig. 1), we consider all Iberian records of L. lacteus as L. marcosii sp. nov. Thus, the description of this species does not increase the recognized biodiversity of the catalogue of nemerteans in this region (Herrera-Bachiller et al. 2015). In summary, the integration of molecular markers and species delimitation methods demonstrates that the Mediterranean and Cantabrian specimens previously ascribed to Lineus lacteus represent a distinct, valid species. Because no available synonyms exist within the known distribution of this lineage and the type locality of L. lacteus is outside this area, this taxon is described herein as Lineus marcosii sp. nov. These two lineages form an allopatric cryptic species complex that can be effectively differentiated using the molecular diagnostic characters of the cytochrome c oxidase subunit I gene defined in this study.

ACKNOWLEDGEMENTS

This work is dedicated to the memory of Marcos Fernández Iglesias (1964-2022), a lawyer and amateur naturalist who spent a great part of his free time collecting, recording, and studying marine animals, as well as helping professional scientists with his vast knowledge and excellent sampling skills. We thank the two anonymous reviewers and the Editor-in-Chief, Ricardo Moratelli, for their helpful comments. DNA barcoding analyses were carried out by AllGenetics & Biology SL. We thank Elena Guerrero (ICM-CSIC) for her curatorial services at the Marine Biological Reference Collection of ICM-CSIC (CBMR-ICM).

