Introduction

Nemerteans belonging to the family Carcinonemertidae are voracious egg-predators that infect a variety of decapod crustaceans1,2. Some species are responsible for the collapse of crustacean fisheries in North America3,4,5,6 given their negative effect on female reproductive performance2,5,7,8. Several species of commercial interest, such as the Caribbean spiny lobster Panulirus argus whose eggs are consumed by C. conanobrieni9,10, the Dungeness crab Metacarcinus magister infected by C. errans5, the Red king crab Paralithodes camtschaticus parasitized by C. regicides, and the sand crab Portunus pelagicus infected by C. mitsukurii11 are used as hosts by carcinonemertid worms. One of the most studied nemertean worms is C. errans that during outbreaks causes considerable egg mortality in 50% or more of the brooding female crabs of Metacarcinus magister. Carcinonemertes errans has been tied to the collapse of this crab fishery in Central California early during the 1960s1,5,12.

The family Carcinonemertidae is comprised of two genera, Carcinonemertes with sixteen described species, and Ovicides with five described species10. Members of the genus Carcinonemertes have a single stylet and no accessory pouches while the genus Ovicides invariably exhibit accessory pouches and more than one stylet. All Carcinonemertes are gonochoric (separate sexes) while representatives of Ovicides can be either gonochoric or simultaneously hermaphroditic13,14. The life cycle of nemertean parasites is variable and appears to be adapted to the reproductive cycle and brooding pattern of their respective crustacean hosts15. At one extreme, some nemertean parasites exhibit a rather simple and short life cycle in which the worms feed, mature, and reproduce on a single ovigerous female host. In this case, host autoinfection is common (i.e., C. regicides in the Red king crab, Paralithodes camtschatica)15. At the other extreme, some Carcinonemertes are long lived and their life cycle involve the infection of both juvenile (males and females) and adult (males and gravid females) host individuals. In these long lived Carcinonemertes, autoinfection is rare and worms are also capable of transferring from male to female hosts when the host crabs mate (i.e., C. errans in the Dungeness crab Metacarcinus magister)15. Host-specificity also varies considerably in the family Carcinonemertidae. Some species of Carcinonemertes are specialists, using a single host species16 while other species are generalists, such as C. carcinophila, reported from as many as 28 different species of decapod hosts17.

Currently, 17 species of Carcinonemertes are known to science: four species inhabiting the Brazilian coast, South-western Atlantic (C. carcinophila inminuta, C. divae, C. caissarum, and C. sebastianensis)13, one species from the Caribbean Sea (Florida, USA and Santa Marta, Colombia) and North-western Atlantic Ocean (C. conanobrieni)9,10, one from the Southwestern Indian Ocean (C. mitsukurii, western Australian coast)18,19, eight species from the North Pacific Ocean (C. coei, C. errans, C. regicides, C. epialti, C. kurisi, C. pinnotheridophila, C. c. carcinophila, and C. wickhami; USA, Baja California, Mexico)1,18,20,21,22,23, and three species from the Southwestern Pacific Ocean (C. australiensis, C. tasmanica, and C. humesi; Australian coast)21,24. Not a single representative of the family Carcinonemertidae has been described so far from the South-eastern Pacific Ocean (SEP).

In the SEP, several species of brachyuran crabs, most of them belonging to the family Cancridae, are of commercial value25,26. In the Chilean coast, eight species with a wide distribution along the continental shelf are targeted by artisan fisheries: Cancer edwardsi, C. coronatus, C. porteri, Homalaspis plana, Ovalipes trimaculatus, Taliepus marginatus, T. dentatus, and Romaleon setosum (previously, C. setosus). Importantly, R. setosum and C. porteri represent two of the most heavily targeted species by local fisheries along the coast of Chile. Cancer porteri is distributed from Panama (9° S) to Concepción Bay (36° S), Chile27, and is commonly extracted by artisan fishermen in central Chile (32°–38° S). In turn, R. setosum is distributed from the coast of Ecuador (5° S) to Peninsula de Taitao (47° S), Chile and is extracted between 24° S and 40° S28,29.

