Research Article |
Corresponding author: Hiroki Kise ( hkm11sea@yahoo.co.jp ) Academic editor: Bert W. Hoeksema
© 2017 Hiroki Kise, Takuma Fujii, Giovanni Diego Masucci, Piera Biondi, 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:
Kise H, Fujii T, Masucci GD, Biondi P, Reimer JD (2017) Three new species and the molecular phylogeny of Antipathozoanthus from the Indo-Pacific Ocean (Anthozoa, Hexacorallia, Zoantharia). ZooKeys 725: 97-122. https://doi.org/10.3897/zookeys.725.21006
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In this study, three new species of macrocnemic zoantharians (Hexacorallia, Zoantharia) are described from localities in the Indo-Pacific Ocean including the Red Sea, the Maldives, Palau, and southern Japan: Antipathozoanthus obscurus sp. n., A. remengesaui sp. n., and A. cavernus sp. n. Although the genus Antipathozoanthus is currently restricted to species living on antipatharians, A. obscurus sp. n. is not associated with any living substrate and instead is found on coral reef carbonate substrate within narrow caves or cracks. The two new species that have association with antipatharians, A. remengesaui sp. n. and A. cavernus sp. n., can be distinguished by their relative coenenchyme development and the antipatharian species that each uses as substrate. Additionally, all new species described in this study have unique nuclear internal transcribed spacer region of ribosomal DNA (ITS-rDNA) sequences. Our results indicate that more phylogenetic studies focusing on increasing the numbers of species examined within each of the genera of Parazoanthidae are required in order to better understand the evolutionary history of substrate specificity within the family Parazoanthidae.
antipatharian, cave-dwelling, diversity, evolution, new species, substrate specificity
Zoantharia Rafinesque, 1815 is the third most speciose order within the subclass Hexacorallia Haeckel, 1896. Zoantharians can be found in a wide variety of marine environments from intertidal zones to deep-sea cold seeps (e.g.,
One of these recently erected genera is Antipathozoanthus Sinniger, Reimer & Pawlowski, 2010. As the generic name indicates, species in this genus utilize antipatharians (Hexacorallia, Antipatharia) as their obligate substrate. The genus currently includes two valid species; A. macaronesicus (Ocaña & Brito, 2003) from the eastern Atlantic and A. hickmani Reimer & Fujii, 2010 from the Galapagos Islands. Additionally, several potentially undescribed species have been reported from the Red Sea (
Specimen collection. Antipathozoanthus specimens were collected between 2009 to 2016 from three localities in the Red Sea, three localities in the Maldives, five localities in Japan, and two localities in Palau (Fig.
Sampling location in the Indian Ocean and Pacific Ocean of specimens used in this study. Location of specimens collected in this study represented by closed symbols. Species abbreviations after locations: (Ao) Antipathozoanthus obscurus sp. n.; (Ar) A. remengesaui sp. n.; (Ac) A. cavernus sp. n.
