The Model Organism Hermissenda crassicornis (Gastropoda: Heterobranchia) Is a Species Complex

Abstract

Hermissenda crassicornis is a mould organism used in various fields of study including neurology, ecology, pharmacology, and science of poisons. In order to investigate the systematics of this assemblage and the presence of cryptic shape in H. crassicornis, we conducted a wide-embracing molecular and morphological analysis of this assemblage covering its entire range across the North Pacific Ocean. We determined that H. crassicornis constitutes a fashion complex of three distinct species. The entitle Hermissensa crassicornis is retained for the northeast Pacific form, occurring from Alaska to Northern California. The celebrity H. opalescens is reinstated for a collection occurring from the Sea of Cortez to Northern California. Finally, the phrase H. emurai is maintained for the northwestern variety, found in Japan and in the Russian Far East. These three collection have consistent morphological and color shape differences that can be used during identification in the field.

Citation: Lindsay T, Valdés Á (2016) The Model Organism Hermissenda crassicornis (Gastropoda: Heterobranchia) Is a Species Complex. PLoS ONE 11(4): e0154265. doi:10.1371/periodical.pone.0154265

Editor: Manabu Sakakibara, Tokai University, JAPAN

Received: February 1, 2016; Accepted: April 11, 2016; Published: April 22, 2016

Copyright: © 2016 Lindsay, Valdés. This is ~y open access article distributed under the stipulations of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the source author and source are credited.

Data Availability: All sequences are useful from GenBank (http://www.ncbi.nlm.nih.gov/genbank/) addition numbers provided.

Funding: Funded by California State University Program conducive to Education and Research in Biotechnology (http://www.calstate.edu/csuperb/) to AV, Prete Foundation to TL, Conchologists of America (http://www.conchologistsofamerica.org/home/) to TL, and the California State Polytechnic University (http://www.cpp.edu/) to TL. The funders had ~t one role in study design, data accumulation and analysis, decision to publish, or state of being prepared of the manuscript.

Competing interests: The authors bear declared that no competing interests endure.

Introduction

The repeatability of experiments involving existing organisms heavily relies on the accurateness of species identifications. For instance, admitting that separate studies on the same protoplast organism use specimens that actually belong to contrary taxa, the results of those studies may not be comparable. Taxonomic accuracy is generally not one issue when dealing with laboratory strains or design species raised in captivity for generations of the like kind as Caenorhabditis elegans, Drosophila melanogaster, or Aplysia californica, goal it can be important when inquiry animals are collected in the ~ of battle.

Hermissenda crassicornis (Eschscholtz, 1831) is ~y important model organism in neuroscience, including studies in successi~ classical conditioning [1–3], memory solidification and associative learning [4–8], the texture of neural circuits [9–10] and neural physiology [11–13]. Additionally, H. crassicornis has been used to follow up ultrastructure and anatomy [14–15], larval and reproductive ecology [16–17], behavioral ecology [18–20] and pharmacology and toxicology [21–22], resulting in a pelf of papers and information widely cited in modern literature. Because H. crassicornis has ~y unusually broad geographic range, across the North Pacific Ocean [23], specimens collected for applied studies have diverse origins, typically from distinct locations between Southern California and Washington, still also from Russia. In many cases specimens were purchased from engaged in traffic suppliers and their exact origin is unknown or difficult to determine.

The taxonomy of H. crassicornis has not been reviewed on the side of decades. In 1922 O’Donoghue [24] concluded that Hermissenda opalescens (Cooper, 1863), originally described from San Diego, California was a junior synonym of H. crassicornis, originally described from Sitka, Alaska, and this estimate became universally accepted [23, 25]. More recently the Japanese species Cuthona emurai Baba, 1937 was synonymized through H. crassicornis [25], establishing the generally recognized transpacific range for this figure.

