was gordon robilliard right? integrative systematics valid ... · draft 4 sea slug species are...

Draft

Was Gordon Robilliard right? Integrative systematics

suggests that Dendronotus diversicolor Robilliard, 1970 is a

valid species

Journal: Canadian Journal of Zoology

Manuscript ID cjz-2016-0096.R1

Manuscript Type: Note

Date Submitted by the Author: 03-Aug-2016

Complete List of Authors: Ekimova, Irina; Lomonosov Moscow State University Valdés, Ángel; California State Polytechnic University Pomona Schepetov, Dimitry; Koltzov Institute of Developmental Biology RAS Chichvarkhin, Anton; A.V. Zhirmunsky Institute of Marine Biology, Russian Academy of Sciences, Palchevskogo 17, 690041 Vladivostok, Russia

Keyword: Dendronotus albus, Dendronotus diversicolor, Integrative taxonomy, Nudibranchia, Dendronotina, species delimitation, molecular phylogeny

https://mc06.manuscriptcentral.com/cjz-pubs

Canadian Journal of Zoology

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Was Gordon Robilliard right? Integrative systematics suggests that Dendronotus diversicolor

Robilliard, 1970 is a valid species

Ekimova I.1,2, Valdés Á.3, Schepetov D.4, Chichvarkhin A.2,5

1Biological Faculty, Lomonosov Moscow State University, Leninskiye Gory 1-12, 119234

Moscow, Russia. E-mail: [email protected]

2Far Eastern Federal University, 690950 Vladivostok, Russia

3Department of Biological Sciences, California State Polytechnic University, 3801 West Temple

Avenue, Pomona, California 91768, USA. E-mail: [email protected]

4Koltzov Institute of Developmental Biology RAS, Vavilov Str. 26, 119334 Moscow, Russia. E-

mail: [email protected]

5A.V. Zhirmunsky Institute of Marine Biology, Russian Academy of Sciences, Palchevskogo 17,

690041 Vladivostok, Russia. E-mail: [email protected]

Correspondence: Irina Ekimova; Biological Faculty, Lomonosov Moscow State University,

Leninskiye Gory 1-12, 119234 Moscow, Russia. Telephone and fax number: +74959393656; E-

mail: [email protected]

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Abstract

Nudibranch molluscs of the genus Dendronotus Alder and Hancock, 1845 are widely distributed in

the Northern Hemisphere. Taxonomic studies on the genus Dendronotus have been problematic due

to high variability in colour pattern in many species, as well as in external morphology and

anatomy. In the present paper, we studied specimens of Dendronotus from northern Pacific

presumably belonging to the species Dendronotus albus MacFarland, 1966. Molecular and

morphological data revealed the existence of two distinct species among the material examined: D.

albus, which has a wide range from Kamchatka and the Kurile Islands (from where we report this

species for the first time) to California in North America, and the pseudo-cryptic species

Dendronotus diversicolor Robilliard, 1970, which has been previously considered a junior synonym

of D. albus. D. diversicolor occurs from California to British Columbia in sympatry with D. albus.

D. albus and D. diversicolor can be clearly distinguished by colour pattern, internal and external

morphology and molecular sequence data. Despite some similarities in radular and external

morphology between D. albus and D. diversicolor, these two species are phylogenetically distant

and belong to different clades within the genus Dendronotus which suggests convergent evolution.

Key words: Dendronotus albus, Dendronotus diversicolor, Integrative taxonomy, Nudibranchia,

Dendronotina, DNA taxonomy, species delimitation, molecular phylogeny.

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Nudibranch molluscs of the genus Dendronotus Alder and Hancock, 1845 are widely distributed

in the Northern Hemisphere and can be commonly found in shallow-water fouling communities. At

present, this genus includes 20 extant valid species (Ekimova et al. 2015; Ekimova et al. 2016).