LITERATURE CITED

  • Alfaya JEF, Chernyshev AV, Lozada M, Bigatti G (2025) Nemertean diversity at the end of the world: histology-free description of three new species and evidence of genetic isolation between Antarctica and South America. Polar Biology 48: 77(2025). https://doi.org/10.1007/s00300-025-03395-5
    » https://doi.org/10.1007/s00300-025-03395-5
  • Ament-Velásquez SL, Figuet E, Ballenghien M, Zattara EE, Norenburg JL, et al. (2016) Population genomics of sexual and asexual lineages in fissiparous ribbon worms (Lineus, Nemertea): hybridization, polyploidy and the Meselson effect. Molecular Ecology 25: 3356-3369. https://doi.org/10.1111/mec.13717
    » https://doi.org/10.1111/mec.13717
  • Anadón N (1980a) Primeros datos sobre la fauna de nemertinos de la Península Ibérica: Asturias y sur de Galicia (N y NW de España). Boletín de la Real Sociedad Española de Historia Natural, Sección Biológica 78: 337-345.
  • Anadón N (1980b) Contribución al conocimiento de la fauna bentónica de la ría de Vigo III. Estudio de los arrecifes de Sabellaria alveolata (L.) (Polychaeta, Sedentaria). Investigación Pesquera 45: 105-122.
  • Anadón N (1981) Los acelomados: platelmintos y nemertinos. In: Ocaña S (Ed.) Enciclopedia Temática de Asturias. Encuadernaciones Belgas, Bilbao, 36-44.
  • Anadón N, Lastra C (1986) Los “arrecifes” de Sabellaria alveolata (L.) (Anélidos Poliquetos) como refugio de invertebrados. In: Principado de Asturias (Ed.) Jornadas sobre la conservación de la Naturaleza en España. Naturaleza y Sociedad. Principado de Asturias, Oviedo, 215-218.
  • Andrade SCS, Strand M, Schwartz M, Chen H, Kajihara H, et al. (2012) Disentangling ribbon worm relationships: multi-locus analysis supports traditional classification of the phylum Nemertea. Cladistics 28: 141-159. https://doi.org/10.1111/j.1096-0031.2011.00376.x
    » https://doi.org/10.1111/j.1096-0031.2011.00376.x
  • Arnold S, Nos D, Sáez-Liante R, Fernández-Álvarez FÁ (2025) Diversity in the squid family Ancistrocheiridae and description of a new family of the order Oegopsida (Cephalopoda). Zoological Journal of the Linnean Society 204(3): zlaf074. https://doi.org/10.1093/zoolinnean/zlaf074
    » https://doi.org/10.1093/zoolinnean/zlaf074
  • Beckers P, Bartolomaeus T, von Döhren J (2015) Observations and experiments on the biology and life history of Riseriellus occultus (Heteronemertea: Lineidae). Zoologica Scripta 32(6): 531-546. https://doi.org/10.2108/zs140270
    » https://doi.org/10.2108/zs140270
  • Bickford D, Lohman DJ, Sodhi NS, Ng PKL, Meier R, et al. (2007) Cryptic species as window on diversity and conservation. Trends in Ecology and Evolution 22: 148-155. https://doi.org/10.1016/j.tree.2006.11.004
    » https://doi.org/10.1016/j.tree.2006.11.004
  • Bürger O (1892) Zur Systematik der Nemertinenfauna des Golfs von Neapel. Nachrichten von der Königlichen Gesellschaft der Wissenschaften und von der Georg-Augusts-Universität 5: 137-178.
  • Bürger O (1895) Die Nemertinen des Golfes von Neapel und der angrenzenden Meeres-Abschnitte. Fauna und Flora des Golfes von Neapel 22: 1-743.
  • Bürger O (1904) Nemertini. Das Tierreich 20: 1-151.
  • Carrera-Parra LF, Fauchald K, Gambi C (2011) Revision of the taxonomic status of Lysidice (Polychaeta, Eunicidae) in the western Caribbean Sea with observation on species reproductive features and habitat preference. Italian Journal of Zoology 78(Suppl. 1): 27-40. https://doi.org/10.1080/11250003.2011.593850
    » https://doi.org/10.1080/11250003.2011.593850
  • Chen H, Strand M, Norenburg JL, Sun S, Kajihara H, et al. (2010) Statistical parsimony networks and species assemblages in cephalotrichid nemerteans (Nemertea). PLOS One 5(9): e12885. https://doi.org/10.1371/journal.pone.0012885
    » https://doi.org/10.1371/journal.pone.0012885
  • Cherneva I, Ellison CI, Zattara EE, Norenburg JL, Schwartz ML, et al. (2023) Seven new species of Tetranemertes Chernyshev, 1992 (Monostilifera, Hoplonemertea, Nemertea) from the Caribbean Sea, western Pacific, and Arabian Sea, and revision of the genus. ZooKeys 1181: 167-200. https://doi.org/10.3897/zookeys.1181.109521
    » https://doi.org/10.3897/zookeys.1181.109521