Specimen ID | Genus | Species | Locality | Coordinates | Collecter | Sampling date | Depth (m) | Accession number (COI) | Accession number (16S–rDNA) | Accession number (ITS–rDNA) | |
---|---|---|---|---|---|---|---|---|---|---|---|
Latitude / Longitude | |||||||||||
AZCN | Antipathozoanthus | macaronesicus | Pico Island, Azores, Portugal | N38°28"3.8", W28°24′0″ | P Wirtz | 13-May-16 | 43 | MG384664 | MG384684 | MG384696 | |
BISE1 | Antipathozoanthus | obscurus | Bise, Motobu, Okinawa, Japan | N26°42'34.4", E127°52'49.2" | JD Reimer, I Kawamura | 14-Aug-14 | 5 | MG384644 | MG384685 | MG384691 | |
BISE3 | Antipathozoanthus | obscurus | Bise, Motobu, Okinawa, Japan | N26°42'34.4", E127°52'49.2" | JD Reimer, I Kawamura | 14-Aug-14 | 5 | – | – | MG384693 | |
MAL46 | Antipathozoanthus | remengesaui | Coral Garden, Maldives | N3°05'24.3", E72°58'04.5" | JD Reimer | 06-May-14 | 24 | MG384658 | MG384679 | – | |
MAL82 | Antipathozoanthus | remengesaui | Wall Street, Maldives | N3°07'14.2", E72°58'46.5" | JD Reimer | 07-May-14 | 9 | MG384657 | – | – | |
MAL83 | Antipathozoanthus | remengesaui | Wall Street, Maldives | N3°07'14.2", E72°58'46.5" | JD Reimer | 07-May-14 | 9 | MG384656 | – | – | |
MAL84 | Antipathozoanthus | remengesaui | Wall Street, Maldives | N3°07'14.2", E72°58'46.5" | JD Reimer | 07-May-14 | 9 | MG384655 | – | MG384702 | |
MAL85 | Antipathozoanthus | remengesaui | Wall Street, Maldives | N3°07'14.2", E72°58'46.5" | JD Reimer | 07-May-14 | 9 | MG384654 | MG384678 | MG384701 | |
MAL145 | Antipathozoanthus | remengesaui | Wall Street, Maldives | N3°07'14.2", E72°58'46.5" | JD Reimer | 10-May-14 | 12 | MG384653 | MG384677 | – | |
MAL147 | Antipathozoanthus | remengesaui | Wall Street, Maldives | N3°07'14.2", E72°58'46.5" | JD Reimer | 10-May-14 | 10 | MG384652 | – | – | |
MAL2592601 | Antipathozoanthus | cavernus | Capital Reef, Maldives | N3°02'55.8", E72°53'21.2" | M Oliverio | 16-May-14 | 19 | MG384651 | MG384676 | MG384697 | |
MAL2592602 | Antipathozoanthus | remengesaui | Capital Reef, Maldives | N3°02'55.8", E72°53'21.2" | M Oliverio | 16-May-14 | 19 | – | MG384675 | – | |
MAL261 | Antipathozoanthus | remengesaui | Wall Street, Maldives | N3°07'14.2", E72°58'46.5" | JD Reimer | 17-May-14 | 9 | MG384650 | MG384674 | – | |
KINKO1 | Antipathozoanthus | cavernus | Sakurajima, Kagoshima, Japan | N31°35'23.5", E130°35.27.8" | JD Reimer | 20-Sep-15 | 21 | MG384660 | MG384681 | MG384699 | |
KINKO2 | Antipathozoanthus | remengesaui | Sakurajima, Kagoshima, Japan | N31°35'23.5", E130°35'27.8" | JD Reimer | 20-Sep-15 | 21 | MG384659 | MG384680 | – | |
PALAU2 | Antipathozoanthus | remengesaui | Blue Hole, Palau | N7°8'29.4", E134°13'23.3" | JD Reimer | 15-Sep-14 | 23 | MG384649 | MG384673 | MG384703 | |
PALAU3 | Antipathozoanthus | remengesaui | Siaes Tunnel. Palau | N7°18'54.8", E134°13'13.3" | JD Reimer | 15-Sep-14 | 37 | MG384648 | – | – | |
PALAU4 | Antipathozoanthus | remengesaui | Blue Hole, Palau | N7°8'29.4", E134°13'23.3" | JD Reimer | 12-Sep-14 | 28 | MG384647 | MG384672 | – | |
PALAU5 | Antipathozoanthus | cavernus | Siaes Tunnel. Palau | N7°18'54.8", E134°13'13.3" | JD Reimer | 15-Sep-14 | 39 | – | – | MG384698 | |
HK70 | Antipathozoanthus | remengesaui | Siaes Tunnel. Palau | N7°18'54.8", E134°13'13.3" | H Kise | 12-Sep-14 | NA | MG384663 | MG384683 | – | |
HK90 | Antipathozoanthus | remengesaui | Blue Hole, Palau | N7°8'29.4", E134°13'23.3" | H Kise | 15-Sep-14 | 22 | MG384662 | – | – | |
TF54 | Antipathozoanthus | obscurus | Cape Zanpa, Yomitan, Okinawa, Japan | N26°26'26.5", E127°42'43.7" | T Fujii | 06-Apr-09 | 3 | MG384641 | – | MG384689 | |