The capture of gravid female crabs of both R. setosum and C. porteri is prohibited year-round, these fisheries are closed during the reproductive season, and a minimum catch body size (12 mm carapace width) has been established by the Fisheries and Aquaculture Sub-Secretary of the government of Chile since 199130,31. Importantly, during the last six years, crab landings (tons) have decreased significantly in Chile25. For instance, C. porteri landings decreased from 667 tons in 2014 to 2.62 tons in 2019. In turn, landings diminished from 548 to 216 tons in the same period for R. setosum32. This steady temporal decrease in landed biomass has forced the implementation of specific management strategies for the two crab species, including a full fishery ban in the case of C. porteri28.

Despite the importance of R. setosum and C. porteri along the Chilean coast, several biological aspects of these crabs are not known, and the presence of nemertean egg-predators has not been previously studied. Nemertean egg-predators, if present, could affect the reproductive performance and population dynamics of these crabs and result in the collapse of their fisheries, as it has been reported before in the north Pacific coast12. Consequently, the objective of this study was to explore, for the first time, the presence and prevalence of nemertean egg-predators in the commercial crabs Cancer porteri and Romaleon setosum, in the central and northern coast of Chile (SEP). Importantly, detailed morphological and molecular analyses of the nemertean worms infecting the aforementioned crabs revealed that they belong to a single species of Carcinonemertes not yet described. Here, we formally describe this new species as Carcinonemertes camanchaco sp. nov.

Results

Prevalence of nemerteans per host species and locality

A total of 48 female crabs (non-ovigerous) of Cancer porteri from Iquique (20° S), 50 gravid females of Romaleon setosum from Coquimbo (30° S), and 21 gravid females of C. porteri from Valparaíso (33° S) were examined. Nemerteans were only detected in egg masses of gravid female crabs (Fig. 1); therefore, the prevalence of nemerteans in specimens from Iquique was 0%. The prevalence of nemerteans in C. porteri and R. setosum from Coquimbo and Valparaíso was 24.0% and 38.1%, respectively. We found several nemertean developmental stages (eggs, juveniles, and adult worms) infecting the egg masses of the infected crabs. Juvenile worms were found either roaming among the crab eggs or encapsulated on the surface of the crab’s abdomen. Adult worms were found only in egg masses, covered by a mucus sheath (not ornamented). Long strands of worm eggs covered by translucent mucus were also observed among the eggs of ‘berried’ (= brooding) female crabs.

Figure 1
figure 1

Host crab species Romaleon setosum (a) and Cancer porteri (b) infected by Carcinonemertes camanchaco sp. nov. present in their egg masses (c, d).

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Morphometric analyses

The studied male worms had a mean ± SD body size of 3.64 ± 0.59 mm and the female worms had a mean body size of 5.84 ± 1.25 mm. A t-test demonstrated statistically significant differences in average body size between male and female worms (t = 4.42, d.f. = 19, P < 0.01), with the females being larger than the males. For both male and female worms, body length was not correlated with stylet length, basis length, and stylet-basis ratio (Spearman correlations; all P > 0.05).

The PCAs performed separately for female and male worms infecting the studied crabs in the SEP and those from other four closely related species of Carcinonemertes, characterized by having two gonad rows on each side of the intestine, indicated that > 95% of the variability in body proportions among the studied specimens and species was explained by the first three PCA axes (Table 1). EW/BL, DW/BL, BW/BL, and SL/SBL/BL (factor loads > 0.95; Table 1) were the most important measures that distinguished both male and female nemertean worms collected during this study from specimens belonging to the other four similar, previously described species (Fig. 2a, b). The DFA, using the orthogonal components of the PCA, revealed significant differences among female (Wilks’ Lambda = 0.01; F (12, 21) = 7.62; p < 0.01) and male (Wilks’ Lambda = 0.001; F (12, 21) = 24.96; p < 0.01) nemertean specimens of the four species. Additionally, the DA showed that 93% of the female specimens and 100% of the male specimens were correctly assigned to their respective nemertean species.

