Research Article |
Corresponding author: Yuka Irei ( yuka.irei.mugi@gmail.com ) Academic editor: Bert W. Hoeksema
© 2015 Yuka Irei, Frederic Sinniger, James Davis Reimer.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Irei Y, Sinniger F, Reimer JD (2015) Descriptions of two azooxanthellate Palythoa species (Subclass Hexacorallia, Order Zoantharia) from the Ryukyu Archipelago, southern Japan. ZooKeys 478: 1-26. https://doi.org/10.3897/zookeys.478.8512
|
Two new species of zoantharians (Hexacorallia, Zoantharia, Sphenopidae), Palythoa mizigama sp. n. and P. umbrosa sp. n., are described from the Ryukyu Archipelago, southern Japan. Unlike almost all other known Palythoa spp., both species are azooxanthellate and inhabit low-light environments such as floors or sides of caves, crevasses, or hollows of shallow coral reefs. The two species were initially considered to be the same species from their similar habitat environments and highly similar morphological features. However, phylogenetic analyses of nuclear internal transcribed spacer (ITS) ribosomal DNA, mitochondrial 16S ribosomal DNA, and cytochrome oxidase subunit I (COI) sequences revealed that these two species have a genetically distant relationship within the genus Palythoa. Morphological characteristics, including polyp size, tentacle number, external/internal coloration, and types and sizes of cnidae were examined in this study. As a result, only tentacle coloration was found to be useful for the morphological distinction between the two species. Palythoa mizigama possesses white tentacles with black horizontal stripes while P. umbrosa possesses white tentacles without any stripe patterns. Considering their distant phylogenetic relationship, it can be assumed that their unique yet similar morphological and ecological characteristics developed independently in each species as an example of parallel evolution.
Cave-dwelling, cryptic species, ITS-rDNA, Ryukyu Archipelago, zoantharian
Zoantharia are sessile marine cnidarians within the class Anthozoa, subclass Hexacorallia. This taxon is often referred to as intermediate in form between hard corals (Scleractinia) and sea anemones (Actiniaria), as most species lack a skeleton and yet are colonial. Zoantharians are widely distributed and are particularly common in subtropical and tropical regions, where they are one of the major benthic components of coral reefs. Some species contain unique chemicals such as palytoxin or norzoanthamine (
The suborder Brachycnemina currently contains three families; Sphenopidae, Zoanthidae, and Neozoanthidae. In the Ryukyu Archipelago in southwestern Japan, one genus of family Sphenopidae (Palythoa Lamouroux, 1816), two genera of family Zoanthidae (Isaurus Gray, 1828, Zoanthus Lamarck, 1801), and one genus in family Neozoanthidae (Neozoanthus Herberts, 1972) have been reported (
The two genera in the family Sphenopidae, Sphenopus and Palythoa, can easily be distinguished from each other by morphological and ecological characteristics. Unusually for a zoantharian, Sphenopus is azooxanthellate, has a solitary (=unitary, not forming colonies) life form and inhabits muddy or sandy bottoms without usually attaching to hard substrates (
In this study, we describe two new Palythoa species discovered from the Ryukyu Archipelago, southern Japan. These two species were initially considered to be the same species as they were found in similar environments and exhibit highly similar morphological features. However, molecular analyses demonstrated considerable differences between these two groups and subtle morphological differences distinguish the two groups. Uniquely for Palythoa, both species are azooxanthellate and are found in caves and cracks in coral reefs. These results lead to a reconsideration of the definition of both Palythoa and Sphenopus, and provide an example of parallel evolution within Zoantharia.
All specimens were collected between 2008 and 2012 by scuba or snorkeling from depths of 2.6 to 12 m, primarily from the Ryukyu Archipelago, southwestern Japan, with a few additional specimens collected from two locations in Taiwan and one location in New Caledonia (Figure
Sampling locations of Palythoa mizigama sp. n. and P. umbrosa sp. n. specimens. a Okinawa Island b Tokashiki Island c Yaeyama Islands d Lyudao (Green Island) e Kenting f Poindimié. Palythoa mizigama was found from Ryukyu Archipelago, Taiwan and New Caledonia (a, b, c, e, f), while P. umbrosa was found from the Yaeyama Islands and Taiwan (c, d).