Traditionally, taxonomic studies on the genus Dendronotus have been problematic due to high

variability in colour pattern in many species, as well as in external morphology and anatomy. In the

mid-20th century Frank MacFarland and Gordon Robilliard published consecutive systematic

revisions of Dendronotus in the Northeastern Pacific (MacFarland 1966; Robilliard 1970). They

conducted detailed anatomical and morphological studies that led to description of four new

species, including Dendronotus albus MacFarland, 1966 and Dendronotus diversicolor Robilliard,

1970. These two species possess very similar colour patterns (white and yellow-white) and their

internal features (radular morphology), and are difficult to distinguish. However, Robilliard (1970)

pointed to some important characters that were consistently different between these two species: the

body size at maturity, the number of pairs of cerata, the body texture, the colour pattern, the number

and location of the hepatic diverticula, the denticulation of the radula, and the reproductive system

morphology. In addition, he observed lack of sexual activity between these two species and

considered it crucially important (Robilliard 1970). Nevertheless, during last two decades, the

validity of D. diversicolor as a distinct species has been questioned. Some authors, including D.

diversicolor discoverer Gordon Robilliard, reported on findings of intermediate forms between

these two species (Behrens 2006). In 2010 the first molecular analysis of the North Pacific species

of the genus Dendronotus was implemented by Stout et al. (2010). These results were compared

with morphological cladistics analysis. Both analyses showed lack of significant genetic or

morphological differences between D. diversicolor and D. albus, which led to the consideration of

D. diversicolor as a junior synonym of D. albus (Stout et al. 2010). Dendronotus albus is

distributed in the NE Pacific from Alaska to California (MacFarland 1966; Robilliard 1970; Stout et

al. 2010) and also was reported from Korea (Koh 2006). However, it has never been found along

the Pacific coast of Russia, although its finding was plausible since overlooked northeastern Pacific

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sea slug species are being recorded from Asian shore till recently (e.g. Chichvarkhin et al. 2016). In

the present paper, we studied specimens of the genus Dendronotus collected off Kamchatka

peninsula that are externally similar to D. albus, and compared them to the NE Pacific specimens

using morphological and molecular markers.

Samples were collected by SCUBA diving techniques; voucher specimens of Dendronotus spp.,

deposited at Natural History Museum of Los Angeles County (LACM), at the California Academy

of Sciences Department of Invertebrate Zoology and Geology in San Francisco (CASIZ) and at

California State Polytechnic University Invertebrate Collection (CPIC) were also used for this study

(Table S1). We studied eight specimens of the genus Dendronotus which were previously identified

as D. albus: three from Kamchatka (IE251-253), one from the Kurile Islands and four specimens

from the NE Pacific (LACM 174845, LACM 174846, LACM 2004-2.2 and CPIC 00948). Fifty-one

new sequences of different Dendronotus species were obtained to investigate the phylogenetic

relationships within the genus (see Table S1). We also used for the analysis 100 previously

published sequences, which have been retrieved from GenBank. DNA extraction, amplification and

sequencing techniques of molecular markers COI, 16S, H3, and 28S that correspond to partial

cytochrome c oxidase subunit I, 16S rRNA, Histone H3, and 28S rRNA genes, respectively,

followed the methods described in Ekimova et al. (2015). All new sequences were deposited in

GenBank.

Sequence assembling, editing and aligning followed methods described in Ekimova et al. (2015).

Individual gene analyses, separate mitochondrial and nuclear genes analyses, and a concatenated

analysis (included all four markers) were performed in this study. The best-fitting nucleotide

evolution models were tested in MEGA6 (Tamura et al. 2013) toolkit using Bayesian information

criterion (BIC). The best-fitting model for COI and 16S partitions was GTR+G+I. H3 partition

scored the lowest BIC for K80+G model and 28S partition for HKY. Phylogeny reconstructions of

individual gene datasets were conducted by maximum likelihood method, implemented in MEGA6

with 2000 bootstrap pseudoreplications and in MrBayes 3.2 (Ronquist and Huelsenbeck 2003).

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Reconstructions based on combined datasets (mitochondrial, nuclear, and concatenated analyses)

were performed applying evolutionary models for partitions separately. The Bayesian estimation of

posterior probability was also performed in MrBayes 3.2. Markov chains were sampled at intervals

of 500 generations. The analysis was started with random starting trees and 107 generations.