Table 1 Factor loadings of the PCA using morphometric data obtained from females and males of Carcinonemertes camanchaco sp. nov., based on a correlation matrix.
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Figure 2
figure 2

Principal components analysis based on morphometric measurements standardized by total length from five species of Carcinonemertes. (a) Female worms, (b) Male worms. White circles = C. camanchaco sp. nov., square = C. coei, diamond = C. errans, triangle = C. regicides, black circle = C. wickhami.

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Phylogenetic analyses

The phylogenetic analyses included 21 cox1 sequences 620 bp in length (after alignment): 8 sequences obtained during this study and 13 sequences retrieved from GenBank. The data matrix comprised a total of 205 parsimony informative sites.

ML and BI analyses (bootstrap support of 89 and 1.0 for ML and BI, respectively), demonstrated that the specimens of Carcinonemertes infesting R. setosum (three sequences) and C. porteri (five sequences) belong to the same species. Furthermore, both ML and BI phylogenetic reconstructions indicated that the studied specimens of Carcinonemertes form a clade sister to C. errans, a worm that infects Metacarcinus magister in the coast of California (Fig. 3). The intra-specific genetic distances calculated for the 8 sequences of this study varied between 0 and 0.3% (Table 2). The mean genetic distance between specimens of Carcinonemertes collected for this study and other species belonging to the genus Carcinonemertes was 3.6% (3.6–4.2%, n = 3 sequences) versus C. errans (ex Metacarcinus magister), 17% (16.8–17.2%, n = 3 sequences) versus Carcinonemertes sp. from an unknown host species previously collected from Chile, and 23.6% (23.6–23.7%, n = 2 sequences) versus C. conanobrieni (ex Panulirus argus) (Table 2). The phylogenetic trees also indicated that the genus Carcinonemertes is paraphyletic, given the inclusion of Ovicides in the same well-supported phylogenetic clade containing all species of Carcinonemertes (Fig. 3).

Figure 3
figure 3

Phylogenetic tree based on the cox1 gene for Carcinonemertes spp. from different host species, inferred using Bayesian inference (BI) and Maximum Likelihood (ML) analyses. Numbers near internal nodes denote posterior probabilities or bootstrap values from the BI and ML phylogenetic analyses (BI/ML), respectively. Sequences for Nipponnemertes bimaculata and N. punctatulus were used as outgroups. SEP South eastern Pacific, NWA North western Pacific, SWA South western Atlantic, NWP North western Pacific.

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Table 2 Genetic distance between sequences of Carcinonemertes spp. available in GenBank and sequences of C. camanchaco sp. nov. provided in this study.
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Morphological characterization of specimens from the SEP

We described morphological characteristics including body color and body measurements of female and male nemertean worms. Comparisons of morphology, morphometry, host species and geographical distributions of Carcinonemertes camanchaco sp. nov. and the other 16 Carcinonemertes species described around the world are presented in Supplementary Table S1. Detailed morphological description for Carcinonemertes spp. is provided in the Taxonomy section.

TAXONOMY : Carcinonemertes camanchaco

N.V. Leiva, L. Ñacari., J.A. Baeza, & M.T. Gonzalez, sp. nov. (Figs. 4, 5 and 6). ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN), details of the new species have been submitted to ZooBank. The Life Science Identifier (LSID) of the article is urn:lsid:zoobank.org:pub:DCF78A3D-19E6-407F-9ED9-47DADAF2521A. The LSID for the new name Carcinonemertes camanchaco is urn:lsid:zoobank.org:act:0DCA7DF8-1E8D-4AF5-A3E9-141BEC061BDD.