Abbreviations of sampling sites:
ADA Ada (Okinawa Island)
IRI Iriomote (Yaeyama Islands)
MIZ Mizugama (Okinawa Island)
NC New Caledonia
ODO Odo (Okinawa Island)
SUN Sunabe (Okinawa Island)
TEN Teniya (Okinawa Island)
TOK Tokashiki (Kerama Islands)
TW Taiwan
YON Yona (Okinawa Island)
DNA extraction was performed with guanidine following the protocol described in
Color patterns were noted from images taken in situ or observed under stereomicroscope. Relative tentacle length (i.e. ratio of tentacle length to oral disk diameter) was estimated from images taken in situ. Additional in situ images (not shown) were taken with small rulers placed next to colonies and polyps. Height (length) and diameter of polyp column (width) of individual polyps were measured by using calipers after fixation, and numbers of tentacles were counted under stereomicroscope. Additionally, cnidae were observed, characterized and measured from three parts of each polyp; tentacles, pharynx, and mesenterial filaments. Portions of each tissue were removed and put on a separate slide glass and a cover glass was gently placed over tissue after adding a drop of glycerin solution (seawater:glycerin=1:1). The cover glass was slightly pressed to squash and disperse tissue and the edges of the cover glasses were sealed by clear nail polish. Prepared slides were then observed under a differential interference contrast (DIC) microscope (Nikon Eclipse80i, Nikon, Tokyo) and photographs of each type of cnidae were taken with a digital camera (Canon Powershot G11, Canon, Tokyo). Types of cnidae were determined with reference to
Palythoa mammillosa (Ellis & Solander, 1786).
Colonial brachycnemic zoantharians with heavily sand-incrusted ectoderm and mesoglea. Occasionally solitary polyps are also seen.
Various authors have suggested to include the genus Protopalythoa Verrill, 1900 over the past century based on both morphological (
Synonymy: “Palythoa sp. tokashiki” –
Type-specimens. Holotype. Specimen number NSMT-Co1560 (original number MIZ_33). Fixed in 99.5% ethanol, deposited in National Museum of Nature and Science, Tokyo, Japan. Original label: “HOLOTYPE Palythoa mizigama, Japan, Okinawa Island, Kadena, Mizugama, 5 m depth, 13 May 2008, Y. Irei leg. Paratypes: Paratype 1. Specimen number RMNH Coel. 41729 (original number TOK_2), Japan, Okinawa Prefecture, Kerama Islands, Tokashiki Island, Mutizuni, 26°09'07"N, 127°21'11"E, in a hollow of reef slope at 5 m depth, 5 January 2008, Y. Irei and F. Sinniger leg. Fixed in 99.5% ethanol, deposited in Naturalis Biodiversity Center, Leiden, The Netherlands. Paratype 2. Specimen number USNM 1231375 (original number ODO_25), Japan, Okinawa Prefecture, Okinawa Island, Itoman, Odo, 26°05'06"N, 127°42'32"E, on the wall of reef cave at 11 m depth, 13 January 2008, Y. Irei and F. Sinniger leg. Fixed in 99.5% ethanol, deposited in Smithsonian Institution National Museum of Natural History, Washington, D.C., USA. Paratype 3. Specimen number RUMF-ZG-04375 (original number IRI_JR2829), Japan, Okinawa Prefecture, Yaeyama Islands, Taketomi, Iriomotesuido, 24°21'51"N, 123°57'25"E, at 4.4–5.3 m depth, 1 September 2012, J. Reimer leg. Fixed in 99.5% ethanol, deposited in University Museum, University of the Ryukyus (Fujukan).
Japan, Okinawa Prefecture, Okinawa Island, Kadena, Mizugama, 26°21'35"N, 127°44'18"E, on wall of reef cave at 5 m depth, 13 May 2008, Y. Irei leg.
Size of colony approximately 2 cm × 3 cm, consisting of six polyps, 3.8–11.2 mm in height and 2.0–3.8 mm in diameter. External color brownish gray to yellowish white with small black blotches on polyp heads and columns. Horizontal wrinkles (1–7 in number) of approximately half the length of column periphery, mostly on inner side of bent polyps. Tentacles approximately 32 in number, color same as column with 7–10 narrow black horizontal lines on tentacles. Columns heavily incrusted with irregularly-sized sand grains.
This species inhabits low-light environments such as floors or sides of caves, crevasses or under reef overhangs on coral reef flats and reef slopes. In general, polyps are open at night with extended tentacles, and are closed in daytime. Polyps tend to bend parallel to the surrounding substrate when closed although polyps become somewhat erect in a diagonal direction at an acute angle (e.g. not perpendicular to substrate) when open, with oral disks generally facing the opening of the cave or crevasse (Figure
In situ images of Palythoa mizigama sp. n. and P. umbrosa sp. n. a P. mizigama; single radial lines visible on oral disks. Image taken by Y. Irei on September 28, 2009, at Mizugama, Okinawa Island, Japan b P. mizigama; several radial lines visible on oral disks. Image taken by J. Reimer on April 22, 2010, at Mizugama c P. mizigama; with no radial lines on oral disks. Image taken by Y. Irei on April 12, 2009, at Mizugama d P. mizigama; closed polyps. Image taken by Y. Irei on June 16, 2009, at Mizugama e P. umbrosa (TW_18); closed polyps. Image taken by Y. Irei on November 2, 2009, at Lyudao, Taiwan f P. umbrosa (IRI_TF2); closed polyps. Image taken by T. Fujii on November 9, 2012, at Sotobanari Island, Yaeyama Islands, Japan.