Maximum likelihood-based phylogeny inference for all combined data sets was performed in

GARLI 2.0 (Zwickl 2006) with bootstrap in 1000 pseudoreplications. Bootstrap values were placed

on the best tree found with SumTrees 3.3.1 from DendroPy Phylogenetic Computing Library

Version 3.12.0 (Sukumaran and Mark 2010). Final phylogenetic tree images were rendered in

FigTree 1.4.0.

Molecular delimitation analysis was performed using the Automatic Barcode Gap Discovery

(ABGD) method (Puillandre et al. 2012), which is commonly used for species delimitation,

including the latest works on heterobranch sea slug taxa (Jörger and Schrödl 2013; Cámara et al.

2014; Carmona et al. 2014a, 2014b; Churchill et al. 2014; Ortigosa et al. 2014; Padula et al. 2014

and others). We analyzed COI alignment (excluding outgroups) using all proposed models: p-

distance (simple distance), Jukes-Cantor (JK69) and Kimura (K80)

(http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html). Pmax was increased to 0.20 and the

number of steps was increased to 15.

The external morphology was studied under a stereomicroscope and using photos of living

specimens. Buccal masses of the NE Pacific specimens were soaked in sodium hydroxide (10%)

leaving only the radula and jaws. For radula extraction of the NW Pacific specimens, we soaked

buccal mass in Proteinase K (Amresco) solution and then heated it at 65oC for 1 hour. The radular

morphology was studied under Scanning Electron Microscopes EVO-40 (Zeiss, Germany) and

JSM-6010 (Jeol, Japan).

Our final combined dataset (1780 bp) resulted from concatenated alignments of 638 bp for COI,

463 bp for 16S, 329 bp for H3 and 350 bp for 28S. Single-gene trees of COI and 16S (not shown)

and the concatenated tree (Fig. 1) showed the same topology. Single-gene trees of H3 and 28S were

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poorly resolved. This could be explained by the slower rates of evolution of these two nuclear genes

as suggested in previous studies of the genus (Stout et al. 2010; Ekimova et al. 2015) or to

incomplete lineage sorting. Nevertheless, all species-level clusters were recovered in all analyses

and were not affected by missing of some markers in the dataset (see Table S1 for missing data). In

all analyses Dendronotus albus, collected in the NW Pacific, formed a separate, high supported

clade (posterior probabilities (PP) = 1; maximum likelihood bootstrap values (ML) = 93) from the

NE Pacific populations of D. albus (PP = 1; ML = 87). The NE Pacific D. albus nests with D.

subramosus MacFarland, 1966 (PP = 1; ML = 74). This clade is a sister to most of boreal

Dendronotus species, including D. dalli Bergh, 1979, D. niveus Ekimova, Korshunova, Schepetov,

Neretina, Sanamyan et Martynov, 2015; D. lacteus (Thompson, 1840); D. rufus O’Donoghue, 1921,

D. kamchaticus Ekimova, Korshunova, Schepetov, Neretina, Sanamyan et Martynov, 2015 and D.

albopunctatus Robilliard, 1972 (PP = 1; ML = 65). NW Pacific specimens of D. albus appear in the

clade grouping all Dendronotus boreal shallow-water species (PP = 0.96), but placed basal to all of

them, excluding D. iris Cooper, 1863. Two specimens of D. albus (LACM 174845 and LACM

174846), for which only 1 or 3 markers were obtained (Table S1), also showed high similarity to

other NE Pacific specimens based on single-gene trees topologies.

Based on the ABGD delimitation analysis using COI, the NE Pacific and the NW Pacific

populations of D. albus are distinct species. Three specimens from the NW Pacific and two

specimens from the NE Pacific appear in two distinct groups, other Dendronotus species form 13

groups in accordance with their preliminary identification. The prior maximum distance ranged

between 0.001 and 0.035. Uncorrected and corrected p-distances between two populations ranged

were 10.2%-11.0% and 8.4%-9.3% respectively. Therefore, according to the molecular data, the NE

Pacific and the NW Pacific specimens identified as D. albus belong to the two distinct species.