TF78 | Antipathozoanthus | obscurus | Cape Manza, Onna, Okinawa, Japan | N26°30'18.3", E127°51'02.3" | T Fujii | 02-Oct-09 | 5 | MG384640 | MG384668 | MG384687 | |
TF102 | Antipathozoanthus | remengesaui | Sakurajima, Kagoshima, Japan | N31°35'23.5", E130°35'27.8" | T Fujii | 26-Jul-11 | 20 | MG384646 | – | MG384704 | |
TF103 | Antipathozoanthus | remengesaui | Sakurajima, Kagoshima, Japan | N31°35'23.5", E130°35'27.8" | T Fujii | 26-Jul-11 | 40 | MG384645 | – | MG384705 | |
TF148 | Antipathozoanthus | obscurus | Cape Manza, Yomitan, Okinawa, Japan | N26°30'18.3", E127°51'02.3" | T Fujii | 22-Oct-12 | 10 | MG384642 | MG384669 | MG384688 | |
TF173 | Antipathozoanthus | remengesaui | Onna, Okinawa, Japan | N26°26'20.9", E127°47'7.2" | T Fujii | 27-Jun-14 | 15 | – | – | – | |
JDR190 | Antipathozoanthus | obscurus | Al Wajh Shaybarah, Saudi Arabia | N25°21', E36°54' | JD Reimer | 03-Oct-13 | 3 | – | MG384667 | MG384692 | |
JDR191 | Antipathozoanthus | obscurus | Al Wajh Shaybarah, Saudi Arabia | N25°21', E36°54' | JD Reimer | 03-Oct-13 | 3 | – | MG384666 | MG384694 | |
JDR192 | Antipathozoanthus | obscurus | Al Wajh Shaybarah, Saudi Arabia | N25°21', E36°54' | JD Reimer | 03-Oct-13 | 3 | MG384643 | MG384665 | MG384695 | |
JDR209 | Antipathozoanthus | remengesaui | Yanbu , Saudi Arabia | N24°26', E37°14' | JD Reimer | 04-Oct-13 | 11 | – | – | MG384700 | |
JDR211 | Antipathozoanthus | remengesaui | Yanbu , Saudi Arabia | N24°26', E37°14' | JD Reimer | 04-Oct-13 | 12 | – | MG384682 | – | |
JDR214 | Antipathozoanthus | remengesaui | Yanbu , Saudi Arabia | N24°26', E37°14' | JD Reimer | 04-Oct-13 | 12 | MG384661 | – | – | |
JDR279 | Antipathozoanthus | obscurus | Shib Nazar, Saudi Arabia | N22°19', E38°51' | JD Reimer | 10-Oct-13 | 4 | – | MG384671 | MG384690 | |
KU1 | Antipathozoanthus | obscurus | Ara, Kumejima Island, Okinawa, Japan | N26°19'15.0", E126°45'21.3" | T Fujii | 20-Nov-09 | 15 | MG384639 | MG384670 | MG384686 |
Molecular analyses. Antipathozoanthus DNA was extracted using the guanidine protocol following
Molecular phylogenetic analyses. Newly obtained sequences were inspected by eye and manually edited using Geneious v8.1 (
The generated alignments of each marker were used to construct a concatenated alignment. All missing data, including gaps, were replaced with “N". All specimens of Antipathozoanthus included in the concatenated alignment included at least ITS-rDNA sequences. The concatenated alignment consisted of 1957 positions and 54 sequences. Phylogenetic analyses of the concatenated alignment were performed using maximum likelihood (ML) and Bayesian inference (BI), with gene partitions set for ML in RAxML v8 (
The NJ phylogeny reconstruction was performed using Geneious v8.1 (
Morphological analyses. Numbers of tentacles, polyp coloration, oral disk coloration, relative tentacle lengths, and polyp dimensions (oral disk diameter/polyp height) were examined using in situ images. Additionally, the relative development of the coenenchyme was examined using a dissecting microscope. Coenenchyme development was classified as 1) “highly developed coenenchyme" when polyps covered the antipatharian substrate completely, or 2) “poorly developed coenenchyme" when polyps did not completely cover the antipatharian substrate and the antipatharians were clearly visible. For internal morphological analyses, we observed mesentery arrangement and numbers, and location and shape of marginal muscle. Histological sections of 8 µm thickness were made and stained with hematoxylin and eosin after decalcification with Bouin’s fluid for 24h.
