Sea star Henricia spiculifera (Clark, 1901) in the northwestern Pacific: one species or three?

Three species of the sea stars are reported from the waters of the northwestern Pacific. These species were referred by earlier authors as Henricia spiculifera or H. leviuscula spiculifera. Two of them, H. lineata and H. uluudax, were recently described from the Aleutian Islands. These species are reported for the first time from the western Pacific (southeastern Kamchatka shore, Commander Islands, and the northern Kurile Islands). The third species, H. olga sp. n. is herein described from the northern Sea of Japan. It is very likely that similar sea stars recorded in Yellow Sea and the southern Kurile Islands belong to H. olga sp. n. These three species are a part of a phylogenetic clade within the subgenus Setihenricia, which also includes H. sanguinolenta, H. multispina, and several undescribed species occurring in the northeastern Pacific.

Here, I present three distinct species from Russian waters of the Pacific, that were formerly identified and referred to as H. spiculifera. Since H. spiculifera is recognized as an invalid name (Clark & Jewett, 2010), all reported species are new records for the waters of the northwestern Pacific.

MATERIAL AND METHODS
Observations and sample collections were taken by SCUBA-diving in 2014 through 2017 in Rudnaya Bay of the Sea of Japan and in the Gulf of Avacha (Avacha Bay and Starichkov Is.), Kamchatka (Fig. 1). Underwater images were taken with a Nikon D810 camera equipped with Nikkor 105/2.8G lens and appropriate Sea & Sea underwater housing. The other images were taken with a Nikon D810 or D7000 cameras and a Nikkor 60/2.8 lens. The specimens are in 96% ethanol and deposited in the Museum of National Scientific Center of Marine Biology, Russian Academy of Sciences, Vladivostok. Skeletal plates and spines were denuded using 5-15% sodium hypochlorite solution. Scanning electron images of the spines were obtained using Zeiss Sigma and Zeiss Evo electron microscopes after carbon coating. Other studied specimens are preserved in the collections of Zoological Institute of Russian Academy of Sciences, St. Petersburg, Russia (ZIN), and National Science Center of Marine Biology, Russian Academy of Sciences (MIMB), and Hokkaido University, Sapporo, Japan.
DNA was extracted using the Diatom TM DNA Prep 100 kit (Isogene Lab, Moscow, Russia) according to manufacturer's protocol. Partial sequence of mitochondrial 16S rRNA gene (16S) was used in this study. The author successfully used this marker earlier for delimitation of Henricia species (Chichvarkhin, Chichvarkhina & Wakita, in press). The primers used to amplify that fragment (Palumbi, 1996) were used here to amplify the region of interest. The master mix for each sample was prepared using 34.75 µL H 2 O, 5.00 µL PCR Buffer (Evrogen, Moscow), 5.00 µL 25 mM MgCl 2 , 1.00 µL 40 mM dNTPs, 1.00 µL 10 mM primer 1, 1.00 µL primer 2, 0.25 µL 5 mg/mL Taq, and 1.00 µL extracted DNA. Reaction conditions were an initial denaturation for 3 min at 95 • C, 39 cycles of (1) denaturation for 45 s at 94 • C, (2) annealing for 45 s at 50 • C, and (3) elongation for 2 min at 72 • C, and a final elongation for 10 min at 72 • C. PCR products yielding bands of ca. 600 b.p. were purified using ethanol precipitation. Sequencing was conducted by Sanger ddNTP termination method using BrightDye reagent (Nimagen) and ABI 3130 Genetic Analyser (Applied Biosystems) at Far Eastern Federal University, Vladivostok. The sequences were assembled and edited using BioEdit (Hall, 1999). BioEdit was also used to extract the consensus sequences. The sequences used in this study, including those mined from GenBank, are listed in the Table 1.
Two methods for species delimitation and identification were used: comparing tree topologies, and Automatic Barcode Gap Discovery (ABGD). The p-distances (i.e., the proportion of variable positions) and Neighbor-Joining (NJ) (Saitou & Nei, 1987) and Maximum Likelihood (ML) gene trees were calculated using MEGA 7 software (Kumar, Stecher & Tamura, 2016). Hasegawa-Kishino-Yano (HKY + + I) (Hasegawa, Kishino & Yano, 1985) evolutionary model was suggested by -lnL value found using Model Selection analysis implemented in MEGA. ABGD method (Puillandre et al., 2012) is based on pairwise distances, detecting the breaks in the distribution referred to as the ''barcode gap'' (Hebert et al., 2003) without any prior species hypothesis. The ABGD program is available at http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html. I analyzed 16S alignment using either uncorrected p-distance or Kimura-2-Parameter (K2P) (Kimura, 1980) and Jukes-Cantor (JC) (Jukes & Cantor, 1969) distances. X (relative gap width) was set to 1.4, the other settings remained as default. Single pure (SPu) character attributes, i.e., species-specific barcoding positions (Sarkar et al., 2002;Sarkar, Planet & DeSalle, 2008;Bergmann et al., 2009), were detected manually because of low number of variable sites in H . cf. spiculifera group alignment. The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:398553F6-F96E-4B82-A6C5-366D7200AF0D. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.

