Two new species of Acanthocotyle Monticelli, 1888 (Monogenea: Acanthocotylidae), parasites of two deep-sea skates (Elasmobranchii: Rajiformes) in the South-East Pacific

Background Parasites of deep-sea fishes from the South-East Pacific (SPO) are poorly known. Of c.1030 species of fish found in this area, 100–150 inhabit the deep-sea (deeper than 200 m). Only six articles concerning metazoan parasites of fish from deep-waters of SOP are known, and nine monogenean species have been reported. Currently, ten species are known in Acanthocotyle Monticelli, 1888 (Monogenea) and when stated, all of them are found in shallow waters (10–100 m). Acanthocotyle gurgesiella Ñacari, Sepulveda, Escribano & Oliva, 2018 is the only known species parasitizing deep-sea skates (350–450 m) in the SPO. The aim of this study was the description of two new species of Acanthocotyle from two Rajiformes. Methods In September 2017, we examined specimens of two species of deep-sea skates (Rajiformes), Amblyraja frerichsi (Krefft) and Bathyraja peruana McEachran & Myyake, caught at c.1500 m depth off Tocopilla, northern Chile, as a by-catch of the Patagonian tooth fish Dissostichus eleginoides Smitt fishery. Specimens of Acanthocotyle were collected from the skin of the skates. Morphometric (including multivariate analysis of proportional measurements, standardized by total length), morphological and molecular analyses (LSU rRNA and cox1 genes) were performed in order to identify the collected specimens. Results The three approaches used in this study strongly suggest the presence of two new species in the genus Acanthocotyle: Acanthocotyle imo n. sp. and Acanthocotyle atacamensis n. sp. parasitizing the skin of the thickbody skate Amblyraja frerichsi and the Peruvian skate Bathyraja peruana, respectively. The main morphological differences from the closely related species Acanthocotyle verrilli Goto, 1899 include the number of radial rows of sclerites, the non-discrete vitelline follicles and the number of testes. Conclusions The two species of monogeneans described here are the only recorded parasites from their respective host species in the SPO. Assessing host specificity for members of Acanthocotyle requires clarifying the systematics of Rajiformes.

Background The deep-sea is one of the most fascinating ecosystems on earth [1], covering more than two-thirds of the world's surface with an average depth of 3800 m and a maximum depth of c.11,000 m in Mariana Trench [2], but knowledge of biodiversity in this environment is still scarce [3]. Knowledge of biodiversity in the Atacama Trench, closely associated to the high productive Humboldt Current Marine Ecosystem is limited; the assemblage of deepsea nematodes, the community of soft-shelled benthic foraminiferans and the presence of some amphipods have been described [4][5][6]. Surprisingly, the Atacama Trench is characterized by very high concentrations of nutritionally-rich organic matter up to depths of 7800 m, displaying characteristics typical of eutrophic systems [4]. The near-total lack of research on the parasites of deep-sea fish in the Atacama Trench represents an important gap in our knowledge of the biodiversity and structure of deep-sea communities in this trench [7] because hostparasite interactions may shape components of ecological communities [8]. Studies of the diversity of parasites of deep-sea fishes in the South-East Pacific (SPO), particularly for monogeneans are limited, and to date 11 monogenean species were recorded from deep-sea fishes in the SPO [7,9,10].
A detailed morphological and molecular study revealed that monogeneans obtained from the skin of two deepsea skates Amblyraja frerichsi (Krefft) and Bathyraja peruana McEachran & Miyake, from SPO represent new species. These are described and differentiated below.

Sample collection and processing
In September 2017, ten specimens of both species, the thickbody skate A. frerichsi and Peruvian skate B. peruana (Rajiformes) were obtained as by-catch from the local demersal long-line fishery on Patagonian tootfish Dissostichus eleginoides Smitt (Nototheniidae) in SPO (off Tocopilla, northern Chile; 22°16′S, 70°38′W-23°26′S, 70°43′W) caught at depths of c.1500 m. The fish were immediately frozen (at − 18 °C) on board and transported to the laboratory for parasitological analysis. The dorsal surface was washed in tap water, and the mucus was sieved and examined for monogeneans using a dissecting microscope with transmitted light. Some monogeneans were fixed in AFA (alcohol: formalin: acetic acid; 1:1:8) or 4% neutral buffered formaldehyde and then transferred and stored in 70% ethanol for further morphological analyses (light microscopy). Selected monogeneans from each of the two hosts were transferred to 96% ethanol for DNA analyses.
Population descriptors, prevalence and mean intensity [13] were recorded for both parasite species.