Cnidae analyses. Cnidae analyses were conducted using undischarged cnidocysts from tentacles, column, actinopharynx, and mesenteries filaments of holotype polyps (n = 6) for all new species under a Nikon Eclipse80i stereomicroscope (Nikon, Tokyo). Cnidae sizes were measured using ImageJ v1.45s (
MISE Molecular Invertebrate Systematics and Ecology Laboratory, University of the Ryukyus, Nishihara, Okinawa, Japan
Antipathozoanthus macaronesicus (Ocaña & Brito, 2003)
Macrocinemic zoantharians with cteniform endodermal muscle or endo-meso transitional sphincter muscle (
Four of five formally described species grow mainly on antipatharians, but this character is not exclusive to all species in the genus as A. obscurus sp. n. is not associated with any host organism. Results of the current study showed that A. obscurus sp. n. is clearly placed within this genus according to COI and 16S-rDNA sequence analyses. Thus, these non-associated species/specimens are within the genus based on their phylogenetic position but do not fit the original definition of the genus by
Antipathozoanthus
sp. 3 sensu
Holotype:
Paratypes:
Other materials examined: MISE-BISE3, collected from Cape Bise, Motobu, Okinawa-jima Island, Japan (26°42'34.4"N, 127°52'49.2"E) at a depth of 5 m by JDR, 14 August 2014.
External morphology: Open oral disks are approximately 5–10 mm in diameter, and polyps approximately 5–10 mm in height when open (Fig.
Polyp images of Antipathozoanthus obscurus sp. n., A. remengesaui sp. n. and A. cavernus sp. n. in situ. a A. obscurus sp. n. .
Internal morphology: Azooxanthellate. Fine sand particles and silica heavily encrusted into ectoderm and mesoglea. We could not obtain cross-sections or images to observe internal morphology such as mesenterial arrangement, marginal muscle or siphonoglyph due to heavy sand and silica encrustation.
Cnidae: Holotrichs (large), basitrichs and microbasic p-mastigophores (usually difficult to distinguish), spirocysts (Fig.
Cnidae types and sizes observed in three new Antipathozoanthus species. Frequency: relative abundance of cnidae type in decreasing order; numerous, common, occasional, rare (n = number of cnidae).
Antipathozoanthus obscurus sp. n. | A. remengesaui sp. n. | A. cavernus sp. n. | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Length (min-max, average) | Width (min-max, average) | n | Frequency | Length (min-max, average) | Width (min-max, average) | n | Frequency | Length (min-max, average) | Width (min-max, average) | n | Frequency | ||
Tentacles | Spirocysts | 11–20, 16.3 | 2–5, 3.1 | 48 | Occasional | 11–25, 18.0 | 2–7, 3.4 | 98 | Numerous | 14–25, 18.0 | 2–5, 2.9 | 137 | Numerous |
Holotrichs (L) | 21–33, 28.1 | 10–15, 11.9 | 39 | Occasional | 20–29, 21.8 | 7–14, 10.1 | 27 | Occasional | 20–31, 22.7 | 9–17, 11.4 | 28 | Occasional | |
Holotrichs (M) | – | – | – | – | 10–19, 17.0 | 5–17, 9.9 | 105 | Numerous | 14–19, 17.9 | 7–14, 10.0 | 95 | Numerous | |
Bastrichs and Mastigophores | – | – | – | – | 10–23, 16.3 | 2–6, 4.2 | 31 | Occasional | 15–19, 16.7 | 4–6, 4.7 | 7 | Rare | |
Column | Spirocysts | 12–22, 18.2 | 2–5, 3.1 | 57 | Numerous | 17–25, 19.7 | 2–4, 3.1 | 20 | Occasional | 11–28, 17.5 | 2–16, 5.6 | 44 | Numerous |
Holotrichs (L) | 22–34, 28.2 | 9–15, 11.8 | 78 | Numerous | 20–29, 25.1 | 9–17, 12.5 | 40 | Occasional | 20–32, 25.6 | 9–16, 11.2 | 24 | Occasional | |
Holotrichs (M) | – | – | – | 11–19, 17.0 | 8–12, 9.9 | 21 | Occasional | 12–19, 16.5 | 5–13, 8.8 | 40 | Occasional | ||
