Alcyonidium kuklinskii sp. nov., a new species of Antarctic ctenostome bryozoan with a key to all Antarctic species of the genus

Recent surveys of Antarctic waters in the Terra Nova Bay (Ross Sea) revealed numerous bryozoan species including ctenostome bryozoans. Whereas cheilostome bryozoans are well-studied in these latitudes, ctenostomes remain highly neglected. Large ctenostomes are easily recognized by their lack of calcified skeletons, but this lack also renders them difficult and tedious to identify. As a result, histology and reconstructions of internal soft tissues are required to classify this group of bryozoans. Thanks to the availability of new specimens from Terra Nova Bay, a detailed analysis of growth form, gut morphology and tentacle number of two colonies, initially ascribed to the ctenostome bryozoan genus Alcyonidum Lamouroux, 1813, turned out to be a new species, Alcyonidium kuklinskii sp. nov., which we described in this study. These specimens were also barcoded (COI) and sequences compared to available ones. Together with the new species described here, a total of ten species of Alcyonidium is now known for the Southern Ocean, accounting for one eighth of the entire genus diversity. All Southern Ocean species appear to be endemic. In order to speed the identification of the Antarctic Alcyonidium species, we provide an identification key and a distribution map of all type species. In brief, colony morphology, zooidal size and, in particular tentacle number represent the most suitable characters for identifying species within this genus. Supplementary Information The online version contains supplementary material available at 10.1007/s13127-023-00629-4.


Introduction
Bryozoans are a phylum of colonial suspension feeders belonging to Lophotrochozoa among Protostomia (Bleidorn et al. 2019).Colonies are formed by asexual buds resulting in clonal individuals, termed zooids, that comprises a tentacle crown or lophophore, u-shaped gut and diverse, associated neuromuscular tissue that is generally referred to as 'polypide'.The protective body-wall, the cystid (Mukai et al., 1997;Schwaha et al., 2020), can be mineralized in the clades Stenolaemata and Cheilostomata, which represent the most speciose and dominant taxa among bryozoans (Taylor, 2020).
Ctenostome bryozoans represent a small group of unmineralized bryozoans with approximately 350 recent species (Schwaha 2020).They show a multitude of different colony and zooid morphologies ranging from erect, encrusting to even boring/endolithic forms; and boxshaped to highly elongated, tubular zooids.One of the largest, more easily encountered and addressed ctenostome genera is Alcyonidium Lamoroux, 1813.It forms rather large colonies that can be erect or encrusting.Species are often transparent, but in several cases yellowish to brown coloration are found (D'Hondt 1983).Species identification is a long-lasting problem in the genus and superficial observations led to many misidentifications in the past (see e.g.Cadman & Ryland, 1996;Porter et al., 2001;Ryland & Porter, 2003;Porter, 2004).While molecular sequencing has made some advances in the past decades (see also references above), morphological characters still remain the most widely used tool for identification and involves typical characters such as colony and zooidal morphology, presence of kenozooids, but also features such as tentacle number, reproductive mode (Porter & Hayward, 2004) and even details of gut morphology (Le Brozec, 1955, D'Hondt, 1983).The latter usually requires either fine dissections or histological analyses.
A recent survey on the bryozoan species of Terra Nova Bay in the Ross Sea, housed in the collection of the Italian National Antarctic Museum, revealed 127 different species from 75 different sampling sites.Most species belong to cheilostome bryozoans (80%) and cyclostomes (18%) whereas ctenostomes account for just 2% of the entire collection (Cecchetto et al., 2019).In this first survey, a species of Alcyonidium remained unidentified (Fig. 1 in Cecchetto et al., 2019), and preliminary analysis by the authors of this study revealed it to represent a yet undescribed species.
In this study, we provide a formal description and a detailed analysis of the new species, Alcyonidium kuklinskii sp.nov., together with an identification key and a short biogeographical assessment of the species occurring in the Southern Ocean.Thereby, we hope to provide a helpful tool in species identification in all future studies of Antarctic Alcyonidium and to give a better tool for biodiversity assessment of Antarctic faunas.

