Two new species of Neozoanthus (Cnidaria, Hexacorallia, Zoantharia) from the Pacific

Abstract The zoanthid genus Neozoanthus was originally described in 1972 from a single species in Madagascar. This monotypic genus was placed within its own family, Neozoanthidae, given its unusual characters of only partial sand encrustation, and an endodermal sphincter muscle combined with a brachycnemic mesenterial arrangement. Recently, undescribed specimens of Neozoanthus were discovered thousands of kilometers away in both Australia and Japan. While the phylogenetic and evolutionary aspects of Neozoanthus spp. are now somewhat well understood, the new specimens remained undescribed. Here we describe the specimens as two new species, Neozoanthus uchina sp. n. from the Middle Ryukyu Islands of southern Japan, and Neozoanthus caleyi sp. n. from the waters around Heron Island, on the Great Barrier Reef in Australia. Both species can be distinguished from each other and the type species, Neozoanthus tulearensis, by their distributions, oral disk colors, and average numbers of tentacles. Additionally, each species appears to have subtle differences in their cnidae. The division of Japanese and Australian specimens into two species is strongly supported by recently reported phylogenetic data. The discovery and description of these two species highlights how little is known of zoanthid species diversity in the Indo-Pacific.


introduction
Zoanthids are a hexacorallian order (Zoantharia =Zoanthidea) of benthic anthozoans with gross morphological characteristics partially reminiscent of both hard corals and sea anemones. Similar to many Scleractinia, most zoanthid species are colonial, with individual polyps connected by common tissue (=coenenchyme). However, like actiniarians, most zoanthids do not secrete hard skeletons. Instead most zoanthids incorporate sand and/or detritus into their body walls to help strengthen their structure. There are, however, exceptions to these general characters within Zoantharia, with some solitary (e.g. the genus Sphenopus), non-encrusting (Zoanthidae; Zoanthus, Acrozoanthus, Isaurus), and skeleton-secreting (Savalia) taxa.
While zoanthids can be found in a wide variety of marine environments from shallow waters to the deep sea, much of their diversity is found in subtropical and tropical coral reef ecosystems, particularly within the suborder Brachycnemina (Swain 2010). The suborder Brachycnemina includes both encrusting (Sphenopidae) and non-encrusting (Zoanthidae) families, grouped together by possession of a mesodermal sphincter, unlike species in suborder Macrocnemina, which generally possess an endodermal sphincter (but see Swain 2010). Additionally, most coral reef brachycnemic zoanthids are in symbiosis with endosymbiotic dinoflagellate zooxanthellae (=Symbiodinium spp.). The most common genera are Zoanthus (Zoanthidae) and Palythoa (Sphenopidae), which are a major component of coral reef fauna in both the Atlantic and Indo-Pacific. The other coral reef zoanthid genera in Brachycnemina have been little studied due in part to their rarity or cryptic nature, and include the genera Acrozoanthus (Ryland 1997;Reimer et al. 2011c) and Isaurus (Reimer et al. 2008) in the family Zoanthidae, and the genus Sphenopus (Soong et al. 1999;Reimer et al. 2012) in the Sphenopidae.
There is one additional family of brachycnemic zoanthids, the monotypic Neozoanthidae. Neozoanthidae was erected by Herberts in 1972 to contain the genus and species Neozoanthus tulearensis, described from unusual zoanthid specimens found in coral reefs of Madagascar. The specimens were notable for zoanthids in that they had an endodermal sphincter (Herberts 1972: 139, fig. 11) but brachycnemic mesentery arrangement, unlike all other Brachycnemina. Furthermore, specimens were only partially encrusted with sand by having no encrustations around the oral ends (=tops) of polyps (Herberts 1972: 139, fig. 10). This genus has remained monotypic, and until the recent rediscovery of undescribed Neozoanthus specimens from the Indo-Pacific (Reimer et al. 2011a), no additional specimens had been noted in the literature.
Neozoanthus represents a unique evolutionary step in the zoanthid phylogeny as the only partially encrusted group of zoanthids (Reimer et al. 2011a) (Figure 1) ( Table 1). Surprisingly, from mitochondrial 16S ribosomal DNA phylogenetic analyses, this group appears to be very closely related to the genus Isaurus, yet also has an indel unique to tropical macrocnemic Hydrozoanthidae (Reimer et al. 2011a), indicating Neozoanthus has a unique and perhaps complex evolutionary history.
In this study, utilizing both morphological and molecular techniques, we formally describe two new Neozoanthus species from subtropical regions of the Great Barrier showing encrustation in outer mesoglea and ectoderm, characteristic of Neozoanthus spp., with irregularly-sized encrustation A light microscope histological cross-section, and B scanning electron microscope image. Both images of specimen RMNH Coel 40098 (Table 2). Abbreviations: cm=complete mesenteries, e= sand/detritus encrustation (in B) or where encrustation existed before decalcification (in A), im=incomplete mesenteries, ec=ectoderm, en=endoderm, m=mesoglea, ss=encrusted sponge spicules. Scales: A=100 µm, B=200 µm.
Reef, Australia and the Ryukyu Archipelago, Japan by examining specimens recently reported in Reimer et al. (2011a). It is hoped these formal descriptions will provide a basis for future research into this enigmatic genus of zoanthids.

