Previous phylogenetic studies of the family Gorgonocephalidae (brittle stars and basket stars) have identified three subfamilies, Astrotominae, Astrothamninae, and Gorgonocephalinae. The genus Astroclon was tentatively assigned to the subfamily Astrothamninae in previous studies, but its morphology is enigmatic and molecular data of the genus was insufficient. Therefore, the systematic position of Astroclon required confirmation to reconstruct the accurate systematics of the Euryalida. In the present study, we sought to clarify the subfamilial classification in the family Gorgonocephalidae (Echinodermata: Ophiuroidea: Euryalida). We revisited molecular phylogenetics of the Gorgonocephalidae including Astroclon and the major taxa in the family. The molecular analysis supported monophyly of the two species of Astroclon and its clear separation from Astrothamninae. The two Astroclon species were also distinguished from the other two subfamilies, Astrotominae and Gorgonocephalinae. Astroclon and three other subfamilies were divided in the basal position of the Gorgonocephalidae, and were assigned to subfamilies. A new subfamily, Astrocloninae was monotypically established for Astroclon in addition to the previous three subfamilies. Morphology of the first ventral arm plates and tentacle pores also supported distinctiveness of the new subfamily.
Gorgonocephalidae is the largest family of the order Euryalida and currently includes 34 genera (Döderlein, 1927; Fell, 1960; Okanishi and Fujita, 2013; Okanishi, 2016). In a recent molecular study, the family was identified as monophyletic with three recognized subfamilies, Astrotominae, Gorgonocephalinae, and Astrothamninae (Okanishi and Fujita, 2013). The subfamilies were distinguished by the morphology of madreporites, teeth, and genital slits. The presence/absence of supplementary oral plates were also emphasized by Matsumoto (1917), however, the plates have only been observed for Astrochele pacifica (Mortensen, 1933), Gorgonocephalus arcticus, Astrochele sp., and Astrotoma sp. (Ezhova et al., 2016). Observation of supplementary oral plates requires destructive dissection of surficial skins and/or external ossicles in the oral area; thus, no other work has recorded the nature of this structure. As a result, Okanishi and Fujita (2013) did not take this character into account in their systematics.
The enigmatic and rare genus Astroclon was monotypically established for Astroclon propugnatoris Lyman, 1879 in Gorgonocephalidae by Lyman (1879) and later another species A. suensoni Mortensen, 1911 was described (Mortensen, 1911). The former species has been recorded from Indonesia, Philippines, and Japan in the literature (Lyman, 1879; Mortensen, 1933; Murakami, 1944; Guille, 1981; Irimura and Kubodera, 1998; Okanishi et al., 2011) and the latter has been recorded from Australia and Japan (Mortensen, 1911, 1933; Baker, 1980). In the current systematics, Astroclon is classified as Astrothamninae in having a madreporite on the disc periphery as well as slit-shaped genital slits, and by the absence of oral papillae (Okanishi and Fujita, 2013).
Nuclear 28S rRNA, 18S rRNA, and mitochondrial 16S rRNA genes of Astroclon propugnatoris were sequenced by Okanishi et al. (2011). The phylogenetic tree of 49 species of Euryalida, including 27 gorgonocephalid species, showed A. propugnatoris was clearly included in Gorgonocephalidae. However, its exact systematic position was unclear (Okanishi et al., 2011). Okanishi and Fujita (2013) analyzed the phylogeny of 83 species of Euryalida including 38 gorgonocephalid species, mainly using mitochondrial COI, but A. propugnatoris was not included in this analysis (Okanishi and Fujita, 2013) and no material for sequencing of A. suensoni has been obtained.
In this study, to confirm the systematic position of the genus Astroclon within the Gorgonocephalidae, we obtained nuclear 18S rRNA, and mitochondrial COI and 16S sequences of a fresh specimen of A. suensoni collected from central Japan for the phylogenetic analysis. We also review important morphological characters including supplementary oral plates of many gorgonocephalid species to compare them with those of other Astroclon species.
MATERIALS AND METHODS
Sample collections for molecular analysis
One specimen of Astroclon suensoni was collected by a fishing line, off Kashino-zaki, Kushimoto, Wakayama, central Japan, 100–140 m depth, 9 January 2014 (NSMT E-10717), and an arm tip specimen of Astroclon propugnatoris was collected by ROV Hakuyo 2000 of M/S Shinsei-maru (Fukada Salvage and Marine Works Co. Ltd.), off Tarama-jima Island, Okinawa, southwestern Japan, 24°29.72′N, 124°33.78′E. 284–290 m depth, 8 March, 2005 (NSMT E-6274). These two specimens were directly immersed in 99% ethanol for molecular analysis.
