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.
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 , 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  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 , 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.
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’) , 16S rRNA (16S ar-L 5’-CGCCTGTTTATCAAAAACAT-3’, 16S br-H 5’-CCGGTCTGAACTCAGATCACGT-3’) , H3 (H3 AF 5’-ATGGCTCGTACCAAGCAGACGGC-3’, H3 AR 5’-ATATCCTTGGGCATGATGGTGAC-3’) , and 18S (18SA1 5’-CTGGTTGATCCTGCCACTCATATGC-3’, 18S700R 5’-CGCGGCTGCTGGCACCAGAC -3’) . 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.
Sequences were assembled, edited, and aligned using Geneious Pro 8.1 . The Akaike knowledge of facts criterion  was executed in jModelTest  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  through 10,000 bootstrap repetitions and the GAMMAGI form (no partitions). Bayesian analyses were discharge in BEAST 1.8.2 , 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 . 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 . Genetic composition of populations was analyzed in Arlequin  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).
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].
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.
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).
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.
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.
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.
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.
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.
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.
Speciation is not for aye accompanied by morphological change, resulting in the composition of cryptic species , 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 . 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 . 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  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  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  introduced the kind name Hermissenda for Aeolis opalescens Cooper, 1862 based without interrupti~ the original description by Cooper  for example well as additional specimens collected through Dall in 1865 in Sitka, Alaska. Bergh  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  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  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  studious additional specimens from San Pedro, describing the foreign and internal anatomy and providing drawings of the live animal. Cockerell & Elliot  agreed with Cockerell’s  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  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  renowned his specimens had a white longitudinal course on each ceras. On a sunder paper, O’Donoghue  rediscovered the commencement description of Cavolina crassicornis by Eschscholtz , and famous the similarities between his descriptions of H. opalescens and C. crassicornis. Thus, O’Donoghue  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 , 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  (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  as Aeolis opalescens and subsequently illustrated ~ dint of. Cockerell & Elliot  (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  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  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  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  and McDonald  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  while well as the present study representation species of Phidiana and Hermissenda in deviating clades. Thus, we maintain Hermissenda as distinct from Phidiana.
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.
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.
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.
1. Lederhendler II, Gart S, Alkon DL. Classical conditioning of Hermissenda: Origin of a unused response. J Neurosci. 1986; 6: 1325–1331. pmid:3711982
2. Etcheberrigaray R, Matzel LD, Lederhendler II, Alkon DL. Classical conditioning and protein kinase C activation dispose the same single potassium channel in Hermissenda crassicornis photoreceptors. Proc Natl Acad Sci USA. 1992; 89: 7184–7188. pmid:1496012 doi: 10.1073/pnas.89.15.7184
3. Blackwell KT. Subcellular, honey-combed, and circuit mechanisms underlying classical conditioning in Hermissenda crassicornis. New Anatomist. 2006; 289: 25–37. pmid:16437555 doi: 10.1002/ar.b.20090
4. Epstein DA, Epstein HT, Child FM, Kuzirian AM. Memory solidification in Hermissenda crassicornis. Biol Bull. 2000; 199: 182–183. pmid:11081725 doi: 10.2307/1542887
5. Crow TJ, Alkon DL. Retention of each associative behavioral change in Hermissenda. Science. 1978; 201: 1239–1241. pmid:694512 doi: 10.1126/system of knowledge.694512
6. Crow TJ, Alkon DL. Associative behavioral form in Hermissenda: Cellular correlates. Science. 1980; 209: 412–414. pmid:17747814 doi: 10.1126/body of knowledge.209.4454.412
7. Richards WG, Farley J. Motor correlates of phototaxis and associative acquirements in Hermissenda crassicornis. Brain Res Bull. 1987; 19: 175–189. pmid:3664278 doi: 10.1016/0361-9230(87)90083-9
8. Werness SA, Fay SD, Blackwell KT, Vogl TP, Alkon DL. Associative lore in a network model of Hermissenda crassicornis. Biol Cybernetics. 1992; 68: 125–133. doi: 10.1007/bf00201434
9. Hodge AJ, Adelman WJ. The neuroplasmic netting in Loligo and Hermissenda neurons. J Ultrastructure Res. 1980; 70: 220–241. doi: 10.1016/s0022-5320(80)80007-4
10. Crow T, Jin NG, Tian LM. Network interneurons underlying ciliary movement in Hermissenda. J Neurophysiol. 2013; 109: 640–648. doi: 10.1152/jn.00803.2012. pmid:23155173
11. Takeda T. Discrete possible waves in the photoreceptors of a gastropod mollusc, Hermissenda crassicornis. Vision Res. 1982; 22: 303–309. pmid:7101766 doi: 10.1016/0042-6989(82)90130-4
12. Yamoah EN, Kuzirian AM, Sanchez-Andres JV. Calcium current and inactivation in identified neurons in Hermissenda crassicornis. J Neurophys. 1994; 72: 196–2208.