Figure 4
figure 4

Adult Carcinonemertes camanchaco sp. nov. (a) Complete female worm, (b) anterior part of a female worm, (c) anterior part of a male worm, (d) complete male worm and (e) Stylet. ey eyes, br brain lobe, st stylet, ac anterior chamber, mc medium chamber, fo foregut, pc proboscis chamber, in intestine, ov ovary, te testes, sv seminal vesicle.

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Figure 5
figure 5

Photograph of complete worm of Carcinonemertes camanchaco sp. nov. (a) Complete female worm, (b) complete male worm; (c) anterior; (d) trunk and (e) posterior female body; (f) anterior; (g) trunk and (h) posterior male body. Body organs nomenclature is given in Fig. 4.

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Figure 6
figure 6

Photographs of anterior body section of Carcinonemertes camanchaco sp. nov (a); showing details of proboscis chamber, median chamber, and anterior chamber (b) and stylet (c).

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Phylum Nemertea.

Class Hoplonemertea Hubrecht, 1879.

Family Carcinonemertidae Sumner, Osburn & Cole, 1913.

Genus Carcinonemertes Coe, 1902.

Etymology The specific name refers to the 'Camanchaco’ ethnic group, indigenous people that inhabited the north and central coast of Chile and whose descendants are legally recognized as original indigenous people since 2020 in Chile.

Site of infection/host Adult specimens were found in the egg masses of hosts Cancer porteri (Rathbun, 1930) and Romaleon setosum (Molina, 1782) (Crustacea, Decapoda).

Prevalence and mean intensity (± SD) 24.0% and 2.6 (± 2.07) in Cancer porteri (Valparaiso) and 38.1% and 3.8 (± 2.4) in Romaleon setosum (Coquimbo).

Host habitat and sampling locality Subtidal, Puerto Aldea, Coquimbo (30°17´37´´S, 71°36´36´´W) and Caleta Portales, Valparaíso (33°1′52.74″S, 71°35′25.18″W), north and central Chilean coast, collected during November 2020.

Materials examined Holotype (one female): MNHNCL NEME-15002; paratype (one male): MNHNCL NEME-15003; paratype (three males): MNHNCL NEME-15004, and paratype (three females): MNHNCL NEME-15005.

Description Body color varies from translucent to pale orange due to gonads and internal organs’ coloration. The anterior and posterior end of the body is rounded (Fig. 4). Worms are filiform in shape and range from 2.381 mm to 8.922 mm in total length. The anterior proboscis chamber could not be distinguished. The middle chamber contains a single stylet (Figs. 5, 6). The stylet appears like a dagger with a broad- stylet basis (Figs. 4e, 5 and 6). Accessory stylets are not present. Posterior proboscis chamber is elongated. Foregut (anterior portion of the esophagus) joins the rhynchodaeum at or just anterior to the brain (Figs. 4, 5). Stomach opens into the intestine, which presents numerous paired unbranched diverticula (Figs. 4, 5). The anus is located at the terminal posterior portion of the body. All body measurements are given in Table 3.

