Integrative systematics illuminates the relationships in two sponge-associated hydrozoan families (Capitata: Sphaerocorynidae and Zancleopsidae)

An integrated approach using morphological stages of the genera Euphysilla and Zancleopsis , which were originally described exclusively based on the medusa stages, describe a new sphaerocorynid genus and species, and assess the phylogenetic position of the two families within the Capitata. The monotypic genus Astrocoryne was found to be a synonym of Zancleopsis . Astrocoryne cabela was therefore transferred to the genus Zancleopsis as Zancleopsis cabela comb. nov. The new polyp-based genus and species Kudacoryne diaphana gen. nov. sp. nov. was erected within the Sphaerocorynidae. Both taxa are primarily based on genetic data, but the introduction of this new genus was made necessary by the fact that it clustered with the genera Heterocoryne and Euphysilla, despite showing Sphaerocoryne -like polyps. Interestingly, the species analysed in this work showed contrasting biogeographical patterns. Based on our data and literature records, some species appear to have a wide circumtropical range, whereas others are limited to few localities. Overall, these results lay the ground for future investigations aimed at resolving the taxonomy and systematics of these two enigmatic families.


Introduction
Hydrozoan life cycles generally comprise three life stages, namely a larva (planula), a polyp, and a medusa, but exceptions to this pattern occur in many hydrozoan groups (Boero et al., 1992(Boero et al., , 1997Bouillon et al., 2006). Because medusae and polyps have different morphologies and require different collecting techniques, early naturalists often adopted two parallel classification systems for hydrozoans, due to the difficulties in linking the two life stages (Edwards, 1973;Boero, 1980). Later attempts were made to unify these classification systems (e.g., Rees, 1957;Brinckmann-Voss, 1970), but the advent of molecular approaches, including dna barcoding and molecular phylogenetics, has facilitated this task, allowing taxonomists to more easily link polyps and medusae previously assigned to different species, genera, or even families (e.g., Pyataeva et al., 2016;Schuchert et al., 2017;Schuchert, 2018). Notably, this integrative approach also revealed previously unsuspected relationships, such as for the hydrozoan Microhydrula limopsicola Jarms & Tiemann, 1996, discovered to be an early stage of the staurozoan Haliclystus antarcticus Pfeffer, 1889(Miranda et al., 2010, or the mysterious genus Dendrogramma Just, Kristensen & Olesen, 2014, which is not classifiable into any metazoan phylum based on morphology alone (Just et al., 2014), but is now recognised as part of a siphonophore (O'Hara et al., 2016).
Recently, Schuchert & Collins (2021) published a study on the hydromedusae of the Gulf Stream off Florida, providing dna barcodes for many species previously not characterised genetically, and establishing the link between polyps and medusae for a number of species. Among others, they found that Euphysilla pyramidata Kramp, 1955 was genetically very similar to a Sphaerocoryne polyp from the Maldives, and that Zancleopsis dichotoma (Mayer, 1900) was closely related to Astrocoryne cabela Maggioni et al., 2017 from the Maldives and the Red Sea, for which only the polyp and the young medusa are currently known (Maggioni et al., 2017). Sphaerocoryne Pictet, 1893, Astrocoryne Maggioni et al., systematics of the sphaerocorynidae and zancleopsidae Downloaded from Brill.com04/05/2022 08:52:11AM via free access 2017, Heterocoryne Larson, 1986, andEuphysilla Kramp, 1955 currently constitute the family Sphaerocorynidae Prévot, 1959, whereas Zancleopsis Hartlaub, 1907and Dicnida Bouillon, 1978 are the two genera of the family Zancleopsidae Bouillon, 1978. Both families are in the suborder Capitata, even though the phylogenetic relationships with the other Capitata members were assessed only for the Sphaerocorynidae (Nawrocki et al. 2010;Maggioni et al. 2017Maggioni et al. , 2018Maggioni et al. , 2021. Species in this latter family are characterized by a rather complex taxonomic history, exacerbated by the incomplete knowledge of the life cycles of some species and by nomenclatural issues (see Calder, 2010;. This is the case, for example, of the genus Sphaerocoryne, currently comprising three valid species, the type species Sphaerocoryne agassizii (McCrady, 1859), Sphaerocoryne bedoti Pictet, 1893, and Sphaerocoryne peterseni Bouillon, 1984, with the latter known only from the medusa stage (Bouillon, 1984). Following Calder (2010), if all species are congeneric, then Sphaerocoryne will become a junior synonym of Corynetes Haeckel, 1879. However, if S. agassizii is not congeneric with the other two species, it will become Corynetes agassizii, whereas the genus Sphaerocoryne will remain valid for S. bedoti and S. peterseni. As already highlighted by , only a genetic assessment can eventually clarify the status of the two generic names. Sphaerocorynid polyps live generally in association with sponges, although some exceptions are known, and the main differences among genera rely on the tentacle organisation (Maggioni et al., 2017). Other differences are in the reproductive stage, with fixed eumedusoids in Heterocoryne and free medusae in Sphaerocoryne, Astrocoryne, and Euphysilla. However, the adult medusa of Astrocoryne and the polyp stage of Euphysilla were unknown until now.