  • Chernyshev AV, Polyakova NE, Vignesh MS, Jain RP, Sanjeevi P, et al. (2020) A histology-free description of a new species of the genus Tetrastemma (Nemertea: Hoplonemertea: Monostilifera) from Hawaii and India. Zootaxa 4808: 379-383. https://doi.org/10.11646/zootaxa.4808.2.10
    » https://doi.org/10.11646/zootaxa.4808.2.10
  • Church SH, Abedon RB, Ahuja N, Anthony CJ, Destanović D, et al. (2025) Population genomics of a sailing siphonophore reveals genetic structure in the open ocean. Current Biology 35(15): 3556-3569.e6. https://doi.org/10.1016/j.cub.2025.05.066
    » https://doi.org/10.1016/j.cub.2025.05.066
  • Czerniavsky W (1881) Materialia ad zoographiam Ponticam comparatum. Fasc. 3 Vermes. Bulletin de la Société des Naturalistes de Moscou 55: 338-420.
  • Da Costa EM (1778) Historia naturalis testaceorum Britanniæ, or, the British conchology; containing the descriptions and other particulars of natural history of the shells of Great Britain and Ireland: illustrated with figures. Millan, White, Emsley & Robson, London, 254 pp.
  • Dalyell JG (1853) The Powers of the Creator. Van Voorst, London, 327 pp.
  • De Queiroz K (2007) Species concepts and species delimitation. Systematic Biology 56: 879-886. https://doi.org/10.1080/10635150701701083
    » https://doi.org/10.1080/10635150701701083
  • Dierckxsens N, Mardulyn P, Smits G (2016) NOVOPlasty: de novo assembly of organelle genomes from whole genome data. Nucleic Acids Research 45: e18. https://doi.org/10.1093/nar/gkw955
    » https://doi.org/10.1093/nar/gkw955
  • Fernández-Álvarez FÁ, Braid HE, Nigmatullin CM, Bolstad KSR, Haimovici M, et al. (2020) Global biodiversity of the genus Ommastrephes d’Orbigny, 1834 (Ommastrephidae: Cephalopoda): an allopatric cryptic species complex. Zoological Journal of the Linnean Society 190: 460-482. https://doi.org/10.1093/zoolinnean/zlaa014
    » https://doi.org/10.1093/zoolinnean/zlaa014
  • Fernández-Álvarez FÁ, Machordom A (2014) Hallado el nemertino invasor Cephalothrix cf. simula en aguas costeras españolas. Quercus 339: 34-37.
  • Folmer O, Hoeh W, Black M, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial Cytochrome C oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294-299.
  • Gibson R (1982) British nemerteans. Cambridge University Press, Cambridge, 212 pp.
  • Gibson R (1995) Nemertean genera and species of the world: an annotated checklist of original names and description citations, synonyms, current taxonomic status, habitats and recorded zoogeographic distribution. Journal of Natural History 29(2): 271-561. https://doi.org/10.1080/00222939500770161
    » https://doi.org/10.1080/00222939500770161
  • Gmelin JF (1791) Vermes. In: Gmelin JF (Ed.) Caroli a Linnaei, Systema Naturae per Regna Tria Naturae, Ed. 13. Tome 1(6). G.E. Beer, Leipzig, 3021-3910.
  • Gontcharoff M (1951) Biologie de la regeneration et la reproduction chez quelques Lineidae de France. Ph.D. Thesis, Université de Paris, Paris. https://primo.sorbonne-universite.fr/discovery/fulldisplay?docid=alma991002078959806616&context=L&vid=33BSU_INST:33BSU⟨=fr&adaptor=Local%20Search%20Engine
    » https://primo.sorbonne-universite.fr/discovery/fulldisplay?docid=alma991002078959806616&context=L&vid=33BSU_INST:33BSU⟨=fr&adaptor=Local%20Search%20Engine
  • Grube E (1855) Bemerkungen über einige Helminthen und Meerwürmen. Archiv für Naturgeschichte 21: 137-158.
  • Guerrero E, Abelló P, Lombarte A, Villanueva R, Ramón M, et al. (2020) Biological Reference Collections ICM-CSIC. v1.28. Institute of Marine Sciences (ICM-CSIC). Dataset/occurrence. Available at: https://www.gbif.org/en/dataset/1d743188-1e65-4d99-a814-fa3fd51f1490
    » https://www.gbif.org/en/dataset/1d743188-1e65-4d99-a814-fa3fd51f1490
  • Gunnerus JE (1770) Nogle smaa rare og meestendeelen nye Norske Soedyr. Skrifter, som udi det Kiöbenhavnske Selskabets af laerdoms og Videnskabers Elskere, 1765-1769 (Actae Havniae) 10: 166-176.
  • Herrera-Bachiller A, Fernández-Álvarez FÁ, Junoy J (2015) A taxonomic catalogue of the nemerteans (Phylum Nemertea) of Spain and Portugal. Zoological Science 32: 507-522. http://doi.org/10.2108/zs140242
    » http://doi.org/10.2108/zs140242
  • Herrera-Bachiller A, Junoy J (2014) Occurrence of the interstitial nemertean Ototyphlonemertes duplex Bürger, 1895 (Nemertea: Hoplonemertea) in the Cabo de Gata Natural Park (Mediterranean, Southeast Spain). Marine Biodiversity Records 7: e126. https://doi.org/10.1017/S1755267214001225