Recent integrative taxonomic studies have revealed that other widely distributed figure of nudibranchs resulted to be shape complexes composed of multiple species with much more restricted ranges [26–28]. In this paper we use similar methodologies to inquire into the genetic structure and morphological modification of H. crassicornis over its not toothed range in an attempt to settle the validity of previously described species. For this purpose we use a mixture of molecular phylogenetics (based on four genes), fashion delimitation analyses, population genetics, and morphological comparisons.

Materials and Methods

Source of Specimens

All Hermissenda crassicornis specimens were obtained through SCUBA, on floating docks or for the period of low tide by the authors or donated ~ the agency of colleagues. Specimens from California were collected subject to California Department of Fish and Game let SC-9153. Specimens from Japan were collected in subordination to the permits of the Mouran and Oshoro Marine Stations. Specimens obtained through the authors were photographed and preserved in 95% ethanol. Specimens were deposited in the Cal Poly Pomona Invertebrate Collection (CPIC) and the Natural History Museum of Los Angeles County (LACM). Sequences of Dondice occidentalis, Nayuca sebastiani, Godiva quadricolor, and Phyllodesmium jakobsenae were obtained from Genbank and included in the separation for comparison. Specimens of Phidiana lascrusensis were obtained from the Natural History Museum of Los Angeles County (LACM) and sequenced to have existence used as the outgroup.

Morphological Analyses

At smallest three specimens of each clade were dissected using a Leica EZ4D stereo microscope. The buccal mass was extracted from one side a ventral incision and placed into a 10% NaOH re~ for approximately 1 hour. The enclosing walls were then removed from the buccal mass and placed in DI furnish with ~ for 5–10 minutes to withdraw excess NaOH. The jaws were hereafter mounted, with masticatory boarder showing in c~tinuance an SEM stub. The remaining buccal mass was left in the 10% sodium hydroxide liquefaction for 2–3 days to to the full dissolve the tissue. The radula was in consequence carefully removed from the solution and placed into DI give ~ to for 5–10 minutes to put an end to excess NaOH. The radula was in that case mounted on an SEM stub. SEM images were taken with a Hitachi S-3000N variable pressure scanning electron microscope.

DNA Extraction, Amplification and Sequencing

A complete of 42 specimens were sequenced concerning this study (Table 1), collected from separate localities across the range of Hermissenda crassicornis. A connection of four gene fragments were sequenced towards this project: mitochondrial 16S and COI and nuclear H3 and 18S.

DNA determination was performed using DNeasy Blood and Tissue Kit (Qiagen) or gauge Chelex extraction. A small 1mm part of tissue was cut from the stand or mantle or used from web samples and macerated using a sterilized razor spark; gay. For Chelex extraction, the macerated woven stuff was transferred using sterilized forceps into a 1.7mL microcentrifuge tube containing 1mL of 1X TE Buffer and placed adhering a rotation block for at smallest 20 minutes to rehydrate the preserved combination and allow cells to begin disassociating. Samples were that time removed from the rotation block, vortexed beneficial to roughly 5 seconds, and centrifuged at 23,897.25 g since 3 minutes. Next, 975μL of 1X TE Buffer was sequestered from each sample being careful to not vex the pellet of tissue in both tube. 175μL of 10% Chelex dis~ was added to each sample and vortexed. The samples were afterward placed in a 56°C hot sprinkle and calender bath for 20 minutes. Samples were removed, vortexed for roughly 5 seconds and placed into a 100°C race block for exactly 8 minutes. Each illustration was vortexed for roughly 5 seconds and afterwards centrifuged at 23,897.25 g as far as concerns 3 minutes. The resultant supernatant was used as antidote to PCR. For Dneasy extraction, the manufacturer’s protocol toward tissue samples was followed. The end products were used in opposition to PCR amplification.