Southern Ryukyu Archipelago (Okinawa Island, Tokashiki Island, Yaeyama Islands), Taiwan (Kenting), and New Caledonia (Tibarama, Poindimié) (Figure
General. Azooxanthellate brachycnemic zoantharian with heavily sand-incrusted ectoderm and mesoglea. Colonies usually composed of several to 20 polyps, with each polyp loosely connected to others by a thin stolon (=“liberae”,
Palythoa mizigama sp. n. after fixation in 70–99.5% ethanol (a, c) or 4–10% SW formalin (b, d). a MIZ_33; closed polyps. Scale bar=0.5 cm. Collected by Y. Irei on May 13, 2008 at Mizugama, Okinawa Island, Japan b MIZ_071110_2; close up of a partially open polyp. Scale bar=0.5 cm. Collected by Y. Irei on November 7, 2010 at Mizugama c MIZ_33; tentacles (isolated). Horizontal black stripes visible. Scale bar=0.25 cm d MIZ_260910; close up image of a longitudinal section. Tentacles visible with faint stripes. Note the column wall densely incrusted with sand grains. Scale bar=0.25 cm. Collected by Y. Irei on September 26, 2010 at Mizugama. All images taken through stereomicroscope.
Polyp size. Approximately 0.5–1.0 cm in length and 0.2–0.4 cm in width after fixation in 4–10% seawater formalin or 70–99.5% ethanol.
Coloration of polyp column and oral disk. The color of external polyp columns varies from ivory to tan in life, occasionally with irregular black blotches. The color of oral disk is also ivory to tan, often with black radial lines extending outwards from the mouth (Figure
Tentacles. 32–40 in number. Extended tentacles are of the same length or longer than the diameter of oral disks (Figure
Cnidom. Four major cnidae types observed; spirocysts, basitrichs, p-mastigophores, and holotrichs. The dominant types of cnidae were spirocysts (length 20.5±3.5 μm, width 3.4±0.7 μm, n=140) in the tentacles, basitrichs (length 23.7±2.3 μm, width 3.6±0.4 μm, n=139) in the pharynx, and p-mastigophores (length 15.7±3.6 μm, width 4.7±0.7 μm, n=140) and holotrichs (length 13.5±1.9 μm, width 4.9±0.7 μm, n=140) in the mesenterial filaments (Figure
The specific epithet “mizigama” was named after the type locality of Mizugama. Additionally, “mizi” and “gama” mean “water” and “cave”, respectively, in the Okinawan language. Japanese name: Shimate-yami-iwasunaginchaku. “Shimate”, “yami”, and “iwasunaginchaku” mean “striped hands”, “dark”, and “Palythoa”, respectively, in Japanese.
Simultaneous hermaphrodite. Reproductive polyps containing ovaries and spermaries were found from specimens collected in August - September 2010 at Mizugama, Okinawa Island, Japan (Figure
Synonymy: “Palythoa sp. tokashiki” –
Type-specimens. Holotype. Specimen number NSMT-Co1561 (original number IRI_TF3). Split into two pieces and fixed in 99.5% ethanol and 4-10% formalin, respectively. Deposited in National Museum of Nature and Science, Tokyo, Japan. Original label: “HOLOTYPE Palythoa umbrosa, Japan, Yaeyama Islands, Sotobanari Island, 8 m depth, 9 November 2012, J. Reimer leg.” Paratypes. Paratype 1. Specimen number RMNH Coel. 41730 (original number IRI_TF1), Japan, Okinawa Prefecture, Yaeyama Islands, Taketomi, Sotobanari Island, 24°22'46"N, 123°43'53"E, at 9 m depth, 9 November 2012, T. Fujii leg. Split into two pieces and fixed in 99.5% ethanol and 4–10% formalin, respectively. Deposited in Naturalis Biodiversity Center, Leiden, The Netherlands. Paratype 2. Specimen number USNM 1231376 (original number IRI_TF2), Japan, Okinawa Prefecture, Yaeyama Islands, Taketomi, Sotobanari Island, 24°22'46"N, 123°43'53"E, at 10 m depth, 9 November 2012, T. Fujii leg. Split into two pieces and fixed in 99.5% ethanol and 4–10% formalin, respectively. Deposited in Smithsonian Institution National Museum of Natural History, Washington, D.C., USA. Paratype 3. Specimen number RUMF-ZG-04376 (original number IRI_31), Japan, Okinawa Prefecture, Yaeyama Islands, Taketomi, Iriomote-suido, 24°21'51"N, 123°57'25"E, at 6 m depth, 7 May 2008, Y. Irei leg. Fixed in 99.5% ethanol, deposited in University Museum, University of the Ryukyus (Fujukan).