    » https://doi.org/10.1017/S1755267214001225
  • Hiebert TC, Maslakova S (2015) Integrative taxonomy of the Micrura alaskensis Coe, 1901 species complex (Nemertea: Heteronemertea), with descriptions of a new genus Maculaura gen. nov. and four new species from the NE Pacific. Zoological Science 32: 615-637. https://doi.org/10.2108/zs150011
    » https://doi.org/10.2108/zs150011
  • Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS (2018) UFBoot2: improving the ultrafast bootstrap approximation. Molecular Biology and Evolution 35: 518-522. https://doi.org/10.1093/molbev/msx281
    » https://doi.org/10.1093/molbev/msx281
  • Jörger KM, Schrödl M (2013) How to describe a cryptic species? Practical challenges of molecular taxonomy. Frontiers in Zoology 10: 59. https://doi.org/10.1186/1742-9994-10-59
    » https://doi.org/10.1186/1742-9994-10-59
  • Jörger KM, Schrödl M (2014) How to use CAOS software for taxonomy? A quick guide to extract diagnostic nucleotides or amino acids for species descriptions. Spixiana 37: 21-26. https://www.zobodat.at/pdf/Spixiana_037_0021-0026.pdf
    » https://www.zobodat.at/pdf/Spixiana_037_0021-0026.pdf
  • Kajihara H (2015) A histology-free description of the branched-proboscis ribbonworm Gorgonorhynchus albocinctus sp. nov. (Nemertea: Heteronemertea). Publications of the Seto Marine Biological Laboratory 43: 92-102. https://doi.org/10.5134/199852
    » https://doi.org/10.5134/199852
  • Kajihara H, Tamura K, Tomioka S (2018) Histology-free descriptions for seven species of interstitial ribbon worms in the genus Ototyphlonemertes (Nemertea: Monostilifera) from Vietnam. Species Diversity 23: 13-37. https://doi.org/10.12782/specdiv.23.13
    » https://doi.org/10.12782/specdiv.23.13
  • Kalyaanamoorthy S, Minh BQ, Wong TKF, Haeseler Av, Jermiin LS (2017) ModelFinder: fast model selection for accurate phylogenetic estimates. Nature Methods 14: 587-589. https://doi.org/10.1038/nmeth.4285
    » https://doi.org/10.1038/nmeth.4285
  • Kang X-X, Fernández-Álvarez FÁ, Alfaya JEF, Machordom A, Strand M, et al. (2015) Species diversity of Ramphogordius sanguineus/Lineus ruber-like nemerteans (Nemertea: Heteronemertea) and geographic distribution of R. sanguineus Zoological Science 32: 579-589. https://doi.org/10.2108/zs150064
    » https://doi.org/10.2108/zs150064
  • Katoh K, Asimenos G, Toh H (2009) Multiple alignment of DNA sequences with MAFFT. Methods in Molecular Biology 537: 39-64. https://doi.org/10.1007/978-1-59745-251-9_3
    » https://doi.org/10.1007/978-1-59745-251-9_3
  • Kawauchi GY, Giribet G (2010) Are there true cosmopolitan sipunculan worms? A genetic variation study within Phascolosoma perlucens (Sipuncula, Phascolosomatidae). Marine Biology 157: 1417-1431. https://doi.org/10.1007/s00227-010-1402-z
    » https://doi.org/10.1007/s00227-010-1402-z
  • Knowlton N (1993) Sibling species in the sea. Annual Review of Ecology and Systematics 24: 189-216. https://doi.org/10.1146/annurev.es.24.110193.001201
    » https://doi.org/10.1146/annurev.es.24.110193.001201
  • Krämer D, Schmidt C, Podsiadlowski L, Beckers P, Horn L, von Döhren J (2017) Unravelling the Lineus ruber/viridis species complex (Nemertea, Heteronemertea). Zoolica Scripta 46: 111-126. https://doi.org/10.1111/zsc.12185
    » https://doi.org/10.1111/zsc.12185
  • Kvist S, Laumer CE, Junoy J, Giribet G (2014) New insights into the phylogeny, systematics and DNA barcoding of Nemertea. Invertebrate Systematics 28: 287-308. http://doi.org/10.1071/IS13061
    » http://doi.org/10.1071/IS13061
  • Martins CPP, Fernández-Álvarez FÁ, Villanueva R (2018) Invertebrate predation on egg masses of the European cuttlefish, Sepia officinalis: an experimental approach. Estuarine, Coastal and Shelf Science 200: 437-448. https://doi.org/10.1016/j.ecss. 2017. 11. 016
    » https://doi.org/10.1016/j.ecss. 2017. 11. 016
  • McIntosh WC (1873-1874) A Monograph of the British Annelids. Part I. The Nemerteans. Ray Society, London, 214 pp.
  • Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ (2015) IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution 32: 268-274. https://doi.org/10.1093/molbev/msu300
    » https://doi.org/10.1093/molbev/msu300