Sample collection
Specimens were collected as detailed in Reimer et al. (2011a) from locations in the Heron Island region of the Great Barrier Reef, Australia, and from the Ryukyu Islands in southern Japan (Table 2). In situ observations were also performed as detailed in Reimer et al. (2011a). Specimens were initially preserved in 70-99% ethanol.

Specimen examination/decalcification/histology
Specimens were examined, decalcified, and sectioned as detailed in Reimer et al. (2011a), with additional analyses as detailed below. As detailed in Reimer et al. (2011a), two polyps from two specimens each (Australia and Japan) were examined (total n=8).

Morphological analyses
External morphology of specimens was examined using both preserved specimens and in situ images. Polyp dimensions (oral disk diameter, polyp height) for both in situ and preserved specimens were obtained, as were the following data: tentacle number, color of polyp, color(s) of oral disk, relative amount of sand encrustation, associated/substrate species. Additionally, the relative development of the coenenchyme was examined.
For internal examinations, the following data were obtained: mesentery form (brachycnemic or macrocnemic arrangement), mesentery numbers, presence/absence of encrustations, location of encrustations, location and development of the sphincter muscle, presence/absence of gonads. Decalcification, histology and electron microscopy were performed as described in Reimer et al. (2011a).

Nematocyst observation
Undischarged nematocysts were measured from tentacles, column, actinopharynx, and mesenterial filaments of polyps (specimens examined n=2-4 colonies/species) for both new species. 400x images of the nematocysts were obtained by optical microscope, and measured using the software ImageJ (National Institutes of Health, USA). Nematocyst nomenclature generally followed England (1991), however both Schmidt (1974) and Hidaka and co-workers (1987;1992) have previously suggested basitrichs and mastigophores are same type of the nematocyst, and thus in this study, these two types were dealt with as the same type (basitrichs and b-mastigophores), unless they could be clearly distinguished from one another (basitrichs and p-mastigophores), in which case they were analyzed separately. Both holotypes and all paratypes of both newly described species were examined.

DNA extraction and PCR amplification/Phylogenetic analyses
Phylogenetic analyses are detailed and were performed on both species in Reimer et al. (2011a). No new genetic analyses were performed in this study. DNA from specimens were extracted, DNA target regions (cytochrome oxidase subunit I; mitochondrial 16S ribosomal DNA) amplified by PCR, and sequences analysed as detailed in Reimer et al. (2011a). DNA alignments from Reimer et al. (2011a) are available from the corresponding author.

MTQ
Museum of Tropical Queensland, Townsville, Australia. USNM Smithsonian National Museum of History, Washington D.C., USA NSMT National Museum of Nature and Science, Tokyo, Japan RMNH Naturalis Biodiversity Center, Leiden, the Netherlands MISE Molecular Invertebrate Systematics and Ecology Laboratory, University of the Ryukyus, Nishihara, Okinawa, Japan

systematics
Additional data related to both species, including tables, phylogenetic trees, and histological images, are reported in Reimer et al. (2011a). The specimens in Reimer et al. (2011a) were placed into the genus Neozoanthus Herberts, 1972 based on the summary of morphological characters (Table 1).