Table 3 Measurements of Carcinonertes camanchaco sp. nov.
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Females (18 living specimens) Dimensions of relaxed worms were 5.837 ± 1.254 (mean ± SD, range = 4.880–8.922) long and 0.313 ± 0.121 (0.167–0.574) wide. Two irregular black eyes, 0.030 ± 0.009 (0.020–0.049) long and 0.019 ± 0.006 wide (0.009–0.030) (Fig. 4). Distance between eyes 0.099 ± 0.030 (0.050–0.163 mm). Distance from eyes to tip of head 0.120 ± 0.028 (0.080–0.177). The brain is 0.186 ± 0.047 (0.109–0.247) long and 0.049 ± 0.008 (0.040–0.060) wide (Fig. 4b, 4c). Distance from brain to tip of head 0.176 ± 0.030 (0.155–0.229). Middle chamber is 0.069 ± 0.014 (0.051–0.100) long and 0.052 ± 0.009 (0.043–0.06) wide (Figs. 4b, 6). Stylet 0.009 ± 0.001 (0.006–0.010) long. Stylet basis 0.041 ± 0.010 (0.026–0.060) long and 0.010 ± 0.002 (0.008–0.014) wide (Figs. 4e; 6). Stylet: basis ratio 0.235 ± 0.042 (0.167–0.33). Distance from central stylet to tips head 0.263 ± 0.048 (0.183–0.331). Proboscis bulb 0.044 ± 0.010 (0.025–0.060) long and 0.043 ± 0.003 (0.039–0.048) wide. Posterior proboscis chamber is elongated, 0.143 ± 0.056 (0.099–0.260) long and 0.069 ± 0.010 (0.055–0.080, n = 6) wide (Figs. 4b, 6). Ovaries are orange and numerous (n = 72 ± 25 [range: 40–126] per worm); regularly distributed in two rows on each side of the intestinal diverticule (Figs. 4a, 5a, d, e). Distance from first gonad to tip of head 1.074 ± 0.437 (0.341–2.016).

Males (9 living specimens) Dimensions of relaxed worms were 3.643 ± 0.857 (2.381–4.929) long and 0.222 ± 0.048 (0.143–0.280) wide. Two irregular black eyes, 0.021 ± 0.006 (0.014–0.030) long and 0.018 ± 0.009 wide (0.008–0.030) (Fig. 4c, d). Distance between eyes 0.101 ± 0.028 (0.070–0.162). Distance from eyes to tip of head 0.101 ± 0.014 (0.090–0.133). Brain is 0.154 ± 0.028 (0.117–0.200) long and 0.047 ± 0.014 (0.026–0.075) wide (Fig. 4c). Distance from brain lobe to tip of head 0.162 ± 0.037 (0.102–0.222). Middle chamber is 0.067 ± 0.015 (0.048–0.100 mm) long and 0.057 ± 0.011 (0.045–0.080) wide (Figs. 4c, 6). Single stylet on basis, 0.011 ± 0.004 (0.006–0.020) long (Figs. 4e, 6). Stylet basis 0.030 ± 0.006 (0.020–0.037) long and 0.013 ± 0.003 (0.010–0.015) wide. Stylet: basis ratio 0.366 ± 0.170 (0.167–0.667). Distance from central stylet to tips head 0.243 ± 0.061 (0.183–0.305). Proboscis bulb 0.042 ± 0.016 (0.026–0.075) long and 0.040 ± 0.012 (0.030–0.061) wide (Figs. 4c, 6). Posterior proboscis chamber elongated 0.147 ± 0.061 (0.100–0.256) long and 0.048 ± 0.004 (0.042–0.052) wide (Figs. 4c, 6). Testes are numerous and translucent, occupying all the middle and rear section of body, between intestinal diverticula. A single seminal vesicle was observed near the posterior end, 0.183 ± 0.040 (0.125–0.240, n = 2) (Table 3; Fig. 6).

Differential diagnosis Carcinonemertes camanchaco sp. nov. closely resembles C. errans, C. coei, C. wickhami, and C. regicides due to the presence of two gonad rows on each side of the intestine. The other 12 described species of Carcinonemertes have only a single gonad row (Table S1). Carcinonemertes camanchaco sp. nov. can be distinguished from C. errans due to the presence of a mucus sheath, present in C. camanchaco sp. nov. but absent in C. errans. Carcinonemertes wickhami has been reported to build mucus sheaths as here reported for C. camanchaco sp. nov. However, in C. camanchaco sp. nov. the mucus sheath is simple, while in C. wickhami the mucus sheath is ornamented. C. camanchaco sp. nov. differs from C. coei in body colour, described as translucent white to pale orange in C. camanchaco sp. nov. and yellowish-white in C. coei. The ratio of the stylet/basis is smaller in C. camanchaco sp. nov. (0.233) than in C. errans (0.321), C. coei (0.381), C. wickhami (0.528) and C. regicides (0.432). A summary of the morphological and morphometric characteristics of the different species of Carcinonemertes are given in Supplementary Table S1.