Molecular analysis
Partial 16S rRNA nucleotide sequences obtained in this study and mined from GenBank were 597-609 b.p. long producing a 614 b.p. alignment, including gaps. Whole analyzed dataset included 101 variable, 510 conserved, and 60 parsimony-informative positions. The sequences of H . cf. spiculifera (i.e., H. lineata, H. uluudax, and H. olga sp. n.) included 10 variable/parsimony-informative, and 510 conserved sites. There is one (site #202: G for H. lineata, T for H. uluudax, and A for H. olga sp. n.) species-specific SPu character attribute for each of these three species, which allows to distinguish these species. H. lineata possesses two SPu (sites #424-C, #453-A), which barcodes this species against the other analyzed Henricia species, while H. uluudax possesses one such SPu (site #450-A). H. olga sp. n. does not possess any SPu character attribute, which may barcode it against the other analyzed species.

Standardized descriptions of Henricia species
In this paper, I am using modified morphological description of Henricia uluudax by Clark & Jewett (2010) to elaborate a standard template for taxonomic descriptions of species the genus Henricia. For centuries, various authors have used different sets of characters and different terminology. In some cases, different terms were used for the same structure within one publication: e.g., Clark & Jewett (2010) used the terms 'spine', 'spinelet', 'thorn', and 'spinule' for a structure, which I call a 'spine' below, while a 'thorn' is a minor denticle on a spine. Such practice often obstructs side-by-side comparison of described entities belonging to this very diverse genus. To avoid this problem, I am proposing a standardized uniform description scheme and terminology based on a trade-off of the recent valuable publication by Clark & Jewett (2010) and keystone works by earlier authors. The main principle of this scheme is using strict consequence of described characters, verbatim overlaps in the descriptions of homologous characters, and using identical terms throughout all the descriptions in order to assist a reader to locate and compare the traits of interest. This template may and should be improved in the future by incorporating additional characters and terms.  Djakonov, 1961
Distribution. Present at Commander Islands, Russia and along the Kamchatka and Kurile Islands. Also found throughout the Aleutians from Fox Islands Avatanak Island to Near Islands, Attu Island, Chichagof Harbor (type locality) at depths of 6-25 m.
Remarks. The sea stars obtained in this study are very similar to original description by Clark & Jewett (2010), although they described higher maximal number of spines in abactinal and superomarginal pseudopaxillae. Also, all sea stars examined and encountered by me in the wild possess bright to pale orange coloration, whereas the stars from the Aleutians were described as bright red. Two lateral lines of coalescing plates on the rays are not discernible in Aleutian specimens.
Description. Relatively small, R to 4.8 cm, r to 1.0 cm, R:r 5.3-5.6; disc small, rays long, slender, tapering to blunt tips (Figs. 5E and 5F). Abactinal plates elongated, wide-set (Figs. 5A and 5B), with low tubercles, rare additional plates present connected with other plates; pseudopaxillae form a tight reticulation, some apical plates close-set or fused into linear series, lacking papular areas between, forming three well discernible lines or ridges at apex of ray; plates crowned with slender, divergent, thorn-tipped spines (Fig. 5G), 5-15 on rays and 25-35 spines on disk, about 300 µm in length; three slender thorns per spine; papular areas small, with 2-3 papulae; madreporite small, circular, irregularly radially spinose, located about 1/3 of the distance between the anus and the edge of the disc. Superomarginals as large as abactinal plates, bearing 15-20 spines, superomarginal row poorly discernible in non-denuded state; inferomarginals about 1/3 third larger than superomarginals, and bearing 30-35 thorny spines; first intermarginal series with 10-15 spines in pseudopaxillae about 1/2 as large as inferomarginals at base of rays, grading smaller aborally, extending about 40-50% of R, second series ending just past the base of the rays; ventrolateral series bearing 25-30 spines on plate extending 95% of R. Adambulacrals (Fig. 5D) each with a single deep furrow spine, and 13-16 actinal spines, arranged in two clearly distinct groups: 4-5 large, thick, blunt spines at edge of furrow, followed by a group of 9-13 three-fold shorter spines behind, arranged in three rows. Oral plates (Fig. 5D) with 3-4 thick, blunt marginal spines, and 2-3 suboral spines; in addition, there are two thick, blunt, triangular, teeth deep in the furrow near the distal edge of the plate. Color in life (Figs. 5E and 5F) striped: orange lines over pale brown background; juvenile paratype looks negative with dominating orange color. Preserved specimens possess almost indistinguishable the lined pattern.
Distribution: Found on the western coast of the Sea of Japan in Rudnaya Bay. Occurrence in the Yellow Sea, southern Kurile Islands and near western Sakhalin Island must be confirmed.
Ecology. The types were found on rocks at the depth of 15 m. In Sakhalin and the southern Kuriles found at 50-414 m depths.
Etymology. Female name Olga, common in Russia and northern Europe, which follows me throughout my life being shared by many of my good friends, colleagues, also by my wife, grandmother, and two aunts.