The family Zancleopsidae currently comprises six Zancleopsis and one Dicnida species, all of which were described based on the medusa stage alone, and the polyps remain unknown (Wang et al., 2016). The phylogenetic position of the family and the internal relationships have never been investigated thoroughly, but a recent phylogenomic work placed the species Zancleopsis tentaculata  within Capitata, together with Millepora and Pennaria (Bentlage & Collins, 2021).
In this work, several sphaerocorynid and zancleopsid polyps and medusae were analysed using an integrative approach in order to disentangle the taxonomy and systematics of the two families. Specifically, morphological and genetic analyses were performed to assess the diversity and the phylogenetic relationships of the two families, and to eventually link polyp and medusa stages of species with partially unknown life cycles.

Material and methods
Sampling of the hydroids was carried out by snorkelling (0-5 m depth) and diving (5-30 m depth) between March 2008 and June 2020 in several localities across the Red Sea, the Indo-Pacific, and the Atlantic Ocean (supplementary table S1). Small fragments of the host sponges (or other substrates) and associated hydroids were collected. Identification of the host sponge to the species or genus level was possible only in a limited number of samples (supplementary table S1). Where possible, live animals were reared in constantly oxygenated bowls filled with seawater, at room temperature, under artificial light, and fed with Artemia nauplii to observe medusa buds maturation and release. Animals were anesthetised with menthol crystals and directly observed and photographed under a Leica maggioni et al. ez4 D stereo microscope and a Zeiss Axioskop 40 compound microscope. Subsequently, polyps were detached from their substrate and fixed in both 99% ethanol for molecular analyses and 10% formalin for further morphological analyses. All measures were taken using the ImageJ 1.52p software. Medusae were collected, photographed, and fixed as described by Schuchert & Collins (2021). Voucher material was deposited at the Muséum d'Histoire Naturelle (Geneva, Switzerland) and is indicated by the catalogue number mhnginve-###. Other acronyms for voucher material are msnm-Coe-### (Museo Civico di Storia Naturale, Cnidaria Collection, Milano, Italy) and uf-### (Florida Museum of Natural History, Gainesville, Florida, USA).
Total genomic dna was extracted from ethanol-fixed samples using the protocols described in Maggioni et al. (2020a) and Schuchert (2005). Briefly, dna was extracted from a single polyp or medusa by digesting the specimen in a mixture of proteinase K and MilliQ water in the first case, or following the CTAB protocol (Coffroth et al., 1992) in the second case. Six molecular markers were amplified using the primers and protocols described in Maggioni et al. (2020b), including portions of the mitochondrial large ribosomal rRNA gene (16S rRNA), cytochrome oxidase subunit I gene (cox1), cytochrome oxidase subunit iii gene (cox3), and nuclear small ribosomal rna gene (18S rRNA), large ribosomal rna gene (28S rRNA), and internal transcribed spacer region (its; including partial its1, 5.8S, and partial its2 regions). The success of polymerase chain reactions (pcr s) was checked through 1.5% agarose electrophoretic runs and pcr products were then purified with Illustra ExoStar (ge Healthcare) and sequenced in both directions by Macrogen Europe (Amsterdam, The Netherlands).
Geneious 6.1.6 (Kearse et al., 2012) was used to check, correct, and assemble the obtained chromatograms and to assess the presence of open reading frames in protein-coding genes. All consensus sequences obtained in this study were deposited in GenBank with the accession numbers mz457346-mz457408, mz457564-mz457713, mz457741-mz457898, and mz463764-mz463844, as listed in table S1. Sequences were aligned using mafft 7.110 (Katoh & Standley, 2013) with the E-ins-i option, after adding outgroup sequences following Maggioni et al. (2017Maggioni et al. ( , 2018) (supplementary table S1). Additionally, except for the protein-coding genes, the alignments were run through Gblocks (Castresana, 2000;Talavera & Castresana, 2007) using the 'less stringent' settings to remove poorly aligned regions (supplementary table S2). All alignments were analysed separately and then concatenated using Mesquite 3.2 (Maddison & Maddison, 2006). Substitution models were determined using jModelTest2 (Darriba et al., 2012) and PartitionFinder 1.1.1 (Lanfear et al., 2012) for the single-locus and multi-locus datasets, respectively, using the Akaike Information Criterion (aic) (supplementary tables S2 and S3). Phylogenetic reconstructions were performed using maximum parsimony (mp), maximum likelihood (ml), and Bayesian inference (bi) using both single-and multi-locus datasets. mp analyses were performed using paup 4.0b10 (Swofford, 2003) with heuristic searches stepwise addition and tree-bisection reconnection branch swapping. Node support was assessed using 1,000 bootstrap replicates with randomly added taxa. ml analyses were run in RAxML 8.2.12 (Stamatakis, 2014), using gtr+G as the substitution model, and with 1,000 bootstrap replicates. bi analyses were run using MrBayes 3.2.6 (Ronquist et al., 2012), and two independent runs for four Markov chains were conducted for 50 million generations, with trees sampled each 5,000th generation. A species tree was also obtained using *beast (Heled & Drummond, 2009) (Bouckaert et al., 2014), considering mitochondrial loci as a single partition, and keeping nuclear loci separate. Yule process prior, together with a linear and constant-root population-size model were used, and each analysis was run for 108 generations, sampling each 10,000th generation. For both MrBayes and beast analyses, parameter estimates and convergence were checked using Tracer 1.6 , and the burn-in was set at 25%. All phylogenetic analyses were run on the cipres server (Miller et al., 2010). Finally, mega X (Kumar et al., 2018) was used to compute genetic distances within and among the obtained clades, calculated as % uncorrected p-distances with 1,000 bootstrap replicates.