Discussion

This is the first study that has tested for the presence/absence of nemertean egg-predators in commercial crabs from the SEP coast. We did find nemertean worms infecting two species of crabs from the SEP and our morphometric and molecular analyses (based on the mitochondrial cox1 gene) indicated that these nemerteans parasitizing eggs of Romaleon setosum and Cancer porteri belong to the same species of Carcinonemertes, here named C. camanchaco sp. nov. The same analyses also indicate that this newly discovered entity is genetically different from all other known species of Carcinonemertes with information available in GenBank. We note that the cox1 is not useful to distinguish among species in some clades within the Nemertea (Sundberg et al., 2016). An integrative taxonomic approach, including the (classical) comparison of morphological traits, multivariate morphometric analyses, and molecular data (cox1 mitochondrial gene) demonstrated that the nemertean inhabiting two sympatric crab species is a new species. The nemertean species recorded in our study is characterized by the presence of two ovaries on each side of the intestine and the production of a simple mucus sheath (not ornamented). The combination of these morphological characters plus genetic characters clearly distinguished our specimens from previously described species in the same genus.

The genus Carcinonemertes now comprises 17 valid species. However, genetic information (i.e., cox1) exist for only five species (plus the new species), which were used here to construct a phylogenetic tree. This tree showed that C. conanobrieni, parasitizing Caribbean spiny lobsters, is sister to a clade comprised of congeneric species infecting brachyuran crabs (Fig. 3). Only two nemertean species have been reported before from crabs belonging to the family Cancridae: C. errans described from Metacarcinus magister33 and C. camanchaco sp. nov. from Cancer porteri and Romaleon setosum (this study). The phylogenetic tree indicated that C. camanchaco sp. nov. and C. errans are sister species (with 3.6% of genetic distance). These nemertean species parasitizing two phylogenetically close host species, belonging to the family Cancridae, implies that the genus Carcinonemertes could be coevolving together with different species used as hosts6,14.

Estimating the prevalence of nemertean egg-predators is exceedingly relevant considering the impact that Carcinonemertes and Ovicides have on the reproductive performance and population dynamics of their host species34,35. Notably, studies reporting the prevalence of nemerteans are rare. Santos et al.13 found that the prevalence of C. divae in female and male crabs of Libinia spinosa was 87% and 43.2%, respectively. In turn, C. errans prevalence in Metacarcinus magister is remarkably high; up to 97% of the gravid females can be infected by this nemertean during one year/ fishing season. Importantly, the intensity of C. errans on infected Metacarcinus magister can also be very high5. In the 70’s, C. errans became an epidemic, resulting in the direct mortality of an average of 55% of the eggs produced by females of M. magister. The above suggests that C. errans might probably be one of the most numerically significant predators on these crabs and was responsible for the collapse of this fishery in Central California5,12,33. The prevalence of C. camanchaco sp. nov. was lower than that reported in the aforementioned species (38% in R. setosum and 24% in C. porteri). Given the previously demonstrated impact of nemerteans on the fecundity of their respective host species, and the considerable and steady decreases in landings reported for R. setosum and C. porteri along the coast of Chile in the last four years, we argue that additional studies are needed to evaluate the effect of C. camanchaco sp. nov. on the fertility and population dynamics of these crabs.