Material examined.
Type specimens. Holotype, specimen number MTQ G65793. Colony in two pieces, 5 polyps on a 2.5 × 2.0 cm stone and 4 polyps on a 2.0 × 1.0 cm stone (originally one colony). Polyps approximately 2.3-5.0 mm in diameter, and approximately 2.5-3.0 mm in height from stoloniferous coenenchyme. Polyps and coenenchyme encrusted with irregularly sized and colored sand grains. There was no noticeable variation between holotype and other specimens. Preserved in 99.5% ethanol.
Paratype (  Morphology: Neozoanthus caleyi sp. n. has 28 to 40 (average 33±3.9, n=18 polyps on 8 colonies) conical tentacles. Tentacles are usually shorter than the expanded oral disk diameter (e.g. 50-80% of oral disk width). Tentacles may be grayish-blue, yellow, or transparent, often with black, white, or fluorescent blue bands or patterning ( Figure  3). Well-developed, simple endodermal sphincter. No bractae are visible. All specimens are zooxanthellate. Polyps are externally heavily encrusted with sand and other particles of irregular sizes, excepting the oral end, which is free of encrustation and appears a bluish-gray similar to as seen in some Zoanthus species. When fully contracted, the sand-free oral end is often not visible, and polyps resemble small balls of sand. Polyps extend well clear of reduced or stoloniferous coenenchyme (Figure 3). Oral disks may be a variety of colors, including light gray-blue, white, or deep wine red. Occasionally, white, yellow, or light blue dots may be seen on the oral disk in regular circular patterns, and the oral opening (mouth) is often white in color. A "skirt" of different coloration (usually white or lighter coloration than remainder of oral disk) covering up to approximately 90 degrees of the oral disk is often seen in the area of the dorsal directive. Colonies consist of tens to <100 polyps, connected by stolons with no welldeveloped coenenchyme.
Etymology. Named for Dr. Julian Caley, the leader of the Australian Census of Coral Reef Ecosystems (CReefs) project. Dr. Caley's acceptance of the first author's participation in CReefs led to the discovery of this species. Noun in genitive. Habitat, ecology and distribution. Specimens from the Great Barrier Reef were found at depths from 4 to 29 m. Despite repeated surveys, no N. caleyi sp. n. have been found further north around Lizard Island despite zoanthid-focused surveys (Burnett et al. 1997;J.D. Reimer & T. Fujii, unpublished data), and it may be that this species is limited to a subtropical distribution in the Great Barrier Reef.
Neozoanthus caleyi sp. n., although not found at many locations surveyed, was locally common, particularly at locations that were characterized by strong currents and some sedimentation, with large coarse sand particles scattered over the bottom or rocks, for example on the bottom of Heron Channel. Preference for such environments may be related to its encrustation patterns. Colonies were never found in locations completely exposed to light, yet all colonies were zooxanthellate. Most colonies were relatively small, consisting of tens (not hundreds) of polyps, with polyps spread out and connected by thin stolons (Figure 3).
Notes. This species can close its polyps much more rapidly than those of other zooxanthellate zoanthid genera (Reimer pers. obs).
DNA Sequences. Originally listed in Table S1 in Reimer et al. (2011a).  Morphology: Neozoanthus uchina sp. n. has 32 to 42 (average 38±3.0, n=24 polyps on 9 colonies) conical tentacles. Tentacles are usually shorter than the expanded oral Figure 5. Cnidae of Neozoanthus caleyi sp. n. and N. uchina sp. n. from the tentacles, pharynx, and filaments showing their relative size. Type abbreviations: Sp=spirocysts, H=holotrichs, LH=large holotrichs, SH=small holotrichs, B=basitrichs, SH?=potential small holotrichs, pM=p-mastigophores. Size and frequency data are given in Table S1. disk diameter (e.g. 50-80% of oral disk width). Tentacles may be grayish-blue, rust red, or transparent, often with black, white, or fluorescent blue bands or patterning ( Figure  4). No bractae are visible, and all specimens were zooxanthellate. Polyps are externally heavily encrusted with sand and other particles of irregular sizes, excepting the oral end, which is free of encrustation and appears a bluish-gray similar to as seen in some Zoanthus species. When fully contracted, the sand free oral end is often not visible, and polyps resemble small balls of sand. Polyps extend well clear of reduced or stoloniferous coenenchyme (Figure 4). Oral disks may be a variety of colors, such as light gray-blue, white, rust or deep wine red. Occasionally, white or light blue dots may be seen on the oral disk in regular circular patterns, and the oral opening (mouth) is often white or cream in color. A "skirt" of different coloration (usually white or lighter coloration than remainder of oral disk) covering up to approximately 90 degrees of the oral disk is often seen in the area of the dorsal directive. Colonies consisted of tens to <100 polyps, connected by stolons with no well-developed coenenchyme.
Neozoanthus uchina sp. n. is currently the only partially encrusted zoanthid described from the Ryukyu Archipelago.
Etymology. Named for the Okinawan dialect word for Okinawa, "uchina", the prefecture where this species was first found. Noun in apposition.
Habitat, ecology, and distribution. Specimens from the Ryukyu Archipelago were found at depths from the intertidal zone to 25 m. Despite repeated surveys focused on zoanthids, no N. uchina sp. n. have been found further north on Yakushima Island or mainland Japan, nor further south in the Miyako and Yaeyama Islands of southern Okinawa, and it may be that this species is limited to a subtropical distribution in the Middle Ryukyu Islands. Additionally, despite surveys, thus far no specimens have been reported from neighboring Taiwan (Reimer et al. 2011d) or the Ogasawara Islands (Reimer et al. 2011b).
Neozoanthus uchina sp. n., although not found at many locations surveyed, was locally common, particularly at locations that were characterized by strong currents and some sedimentation, with large coarse sand particles scattered over the bottom or rocks. Preference for such environments may be related to its encrustation patterns. Colonies were almost always found in cracks and holes in rocks partially exposed to light, and usually not in locations completely exposed to light. Most colonies were relatively small, consisting of tens (not hundreds) of polyps, with polyps spread out and connected by thin stolons (Figure 4).
Notes. This species can close its polyps much more rapidly than species of other zooxanthellate zoanthid genera (Reimer pers. obs).