Bastrichs and Mastigophores | 12–25, 18.2 | 2–5, 3.6 | 29 | Occasional | 14–18, 15.9 | 4–8, 5.9 | 16 | Common | 2 | 5 | 1 | Rare | |
Pharynx | Spirocysts | 13–25. 16.5 | 2–5, 3.0 | 69 | Numerous | 11–24, 17.7 | 2–6, 3.4 | 65 | Numerous | 13–23, 18.2 | 2–5, 3.2 | 101 | Numerous |
Holotrichs (L) | 20–34, 28.5 | 8–15, 11.5 | 76 | Numerous | 20–31, 23.4 | 7–18, 11.7 | 35 | Occasional | 20–31, 22.7 | 9–15, 12.0 | 33 | Occasional | |
Holotrichs (M) | – | – | – | 10–19, 16.8 | 6–13, 9.5 | 76 | Numerous | 13–19, 17.6 | 6–13, 10.0 | 85 | Numerous | ||
Bastrichs and Mastigophores | 13–18, 16.1 | 3–6, 3.4 | 18 | Common | 13–21, 16.5 | 2–8, 4.5 | 52 | Numerous | 12–21, 16.9 | 2–5, 3.4 | 37 | Occasional | |
Mesenteries | Spirocysts | 13–21, 17.4 | 2–6, 3.4 | 64 | Numerous | 13–25, 17.9 | 2–6, 3.3 | 60 | Numerous | 3–26, 18.0 | 2–5, 3.1 | 61 | Numerous |
Holotrichs (L) | 23–38, 28.2 | 7–14, 11.5 | 27 | Occasional | 20–34, 24.3 | 8–15, 10.8 | 31 | Occasional | 20–36, 27.6 | 10–15, 11.8 | 52 | Numerous | |
Holotrichs (M) | – | – | – | 10–19, 16.5 | 4–15, 9.4 | 86 | Numerous | 12–19, 16.2 | 6–13, 8.3 | 61 | Numerous | ||
Bastrichs and Mastigophores | 13–18, 16.0 | 3–5, 3.8 | 16 | Common | 13–22, 16.9 | 3–9, 4.6 | 71 | Numerous | 10–18, 14.6 | 2–5, 2.7 | 21 | Occasional |
Antipathozoanthus obscurus sp. n. is found in low-light environments such as within crevasses of reef slopes and reef floors, and coral reef caves. Specimens were found from 3 to 15 m. This species has been found from the Red Sea and Okinawa.
Antipathozoanthus obscurus sp. n. is easily distinguished from all other Antipathozoanthus species, including the two other new species in this study, which all have associations with antipatharians. A. obscurus sp. n. is not associated with antipatharians and instead is found on coral reef carbonate substrate within caves or cracks. Additionally, the cnidome of A. obscurus sp. n. is different from all other known Antipathozoanthus species, including the other new species in this study, as there are no medium holotrichs in any tissue of A. obscurus sp. n., and instead only large holotrichs are found in all tissues.
Although A. obscurus sp. n. is not associated with antipatharians, phylogenetic data indicate that A. obscurus sp. n. is very closely related to other Antipathozoanthus species associated with antipatharians, with identical COI and 16S-rDNA sequences to those of A. macaronesicus (EU591618).
The samples of Antipathozoanthus obscurus sp. n. in the present study contain two morphotypes; one with bright brown tentacles that are longer than the oral disk (MISE-TF54); and the other morphotype with orange tentacles that are only as long as the oral disk (MISE-BISE1, MISE-BISE3, MISE-JDR190, MISE-JDR191, MISE-JDR192, MISE-JDR279, MISE-KU1, MISE-TF78, MISE-TF148). However, the sequences of all specimens formed a monophyletic clade and therefore we have described A. obscurus sp. n. in this study as containing two morphotypes. Genetic variation in all three genetic markers in the samples of A. obscurus sp. n. was observed, and the possibility remains that A. obscurus sp. n. may contain cryptic species. Thus, we have excluded specimen MISE-BISE3 from the type series, although it was tentatively identified as A. obscurus sp. n. Further specimens and fine-scale genetic analyses are required to better understand if there is any cryptic diversity within this species.