The polymerase chain reverse action (PCR) was used to amplify portions of the mitochondrial cytochrome c oxidase 1 (COI) and 16S ribosomal RNA (16S) genes, taken in the character of well as the nuclear histone 3 (H3) gene and the elementary 500bp of 18S ribosomal RNA (18S) gene. The following whole primers were used to amplify the regions of touch for all specimens: COI (LCOI490 5’-GGTCAACAAATCATAAAGATATTGG-3’, HCO2198 5’TAAACTTCAGGGTGACCAAAAAATCA-3’) [29], 16S rRNA (16S ar-L 5’-CGCCTGTTTATCAAAAACAT-3’, 16S br-H 5’-CCGGTCTGAACTCAGATCACGT-3’) [30], H3 (H3 AF 5’-ATGGCTCGTACCAAGCAGACGGC-3’, H3 AR 5’-ATATCCTTGGGCATGATGGTGAC-3’) [31], and 18S (18SA1 5’-CTGGTTGATCCTGCCACTCATATGC-3’, 18S700R 5’-CGCGGCTGCTGGCACCAGAC -3’) [32]. Amplification of DNA was confirmed using agarose gel electrophoresis through ethidium bromide to detect the air of DNA. PCR products were sent to Source Bioscience Inc. (Santa Fe Springs, CA, USA) with regard to sequencing.

Phylogenetic Analyses

Sequences were assembled, edited, and aligned using Geneious Pro 8.1 [33]. The Akaike knowledge of facts criterion [34] was executed in jModelTest [35] to make out the best-fit model of evolving for each gene (COI and 16S were portioned ~ the agency of codon position): GTR + I (H3 and COI 1st-2nd codon positions), GTR + G (H3 3rd codon positions), HKY + G (COI 3rd codon positions), GTR+I+G (18S, 16S), and GTR+I+G concerning the entire concatenated dataset. Phylogenetic analyses were conducted with Phidiana lascrucensis as the outgroup and using a limited contain of specimens of H. crassicornis on this account that which all four genes were useful. Maximum likelihood analyses were conducted on the side of the entire concatenated alignment with RaXML [36] through 10,000 bootstrap repetitions and the GAMMAGI form (no partitions). Bayesian analyses were discharge in BEAST 1.8.2 [37], partitioned by gene and codon position (unlinked), by two runs of six chains for 10 million iterations with a sampling interval of 1,000 iterations and consume -in of 10%.

Automatic Barcode Gap Discovery (ABGD) Analysis

ABGD separation was run on the ingroup sequences to prepare further corroboration for the delimitation of collection identified through the phylogenetic and morphological analyses. ABGD infers the contain of species present in a place upright of sequence data (and assigns individuals to the reported species) based on gaps in the grouping of pairwise distances between each arrangement in a dataset [38]. The algebra was run twice for each gene individually, once using Kimura 2-parameter (K2) and one time using Tamura-Nei (TN) distance matrices. The matrices were loaded into the online ABGD webtool (http://wwwabi.snv.jussieu.fr/the community/abgd/abgdweb.html). The default connection gap width (x) of 1.5 and a stroll of prior values for maximum forking of intraspecific diversity (P) from 0.001 to 0.1 were used.

Haplotype Network and Population Genetics Analyses

A haplotype reticulated was constructed for CO1 using TCS 1.21 [39]. Genetic composition of populations was analyzed in Arlequin [40] using separation of molecular variance (AMOVA) and to proof for genetic differentiation between populations (FST). Two groups (oriental Pacific and western Pacific) were steal away using 7 populations (see Table 2) and three groups (Sea of Japan, northeastern Pacific and southeastern Pacific) using the identical 7 populations. Significance of the AMOVA and ΦST analyses was assayed using 16,000 permutations. AMOVA is a hierarchical bring near analogous to ANOVA where the correlations amidst haplotypes at various hierarchical levels are used considered in the state of F-statistics analogs. AMOVA computes the adjustment of variation among groups (FCT), the proportion of variation among populations within groups (FSC) and the put in ~ of variation within populations (FST).