Material
Two specimens of Alcyonidium sp. were collected in the framework of the XXV expedition of the Italian National Antarctic Program (PNRA) by one of the authors (SS) in Terra Nova Bay of Ross Sea during two separate SCUBA dives at the same location, named "Zecca" (74°41′24.972″S, 164°6′9.18″E), at a depth of 26 m.This site is already the type locality of another species, the calcareous sponge Megapogon schiaparellii Alvizu et al., 2019(Alvizu et al., 2019).One portion of a specimen (MNA voucher code MNA-02733, field code XXV-20, collected on 10 December 2009) was stored in paraformaldehyde, another in absolute ethanol and the rest kept at − 20 °C, whereas the other specimen (voucher code MNA-02904, field code XXV-91, collected on 12 December 2009) was stored in absolute ethanol.Both specimens are now stored at the Italian National Antarctic Museum (MNA, section of Genoa).
Samples of Alcyonidium gelatinosum were collected by dredging of dead bivalve shells at the Bay of Morlaix, Chateau du Taureau (France) in 2021.Samples were fixed in 96% ethanol.

Morphological methods
Documentation of colony pieces was conducted with a Nikon SMZ25 (Nikon, Tokyo, Japan) equipped with a Nikon DsRi-2 microscope camera, or a Hirox RH2000 microscope.For histological analysis, short branches of the colony were cut off, dehydrated in a graded ethanol series and afterwards infiltrated and embedded into Agar Low Viscosity Resin (Agar Scientific, Stansted, UK).Serial sections were conducted according to Ruthensteiner (2008).Sections were stained with toluidine blue, documented and analysed with a Nikon NiU microscope.

DNA extraction and sequencing
One portion of specimen MNA-02904 was clipped from the colony and transferred to a "plant tube rack" following the instructions provided by the Canadian Centre for DNA Barcoding (CCDB, https:// ccdb.ca/ site/ wp-conte nt/ uploa ds/ 2019/ 07/ Instr uctio ns_ Plant.pdf).The DNA extraction was performed according to the CTAB protocol (https:// ccdb.ca/ site/ wp-conte nt/ uploa ds/ 2016/ 09/ CCDB_ DNA_ Extra ction-Plants.pdf).Amplification and sequencing were carried out at the CCDB, targeting the partial cytochrome c oxidase subunit 1 (COI-5P) with the primer pair LCO1490_t1 and HCO2198_t1 (forward and reverse respectively, Foottit et al. 2009).
For Alcyonidium gelatinosum DNA was extracted using the QIAamp DNA Micro Kit (QIAGEN, Hilden, Germany) following the manufacture's protocol.Approximately 650 nucleotides of the COI gene were PCR amplified using primers LCO 1490 and HCO 2198 (Folmer et al., 1994).The PCR amplification of COI gene was performed using Red HS Taq Master Mix (Biozym, Oldendorf, Germany) (30 µl reaction with 1 µm of 20 µM primer, 1-3 µl of extracted DNA and 15 µl of Red HS Taq Master Mix).PCR products were cleaned using an enzymatic clean up reagent A'SAP (ArcticZymes Technologies ASA, Tromsø, Norway) and send to Microsynth Austria GmbH for sequencing.

