**Zooxanthellate, Sclerite-Free, and Pseudopinnuled Octocoral** *Hadaka nudidomus* **gen. nov. et sp. nov. (Anthozoa, Octocorallia) from Mesophotic Reefs of the Southern Ryukyus Islands**

**Yee Wah Lau 1,\* and James Davis Reimer 1,2**

http://zoobank.org/urn:lsid:zoobank.org:pub:1AB2F0C1-FAB0-40B0-AB7A-C07A296E9C50 Received: 25 August 2019; Accepted: 18 September 2019; Published: 22 September 2019

**Abstract:** Shallow water coral reefs are the most diverse marine ecosystems, but there is an immense gap in knowledge when it comes to understanding the diversity of the vast majority of marine biota in these ecosystems. This is especially true when it comes to understudied small and cryptic coral reef taxa in understudied ecosystems, such as mesophotic coral reef ecosystems (MCEs). MCEs were reported in Japan almost fifty years ago, although only in recent years has there been an increase in research concerning the diversity of these reefs. In this study we describe the first stoloniferous octocoral from MCEs, *Hadaka nudidomus* gen. nov. et sp. nov., from Iriomote and Okinawa Islands in the southern Ryukyus Islands. The species is zooxanthellate; both specimens host *Cladocopium* LaJeunesse & H.J.Jeong, 2018 (formerly *Symbiodinium* 'Clade C') and were collected from depths of ~33 to 40 m. Additionally, *H. nudidomus* gen. nov. et sp. nov. is both sclerite-free and lacks free pinnules, and both of these characteristics are typically diagnostic for octocorals. The discovery and morphology of *H. nudidomus* gen. nov. et sp. nov. indicate that we still know very little about stoloniferous octocoral diversity in MCEs, their genetic relationships with shallower reef species, and octocoral–symbiont associations. Continued research on these subjects will improve our understanding of octocoral diversity in both shallow and deeper reefs.

**Keywords:** *Cladocopium*; cryptofauna; marine biodiversity; mesophotic coral reef environments (MCEs); Octocorallia; stoloniferous octocorals; Symbiodiniaceae; taxonomy

#### **1. Introduction**

Coral reefs make up only 0.2% of the earth's ocean but are estimated to harbor a quarter of all marine species [1,2] and are the most diverse marine ecosystems on the planet. Unfortunately, these diverse marine communities are also one of the most threatened [3–6]. The 'hotspot' concept, a term used to mark a relatively restricted geographic area accommodating exceptionally high concentrations of biodiversity and endemism [7–9] has highlighted the wealth of species that are at risk and how localized such areas of richness can be [10]. However, there are vast gaps in knowledge concerning the majority of marine biota [11,12], making the recognition of biodiversity geographic patterns and hotspots questionable [13,14], as priorities identified for one taxon may not reflect the diversity of other taxa [14,15]. This is especially true for understudied localities and environments, such as understudied coral reef ecosystems.

Mesophotic coral reef ecosystems (MCEs) occur at depths below 30–40 m to 100 m or deeper in tropical and sub-tropical regions [16–19]. MCEs are considered understudied, as their depths make them difficult to access via normal SCUBA technology, yet too shallow for most submersibles [19,20]. However, research regarding MCEs has increased in recent years, along with calls for increased awareness and protection of these ecosystems [21]. Additionally, studies have demonstrated that MCEs can accommodate high levels of endemism [19,22] and harbor distinct geographical communities [19].

The coral reefs of southern Japan are at the top of the list in terms of global marine conservation priority, when considering the region's high levels of multi-taxon endemism and the high risk of biodiversity loss due to overexploitation and coastal development [23]. The Ryukyus Islands (RYS), i.e., Ryukyu Archipelago, encompass the southernmost region of Japan and include islands of different geological formations, ages, and sizes [24,25]. The surrounding waters and coral reefs fringing the islands are strongly influenced by the warm water brought from tropical areas around the Philippine islands by the Kuroshio Current, which flows towards the north along the west side of the island chain [24–26], extending warm water conditions northerly. As such, the RYS experience higher sea temperatures compared to other areas at similar latitudes, such as eastern Australia [27,28], thus creating unique coral reef conditions. Serious taxonomic and geographic biases are present in marine biodiversity research in the RYS. Most work in the RYS has been conducted on the phyla Pisces, Crustacea, and Cnidaria, with the majority of research on hermatypic hard corals (Scleractinia) and, surprisingly, far less work on other commercially important groups such as Echinodermata and Mollusca, as well as on other understudied small and cryptic coral reef taxa [25].

One such understudied small and cryptic group are octocorals belonging to the subordinal group, Stolonifera. Stoloniferan octocorals are characterized by having relatively simple colony growth forms, where the polyps are united basally by ribbon-like stolons, instead of being embedded side by side within a common coenenchymal mass [29–31]. There are seven families that are considered to belong to Stolonifera: Acrossotidae Bourne, 1914; Arulidae McFadden & Van Ofwegen, 2012; Clavulariidae Hickson, 1894; Coelogorgiidae Bourne, 1900; Cornulariidae Dana, 1846; Pseudogorgiidae Utinomi & Harada, 1973; and Tubiporidae Ehrenberg, 1828. The most speciose as well as the most studied family is Clavulariidae, which comprises approximately 30 genera and over 60 species. Until recently, all other families are all either monospecific or monogeneric, with no more than a few described species; recent studies have additionally introduced new genera and species for Arulidae [32,33], which is the most recently erected family.

Stoloniferous octocorals often have inconspicuous small colonies and polyps, which makes them hard to detect [32–34]. There are critical gaps that remain in the understanding of the functional and ecological significance of octocoral–zooxanthellae symbioses [35]. To date, only a handful of data are available on stoloniferous octocoral–symbiont relationships, which all concern members of the speciose genus *Clavularia* Blainville, 1830. *Clavularia* spp. from Australia all hosted *Durusdinium* LaJeunesse, 2018 [36,37]. On one other occasion, a single *Clavularia* sp. specimen from the Caribbean was found to host *Durusdinium* [38].

Obligate mutualistic symbioses play important roles in extending available energy resources and thus potentially influence biodiversity on reefs [36,39]; however, stoloniferous octocorals and their host–symbiont associations are a relatively underexamined fauna in the RYS, particularly from within MCEs. In this study we formally describe the zooxanthellate, sclerite-free, and pseudopinnuled octocoral *Hadaka nudidomus* gen. nov. et sp. nov. from MCEs around Okinawa and Iriomote Islands.

#### **2. Materials and Methods**

#### *2.1. Specimen Collection and Morphological Examinations*

One specimen was collected from one location each around Okinawa (August 2017; 26.856412 N, 128.245093 E) and Iriomote (December 2016; 24.370413 N, 123.736428 E) Islands (Figure 1). The specimens were found at depths of 33 and 40 m, respectively, by means of SCUBA (atmospheric

air) and were preserved in 70–90% ethanol and subsamples in 95% ethanol. The current study is part of an ongoing survey of mesophotic and deep reef work. Vouchers and type material were deposited at the National Museum of Nature and Science (NSMT), Tokyo, Japan (Table 1). Both specimens were examined for the presence of sclerites by dissolving entire polyps and stolons in 4% hypochlorite (household bleach). Additionally, to visualize polyp tentacles and pseudopinnules, polyps were fixed in 20% formalin and embedded in methylene blue (1%).

#### *2.2. DNA Extraction, Amplification, and Sequencing*

DNA was extracted from polyps using a DNeasy Blood and Tissue kit (Qiagen, Tokyo, Japan). PCR amplification and sequencing were performed for four markers, of which three were mitochondrial (cytochrome c oxidase subunit I (COI), the MSH homologue mtMutS, and subunit ND6) and the fourth was the nuclear ribosomal marker (28S rDNA). Additionally, for Symbiodiniaceae, the nuclear internal transcribed spacer (ITS) region of ribosomal DNA was amplified. Protocols in [34] were followed and PCR products were treated with Exonuclease I and alkaline phosphate (shrimp) and sent for bidirectional sequencing on an ABI 3730XL (Fasmac, Kanagawa, Japan). Sequences were assembled and edited using Geneious R11 [40] and BioEdit [41]. COI, mtMutS, and ND6 were checked for introns, exons, and stop-codons in AliView [42].

**Figure 1.** Map of the Ryukyus Islands (RYS), with the six island group divisions (grey dotted lines) and the two dive locations where *Hadaka nudidomus* gen. nov. et sp. nov. specimens were found (red dots) at Iriomote (NSMT-Co 1681, holotype) and Okinawa (NSMT-Co 1682, paratype) Islands.

**Table 1.** Overview of information on octocoral specimens collected from mesophotic coral reef ecosystems (MCEs) at Iriomote and Okinawa Islands, Okinawa Prefecture, Japan, including GenBank accession numbers and locality. Catalogue number: NSMT = National Museum of Nature and Science, Tokyo, Japan; n.a. = not available.

#### *2.3. Molecular Phylogenetic Analyses*

Multiple sequence alignments were performed using MAFFT 7 [43] and coding markers were aligned using MACSE [44] under default parameters. The phylogenetic position of the collected specimens (*n* = 2) was determined by aligning the consensus sequences for markers 28S rDNA, COI, and mtMutS to a reference dataset of 124 octocoral genera, including *Cornularia pabloi* and *Cornularia cornucopiae* as outgroup (total *n* = 144), as used in Lau and Reimer [33]. This resulted in alignments of 887 bp for 28S rDNA, 717 bp for COI, and 714 bp for mtMutS, and a total concatenated three-marker dataset of 2318 bp. The separate markers were run in ML analyses, to check for contamination and congruency (Supplementary Materials Figures S1–S3).

A separate phylogenetic analysis was made to examine the lower level phylogenetic relationships of the collected mesophotic specimens, using a concatenated four-marker dataset. The concatenated four-marker dataset resulted in an alignment of 2670 bp (total *n* = 12). A total of seven reference species were included in the analysis, which clustered in nearby clades with the specimens in the three-marker dataset, including *Rhodelinda* sp. and *Telesto* sp. as outgroup. The four separate markers (28S rDNA, 787 bp; COI, 708 bp; mtMutS, 734 bp; ND6, 441 bp) were also run in ML analyses, to check for contamination and congruency (Supplementary Materials Figures S4–S7).

Additionally, ITS sequences from the two specimens were aligned with a total of 25 reference sequences (*Cladocopium* spp. and *Durusdinium* spp.), including *Gerakladium* sp. as outgroup. The resulting dataset comprised 641 bp and a total of 27 sequences and was run in ML analyses (Supplementary Materials Figure S8).

Alignments of the separate markers were concatenated using SequenceMatrix 1.8 [45]. ML analyses were run with RAX-ML 8 [46], using the GTRCAT model. The best ML tree was calculated using the –D parameter. A multi-parametric bootstrap search was performed, which automatically stopped based on the extended majority rule criterion. The Bayesian inference was performed with ExaBayes 1.5 [47] using the GTR substitution model. Four independent runs were run for 10,000,000 generations during which convergence (with a standard deviation of split frequencies < 2%) was reached. Bootstrap supports and posterior probabilities were depicted on the branches of the best ML tree using P4 [48]. The resulting trees were visualized in FigTree 1.4.2 [49]. Additionally, average distance estimations within species and within genera were computed using MEGA X [50] by analyzing pairwise measures of genetic distances (uncorrected *P*) among sequences (Supplementary Materials Tables S1–S3).

#### **3. Systematic Account**

Class Anthozoa Subclass Octocorallia Ehrenberg, 1831 Order Alcyonacea Lamouroux, 1812 Family Clavulariidae Hickson, 1894

#### *3.1.* Genus *Hadaka* gen. nov.

*Type species*: *Hadaka nudidomus* sp. nov. by original designation and monotype.

*Diagnosis*: Colony with polyps connected through flattened ribbon-like stolons, which are loosely attached to a hard substrate. Polyps retract fully into the calyx, which is cylindrical to conical in shape, narrowing at the base and does not retract fully into the stolon. Tentacles have a wide rachis with a protruding ridge and pseudopinnules of different lengths arranged on either side, giving the polyps feather shaped tentacles. No sclerites. Zooxanthellate.

*Remarks*: *Hadaka* gen. nov. et sp. nov. shows gross resemblance to *Hanabira* Lau, Stokvis, Imahara & Reimer, 2019 in having a similar polyp shape with feather or petal shaped tentacles and fused pinnules, which can still be distinguished by shallow furrows. *Hadaka* gen. nov. et sp. nov. differs from *Hanabira* in having no sclerites in any part of the colony and having a protruding ridge on the upper side of the tentacle. Genetically, *Hadaka* gen. nov. is well-supported and positioned in a different phylogenetic clade from *Hanabira*. The closest sister taxa of *Hadaka* gen. nov. is *Acrossota* Bourne, 1914, which is also sclerite-free, but morphologically very different; *Acrossota* lacks pinnules completely.