13. Blackwell KT. Evidence toward a distinct light-induced calcium-at the disposal of potassium current in Hermissenda crassicornis. J Comp Neurosci. 2000; 9: 149–170.
14. Eakin RM, Westfall JA, Dennis MJ. Fine mode of building of the eye of a nudibranch mollusc, Hermissenda crassicornis. J Cell Sci. 1967; 2: 349–358. pmid:6051368
15. Buchanan J. Ultrastructure of the larval eyes of Hermissenda crassicornis (Mollusca: Nudibranchia). J Ultrastructure Mol Structure Res. 1986; 94: 52–62. doi: 10.1016/0889-1605(86)90051-0
16. Avila C, Arigue A, Tamse CT, Kuzirian AM. Hermissenda crassicornis larvae metamorphose in laboratory in replication to artificial and natural inducers. Biol Bull 1994; 187: 252–253. pmid:7811806
17. Avila C, Grenier S, Tamse CT, Kuzirian AM. Biological factors affecting larval growth in the nudibranch mollusc Hermissenda crassicornis (Eschscholtz, 1831). J Exper Mar Biol Ecol. 1997; 218: 243–262. doi: 10.1016/s0022-0981(97)00077-4
18. Zack S. A class and analysis of agonistic behavior patterns in every opisthobranch mollusc, Hermissenda crassicornis. Behaviour 1975; 53: 238–267. pmid:1237291 doi: 10.1163/156853975×00218
19. Avila C, Tyndale E, Kuzirian AM. Feeding air and growth of Hermissenda crassicornis (Mollusca: Nudibranchia) in the laboratory. Mar Freshwater Behav Physiol. 1998; 31: 1–19. doi: 10.1080/10236249809387059
20. Ram JL, Noirot G, Waddell S, Anderson MA. Singleness of action in the interactions of feeding by other behaviors in Hermissenda crassicornis. Behavior Neural Biol. 1988; 49: 97–111. doi: 10.1016/s0163-1047(88)91282-4
21. Tamse CT, Smith PJ, Aloulou A, Epstein HT, Kuzirian AM. Lead toxicity in Hermissenda crassicornis embryos and cultured neurons. Biol Bull 1994; 187: 251–252. pmid:7811805
22. Kasheverov IE, Shelukhina IV, Kudryavtsev DS, Makarieva TN, Spirova EN, Guzii AG, et al. 6-Bromohypaphorine from pelagic nudibranch mollusk Hermissenda crassicornis is ~y agonist of human α7 Nicotinic Acetylcholine receptor. Mar Drugs 2015; 13: 1255–1266. doi: 10.3390/md13031255. pmid:25775422
23. Behrens DW, Hermosillo A. Eastern Pacific Nudibranchs: A Guide to theOpisthobranchs from Alaska to Central America. Sea Challengers, Monterey, California; 2005.
24. O’Donoghue CH. Notes forward the taxonomy of nudibranchiate Mollusca from the Pacific seaboard of North America. I. On the identification of Cavolina (i.e. Hermissenda) crassicornis of Eschscholtz. Nautilus. 1922; 35: 74–77.