Japan, Okinawa Prefecture, Yaeyama Islands, Taketomi, Sotobanari Island, 24°22'46"N, 123°43'53"E, on wall of reef cave at 8 m depth, 9 November 2012, J. Reimer leg.
Size of colony approximately 2 cm × 2 cm, consisting of three polyps, 6.1 mm in height and 2.7–3.0 mm in diameter. Columns yellowish white with irregular black blotches. Dark brown polyp heads. Horizontal wrinkles (3–5 in number) of approximately half the length of column periphery, on mostly inner side of bent polyps. Tentacles approximately 32 in number, white in color. Columns heavily incrusted with irregularly-sized sand grains.
This species inhabits low-light environments such as floors or sides of caves, crevasses or under reef overhangs on coral reef flats and reef slopes. In general, polyps are open at night with extended tentacles, and are closed during the daytime. Polyps tend to bend parallel to the surrounding substrate when closed (Figures
Palythoa umbrosa sp. n. after fixation in 4-10% SW formalin. a IRI_TF2; slightly expanded polyp. Scale bar=0.5 cm. Collected by T. Fujii on November 9, 2012 at Sotobanari Island, Yaeyama Islands, Japan b IRI_TF1; closed polyp. Scale bar=0.5 cm. Collected by T. Fujii on November 9, 2012 at Sotobanari Island c IRI_TF2; close up of a longitudinal section. Note the white tentacles with small black dots. Scale bar=0.2 cm d IRI_TF2; close up of a polyp capitalum. Column incrusted with irregularly-sized particles. Scale bar=0.25 cm. All images taken through stereomicroscope.
Southern Ryukyu Archipelago (Yaeyama Islands) and Taiwan (Lyudao) (Figure
General. Azooxanthellate brachycnemic zoantharian with heavily sand-incrusted ectoderm and mesoglea. Colonies usually composed of several to 20 polyps with each polyp loosely connected to neighbor(s) by a thin stolon (=“liberae”,
Polyp size. 0.5–0.9 cm in length and 0.3–0.5 cm in width after fixation in 4–10% seawater formalin or 70–99.5% ethanol.
Coloration of polyp column and oral disk. Since sampling of P. umbrosa was performed only during the daytime, images of expanded polyps in situ were not available and observations were performed under a stereomicroscope. The color of external polyp columns was ivory to tan, occasionally with irregular black/brown blotches (Figures
Tentacles. 32–38 in number and white to ivory in color (Figure
Cnidom. There were four major cnidae types observed (Figure
Named for habitat environment of this species. The specific epithet “umbrosa” means “shadowy” in latin. Japanese name: Shirote-yami-iwasunaginchaku. “Shirote” means “white hands” in Japanese.
The azooxanthellate species Palythoa macmurrichi has previously been described from the Torres Strait, northern Australia. It was described based on a single polyp collected from a channel at 20 fathoms (approximately 36.5 m), and the holotype was lost when it was used for histological examination in
Palythoa mizigama and P. umbrosa were included in a monophyletic clade along with other Palythoa spp. in the phylogenetic trees for all three DNA markers (Figures
Maximum likelihood tree based on internal transcribed spacer region of ribosomal DNA for family Sphenopidae. Numbers on nodes represent ML and neighbor-joining (NJ) bootstrap values (>65% are shown). Thick branches indicate high supports of Bayesian posterior probabilities (>0.95). Sequences obtained from GenBank are shown with accession numbers.
Maximum likelihood tree based on mitochondrial 16S ribosomal DNA for family Sphenopidae. Numbers on nodes represent ML and neighbor-joining (NJ) bootstrap values (>50% are shown). Thick branches indicate high supports of Bayesian posterior probabilities (>0.95). Sequences obtained from GenBank are shown with accession numbers.
