First record of the genus Staurocladia (Hydrozoa: Cladonematidae) in the Gulf of Mexico with the description of Staurocladia dzilamensis sp.

. The Cladonematidae are a family of hydrozoans with a worldwide distribution and morphological adaptations for a benthic mode of life. Species of this family are characterized by high morphological variability, which has caused many taxonomical debates, mainly for the species of the genera Eleutheria Quatrefages, 1842 and Staurocladia Hartlaub, 1917. Herein, we describe Staurocladia dzilamensis sp. nov., a new species of crawling hydromedusa from the southern Gulf of Mexico. This finding also constitutes the first record of the genus Staurocladia for the Gulf of Mexico. The presence of additional nematocyst clusters, supplementing the apical one on the upper branch of the tentacles, places it within Staurocladia . The presence of exumbrellar buds, a conspicuous marginal ring of nematocysts, 6–11 bifid tentacles with lower branches longer than their upper counterpart, the cnidome with stenoteles of two size classes, and two nematocyst clusters on the upper branch supplementing the apical one, opposite placed alternately on its aboral and oral sides permits to differentiate S. dzilamensis from its congeners. A taxonomic key for the species of Staurocladia is provided.


Introduction
Medusae of the family Cladonematidae Gegenbaur, 1857 are characterized by having adaptations for a benthic mode of life.The individuals often show extensive morphological variability, which has generated much uncertainty in their taxonomy (Brinckmann-Voss 1970;Bouillon et al. 2006;Schuchert 2006;Ahuatzin-Hernández et al. 2022).Three genera are currently recognized within this family: Cladonema Dujardin, 1843, Eleutheria Quatrefages, 1842and Staurocladia Hartlaub, 1917 (Schuchert 2023).The polyphyletic origin of some of its members (i.e., Staurocladia, Eleutheria), together with the limited morphological studies, have caused the taxonomic boundaries of the group to be uncertain (Nawrocki et al. 2010;Fang et al. 2022;Zhou et al. 2022).The main taxonomic debates have been on the genus level, mainly focusing on members of Eleutheria and Staurocladia, which show adaptations for a fully benthic lifestyle.Among these adaptations are a flattened umbrella, mesoglea reduction, and bifid tentacles with adhesive pads (Mayer 1910;Russell 1953;Nawrocki et al. 2010).Currently, both genera are separated based on the number of nematocyst clusters on their tentacles, i.e., one for Eleutheria and more than one for Staurocladia (Nawrocki et al. 2010).Hartlaub (1917) established the genus Staurocladia to separate Eleutheria vallentini Browne, 1902, E. claparedii Hartlaub, 1889 and others from E. dichotoma Quatrefages, 1842, due to the hermaphroditism, the presence of a brood chamber, and the gonostyle in the polyp stage in the latter (Schuchert 2006).Hartlaub therefore used the combination Staurocladia claparedii (Hartlaub, 1889) since, like the other members of Staurocladia, this species lacks a brood chamber (Schuchert 2006).Gilchrist (1919) suggested the genus Cnidonema Gilchrist, 1919 for all the species of Eleutheria of the southern hemisphere due to the need to introduce a new genus for E. claparedii, which lacks a brood chamber (Schuchert 2006).Later studies (Browne & Kramp 1939;Kramp 1959Kramp , 1961Kramp , 1968;;Brinckman-Voss 1970) modified this taxonomy and differentiated Eleutheria from Staurocladia by the number of nematocyst clusters on the tentacles (Schuchert 2006).Staurocladia currently comprises 11 valid species (Schuchert 2023), which can be distinguished mainly by the arrangement of their nematocyst clusters on the upper branch.Additional features to distinguish among the species of this genus are the presence/ absence of exumbrellar buds, the number of tentacles and the length of their branches, and the features of the manubrium (Kramp 1961(Kramp , 1968;;Bouillon 1978).The hydroid of few species of Staurocladia is known, and the characteristics of the known species are very similar to each other (3-4 oral capitate tentacles and 4-6 aboral filiform tentacles) (Briggs 1920;Kakinuma 1963;Brinckmann-Voss 1970), so it seems there are not reliably diagnostics features to differentiate species only with this stage.

Collection site
The town of Dzilam de Bravo is located in the north-central region of the Yucatan coast in the southern Gulf of Mexico.The region is characterized by diverse ecosystems, such as coastal lagoons, grass beds, and mangrove areas (Herrera-Silveira & Morales-Ojeda 2010).The depth of the zone ranges from 2 to 8.7 m (Marina et al. 2017).On average, the values of temperature (28.4°C) and salinity (36.1 PSU) show tropical features, being relatively homogeneous temporarily (Marina et al. 2017), although they can be sharply heterogeneous spatially (i.e., with sharp changes in the physicochemical characteristics of the water column over a relatively short distance, regardless of seasonality) due to the presence of freshwater outcrops (Herrera-Silveira 2006;Marina et al. 2017).The substratum is diverse, which can be sandy, sandy with shells, covered by grasses and macroalgae, or hard bottom of flagstones and rocks AHUATZIN-HERNÁNDEZ J.M. et al., New species of Cladonematidae (Anthoathecata) (Rosado-Espinosa et al. 2012).The ocean circulation is mainly due to the influence of the Caribbean Current System, which enters the Gulf through the Yucatan Channel and plays an important role in the distribution of various organisms (Candela et al. 2002;Manzanilla-Domínguez & Gasca 2004;Sanvicente-Añorve et al. 2018).