Results

Phylogenetic Analyses

Bayesian and greatest likelihood consensus trees (Fig 1) be in actual possession of similar topologies and recovered the sort clades. Bayesian pp values greater than or like to 0.95 and mlb values greater than or alike to 70 were considered significant [41–42]. Specimens antecedently identified as Hermissenda crassicornis are break into three main clades in the couple trees. One clade includes specimens with a restricted range from the Sea of Japan [pp = 0.99; mlb = 81]. A abet clade covers specimens with a disposition from Alaska through northern California [pp = 0.96; mlb = 80]. The third part clade includes specimens with a bend from northern California through the Sea of Cortez [pp = 0.95; mlb = 83].

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Fig 1. Bayesian unison tree of the concatenated analysis including hind probabilities (pp) and bootstrap values from the maximum likelihood (mlb) analysis.

Only values >0.5 (pp) or 50 (mlb) are by stipulation.

http://dx.doi.org/10.1371/periodical.pone.0154265.g001

Automatic Barcode Gap Discovery (ABGD) Analysis

Using the one and the other K2 and TN distance matrices, the CO1 following showed a major barcode gap betwixt a priori genetic distance thresholds of 0.01 and 0.02. Using a import of P between this range (.0129), three group were identified for CO1. Assignment of individuals in the compass of the three groups for CO1 matched the Bayesian and greatest likelihood phylogenies.

Haplotype Network and Population Genetics Analyses

The haplotype netting was unable to resolve all samples of CO1 of Hermissenda crassicornis specimens into a unmixed network, suggesting the presence of in addition than one species. The analysis resolved three defined haplotype networks (Fig 2). The pattern composition of the three networks coincides with the three clades recovered in the phylogenetic separation and the three species found in the fashion delimitation analysis. An AMOVA analysis was discharge to compare the genetic structure of western Pacific and eastern Pacific populations, and once more the groups resolved in the Bayesian analytics and ABGD analysis, using seven predetermined populations (Table 2). For the relative estimate of eastern Pacific and western Pacific populations, the majorship of genetic variation (60.54%) occurred within populations, whereas the variation among groups was 14.86% and the deviation among populations within groups was 24.60%. For simile of the three species identified in the Bayesian and maximum likelihood phylogenetic trees, the majority of genetic change (60.48%) occurred among groups, since the variation among populations within groups was 2.86% and the difference within populations was 36.65% (Tables 3 and 4).

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Fig 2. Haplotype netting showing three distinct groups.

Each ball represents a unique haplotype and the magnitude of each circle indicates how ~ people specimens share that haplotype, the larger the bounds the more specimens sharing an selfsame haplotype. Each line between haplotypes indicates a one only nucleotide polymorphism.

http://dx.doi.org/10.1371/daily register.pone.0154265.g002

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Table 3. Results of the couple AMOVA analyses conducted using seven already settled populations listed in Table 2; in the elementary analysis western Pacific populations and toward the east Pacific populations were grouped separately (East vs. West) to touchstone for genetic differentiation across the Pacific Ocean; in the abet analysis the populations were grouped according to the three clades (assemblage) recovered in the phylogenetic and ABGD analyses (three figure) to test for genetic differentiation mixed and between the three species in the present state recognized.

http://dx.doi.org/10.1371/diary.pone.0154265.t003

Morphological Analyses

External morphology was examined and compared betwixt specimens of the three groups (group ) recovered by phylogenetics, species delimitation, and haplotype network analyses (Figs 1 and 3). Consistent differences in external coloration and morphology were confirmed using images taken in the expanse of specimens and examining photographs from the Sea Slug Forum (www.seaslugforum.trap) and www.wallawalla.edu. Both, the variety found in the Sea of Japan and the group found in the Sea of Cortez through Oregon have cerata with light brown to untaught brown to bright orange background hue, which may or may not live in continence reddish to brown tipping with ~t one apparent white stripe extending along the prior surface of each ceras. In wholly three species the cerata are arranged into separate groups, but in the species from the Sea of Japan the gaps betwixt the groups of cerata tend to exist much longer than in the other sum of ~ units species, making the ceratal groups much more obvious in a dorsal scan; also the body of this description is much more elongate than the couple eastern Pacific species. The entire visible form of the Sea of Japan animals exhibits each orange hue, while the specimens from the orient Pacific show a more white or semi-transparent body. The longitudinal strip between the rhinophores appears ignorant orange to an almost reddish show ~, while it is light orange to effulgent orange on specimens from the eastern Pacific. The species ranging from Alaska to arctic California has light brown to want of knowledge brown to bright orange cerata by a distinct white stripe extending beside the anterior surface of each ceras. These illustrious ceratal white lines are the ~ly characteristic external trait of this figure and are never present in the description ranging from the Sea of Cortez from one side Oregon, making these two species easily distinguishable.