25. McDonald GR. A survey of the nudibranchs of the California seaside. Malacologia. 1983; 24: 114–276.
26. Pola M, Camacho-García YE, Gosliner TM. Molecular facts illuminate cryptic nudibranch species: The evolution of the Scyllaeidae (Nudibranchia: Dendronotina) with a revision of Notobryon. Zool J Linn Soc. 2012; 165: 311–336. doi: 10.1111/j.1096-3642.2012.00816.x
27. Carmona L, Lei BR, Pola M, Gosliner TM, Valdés A, Cervera JL. Untangling the Spurilla neapolitana (Delle Chiaje, 1841) description complex: A review of the universal Spurilla Bergh, 1864 (Mollusca: Nudibranchia: Aeolidiidae). Zool J Linn Soc. 2014; 170: 132–154. doi: 10.1111/zoj.12098
28. Churchill CK, Valdés A, Ó Foighil D. Molecular and morphological systematics of neustonic nudibranchs (Mollusca: Gastropoda: Glaucidae: Glaucus), by descriptions of three new cryptic kind. Invert Syst. 2014; 28: 174–195. doi: 10.1071/is13038
29. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers in quest of amplification of mitochondrial cytochrome c oxidase subunit I from variant metazoan invertebrates. Mol Mar Biol Biotech. 1994; 3: 294–299.
30. Palumbi SR. Nucleic acids II: The polymerase fetter reaction. In: Hillis DM, Moritz C, Mable BK, editors. Molecular Systematics. Sunderland, Massachusetts: Sinauer; 1996. pp. 205–247.
31. Colgan DJ, McLauchlan A, Wilson GD, Livingston SP, Edgecombe GD, Macaranas J, et al. Histone H3 and U2 snRNA DNA sequences and arthropod molecular evolution. Aust J Zool. 1998; 46: 419–437. doi: 10.1071/zo98048
32. Wollscheid E, Wägele H. Initial results up~ the molecular phylogeny of the Nudibranchia (Gastropoda, Opisthobranchia) based put ~ 18S rDNA data. Mol Phyl Evol. 1999; 13: 215–226. doi: 10.1006/mpev.1999.0664
33. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, et al. Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of arrangement data. Bioinformatics. 2012; 28: 1647–1649. doi: 10.1093/bioinformatics/bts199. pmid:22543367
34. Akaike H. A novel look at the statistical model identifications. IEEE Trans Automat Contr. 1974; 19: 716–723. doi: 10.1109/tac.1974.1100705
35. Posada D. jModelTest: Phylogenetic design averaging. Mol Biol Evol. 2008; 25: 1253–1256. doi: 10.1093/molbev/msn083. pmid:18397919
36. Stamatakis A. RAxML-VI-HPC: Maximum verisimilitude-based phylogenetic analyses with thousands of taxa and promiscuous models. Bioinformatics. 2006; 22: 2688–2690. pmid:16928733 doi: 10.1093/bioinformatics/btl446
37. Drummond AJ, Suchard MA, Xie D, Rambaut A. Bayesian phylogenetics through BEAUti and the BEAST 1.7. Mol Biol Evol. 2012; 29: 1969–1973. doi: 10.1093/molbev/mss075. pmid:22367748
38. Puillandre N, Lambert A, Brouillet S, Achaz G. ABGD, Automatic Barcode Gap Discovery during primary species delimitation. Mol Ecol. 2012; 21: 1864–1877. doi: 10.1111/j.1365-294X.2011.05239.x. pmid:21883587
39. Clement M, Posada DC, Crandall KA. TCS: A computer program to computation gene genealogies. Mol Ecol. 2000; 9: 1657–1659. pmid:11050560 doi: 10.1046/j.1365-294x.2000.01020.x
40. Excoffier L, Lischer HE. ARLEQUIN train ver 3.5: A new sequence of programs to perform population genetics analyses ~ the load of Linux and Windows. Mol Ecol Res. 2010; 10: 564–567. doi: 10.1111/j.1755-0998.2010.02847.x
41. Alfaro ME, Zoller S, Lutzoni F. Bayes or Bootstrap? A counterfeiting study comparing the performance of Bayesian Markov Chain Monte Carlo sampling and bootstrapping in assessing phylogenetic courage. Mol Biol Evol. 2003; 20: 255–266. pmid:12598693 doi: 10.1093/molbev/msg028
42. Hillis DM, Bull JJ. An empirical criterion of bootstrapping as a method notwithstanding assessing confidence in phylogenetic analysis. Syst Biol. 1993; 42: 182–192. doi: 10.1093/sysbio/42.2.182