Regarding the external morphology, specimens from the NW Pacific showed high similarity in

external features with the original description of Dendronotus albus by MacFarland (1966) and its

redescription by Robilliard (1970): size of adult animals was approximately 20 mm; 5-6 pairs of

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cerata (Fig. 2D, E), even in juveniles the number of cerata is 5 (Fig. 2F); digestive gland diverticula

in 4-5 pairs of cerata; cerata with internal yellow pigment near the base; tips with white pigment.

On the other hand, two specimens of D. albus from the NE Pacific available for morphological

study, had a different external morphology (Fig. 2A-C): size of mature specimens was

approximately 40-50 mm; 4 pairs of cerata (one specimen with a reduced 5th pair), digestive gland

only in 2 anterior pairs of cerata; yellow pigment on cerata only on the tips, pigment occurs in

epidermal cells. These external features correspond to original description of D. diversicolor, but

are clearly distinct from the original description of D. albus. For example, based on Robilliard

(1970) description, D. albus is characterized by having “5-8 pairs of tall, delicate, moderately

branched cerata; […] the hepatic diverticula in the first to fourth (1-6) pairs of cerata”; “the brown

hepatic diverticula become a very dark, rich brown about ¼ - ½ way up the branch. From about ½ -

1/3 of the way up, this brown merges with a very beautiful metallic orange or copper pigment, and

this in turn merges with the opaque, dead-white pigment near the tip”. In contrast, D. diversicolor

possesses “4, sometimes 5, pairs of tall, slender, sparsely branched cerata. […] If present, the fifth

pair is reduced to simple, short papillae”; digestive gland diverticula “extending into the first two

pairs of cerata”; colour pattern is represented by “an opaque, dead-white or striking, opaque orange

pigment, or both, which may be found on the distal third of main branches of the cerata” (Robilliard

1970).

All studied specimens have very similar radular morphology: large rachidian teeth with 10-17

denticles and reduced furrows, and lateral teeth with 4-10 denticles (Fig. 3). Radular formula: 34-38

x 7-9.1.7-9 in the NW Pacific specimens and 34 x 8.1.8 in the NE Pacific specimen (CPIC 00948).

Robilliard (1970) stated some minor differences between Dendronotus albus and D. diversicolor,

which is based on denticulation pattern of lateral teeth (e.g. number of denticles on different lateral

teeth). Probably for this reason, in the studies by Pola and Stout (2008) and Stout et al. (2010) one

specimen deposited at the LACM (LACM 174846) was identified as D. diversicolor while another

individual (LACM 174845) was identified as D. albus. However, it has been shown by Ekimova et

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al. (2015) that these differences are variable even in a single specimen on anterior and posterior

rows of radula and therefore cannot be used for species delimitation and identification. Moreover,

collector of specimen LACM 174845 pointed out that it possessed only 4 pairs of cerata and its

coloration was similar to that described by Robilliard (1970) for D. diversicolor (J. Goddard,

personal communication, 2016).

The morphology of the reproductive system in the NW Pacific specimens is quite similar to that

described by MacFarland (1966) and Robilliard (1970) (Fig. 4A): the ampulla is wide and short,

crescent-shaped; small prostate consists of 10 alveoli; distal part of vas deferens is narrow;

insemination duct is short and transparent, arising from the base of spherical seminal receptaculum.

Only one specimen from the NE Pacific was properly preserved for reproductive system study

(CPIC 00948). This specimen is a sub-adult with underdeveloped genitalia (Fig. 4B). However, it

possesses well-developed ampulla, which is folded against itself for most of its length; number of

prostatic alveoli is about 10 and the prostate itself is quite large; distal portion of the vas deferens is

short and wide; insemination duct is long and wide, well-developed. These features were described

by Robilliard (1970) for D. diversicolor as important for its separation from D. albus.