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Fig 3. Morphological differences in specimens of H. crassicornis to this place examined.

(A) Long Beach, California. (B) Bodega Bay, California. (C) Bodega Bay, California. (D) Sitka, Alaska. (E) Victoria, British Columbia. (F), Chiba, Japan. (G) Muroran, Japan.

http://dx.doi.org/10.1371/journal.pone.0154265.g003

Using SEM images, the radula rule was determined for each group (fashion) using at least two specimens to narration for variation. The radula formula during the Sea of Japan species is 25 × (0.1.0), the radula form for the northeastern Pacific species is 31 × (0.1.0), and the radula ~ry for the South Northeastern Pacific class is 28–30 × (0.1.0). The radular formula is not substantially different between the three assemblage, however, there are very slight morphological differences in the masticatory limit of each species. The South Northeastern class (Fig 4) has the largest purport of denticles, about eight, that come in sight as large, round projections, while the Sea of Japan sort (Fig 5) has fewer denticles, end for end six to seven, which are not for the re~on that large, and have a blunt end as opposed to a rounded extreme point. The North Northeastern Pacific species (Fig 6) has the in the smallest degree amount of denticles, about four to five, what one. are smaller and appear more for example slight bumps on the masticatory make a ~ for instead of strong denticles protruding from the boundary.

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Fig 4. SEM images of H. opalescens from south California.

(A) Radula, dorsal view with ventral denticles of the cusp apparent in some teeth. (B) Jaw (B). (C, E) Jaw masticatory skirt. (D) Lateral view of the radular teeth through ventral denticles of the cusp palpable.

http://dx.doi.org/10.1371/magazine.pone.0154265.g004

Discussion

Speciation is not for aye accompanied by morphological change, resulting in the composition of cryptic species [43], which are variety physically indistinguishable from each other. Morphological deviation associated with speciation can be such subtle that differences are difficult to quantify and set forth the character of. Species that can only be supreme a posteriori (after molecular data becomes suitable) are called pseudocryptic [44]. The subsisting of cryptic and pseudocryptic species constitutes a greater challenge to organismal biology research and underpins the concern of modern taxonomy and systematics. The taxonomic obstacle [45–48], or the lack of funding and practised taxonomists for numerous groups of organisms has harmful consequences for conservation, and hampers progress in other according to principles disciplines, such as ecology and evolutionary biology [45–48]. The arrival of molecular techniques and the integrative grain of modern taxonomy have helped to make plain this problem by providing more belonging to methodologies and faster procedures for shape delineation [49]. At the same time, greater nicety in species descriptions has revealed the existing of numerous cryptic and pseudocryptic taxa [43–44], what one. challenge studies that relied on pre-molecular taxonomic work. The present study is a sharp example of this problem. Molecular and morphological data supports the hypothesis that the present use of the binominal name Hermissenda crassicornis includes three separate species. Therefore, experiments based on H. crassicornis for example a model organism and published anterior to this study might need to exist re-evaluated in light of these results. Although the three kind are closely related, fundamental differences in their biology efficiency produce biases when comparing results from distinct studies. The results of this news~ raise questions on the repeatability of gone experiments based on H. crassicornis, supposing that not the identity of the specimens can be verified, and highlight the privation for careful taxonomic evaluation of design organisms collected in the wild. Because the three fashion in the H. crassicornis species tangled skein are pseudocryptic and rarely overlap in lie, it should be relatively straightforward to adjust the identity of specimens used in preceding studies, with the exception perhaps of specimens collected adjoining the San Francisco Bay Area, whither two of the species coexist.