*Etymology*: From the Japanese word *hadaka* (裸), meaning naked, bare, nude; denoting the absence of two characteristic features of octocorals, sclerites, and free pinnules. Gender: feminine.

http://zoobank.org/39430672-5ADA-4EFF-9F5A-B4076B6B90C0

#### *3.2. Hadaka nudidomus* sp. nov.

#### See Figure 2.

*Material examined*: All specimens were collected from Okinawa Prefecture, Japan. *Holotype*: NSMT-Co 1681, northeast Uchibanare, Iriomote Island (24.370413 N,123.736428 E), ~40 m depth, 19 December 2016, coll. D. Uyeno. GenBank accession numbers: 28S rDNA, MN488601; COI, MN488603; mtMutS, MN488605. *Paratype*: NSMT-Co 1682, entrance to Hedo Dome, Cape Hedo, Okinawa Island (26.856412 N, 128.245093 E), 33 m depth, 18 August 2017, coll. J.D. Reimer. GenBank accession numbers: 28S rDNA, MN488602; COI, MN488604; ND6, MN488606.

*Description*: Holotype colony consists of 15 polyps with flattened ribbon-like stolons encrusting a sponge. Polyps can be seen individually or clustered in groups and are spaced apart irregularly, 3 mm to 2 cm in between polyps and clusters. Stolons are 0.5 mm at their narrowest and 1 mm at their widest point. Polyps retract fully into the calyx (~1.8 mm wide and ~3.55 mm in length), which is cylindrical to conical shaped, narrowing at the base, and does not retract fully into the stolon. Expanded polyps are ~4–5 mm diameter in life. Tentacles have a wide rachis with a protruding ridge on the upper side and long pseudopinnules arranged on either side (~24–26 pseudo-pairs), giving the polyps feather shaped tentacles. When stained with methylene blue, the outline of the tentacles can be observed. Structures of the pinnule axis are visible; however, the notches that distinguish the pseudopinnules are not observed in the contour of the tentacle (Figure 2d). No sclerites were found in any parts of the specimens. Polyps are brown in life and yellowish-white in ethanol (Figure 2c). Zooxanthellate.

*Morphological variation*: There is a difference in color between the polyps of the holotype (NSMT-Co 1681) and paratype (NSMT-Co 1682); the polyps of the holotype are brown with a white oral disc and base of the tentacles and the polyps of the paratype are whitish yellow with a bright blue oral disc (Figure 2a,b).

*Distribution*: Southwestern Japan, southern Ryukyus Islands, around northern Okinawa Island, and inside the bay of western Iriomote Island in the East China Sea. Specimens were collected from depths of ~33–40 m.

*Remarks*: The polyps of paratype NSMT-Co 1682 were all used for DNA extraction and sclerite examination, as they were initially thought to be a *Hanabira yukibana* specimen; three fragments of rock with stolon remain. The holotype colony (NSMT-Co 1681) was attached to sponge tissue, but this epibiont is not obligate, as the paratype was attached to rock.

 The paratype (NSMT-Co 1682) was found on consolidated hard carbonate bottom. Both colonies were on the upward-facing side of the bottom.

*Etymology*: From Latin *nudus*, meaning naked or bare, and *domus*, meaning home or house; denoting the 'naked' host habitat in which the zooxanthellae reside, as the species is sclerite-free. http://zoobank.org/71620752-8C33-4DCE-9B6E-DD7FC2DA3E20

### **4. Molecular Results**

This study added a total of six sequences of *Hadaka nudidomus* gen. nov. et sp. nov. to the public reference database GenBank and no barcodes were available before. For the family Symbiodiniaceae, two *Cladocopium* spp. sequences were added. The phylogenies resulting from the ML analyses of the separate markers (COI, 28S rDNA, mtMutS, ND6) were highly congruent with those from the concatenated alignments for both the three- and four-marker datasets (Supplementary Materials Figures S1–S7). ML and Bayesian analyses for the concatenated datasets yielded almost identical tree phylogenies (Supplementary Materials Figure S9). Sequences of *Hadaka nudidomus* gen. nov. et sp. nov. collected from Okinawa and Iriomote Islands formed a completely-supported clade, containing sclerite-free species only: species of clavulariid genus *Phenganax* Alderslade & McFadden, 2011 and monospecific acrossotid genus *Acrossota* Bourne, 1914 in both the three- and four-marker analyses (Figures 3 and 4).

**Figure 2.** Photographs of *Hadaka nudidomus* gen. nov. et sp. nov.: (**a**) in situ holotype NSMT-Co 1681, scale bar approximately 1 mm; (**b**) in situ paratype NSMT-Co 1682, scale bar approximately 1 mm; (**c**) holotype in ethanol, scalebar 1 mm; (**d**) holotype in methylene blue staining, scale bar 0.1 mm.

**Figure 3.** Phylogenetic relationships among 122 octocoral genera (total *n* = 144), including two species, *Hadaka nudidomus* gen. nov. et sp. nov. (highlighted red), collected at Iriomote and Okinawa Islands using the combined 28S rDNA + COI + mtMutS dataset. The best maximum likelihood tree is shown, with values at branches representing bootstrap probabilities (shown when >70%; top/left) and Bayesian posterior probabilities (shown when >0.80; bottom/right; A = 1.00, B = 0.95–0.99, C = 0.90–0.94, D = 0.80–0.89). \* represents 100%/1.00 for both analyses. Non-stoloniferous families are shown with family classification only and stoloniferous families are highlighted in grey. Sclerite-free species are indicated with a blue dot. Species that are both sclerite-free and lack free pinnules are indicated with a blue circle. *Cornularia* spp. were used as outgroup.

*Hadaka* gen. nov. was sister to the remaining clade in the three-marker analyses; however, in the four-marker analyses, *Phenganax* was sister to *Hadaka* gen. nov. and *Acrossota*. Nonetheless, in both phylogenies, the two specimens of *Hadaka* gen. nov. formed a completely-supported clade.

Additionally, genetic distances gave further support to phylogenetic affinities and morphological features justifying the establishment of a new genus. Between-genus distances (*Hadaka* compared to *Acrossota* and *Phenganax*) for COI were 2.52–2.54% and 7.69–13.33% for mtMutS, which are well above the intergeneric range for octocorals [51]. Additional comparisons between *Hadaka nudidomus* sp. nov. specimens, *Acrossota amboinenesis* Burchardt, 1902 and *Phenganax* spp.; *Phenganax parrini* Alderslade & McFadden, 2011, *Phenganax marumi* Lau & Reimer, 2019, *Phenganax subtilis* Lau & Reimer, 2019, *Phenganax stokvisi* Lau & Reimer, 2019, also resulted in ranges (COI: 2.15–2.97%, MSH: 7.00–13.33%) that indicated that *Hadaka* gen. nov. specimens belong to a different genus (Supplementary Materials Tables S1–S3). There were no differences (0%) when comparing genetic distances within the two *Hadaka* specimens, indicating that the specimens are of the same species.

*Hadaka nudidomus* gen. nov. et sp. nov. specimens were analyzed for the presence of zooxanthellae, and identical sequences of Symbiodiniaceae were found. Both *Hadaka nudidomus* specimens collected from Okinawa and Iriomote Island hosted *Cladocopium* LaJeunesse & H.J.Jeong, 2018 (formerly *Symbiodinium* 'Clade C').

**Figure 4.** Phylogenetic reconstruction using a four-marker concatenated dataset (28S rDNA + COI + mtMutS + ND6) among *Hadaka nudidomus* gen. nov. et sp. nov., closest sister species *Phenganax* spp. and outgroup specimens *Rhodelinda* sp. and *Telesto* sp. (total *n* = 12). The best maximum likelihood tree is shown, with values at branches representing bootstrap probabilities in percentages (top/left) and Bayesian posterior probabilities (bottom/right). In situ photographs are shown for the two octocoral species that are sclerite-free and lack free pinnules, *Hadaka nudidomus* gen. nov. et sp. nov. and *Acrossota amboinensis*. Photograph credit: in situ image RMNH Coel. 40798 *Acrossota amboinensis*, by Daniel Knop (modified from [52]; reproduced with permission from copyright holder).

#### **5. Discussion**

In the three- and four-marker phylogenies, there was disparity in the position of *Hadaka nudidomus* gen. nov. et sp. nov. and *Acrossota amboinensis*. It remains unresolved how these genera and genus *Phenganax* are related to one another. A possible explanation could be that there is no sufficient signal in the sequences of both *Hadaka nudidomus* gen. nov. et sp. nov. and *Acrossota amboinenesis* due to the fact that the closest relatives for these genera are yet to be discovered.

Morphologically, there was a difference between the coloration of the polyps of the holotype and paratype found at Iriomote and Okinawa Islands, respectively. The differences in coloration suggested that perhaps the specimens hosted different members of Symbiodiniaceae. However, both specimens hosted genus *Cladocopium* and thus no biogeographical distinction in Symbiodiniaceae was observed. Members of *Cladocopium* spp. are known to be adapted to a wide range of temperatures and irradiances [53], which would be expected from MCEs, where irradiances are not only subject to seasonal variations but are already reduced.

*Hadaka nudidomus* gen. nov. et sp. nov is the first zooxanthellate stoloniferous octocoral described from mesophotic depths. Only one other zooxanthellate octocoral, an alcyoniid species, *Sinularia mesophotica* Benayahu, McFadden, Shoham & van Ofwegen, 2017, has been explicitly described from mesophotic depths [54]. However, it was not further specified which genus or species of Symbiodiniaceae was hosted by *S. mesophotica* and therefore, we cannot yet hypothesize differences of zooxanthellae hosted by octocorals from MCEs.

Nonetheless, another recent study has shown that there are geographical differences in the genera of Symbiodiniaceae in *Hanabira yukibana* Lau, Stokvis, Imahara & Reimer, 2019 from shallow coral reefs, as specimens found from Okinawa Island hosted *Cladocopium* while *Durusdinium* LaJeunesse, 2018 was hosted in specimens from Iriomote Island. However, in this previous study, similar to the current study, no consistently different patterns of polyp coloration related to symbiont associations were observed [34]. To this end, finer-scale examinations of Symbiodiniaceae using faster-evolving DNA markers [55] may reveal patterns yet unseen.

*Hadaka nudidomus* gen. nov. et sp. nov. is the second species within Octocorallia after *Acrossota amboinensis* that has no sclerites in any part of the colony and also no free pinnules; both species are taxonomically placed within family Clavulariidae. *Acrossota amboinensis* differs from *Hadaka nudidomus* gen. nov. et sp. nov. in colony form, polyp morphology, and habitat (Figure 4); *A*. *amboinensis* does not have pseudopinnules, but instead lacks pinnules completely and has, so far, not been found at mesophotic depths. When comparing *A*. *amboinensis* to *Phenganax* spp. there are also distinct morphological differences; all *Phenganax* species have free pinnules and have completely different polyp and tentacle shapes. Nonetheless, the genera *Acrossota* and *Phenganax* are phylogenetically closely related. In a recent study, the phylogenetic topology for these genera was different from that generated in the current study [33], in which *Acrossota* is placed basally to all *Phenganax* species. As a result of the unresolved phylogenetic location of *Acrossota* within Clavulariidae, while it is clear these three genera are distinct, it remains unclear how *Hadaka* and *Phenganax* are related to *Acrossota*.

Moreover, it can be concluded that several octocoral species lack both sclerites and free pinnules, and thus, that such features are not completely rare in octocorals, which raises important implications for the definition of subclass Octocorallia, as sclerite characterization and the presence of pinnated tentacles are two of the major diagnostic features of the group [52,56,57].

It is clear that more species diversity data from many marine regions are needed before we can state with certainty that the southern Ryukyus harbor high levels of stoloniferous octocoral diversity and endemism, but at least it can be said that this region potentially harbors many undiscovered species, not only in shallow coral reefs [33,34], but also among the many unexplored MCEs in this region.

Recent studies have shown that MCEs harbor distinct and independent biological communities when compared to shallower reefs [21]. MCEs are not only affected by anthropogenic and natural impacts as are shallow reefs but have seldom been the focus of specific conservation efforts [21,58]. Thus, researchers have only begun to scratch the surface of what we know about mesophotic marine life [21,58,59], including information on stoloniferous octocoral diversity and octocoral–zooxanthellae relationships in MCEs. The discovery of *Hadaka nudidomus* gen. nov. et sp. nov. and other recent discoveries [54,57] emphasize the need for continued studies on MCE octocoral diversity, as undescribed species may disappear before we have the opportunity to discover and study them [21].