Unfortunately, we had no access to specimens of Dendronotus albus collected from the type

locality (Monterey Bay, CA), but the morphology of the studied specimens from the NW Pacific

strongly corresponds to the features mentioned the descriptions of D. albus by MacFarland (1966)

and Robilliard (1970) (Figs 2-4). For this reason, we identify the NW Pacific specimens as the

“true” D. albus. Consequently, the range of this species includes both Pacific boreal coasts, but

probably has a wider distribution. For instance, a specimen of D. albus reported from Korea (Koh

2006) possesses a quite similar external morphology according to the photo provided in this report.

Molecular data indicate that the NE Pacific specimens studied here belong to a separate species.

Due to significant differences in external and internal morphology with D. albus, and similarities to

the original description of D. diversicolor, we propose that the NE Pacific specimens here studied

belong to this species, and therefore its taxonomic status as a valid species should be restored. The

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misidentification of D. albus specimens (Voucher LACM 174845) in previous studies led to

erroneous conclusion about synonymy of D. albus and D. diversicolor.

The absence of Dendronotus albus in newer collections could be explained by its rarity, as it has

been already mentioned by MacFarland (1966): “this strikingly beautiful species is rather rare in the

Monterey region and has been taken during the summer months in the neighborhood of Point

Pinos”. However, specimens with similar external morphology have been reported in notes and

photographs in different SCUBA diver’s sites (e.g.

http://www.diver.net/bbs/posts003/89747.shtml). Probably specimens of D. albus are often

confused with D. diversicolor. For instance, specimens with similar external morphology to D.

albus were identified as D. diversicolor even before the synonymization of these two species

(Hildering, 2007). Due to the difficulties in their distinguishing, a more comprehensive study and a

wider collecting effort are necessary to determine the precise range and relative abundance of D.

albus and D. diversicolor.

According to our molecular phylogenetic reconstruction (Fig. 1), Dendronotus albus and D.

diversicolor are not closely related as it has been proposed based on morphology (Robilliard 1970;

Stout et al. 2010). D. diversicolor is a sister species to D. subramosus, which also has a similar

radular and reproductive system morphology. On the other hand, D. albus has a more basal position

with respect to all other shallow-water, northern hemisphere Dendronotus species, excluding D.

iris. Such distant phylogenetic relationships between D. albus and D. diversicolor suggest that their

external similarities are probably due to convergent evolution. Also, this result is important for

understanding of the evolution within this genus. Dendronotus kalikal Ekimova, Korshunova,

Schepetov, Neretina, Sanamyan, Martynov, 2015, D. venustus MacFarland, 1966 and D. frondosus

(Ascanius, 1774) also show basal placements in the tree, as well as D. albus. Dendronotus kalikal

possesses very similar radular morphology to D. albus and D. diversicolor (Ekimova et al. 2015),

while in D. frondosus and D. venustus the rachidian tooth of the radula possesses deep furrows and

large denticles (Ekimova et al. 2015). Such radular morphology has been observed in juvenile

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specimens of D. kalikal, D. albus and other boreal species e.g. Dendronotus niveus. It has been

proposed earlier (Ekimova and Malakhov 2016) that the D. frondosus linage probably has a

paedomorphic origin, based on the analysis of radula development through the late ontogenesis. The

basal placement of D. albus and D. kalikal, in correspondence with the D. diversicolor position,

supports this suggestion. However, new data on the radula development and larger taxon sampling

for molecular study are necessary for more precise conclusions on the phylogenetic relationships

within the genus Dendronotus.

Acknowledgments

We are deeply grateful to Tabitha Lindsay, Jeffrey Goddard and Alexander Semenov for

collecting and providing specimens and their photos for this study. Molecular analysis of specimens

from Russia was supported to DS by Russian Foundation for Basic Research (№16-34-00955),

molecular analysis of the NE Pacific specimens was supported to IE by Russian Science Foundation

Grant №14-50-00034, field explorations were supported to AC by Far East Program 15-I-6-014o.

SEM work was conducted at the California State Polytechnic University SEM laboratory supported

by the US National Science Foundation (NSF) grant DMR-1429674.