DNA extraction, PCR amplification, and DNA sequencing
We sequenced nuclear 18S rRNA and mitochondrial COI and 16S rRNA genes for Astroclon suensoni and mitochondrial COI for Astroclon propugnatoris. The method of DNA extraction and PCR parameters followed that by Okanishi and Fujita (2013). Total DNA solution of Astroclon was diluted by distilled water 50 times for PCR of 18S rRNA, 100 times for COI and 16S rRNA. Primer sets of SSU001 (5′-GCTTGTCTTAAAGACTAAGCCATGC-3′) and SSU002 (5′-CCGTGTTGAGTCAAATTAAGCCGC-3′) (Okanishi et al., 2011) was used for PCR of 18S rRNA, COI005 (5′-TTAGGTTAAHWAAACCAVYTKCCTTCAAAG-3′) and COI008 (5′-CCDTANGMDATCATDGCRTACATCATTCC-3′) (Okanishi and Fujita, 2013) was used for PCR of COI, and 16Sar (5′-CGCCTGTTTATCAAAAACAT-3′) and16Sbr (5′-CCGGTCTGAACTCAGATCACGT-3′) (Palumbi, 1996) was used for PCR of 16S rRNA. The PCR products were separated from excess primers and oligonucleotides using Exo-SAP-IT (GE Healthcare), following the manufacturer's protocol. All samples were sequenced bidirectionally and sequence products were run on a 3730xI DNA Analyzer (Thermo Fisher Scientific). The accession numbers of DNA Data Bank of Japan (DDBJ) of the Astroclon are LC272069 (18S rRNA), LC272067 (COI), and LC272070 (16S rRNA) for A. suensoni, and LC272068 (COI) for A. propugnatoris.
Sampling locality and voucher depository of the examined specimens. Asterisks indicate type species.
We analyzed sequences of both species of Astroclon, with the sequence data of other 38 gorgonocephalid species obtained by Okanishi and Fujita (2013). For outgroups, we selected five euryalid species with the shortest genetic distance from Gorgonocephalidae, two from Asteronychidae and three from Euryalidae, to avoid long branch attraction (Bergsten, 2005; Okanishi and Fujita, 2013). Additionally, we selected Ophiopholis aculeata (Ophintegrida; Ophiactidae) as a representative of Ophintegrida. This species is also used in previous molecular phylogeny (Okanishi and Fujita, 2013) with sequences of 18S rRNA, COI, and 16S rRNA genes.
All sequences were aligned using the Clustal W algorithm in MEGA5 (Thompson et al., 1994; Tamura et al., 2011). Gene regions where the alignment was ambiguous, including ribosomal loops, were excluded by eye, and all missing sequences were scored as gaps. Overall average of substitution rate was computed using MEGA5 according to the Kimura 2-parameter model (Kimura, 1980) to compare the evolutionary rate of each gene. The Kimura 2 parameter model (Kimura, 1980) with a gamma distribution was selected as best-fit model of 18S rRNA gene, and general time reversible model (GTR; Yang, 1994a) assumed to be invariable (I; Hasegawa et al., 1985) with a gamma distribution approximated by discrete categories (γ Yang, 1994b) being selected as the best-fit model of nucleotide substitution with COI and 16S rRNA genes, by using the “Find best fit models” option of MEGA5. BioEdit ver. 184.108.40.206. (Hall, 1999) and Seaview ver. 4.3.0 (Gouy et al., 2010) were used in preparing the data matrices in FASTA and PHYLIP format, respectively. Seaview ver. 4.3.0 was also used in concatenating sequences of three gene sequences. Tree View for Win 16 (Page, 1996) was used in exploring tree files, in preparing NEWICK format and exploring alternative tree topologies. The phylogenetic tree was constructed with MrBayes ver. 3.1.2 (Ronquist and Huelsenbeck, 2003) to obtain Bayesian posterior probabilities (BPP) and RAxML ver. 8.1.20 (Stamatakis, 2014) for maximum likelihood analysis (ML) to obtain bootstrap support values. The three gene sequences were placed in separate partitions. We set parameters in MrBayes as follows; two substitution types (nst = 2, statefreqpr = fixed; K2P) for 18S rRNA and six substitution types (nst = 6; GTR) for COI and 16S rRNA were employed; rate variation across sites was modeled using a gamma distribution (rate = gamma) for 18S rRNA and using a gamma distribution with a proportion of sites being invariant (rate = invgamma) for COI and16S rRNA; the shape, proportion of invariable sites, state frequency, and substitution rate parameters were estimated for each partition separately. The Markov-Chain Monte-Carlo (MCMC) process was run with four chains for 4,000,000 generations, with trees being sampled every 100 generations. The first 10,000 trees were discarded as burn-in. Data sets were partitioned by gene region for the maximum likelihood analysis to allow for separate optimization per-site substitution rates. The best-supported likelihood tree was found by performing 1000 replications. Nodes in the phylogenetic trees were considered as supported if BPP values were larger than 0.98 and bootstrap value larger than 85%. BPP values lower than 0.97 and bootstrap lower than 74% for each node were considered as not significant (Fig. 1).