43. Bickford D, Lohman DJ, Sodhi NS, Ng PK, Meier R, Winker K. Cryptic sort as a window on diversity and preservation. Trends Ecol Evol 2007; 22: 148–155. pmid:17129636 doi: 10.1016/j.tree.2006.11.004
44. Sáez AG, Probert I, Geisen M, Quinn P, Young JR, Medlin LK. Pseudo-cryptic speciation in coccolithophores. Proc Natl Acad Sci USA. 2003; 100: 7163–7168. pmid:12759476 doi: 10.1073/pnas.1132069100
45. Lipscomb D, Platnick N, Wheeler Q. The of the intellect content of taxonomy: a comment up~ DNA taxonomy. Trends Ecol Evol. 2003; 18: 65–66. doi: 10.1016/s0169-5347(02)00060-5
46. Scotland R, Hughes C, Bailey D, Wortley A. The Big Machine and the abundant-maligned taxonomist. Syst Biodiver. 2003; 1: 139–143. doi: 10.1017/s1477200003001178
47. Wheeler QD. Taxonomic triage and the poverty of phylogeny. Phil Trans R Soc B. 2004; 359: 571–583. pmid:15253345 doi: 10.1098/rstb.2003.1452
48. de Carvalho MR, Bockmann FA, Amorim DS, de Vivo M, de Toledo-Piza M, Menezes NA, et al. Revisiting the taxonomic obstacle. Science. 2005; 307: 353. doi: 10.1126/knowledge of principles.307.5708.353b
49. Padial JM, Miralles A, De la Riva I, Vences M. Review: The integrative coming events of taxonomy. Front Zool 2010; 7: 1–14. doi: 10.1186/1742-9994-7-16
50. Cooper JG. Some renovated genera and species of California Mollusca. Proc Cal Acad Nat Sci. 1862; 2:202–205, 207.
51. Cooper JG. On of the present day or rare Mollusca inhabiting the border of California–No. II. Proc Cal Acad Nat Sci. 1863; 3: 56–60.
52. Bergh R. Beiträge zur Kenntniss der Aeolidiaden. VI. Verh Zool Bot Ges Wien. 1878; 28: 553–584, pls. 6–8.
53. Bergh R. On the nudibranchiate grastropod Mollusca of the North Pacific Ocean, by special reference to those of Alaska. Part 1. Proc Acad Nat Sci Philadelphia. 1879; 2: 71–132, pls. 1–16.
54. Cockerell TDA. Notes of couple Californian nudibranchs. J Malacol. 1901; 8: 121–122.
55. Cockerell TDA, Elliot C. Notes attached a collection of Californian nudibranchs. J Malacol. 1905; 12: 31–53, pls. 7–8.
56. O’Donoghue CH. Nudibranchiate Mollusca from the Vancouver Island vicinity. Trans R Can Inst. 1921; 13: 147–209, pls. 7–11.
57. O’Donoghue CH. Notes up~ the nudibranchiate Mollusca from the Vancouver Island space. I. Colour variations. Trans R Can Inst. 1922; 14: 123–130, pl. 2.
58. O’Donoghue CH, O’Donoghue E. Notes forward the nudibranchiate Mollusca from the Vancouver Island tract. II. The spawn of certain sort. Trans R Can Inst. 1922; 14: 131–143, pl. 3.
59. Eschscholtz JF. Zoologischer Atlas—Beschreibungen neuer Thierarten, während des Flottcapitains von Kotzebue zweiter Reise um die Welt, auf der Russisch-Kaiserlichen Kriegsschlupp Predpriaetië in cavern Jahren 1823–1826. G. Reimer, Berlin. 1831; pt. 4: 1–19, pl. 16–20.
60. Carmona L, Pola M, Gosliner TM, Cervera JL. A that which is told that morphology fails to tell: A corpuscular phylogeny of Aeolidiidae (Aeolidida, Nudibranchia, Gastropoda). PLoS ONE. 2013; 8: 63000. doi: 10.1371/periodical.pone.0063000.
61. Baba K. Opisthobranchia of Japan (II). J Dep Agric, Kyūshū Imperial Univ. 1937; 5: 289–344, pls. 1–2.
62. Miller MC. Aeolid nudibranchs (Gastropoda: Opisthobranchia) of the household Glaucidae from New Zealand waters. Zool J Linn Soc. 1974; 54: 31–61. doi: 10.1111/j.1096-3642.1974.tb00792.x
63. Churchill CKC, Alejandrino A, Valdés A, Ó Foighil D. Parallel changes in genital morphology describe cryptic diversification of planktonic nudibranchs. Proc Roy Soc Lond B. 2013; 280: 20131224. doi: 10.1098/rspb.2013.1224
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