Antipathozoanthus obscurus sp. n. is named from the Latin “obscura" meaning “dark", as this species can be found in dark environments.
Tsuno-nashi-sunaginchaku (new Japanese name).
Antipathozoanthus
sp. sensu
Antipathozoanthus
sp. 1 sensu
Holotype:
Paratypes:
Other materials examined: MISE-PALAU3, collected from Siaes Tunnel, Palau (7°18'54.8"N, 134°13'13.3"E) at a depth of 37 m by JDR, 15 September 2014; MISE-PALAU4, collected from Blue Hole, Palau (7°8'29.4"N, 134°13'23.3"E) at a depth of 28 m by JDR, 12 September 2014; MISE-KINKO2, collected from Hakamagoshi, Sakurajima, Kagoshima, Japan (31°35'23.5"N, 130.35.27.8"E) at a depth of 21 m by JDR, 20 September 2015; MISE-TF173, collected from Onna, Okinawa, Japan (26°26'20.9"N, 127°47'7.22"N) at depth of 15 m by TF, 27 June 2014; MISE-MAL46, collected from Coral Garden, Maldives (3°05'24.3"N, 72°58'04.5"E) at a depth of 24 m by JDR, 6 May 2014; MISE-MAL82, collected from Wall Street, Maldives (3°07'14.2"N, 72°58'46.5"E) at a depth of 9 m by JDR, 7 May 2014; MISE-MAL83, collected from Wall Street, Maldives (3°07'14.2"N, 72°58'46.5"E) at a depth of 9 m by JDR, 7 May 2014; MISE-MAL2502602, collected from Capital Reef, Maldives (3°02'55.8"N, 72°53'21.2"E) at a depth of 19 m by Marco Oliverio, 16 May 2014; MISE-MAL145, collected from Wall Street, Maldives (3°07'14.2"N, 72°58'46.5"E) at a depth of 12 m by JDR, 10 May 2014; MISE-MAL147, collected from Wall Street, Maldives (3°07'14.2"N, 72°58'46.5"E) at a depth of 10 m by JDR, 10 May 2014; MISE-MAL261, collected from Wall Street, Maldives (3°07'14.2"N, 72°58'46.5"E) at a depth of 9 m by JDR, 17 May 2014; MISE-HK70, collected from Siaes Tunnel, Palau (7°18'54.8"N, 134°13'13.3"E) by Hiroki Kise (HK), 12 September 2014, depth not available; MISE-HK90, collected from Blue Hole, Palau (7°8'29.4"N, 134°13'23.3"E) at a depth of 22 m by HK, 15 September 2014; MISE-JDR211, collected from Yanbu, Saudi Arabia, (24°26'N, 37°14'E) at a depth of 12 m by JDR, 4 October 2013; MISE-JDR214, collected from Yanbu, Saudi Arabia, (24°26'N, 37°14'E) at a depth of 12 m by JDR, 4 October 2013.
External morphology: Polyps in situ are approximately 4–8 mm in diameter, and approximately 3–8 mm in height in situ when oral disks expanded (Fig.
Internal morphology: Cteniform endodermal marginal muscle sensu
Cnidae: Holotrichs (large and medium), basitrichs and microbasic p-mastigophores (usually difficult to distinguish), spirocysts (Fig.
Antipathozoanthus remengesaui sp. n. has been found on the sides and/or floors of cave entrance, and always on Antipathes. Specimens were collected from depths of 9 to 40 m. This species is known from Palau, Kagoshima in Japan, the Maldives, and the Red Sea.