To confirm the morphology-based species boundaries, dna-based species delimitation approaches were applied to the single-locus 16S rRNA dataset. Prior to performing the analyses, identical sequences were collapsed into representative sequence types using FaBox (Villesen, 2007), following Fontaneto et al. (2015). The distance-based Assemble Species by Automatic Partitioning (asap; Puillandre et al., 2021) method was run on the website 'asap web' (https://bioinfo.mnhn.fr/ abi/public/asap/) using all the three available models (p-distance, Kimura 2-parameter, and Jukes Cantor) and considering only the partitions showing the lowest asap-score. The tree-based Poisson Tree Process (ptp) and Generalised Mixed Yule Coalescence (gmyc) approaches were also used. Specifically, a 16S rRNA ml tree was obtained as described above and was used as input for multiple-threshold ptp analyses (mtPTP: Kapli et al., 2017) on the website 'mPTP Webservice' (https://mptp.hits.org). For gmyc analyses, a 16S rRNA ultrametric Bayesian tree was obtained with beast 1.8.2 , setting a coalescent tree prior and an uncorrelated lognormal relaxed clock. Three replicate analyses were run for 108 million generations, sampling trees every 10,000 generations, and were combined using LogCombiner 1.8.2  with a burn-in set to 25%. A maximum clade credibility tree was then obtained using TreeAnnotator 1.8.2 . gmyc analyses were run in the R environment (R Core Team, 2020), using the single-threshold (stGMYC; Pons et al., 2006) and bGMYC (Reid and Carstens, 2012) implementations, using the packages 'Splits' (Ezard et al., 2009), 'Ape' (Paradis et al., 2004), and 'bGMYC' (Reid and Carstens, 2012).
dna diagnostic characters were searched for each genus in the family Sphaerocorynidae, given the difficulties in defining generic diagnoses based on morphology, using the package quiddich (Kühn & Haase, 2020) in the R environment (R Core Team, 2020). Specifically, character attributes (i.e., single nucleotides) present in all members of a defined clade but absent in members of other clades were searched, and the analyses were run for each molecular marker, apart from its since it showed a high number of insertions or deletions. It must be specified that this approach provide diagnostic characters that are provisional, since increasing the geographic scale and the sampling effort could introduce variability in the dataset, and therefore influence the reliability of some of the detected diagnostic characters. The alignments used to detect dna diagnostic characters were deposited at www.figshare.com (https://doi.org/10.6084/ m9.figshare.14932662) and the diagnostic positions refer to reference sequences belonging to S. bedoti (mhng-inve-0137431), with the following GenBank accession numbers: mz457614 for 16S, mz457396 for cox1, mz463827 for cox3, mz457880 for 18S, and mz457801 for 28S. When diagnostic positions differed between reference sequences and alignments (due to the presence of gaps in the reference sequences), the position in the alignments was also reported in parentheses. maggioni et al.
To visualise possible geography-related genetic structure within selected clades, median-joining haplotype networks based on the most complete single-locus dataset (16S rRNA) were built using the software PopART 1.7 (Leigh & Bryant, 2015). Finally, maps were produced in Python using the Matplotlib Basemap Toolkit (Hunter, 2007). All images were edited with CorelDRAW X7.

Results
A total of 84 samples (supplementary table S1) were analysed from the studied localities ( fig.  1A), allowing the morpho-molecular assessment of five species and four genera belonging to the Sphaerocorynidae (i.e., Sphaerocoryne bedoti, Kudacoryne diaphana gen. nov. sp. nov., Euphysilla pyramidata, Euphysilla sp., and Heterocoryne caribbensis) and three species and one genus of the Zancleopsidae (i.e., Zancleopsis dichotoma, Zancleopsis cabela comb. nov., and Zancleopsis sp.). A phylogeny reconstruction of the two families, based on six loci, is presented in the 'Molecular results' section, whereas taxonomic accounts for each species are reported in the 'Taxonomy' section, including the establishment of Kudacoryne diaphana gen. nov. sp. nov. and Zancleopsis cabela comb. nov. Finally, the distribution of each species and the genetic structure of selected species across the sampling localities are addressed in the 'Distribution and geographic structure' section.

