Zoanthid species identities and comparisons with previous morphological identifications
Results from the phylogenetic analyses of zoanthid specimens in this study generally confirm conclusions previously reached by Reimer et al. (2011b). Identifications in the previous study of specimens utilizing morphological features corresponded to phylogenetic species groupings observed in this study in almost all cases. In particular, results for specimens of Z. sansibaricus, Z. gigantus, Z. kuroshio, A. australiae (also reported in Reimer et al. 2011a), P. heliodiscus, P. tuberculosa, and P. mutuki matched the 2011 identifications. As previously reported (Reimer et al. 2006c, 2007c), P. mutuki may consist of more than one species, and evidence for this can be seen in the mt 16S rDNA tree with two separate groupings in this species, and in the large variety within the species' ITS-rDNA clade. Palythoa tuberculosa ITS-rDNA sequences from Taiwan were often distinct from those of Japanese specimens but formed multiple clades. Given previous reports on P. tuberculosa (Reimer et al. 2007c) showing that this species has large amounts of ITS-rDNA variation, we feel that the explanation that this is one species is much more likely than the alternate hypothesis that there are many species in Taiwan (and Japan and the Indo-Pacific) with overlapping morphology and distributions.
One important finding from this study was the confirmation of subtle phylogenetic differences between Z. kuroshio and Z. vietnamensis (referred to as Z. aff. vietnamensis in Reimer et al. 2011b). It is known that these two species are phylogenetically very closely related, and the status of Z. kuroshio as a valid independent species has been in question (Reimer et al. 2006b). However, results from this study confirm the separation of these two species. A small phylogenetic difference (1 to 2 bp in mt 16S rDNA + COI sequences combined) was shown to be adequate to separate other zoanthid species (e.g., P. tuberculosa and P. mutuki) due to the slow rate of mtDNA mutations in Anthozoa (Shearer et al. 2002; Huang et al. 2008), and given the obvious morphological differences between these two groups (reminiscent of P. tuberculosa and P. mutuki), it is almost certain that these are two separate species. Further evidence comes the distribution of these two species in Taiwan, with Z. kuroshio only observed in coral reef environments (Kenting and Green Island) and Z. vietnamensis appearing most common at Keelung, a non-reef location. This is similar to the species' distribution in Japan, with Z. kuroshio most commonly seen in the southern Ryukyu Islands, and Z. vietnamensis reported from colder, more northerly locations and very rarely from the Ryukyus (Reimer 2010). Z. vietnamensis was described from Vietnam (Pax and Müller 1957) in the tropical South China Sea, but the coastline of Vietnam occasionally experiences winter sea surface temperatures of <20°C (Japan Oceanographic Data Center; JODC), and morphologically, the Z. vietnamensis specimens reported here agree well with the original description, while Z. kuroshio was described from the Ryukyu Archipelago, which is influenced by the warm Kuroshio Current, and the type locality of Yakushima also has low winter sea surface temperatures of approximately 20°C (JODC). Zoanthus vietnamensis may be a subtropical/temperate species, and Z. kuroshio a tropical/subtropical species, with some overlap in their distributions. Another possibility is that these two species prefer different environments, with the “immersae” (= polyps embedded in well-developed coenenchyme; Pax 1910) Z. kuroshio on exposed reef crests and reef slopes (Irei et al. 2011) and the “liberae” (= polyps free and clear of coenenchyme) Z. vietnamensis in more sheltered locations. However, no investigation into the habitat preference of Z. vietnamensis has been conducted, and therefore, research into the sexual reproduction, ecology, and distribution of these two species should help further clarify the differences between these closely related species.
One additional finding of this study is that there is likely more than one undescribed Palythoa species in the northwestern Pacific. Some specimens in this study were seen in the phylogenies to be closely related to undescribed Palythoa sp. “sakurajimensis”, with other specimens previously reported from southern Japan (Reimer et al. 2007c) and the Ogasawara Islands (Reimer et al. 2011a). These specimens were initially identified in Reimer et al. (2011b) as either P. mutuki or Palythoa sp. In this study and in previous studies, Palythoa sp. “sakurajimensis” specimen numbers were low compared to other Palythoa species, and colonies consisted only of one or a few polyps. Distinguishing this putative species from P. mutuki based on morphology is currently very difficult, with Palythoa sp. “sakurajimensis” being found at slightly deeper depths than P. mutuki. A concerted effort to collect more Palythoa sp. “sakurajimensis” specimens from each region and their examination are needed to formally describe this species.
Another potentially undescribed species is represented by specimen YI2, which was identified as P. heliodiscus based on COI and mt 16S rDNA phylogenies, but it was previously identified as Palythoa sp. or Palythoa aff. heliodiscus based on morphology (Reimer et al. 2011b). A differing oral disc color (purple and/or green opposed to brown of P. heliodiscus) and ITS-rDNA results (Figure 3) indicate some differences between this specimen and P. heliodiscus. Similar insertions were seen in morphologically similar specimens from Singapore and Japan (P.A. Todd, T. Nishimura, and J.D. Reimer, unpublished data). As with Palythoa sp. “sakurajimensis”, colonies of this putative species are likely to be rare, small, and cryptic.
For Isaurus tuberculatus reported in Reimer et al. (2011b), specimens were only observed by a third party and not collected, and no unforeseen results (e.g., undescribed species) are expected regarding their identity based on previous results (Muirhead and Ryland 1985; Reimer et al. 2008). Specimens of the “unknown zoanthid” from Reimer et al. (2011b) were also only observed but not collected, and these are very likely species within the recently described genus Microzoanthus Fujii & Reimer 2011, based on their extremely small size and morphological similarity to that genus.