In the Pacific, Antipathozoanthus remengesaui sp. n. can be distinguished from A. hickmani by the development of the coenenchyme and in part by polyp size; the larger polyps (4–12 mm in diameter and 4–15 mm in height) of A. hickmani are connected by a well-developed coenenchyme on Antipathes galapagensis, while the slightly smaller polyps (4–8 mm in diameter and 3–8 mm in height in situ) of A. remengesaui sp. n. are either connected by a poorly developed coenenchyme or may even be solitary on Antipathes. Additionally, the cnidomes of these species are different; A. hickmani does not have spirocysts in the column, while A. remengesaui sp. n. has spirocysts in the column.
The Antipathozoanthus remengesaui sp. n. specimens found in Kagoshima, Japan have different morphological features compared to the specimens found in all other regions. Specimens collected from Kagoshima have relatively large polyps (6–8 mm in diameter, and approximately 5–8 mm in height in situ) compared to specimens from other regions. The coloration of oral disks is also different between Kagoshima and other regions; A. remengesaui sp. n. from Kagoshima has a bright brown oral disk, while those from other regions have pink oral disks. However, sequences of these specimens collected from all regions formed a monophyletic clade for all genetic markers including ITS-rDNA. In terms of substrate organisms, A. remengesaui sp. n. collected from all regions in this study was associated with black corals of the genus Antipathes. Here, we have described this group as a single species, A. remengesaui sp. n., based on phylogeny and substrate specificity, although we have excluded some specimens for which we could not amplify ITS-rDNA successfully from the type series.
Antipathozoanthus remengesaui sp. n. is named after Tommy Esang Remengesau, Jr., the current president of the Republic of Palau, who has greatly contributed to marine research and conservation in Palau.
Common name. Momoiro-mame-tsuno-sunaginchaku (new Japanese name).
Holotype:
Paratypes:
External morphology: Polyps in situ are approximately 4–15 mm in diameter when oral disk is expanded, and approximately 3–10 mm in height (Fig.
Internal morphology: Cteniform endodermal arrangement marginal muscle sensu
Cnidae: Holotrichs (large and medium), basitrichs and microbasic p-mastigophores (usually difficult to distinguish from each other), spirocysts (Fig.
Antipathozoanthus cavernus sp. n. is found on the sides and/or floor of cave entrances, and on steep slopes, and always on Myripathes. Specimens were collected from depths of 19 to 39 m.
Antipathozoanthus cavernus sp. n. occurs in similar environments as A. remengesaui sp. n., but these species can be distinguished by their coenenchyme development and by the generic identity of the antipatharian host. A. remengesaui sp. n. is associated with genus Antipathes (family Antipathidae) covered by a poorly developed coenenchyme, while A. cavernus sp. n. is associated with genus Myripathes (family Myripathidae) covered by a highly developed coenenchyme. A. cavernus sp. n. can be distinguished from A. hickmani by a different coloration and by its antipatharian association; A. cavernus sp. n. does not have red or cream colored polyps as seen in A. hickmani. Additionally, A. hickmani is associated with Antipathes galapagensis, while A. cavernus sp. n. is associated with genus Myripathes. A. macaronesicus is easily distinguishable from A. cavernus sp. n. by their polyp coloration (orange and light orange versus pinkish and yellowish, and their antipatharian host (genus Antipathes versus genus Myripathes). Finally, all species above have unique ITS-rDNA sequences.
Antipathozoanthus cavernus sp. n. is named from the Latin “caverna" meaning “cave", as this species is found in caves.
Hana-tsuno-sunaginchaku (new Japanese name).
Concatenated alignment. All Antipathozoanthus species together formed a large monophyletic clade within the Parazoanthidae with complete support (ML = 100%, BI = 1) in the concatenated (COI+16S-rDNA+ITS-rDNA) alignment phylogeny (Fig.
COI. All Antipathozoanthus species formed a large monophyletic clade within the Paraozoanthidae with a very strong support (NJ = 99%, ML = 99%, BI = 1) in the COI phylogeny (Suppl. material
16S-rDNA. All Antipathozoanthus species formed a large monophyletic clade within the Parazoanthidae with generally high support (NJ = 99%; ML = 85%; BI = 1) in the 16Sr-DNA phylogeny (Suppl. material
ITS-rDNA. All Antipathozoanthus species formed a large monophyletic clade within the Parazoanthidae with complete support (NJ = 100%, ML = 100%, BI = 1) in the ITS-rDNA phylogeny (Suppl. material
Distribution of Antipathozoanthus species in the Indo-Pacific Ocean. Antipathozoanthus hickmani is found in only the Galapagos with A. cf. hickmani reported from the coast of Ecuador (
Evolution of macrocnemic zoantharians in caves. Antipathozoanthus obscurus sp. n. without host was found in similar environments as the ‘associated’ Antipathozoanthusspecies, but this species does not associate with antipatharians and is instead directly attached to coral reef carbonate.