For morphological comparison between the two species of Astroclon (A. propugnatoris and A. suensoni) and other gorgonocephalids, 19 species in 16 genera covering the three subfamilies of Gorgonocephalidae were examined (Table 1). Specimens identifiable as the type genera of the three subfamilies, Astrothamnus, Astrotoma, and Gorgonocephalus, were included. From Astrothamninae and Astrotominae, we examined one or more species from each of the five genera (Astrothamnus, Astrothrombus, Astrocrius, Astrohamma, and Astrotoma). Since we did not obtain any dissectable specimens of Astrotoma drachi and A. agassizii (which were only used for obtaining molecular data), we examined a specimen of A. manilense as a representative of this genus; according to O'Hara and Harding (2014), A. drachi and A. manilense are potentially synonymous. In Gorgonocephalinae, some genera were recovered to be non-monophyletic (Okanishi and Fujita, 2013). Therefore, we selected species so that they represent all the gorgonocephaline subclades recognized by molecular analysis in this study (see Fig. 1, nodes S1–S7). We examined one species of Astrochele, one species of Astrodendrum, and two species of Gorgonocephalus from clade S1, and one species each from clades S2–S7 (Astrosierra from S2, Asterporpa from S3, Astrothorax from S4, Astrodendrum S5, Astroboa from S6, Astroglymma from S7). Because Asteroporpa muricatopatella and three species of Astrocladus formed a clade despite with the relatively lower support value of ML, we selected Astrocladus coniferus as a representative of this species group (see Fig. 1, node marked by a star).
In morphological observation, we focused on supplementary oral plates, adoral plates, and the first ventral arm plates, all recognized as diagnostic characters of subfamilies in Gorgonocephalidae since Matsumoto (1917). The presence/absence of supplementary oral plates have been observed only for Astrocrius sobrina (Astrotominae) (Matsumoto, 1917). Adoral plates and the first ventral arm plates have been observed for a limited number of species (e.g., Astrochele pacifica, Mortensen, 1933), but never for Astroclon. We also examined the following external characters used in generic diagnoses in Gorgonocephalidae: presence/absence of tubercles, spines, special calcareous plates, and concentric transverse ridges on disc; maximum number of arm spines; maximum numbers of secondary teeth on hooklets; and patterns of arm branching (see Matsumoto, 1917; Döderlein, 1927; Mortensen, 1933; Fell, 1960; Turner and Boucher, 2010).
To observe the supplementary oral plates, adoral plates, and the first ventral arm plates, skin and external ossicles were removed using domestic bleach (~5% sodium hypochlorite solution). The aboral plates and the first ventral arm plates were isolated, washed in deionized water, observed and photographed with Keyence VHX 600. The terms used in this study to describe brittle stars follow Matsumoto (1917), Martynov (2010), Stöhr et al. (2012), Okanishi and Fujita (2013), and Okanishi (2016). All the specimens with isolated ossicles are deposited in the National Museum of Nature and Science, Japan (NSMT).
Morphological characters of examined species. Abbreviations: AS, arm spine; SOP, supplementary oral plate; SCP, special calcareous plate; ST, secondary teeth of hooklet; VAP, ventral arm plate. Adoral plates are not included because no morphological differences were observed between species.
After removal of ambiguously aligned sites, we were left with 949 bp of 18S rRNA, 1511 bp of COI, and 429 bp of 16S rRNA. Overall averages of nucleotide substitution rates within nuclear 18S rRNA, mitochondrial COI and 16S rRNA genes were 0.019, 0.261, and 0.186, respectively.