Morphological and statistical analyses
Fixed specimens were stained with Gomori's trichrome and cleared with clove oil (Sigma-Aldrich, Taufkirchen, Germany), then mounted in Eukitts ® (O. Kindler GmBH, Freiburg, Germany). The specimens were photographed (Leica M125 camera, Wetzlar, Germany) and measured using ImageJ [14]. Figures were made with a drawing tube. Measurements are in micrometers and are given as the range followed by the mean and the number of structures measured or counted in parentheses. The type-material was submitted to the National Museum of Natural History of the Smithsonian Institution, Washington, USA (NMNH-SI), Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Lima, Perú (MHN-UNMSM) and the Natural History Museum, London, UK (NHMUK).
To comply with the regulations set out in article 8.5 (amended version 2012) of the International Code of Zoological Nomenclatures (ICZN), details of the paper have been submitted to ZooBank. The LSID (Life Science Identifier) is urn:lsid:zoobank. org:pub:61DF198B-CF21-4B5E-B9BF-D7B80B187E49.
Principal components analysis (PCA) was performed for proportional morphometric measurements [15]. The ratios body width/total length (TL), body length (excluding posthaptor)/TL, pharynx length/TL, pharynx width/ TL, diameter of the pseudohaptor/TL, number of sclerite rows/TL, testes maximum width/TL, germarium length/TL and germarium width/TL were used instead of the original measurements because previous results indicated a correlation between the morphological variables and the total length [16]. Subsequently, the first five main components of the PCA, explaining 90.4% of the variance, were used in a multivariate discriminant analysis (MDA). Statistical analyses were performed with Statistica 10.0.

Molecular data and phylogenetic analyses
Parasites were preserved in 95% ethanol and placed individually into 1.5 ml Eppendorf tubes for DNA extraction. The DNA of each individual was isolated following a modified protocol [17], involving treatment with sodium dodecyl sulfate, digestion with Proteinase K, NaCl protein precipitation, and subsequent ethanol precipitation of the DNA.
Each PCR reaction had a final volume of 35 μl including: 5 standard units of GoTaq DNA polymerase (Promega, Madison, USA), 7 μl 5× PCR buffer, 5.6 μl MgCl 2 (25 mM), 2.1 μl BSA (10 mg/ml), 0.7 μl of deoxynucleotide triphosphate (dNTP) (10 mM), 10 pM of each primer and 7 μl template DNA. A Boeco Ecogermany M-240R Thermal Cycler (Boeckel, Hamburg, Germany) was used with a cycling profile as follows: 30 temperature cycles programmed on a slow temperature ramp rate. Cycle 1 was 95 °C for 3 min, 45 °C for 2 min and 72 °C for 90 s. This was followed by four cycles of 95 °C for 45 s, 50 °C for 45 s and 72 °C for 90 s, then a further 25 cycles of 95 °C for 20 s, 52 °C for 20 s and 72 °C for 90 s. The mix was held at 72 °C for 5 min to complete extension and then dropped to 4 °C. For cox1 PCR, there was an initial denaturation step at 95 °C (5 min) followed by 35 cycles of 95 °C for 1 min, 48 °C for 2 min and 72 °C for 21 min) with a final extension step at 72 °C for 10 min. PCR products were directly sequenced (Macrogen, Seoul, Korea; http://www.macro gen.com).
Sequences were edited and assembled using ProSeq v2.9 [20]. The fragments obtained from the LSU rRNA gene were aligned using the Clustal 2 [21] software package with sequences of related monogeneans retrieved from GenBank (Table 1). All new DNA sequences were deposited in the GenBank database, and the accession numbers are given in Table 1.
Phylogenetic reconstruction was performed using Bayesian inference (BI) and maximum-likelihood (ML) analyses. jModelTest 0.1.1 software [22] was employed to determine the best-fit nucleotide substitution model under the Akaike information criterion AIC [23]. For LSU rRNA and cox1 genes, the models GTR + G and GTR + I, respectively, were used as optimal models. For BI, unique random starting trees were used in the Metropolis-coupled Markov chain Monte Carlo (MCMC [24]. For both genes, independent MCMC chains were run with 50,000,000 of generations, sampling every 1000 generations, obtaining effective samples sizes (ESS) of parameter estimates over 200. Results were visualized in TRACER v. 1.7 [25]. ML analysis was performed using the MEGA v.6 considering gaps [26], missing data, pairwise deletions, codon positions, and 1st + 2nd + 3rd + noncoding positions. Nodal support was estimated by bootstrapping (n = 1000).
The sequences of the monogeneans Amphibdellatidae gen. sp. (GenBank: FJ971964) and Neocalceostoma sp. (GenBank: AF387510) were used as the outgroup for LSU rRNA phylogenetic tree [27,28]. No sequences for cox1 gene are available on GenBank for potential outgroups in the phylogenetic tree. Pairwise p-distances were also calculated with MEGA v6.

