First record of Zoantharia in the Black Sea: Isozoanthus cf. sulcatus reared from planulae

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Zoantharia is an order within hexacorallian Anthozoa (Sinniger et al., 2008), comprising primarily colonial species with polyps characterized by two rows of tentacles, a single siphonoglyph, and presence of incrustations in their body walls (Low et al., 2016). The incrustations pose challenges when applying traditional morphological identification methods. Consequently, in recent years, molecular barcoding has emerged as an approach broadly used within Zoantharia for species identification and phylogenetic reconstructions (Sinniger et al., 2008; Fujii, Reimer, 2013; Low et al., 2016).

Despite zoantharians being found in many marine ecosystems from Arctic to Antarctic (Low et al., 2016), they have never been documented in the Black Sea. In September 2017, hundreds of early cnidarian larvae were collected in a planktonic net haul taken from the pier of the Southern Branch of the Shirshov Institute of Oceanology of Russian Academy of Sciences (SO IO RAS), Golubaya (Blue) Bay, NW Black Sea. Initially regarded as juvenile actiniarians, settled larvae were left unattended in a glass container in a sea water aquarium. Within a few weeks, settled larvae unexpectedly developed into small colonies exhibiting the characteristic appearance of zoantharians (two rings of tentacles, colonial growth form). The primary objective of our work was to document the first record of Zoantharia in the Black Sea and to provide preliminary description and identification.

MATERIAL AND METHODS

Zoanthid larvae (several hundred) were collected from a haul of a simple Apstein plankton net (max diameter 50 cm, mesh size 100 µm) taken from the pier of SO IO RAS, 44°34.5’N, 37°58.7’E (15 km northwest of Gelendzhik, NW Black Sea). Twenty larvae were fixed in 2.5% glutaraldehyde/0.1 M cacodylate buffer (pH 7.2). Remaining larvae were transported alive in a Falcon tube filled with filtered seawater to the Invertebrate Zoology Department at Lomonosov Moscow State University. The larvae were kept in a glass container with mesohaline salinity water at ambient temperature (18 psu, 18 °C), where some of planulae have settled. Settled larvae in the same glass container were transferred into a sea water aquarium where they were maintained at the same conditions (18 psu, 18 °C) for several months. In April and June 2018, polyps were sampled from three largest colonies and fixed in 96% ethanol for DNA analysis. In addition, we used 96% ethanol fixed fragment of Isozoanthus sulcatus (Gosse 1860) from the Plymouth area (UK) ~55 km from the type locality.

For SEM (scanning electron microscopy) observations, the larvae fixed in glutaraldehyde were dehydrated in an increasing ethanol series, critical point dried with CO₂, mounted on aluminum stubs, coated with an Au-Pd mixture and examined using a CAM SCAN S-2 (Cambridge Instruments, Cambridgeshire, UK) of the Shared Facilities center “Electron Microscopy for Life Sciences” at Lomonosov Moscow State University.

Genomic DNA was extracted from 96% ethanol fixed specimens. Fragments were exsiccated at 60 °C before lysis and genomic DNA was extracted using LumiPure kit from AnySample (Lumiprobe) according to the manufacturer’s recommendations. PCR amplification was accomplished for the mitochondrial COI sequenced with universal primers jgHCO2198 and jgLCO1490 (Geller et al., 2013) using HS-ScreenMix kit (Evrogen) according to the manufacturer’s protocol. The resulting PCR product was sequenced with same primers using ABI PRISM® BigDye™ Terminator v. 3.1 on Applied Biosystems (Foster City, CA, USA) DNA Analyzer 3500 ABI. Sequences of Isozoanthus sulcatus (OR785060) and I. cf. sulcatus (OR785057, OR785058, OR785059) generated in the study were deposited in the NCBI database. Additional sequences of species representing several families of Zoantharia were obtained from NCBI (GenBank) database (Benson et al., 2012). Sequences were aligned using the MAFFT v7.450 (Katoh, Standley, 2013) with the manual check and correction.