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Figure captions

Figure 1. Phylogenetic hypothesis based on combined molecular data (COI+16S+H3+28S)

represented by Bayesian inference. Numbers above branches indicate posterior probabilities from

BI, numbers below branches – bootstrap values for Maximum Likelihood. Branches colour

represents by posterior probabilities from BI.

Figure 2. Dendronotus diversicolor Robilliard, 1970 and Dendronotus albus MacFarland, 1966.

Living animals. A–C. D. diversicolor, California, photos by J. Goddard. D. D. albus, IE251, photo

by A. Chichvarkhin. E. D. albus, IE252, photo by A. Chichvarkhin. F. D. albus, Kurile islands,

photo by A. Semenov.


Scanning electron micrographs of radula. A. D. diversicolor, CPIC 00948, posterior radular portion,

rachidian and lateral teeth. B. D. diversicolor, CPIC 00948, rachidian and lateral teeth. C. D.

diversicolor, CPIC 00948, anterior radular portion, rachidian and lateral teeth. D. D. albus, IE251,

rachidian and lateral teeth. E. D. albus, IE251, rachidian teeth. F. D. albus, IE253, rachidian and

lateral teeth. G. D. albus, IE251, lateral teeth. Scale bars: A–C = 50 µ; D–F = 20 µ; G = 10 µ.


Reproductive system morphology. A. D. albus, mature specimen, IE251. B. D. diversicolor,

subadult specimen, CPIC 00948. Abbreviations: amp, ampulla; bc, bursa copulatrix; fgm, female

gland mass; hd, hermaphroditic duct; id, insemination duct; ov, oviduct; p, penis; pr, prostata; sr,

seminal receptaculum; va, vagina; vd, vas deferens, ve, vestibulum. Scale bars: 500 µ.

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Figure 1. Phylogenetic hypothesis based on combined molecular data (COI+16S+H3+28S) represented by Bayesian inference. Numbers above branches indicate posterior probabilities from BI, numbers below

branches – bootstrap values for Maximum Likelihood. Branches colour represents by posterior probabilities from BI.

160x177mm (300 x 300 DPI)

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Figure 2. Dendronotus diversicolor Robilliard, 1970 and Dendronotus albus MacFarland, 1966. Living animals. A–C. D. diversicolor, California, photos by J. Goddard. D. D. albus, IE251, photo by A.

Chichvarkhin. E. D. albus, IE252, photo by A. Chichvarkhin. F. D. albus, Kurile islands, photo by A.

Semenov.

175x221mm (300 x 300 DPI)

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Figure 3. Dendronotus diversicolor Robilliard, 1970 and Dendronotus albus MacFarland, 1966. Scanning electron micrographs of radula. A. D. diversicolor, CPIC 00948, posterior radular portion, rachidian and lateral teeth. B. D. diversicolor, CPIC 00948, rachidian and lateral teeth. C. D. diversicolor, CPIC 00948, anterior radular portion, rachidian and lateral teeth. D. D. albus, IE251, rachidian and lateral teeth. E. D. albus, IE251, rachidian teeth. F. D. albus, IE253, rachidian and lateral teeth. G. D. albus, IE251, lateral

teeth. Scale bars: A–C = 50 µ; D–F = 20 µ; G = 10 µ.

175x154mm (300 x 300 DPI)

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Caption : Figure 4. Dendronotus diversicolor Robilliard, 1970 and Dendronotus albus MacFarland, 1966. Reproductive system morphology. A. D. albus, mature specimen, IE251. B. D. diversicolor, subadult

specimen, CPIC 00948. Abbreviations: amp, ampulla; bc, bursa copulatrix; fgm, female gland mass; hd,

hermaphroditic duct; id, insemination duct; ov, oviduct; p, penis; pr, prostata; sr, seminal receptaculum; va, vagina; vd, vas deferens, ve, vestibulum. Scale bars: 500 µ.

206x260mm (300 x 300 DPI)

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was gordon robilliard right? integrative systematics valid ... · draft 4 sea slug species are...

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