Molecular results
The single-and multi-locus phylogeny reconstructions were broadly concordant in recovering all the investigated species as monophyletic groups, with a few exceptions in single-locus analyses based on nuclear loci ( fig. 1b, supplementary figs. S1 and S2). As shown in the multi-locus phylogenetic tree and in the species tree ( fig. 1B, C), both the Sphaerocorynidae and Zancleopsidae were demonstrated to be highly supported and monophyletic sister groups. Moreover, the two families were placed as sister group to a clade composed of the families Cladocorynidae Allman, 1872, Zancleidae Russel, 1953, Asyncorynidae Kramp, 1949, Milleporidae Fleming, 1828, and Solanderiidae Marshall, 1892, similarly to previous analyses focusing on the Cladocorynidae (Maggioni et al., 2021) (fig. 1B). Within the Sphaerocorynidae, Sphaerocoryne bedoti was recovered as the sister group to all other species, whereas Kudacoryne diaphana gen. nov. sp. nov. (see 'Taxonomy' section) was sister to the clade Euphysilla + Heterocoryne, even though the latter showed low ml and mp bootstrap values (figs. 1B, C). The genetic data enabled linking of the polyp and medusa stages of E. pyramidata, since medusa-based sequences from Florida consistently clustered with polyp-based sequences from Indian Ocean and Caribbean localities.
Within the Zancleopsidae, genetic data also helped establish the link between the polyp and medusa stages of two species. Specifically, an Astrocoryne-like colony from Singapore was linked to Zancleopsis dichotoma medusae from Florida. Similarly, sequences of Astrocoryne cabela polyps from the Maldives and Red Sea clustered with medusae from Florida, provisionally ascribed to Z. dichotoma by Schuchert & Collins (2021), requiring the synonymisation of Astrocoryne with Zancleopsis. Schuchert & Collins (2021) divided their Z. dichotoma medusae from Florida into two morphs which also showed high genetic divergence and suggested the possible presence of two species, namely a small morph, herein linked to systematics of the sphaerocorynidae and zancleopsidae Downloaded fig. S3), seven belonging to the Sphaerocorynidae and three to the Zancleopsidae, with no discordance among methods. Genetic distance analyses revealed limited intraspecific variation in the recovered species hypotheses (range: 0-1.5 %), not overlapping with interspecific distances (range: 3.5-15.2 %) (table 1). Notably, other sphaerocoynid sequences recovered from GenBank were included in the 16S rRNA analyses, consisting of S. bedoti and S. agassizii examined in Nawrocki et al. (2010) and S. agassizii examined in Mendoza-Becerril et al. (2018). Species delimitation analyses found that these samples likely belong to two species not sampled in this study. Sequences from Nawrocki et al. (2010) from Florida and Panama proved to be almost identical, despite having been allocated to different nominal species, and were placed as the sister group of the Euphysilla clade (supplementary figs. S1 and S3). The identity of this species remains unknown and may represent another Euphysilla species. The sequence from Mendoza-Becerril et al. (2018) was obtained from polyps collected in the Gulf of Mexico, and was sister to the H. caribbensis clade, from which it showed a divergence of 3.5%. Despite having been identified as S. agassizii, this colony showed a morphology similar to H. caribbensis (Mendoza-Becerril, personal communication) and may therefore represent another Heterocoryne species. Finally, the species hypothesis Euphysilla sp. was discriminated from E. pyramidata based exclusively on phylogenetic, species delimitation, and genetic distance results, since only a few EtOH-preserved Euphysilla sp. polyps were available and no morphological differences were detectable between the two species (see 'Taxonomy' section).

Taxonomy
Because most sphaerocorynid genera have hydroids that are practically inseparable from each other, a genus diagnosis based on morphology alone appears insufficient. Therefore, for each genus in the family Sphaerocorynidae a genetic diagnosis based on diagnostic molecular characters for 16S, cox1, cox3, 18S, and 28S was provided. its was not included due to the large number of insertions or deletions. For details on the analysed material (e.g., dna name, life stage, precise sampling locality and coordinates, GenBank accession numbers), see supplementary table S1.
Adult medusa with thick ovoid to bellshaped umbrella; with or without apical projection, when present, conical or domeshaped, with broad apical chamber; manubrium flask-shaped, quadrate, or cruciform, mouth simple, round or cruciform; four tentacles, when present, with adaxial, evenly distributed, or spirally arranged nematocyst clusters, with a terminal, ellipsoid or spherical capitation; with abaxial ocelli; gonads adradial, confluent in perradii in mature specimens.
Genetic diagnosis: 79 molecular diagnostic characters differentiate Sphaerocoryne from other genera in the family, specifically 16 in the 16S, 20 in the cox1, 22 in the cox3, 10 in the 18S, and 11 in the 28S (table 2).
14.7 ± 1.5 15.2 ± 1.   2 Molecular diagnostic characters for each sphaerocorynid genus and molecular marker, except for its. Positions refer to reference sequences belonging to S. bedoti (mhng-inve-0137431), with the GenBank accession numbers mz457614 (16S), mz457396 (cox1), mz463827 (cox3), mz457880 (18S), and mz457801 (28S). When positions in the alignments differ from reference sequences they are also reported in parentheses. If a diagnostic position corresponds to a position in which there is a gap in the reference sequence, it is only reported in parentheses Therefore, if S. bedoti and S. agassizii are not congeneric, the latter should be transferred to the genus Corynetes and Sphaerocoryne will remain valid at least for S. bedoti. On the other hand, if the two species are demonstrated to be congeneric, the widely used generic name Sphaerocoryne will not be valid anymore, and a case should be submitted to the International Commission on Zoological Nomenclature (iczn) to assess the merits of its conservation.