The Bayesian tree of concatenated sequences of the three genes, 18S rRNA, COI, and 16S rRNA is shown in Fig 1. The ML tree also showed the same topology. Both Bayesian and ML analyses supported monophyly of Gorgonocephalidae (Fig. 1, node 1, bootstrap value 85%, BPP 1.00).
Two species of Astroclon formed a clade (Fig. 1, node 2, bootstrap 99%, BPP 1.00). The Gorgonocephalidae clade was divided into the following four clades: two species of Astroclon (Fig. 1, node 2, bootstrap 99%, BPP 1.00), Astrothamninae (Fig. 1, node 3, bootstrap 100%, BPP 1.00), Astrotominae (Fig. 1, node 4, bootstrap 99%, BPP 1.00), and Gorgonocephalinae (Fig. 1, node 5, bootstrap 88%, BPP 1.00). In Gorgonocephalinae, monophyly of seven subclades were well supported (Fig. 1, node S1–S7, bootstrap 85–100%, BPP 1.00), whereas support value of a subclade of three species of Astrocladus and A. muricatopatella was lower in our ML analysis (Fig. 1, node star).
Adoral plates were flat and plate-shaped for all examined species, with no distinct morphological differences being confirmed between Astroclon and the other gorgonocephalids. However, the first ventral arm plates were different in shape between Astroclon and the other gorgonocephalids. The oral surface of the first ventral arm plates of Astroclon propugnatoris (Fig. 3A) and A. suensoni were polygonal and flat, with the wedge-like projections protruding from the aboral surface (Fig. 3A). The first ventral arm plates of the other 19 gorgonocephalid species were plate-shaped, and both oral and aboral surfaces were polygonal and flat with no projections (Fig. 3B–D; Table 2).
We observed no distinctive features for Astroclon in the six external characters used as generic diagnoses in Gorgonocephalidae: presence/absence of tubercles, spines; presence/absence of special calcareous plates; presence/absence of concentric transverse ridges on disc; maximum number of arm spines; maximum number of secondary teeth on hooklets; and patterns of arm branching (Table 2).
In the molecular tree of Okanishi and Fujita (2013), three clades were recognized in Gorgonocephalidae and classified as three subfamilies, Astrothamninae (including Astroclon), Astrotominae, and Gorgonocephalinae. In the present study, the two species of Astroclon were distinctly separated from the other genera of Astrothamninae and formed a clade (Fig. 1). The Astroclon was also separated from Astrotominae and Gorgonocephalinae clades, and monophyly of the two subfamilies and Astrothamninae, except Astroclon, was well supported. The Astroclon and the three subfamily clades were diverged near the base of the family Gorgonocephalidae. Branch length between the Astroclon and Astrothamninae was longer than those between Astrothamninae and Astrotominae, and between Astrothamninae and Gorgonocephalinae. These molecular evidences suggest that Astroclon is well genetically separated from other three subfamilies and the systematic position of Astroclon should be elevated to the same rank as the three subfamilies.
By our morphological observations, previously used taxonomic characters by Matsumoto (1917) did not distinguish the three subfamilial clades and Astroclon recognized by our molecular analysis. Presence/absence of supplementary oral plate was considered to distinguish Matsumoto (1917)'s Gorgonocephalinae (=current Gorgonocephalinae) and Astrotominae (= current Astrotominae + Astrothamninae + Astroclon) in Gorgonocephalidae; however, they were absent only in Asteroporpa annulata and Schizostella sp. of Gorgonocephalinae. The six generic characters mentioned above also did not distinguish the subfamilial clades (Table 2). Therefore, traditionally known morphological characters have been confirmed unavailable for subfamilial taxonomy of Gorgonocephalidae.
Diagnostic characters distinguishing four subfamilies of Gorgonocephalidae in the proposed classification.