Maximum likelihood tree based on mitochondrial cytochrome oxidase subunit I gene for family Sphenopidae. Numbers on nodes represent ML and neighbor-joining (NJ) bootstrap values (>50% are shown). Thick branches indicate high supports of Bayesian posterior probabilities (>0.95). Sequences obtained from GenBank are shown with accession numbers.
This study revealed a wide distribution for P. mizigama in the Central Indo-Pacific from the Ryukyu Archipelago in the north to New Caledonia in the south. Palythoa umbrosa was found from a more limited region including the Yaeyama Islands (southwest Ryukyu Archipelago) and Lyudao (= Green Island, southeast of Taiwan) (Figure
Palythoa mizigama and/or P. umbrosa may possibly also occur in the Great Barrier Reef, Australia.
Both morphological and molecular characteristics clearly indicate the two new species belong within the genus Palythoa. Phylogenetic analyses showed that P. mizigama and P. umbrosa are relatively distantly related to each other within the genus despite their morphological features being almost identical. We detected statistically significant differences in sizes of cnidae between the two species, however, due to large overlaps and relatively high standard deviation values (Suppl. material
The only morphological characteristic that was consistently found to be useful for species identification was tentacle coloration. Specimens of P. mizigama and P. umbrosa both possessed white tentacles, but black horizontal stripes were always present on the tentacles of P. mizigama (Figure
In general, species identification based on coloration has been thought of as not generally reliable for brachycnemic zoantharians as much intraspecific variation is present (
This study showed a high morphological similarity between P. mizigama and P. umbrosa in spite of their relatively distant genetic relationship within genus Palythoa. Two possibilities can be considered as the potential cause of this situation. One hypothesis is that P. mizigama and P. umbrosa have recently diverged from each other but their morphological characteristics have not yet diverged. However, our phylogenetic results showed that P. mizigama and P. umbrosa do not form a monophyletic group with any of three DNA markers used in this study, and even conservative mitochondrial DNA regions (COI and 16S rDNA) differentiated between the two groups. Thus, there is no support for the idea that P. mizigama and P. umbrosa have recently diverged.
The other hypothesis is that P. mizigama and P. umbrosa diverged at relatively the same time as many other zooxanthellate Palythoa spp. (e.g. P. mutuki, P. heliodiscus, P. tuberculosa in Figures
Sphenopus has an unusual morphology and ecology among zoantharians, as it is solitary and buried in sand or soft substrate usually without attaching to the substrate. From these unique characteristics, Sphenopus has been considered to be a different genus from Palythoa. However, the general morphological characters (e.g. mesenterial arrangement, heavy sand incrustation etc.) of Sphenopus asides from the lack of colonial form and lack of attachment are the same as for Palythoa. In fact, there are some Palythoa species that have somewhat similar ecological features to Sphenopus, such as Palythoa psammophilia Walsh & Bowers, 1971, which inhabits sandy areas with its polyps partially buried (
Planktonivory (
The two new species have very similar morphological features although their phylogenetic positions are relatively distant and without molecular analyses they could have been misdiagnosed as a single species. To avoid taxonomic confusion of zoantharian identification caused by their morphological plasticity, a combination of molecular and morphological data is recommended (
The authors thank Dr T. Fujii (University of the Ryukyus) for his great help providing specimens from Yaeyama Islands, Japan. Drs Y. Nozawa and CA. Chen (Biodiversity Research Center, Academia Sinica; BRCAS) and members of their laboratories for their invaluable assistance in the field in Taiwan. Prof. E. Hirose (University of the Ryukyus), Dr S. Keshavmurthy, CY. Kuo, HJ. Hseih, and AC. Chung (all BRCAS) also greatly assisted with the sample collection in Taiwan. Dr C. Payri, Dr B. Richer-de-Forges and Tiéti diving in Poindimié are thanked for the assistance in the sampling of the New Caledonian specimen. F. Sinniger was partially financed by the Japan Society for Promotion of Science and the senior author was funded by a grant-in-aid from the Japan Society for the Promotion of Science (“Wakate B” #217700896), as well as the Rising Star Program and the International Research Hub Project for Climate Change and Coral Reef/Island Dynamics, both at the University of the Ryukyus. Support was also provided by a Japan Society for the Promotion of Science ‘Zuno-Junkan’ grant entitled ‘Studies on origin and maintenance of marine biodiversity and systematic conservation planning’ to the senior author.
Table 1
Data type: Microsoft Excel File (xlsx)
Explanation note: List of Palythoa mizigama sp. n. and P. umbrosa sp. n. specimens used in molecular analyses.
Table 2
Data type: Microsoft Excel File (xlsx)
Explanation note: Length and widths of four major cnidae types found in Palythoa mizigama sp. n. and P. umbrosa sp. n.