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1424-2818/11/10/176/s1, Figure S1: Maximum Likelihood phylogeny reconstruction of 28S rDNA gene region of *Hadaka nudidomus* gen. nov. et sp. nov. from Okinawa and Iriomote Islands (Japan) and octocoral references from 123 genera, including outgroup *Cornularia* spp., Figure S2: Maximum Likelihood phylogeny reconstruction of COI gene region of *Hadaka nudidomus* gen. nov. et sp. nov. from Okinawa and Iriomote Islands (Japan) and octocoral references from 123 genera, including outgroup *Cornularia* spp., Figure S3: Maximum Likelihood phylogeny reconstruction of mtMutS gene region of *Hadaka nudidomus* gen. nov. et sp. nov. from Okinawa and Iriomote Islands (Japan) and octocoral references from 123 genera, including outgroup *Cornularia* spp., Fiugre S4: Maximum Likelihood phylogeny reconstruction of 28S rDNA gene region of *Hadaka nudidomus* gen. nov. et sp. nov. from Okinawa and Iriomote Islands (Japan) and five octocoral references (*Phenganax* spp., *Acrossota amboinensis*), and outgroup (*Telesto* sp., *Rhodelinda* sp.)., Figure S5: Maximum Likelihood phylogeny reconstruction of COI gene region of *Hadaka nudidomus* gen. nov. et sp. nov. from Okinawa and Iriomote Islands (Japan) and five octocoral references (*Phenganax* spp., *Acrossota amboinensis*), and outgroup (*Rhodelinda* sp.)., Figure S6: Maximum Likelihood phylogeny reconstruction of mtMutS gene region of *Hadaka nudidomus* gen. nov. et sp. nov. from Okinawa and Iriomote Islands (Japan) and four octocoral references (*Phenganax* spp., *Acrossota amboinensis*), and outgroup (*Telesto* sp.)., Figure S7: Maximum Likelihood phylogeny reconstruction of ND6 gene region of *Hadaka nudidomus* gen. nov. et sp. nov. from Okinawa and Iriomote Islands (Japan) and two octocoral references (*Phenganax* spp.)., Figure S8: Maximum likelihood phylogenetic reconstruction of gene region ITS of Symbiodiniaceae hosted by *Hadaka nudidomus* gen. nov. et sp. nov. specimens from Okinawa and Iriomote Islands (Japan) and reference taxa *Durusdinium* sp. (= former *Symbiodinium* 'Clade D', n = 14) and *Cladocopium* sp. (former Symbiodinium 'Clade C', n = 10) and outgroup sister taxa, *Gerakladium* sp. (= former *Symbiodinium* 'Clade G') as used in Lau et al (2019)., Figure S9: Bayesian inference phylogeny reconstruction of the combined 28S rDNA+COI+mtMutS gene regions of *Hadaka nudidomus* gen. nov. et sp. nov. from Okinawa and Iriomote Islands (Japan) and octocoral references from 123 genera, including outgroup *Cornularia* spp., Table S1: Number of base differences per site from averaging over all sequence pairs between stoloniferous octocoral genera (*Hadaka* gen. nov., *Phenganax*, *Acrossota*) is shown (p expressed as percentage) for COI and mtMutS gene regions. Standard error estimates (S.E.) are shown above the diagonal. Analysis involved 9 and 6 nucleotide sequences for COI and mtMutS, respectively. All positions containing gaps and missing data were eliminated. There were totals of 708 and 734 positions in the final dataset for COI and mtMutS, respectively. Evolutionary analyses were conducted in MEGA X (Kumar et al. 2018)., Table S2: Number of base differences per site from averaging over all sequence pairs between stoloniferous octocoral taxa (*Hadaka nudidomus* gen. nov. sp. nov., *Phenganax* spp., *Acrossota amboinensis*) is shown (p expressed as percentage) for gene regions COI and mtMutS. Standard error estimates (S.E.) are shown above the diagonal. Analysis involved 7 nucleotide sequences for both COI and mtMutS. All positions containing gaps and missing data were eliminated. There were totals of 717 and 881 positions in the final dataset for COI and mtMutS, respectively. Evolutionary analyses were conducted in MEGA X (Kumar et al. 2018)., Table S3: Estimates of average evolutionary divergence over sequence pairs within stoloniferous octocoral genera (*Hadaka* gen. nov., *Phenganax*, *Acrossota*, *Rhodelinda*, *Telesto*) for gene regions COI and mtMutS. The numbers of base differences per site from averaging over all sequence pairs within each group (d) are shown (p expressed as percentage). Standard error estimates (S.E.) are shown in the second column and were obtained by a bootstrap procedure (1000 replicates). Analyses involved 9 and 6 nucleotide sequences for COI and mtMutS, respectively. All positions containing gaps and missing data were eliminated. There were totals of 708 and 734 positions in the final dataset for COI and mtMutS, respectively. Evolutionary analyses were conducted in MEGA X (Kumar et al. 2018).

**Author Contributions:** Conceptualization, methodology, and writing—review and editing, Y.W.L. and J.D.R.; formal analysis, investigation, data curation, writing—original draft preparation, and visualization, Y.W.L.; supervision and resources, J.D.R.

**Funding:** This research received no external funding.

**Acknowledgments:** Daisuke Uyeno (Kagoshima University) is thanked for providing the holotype specimen NSMT-Co 1681. Tohru Naruse (University of the Ryukyus) is thanked for logistical support in the field in Iriomote Island. Kristen G.Y. Soong (University of the Ryukyus) is thanked for her help with laboratory work. Frank R. Stokvis (Naturalis Biodiversity Center) is thanked for providing help with phylogenetic analyses.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Di**ff**erential Occupation of Available Coral Hosts by Coral-Dwelling Damselfish (Pomacentridae) on Australia's Great Barrier Reef**

#### **Tory J Chase \* and Mia O Hoogenboom**

Marine Biology and Aquaculture Group, College of Science and Engineering, and ARC Centre of Excellence for Coral Reef Studies, James Cook University, Townsville QLD 4811, Australia<EMAIL_ADDRESS>**\*** Correspondence<EMAIL_ADDRESS>
Received: 14 October 2019; Accepted: 8 November 2019; Published: 15 November 2019

**Abstract:** Associations between habitat-forming, branching scleractinian corals and damselfish have critical implications for the function and trophic dynamics of coral reef ecosystems. This study quantifies how different characteristics of reef habitat, and of coral morphology, determine whether fish occupy a coral colony. In situ surveys of aggregative damselfish–coral associations were conducted at 51 different sites distributed among 22 reefs spread along >1700 km of the Great Barrier Reef, to quantify interaction frequency over a large spatial scale. The prevalence of fish–coral associations between five damselfish (*Chromis viridis*, *Dascyllus aruanus*, *Dascyllus reticulatus*, *Pomacentrus amboinensis* and *Pomacentrus moluccensis*) and five coral species (*Acropora spathulata*, *Acropora intermedia*, *Pocillopora damicornis*, *Seriatopora hystrix,* and *Stylophora pistillata*) averaged ~30% across all corals, but ranged from <1% to 93% of small branching corals occupied at each site, depending on reef exposure levels and habitat. Surprisingly, coral cover was not correlated with coral occupancy, or total biomass of damselfish. Instead, the biomass of damselfish was two-fold greater on sheltered sites compared with exposed sites. Reef habitat type strongly governed these interactions with reef slope/base (25%) and shallow sand-patch habitats (38%) hosting a majority of aggregative damselfish-branching coral associations compared to reef flat (10%), crest (16%), and wall habitats (11%). Among the focal coral species, *Seriatopora hystrix* hosted the highest damselfish biomass (12.45 g per occupied colony) and *Acropora intermedia* the least (6.87 g per occupied colony). Analyses of local coral colony traits indicated that multiple factors governed colony usage, including spacing between colonies on the benthos, colony position, and colony branching patterns. Nevertheless, the morphological and habitat characteristics that determine whether or not a colony is occupied by fish varied among coral species. These findings illuminate the realized niche of one of the most important and abundant reef fish families and provide a context for understanding how fish–coral interactions influence coral population and community level processes.

**Keywords:** coral-fish association; symbiosis; habitat structure; prevalence; damselfish; coral reefs; biological interactions

#### **1. Introduction**

Scleractinian corals are the predominant habitat-forming organisms within coral reef ecosystems contributing to the (i) overall structure of reef habitats [1], (ii) co-existence and biodiversity of reef associated species [2–4], and (iii) providing critical microhabitats used by specialist species [5–8]. Consequently, the abundance of coral-dwelling and reef-associated species (e.g., crustaceans, sponges, bryozoans, fishes) is influenced by the abundance of habitat-forming corals [2,9], as well as by the structural complexity provided by coral-rich habitats [4,10–12], and the diversity of corals [13]. Importantly, high coral cover and habitat complexity moderate predation [14] and competition [15]

among reef fish species. Meanwhile, fishes that have an intimate and obligate reliance on live corals for shelter (e.g., coral Gobiidae spp. [16], coral-dwelling Pomacentridae spp. [7]) or food (e.g., coral-feeding Chaetodontidae spp. [17]), often have specific preferences for select coral species which, themselves, might occur only in certain habitats (under certain environmental conditions or shelf positions [18]). Ultimately, corals might be a limiting resource that regulates the distribution and abundance of many reef fishes [6,19], depending on their specificity to particular coral species and their reliance on live coral habitats. Understanding this process requires intensive and broad-scale quantification of fish–coral interactions to distinguish effects of habitat types from the effects of coral cover.

The abundance of suitable coral, that enable long-term usage or residency of associated fauna across various life stages, is one of the most importance factors dictating damselfish presence [20,21], evident by the fact that abundances of fishes and motile invertebrates' abundances decline sharply following coral mortality [19,22]. Despite a strong dependence on corals by several fish families, not all coral colonies are occupied by fishes due to physical and behavioural limitations [7,21]. At a larger spatial scale of reefs and latitude, local availability of specific types of habitat determine spatial distribution patterns in habitat-specialized fish, (i.e., *Gobiodon* spp., [23,24]). However, to determine the extent to which the availability of specific coral habitats constrain the abundance of reef fishes, direct measurement of the abundance of fishes on individual coral colonies is required. Previous studies have linked variation in damselfish's abundance and diversity with habitat-related variation in the percentage cover or functional diversity of corals [18,25], but have not assessed whether and how features of coral colonies within habitats also influence fish abundance. Assessment of fish-coral interactions at the colony level is important, because this is the scale at which impacts of damselfish on corals are the most prevalent [26,27]. Services that fish provide to corals are often density-dependent (such as nutrient provision) and are heavily dependent on fish biomass [26,28]. Furthermore, understanding the spatial variation in coral-dwelling fish provides a context for understanding how these fish influence coral populations and communities, and how these mutualisms are likely to change during external disturbances and degradation.

Habitat type and colony morphology influence the suitability of coral hosts for nearly all types of coral-associated fauna, especially fishes, as observed for both Scleractinian and Alcyonacean corals [29–32]. This colony-scale association correlates with fish size [33], with how fish utilize the coral, and with fish diet preferences, and social and spatial niches [29,34,35]. Indeed, seascape and colony features strongly influence feeding behaviour, especially for zooplankton feeding damselfishes for which among-species partitioning of planktonic prey dictates how different fish species are distributed among reef zones [36–38]. Moreover, for other fish taxa, including Pomacentridae, Gobiidae, and Blennidae, fine-scale (1 to 10 cm2) differences in the suitability of coral hosts depends on much more than just the coral taxonomic identity. For example, *Dascyllus marginatus* and other aggregating damselfish are more likely to inhabit colonies with fine branches, compared with lobed branching morphologies, as inter-branch space is a limiting determinant for these fishes [21,39]. Furthermore, seascape features such as distance from the reef's edge and water flow velocities govern species-specific patterns and biomass due to fishes' swimming ability, plankton/prey availability, trophic specializations [37], and colony arrangement requirements; this has been demonstrated in habitat-specialist/coral dwelling and non-coral-dwelling fishes [38,40–43]. However, the specific reef habitat characteristics, and with both within- and among-species variation in coral colony structures, that promote occupancy and residency by aggregative damselfish has yet to be fully understood, with most of this work to date focusing on Blennidae and Gobiidae that usually inhabit corals as individuals or pairs rather than in large aggregations [23]. Aggregative species are likely to utilize different resources and have different association levels compared with large transient and/or small solitary species [44].