All new species in the present study are azooxanthellate, and this trait is common within macrocnemic zoantharians to the exception of some species such as Bergia cutressi (West, 1979) and Nanozoanthus harenaceus Fujii & Reimer, 2013.
Substrate specificity within Antipathozoanthus. Within the family Parazoanthidae, different generic lineages likely have long evolutionary histories associated with their substrate organisms, based on the fact that many parazoanthid genera form monophyletic clades in accord to their substrates (
We thank the following people and institutions for supporting fieldwork and logistics; in Okinawa, all members of the Molecular Invertebrate Systematics and Ecology Laboratory (MISE) at the University of Ryukyus (UR), the KUMEJIMA 2009 expedition organized by the Transdisciplinary Research Organization for Subtropical and Island Studies of the University of the Ryukyus, the Center for Marine Bioscience & Biotechnology of the National Taiwan Ocean University, National University of Singapore, and the Biodiversity Research Center of the Academia Sinica; in Kagoshima, S. Dewa (Diving Service Umiannai), M. Matsuoka, R. Terada (both Kagoshima University) and D. Probizanski; in Palau, G. Mereb and A. Merep at the Palau International Coral Reef Center (PICRC) supported by the SATREPS P-CoRIE Project “Sustainable management of coral reef and island ecosystem: responding to the threat of climate change", funded by the Japan Science and Technology Agency (JST) and the Japan International Cooperation Agency (JICA) in cooperation with PICRC and Palau Community College. H. Takaoka (Okinawa Churaumi Aquarium, Okinawa) is acknowledged for identification of antipatharians. We also thank the University of Milano-Bicocca Marine Research and High Education Centre in Magoodhoo, the Ministry of Fisheries and Agriculture, Republic of Maldives and the community of Maghoodhoo, Faafu Atoll, for field work in the Maldives, and M.L. Berumen at the King Abdullah University of Science and Technology (KAUST), Saudi Arabia for Red Sea field work. The second author was partially supported by the “Establishment of Research and Education Network on Biodiversity and Its Conservation in the Satsunan Islands" project of Kagoshima University adopted by the Ministry of Education, Culture, Sports, Science and Technology, Japan and JSPS KAKENHI Grant numbers 17K15198 and 17H01913. Comments by Dr. S. Stampar and the editor improved the manuscript.
List of GenBank accesion number
Data type: GenBank accession numbers
Explanation note: GenBank accession numbers, names and details of the sequences used in phylogenetic analyses of COI, 16S-rDNA and ITS-rDNA in this study. Sequences that were concatenated are indicated by bold text.
Phylogenetic tree of COI
Data type: phylogenetic tree
Explanation note: Maximum likelihood (ML) tree based on cytochrome oxidase subunit I sequences. Numbers on nodes represent ML and neighbor-joining (NJ) bootstrap values (> 50% are shown). Bold branches indicate high supports of Bayesian posterior probabilities (> 0.95).
Phylogenetic tree of 16S-rDNA
Data type: phylogenetic tree
Explanation note: Maximum likelihood (ML) tree based on mitochondrial 16S ribosomal DNA sequences. Numbers on nodes represent ML and neighbor-joining (NJ) bootstrap values (> 50% are shown). Bold branches indicate high supports of Bayesian posterior probabilities (> 0.95).
Phylogenetic tree of ITS-rDNA
Data type: phylogenetic tree
Explanation note: Maximum likelihood (ML) tree based on internal transcribe spacer region of ribosomal DNA sequences. Numbers on nodes represent ML and neighbor-joining (NJ) bootstrap values (> 50% are shown). Bold branches indicate high supports of Bayesian posterior probabilities (> 0.95).