Remarks: This species was initially described by Pictet (1893) based on polyp stage alone. The most striking difference to other sphaerocorynid species is the typical colouration of the polyp and the medusa buds forming large clusters, as already noted by Warren (1908 (1893), Hargitt (1904), andCalder (1971), something never observed in the species investigated herein. Medusa buds and newly released medusae in S. bedoti are small in size and with no developed tentacles or ocelli, as depicted by Yamada & Konno (1973) and in the present work. Most of the differences between S. bedoti and S. agassizii occur in the adult medusa, specifically in the exumbrellar nematocyst rows and in the tentacular nematocyst clusters . However, adult medusae clearly ascribable to S. bedoti and S. agassizii could not be collected for the present work, and, therefore, the status of the two species and the consequent taxonomic implications related to the validity of the genera Sphaerocoryne and Corynetes must remain unresolved. The paucity of reliable diagnostic characters in the polyp stage of sphaerocorynid species has led to subsequent confusion in species identifications in the family, and many records should be considered dubious due to the lack of thorough descriptions, drawings and photos of colonies, or genetic data. Therefore, we abstain from including these records as synonyms.   Newly liberated medusa. Bell-shaped umbrella, 370-410 μm wide and 405-420 μm high, with several nematocysts scattered on the exumbrella (fig. 3H). Manubrium cylindrical, about 300 μm long, 2/3 to 3/4 of the bell height, distally provided with a circular mouth. Four radial canals ending in four bulbs with a diameter of 70-80 μm, containing nematocysts. When released, medusae with no tentacles and no ocelli. Living medusae transparent with reddish manubrium. Microbasic mastigophores and small stenoteles scattered on the exumbrella and large stenoteles in bulbs.
Etymology: The species name derives from Greek diaphanes, referring to the absence of a typical colouration of polyps, being almost completely transparent.
Distribution: Red Sea and Indo-West Pacific (Saudi Arabia; Maldives).
Remarks: The polyps of this species largely agree with the description of other sphaerocorynid polyps. The main differences to other species rely on the colour and size of polyps, being transparent, smaller, and with shorter pedicels in Kudacoryne diaphana. Additional differences are in medusa buds, being in this species up to two in number, not in clusters, and giving rise to free-swimming medusae. Given these differences, and the divergent position in the proposed phylogenetic hypotheses, the new genus Kudacoryne was established (authored by Maggioni). However, we admit that the morphological differences are minimal and likely not useful to distinguish the genera in practice. Therefore, we resorted to a more objective genetic diagnosis.
Since the adult medusa of Kudacoryne diaphana is currently unknown, it is possible that this species will later be synonymised with a Sphaerocoryne or Euphysilla species with a name that is based on the medusa stage, but for which no genetic data are available yet.
Medusa umbrella ovoid, with apical chamber; manubrium with quadratic base; mouth circular; no gastric peduncle; mature gonads surrounding the whole manubrium; four equally developed tentacles with adaxial or abaxial nematocyst clasps and a terminal cluster; ocelli absent or underdeveloped and very inconspicuous. Medusa buds on manubrium usually present.
Distribution: Atlantic Ocean and Caribbean Sea (Benin, Florida; Sint Eustatius), Red Sea and Indo-West Pacific (Northern Red Sea; Australia, India, Indonesia, Madagascar, Maldives, South Africa, Taiwan Strait), and East Pacific. The records from Papua New Guinea may refer to another species (Schuchert & Collins, 2021).
Remarks: The polyp stage of E. pyramidata is herein described for the first time, and largely resembles Sphaerocoryne and Kudacoryne polyps. However, differences are found in the colouration of the hydranth, tentacle organisation, lack of inclusions in the capitula, and organisation and number of medusa buds. It is possible that polyps of this species were assigned to the genus Sphaerocoryne in previous works, as for instance Sphaerocoryne sp. from Indonesia by Di Camillo et al. (2008) (cg Di Camillo, pers. comm.). The medusa shows strong similarities to Sphaerocoryne medusae as well, but is distinguished by the absence or reduction of ocelli and the common presence of medusa buds on the manubrium. Schuchert & Collins (2021) initially detected the genetic similarities in the 16S rRNA region between a Sphaerocoryne polyp from Maldives (Maggioni et al., 2017) and E. pyramidata medusae from Florida, here confirmed on the basis of multiple dna regions and phylogenetic analyses. They also suggested that E. pyramidata in the sense of Bouillon (1978) may be a species complex due to morphological differences, but further sampling is still needed to address this issue.
Distribution: Currently only known from the Caribbean Sea (Martinique, Panama).