For phylogenetic reconstructions we employed the Maximum Likelihood (ML) method implemented in the IQ-TREE 1.6.12 software (Nguyen et al., 2015). To assess branch support we used ultrafast bootstrap (Hoang et al., 2018) approximation (UFboot) and the SH-like approximate likelihood ratio test (SH-aLRT) (Guindon et al., 2010) with 10000 bootstrap replicates. The substitution model was chosen according to the Bayesian information criterion (BIC) with the help of the ModelFinder (Kalyaanamoorthy et al., 2017) implemented in the IQ-TREE software. Obtained phylogenetic trees were visualized with the help of ITOL v.6.7.5 (Letunic, Bork, 2021).

To test the presence of Symbiodiniacea in specimens of Isozoanthus sulcatus from Plymouth (1) and I. cf. sulcatus from the Black Sea (3) we used specific primers and protocols developed by Manning and Gates (2008) for Symbiodiniacea (Symbiodinium on original paper). The results of PCR were visualized with 2% agarose gel.

RESULTS AND DISCUSSION

The larvae were elongated ciliary planulae, 0.3 mm in length, with a cluster of longer cilia forming an apical tuft at the aboral pole (Fig. 1A). The vast majority of larvae settled in a small glass container and developed into small anemone-like polyps. The polyps were left unattended, and in few weeks settled polyps developed into small colonies few millimeters across comprised of flat horizontal stolons bearing diminutive cylindrical polyps. Each polyp had a small number (18–21) of alternating longer and shorter tentacles arranged in two rings distinguishable by their size and position. Siphonoglyph was not detected. Measured stolons were 1.2–2.0 mm in width, with 3–5 polyps per 1 cm. At thinner stolons (up to 1.5 mm) polyps were arranged in one row, and in two rows and more on thicker (1.5–2.0 mm) ones. In the middle of colonies, most of stolons were interconnected and fused, and polyps were denser set. By April–May 2018, larger irregularly shaped colonies reached a size up to 55–60 mm across.

 

Fig. 1. Isozoanthus cf. sulcatus from the Black Sea: A – Planula, SEM; B–D – Close-ups of colonies reared in aquarium.

 

The larger polyps were 3–4 mm high, with an oral disk up to 1.5–2.5 mm in diameter. Tentacles of undisturbed, fully expanded polyps reached up to 2.5–4 mm, with basal diameter 0.2–0.25 mm (Fig. 1B–1D). By May 2018, in the grown colonies brownish-green globular bodies were clearly visible within the polyp walls, oral disks, and tentacles, giving the tissues a brownish color (Fig. 1B–1D). Small growing polyps (<1 mm in diameter) in the distal part of stolons were almost transparent (Fig. 1B). Same structures have been seen observed at early developmental stages (planulae and settled larvae). Based on PCR with Symbiodiniacea-specific primers (Manning, Gates, 2008), we consider these structures to be symbiotic algae of the family Symbiodiniacea. A specific PCR product was obtained for two of three zoantharian specimens from the Black Sea and Isozoanthus sulcatus from the Plymouth area. The latter is reported to harbor “zooxanthellae” (Williams, 2000). Genetic identification of symbionts from Isozoanthus cf. sulcatus from the Black Sea is currently under way.

The colonies survived in the aquarium for approximately nine months, but in June 2018 they all died due to algal bloom.

The resulting ML reconstruction using COI sequences (Fig. 2) supported the monophyly of specimens from the Black Sea with Isozoanthus sulcatus (Gosse 1860) from the North Atlantic Ocean (Mediterranean Sea, Sweden and Plymouth area). Three COI sequences of the Black Sea specimens were identical to each other. Sequences of I. sulcatus from the Plymouth area were identical to those of GenBank I. sulcatus from the Medditerranean Sea and Skagerrak (Sweden). The similarity between the two Isozoanthus groups of specimens was 98.8%. Variability of COI in Zoantharia is rather low, thus, specimens from the Black Sea may represent a new species closely related to I. sulcatus.

 

Fig 2. The ML Phylogenetic tree COI gene (658 bp). Only reliable supports values (SH‒aLRT/UFboot greater than or equal to 80/95%) are shown. Data generated in our study are marked in bold.