Differential diagnosis
Acanthocotyle imo n. sp. is morphologically similar to A. verrilli and A. gurgesiella, species with a pseudohaptor armed with 21-39 radial rows of sclerites, having a dextral opening of uterine atrium, non-discrete vitelline follicles, and more than 20 testes [11]. The number of radial rows of sclerites has been reported to range from 30 to 34 in A. verrilli [11] and between 36-40 (mode 40) [24] to c.57) [29,31] and A. gurgesiella (28-43) [10], but the mode (30) for the latter species is lower than for A. imo n. sp. The testes in A. verrilli are arranged in numerous rows (vs mainly in two rows in A. imo n. sp. and A. gurgesiella). The ratio total length/pseudohaptor is higher in the new species (4.61-5.86) compared with A. verrilli (2.48-2.86) [29,31], but similar to the ratio in A. gurgesiella (4.37-5.10) [10]. The presence of a smooth marginal valve of the pseudohaptor in A. imo n. sp. instead of a marginal valve with a distinct fringe in A. verrilli is an additional difference between the two species. Acanthocotyle imo n. sp. can be readily differentiated from A. gurgesiella by the lack of a spear-shaped spine in penis (present in the latter species) (see Fig. 2 and Additional file 2: Table S2).

Etymology:
The specific name of the new species refers to the Atacama trench where samples were obtained.
Notably, the three species of Acanthocotyle from the SPO harbored a single egg and not egg bundles as indicated for other species in the genus. Figure 5 presents a plot of the specimens in the twodimensional plane of the PCA. The first and second components of the PCA explained 64.86% of the total variance. The first component explaining 49.67% of the variance was associated with the proportional morphometric measurements of body width/TL, body length (excluding pseudohaptor)/TL, pharynx width/TL and germarium length/TL, whereas the second component explaining 15.19% of the variance was associated with pharynx length/TL, testes width/TL and germarium width/TL.

Morphometric analysis
The results of the multivariate discriminant analysis (MDA) ( Table 3) showed a correct assignment for the three species that, on average, reached 93.5% of the studied specimens of Acanthocotyle (Wilk's lambda = 0.78, F (10, 78) = 20.17, P < 0.001). The probability of correct assignment by chance alone was 36.2%.

Molecular and phylogenetic analyses
For the LSU rRNA region, seven sequences of 862 bp were obtained (4 from A. atacamensis n. sp. and 3 from A. imo n. sp.). Intraspecific genetic variability for both A. imo and A. atacamensis was 0%. Sequences were aligned and trimmed to 409 bp (the size of sequences available on GenBank) in order to compare the new species with   Table 4.
The trees in Fig. 6 show the phylogenetic relationships based on the LSU rRNA and cox1 genes for members of Acanthocotyle. LSU rRNA gene suggest that sequences from conspecific specimens of Acanthocotyle spp. clustered together in a single monophyletic clade, supported by high posterior probability (BI = 1) and high bootstrap support value from the ML analysis (ML = 100). LSU rRNA data did not support reciprocal monophyly of A. imo n. sp. but this was supported by the more variable mitochondrial gene, cox1. Unrooted cox1 tree in Fig. 6 shows the sequences of the two new species from South-East Pacific (A. atacamensis n. sp. and A. imo n. sp.) and A. gurgesiella forming three well supported clades.