Remarks: This species was identified as belonging to the genus Euphysilla based on genetic data alone, since no comprehensive information on its polyp stage was available (e.g., colour of living polyps). Additionally, the medusa stage is currently unknown. However, the general morphology and the cnidome composition and size are comparable to those of E. pyramidata. Other than E. pyramidata, the genus Euphysilla currently includes two additional species, namely E. peterseni Allwein, 1967, andE. tubularia Huang, Xu &Lin, 2015, from nw Atlantic and China Sea, respectively (Allwein, 1967;Huang et al., 2015). Morphological differences among the medusae of Euphysilla species are only slight and they might represent intraspecific variation. However, the finding of another Euphysilla species based on genetic data may support the presence of multiple species, and our material may be later demonstrated to  Genetic diagnosis: 87 molecular diagnostic characters differentiate Heterocoryne from other genera in the family, specifically 21 in the 16S, 23 in the cox1, 33 in the cox3, 6 in the 18S, and 4 in the 28S (table 2).
Remarks: Heterocoryne is a monotypic genus.
Distribution: Presumably endemic to the Caribbean Sea (currently only known from Guadeloupe, Martinique, Puerto Rico, Sint Eustatius, US Virgin Islands).
Remarks: The analysed colonies fully agree with the specimens analysed by previous authors, both in general morphology and cnidome composition (Wedler & Larson, 1986;Galea, 2013). A minor difference is the presence, in some polyps, of four instead of three tentacles in the aboral whorl.

Family Zancleopsidae Bouillon, 1978
Diagnosis: Polyps known for Zancleopsis only; colonies stolonal; hydrocaulus short to moderately long, simple, arising from a hydrorhiza embedded in host sponge; perisarc thin, covering both the hydrorhiza and the hydrocaulus; hydranth slightly pyriform to cylindrical, with proboscis-like hypostome and dicapitate tentacles in one or two alternating whorls around broadest part of body; gonophores developing among tentacles as free medusae.
Medusa with conical or dome-shaped umbrella, with or without apical projection; manubrium flask-shaped, with quadrate or cruciform base, mouth simple, cruciform or circular, with or without lips; two or four tentacles, with or without capitate branches; bulbs with adaxial hemispherical projections equipped with nematocysts; with or without abaxial ocelli; gonads aradial to interradial, with interradial grooves.

Zancleopsis Hartlaub, 1907
Diagnosis: As for the family, but medusae when fully mature with four tentacles, the two opposite pairs differing also in size and morphology.
Adult medusa (from Schuchert & Collins, 2021, small form only). Total bell height up to 3 mm, 1/4 to 1/3 of the height taken by pointed apical process ( fig. 7F, G); umbrella bellshaped to conical, relatively thick walls, with shallow interradial subumbrellar pockets, tip of apical process green. Manubrium height about half the subumbrellar height when gonads start to develop, pear-shaped, short tubular oral part, mouth rim with four perradial white regions, upper part of manubrium (stomach) ochre coloured, with about 10 longitudinal, shallow gonad folds, folds mostly adradial, irregular ( fig. 7H). Radial canals not forming mesenteries, smooth. Tentacle bulbs all equally developed, almost spherical, placed adaxial of origin of tentacles, white or faintly yellow. Two long, opposite tentacles, much extendable/contractible, with up to 25 short, abaxial, side branches ending in capitula, size of capitula gradually increasing towards distal ( fig. 7I). The other tentacle pair very short, ending in spherical nematocyst knob. In young animals these short tentacles either missing or just beginning to develop. All tentacle bases with a red ocellus on abaxial side.
Remarks: Schuchert & Collins (2021) found that Z. dichotoma medusae were genetically similar to A. cabela polyps. However, they found two genetically divergent morphs, one smaller (bell height of 3 mm) and the other larger (bell height up to 15 mm). The small morph better agrees with the original description of Z. dichotoma by Mayer (1900), and it was here linked through genetic data to Astrocoryne-like polyps from Singapore, supporting the synonymisation of the genus Astrocoryne with Zancleopsis.
Zancleopsis cabela (Maggioni et al., 2017) comb. nov. Zancleopsis dichotoma. Bigelow, 1938:  Description: Polyp. Colonies monomorphic, living in association with sponges (figs. 8A-C). Hydrorhiza tubular, covered by moderately thick and slightly wrinkled perisarc, embedded by the sponge host. Pedicels short to moderately long (up to 580 μm), unbranched, covered by a smooth, thin, cup-shaped or elongated perisarc. Hydranth pyriform or slightly pyriform, up to 0.8 mm long, with variable diameter (up to 250 μm). Hypostome proboscis-like, contractile. Up to 10 tentacles (range 8-10) arranged in one or two close whorls in the broadest part of the polyp. Each tentacle with terminal and sub-terminal capitula (diameter: 50-110 μm in the distal whorl; 25-45 μm in the proximal whorl) ( fig. 8D). Tentacles up to 550 μm long in the distal whorl, shorter in the proximal whorl (up to 320 μm) when present. Nematocyst clusters about 100 μm distant from one other, closer when tentacles are contracted. Up to 11 medusa buds at different stages of maturation develop among tentacles, singly or in couple on blastostyles ( fig. 8E). Living hydranths transparent, with white mouths and whitish or light orange gastric cavities. Desmonemes, microbasic euryteles, small, large and medium-sized stenoteles (figs. 8F, G) occurring simultaneously in the terminal and proximal capitula, and in the hydrorhiza, rare in the hydranth.