This research explores the large-scale spatial variation in occupancy rates and biomass of coral-dwelling damselfish among predominant habitat-forming scleractinian coral species, and assesses specific habitat and colony features that influence whether or not individual coral colonies are used by coral-dwelling, planktivorous damselfish. The prevalence of fish-coral

interactions is examined for five damselfish (*Chromis viridis*, *Dascyllus aruanus*, *Dascyllus reticulatus*, *Pomacentrus amboinensis*, and *Pomacentrus moluccensis*) on five coral species (*Acropora spathulata*, *Acropora intermedia*, *Pocillopora damicornis*, *Seriatopora hystrix*, and *Stylophora pistillata*). These corals are frequently occupied by coral-dwelling damselfish [5,21,45–47]. Previous research demonstrates that these select coral and fish species can account for >70% of non-cryptic fish-coral interactions within the Great Barrier Reef [21,47] and play important roles in assimilating energy and nutrients from plankton into the reef food web. Each of these Pomacentrid sp. have been documented to be 'coral-dwelling' with a home range of a single coral or similar <2 m structure [18,27,39,48,49]. Finally, the focal fish species represent important prey for meso- and top predators [18] and are therefore important in reef trophic dynamics. Multiple coral colony traits were measured in situ as these traits are hypothesized to correlate with patterns of fish occupation and biomass. This study increases the number studies that have quantified broad-scale abundance of damselfish associated with different colony morphologies. We investigate the distribution of these coexisting damselfish within and among reef zones to (a) determine if suitable coral habitat governs patterns in damselfish's distribution and abundance (large scale, >10 m, based on variation in coral cover among reefs), and (b) quantify variations in fish biomass within and among coral colony species (small scale >1 m, based on observations of individual coral colonies). Evaluating the multiscale spatial variation of fish-coral interactions provides insight into fishes' effects on coral health, and context for predicting the functioning of interspecific and symbiotic associations during global environmental change.

#### **2. Materials and Methods**

#### *2.1. Study Sites and Surveys*

This study was conducted in March–November 2016, in the northern Great Barrier Reef (GBR), Australia. Surveys were conducted at 51 study sites across 20 different reefs (Figure 1) including the far northern sector (*n* = 11 sites), the northern sector (*n* = 24) including Lizard Island sites (*n* = 16 sites), the central sector (*n* = 13 sites) and the southern sector near One Tree Island (*n* = 3 sites, see Table 1).

**Figure 1.** Location of study reefs (for reef seascape surveys and colony level surveys) along the Great Barrier Reef (GBR, 51 sites spread among 22 different reefs), spanning >1700 km (map modified from [50]) with Lizard Island subset including 16 sites, surveyed between February and November 2016. Some reefs contained more than one transect. Map template is provided by Geoscience Australia under a Creative Commons Attribution 4.0 International License.

Along latitudes spanning >10◦, 1–3 transects per site were compared to quantify occupancy and resident damselfish's biomass. Sites were either sheltered or exposed; western facing aspects (sheltered sites, often with sandy lagoons) receive less exposure of wave energy and weather, compared with eastern facing aspects (exposed) sites on the GBR, due to the geomorphology of the surveyed mid-shelf and off-shore reefs [53,54]. Transects were located within different habitat zones (sand patches, flat, crest, wall (distinguished from slope by approximately vertical relief of the substratum), slope/base (gentle gradient or approximately flat), at different distances from shore (mid-shelf and off-shore reefs), and at varying depths (0–14 m, standardized to Lowest Astronomical Tide (LAT)). Herein, occupancy is described as a colony being used as the sole site of shelter/habitat within a damselfish's territory or home range [21,22,55]. Surveys focused on five species of damselfish (*Chromis viridis*, *Dascyllus aruanus*, *Dascyllus reticulatus*, *Pomacentrus amboinensis*, and *Pomacentrus moluccensis*) and five species of branching corals (*Acropora intermedia*, *Acropora spathulata*, *Pocillopora damicornis*, *Seriatopora hystrix*, and *Stylophora pistillata*, see Figure S1 in Supplementary Materials). The host corals were selected for their abundance on the GBR, while also displaying differences in morphology, and particularly, branch spacing patterns due to the hypothesized role of branch spacing in determining colony occupancy [9,56].

At each site, the abundance and occupation of colonies (20–100 cm in diameter) of each study species (*Acropora intermedia*, *Acropora spathulata*, *Pocillopora damicornis*, *Seriatopora hystrix*, and *Stylophora pistillata*) were recorded along a 50 m <sup>×</sup> 5 m belt transect (total area of 250 m2) by scuba diving. We also recorded the size and abundance of focal fish species (*Chromis viridis*, *Dascyllus aruanus*, *Dascyllus reticulatus*, *Pomacentrus amboinensis*, and *Pomacentrus moluccensis*) within each colony through a visual census during scuba diving. Along each transect, each colony was slowly approached and observed for at least 30 s to determine damselfish species presence, size, and abundance for biomass estimates. For consistency, all coral and fish observations were performed by the same observer during daylight hours (between 8:00 and 18:00 h). In addition, four replicate 10 m line intercept transects were completed at each site to measure total coral cover (of all corals not just the 5 focal species [51,57]).

To assess whether occupation of focal coral species by the specific damselfish was influenced by intrinsic or extrinsic factors, we measured a series of colony attributes for a subset of colonies (*n* = 226) at 15 different sites. These colonies were located on 11 exposed and sheltered reefs, spanning habitats at a depth range of 0–13 m, positioned in the Far North, North, Central, and Southern GBR regions as described above (see Tables S3–S5 in Supplementary Materials for details). Colony position was categorized as being either within a crevice, on an overhang, on open carbonate pavement, or on sand [52]. Colony structure traits measured included: colony size (colony diameter, planar area, and colony height), distance from nearby corals (isolation), and branch dimensions (i.e., inter-branch spacing and average branch width [47,58], see Table 1 and Figure S2 in Supplementary Materials). Branch spacing and branch width were averaged for five measurements around each colony, with all branch measurements taken at ~15 mm from the branch tip, while colony isolation being measured as the distance to the closest habitat providing coral (i.e., branching or other complex morphology colonies). For colonies with resident fishes (*n* = 142), the numbers of all fishes on each focal colony were recorded, and all fish were placed into general standard-length size classes of small, medium, and large, for each species respectively. Size class data were subsequently converted into biomass estimates, based on published length/weight relationships generated from damselfish [27,47], where damselfish were collected using hand-nets and a liquid anesthetic (a diluted solution of clove oil, ethanol, and seawater [27,59,60]). Surveys focused on ecologically important damselfish's occupancy and biomass patterns rather than fish numbers, as biomass has been directly linked to fish-derived services and benefits for corals [26,61]. For the purposes of these surveys, fish biomass summarizes both fish numbers and size, and the analysis did not delineate which of these components contribute more to biomass levels. Additional details of transects, sites and colonies are provided in Table 1 and Supplementary Tables S3–S5.

#### *2.2. Data Analysis*

#### 2.2.1. Reefscape Prevalence of Fish–Coral Interactions

At the reef seascape level, the proportion of colonies occupied by fish (all damselfish and coral species pooled, as the independent variable) varied on each transect, and was analysed using a full additive beta regression model with latitude, aspect (exposure level), habitat, and coral cover as fixed dependent factors, and reef as a random factor. A beta regression was deemed appropriate, as it includes a logit transformation which is necessary for proportional data [62,63]. The appropriateness of the models selected were confirmed by assessing quantile, or Q-Q plots for normality and residual plots for homogeneity of variance and linearity, as well as calculations of dispersion. Additive models (latitude + aspect + habitat + coral cover) were used due to the non-factorial nature of the dataset wherein not all habitats and aspects could be sampled at each latitude.

A linear mixed-effect model (LME) was used to analyse effects of latitude, aspect, habitat, and coral cover, on total biomass of focal damselfish species (grams per 250 m2), log +1 transformed, recorded on each transect. The fish biomass LME was fitted using maximum likelihood [64]. Model selection, based on Akaike information criteria (AIC) values, was implemented to determine the importance of latitude, aspect, habitat, and coral cover as predictors of fish biomass (following [65,66], see below) and assumptions for model validity were checked through Q-Q plots (normality) and residual plots (homogeneity of variance and linearity), as well as calculations of dispersion. In addition, the multi-model interference R package *MuMIn*, was used to perform model selection on proportion of colonies occupied and total biomass models based on model weights derived from AICc. *MuMIn* allows for an estimate of the variance explained by all factors included in the model (*R package MuMIn*, [67,68]). A ranking of the possible models to identify the contribution/importance of each variable as well as the number of models in which each variable was completed (function "dredge" in R package *MuMIn*).

To compare differences in total occupancy (only occupied colonies, *n* = 898) among each of the five coral species, binomial generalized linear models (GLMs) with Tukey's honestly significant difference (HSD) post hoc (with Bonferroni adjusted *p*-values) were used. Total damselfish's biomass was analyzed using a Gaussian GLM with Tukey's HSD post hoc. Separate Kruskal–Wallis rank sum tests were performed for each damselfish species to analyse whether coral species identity (independent variable) affected the biomass of different species of resident damselfish (dependent variable) on these 898 occupied colonies. Kruskal–Wallis tests were deemed appropriate as fish biomass data did not meet assumptions of homogeneity of variance and normality. Dunn tests were used for multiple post hoc comparisons between species due to unequal sample sizes, and *p*-values were adjusted with the Benjamini–Hochberg method to decrease type I error; the Benjamini–Hochberg method is a more powerful method than the Bonferroni correction to control the false discovery rate [69] and frequently used with the Kruskal–Wallis test.

#### 2.2.2. Effects of Colony Position and Structure on Damselfish's Occupancy

To compare how colony position and structure impacted occupation and biomass for a subset of colonies, principle component analyses (PCAs) were used to evaluate overall differences in colony morphology between corals with (*n* = 142) and without fish (*n* = 84), both with data pooled over all corals (*n* = 226), and separately for each coral species (using the colony level dataset). These different analyses were conducted to assess whether there were particular colony structure traits that influenced fish presence overall, and whether such features were consistent among coral species. PCAs were deemed appropriate due to the multivariate nature of the data with variables (e.g., branch width and branch spacing) that were likely to be correlated with each other. The PCA ordinated colonies were based on the standardized correlation matrix between colony attributes using the R function princomp [70,71]. Subsequently, the principle component (PC) 1 and 2 scores of each colony were used to represent the overall variation in colony morphology in subsequent linear models (LM) of fish

occupation (presence/absence). To further differentiate occupancy patterns between the colony position, a binomial GLM was used with a Tukey's HSD post hoc to assess between factor level differences. A lognormal linear model was used to quantify total damselfish's biomass (only occupied colonies) with regards to colony position, again with Tukey's HSD post hoc comparisons.

Similarity percentage analysis (SIMPER [71–73]) was used to determine which coral structure traits (colony diameter, planar area, colony height, branch spacing, branch width, and isolation) contributed the most to the differences among corals with and without fish. This analysis compared the importance of these structural traits for all coral species pooled and pooled across the different species of fish occupying these corals. The SIMPER analysis was performed on the PCA standardized data to assess which structure traits were driving the differences (by individual coral species and species pooled) and ranked in order according to their contribution (% or importance ranking). This similarity percentage is based on the decomposition of Bray-Curtis dissimilarity index, giving the overall contribution of individual structure traits.

#### 2.2.3. Effects of Colony Position and Structure on the Biomass of Damselfish

Total biomass of damselfish on colonies located in different reef microhabitats (colony orientation, Table 1) were analysed with lognormal linear models. Model fit was assessed using residual plots, all of which were satisfactory (normal and homogenous). As total pooled damselfishes' biomass is a continuous variable, a series of linear models per individual coral species and for all colonies pooled were completed to determine if total damselfishes' biomass (dependent variable) varied with the two most important structure traits (independent variables) from the SIMPER of colony structure occupancy.

All data analyses were performed in the statistical software R [74] using the *betareg* [62], *multcomp* [75], *lsmeans* [76], *simper* function in *vegan*, and *MuMIn* [67,68] packages. Full datasets are available at [77].