Discussion
Traditional taxonomy based on morphology and morphometry and multivariate analyses based on morphometric data corrected for body length, strongly supports three species of Acanthocotyle detected in three different skates (all members of Rajiformes) from SPO (off Tocopilla, northern Chile). LSU rRNA data did not support reciprocal monophyly for A. imo n. sp. but this was supported by the cox1 gene data. Absence of reciprocal monophyly could be a consequence of a short length of the studied fragment and/or the gene may not be variable enough to reflect recent divergence. The lack of molecular data from almost all members of Acanthocotyle (except for A. urolophi and A. gurgesiella) precludes the molecular confirmation of the species described herein, but morphological characteristics of the new species are robust enough to confirm their distinct status. The two new species are easily differentiated from their congeners by a combination of characteristics that includes morphometric and morphological characters as indicated in the differential diagnosis for each species.
Including the two new species described above, Acanthocotyle, the unique genus in the Acanthocotylidae, now   [11] and Acanthocotyle sp. recorded from Raja clavata but also Narcine maculata (Torpediniformes); no differences between specimens obtained from the two hosts were indicated [12]. The presence of Acanthocotyle sp. in N. maculata could be the result of a transfer of the parasite between fishes during capture. A similar reason was suggested to explain the presence of A. williamsi (as Pseudacanthocotyla williamsi) in the teleost Sebastes alutus (Gilbert) (Scorpaeniformes) [32]. Surprisingly, this species was also recorded from the gills of another teleost fish, Reinhardtius hippoglossoides (Walbaum) (Pleuronectiformes) [33]. Unfortunately, we were unable to find additional records of A. williamsi in order to check the identity of the host. A different picture is evident for A. verrilli, also described originally for a "skate" but at least seven records, from four members of the Rajidae, are available. Species of Acanthocotyle seem to be specific to members of the Rajidae, but this assumption must be treated with caution. During 2016, three major contributions to the taxonomy of elasmobranchs were published [34][35][36]. Accordingly, hosts for Acanthocotyle are included not only in the Rajidae [32] but also in the Arhynchobatidae [34,35]. The specificity of Acanthocotyle, at least at the family level, requires clarifying the systematics of the Rajiformes. The integration of both, molecular and morphological tools and discriminant morphometric characters has strongly strengthened the traditional taxonomy, resolving the existence of cryptic species, identification of new species, and also clarification of species taxonomic status [16,[37][38][39]. Thus, our findings, based on molecular and morphometric multivariate analysis, are strongly consistent. The results of PCA and cox1 genes support the same conclusion: the species that are closer in the first plane of the PCA plot (A. imo n. sp. and A. atacamensis n. sp., see   Fig. 5), also appear closer in the phylogenetic tree based on cox1 but reciprocal monophyly for A. imo is not supported by LSU rRNA (Fig. 6). These results clearly suggest the key importance of integrating molecular and multivariate morphometric analyses for taxonomic studies.
The analysis of the geographical distribution of members of Acanthocotyle suggests a close association with the temperate region (Fig. 7), although this conclusion should be considered with caution. To date, all known species have been described from fishes of two families of the Rajiformes (Rajidae and Arhynchobatidae), except for A. urolophi and Acanthocotyle sp. (Myliobatiformes and Torpediniformes, respectively). Regarding hosts of the Rajiformes, a search in the ISI Web of Sciences  and Scopus (1990-2018), using as search criteria "Rajidae", "Rajiformes" and "parasites", yielded 46 references (excluding records from freshwater Rajiformes) that include parasitological records for just 61 host species. The known species count of Rajiformes is 287 [36], and the geographical range of distribution of members of this order includes from tropical to polar seas, and from shallow to deep-waters in the Atlantic, Indian and Pacific Ocean [32] and only 14 species of Rajiformes have been recorded as host for members of Acanthocotyle. As stated in a previous study [40], Rajidae have particularly been neglected in terms of limited sampling effort when studying cestodes. This limitation also applies to other parasites, such as the monogeneans, and therefore clarifying patterns of distribution will require a substantial increase in research effort, particularly for deep-sea hosts. The current distribution of members of Acanthocotyle (Fig. 7) can thus be explained by intensive sampling effort in some localities, specifically off the Pacific and Atlantic coasts of North America and the English Channel. It is early to consider host specificity of Acanthocotyle even more if c.104 species of Rajidae are considered as deep-sea skates [41], and only four species (including this record) have been studied as host for species in Anthocotyle.

Conclusions
Two new species of the genus Acanthocotyle are described from the skin of two deep-sea skates (Rajiformes) obtained at a depth of c.1500 m off Tocopilla (northern Chile). Both species represent the deepest record for members of Acanthocotyle. Conclusions about host specificity as well as geographical distribution of Acanthocotyle should be treated with caution due to the low proportion of Rajiformes studied for monogeneans. Acanthocotyle spp. have been recorded for 14 of the 287 species in Rajiformes. Future studies regarding parasites of Rajiformes are needed in order to evaluate the real level of host specificity and geographical distribution of members of Acanthocotyle.