  • Okonechnikov K, Golosova O, Fursov M (2012) Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics 28: 1166-1167. https://doi.org/10.1093/bioinformatics/bts091
    » https://doi.org/10.1093/bioinformatics/bts091
  • Puillandre N, Brouillet S, Achaz G (2021) ASAP: assemble species by automatic partitioning. Molecular Ecology Resources 21: 609-620. https://doi.org/10.1111/1755-0998.13281
    » https://doi.org/10.1111/1755-0998.13281
  • R Core Team (2021) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, v. 4.1.5. https://www.r-project.org/
    » https://www.r-project.org/
  • Rambaut A (2018) FigTree. http://tree.bio.ed.ac.uk/software/figtree/
    » http://tree.bio.ed.ac.uk/software/figtree/
  • Rathke H (1799) Jagttagelser henhörende til Indvotdeormenes og Blöddyrenes Naturhistorie. Skrivter af Naturhistorie Selskabet, Kjobenhaven 5: 61-148.
  • Rathke H (1843) Beiträge zur Fauna Norwegens. Verhandlungen der Kaiserlichen Leopoldinisch-Carolinischen Akademie der Naturforseher 20: 1-264.
  • Rogers AD, Junoy J, Gibson R, Thorpe JP (1993) Enzyme electrophoresis, genetic identity and description of a new genus and species of heteronemertean (Nemertea, Anopla) from northwestern Spain and North Wales. Hydrobiologia 266: 219-238. https://doi.org/10.1007/BF00013370
    » https://doi.org/10.1007/BF00013370
  • Santoro M, Bellisario B, Fernández-Álvarez FÁ, Crocetta F, Palomba M (2023) Parasites and prey of the nursehound shark Scyliorhinus stellaris (Linnaeus, 1758): insights into hidden trophic web interactions in the Mediterranean Sea. Journal of Fish Biology 102(1): 271-280. https://doi.org/10.1111/jfb.15259
    » https://doi.org/10.1111/jfb.15259
  • Schultze MS (1851) Beiträge zur Naturgeschichte der Turbellarien. C.A. Koch, Greifswald, 78 pp.
  • Sowerby J (1805) The British miscellany; or, Coloured figures of new, rare, or little known animal subjects: many not before ascertained to be inhabitants of the British Isles: and chiefly in the possession of the author, James Sowerby. R. Taylor & Co., London, vol. 2, 13-21. http://doi.org/10.5962/bhl.title.41623
    » http://doi.org/10.5962/bhl.title.41623
  • Strand M, Sundberg P (2011) A DNA-based description of a new nemertean (phylum Nemertea) species. Marine Biology Research 7: 63-70. https://doi.org/10.1080/17451001003713563
    » https://doi.org/10.1080/17451001003713563
  • Sundberg P, Chernyshev AV, Kajihara H, Kånneby T, Strand M (2009) Character-matrix based descriptions of two new nemertean (Nemertea) species. Zoological Journal of the Linnean Society 157: 264-294. https://doi.org/10.1111/j.1096-3642.2008.00514.x
    » https://doi.org/10.1111/j.1096-3642.2008.00514.x
  • Sundberg S, Gibson R, Strand M (2007) Swedish nemerteans (phylum Nemertea), with description of a new hoplonemertean genus and species. Journal of Natural History 41(37-40): 2287-2299. https://doi.org/10.1080/00222930701589939
    » https://doi.org/10.1080/00222930701589939
  • Tamura K, Stecher G, Kumar S (2021) MEGA11: molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution 38: 3022-3027. https://doi.org/10.1093/molbev/msab120
    » https://doi.org/10.1093/molbev/msab120
  • Thollesson M, Norenburg JL (2003) Ribbon worm relationships: A phylogeny of the phylum Nemertea. Proceedings of the Royal Society of London (B) 270: 407-414. https://doi.org/10.1098/rspb.2002.2254
    » https://doi.org/10.1098/rspb.2002.2254
  • Valdés A, Breslau E, Padula V, Schrödl M, Camacho Y, et al. (2017) Molecular and morphological systematics of Dolabrifera Gray, 1847 (Mollusca: Gastropoda: Heterobranchia: Aplysiomorpha). Zoological Journal of the Linnean Society 184: 31-65. https://doi.org/10.1093/zoolinnean/zlx099
    » https://doi.org/10.1093/zoolinnean/zlx099
  • Vences M, Miralles A, Brouillet S, Ducasse J, Fedosov A, et al. (2021) iTaxoTools 0.1: Kickstarting a specimen-based software toolkit for taxonomists. Megataxa 6: 77-92. https://www.mapress.com/mt/article/view/megataxa.6.2.1
    » https://www.mapress.com/mt/article/view/megataxa.6.2.1
  • Verdes A, Álvarez-Campos P, Conejero M, Riesgo A, Böhne A, et al. (2025) ERGA-BGE reference genome of the lineid heteronemertean Lineus lacteus (Pilidiophora, Nemertea) [version 1; peer review: 1 approved]. Open Research Europe 5: 317. https://doi.org/10.12688/openreseurope.21501.1
    » https://doi.org/10.12688/openreseurope.21501.1