#### **3. Results**

#### *3.1. Range of Damselfish's Occupations across the Great Barrier Reef (GBR)*

In transect-based surveys, a total of 5154 damselfish of the five focal species (*Chromis viridis*, *Dascyllus aruanus*, *Dascyllus reticulatus*, *Pomacentrus amboinensis*, and *Pomacentrus moluccensis*) were counted on 3034 coral colonies of the five focal species (*Acropora intermedia*, *Acropora spathulata*, *Pocillopora damicornis*, *Seriatopora hystrix*, and *Stylophora pistillata*) on 51 transects (with combined sample area of 12,750 m2). Overall, 30% of colonies were occupied by one or more of the focal damselfish species (898 out of 3034, all transects pooled). Single-species groups of *Pomacentrus moluccensis* or *Dascyllus aruanus* were recorded on 80% of occupied colonies. *P. moluccensis* were prevalent (in terms of coral host occupancy) in all habitats, while *Chromis* and *Dascyllus* species almost exclusively inhabited corals on sand-patch and slope habitats (Figure S2 in Supplementary Materials).

Occupancy varied with aspect and habitats, with values ranging from 0% at exposed, flat and crest habitat zones, up to 93% at sheltered sand-patch habitats. In the full model, habitat (1) and aspect (2) were the most important variables in predicting fish occupancy (Table S1 in Supplementary Materials). In general, occupancy levels were higher in western aspect/sheltered sites locations (including lagoons) than eastern aspect/exposed sites (betareg (logit): *p* = 0.002), and highest numbers were observed in sand patches and slope habitats (*p* = 0.016, Figure 2b). Latitude (*p* = 0.051), and coral cover (*p* = 0.735) were not significant predictors of the proportion of colonies occupied (Figure 2a,b).

**Figure 2.** Boxplots (horizontal lines show median; boxes indicate 25th and 75th percentiles; vertical dotted lines show range; data points show outliers) of colonies occupied (reef seascape) (**a**,**b**) and damselfish's biomass (log +1) abundance (**c**,**d**) on five species of branching coral (*Acropora intermedia*, *Acropora spathulata*, *Pocillopora damicornis*, *Seriatopora hystrix*, and *Stylophora pistillata*) in relation to aspect category (exposed or sheltered) and reef habitat (sand patches, flat, crest, wall, and slope/base).

Additionally, occupancy also varied with coral species (binomial GLM, significant effect of species, *p* < 0.05). Both *P. damicornis* (34% occupancy) and *Stylophora pistillata* (33% occupancy) had the highest average occupancy, when compared with *Acropora spathulata* (30%), *S. hystrix* (23%), and *Acropora intermedia* (22%) (see Table 2 for post hoc comparisons and Table S2 for the binomial GLM output). These damselfish species-specific occupancy patterns translated into different damselfish diversity and biomass on each coral species (Tables S3–S5 in Supplementary Materials); for instance, *Acropora intermedia*, *Pocillopora damicornis*, and *Stylophora pistillata* hosted mainly *Dascyllus aruanus* and *Pomacentrus moluccensis* aggregations, while *Acropora spathulata* hosted *Chromis viridis* and *Pomacentrus moluccensis* heterospecific groups.

**Table 2.** Multiple comparisons of coral-species, with *p*-values, (Tukey's honestly significant difference (HSD) post hoc) based on a binomial generalized linear model of colony occupancy with damselfish species pooled (reef seascape dataset): colony occupancy (dependent) and colony species (independent variable). Significant *p*-values are in bold.

#### *3.2. Patterns of Damselfish Biomass across Reefs on Occupied Colonies*

An average of six damselfish were present on each occupied colony. *Pomacentus amboinensis* was the most prevalent damselfish species on the coral colonies considered during this study, present on nearly half of all occupied coral colonies (~2.3 *Pomacentus moluccensis* colony−1), and accounting for ~45% of all damselfish's biomass on coral hosts (Table 3, Figure 3, and Tables S3–S5 and Figure S3 in Supplementary Materials). *Dascyllus aruanus* was the second most abundant with an average 1.8 fish per occupied colony−<sup>1</sup> and the other three species were present at considerably lower abundance (*Chromis viridis*: 0.8 fish occupied colony−1, *Dascyllus reticulatus* 0.2 fish occupied colony−1, and *Pomacentrus moluccensis*: 0.5 fish occupied colony<sup>−</sup>1).

Damselfish's biomass was broadly similar to occupancy patterns, displaying significant differences in biomass per 250 m2 depending on aspect (LME (log+1), aspect, χ<sup>2</sup> = 6.88, *p* = 0.008, Figure 2c,d). Sheltered sites had three-fold higher biomass (250 <sup>±</sup> 71 g 250 m−<sup>2</sup> for all colonies per transect) than exposed sites (86.7 <sup>±</sup> 17 g 250 m−2). Biomass per 250 m2 also varied by habitat zone (LME (log+1), habitat, <sup>χ</sup><sup>2</sup> = 9.54 *p* = 0.0489) with the highest biomass in sand patches (404.9 <sup>±</sup> 166 g 250 m−2) and slope habitats (161.7 <sup>±</sup> 33 g 250 m−2), and lowest biomass on wall habitats (70.1 <sup>±</sup> 42 g 250 m−2). Again, latitude (LME (log+1) χ<sup>2</sup> = 2.81, *p* = 0.42) and coral cover (χ<sup>2</sup> = 0.109, p = 0.740) were not significant predictors of total fish biomass per transect. In the full model, aspect (1) and habitat (2) were the most important variables in predicting fish occupancy (Table S1 in Supplementary Materials).

Damselfish's biomass per occupied colony ranged from 1.3 g (a single *Pomacentrus amboinensis*) to 120 g (a school of ~100 *Chromis viridis* or a large aggregation of ~30 *Dascyllus aruanus*). Among the five fish species, *Pomacentrus moluccensis* exhibited the most consistent and broadest distribution being present in high biomass in every habitat zone. *Seriatopora hystrix* coral colonies hosted the highest fish biomass per occupied colony (12.45 g ± 1.33), with *Acropora intermedia* having the lowest biomass per occupied colony (6.87 g ± 1.33). As a result, total damselfish's biomass was significantly different among occupied coral species (GLM: *p* = 0.012, see Supplementary Table S6 for post hoc comparisons). When data were analysed by fish species, the biomass of each damselfish species significantly varied among host coral species (see Table S7 in Supplementary Materials for post hoc comparisons), except for *Chromis virdis* (Kruskal–Wallis: χ<sup>2</sup> = 9.104, df = 4, *p* = 0.0586). *Seriatopora hystrix* and *Pocillopora damicornis* colonies were favoured by *Dascyllus aruanus* (χ<sup>2</sup> = 45.304, df = 4, *p* < 0.001) and *Dascyllus reticulatus* (χ<sup>2</sup> = 29.962, df = 4, *p* < 0.001). *Acropora spathulata* and *Stylophora pistillata* colonies were favoured by *Pomacentrus amboinensis* (χ<sup>2</sup> = 11.715, df = 4, *p* = 0.019) and *Pomacentrus moluccensis* (χ<sup>2</sup> = 29.962, df = 4, *p* < 0.001).

**Figure 3.** Mean biomass per fish species (g ± standard error (SE) of total biomass of damselfish) per coral species (*Acropora intermedia*, *Acropora spathulata*, *Pocillopora damicornis*, *Seriatopora hystrix*, and *Stylophora pistillata*) for all occupied colonies (*n* = 898) for 5154 fish (*Chromis viridis*, *Dascyllus aruanus*, *Dascyllus reticulatus*, *Pomacentrus amboinensis*, and *Pomacentrus moluccensis*) at 51 sites. Coral sample sizes per species are displayed above the bars. Note the collapse of *Dascyllus reticulatus* and *Pomacentrus amboinensis* sub-bars for the *Acropora spathulata* coral bar, and again for *Dascyllus reticulatus* on the *Seriatopora hystrix* bar, indicating very low biomass values for these fish species on these corals. Further damselfish species-specific and coral species-specific average biomass (±SE) per site aspect, and habitat are displayed in Supplementary Tables S3–S5).

#### *3.3. Colony Orientation as a Determinant of Damselfish's Occupation and Group Biomass*

Higher coral occupancy was observed on corals located in reef microhabitats that were either open carbonate pavement or open sandy substratum habitats (LM: (open) *p* = 0.0068) and (sand) *p* < 0.0001, see Table S8 for post hoc comparisons in Supplementary Materials). Similarly, total damselfish's biomass on occupied colonies (all fish and coral species pooled), averaged 15.3 g ± 2.4 on sand, and 11.4 g ± 1.8 on open colonies; values that were three- to four-fold higher than observed on colonies in underhang (4.9 g ± 0.8) and crevice (3.8 g ± 0.7) colony orientations (LM: F3132 = 5.387, *p* < 0.001, see Table S9 for post hoc comparisons in Supplementary Materials).

#### *3.4. Colony Structure as a Determinant of Damselfish's Occupation and Biomass*

The PCAs of colony attributes (based on the specific subset of corals and study locations where these attributes were measured) of the five coral-dwelling damselfish (species pooled), revealed distinctive groupings of colonies with and without fish both when data were pooled across coral species, and when analysed separately for each coral species. The first two principal components explained 70% of variance for all colonies pooled (Table S10 and Figure S4 in Supplementary Materials), and between 55% and 77% of variance in colony structure when coral species were analysed individually. Overall, colonies (pooled over species) that were occupied by fish had considerably lower PC1 scores than colonies without fish, and lower PC2 scores (Table S6). In this analysis, PC1 scores were associated with variation in colony diameter and planar areas (dictated by *Acropora intermedia* colonies), and PC2 scores were driven by branch spacing and colony isolation. When coral colonies were analysed separately by individual species (Supplementary Table S11) isolation was the most influential colony variable for all coral species, with branch spacing and planar area as secondary variables.

Total damselfish's biomass per colony followed similar trends with fish occupancy (Table 4 and Table S12 in Supplementary Materials), with isolation and colony height as the most influential colony-structure variables for five of the six coral species, and all structure traits were significant except for branch width, which when analysed individually by species, was only important for *Stylophora pistillata*. Branch spacing, colony diameter, and planar colony area were significant for three coral species. Branch width was only important for predicting fish biomass present on *Stylophora pistillata* colonies (Table 4).

**Table 4.** Series of linear models illustrating variation in total biomass of damselfish in small branching coral colonies (*Acropora intermedia*, *Acropora spathulata*, *Pocillopora damicornis*, *Seriatopora hystrix*, and *Stylophora pistillata*), by damselfish (*Chromis viridis*, *Dascyllus aruanus*, *Dascyllus reticulatus*, *Pomacentrus amboinensis*, and *Pomacentrus. moluccensis*) for six fine-scale indicators of colony attributes (colony level dataset). The first two traits, colony isolation and branch spacing (shaded), had the highest importance for determining colony occupation. Significant *p*-values are in bold.

#### **4. Discussion**

This research demonstrates substantial variation in the occupancy rates of small-branching coral hosts by five species of coral-dwelling damselfish, with between 0%–93% of coral colonies being occupied per transect, depending on reef habitat zone and exposure. Within habitats, small-scale differences in the morphology and position of coral colonies also contributed to occupancy and biomass of fishes. Previous studies have suggested that variation in coral colony structure and health are likely to play important roles in determining the population dynamics of coral-associated fishes and invertebrates [78,79], as well as the persistence of these fish assemblages. This study provides new insight into the factors that control the presence and abundance of individual symbiotic damselfish species (and associated group biomass, distribution across parts of the GBR) and provides context for understanding the potential impacts of aggregating damselfish on complex networks of reef species and reef ecosystem function.

The overall rates of occupancy reported in this study (30%) were aspect- and habitat zone-specific, demonstrating patterns of both high occupancy and high biomass on patchy sheltered aspect sites and significantly lower values on continuous, exposed aspect sites. At the transect level, physical conditions of these habitats are congruent with many of the environmental gradients (i.e., water-flow) and niche partitioning requirements that structure damselfish populations [7,18,35,80]. These results suggest that generalist damselfish species may be better able to utilise corals as habitat in high-flow environments than other species that are limited to specific coral species which may be more prevalent in sheltered areas [21,22,41]. For instance, *Pomacentrus moluccensis* was the most prevalent damselfish species

recorded and contributed disproportionally to the fish biomass present on occupied colonies on exposed sites. Consequently, damselfish inhabiting branching corals in exposed or deeper locations may be generalists zooplankton/omnivore feeders with the ability to take advantage of pelagic subsidies [81] rather than more specialized feeders [34,36,37,82]. While most coral-dwelling damselfish are found in sheltered habitats (i.e., flow <21.2 cm s<sup>−</sup>1), the body shape and fin morphology of *Pomacentrus moluccensis* may make them more adapted to higher current velocities, while *Dascyllus aruanus* may be more suited to lower currents [40,41] and was present on only 14% of damselfish-occupied corals, nearly all on sheltered sites. As an omnivorous bentho-pelagic feeder, *Dascyllus aruanus* can consume zooplankton and algae in equal proportions [34,38] which may partially explain its high abundance in slope/base habitats. Environmental factors such as water temperature, salinity, predators, conspecifics, and prey availability are also likely to influence the distribution and abundance of damselfish, independent of the abundance or availability of suitable coral hosts [34,37,83]. Although structural complexity and subsequent coral cover are often positively associated with fish biodiversity [1,4], results of this study showed that these two variables did not predict occupancy or biomass of coral-dwelling damselfish that closely associate with corals, consistent with previous studies [7,9,25,84–86]. Furthermore, latitude did not significantly affect colony occupancy or biomass; consistent with other studies reporting distribution and abundance of planktivorous damselfish along the Great Barrier Reef [18].