Another implication of the results of this study is the strait to conduct a thorough review of the literary productions to determine whether there are beneficial names for the three species. This is conferred in the following paragraphs.

Aeolis opalescens was originally described ~ the agency of Cooper [50] based on specimens collected from San Diego Bay, California since “bluish white, pellucid, somewhat quadrangular, posteriorly wedge-shaped ending in a piercing point.” The foot had brace anterior, “short spreading appendages and dilute and flattened laterally.” The acme was short with two long, sudden tentacles (the lower pair replaced by the appendages of the foot), and “couple erect, club-shaped rhinophores of one opaline color, with an orange blow between them.” The “branchiae” [= cerata] were in “five pairs of fasciculi [= groups] by the upper edges of the back, one and the other bundle of about four rows, longest above their color yellowish, with a purple or relationship-red spot near the end.” There was a “rose-colored tint often visible from the chord of ova shining through the ventral walls.” Cooper [51] reported this variety again as Flabellina opalescens based without ceasing additional specimens collected in San Diego similar to well as new records from Santa Barbara Island, differing from the primeval description by having olive cerata by white tips. Bergh [52] introduced the kind name Hermissenda for Aeolis opalescens Cooper, 1862 based without interrupti~ the original description by Cooper [50] for example well as additional specimens collected through Dall in 1865 in Sitka, Alaska. Bergh [53] more remote expanded the description of Hermissenda and re-described H. opalescens providing because the first time anatomical details based in c~tinuance the Alaskan specimens. Cockerell [54] examined superadded specimens from San Pedro, California, and reported the species from La Jolla, California, describing the superficial coloration as well as some anatomical features. Cockerell [54] notable some color variation between specimens establish on kelp and those collected up~ the substrate, and indicated this kind has two opal blue lines practically fused in concert along the dorsum, but diverging at two or more points, leaving bright orange streaks in betwixt, as well as bright orange streaks steady the sides of the head; he described the vocal tentacles as opalescent blue. Cockerell & Elliot [55] studious additional specimens from San Pedro, describing the foreign and internal anatomy and providing drawings of the live animal. Cockerell & Elliot [55] agreed with Cockerell’s [54] assessment that his specimens from San Pedro belong to the same class as Cooper’s original animals from San Diego, mete considered that the specimens from Alaska are smaller and sundry in coloration, without providing further minutiae.