  • Verdes A, Arias MB, Junoy J, Schwartz ML, Kajihara H (2021) Species delimitation and phylogenetic analyses reveal cryptic diversity within Cerebratulus marginatus (Nemertea: Pilidiophora). Systematics and Biodiversity 19: 895-905. https://doi.org/10.1080/14772000.2021.1950231
    » https://doi.org/10.1080/14772000.2021.1950231
  • Vernet G, Anadón N (1991a) Continental shelf and littoral nemerteans from the North and North-West Spanish Atlantic coasts. Cahiers de Biologie Marine 32: 45-56.
  • Vernet G, Anadón N (1991b) Littoral nemerteans from Spain. North and north-west Atlantic coasts species. Italian Journal of Zoology 58: 91-93.
  • Vernet G, Anadón N (1994) The influence of sea water temperature and the photoperiod on the reproductive cycle of Lineus lacteus (Heteronemertea) from Vigo Bay (NW of Spain). Arquivos do Museu Bocage. Nova Série 2: 417-422.
  • Vernet G, Bierne J (1983) Etude de la couleur du corps de trois némertes: Lineus sanguineus, L. pseudolacteus et L. lacteus Application au marquage des tissus des chimères interspécifiques. Bulletin de la Société Zoologique de France 108: 257-266.
  • Vernet G, Bierne J (1988) Neuroendocrine control of gonadogenesis in regenerating Lineus lacteus (Heteronemertea). Hydrobiologia 156: 53-60. https://doi.org/10.1007/978-94-009-4063-5_7
    » https://doi.org/10.1007/978-94-009-4063-5_7
  • Wickham H (2016) ggplot2: Elegant Graphics for Data Analysis. Springer, Luxembourg. https://doi.org/10.1007/978-3-319-24277-4
    » https://doi.org/10.1007/978-3-319-24277-4
  • Winhoff G (1912) List of nemerteans collected in the neighbourhood of Plymouth from May-September, 1910. Journal of the Marine Biological Association of the United Kingdom 9: 407-434. https://doi.org/10.1017/S0025315400048359
    » https://doi.org/10.1017/S0025315400048359
  • Zattara EE, Fernández-Álvarez FÁ (2022) Collection and rearing methods to study whole-body regeneration in Lineus sanguineus (Nemertea). In: Walker J (Ed). Methods in Molecular Biology - Whole Body Regeneration. Humana, New York, 227-243. https://doi.org/10.1007/978-1-0716-2172-1_12
    » https://doi.org/10.1007/978-1-0716-2172-1_12
  • Zattara EE, Fernández-Álvarez FA, Hiebert TC, Bely AE, Norenburg JL (2019) A phylum-wide survey reveals multiple independent gains of head regeneration in Nemertea. Proceedings of the Royal Society B 286: 20182524. http://doi.org/10.1098/rspb.2018.2524
    » http://doi.org/10.1098/rspb.2018.2524
  • Zhang J, Kapli P, Pavlidis P, Stamatakis A (2013) A general species delimitation method with applications to phylogenetic placements. Bioinformatics 29: 2869-2876. https://doi.org/10.1093/bioinformatics/btt499
    » https://doi.org/10.1093/bioinformatics/btt499
  • ZooBank register
  • Data Availability Statement
    Sequences newly obtained in this manuscript can be accessed at GenBank through the GenBank Accession numbers PX559719-PX559724.
  • Funding
    This study was supported by a Ramón y Cajal fellowship (Ref. RYC2023-043494-I) awarded to FÁFÁ and funded by MCIN/AEI/10.13039/501100011033 and FSE+. Additional support was provided by the Spanish government through the Severo Ochoa Center of Excellence (Grant CEX2024-828 001494-S, funded by AEI).
  • Ethical Statement
    This study did not involve live vertebrate or cephalopod animals and therefore did not require approval by an ethics committee.
  • AI Statement
    The text of this manuscript was refined to improve grammar and linguistic clarity using a generative AI tool (ChatGPT 4 (https://chatgpt.com/), without altering its original meaning or scientific content. The process was conducted paragraph by paragraph, and all AI-generated outputs were subsequently reviewed and manually curated by the authors. This approach ensures full traceability and transparency, in compliance with the European Artificial Intelligence (AI) Act (EU Regulation 2024/1689).
  • How to cite this article
    Fernández-Álvarez FÁ, Menéndez Valderrey JL (2026) A histology-free description of Lineus marcosii (Pilidiophora: Lineidae), a cryptic species resembling Lineus lacteus. Zoologia 43: e25054. https://doi.org/10.1590/S1984-4689.v43.e25054
  • Published by
    Sociedade Brasileira de Zoologia at Scientific Electronic Library Online - https://www.scielo.br/zool