Phylogenetic analysis
The COI sequences were aligned using MAFFT 7.310 (Katoh et al., 2002) with the following options: -auto, -localpair and -maxiterate 1000.The alignment was then trimmed manually in order to remove ambiguously aligned sites.Maximum likelihood (ML) analysis was performed with IQ-TREE2 v2.1.2(Minh et al., 2020) using ModelFinder tree search with 1000 ultrafast bootstraps and SH-aLRT test replicates (Hoang et al., 2018;Kalyaanamoorthy et al., 2017).Bayesian analysis (BI) was performed using the MrBayes (version 3.2.7a)package (Ronquist et al., 2012) with two separate runs of four chains of a Markov Chain Monte Carlo (MCMC) algorithm.BI analysis was conducted for two million generations with tree sampling every 100 generations.The run ended only after the Bayesian MCMC searches had reached a stationary phase (indicating convergence of the chains onto the target distribution) .A consensus tree was calculated using the last 75% best scoring trees and 25% of the sampled trees were discarded as burn-in.Finally, ML-corrected substitutions per site were calculated in MEGA 7 using the maximum composite likelihood parameter with a gamma parameter of 1.0 (Kumar et al., 2008).Type locality: Ross Sea, Terra Nova Bay, "Zecca" diving site, Latitude: 74°41′24.972″S, Longitude: 164°6′9.18″E.

Barcode sequence
The specimen analysed, MNA-02904, was sequenced to obtain a final COI sequence length of 646 bp.Taxonomic assignation was checked through Barcode of Life database (BOLD) and National Center for Biotechnology Information (NCBI) database BLAST (https:// blast.ncbi.nlm.nih.gov/ Blast.cgi, accessed on 09 June 2023) for definitive assignment.A sequence match > 98% with the reference database was considered an 'exact' match (Leray & Knowlton, 2016).The other Antarctic species with available sequences, Alcyonidium flabelliforme, did not match our reported species A. kuklinskii sp.nov..The sequence of A. kuklinskii sp.nov is available at the Barcode of Life Data System (BOLD, Ratnasingham and Hebert, 2007) with the processID BAMBI2007-20.Genbank accession number is OR076125.Sequence data of A. gelatinosum is accessible under OR187863 Genbank accession number.

Molecular phylogeny
Sequences of the COI gene were generated for two Alcyonidium species and combined with five publicly available COI sequences of Alcyonidium.ML phylogenetic tree was constructed using of 646 unambiguously aligned nucleotide sites of COI gene.Pectinatella magnifica: NC_038192 (Gim et al., 2018) was used as an outgroup to root the phylogenetic tree.
The ML and BI phylogenies resulted in highly consistent topologies with little variation in support values as shown in Fig. 5  The pairwise ML-corrected distances over the COI gene sequences between all Alcyonidium species are provided in Table 2. Pairwise ML distances ranged from 0.300 between Alcyonidium kuklinskii sp.nov.and A. mamillatum to 0.565 between A. gelatinosum and Alcyonidium kuklinskii sp.nov..

Geographical distribution of Antarctic alcyonidiids
Five species of Antarctic Alcyondium are recorded from the Straight of Magellan and the Antarctic Peninsula (see Table 3): A. australe, A. eightsi, A. epispiculum, A. scolecideum, A. simulatum.A. antarcticum is found close to the peninsula in the Bellinghausen Sea.The pelagic and still almost unknown A. pelagosphaerum occurs in the Weddell Sea.Types of A. flabelliforme and A. kuklinskii sp.nov.have been found in the Ross Sea, with A. torpedo having a close collection site (Fig. 6).

Diversity of polar species of Alcyonidium
As previously already indicated (Porter & Hayward, 2004), there seems to be a high degree of endemism among Antarctic species of Alcyonidium.So far, all ten species seem to be restricted to the Antarctic and Subantarctic waters, with some occurring in the southernmost tip of South America.With currently ~ 80 species described in the genus, the Southern alcyonidiids would entail one eight of all known species.Although some older species descriptions are very limited and provide little details (e.g.Waters, 1904, D'Hondt 2006), we were able to provide a key to all known Antarctic species of Alcyonidium based on all available information that should allow for easier species discrimination in the future.
Up to 15 species or variants have been reported from Arctic waters (Kluge, 1975), but many were assigned to species known from more temperate, particularly European waters such as Alcyonidium gelatinosum, A. mytili, or A. diaphanum, which were particularly prone to being misidentified in the past (Cadman & Ryland, 1996;Porter et al., 2001, Ryland & Porter 2003).Unfortunately, no detailed analyses employing morphological and basic molecular techniques have been applied to Arctic species yet.This would ultimately clarify the true identity of these species and also show whether a similar degree of endemism is present in Arctic waters.One particularly unique species, however, truly endemic to Arctic areas is A. disciforme, which forms unattached, disc-shaped colonies (Kuklinski & 2004).