 

In absence of material for morphological analysis, we were not able to formally describe a new species. In resulting phylogenetic tree, the clade comprised of I. sulcatus and I. cf. sulcatus was closely related to Nanozoanthus Fujii & Reimer 2013 and less to Microzoanthus Fujii & Reimer 2013. The lack of COI sequences for type species of the genus, Isozoanthus giganteus Carlgren in Chun 1903, does not allow us to discuss further phylogenetic relationships of the genus Isozoanthus Carlgren in Chun 1903. More genetic data is required to address this question.

Information on reproductive strategies in Zoantharia is limited (Ryland, 1997; Previati et al., 2010). Nevertheless, the majority of Macrocnemina (Previati et al., 2010) and particularly I. sulcatus (Carlgren, 1913; Williams, 2000) are reported to have separate sexes. Thus, the discovery of hundreds of competent planulae suggests the presence of a reproductive population.

The question of whether this species is invasive or native remains unresolved. Isozoanthus cf. sulcatus, from the Golubaya Bay demonstrates remarkable tolerance for low (17.5–18 psu) salinity. The invasiveness of the new species could explain this, but there is no direct indication of where it might have come from. The genus Isozoanthus has never been reported from the Black Sea neither it been listed from the Sea of Marmara (Cinar et al., 2014). However, the small size of polyps (1.0–2.5 mm in diameter in studied colonies), and the lack of focused research may contribute to diminutive zoantharians remaining undetected (e. g. Reimer et al., 2017). Further targeted investigations of cryptic habitats, such as shallow sedimented benches, cracks and crevices, hold the potential to enhance our understanding of the Black Sea biodiversity.

ACKNOWLEDGEMENTS

We would like to thank Frederic Sinniger (University of the Ryukyus) for his kind advice with DNA sequencing protocols, James Hall-Spencer (University of Plymouth) for DNA graded material of Isozoanthus sulcatus, and also Galina Kolyuchkina (IORAS), Vitaliy Semin (SO IORAS) and Glafira Kolbasova (WSBS MSU) for their immense however fruitless effort to find source colonies of I. cf. sulcatus in Golubaya Bay. In addition, we express our gratitude to Nadezhda Rimskaya-Korsakova and Stanislav Kremnyov (Lomonosov MSU) for invaluable help in collecting animals, to Maria Rimskaya-Korsakova (IORAS) for support in organizing the expedition, and to the Shared Facilities center “Electron Microscopy for Life Sciences” at Lomonosov Moscow State University for the opportunity to conduct electron microscopic studies and two anonymous reviewers for their constructive and helpful suggestions that resulted in substantial improvements to this study.

FUNDING

This work was supported by the Russian Science Foundation (research grant 22-24-00873 to TM).

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

This work does not include any human or animal studies that meet the criteria of Directive 2010/63/EU.

CONFLICT OF INTEREST

The authors of this work declare that they have no conflicts of interest.

About the authors

Ulyana V. Simakova

Shirshov Institute of Oceanology, Russian Academy of Sciences

Email: tina@ocean.ru
Russian Federation, 36 Nakhimovsky prospekt, Moscow, 117218

Andrey A. Prudkovsky

Lomonosov Moscow State University

Email: tina@ocean.ru

Invertebrate Zoology Department

Russian Federation, Leninskie Gory 1–12, Moscow, 119234

Tatiana S. Lebedeva

Lomonosov Moscow State University

Email: tina@ocean.ru

Embryology Department

Russian Federation, Leninskie Gory 1–12, Moscow, 119234

Alexandra E. Smorygo

Université libre de Bruxelles

Email: tina@ocean.ru
Belgium, Avenue Franklin Roosevelt 50, 1050, Bruxelles

Viktoria N. Moskalenko

Shirshov Institute of Oceanology, Russian Academy of Sciences

Email: tina@ocean.ru
Russian Federation, 36 Nakhimovsky prospekt, Moscow, 117218

Tina N. Molodtsova

Shirshov Institute of Oceanology, Russian Academy of Sciences

Author for correspondence.
Email: tina@ocean.ru
Russian Federation, 36 Nakhimovsky prospekt, Moscow, 117218

References

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