SUPPLEMENTARY MATERIALS

Supplementary S1

Supplementary S2

Supplementary S3

Supplementary S4

Authors: Fernández-Álvarez FÁ

Data type: DNA sequences in FASTA format.

Copyright notice: This dataset is made available under the Open Database License - ODBbL (https://opendatacommons.org/licenses/odbl/1.0/). The ODbL is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Link: https://doi.org/10.1590/S1984-4689.v43.e25054

Edited by

  • Editorial responsibility
    Marcus V. Domingues

Data availability

Sequences newly obtained in this manuscript can be accessed at GenBank through the GenBank Accession numbers PX559719-PX559724.

Data citations

Guerrero E, Abelló P, Lombarte A, Villanueva R, Ramón M, et al. (2020) Biological Reference Collections ICM-CSIC. v1.28. Institute of Marine Sciences (ICM-CSIC). Dataset/occurrence. Available at: https://www.gbif.org/en/dataset/1d743188-1e65-4d99-a814-fa3fd51f1490

Publication Dates

  • Publication in this collection
    27 July 2026
  • Date of issue
    2026

History

  • Received
    28 July 2025
  • Accepted
    27 Jan 2026
Creative Common - by 4.0
This is an open-access article distributed under the terms of the Creative Commons Attribution License
location_on
Sociedade Brasileira de Zoologia Caixa Postal 19020, 81531-980 Curitiba PR Brasil, Tel./Fax: (55 41) 3266-6823 - Curitiba - PR - Brazil
E-mail: sbz@sbzoologia.org.br
rss_feed Acompanhe os números deste periódico no seu leitor de RSS
Reportar erro