The coral species considered within this study (*Acropora intermedia*, *Acropora spathulata*, *Pocillopora damicornis*, *Seriatopora hystrix*, and *Stylophora pistillata*) are among the preferred coral hosts for coral-dwelling damselfish [21,22,46], yet 68% of colonies were unoccupied. This suggests that either abundance of these damselfish is not limited by coral host availability [87,88], or that there are colony attributes beyond species identity that determine their suitability as host corals [78,89]. This research reveals a suite of factors at small scale (<1 m) that influence occupation rates, including colony height, and colony position on the benthos, as well as the distance to other potential host corals. These attributes do not necessarily distinguish suitable versus unsuitable microhabitats, but given the choice of host corals, it would be expected that damselfish would select hosts that maximize individual fitness. Colonies with more elevated growth forms, raised above the seafloor, may also enhance fishes' abilities to stay higher in the water column, containing more enriched plankton, yet still close to refuge [90,91]. Elevated (high overall height) and isolated colonies, often in open position or on sandy substrates, allow for feeding with reduced danger due to visibility and enhanced colony structure complexity for refuge. Furthermore, damselfish species may respond differently to different species and morphologies of corals, with colony structure likely being more important to small-bodied fish [29,30]. For instance, *Dascyllus aruanus* prefer colonies with medium open-branch spacing (i.e., *Pocillopora damicornis* over *Acropora intermedia* or *Acropora spathulata*), while *Pomacentrus moluccensis* showed more equivalent abundance on all colony species. Branch spacing of corals limits occupancy only in tighter branching species (*Acropora spathulata*, *Pocillopora damicornis*, and *Seriatopora hystrix*) and may lead to variations in species interactions [92] between damselfish with their competitors and/or predators, and services (i.e., nutrient retention [26]).

Colony isolation was consistently the most important attribute predicting the presence and biomass of damselfish. Many damselfish species exhibit 'clumped' or 'patchy' distributions, leading to increased fish–coral interactions with increased fish abundance [47]. Sand patch and slope/base habitats, often categorized as edge habitats [43] with lower coral cover, host more fish–coral interactions and allow for more 'open' colonies, rather than nested corals along continuous reefs [39,93]. The isolation and spacing of colonies occupied may allow for: (a) continual use and residency by fish (i.e., distance to nearest available habitat is beyond the fish's home range); (b) increased impacts of association defense and reduction of fish predation [9,94–96]; (c) access to plankton resources and reduced competition [9,97–99]; and (d) larger borders with sandy substrates as an alternative foraging substrate [100]. Competition between damselfish species is also responsible for the ecological partitioning of these species along gradients [98], leading to differential use and fish-derived benefits

to coral hosts [9,101]. Many of these factors may enhance the survival of the coral holobiont in select habitats.

Coral occupancy of 30% may be an underestimate, as it excludes additional common fish families that can inhabit coral colonies (i.e., Apogonidae, Gobiidae, Haemulidae), and coral sizes (>100 cm), and coral species (i.e., *Porites* and *Echinopora*). While damselfish are present in many coral reef habitats [18], fish–coral interactions may vary in the sign or magnitude of the effect on their coral host [47,92], with sand patch, and slope/base zones acting as small-scale interaction hotspots with high occupancy and biomass patterns. These hotspots, areas of high localized nutrient production by fishes [28,102] are generally infrequent across seascapes. Nutrient subsidy, along with other fish-derived services like increased photosynthesis [103], colony growth [28], bleaching susceptibility [27] sediment removal [101], may be density-dependent (i.e., >15 g seen in studies focusing on larger-bodied or more abundant fish species, see [26,104,105]) and fish-species dependent. With 68% of corals vacant, it is clear that many colonies do not receive potential beneficial effects of resident damselfish. However, certain provided benefits, such as increased oxygen input [103] or nutrients [26], may be more necessary within specific habitats (i.e., deeper sand patch and slope habitats) or under specific environmental conditions (i.e., low-flow habitats [47]), thereby having a stronger impact on coral health for a smaller proportion of the population. Finally, different coral species have important effects on the biodiversity and function of resident fishes, with several colony structure traits directly associated with fish-derived services (i.e., hosting fish, retention of nutrients [25]).

By analyzing the occupancy and biomass of damselfish, one of the most abundant reef fish families that make an important contribution to reef food webs [34], this research illustrates that both large-scale features of reef habitats and fine-scale coral morphological traits contribute to fish-coral association. Several coral-dwelling damselfish species are constrained to certain reef habitats likely due to the physical constraints of the habitat, such as high-water energy. However, even after accounting for extrinsic factors, there are important colony traits that influence colony use. Clearly, studies of coral-associated fauna across multiple spatial scales [30] that go beyond simply quantifying fauna presence–absence are necessary to understand the population dynamics of corals and symbiotic fauna. Quantifying the establishment and maintenance of such symbiotic associations with scleractinian corals will be essential to predicting how these complex networks operate under global environmental stress [83]. Indeed, many of these branching coral species, particularly, *Seriatopora hystrix*, which hosts the highest damselfish biomass, are the species most vulnerable to global climate change [51,106]; the loss of these coral species will reduce considerably the habitat for small-bodied fishes [22]. Moreover, the high degree of spatial variation in the strength of fish–coral interactions and other symbiotic interactions will make it challenging to predict their ecological functioning and cost-benefit ratios.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1424-2818/11/11/219/s1; Figure S1: Focal coral-fish interactions of this study focused on (a–e) five common small-branching coral species, Figure S2: Illustration of 7 coral colony structure attributes for five species of branching colonies (15–100 cm diameter) for 226 colonies over 15 sites on 11 reefs, Figure S3: Average biomass (g ± SE) of damselfishes per occupied colony on the different reef habitat zones, Figure S4: Principal component analysis (PCA) of coral colony structure attributes for *n* = 216 branching corals with and without resident damselfishes along mid-shelf and off-shore reefs of the GBR; Table S1: Relative importance of environmental variables influencing fish-coral interactions (reef seascape level), based on *MuMIn* model selection and model averaging, with Akaike information criteria (AICc) weighting schemes, Table S2: Binomial generalized linear model (GLM) output for fishes (species pooled) occupation by coral species (reef seascape level dataset), Table S3: Descriptive statistics of reef seascape biomass estimated (mean grams ± SE) for each damselfish species and total biomass pooled for all coral species (per occupied colony of *Acropora intermedia*, *Acropora spathulata*, *Pocillopora damicornis*, *Seriatopora hystrix*, and *Stylophora pistillata*) by site aspect (sheltered or exposed), Table S4: Descriptive statistics of reef seascape biomass estimated (mean grams ± SE) for each damselfish species (*Chromis viridis*, *Dascyllus aruanus*, *Dascyllus reticulatus*, *Pomacentrus amboinensis*, and *Pomacentrus moluccensis*) and total biomass pooled for all coral species, Table S5: Average reef seascape biomass estimates (mean ± SE) for each damselfish species (*Chromis viridis*, *Dascyllus aruanus*, *Dascyllus reticulatus*, *Pomacentrus amboinensis*, and *Pomacentrus moluccensis*) on each coral species, Table S6: Multiple comparisons of coral-species, with *p*-values, (Tukey's HSD post hoc) based on a Gaussian generalized linear model of total damselfish biomass, Table S7: Multiple coral species comparisons

with *p*-values (post hoc Dunn test for (Benjamini–Hochberg method based off a Kruskal–Wallis rank sum test) for each damselfish species (damselfish-species specific biomass) for only occupied colonies, Table S8: Tukey's HSD post hoc test for multiple comparisons of position of coral on benthos, with *p*-values, based on a binomial generalized linear model of damselfish presence with damselfish species pooled, Table S9: Tukey's HSD post hoc test for multiple comparisons of position of coral on benthos, with *p*-values, based on a lognormal linear model of total biomass with damselfish species pooled for only occupied colonies, Table S10: Variance explained and linear models displaying differences between coral colonies with and without fish along principal component analyses PC1 and PC2, for a subset of coral colonies (*n* = 226) at 15 different sites on 11 reefs, Table S11: Similarity percentage analysis (SIMPER) results displaying the cumulative contributions of the most influential colony structure variables on coral colony occupation (presence or absence) by damselfishes, Table S12: Series of linear models illustrating variation in damselfishes' occupancies on small-branching coral colonies.

**Author Contributions:** Conceptualization, methodology, data collection, formal analysis, resources, data curation, writing—original draft preparation, writing—review and editing, visualization, project administration, funding acquisition all completed by T.JC. and M.OH.

**Funding:** This research was funded by the Australian Research Council to the ARCCOE for Coral Reef Studies CE140100020 and James Cook University. The funders have no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Acknowledgments:** We thank Lizard Island staff, Grace Frank, Margaux Hein, Saskia Jurriaans, Sterling Tebbett, Andrew Baird, and the crew of the RV Kalinda for their field support and assistance. This project was implemented in accordance with the Great Barrier Reef Marine Park Authority permit (G15/37657.1, G15/37950.1, and G16/38437.1), James Cook University Animal Ethics Permit (A2186 and A2207), and James Cook University's General Fisheries Permit (170251).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Green Fluorescence Patterns in Closely Related Symbiotic Species of** *Zanclea* **(Hydrozoa, Capitata)**

**Davide Maggioni 1,2,\*, Luca Saponari 1,2, Davide Seveso 1,2, Paolo Galli 1,2, Andrea Schiavo 1,2, Andrew N. Ostrovsky 3,4 and Simone Montano 1,2**

Received: 7 January 2020; Accepted: 17 February 2020; Published: 18 February 2020

**Abstract:** Green fluorescence is a common phenomenon in marine invertebrates and is caused by green fluorescent proteins. Many hydrozoan species display fluorescence in their polyps and/or medusa stages, and in a few cases patterns of green fluorescence have been demonstrated to differ between closely related species. Hydrozoans are often characterized by the presence of cryptic species, due to the paucity of available morphological diagnostic characters. *Zanclea* species are not an exception, showing high genetic divergence compared to a uniform morphology. In this work, the presence of green fluorescence and the morpho-molecular diversity of six coral- and bryozoan-associated *Zanclea* species from the Maldivian coral reefs were investigated. Specifically, the presence of green fluorescence in polyps and newly released medusae was explored, the general morphology, as well as the cnidome and the interaction with the hosts, were characterized, and the *16S rRNA* region was sequenced and analyzed. Overall, *Zanclea* species showed a similar morphology, with little differences in the general morphological features and in the cnidome. Three of the analyzed species did not show any fluorescence in both life stages. Three other *Zanclea* species, including two coral-associated cryptic species, were distinguished by species-specific fluorescence patterns in the medusae. Altogether, the results confirmed the morphological similarity despite high genetic divergence in *Zanclea* species and indicated that fluorescence patterns may be a promising tool in further discriminating closely related and cryptic species. Therefore, the assessment of fluorescence at a large scale in the whole Zancleidae family may be useful to shed light on the diversity of this enigmatic taxon.

**Keywords:** integrative taxonomy; symbiosis; corals; bryozoans; Maldives; phylogeny

#### **1. Introduction**

Green fluorescence is a diffuse phenomenon in the marine environment, being found in a variety of taxa, including cnidarians, ctenophores, crustaceans, and chordates [1]. Green fluorescence is caused by green fluorescence proteins, which were firstly described in the hydrozoan species *Aequorea victoria* (Murbach and Shearer, 1902) [2]. Lately, similar proteins were detected in several other species, mainly belonging to the Anthozoa [3], and they are currently known to be widespread in the marine metazoans [4]. In most cases, the ecological function of fluorescence is still unclear, even though some hypotheses have been proposed. For instance, in anthozoans associated with unicellular algae, fluorescent proteins may have a role in regulating the light environment of the

symbionts [5], whereas in bioluminescent organisms they seem to be involved in the modification of bioluminescence emission [6]. However, these hypotheses do not apply to non-symbiotic (with algae) and non-bioluminescent species. Other possible roles of fluorescence in marine organisms relate to camouflage, intraspecific communication [7], and prey attraction [8,9]. The latter hypothesis seems to fit better for hydrozoans, since it has been experimentally demonstrated that at least one species, *Olindias formosus* (Goto, 1903), uses fluorescence in tentacles to attract juvenile fish preys [8].