Distribution of Antarctic alcyonidiids
As evident from the localities from all described species, most species have been described from the Straight of Magellan, or locations around the Antarctic Peninsula, such as the South Shetlands Islands, whereas other areas show a distribution over dispersed localities.This is most likely a result of sampling bias because of extensive collections from the US Antarctic Research Program (USAP), which were also thoroughly studied (Porter & Hayward, 2004).Besides sampling, taxonomic expertise is another requirement for ctenostome animals, especially since it usually requires histological information for proper species identification and diagnosis.We estimate that the diversity of Antarctic alcyonidiids might be much higher, but requires more study in the future.

Characters in Alcyonidium systematics
As mentioned, Alcyonidium represents one of the largest genera of ctenostome bryozoans (www.bryoz oa.net) and has traditionally been one of the most difficult to identify and many species have been wrongly addressed or identified in the past (see references above).Particularly, features such as reproduction and tentacle number have proved to be very important for species discrimination.Also, internal features of the digestive tract have long been pointed out as very informative character for species identity (Le Brozec, 1955, see also D'Hondt, 1983).Unfortunately, this information is seldom recorded in more recent species description, but has proven also very useful in closely-related families such as the Pherusellidae (Decker et al., 2021).Soft-tissue or reproductive characters remain the most useful characters for species determination in all ctenostome bryozoans (Jebram, 1986;Schwaha, 2020).The current assessment on Antarctic bryozoans shows that colony morphology and tentacle number essentially can provide the necessary features for species discrimination.In many preserved material or broken pieces of colonies this will practically be difficult to test without dissection or histology.Also, some broken pieces of colonies might either lack polypides or be insufficient altogether for proper species identification (see also Porter & Hayward, 2004).Polypide features especially the variable gut structure would be important to evaluate in the future, as it could represent an easy and reliable tool for species discrimination by simple mounting of bryozoan guts.
Reproduction is likewise a character of important species assignment, but very difficult to assess in most cases as it often required histology to check: size and number of gonads (oocytes), presence of an intertentacular organ (ITO, used in planktotrophic species), presence of brooded large embryos.Also, in many species the mode of reproduction is not evident when lacking embryos or distinct gonads as encountered in our current analysis on Alcyonidium kuklinskii sp.nov..
Ultimately, it will be crucial that more genetic data along with proper morphological identification will be provided for many species of Alcyonidium.This would ease the difficulties in species identification and biodiversity assays in the future.

Fig. 2
Fig. 2 Stereomicroscopic images of Alcyonidium kuklinskii sp.nov.Images a-c, e-f from holotype MNA-02733, d from paratype MNA-02904, a and b Colony pieces of the holotype, c Close-up showing thinner and thicker branches of the colony.d and e Zooidal shapes and few small kenozooids in between.f More cleared colony piece showing internal, functional polypides.Abbre: ap -aperture, az -autozooid, kzkenozooid, ply -polypide in the ML topology and in BI tree in the supplementary information Fig. S1.Two clades were recovered in the Alcyonidium phylogeny, the first clade comprising A. gelatinosum and A. verrilli supported with 0.98 Bayesian posterior probabilities (PP) and 79 bootstrap replicates.The second clade includes the remaining Alcyonidium species and is supported with 0.86 PP and 52 bootstraps.Within the latter clade, the new Alcyonidium species and A. mamillatum have a sister taxon relationship supported in 0.95 PP and 80 bootstraps.

Fig. 6
Fig. 6 Map showing the type localities of all Antarctic species of Alcyonidium

Table 1
Comparison of all Antarctic species of Alcyonidium, modified and extended from Porter et al.