Newly liberated medusa. Newly liberated medusa hemispherical, up to 500 μm wide and high, with nematocysts scattered on the exumbrella. Manubrium cylindrical, up to 200 μm long and 110 wide at the base, spanning from 1/3 to 1/2 of the bell height, distally provided with a circular mouth. Four radial canals ending in four bulbs with a diameter of up to 70 μm, and a circular canal. Bulbs and circular canals containing nematocysts. At release medusae with no tentacles, but with bulbs showing swellings filled with nematocysts. Two opposite tentacles after two days from release. Tentacles up to 300 μm long, with terminal spherical nematocyst-rich capitula with a diameter of up to 85 μm. Ocelli absent at release. Microbasic mastigophores and rarely microbasic euryteles scattered on the exumbrella, medium-sized stenoteles in the circular canal, medium-sized stenoteles and desmonemes in the bulb swellings and in the terminal capitula of tentacles.
Adult medusa (from Schuchert & Collins, 2021, Z. dichotoma large form). Similar but much larger than Z. dichotoma (= small form), height 8 to 15 mm and 5 mm diameter, apical process larger reaching 1/2 of total height, tip of apical process whitish, more and larger vertical gonad folds, approximately up to 15, grouped in 2-3 folds adradial, brownish, oocytes yellow, tentacle bulbs with intense yellow colour, shorter tentacle pair longer than in Z. dichotoma (figs. 8H-K). Remarks: The polyp stage of this species was previously identified as A. cabela, but genetic data allowed us to link it to the large morph of Z. dichotoma described by Schuchert & Collins (2021), further confirming the synonymisation of Astrocoryne with Zancleopsis. The main differences to Z. dichotoma are a larger adult medusa and larger polyps with tentacles less regularly organised. Kramp (1968) suspected that Z. dichotoma and Zancleopsis tentaculata  could be conspecific. Zancleopsis tentaculata (type locality: Banda Sea, Indonesia) generally shows a larger size and the short tentacles are longer than in Z. dichotoma, something shared with Z. cabela. However, given the uncertainties in the morphological identification of Z. tentaculata we prefer to ascribe these specimens to Z. cabela, because genetic data of the type material of A. cabela were included in the analyses.
Distribution: Only known from the Indo-West Pacific (Singapore).
Remarks: This species has polyps that are apparently identical to those of Z. dichotoma in both general morphology and cnidome, and was separated from the latter based on molecular data alone, nevertheless showing high genetic divergence. Since the adult medusa is unknown, it is currently not possible to identify these colonies to the species level using morphology alone. Because it could additionally also belong to an already named medusa, we prefer here not to give it a new species name, also because all the other Zancleopsis species not sampled in this work have their type localities in the Western Pacific, namely in the Banda Sea, Bismarck Sea, South China Sea, and Japan (Uchida, 1927;Bouillon, 1978Bouillon, , 1985Wang et al., 2016).  10D). Zancleopsis dichotoma showed a wide distribution, spanning the Atlantic and Pacific Oceans ( fig. 10F). Similarly, also Z. cabela was demonstrated to have a wide distributional range, being found in both the Atlantic and Indian Oceans and the Red Sea ( fig. 10G). Finally, Zancleopsis sp. is currently known only from the Indo-West Pacific (Singapore) (fig. 10H).
Haplotype networks of Sphaerocoryne bedoti, Kudacoryne diaphana, and Euphysilla pyramidata based on the 16S region revealed a moderate geographic structure for all species (fig. 11). Indeed, no haplotypes were shared among localities, being all haplotypes from different localities well separated from each other in all investigated species.

Discussion
The integrative scope of the present work disentangled some of the taxonomic uncertainties affecting the families Sphaerocorynidae and Zancleopsidae. Indeed, the morpho-molecular approach enabled us to accurately characterise the analysed species, to identify the previously unknown polyp stages of Euphysilla and Zancleopsis, to describe a new sphaerocorynid genus and species (both authored by Maggioni), and to assess the phylogenetic position of the two families within the Capitata. Molecular species delimitations concordantly revealed the presence of ten species hypotheses in the investigated dataset, and also genetic distances between species were always moderate to high, not overlapping with intraspecific distances, with values comparable to other closely related capitate species (e.g., Miglietta et al., 2019;Maggioni et al., 2020aMaggioni et al., , b, 2021. Morphological analyses identified diagnostic and easy-to-detect features of sphaerocorynid polyps, mostly related to the colour of hydranths, tentacles organisation, inclusions in capitula, and organisation of medusa buds. Other than these differences, sphaerocorynid polyps show a consistent morphology, with a pyriform hydranth, proboscis-like hypostome and tentacles organised in close whorls in the broadest part of the hydranth. For these reasons, a genetic diagnosis for each genus was also provided. Polyps of the three Zancleopsis species analysed in this work were very similar to each other, making it difficult to identify them without medusa or genetic information. A common feature of sphaerocorynid and zancleopsid polyps is the frequent association with sponges, also seen among some zoantharians, which belong to Anthozoa (e.g., Reimer et al., 2018;Montenegro et al., 2020). Previous reports and the present study highlighted that occasional associations with other substrates are also possible in the family Sphaerocorynidae, namely with coralline algae, dead