Among hydrozoans, green fluorescence is common and has been reported from polyps and medusae of several species (see [10] and references therein). In medusae, fluorescence is found in the umbrella, radial and circular canals, manubrium, gonads, bulbs, and tentacles (e.g., [11–13]), whereas in polyps in the hydrocaulus, hypostome, and in the epithelium below tentacles [10,13,14]. Green fluorescence patterns were found to differ significantly in closely related species of *Eugymnanthea* Palombi, 1936 [11], and even if these patterns changed during the development, they remained distinguishable from those in the relatives [11]. Moreover, Prudkovsky et al. [10] recently demonstrated that these patterns also differ between cryptic or pseudo-cryptic species of *Cytaeis* Eschscholtz, 1829, indicating that they may be reliable and informative taxonomic characters that could be useful especially when dealing with morphologically undistinguishable species.

Indeed, cryptic species are common in hydrozoans, since morphologically very similar polyps and medusae often show strong genetic diversification, that in many cases relates to host specialization and geography (e.g., [15–17]). This is especially true for the capitate family Zancleidae Russel, 1953, in which the few morphological diagnostic characters available make species identification and description challenging [18,19]. The cnidome is considered a useful character to discriminate among zancleid species, due to the variation of type and size of nematocysts in different species [20]. For instance, the statistical treatment of nematocysts measurements of three *Zanclea* cryptic species resulted in significant differences between the taxa [21], further supporting the importance of the cnidome as a reliable taxonomic character. Another useful character to distinguish closely related symbiotic species is the host specificity, since some species or lineages are specifically associated with one or a few invertebrate taxa (e.g., scleractinian corals) [16,18]. Moreover, some coral-associated *Zanclea* species were found to induce modifications of the host skeletons that could be taxonomically informative [21].

In this work we analyzed the morphology (polyps, newly released medusae, and modifications of the hosts) and genetic diversity (*16S rRNA*) of six symbiotic *Zanclea* species collected in the Maldives. Yet, along with the morpho-molecular analyses, we investigated the informativeness of green fluorescence patterns of polyps and medusae to discriminate between closely related taxa.

#### **2. Materials and Methods**

#### *2.1. Morphological Analyses and Fluorescence Essay*

Colonies of symbiotic *Zanclea* species were collected in reefs around Magoodhoo Island, Faafu Atoll, Republic of the Maldives (3.0782◦ N, 72.9613◦ E), during February 2017. Six *Zanclea* species were collected: *Zanclea sango* Hirose and Hirose, 2011 and *Zanclea* sp. (Clade I, *sensu* [18]) associated with the scleractinians *Pavona varians* (Verril, 1864) and *Goniastrea* sp., respectively; *Zanclea divergens* (Boero, Bouillon, and Gravili, 2000), *Zanclea* sp. 1 and *Zanclea* sp. 2 (*sensu* [22]) associated with the bryozoans *Celleporaria vermiformis* (Waters, 1909), *Celleporaria pigmentaria* (Waters, 1909), and *Celleporaria* sp., respectively; *Zanclea* cf. *protecta* associated with the bryozoans *Parasmittina* cf. *spondylicola* and *Schizoporella* sp. For comparison, *Asyncoryne ryniensis* Warren, 1908 was included in the analyses, since it is closely related to the family Zancleidae [22]. For each *Zanclea* species three colonies were collected, whereas two colonies of *A. ryniensis* were analyzed, for a total of 20 samples. Hydrozoan colonies were collected together with their hosts using hammer and chisel, by snorkeling or SCUBA diving. Colonies were immediately transferred in bowls with seawater after diving, and they were kept in the laboratories of the Marine Research and High Education (MaRHE) Center in Magoodhoo. One colony per species had medusa buds at the time of sampling, and these colonies were reared until

medusae were released. Seawater was replaced daily, approximately two hours after a feeding session with *Artemia* nauplii. Newly released medusae were reared for a few days and then anesthetized with menthol crystals and fixed with 10% formalin for further morphological analyses. Hydrozoan polyps were detached from their hosts using precision forceps and micropipettes, and they were fixed in 10% formalin and 99% ethanol for morphological and genetic analyses, respectively. Formalin-preserved polyps and medusae were analyzed using a Zeiss Axioskop 40 compound microscope to observe their general morphology and characterize their cnidome. Measurements were taken using the software ImageJ 1.52p. All pictures were taken using Canon G7X Mark II camera.

To investigate possible modifications related to the associations with hydroids, the skeletons of the hosts were analyzed under a scanning electron microscope. Specifically, fragments of the *Zanclea*-bearing bryozoan and scleractinian colonies were immersed in a 10% sodium hypochlorite solution for 6–24 h. After rinsing, fragments were sputter-coated with gold and observed under a Zeiss Gemini SEM 500 scanning electron microscope.

Before fixation, all hydrozoan polyps (*n* = 15 for each species and colony) and medusae (*n* = 5–15 for each species) were checked for green fluorescence emission using a Leica EZ4 D stereomicroscope equipped with a Weefine Smart Focus 2300 lamp (excitation wavelength: 420 nm) and yellow filter. All medusae were observed at day one and five after release.

#### *2.2. Molecular Characterization*

Genetic analyses were performed to check the molecular identity of the samples (*n* = 20) and to assess their phylogenetic relationships. DNA was extracted from one polyp per colony using a protocol modified from Zietara et al. [23] and already used proficiently to extract DNA from hydrozoans (e.g., [24]). A portion of the *16S rRNA* was then amplified using the primers and protocol described in Cunningham and Buss [25]. The success of PCRs was assessed through an electrophoretic run in 1% agarose gel. PCR products were purified and sequenced in forward and reverse directions with the same primers used for amplification, with ABI 3730xl DNA Analyzer (Applied Biosystems). The obtained chromatograms were visually checked and assembled using Geneious 6.1.6 and sequences were deposited with the EMBL (GenBank accession numbers: MN923260-MN923279). Each sequence was searched in the NCBI BLASTn database to confirm the morphological identifications. All the obtained sequences were then aligned using MAFFT 7.110 [26], with the *E-INS-i* option and the sequences of *Cladocoryne haddoni* and *Pennaria disticha* (GenBank accession numbers: MG811591 and LT746002, respectively) were included as outgroups. The best-fitting evolutionary model was determined using JModelTest 2 [27] and resulted in GTR+I+G, following the Akaike Information Criterion. Phylogenetic trees were built using both Bayesian inference and maximum likelihood approaches. For Bayesian analyses, MrBayes 3.2.6 [28] was used, and four parallel Markov Chain Monte Carlo runs (MCMC) were run for 107 generations, trees were sampled every 1000th generation, and burn-in was set to 25%. Maximum likelihood trees were built with RAxML 8.2.9 [29] using 1000 bootstrap replicates.

Pairwise genetic distances between and within species were calculated as % uncorrected *p*-distances with 1000 bootstrap replicates using MEGA X [30].

#### **3. Results**

#### *3.1. General Morphology of Polyps and Medusae*

All the analyzed *Zanclea* species showed a similar morphology in both polyp and medusa stages (Figures 1–6). All polyps were colonial, cylindrical, or claviform, with a whorl of oral capitate tentacles and aboral tentacles scattered on the hydranth body wall. Bryozoan-associated species (*Zanclea divergens*, *Zanclea* cf. *protecta*, *Zanclea* sp. 1, and *Zanclea* sp. 2) were monomorphic and deprived of perisarc, whereas the scleractinian-associated *Zanclea sango* and *Zanclea* sp. (Clade I) showed polymorphic polyps, having both gastrozooids and dactylozooids, and the hydrorhiza was surrounded by a thin layer of chitinous perisarc. All species had stenotele capsules in their capitula, and apart from *Zanclea* cf. *protecta*, all had euryteles in their polyps and/or hydrorhiza. Medusa buds arose directly from the hydrorhiza in *Zanclea divergens*, *Zanclea* sp. 1, and *Zanclea* sp. 2, whereas they were borne on both gastrozooids and hydrorhiza in *Zanclea* cf. *protecta*, *Zanclea sango*, and *Zanclea* sp. (Clade I). Medusae had a bell-shaped or globular umbrella, with nematocysts scattered over the surface in all species apart from scleractinian-associated species. *Zanclea* sp. 1 and *Zanclea* sp. 2 did not have canals and exumbrellar nematocyst pouches at release, whereas all other species had four radial and one circular canal and four nematocyst pouches containing stenoteles and euryteles (the latter only in coral-associated species). Manubria were cylindrical and had stenoteles around the mouth in *Z. divergens*, *Z*. cf. *protecta*, *Zanclea sango*, and *Zanclea* sp. (Clade I). *Zanclea* sp. 1 and *Zanclea* sp. 2 had no nematocysts on the manubrium but four short oral arms. All medusae had two opposite tentacles, bearing a variable number of rounded or elongated cnidophores containing bean-shaped macrobasic euryteles.

*Asyncoryne ryniensis* (Figure 7) polyps had a distinct morphology, being characterized by a whorl of capitate oral tentacles and moniliform tentacles scattered on the hydranth body wall. Polyps were monomorphic and had both stenoteles and euryteles. Medusa buds were borne on the distal half of polyps. The medusa stage was very similar to that of *Zanclea* species, showing a bell-shaped umbrella, one circular and four radial canals, four exumbrellar nematocyst pouches, four bulbs, and two opposite tentacles bearing cnidophores with macrobasic euryteles inside.

Detailed characterizations of morphology and cnidome of polyps and medusae of all species are summarized in Tables 1 and 2.

**Figure 1.** *Zanclea divergens*. (**a**) Colony associated with *Celleporaria vermiformis*; (**b**) close-up of a polyp; (**c**) stenoteles in the capitula, and (**d**) euryteles in the hypostome; (**e**) tube-like skeletal modifications of the bryozoan skeleton (arrowheads); (**f**) newly released medusa and close-up of (**g**) manubrium, (**h**) nematocyst pouch, and (**i**) cnidophores. Scale bars: (**a**) 0.5 mm; (**b**,**e**,**f**) 0.1 mm; (**c**,**d**,**g**–**i**) 10 μm.

**Figure 2.** *Zanclea* cf. *protecta*. (**a**) Colony associated with *Parasmittina* cf. *spondylicola*; (**b**) close-up of a polyp; (**c**) stenoteles in the capitula; (**d**) bryozoan skeletal lamina overgrowing the hydrorhiza (arrowheads); (**e**) newly released medusa; close-ups of (**f**) manubrium, (**g**) nematocyst pouch, and (**h**) cnidophores. Scale bars: (**a**) 0.5 mm; (**b**,**d**,**e**) 0,1 mm; (**c**,**f**–**h**) 10 μm.

**Figure 3.** *Zanclea* sp. 1. (**a**) Colony associated with *Celleporaria pigmentaria*; (**b**) close-up of a polyp; (**c**) stenoteles in the capitula, and (**d**) eurytele in the hydrorhiza; (**e**) tube-like modifications of the bryozoan skeleton (arrowheads); (**f**) newly released medusa; close-ups of (**g**) manubrium, (**h**) tentacular bulb, and (**i**) cnidophores. Scale bars: (**a**) 0.5 mm; (**b**,**e**) 0.1 mm; (**c**,**d**,**g**,**h**) 10 μm; (**f**) 20 μm.

**Figure 4.** *Zanclea* sp. 2. (**a**) Colony associated with *Celleporaria* sp.; (**b**) close-up of a polyp; (**c**) stenoteles in the capitula, and (**d**) euryteles in the hydrorhiza; (**e**) tube-like modifications of the bryozoan skeleton (arrowheads); (**f**) newly released medusa; close-ups of (**g**) manubrium, (**h**) tentacular bulb, and (**i**) cnidophores. Scale bars: (**a**) 0.5 mm; (**b**,**e**) 0.1 mm; (**c**,**d**,**g**–**i**) 10 μm; (**f**) 20 μm.

**Figure 5.** *Zanclea sango*. (**a**) Colony associated with *Pavona varians*; close-ups of (**b**) gastrozooid, and (**c**) dactylozooid; (**d**) stenoteles in the capitula, and (**e**) eurytele in the hypostome; (**f**) micro-alteration of the coral skeleton (arrowhead); (**g**) newly released medusa; close-ups of (**h**) manubrium, (**i**) nematocyst pouch, and (**j**) cnidophores. Scale bars: (**a**,**c**) 0.5 mm; (**b**,**f**,**g**) 0.1 mm; (**d**,**e**,**h**–**j**) 10 μm.