DISCUSSION
The three examined sea star species can be well delimited and identified using the 16S molecular marker. However, low number of variable sites in their alignment suggests a discovery of additional markers with more robust barcode. These species form a monophyletic clade with Henricia sanguinolenta within the subgenus Setihenricia.  Morphologically, these species are rather similar but possess several robust diagnostic traits listed in Table 2. Life coloration is probably the best character for their identification: vivid and striped in H. lineata, uniformly orange or red in H. uluudax, and pale striped in H. olga sp. n. Although H. lineata and H. olga sp. n. are similarly colored, the latter species possesses more slender rays, poorly discernible superomarginals, and southern distribution; H. lineata lacks additional abactinal plates. The adambulacral spines can be used to distinguish preserved H. olga sp. n. specimens by two sets of adambulacral spines: 3-5 near-furrow spines are stouter and 2-3-fold longer than the other spines, whereas in two other species, the size of adambulacral spines decreases almost gradually without a conspicuous ''step''. H. uluudax can be easily distinguished by more numerous spines in aboral pseudopaxillae and stout four-thorned abactinal spines. Similar spines are a character of H. densispina but this species does not possess the lines of nearly-coalescing aboral plates extending along the rays. A similar species H. multispina was reported from Aleutian Islands (Jewett et al., 2015): its life coloration is pale orange, pale lavender or pale pink, its adambulacrals bear 35-50 spines, many more than in the other sympatric Henricia species, and it also lacks the rows of coalescing abactinal plates. H. sanguinolenta (Müller, 1776) may bear rather discernible rows of almost fused plates but it can be distinguished by more than 4 thorns on stout abactinal spines and by its pink to violet life coloration. The specimens from Sakhalin Island and the southern Kuriles (Fig. 1, yellow pentagons) preserved in ZIN and identified earlier (mainly by AM Djakonov) as H. spiculifera and H. multispina, also the individual imaged by Xiao, Liao & Liu (2011) as H. leviuscula spiculifera are similar to H. olga sp. n. Although, the sea stars from Sakhalin and Kuriles possess stouter thick rays and discernible superomarginal series of the plates that are similar in size to the abactinals. Their life coloration is unknown. All of them share a near-furrow group of few very long spines and a group of 3-fold smaller ones as in H. olga sp. n. Therefore, a further