Neozoanthidae a valid family?
As stated previously (Reimer et al. 2011a), given Neozoanthus' close phylogenetic affiliation with Isaurus, a genus of the family Zoanthidae, it is unlikely that Neozoanthidae is a valid family grouping. However, given the unique morphological characters of Neozoanthus, as well as a unique mt 16S rDNA indel shared with Hydrozoanthidae, more molecular data from other markers are needed before Neozoanthidae is formally merged into Zoanthidae. These results clearly indicate that at least one of the diagnostic characters for erecting Neozoanthidae by Herberts (1972), sphincter muscle position, does not have utility, as the family was erected based in a large part on an endodermal (macrocnemic) sphincter muscle. As shown by Swain (2010), sphincter muscle position is apparently not diagnostic for higher level (genus, family, suborder) taxonomy in zoanthids, and furthermore, as mentioned in Reimer et al. (2011a), many morphological features in zoanthids, including the presence or absence of sand encrustation and sphincter muscle position can evolve or change relatively rapidly.

Ecology of Neozoanthus
Both new species in this study were found in areas notable for their strong currents. Both species, although zooxanthellate, were found in areas somewhat sheltered from direct sunlight, unlike many Zoanthus and Palythoa spp. The sand encrustation plus Neozoanthus species' preference for cracks and overhangs may have led to their lack of discovery in both Australia and Japan until 2008-2009(Reimer et al. 2011a). Certainly, the species are not "cryptic" in the classic sense, as they have colorful oral disk patterns, but the fact that they had been overlooked until recently demonstrates the lack of attention paid to zoanthids in field surveys. Neozoanthus caleyi sp. n. possesses Symbiodinium (=zooxanthellae) of subclade C1 sensu LaJeunesse (2002) (Reimer and Irei unpubl. data), a "generalist" type of Symbiodinium known to be sensitive to thermal stress, and the distribution patterns of N. uchina sp. n. and N. caleyi sp. n. fit well with this symbiont's physiology, as both species were usually not found at depths of <10 m exposed directly to sunlight and colonies in shallower waters were in cracks or crevices that provided shading. Despite the fact that no studies have yet been conducted on their ecology, the presence of both species from shallow to deeper waters in areas of strong currents combined with somewhat long tentacles (e.g. compared to P. heliodiscus with tentacles only 10% length of oral disk - Ryland and Lancaster 2003) indicates both Neozoanthus species may be mixotrophic, obtaining energy from both prey capture and photosymbiotic Symbiodinium.
As mentioned in the species' descriptions, for now it appears that both N. uchina sp. n. and N. caleyi sp. n. have subtropical distributions, as no colonies were found to regions directly north (both species) or directly south (N. uchina sp. n.) of their distribution. However, due to their small size and preference for semi-cryptic microhabitats, we cannot discount the possibility that there are further populations of both species that await discovery. Furthermore, it is known there are unidentified Neozoanthus species in Indonesia (Reimer & Hoeksema, unpublished data), and specimens are needed to complete work on these.

Conclusions
Two new species of Neozoanthus from the Pacific are formally described, one from the Great Barrier Reef and one from the Middle Ryukyu Islands.
The discovery of these two species (detailed in Reimer et al. 2011a) and their relative commonness at some sampling sites indicates that much work remains to be performed in order to properly understand zoanthid diversity in the Indo-Pacific.
We recommend the utilization of the combination of both molecular results (Reimer et al. 2011a) with the morphological descriptions given in this study for zoanthid identification and description, as seen in many recent studies.
were collected under Great Barrier Reef Marine Park Authority permit #G32313.1 and Queensland Fisheries permit # 95152. Two anonymous reviewers' comments greatly improved the manuscript.