The Astroclon was originally classified in Astrothamninae which shared the following morphological characters: a madreporite located on periphery disc, slit-like genital slits, and absence of oral papillae (Okanishi and Fujita, 2013). Our morphological observations newly showed that Astroclon has a unique wedge-shaped projection on the first ventral arm plate (Fig. 3A) while the other examined genera have no such projections (Fig. 3B–D; Table 2), and has large pits adjacent to tentacle sheath (Fig. 4A) which are also lacking in the other examined genera (Fig. 4B–D). Although the shapes of the first ventral arm plates have not been described previously, our literature surveys for descriptions of tentacle pores of all gorgonocephalid species found that large pits were only possessed by Astroclon in Gorgonocephalidae (Linnaeus, 1758; Retzius, 1783; Lamarck, 1816; Leach, 1819; Risso, 1826; Müller and Troschel, 1842; Duchassaing, 1850; Lütken, 1856; Philippi, 1858; Lyman, 1861, 1869, 1874, 1875, 1879; Verrill, 1867, 1876, 1878, 1899; Bell, 1894; Döderlein, 1896, 1898, 1902, 1911, 1927, 1930; Koehler, 1897, 1898, 1904, 1905, 1910, 1912, 1922, 1923, 1930; Lütken and Mortensen, 1899; Benham, 1909; H. L. Clark, 1909, 1911, 1914, 1915, 1938; Mortensen, 1911, 1912, 1933, 1936; Matsumoto, 1912, 1915, 1918; A. H. Clark, 1916, 1919, 1948, 1952; Guille, 1979; Baker, 1980; McKnight, 2000; Okanishi and Fujita, 2011). Astroclon is clearly distinguished morphologically not only from the other genera of Astrothamninae, but also from Astrotominae and Gorgonocephalinae. Therefore, our comprehensive morphological study also supports treating the Astroclon and the three subfamilies as equally ranked.
Combination of the newly found two unique characters, presence/absence of wedge-shaped projections of the first ventral arm plate and pits besides tentacle pore, by our study (Figs. 3, 4), and previously used three subfamilial characters, shapes of oral papillae, position of madreporite, and shape of genital slits, found by Okanishi and Fujita (2013), clearly distinguishes the genus Astroclon from the three subfamilies in Gorgonocephalidae (Table 3). Therefore, these four clades in a multi-branching relationship (Fig. 1) can be classified as four subfamilies with distinct different morphological characters, and we propose a new subfamily, Astrocloninae, monotypic for the genus Astroclon. The systematic position and definition of the new subfamily are as follows:
Order Euryalida Lamarck, 1816
Family Gorgonocephalidae Ljungman, 1867
Subfamily Astrocloninae subfam. nov.
Diagnosis. Gorgonocephalids with a madreporite located on disc periphery; slit-shaped genital slits, absence of oral papillae; tentacle pores with large pits and wedgeshaped first ventral arm plates.
Type genus. Astroclon Lyman, 1879
Other included genera. none.
Distribution. Australia: off Broome (Baker, 1980). Indonesia: Arafura Sea (Lyman, 1879), off Manado (Mortensen, 1933). Philippines: off Lubang Islands (Guile, 1981). Japan: off Goto Island (Mortensen, 1911, 1933), East China Sea (Irimura and Kubodera, 1998), off Okinawa Island (Okanishi et al., 2011), off Amami and Kushimoto (this study). The bathymetric range is 158–457 m.
Remarks. Astrocloninae is clearly distinguished from the other three subfamilies of Gorgonocephalidae by five morphological characters (Table 3). The monotypic genus Astroclon includes A. propugnatoris Lyman, 1879 (type species) and A. suensoni Mortensen, 1911.
We wish to express our sincere gratitude to Jennifer M. Olbers of Ezemvelo KZN Wildlife for her critical reading of the manuscript and constructive comments. We thank to Shinsuke Ui of Kushimoto Marine Park Center, Wakayama, who collected and donated a fresh specimen of Astroclon suensoni and to Atsushi Kaneko, Senzo Uchida and Takuo Higashiji of Okinawa Churaumi Aquarium who collected and donated a fresh specimen of an arm tip of Astroclon propugnatoris. Thanks are also extended to Osamu Kitade of Ibaraki University for his assistance in examination and photography of the ossicle with the Keyence VHX 600. This work was supported by JSPS KAKENHI Grant Numbers 17K07549, 25440226, 22570104 and a grant from the Director-General of National Museum of Nature and Science, for the project, Imaging of threedimensional morphology of marine invertebrate specimens by nondestructive methods and its application to taxonomical researches. This is a contribution to the integrated research project, Geological, biological, and anthropological histories in relation to the Kuroshio Current, of the National Museum of Nature and Science.
MO performed molecular phylogenetic analysis, reconstruction of molecular tree, morphological observations of specimens. Manuscript including figures and Tables were prepared by MO and brush upped by MO and TF.