Results of this study demonstrate that mt 16S rDNA sequences can more clearly define closely related species of zoanthids than can COI sequences. In this and previous studies, COI sequences failed to resolve the following species groups from each other: Z. vietnamensis + Z. kuroshio and P. mutuki + Palythoa sp. “sakurajimensis”. A COI sequence from one specimen of P. mutuki was also seen within the P. tuberculosa clade. Future phylogenetic work on brachycnemic zoanthids should always include mt 16S rDNA for this reason, and ITS-rDNA if possible.
Symbiodinium spp. diversity in zoanthids in Taiwan
Results of symbiont identification of Taiwanese zoanthids reflect other recent results reported from Japan, with some small but potentially important differences. Overall, association patterns as previously noted from different sites in Japan between various zoanthid host species and their symbionts were generally observed in this study. For example, the majority of Taiwanese P. tuberculosa (42 of 47 colonies or 89%) and P. mutuki (9 of 12 colonies or 75%) were associated with subclade C1 or closely related types, as reported in Japan (Reimer et al. 2006d). No Palythoa spp. were observed in association with clade D, which was reported from the Indian Ocean (Burnett 2002) and Singapore (Reimer and Todd 2009). Symbiodinium subclade C1 was shown to be a 'generalist’ in terms of both associations and preferred environments, and together with subclade C3, is the most common Indo-Pacific type (LaJeunesse 2005). The lone A. australiae examined from Green Island was associated with clade D, as was previously reported from Kenting and Green Island (Reimer et al. 2010). Furthermore, Z. sansibaricus was associated with either C3-derived Symbiodinium (two of three colonies) or clade A (light tolerant) in shallow water, as was seen in Japan (Reimer et al. 2006e, 2007b). Finally, all three Z. kuroshio specimens examined had Symbiodinium ITS sequences identical to previously reported specimens from Japan (Reimer et al. 2006e) and Singapore (Reimer and Todd 2009).
However, some small differences from previous reports were seen. At Penghu, half (four of eight colonies) of P. tuberculosa specimens had a novel Symbiodinium C subclade derived from C1. This subclade had very high bootstrap support. These results suggest that P. tuberculosa can potentially associate with more than 1 clade C type. Palythoa tuberculosa is known to be a generalist species (Irei et al. 2011; Polak et al. 2011) with a high larval dispersal potential (Hirose et al. 2011; Polak et al. 2011), and associations with different symbiont types in different environments would seem to be a good strategy to adapt to different environments. However, while symbioses with clade D were observed in the Indian Ocean and Singapore, in the Pacific until now, P. tuberculosa is only known to associate with clade C1/C3 or very closely related types, even in remote oceanic locations such as the Ogasawara (Reimer et al. 2011a) and Galapagos Islands (Reimer and Hickman 2009). The marine environment of Penghu is quite different from Kenting and other subtropical and tropical areas in southeastern Taiwan, as winter water temperatures may dip to <15°C (Taiwan Central Weather Bureau), and often, the water is relatively turbid during the monsoon season (Huang et al. 2012a, b), and one or more of these factors may be contributed to the presence of this novel symbiont type. The occurrence of this divergent and novel type was somewhat unexpected, and further effort should be put into examining specimens from other areas near Penghu and the South China Sea, for which very few zoanthid data exist.
Another novel clade of symbionts was seen in Z. vietnamensis from Keelung. Additionally, it appeared that while Z. kuroshio from both Taiwan and Japan was associated only with one type of symbiont (C15/C91-derived, see Reimer et al. 2006e, 2007b), Z. vietnamensis is apparently more flexible in its association. Of the seven colonies examined in this study, two were associated with the new novel subclade (both at Keelung) and two with C15/C91 (identical to Z. kuroshio; both from Green Island), two were closely related to C15/C91 (Keelung/Green Island), and one was associated with subclade C3 (Keelung). Despite the examination of only seven colonies, the variety of types observed within Z. vietnamensis is surprising given its very close phylogenetic relationship with Z. kuroshio (Reimer et al. 2006b), and the fact that many other closely related zoanthid species are associated with similar or identical symbiont types, even in different oceans (e.g., Atlantic P. caribaeroum, sibling species to P. tuberculosa, harboring subclade C1 in Figure 1). Increasing specimen numbers with the inclusion of more specimens from the South China Sea (which is the type locality) and the Pacific coast of mainland Japan could also help clarify the nature of Z. vietnamensis' symbiont associations.
Finally, within this study, we examined zoanthid species for which no information on symbionts previously existed. Z. gigantus, P. heliodiscus, and Palythoa sp. “sakurajimensis” are not as common as the above mentioned zoanthid species (e.g., Irei et al. 2011) and are known to be distributed in both Taiwan and some locations in Japan (e.g., Reimer et al. 2011a). Specimen numbers of these species in this study are low (n = 2 to 4), and therefore, speculation on symbiont association patterns would be premature. The data acquired in this study should be compared to future, more in-depth studies. While all three of these species are usually associated with the “generalist” Symbiodinium C1 or C3 type, surprisingly, one Palythoa sp. “sakurajimensis” specimen from Kenting in southern Taiwan was associated with the C15/C91 type. This is the first time a Palythoa species has been reported to harbor this symbiont type. However, this species group contains multiple genotypes, and it is possible that it encompasses more than one species. Thus, any conclusions on host-symbiont associations for this group should be treated with caution.