Canadian Journal of Zoology 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;Draft 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 Dendronotus albus, Dendronotus diversicolor, Integrative taxonomy, Keyword: Nudibranchia, Dendronotina, species delimitation, molecular phylogeny https://mc06.manuscriptcentral.com/cjz-pubs Page 1 of 17 Canadian Journal of Zoology 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] 1 https://mc06.manuscriptcentral.com/cjz-pubs Canadian Journal of Zoology Page 2 of 17 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 beDraft 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. 2 https://mc06.manuscriptcentral.com/cjz-pubs Page 3 of 17 Canadian Journal of Zoology 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-20 th 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 charactersDraft 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 3 https://mc06.manuscriptcentral.com/cjz-pubs Canadian Journal of Zoology Page 4 of 17 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 DendronotusDraft 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). 4 https://mc06.manuscriptcentral.com/cjz-pubs Page 5 of 17 Canadian Journal of Zoology 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 10 7 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 heterobranchDraft sea slug taxa (Jörger and Schr ödl 2013; Cámara et al. 2014; Carmona