The prevalence of several parasite species can vary across short- and long-time scales and can be associated to environmental variability and/or host availability11. Shields and Kuris3 showed that prevalence of C. epialti on the shore crab Hemigrapsus oregonensis varied between years, finding that during non-outbreak periods prevalence was 48%, while during the outbreak periods this prevalence increased to 97%. Campbell et al.24 showed that prevalence of C. australiensis was 67% in females of the spiny lobster Panulirus cygnus. Most recently, Baeza et al.35 determined that the prevalence of C. conanobrieni in the lobster Panulirus argus was 7.4%, and Simpson et al.10 showed that prevalence increased to 93.9% a year later (107 of 114 gravid females). This information suggests that nemertean infection levels can change quickly between consecutive years. Therefore, we argue in favor of additional studies to determine short- and long-term spatial and temporal variability in the prevalence and intensity of C. camanchaco sp. nov. in the different species infected, as well as the effect of this parasite on the reproductive performance and overall health of its crab hosts. Carrying out temporal monitoring of the newly discovered nemertean species across host species is needed to manage these heavily fished species towards the goal of sustainability in Chile.

Methods

Sampling and parasite collection

Females of Cancer porteri and Romaleon setosum were collected by divers from three localities, Caleta Chanavayita, Iquique (20°42′10″S, 70°11′15″W), Puerto Aldea, Coquimbo (30°17´37´´S, 71°36´36´´W), and Caleta Portales, Valparaíso (33°1′52.74″S, 71°35′25.18″W). We collected a total of 48 crabs belonging to Cancer porteri from Iquique, 50 crabs belonging to Romaleon setosum from Coquimbo, and 21 specimens of C. porteri from Valparaíso. The samples of C. porteri collected in Iquique and Valparaíso were transported to the laboratory at the Universidad de Antofagasta, and alive specimens of R. setosum were transported to the laboratory at the Universidad Católica del Norte, Chile. Collection of these specimens was authorized by the Fishery and Aquaculture Sub-Secretary of the government of Chile (R. Ex N° E-2020–481).

In the laboratory, each crab (either alive or frozen [− 10 °C]) was examined for the presence or absence of nemertean worms. All eight pleopods were removed from each studied crab, and all embryos were gently stripped away from the pleopods of each gravid female using fine forceps and placed into Petri dishes with filtered seawater (10 µm). The egg mass of each gravid female, as well as the abdomen, pleopods, gills, and branchial chambers from all analyzed crabs were inspected under a stereomicroscope (Leica M80, Leica Microsystems, Wetzlar, Germany) to determinate the presence or absence of nemertean worms. Nemerteans collected from gravid female crabs were placed in Petri dishes with filtered seawater up to the point in time when measurements and photographs were taken, and morphological characters were studied in detail. Nemerteans were first relaxed in a 1:1 solution of 1 M MgCl2 (prepared with distilled water) and seawater for 1–5 min. The specimens were fixed in formaldehyde at 10% for morphological examination and 99% EtOH solution for genetic analyses.

Morphometric study and morphological characterization

Different body parts were measured in all adult worms (in mm, precision = 0.01 mm) using a digital camera (Mshot MD90) connected to a stereomicroscope (Olympus SZX7, Tokyo, Japan) and the programs MShot Image Analysis System 1.5.2 and ImageJ36. We measured total body length (BL), body width (BW), ocelli length (OL), ocelli width (OW), diaphragm width (DW), stylet length (SL), and stylet basis length (SBL) of each studied specimen. The different body measurements of the studied specimens were compared to those from all other previously described species of Carcinonemertes (n = 16).

Principal component analyses (PCAs) were also performed separately for both male and female worms, using body part measurements standardized by BL. For these analyses, only previously described nemertean species with taxonomic characteristics most similar to the specimens collected during this study were used; only species that exhibit two rows of gonads (i.e., Carcinonemertes coei, C. errans, C. regicides, C. wickhami). The body proportions used were BW/BL, OL/BL, OW/BL, DW/BL, SL/BL, SL/SBL, SBL/BL and SL/SBL/BL. The first two axes of the PCA were also used to evaluate the importance of each measured ratio on the morphometric variability observed among different species of Carcinonemertes. Finally, orthogonal variables (principal components), which preserve the original information of the database, were used to perform a Discriminant Function Analysis (DFA) and to assign specimens to the different worm species37,38. All statistical analyses were conducted using the software Statistica 10.0 (Statsoft, Tulsa, USA).