**Figure 6.** *Zanclea* sp. (Clade I). (**a**) Colony associated with *Goniastrea* sp.; close-ups of (**b**) gastrozooid and (**c**) dactylozooid; (**d**) stenoteles in the capitula, and (**e**) eurytele in the hypostome; (**f**) micro-alteration of the coral skeleton (arrowhead); (**g**) newly released medusa; close-ups of (**h**) manubrium, (**i**) nematocyst pouch, and (**j**) cnidophores. Scale bars: (**a**,**c**) 0.5 mm; (**b**,**f**,**g**) 0.1 mm; (**d**,**e**,**h**–**j**) 10 μm.

**Figure 7.** *Asyncoryne ryniensis*. (**a**) Colony growing on dead coral; (**b**) close-up of a polyp; (**c**) polyp showing green fluorescence before stimulation with blue light; (**d**) stenoteles in the capitulum, and (**e**) eurytele in the hydranth; (**f**,**g**) newly released medusa; close-ups of (**h**) manubrium, and (**i**) nematocysts in the tentacular bulb. Scale bars: (**a**) 0.1 mm; (**b**,**c**,**f**,**g**) 0.1 mm; (**d**,**e**,**h**,**i**) 10 μm.

*Diversity* **2020** , *12*, 78

**Table 1.** *Cont.*

47

**Table 2.** *Cont.*

*Diversity* **2020**, *12*, 78

#### *3.2. Modifications of the Hosts*

In all *Zanclea* samples, modification of the skeletons of the hosts were observed. *Zanclea divergens* polyps 'pierced' the skeleton of *Celleporaria vermiformis* along the border between zooids, and in some cases the bryozoan skeleton overgrew the base of polyps as a tube (Figure 1e). The hydrorhiza of *Zanclea* cf. *protecta* growing over the colony of bryozoan host *Parasmittina* cf. *spondylicola* was surrounded by a thin skeletal lamina produced exactly along the border between zooids (Figure 2d). Polyps of *Zanclea* sp. 1 and *Zanclea* sp. 2, associated with *Celleporaria pigmentaria* and *Celleporaria* sp. respectively, were observed coming out from the colony of the hosts at the borders between zooids, being partially overgrown at their base by the skeleton (Figure 3e, Figure 4e). Scleractinian-associated *Zanclea sango* and *Zanclea* sp. caused micro-alterations in the skeleton of the host corals, due to the skeletal overgrowth of the base of polyps and portions of the hydrorhiza (Figures 5f and 6f, respectively).

#### *3.3. Green Fluorescence Essay*

The six *Zanclea* and the *Asyncoryne* species showed different patterns of green fluorescence in both the polyp and medusa stages (Figure 8). Specifically, three *Zanclea* species (*Zanclea divergens*, *Zanclea* sp. 1, *Zanclea* sp. 2) and *Asyncoryne ryniensis* did not show fluorescence in the medusa stage. By contrast, the other three *Zanclea* species showed a marked green fluorescence in different structures. *Zanclea* cf. *protecta* showed a fluorescence at the level of the subumbrella, manubrium, and bulbs (Figure 8e,f). *Zanclea* sp. (Clade I) medusae released from colonies associated with *Goniastrea* sp. were characterized by a fluorescence of the radial and circular canals, bulbs, and whole manubrium (Figure 8a,b). Finally, *Zanclea sango* medusae displayed a pattern similar to that of *Zanclea* sp. (Clade I), with the exception of the central portion of the manubrium that did not show any fluorescence (Figure 8c,d). Fluorescence in these medusae was also present when still attached to the parental colony, and showed the same patterns displayed by newly released medusae (Figure 8g,h).

Regarding the polyp stages, *Zanclea* species did not show any fluorescence. Contrarily, *Asyncoryne ryniensis* polyps were characterized by a marked fluorescence at the base of moniliform tentacles (Figure 8i,j). In one polyp, green fluorescence was easily detected without excitation with blue light (Figure 7c).

Fluorescence patterns were identical for all medusae belonging to the same species, and no differences were detected between observations carried out at day one and five after release.

Fluorescence patterns of polyps and medusae for each species are summarized in Table 3.

**Table 3.** Summary of green fluorescence (GF) patterns in polyps and medusae of *Zanclea* and *Asyncoryne* species.

**Figure 8.** Green fluorescence in *Zanclea* and *Asyncoryne* species. (**a**,**b**) Medusa of *Zanclea* sp. (Clade I) released from a colony associated with *Goniastrea* sp.; (**c**,**d**) medusa of *Zanclea sango;* (**e**,**f**) medusa of *Zanclea protecta*; (**g**) medusa of *Zanclea* sp. before release, associated with *Goniastrea* sp.; (**h**) *Zanclea* cf. *protecta* medusa buds in the colony associated with *Schizoporella* sp. overgrowing the gastropod *Drupella* sp.; (**i**,**j**) *Asyncoryne ryniensis* polyps. Scale bars: (**a**–**g**) 0.2 mm; (**h**) 5 mm; (**i**,**j**) 1 mm.

#### *3.4. 16S rRNA Phylogeny*

DNA was extracted successfully, and *16S rRNA* sequences were generated for each analyzed sample. BLASTn searches resulted in a 100% match with previously deposited sequences obtained from Maldivian samples for *Zanclea* sp. 1, *Zanclea* sp. 2, *Zanclea* sp. (Clade I), and *Zanclea sango*. *Zanclea divergens* resulted in a match of 90.7% with an Indonesian sequence of the same species (MF000525), and this low value is explained by the fact that *Z. divergens* is a complex of cryptic species [31]. No *Zanclea protecta* sequences have been deposited so far, and the search for this species resulted in a match of 91.3% with *Zanclea costata* from the Mediterranean Sea (FN687559). Sequences of Maldivian *Asyncoryne ryniensis* resulted in a match of 98.4% with a Japanese specimen (EU876552).

The phylogenetic tree was rooted using *Pennaria disticha* [22,32] and, despite the overall poorly supported relationships (Figure 9), it agrees with previous reconstructions of *Zanclea* phylogeny [22]. Specifically, coral-associated *Zanclea* resulted in a fully supported clade, similarly to the clade composed of *Zanclea* sp. 1 and sp. 2 associated with bryozoans. Moreover, *Z. divergens* was well supported as the sister species of the latter clade, and all three species were associated with *Celleporaria* spp. Finally, the family Zancleidae was confirmed to be polyphyletic, due to the position of *Asyncoryne ryniensis*, which divides the family in two main clades, one associated with corals, and the other with bryozoans.

**Figure 9.** *16S rRNA* phylogeny of the species included in the analyses. Numbers at nodes represent Bayesian posterior probabilities and maximum likelihood bootstrap values, respectively. Hosts for each species are in brackets. Schematic drawings of fluorescence patterns in *Zanclea* medusae are also represented.

Inter-specific genetic distances were high in all comparisons, with the lowest level between the two coral-associated species *Z. sango* and *Zanclea* sp. (Clade I) (4%). All other species showed values higher than 10%. Intra-specific distances were equal to 0% in all cases (Table 4).

**Table 4.** Pairwise % uncorrected *p*-distances (*16S rRNA*) between all species analyzed.

#### **4. Discussion**

The genus *Zanclea* and family Zancleidae are challenging taxa both from an evolutionary point of view and for species identification or description [19,22]. Indeed, the genus and family are polyphyletic [22,33], and further analyses are needed to establish new genera or even families. Their taxonomy is complicated by the fact that polyps often have intergrading morphologies, and the adult medusa must be observed and characterized for correct species identification and description [20,22]. Indeed, cryptic or unidentifiable species are common in the *Zanclea* genus [18,19,22].

In this work we analyzed the morphology of six *Zanclea* species, considering the general features of polyps and medusae, the cnidome of both life stages, the alteration of the host skeletal structures, and the green fluorescence patterns. Additionally, we analyzed the molecular identity, phylogenetic relationships and genetic diversity of the species, confirming their possible belonging to six well separated *Zanclea* lineages. Our results show that the characterization of general morphology, and cnidome is in some cases enough to distinguish between *Zanclea* species. For instance, by combining observations on the presence, localization and size of euryteles, and the general appearance of polyps and medusae, it is possible to distinguish the analyzed bryozoan-associated species. By contrast, scleractinian-associated species showed a very similar morphology, as already documented in previous studies [18,21,34].

In all *Zanclea* species here analyzed, alterations of the host skeleton were observed. The bryozoan *Parasimittina* cf. *spondylicola* showed the most evident modification, with the skeletal lamina overgrowing the hydrorhiza of *Zanclea* cf. *protecta*, as already noted by Hasting [35] and Boero et al. [20] for *Zanclea protecta* associated with *Parasmittina crosslandi* (Hastings, 1930) and other unidentified bryozoans. A similar situation was observed for *Celleporaria*–*Zanclea* associations, where the base of polyps was occasionally surrounded by bryozoan skeletal structures. Additionally, scleractinians hosting *Zanclea* showed micro-alterations related to the presence of symbionts, as already observed in *Goniastrea*, *Pavona*, and *Porites* corals [21]. The presence of these modifications may support the hypothesis that at least some *Zanclea* species are mutualistically associated with their hosts, since they may provide additional protection and competitive advantages to their hosts and in turn benefit from being partially enclosed in hard carbonatic structures [36,37].

Differences were found in the green fluorescence patterns of *Zanclea* and *Asyncoryne* species. *Zanclea divergens*, *Zanclea* sp. 1 and *Zanclea* sp. 2 did not show any fluorescence neither in the polyps nor in the medusae. *Zanclea* cf. *protecta*, *Zanclea sango*, and *Zanclea* sp. (Clade I) did not show any fluorescence in the polyps but medusae were characterized by different green fluorescence patterns. Finally, *Asyncoryne ryniensis*, which has different polyps but medusae very similar to those of *Zanclea*, showed fluorescence in polyps but not in medusae. *Zanclea* cf. *protecta* is characterized by a diffuse fluorescence in bulbs, manubrium, and subumbrella, whereas *Z. sango* and *Zanclea* sp. (Clade I) are fluorescent in bulbs, manubrium, and canals. Despite the two latter coral-associated species have overlapping morphologies in both polyp and medusa stages, medusae showed differences in the distribution of green fluorescent proteins at the level of manubrium. Specifically, in *Zanclea* sp. (Clade I) the entire manubrium is fluorescent, and this pattern is visible even in the medusa buds still attached to the parental colony, whereas in *Zanclea sango* fluorescence is concentrated at the extremes

of manubrium (mouth and close to the umbrellar margin), being absent in the middle portion. These conditions were observed in all analyzed medusae and therefore may be taxonomically informative, even if further analyses are needed to confirm this hypothesis. Fontana et al. [38] also found green fluorescence in medusa buds of *Acropora*-associated *Zanclea* species, but the localization was not reported. However, this suggests that, potentially, the medusae of other coral-associated *Zanclea* species may be fluorescent. If this is true, the investigation of fluorescence patterns in all *Zanclea* species associated with scleractinians may help disentangling the cryptic diversity that characterize this group.

The function, if any, of green fluorescent proteins in the analyzed species is still not clear. One of the possible explanations is attraction of prey [8]. The polyps of the six *Zanclea* species observed all live in symbiosis with other organisms, and the lack of fluorescence in this stage may be related to specific feeding interactions with the hosts, as described for *Zanclea divergens*, which seems to feed on mucous aggregates of particles egested by the bryozoan [39]. Moreover, *Asyncoryne ryniensis* is not symbiotic, and fluorescence is found at the base of polyp tentacles. This explanation complicates with the medusa stages, since species with potentially similar feeding behaviors show contrasting fluorescence patterns.

Overall, the results obtained in this work show that the combination of multiple approaches allows one to discriminate closely related *Zanclea* species and provide information on the relationships between these hydrozoans and their hosts. Additionally, the analysis of green fluorescence patterns seems to be a promising tool for hydrozoan taxonomy and should be performed at a large scale to assess its adequacy in exploring and distinguishing the diversity of enigmatic hydrozoan taxa, such as zancleids.

**Author Contributions:** Conceptualization, D.M. and S.M.; investigation, D.M., L.S., A.S., and A.N.O.; writing—original draft preparation, all authors.; writing—review and editing, all authors. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** A. Ostrovsky thanks the Austrian Science Fund (project P19337-B17) for supporting the taxonomical research on the tropical Bryozoa. The authors are grateful to two anonymous reviewers, whose comments greatly improved the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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