In a series of papers, O’Donoghue [56–57] and O’Donoghue & O’Donoghue [58] reported specimens of H. opalescens from the Vancouver Island space, Canada, which were described in rich detail, including the internal anatomy, pigment variation and egg mass. O’Donoghue [56] renowned his specimens had a white longitudinal course on each ceras. On a sunder paper, O’Donoghue [24] rediscovered the commencement description of Cavolina crassicornis by Eschscholtz [59], and famous the similarities between his descriptions of H. opalescens and C. crassicornis. Thus, O’Donoghue [24] transferred C. crassicornis to Hermissenda and regarded, as being the first time, H. opalescens for example a junior synonym of H. crassicornis. This esteem was universally accepted [60], and the memory H. crassicornis became well established in the northeastern Pacific learning [23–24]. The examination of the primary description of C. crassicornis by Eschscholtz [59] (Fig 7A) from Alaska and the descriptions ~ dint of. O’Donoghue [56–57] of specimens from Canada disclose that their characteristics match those of the specimens to this place examined from Alaska to northern California, including the vicinity of white longitudinal lines in the cerata. Therefore, we keep in possession the name Hermissenda crassicornis for this figure. On the contrary, the specimens from San Diego described by Cooper [50] as Aeolis opalescens and subsequently illustrated ~ dint of. Cockerell & Elliot [55] (Fig 7B) shortness white lines in the cerata and suit the characteristics of the specimens hither examined from the Sea of Cortez to north California. Thus, we re-introduced the name Hermissenda opalescens for this second species. Baba [61] described Cuthona (Hervia) emurai based without ceasing specimens collected in Niigata, Niigata Prefecture (Sea of Japan). The kind was described as follows: “The account-colour of the body is of a territory (fleshy) yellow. Along the mid-on the back region there run two bluish two-sided lines which pass forward and proceed right up the rhinophores and verbal tentacles; posteriorly they converge to the end of the tail. A broken middle-dorsal vermilion line runs about moiety way down from the head. The sides of the corpse are each marked with two lines running counterpart with each other, the upper bluish and the humble shorter and vermilion. The branchial papillae [= cerata] are chocolate-coloured through usually a white vein and a bright red marking immediately below the whitish donation, sometimes a white broken vein running up to the tip. The antero-lateral tentaculiform processes of the lower part are each marked with a bluish cover with ~s.” Years later, McDonald [25] proposed that Cuthona emurai was a pervert variation of the Hermissenda crassicornis (while burdened with Phidiana) and formally synonymized these sum of ~ units species. Based on Baba’s [61] type description and illustrations of the radula, the enclosing walls and the external morphology (Fig 7C), which closely match those of the specimens from the Sea of Japan in the present life examined, as well as the thing done that Cuthona emurai was described from Japan, we propose using the name Hermissenda emurai for the species in the present life recognized from in the Sea of Japan.

Miller [62] and McDonald [25] placed Hermissenda crassicornis in the genus Phidiana. However, this opinion was not accepted ~ means of other authors in subsequent publications. A new phylogenetic analysis of aeolid nudibranchs [63] while well as the present study representation species of Phidiana and Hermissenda in deviating clades. Thus, we maintain Hermissenda as distinct from Phidiana.

Conclusions

The design organism Hermissenda crassicornis is a tangled of three pseudocryptic species. Because the praise H. crassicornis was introduced for specimens collected in Alaska, this reputation is retained for the northeast Pacific species, which occurs in Alaska, the Pacific border of Canada, Washington, Oregon as well to the degree that Point Reyes, Northern California (based in c~tinuance the material here examined). The denomination H. opalescens, originally introduced from Southern California, is reinstated in opposition to the southwestern species, found from the Sea of Cortez, Mexico to Bodega Bay, Northern California. Finally, the title H. emurai, introduced for Japanese specimens, is maintained with respect to the northwestern species, found in Japan and in the Russian Far East. Close morphological investigation of the three species revealed suitable accordant morphological differences that can be used for identification in the field. This is especially important where H. crassicornis and H. opalescens overlap in roving, between Point Reyes and Bodega Bay. All specimens of H. crassicornis examined get white, longitudinal lines on their cerata, that are absent in H. opalescens. On the other workmanship, H. emurai is also distinguishable through having the cerata arranged in distinct groups and a more orange overall coloration.

Acknowledgments

Helena Fortunato, Luis Eduardo and Hiroshi Kajihara (Hokkaido University) assisted with fieldwork in Japan and Michelle Ridgway and Sherry Tamone (University of Alaska Southeast) assisted by fieldwork in Alaska. Yayoi Hirano supposing specimens from Chiba Prefecture, Japan. Additional specimens were obtained from the collections of the Natural History Museum of Los Angeles County with the assistance of Lindsey Groves. The SEM moil was conducted at the SEM laboratory of the Natural History Museum of Los Angeles County with the assistance of Giar-Ann Kung. Undergraduate researchers Eric Breslau, Matt McPhillips and Natalie Yedinak assisted by lab work.

Author Contributions

Conceived and designed the experiments: TL AV. Performed the experiments: TL. Analyzed the premises: TL AV. Contributed reagents/materials/decomposition tools: AV. Wrote the paper: TL AV.

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