DNA isolation, PCR amplification and sequencing

A total of eight juvenile worms were dissected with forceps and DNA was isolated from each one of them separately using a protocol modified from Miller et al.39 that included treatment of each specimen with sodium dodecyl sulphate and digestion with Proteinase K. Proteins were removed by precipitation with NaCl, and the DNA was finally precipitated with ethanol. The resultant DNA was eluted in nuclease-free water and quantified using a spectrophotometer until a concentration of 30 to 50 ng/μL was reached.

The mitochondrial gene cytochrome c oxidase 1 (cox1) was amplified using universal primers described in Folmer et al.40. Each PCR reaction had a final volume of 35 μl including: 0.175 U/μl Taq polymerase (GoTaq G2, Promega), 7 μl 5X PCR buffer, 5.6 μl MgCl2 (25 mM), 2.1 μl bovine serum albumin (10 mg/ml), 0.7 μl of deoxynucleotide triphosphate (dNTP; 10 mM), 1.4 ul 10 pM each primer, and 3 μl template DNA. PCR amplifications were performed in a Boeco Ecogermany M-240R thermal cycler using the following optimal cycling conditions: 95 °C (5 min), followed by 40 cycles at 95 °C (45 s), 50 °C (45 s) and 72 °C (1 min), and a final extension step at 72 °C (10 min). PCR products were sent to Macrogen (Seoul, Korea; http://www.macrogen.com) for purification and sequencing of both forward and reverse DNA strands.

Sequences were edited and contigs were assembled using ProSeq 2.9 beta41. All haplotype sequences obtained during this study were deposited in GenBank under the following accession numbers MW596479-MW596486.

Phylogenetic analyses

Four other species of Carcinonemertes were used as ingroup terminals for molecular comparisons with our new species, and two species of Nipponnemertes were included in the analysis as outgroup terminals. All sequences were first aligned using the software Clustal W42. The aligned dataset was analyzed with the software JModelTest243 which compares different models of DNA substitution in a hierarchical hypothesis-testing framework to select a base substitution model that best fits the data for each gene. The best model found by JModelTest2, selected with the corrected Akaike information criterion44, was GTR + I. The model parameters were as follows: assumed nucleotide frequencies A = 0.1988, C = 0.1266, G = 0.2261, and T = 0.4485; substitution rate matrix with A-C substitution = 0.0025, A-G = 11.4049, A-T = 2.8337, C-G = 0.1128, C-T = 7.4120, G-T = 1.000, and p-inv distribution with shape parameter 0.6420. Next, the best model was implemented in MrBayes 3.2.7a45 for Bayesian Inference analysis (BI) and in IQ-TREE46 for Maximum Likelihood analysis (ML). All phylogenetic analyses were conducted in the CIPRES Science Gateway V. 3.3 platform (http://www.phylo.org/)47.

For the BI analysis, unique random starting trees were used in the Metropolis-coupled MCMC45. The analysis was performed for a total of 5,000,000 generations. Visual inspection of log-likelihood scores against generation time indicated that the log-likelihood values reached a stable equilibrium before the 100,000th generations. Thus, a burn-in of 1000 samples was conducted; every 100th tree was sampled from the MCMC analysis, obtaining a total of 100,000 trees, and a consensus tree with the 50% majority rule was calculated for the last 59,000 sampled trees. Support for nodes in the BI tree topology was obtained by posterior probability. For the ML analysis, we used the default options in IQ-TREE run through the Cypress Science Gateway47. The robustness of the ML tree topology was assessed by bootstrap iterations of the observed data 1000 times. Phylogenetic trees were visualized and edited in Figtree 1.4.4.

Pairwise genetic distances (intra- and inter-specific) between the sequences of cox1 gene were calculated in MEGA 648 using the Kimura 2-Parameter model49.