and living corals (Calder et al., 2003), coral rubble (Calder, 2010), bivalves (Calder, 1971), serpulid tubes (Galea, 2008), and other hydrozoans (Mergner & Wedler, 1977). However, with a few exceptions for S. bedoti and E. pyramidata, all other samples analysed in this work were associated with sponges. Capitate hydrozoans often establish symbiotic associations with benthic invertebrates, such as scleractinian corals, octocorals, bryozoans, and bivalves (Cerrano et al., 1997;Maggioni et al., 2016Maggioni et al., , 2020c, in some cases with high specificity (e.g., Montano et al., 2017a;Maggioni et al., 2020a) and hypothesised important ecological roles for their hosts (Montano et al., 2017b). On the other hand, they can also be hosts themselves to anthoathecate hydrozoans (Montano et al., 2020). Regarding sphaerocorynid species, little is known about the specificity and the possible outcomes of their association with sponges. Indeed, the taxonomic confusion that has characterised the family for a long time, together with the difficulties hampering sponge taxonomy (Wörheide & Erpenbeck, 2007) and the low number of previous works reporting the identification of the host sponge (e.g., Calder 1971;Yamada & Konno, 1973;Wedler & Larson, 1986), prevent precise speculations on this aspect. Nevertheless, an overall lack of specificity can be hypothesised, given that the same host can be shared by different species (Maggioni et al., 2017). In the same way, it is also possible that Zancleopsis species are generalist symbionts of poriferans, with Z. cabela hosted by three different sponge species in the Maldives and Red Sea (Maggioni et al., 2017). Future increased efforts in sponge host identification will likely shed light on the degree of specificity of these associations and may also help with the delineation of species boundaries in the associated hydrozoans, as in other species (Maggioni et al., 2020a(Maggioni et al., , 2021.
Regarding the adult medusae, in this work we were able to thoroughly characterise three species, namely E. pyramidata, Z. dichotoma, and Z. cabela, whereas for the other species only the newly released medusae were observed, when found, with the exception of H. caribbensis that has fixed gonophores. Given the lack of material of the medusa stage, two species (i.e., Euphysilla sp. and Zancleopsis sp.) could not be identified to the species level, and may be demonstrated in the future to correspond to already described medusa-based species not analysed in this work. Similarly, the new polyp-based species Kudacoryne diaphana (authored by Maggioni) was herein erected to allow the establishment of the new genus Kudacoryne (authored by Maggioni), based on both morphological and genetic data, and we cannot exclude that this species will later be synonymised with one of the sphaerocoynid species based solely on the adult medusa stage. Notably, the unavailability of these latter species among the analysed samples left unresolved some taxonomic issues. In particular, the morphological re-description and the genetic analysis of S. agassizii material is needed to address the validity of the genus Sphaerocoryne (Calder, 2010;. The phylogenetic hypotheses presented allowed the identification of a sister-group relationship between the Sphaerocorynidae and Zancleopsidae, an aspect already hypothesised in previous morphology-based cladistic analyses (Petersen, 1990), in which the presence of medusa tentacular bulbs with adaxial expansions was considered to be a synapomorphy of the two families. In that analysis, Petersen (1990) also placed these two families together with Paragotoea Kramp, 1942, forming the superfamily Sphaerocorynoidea, and with the Hydrocorynidae Rees, 1957, forming the suborder Sphaerocorynida, finding that the long, capitate, aboral tentacles of polyps, arranged in close-set whorls, could be a synapomorphy of the suborder. However, later dna-based phylogenetic analyses placed the Hydrocorynidae in a divergent position (e.g., Nawrocki et al., 2010), and Paragotoea is currently included in the aplanulate family Corymorphidae Allman, 1872 (Pages & , thus invalidating the classification system proposed by Petersen (1990) for these taxa. Also, Euphysilla was proposed to belong to the Sphaerocorynida suborder as incertae sedis, and with this work we demonstrated that it clearly belongs to the family Sphaerocorynidae.
Interestingly, the species analysed in this work showed contrasting biogeographical patterns. Based on our data and literature records, some species appear to have a wide circumtropical distributional range, such as S. bedoti and E. pyramidata, whereas other are limited to few localities, such as H. caribbensis in the Caribbean Sea, even if in some instances an extended sampling could likely widen their distribution. These mixed distributional patterns seem to be common in closely related hydrozoan species (e.g., Arrigoni et al., 2018;Miglietta et al., 2018). Here, populations from different localities also appeared well separated in some species, suggesting possible limited gene flow among localities. The wide distribution of some of the investigated species may be related to recent human-mediated dispersal, as already hypothesised for other hydrozoans (Miglietta & Lessios, 2009;Miglietta et al., 2015), even though the observed intraspecific geographic structure may hinder this hypothesis.
Overall, the present work has shed new light on the diversity, biogeography, and evolution of the Sphaerocorynidae and Zancleopsidae, by providing the first detailed morpho-molecular account of eight species belonging to these families. Moreover, the results provided lay the ground for future investigations to further resolve the taxonomy and systematics of these two enigmatic taxa.