Field work
Hydromedusae were collected on May 7, 14 and 28, 2022, by snorkeling and manually off the coast of Dzilam de Bravo (21°23.7′N, 88°53.1′W).The collection site was of the rocky intertidal type, with freshwater influence and a maximum depth of 1.5 m.The substratum was covered by macroalgae  Taxonomy 921: 251-275 (2024) with the presence of hydroids of Dynamena sp. (Fig. 2, photo taken with a Go Pro10 Black camera).The collected samples (one 250 mL bottle per day, full of algae and hydroids) were transferred to the Aquatic Pathology Laboratory at CINVESTAV-Mérida for further analysis.Salinity (ppt), dissolved oxygen (mg/L), and temperature (°C) values were recorded at points adjacent to the collection site.

Laboratory analysis
Samples were observed under a Motic SMZ-168 stereoscopic microscope.Hydromedusae were sorted from the rest of the material and fixed in a 4% formalin solution.In total, 110 specimens were analyzed, of which only 100 were in an optimal state of preservation to be measured.The umbrella diameter of specimens was measured with an ocular micrometer mounted on an Olympus BX50 optical microscope.Photographs were taken with a Leica DM2500 microscope equipped with a Leica MC170 HD camera.Squash preparations of tissue from tentacles and subumbrella were made from five specimens to observe their cnidae types.All measurements, observations, and taxonomic drawings were made on preserved specimens.Type material was deposited in the Regional Collection of Cnidarians of the Yucatán Peninsula 'Lourdes Segura', based at the Universidad Nacional Autónoma de México: Facultad de Ciencias, Unidad Multidisciplinaria de Docencia e Investigación-Sisal, Yucatán (YUC-CC-254-11).The organization of the material examined section follows the Darwin Core terms.A map with the distribution of nominal species of Staurocladia was made with QGIS ver.3.28.2,considering the published literature (i.e., Brinckmann-Voss 1970;Bouillon 1978Bouillon , 1991;;Schuchert 1996Schuchert , 2006;;Hirano et al. 2006;Galea & Schories 2012a, 2012b), and occurrences from the Global Biodiversity Information Facility (GBIF) and the Ocean Biodiversity Information System (OBIS) databases.We only retrieved occurrences coming from the basis of record "preserved specimen" and "material citation".Doubtful records were removed.
Histological sections were obtained in the sagittal plane from eight specimens.The tissues were dehydrated in a Histokinette Reichert-Jung and embedded in paraffin (melting point ~56°C).Serial thin sections of 5 μm were obtained using a Kedee microtome, model KD-3358, and then stained with hematoxylin and eosin, following the standard criteria proposed by Estrada-Flores et al. (1982) and Roberts (2012).Histological measurements (i.e., width of the marginal ring of nematocysts, width of the subumbrellar and gastric cavities, and width of the gastrodermis layer) were made with the Leica Application Suite software ver.4.13.0.

Etymology
The name is derived from the type locality, Dzilam de Bravo.

Description
Medusa bell flattened-hemispherical to dome-shaped, ca 0.43 mm wide (0.25 ± 0.60 mm, n = 100); brown-olive coloured, commonly with medusa buds on exumbrella, dorsally located; manubrium conical, extending beyond the margin, occupying a great part of the subumbrellar cavity, distally with simple, circular mouth; gonads surrounding the upper part of manubrium; neither pouches nor nematocyst knobs were observed on the manubrium; some specimens with visible eggs; a continuous and thickened nematocyst ring present on subumbrella; velum fully closing subumbrella; 5-7 short, unbranched radial canals, hard to see in most specimens and rarely corresponding to the number of marginal tentacles; 6-11 (usually 8) bifid tentacles with a black-reddish ocellus at their aboral base; tentacles branching a short distance after their origin, with the lower branch up to 2 times as long as the upper one, the former with a terminal adhesive pad; upper branch short, with 3 nematocyst clusters, one apical, knob-like shaped, and 2 additional, crescent-shaped, placed alternately on the aboral and oral sides of the branch.Cnidome: stenoteles of two size classes, class 1 = (15.00-17.50)× (12.50-15.00)μm, class 2 = 10.00 × 7.50 μm, and desmonemes = 7.50 × 5.00 μm, all in the upper branch, capsules restricted to both the terminal knob and the crescent-shaped clusters; no nematocysts were observed in the adhesive pad of the lower branch; marginal ring with stenoteles and desmonemes.
Histology: mesoglea reduced, not noticeable; velum evident, fully closing subumbrella; subumbrellar cavity wide, ca 149.18 µm (SD = 33.60);gonads like an external thin layer that surrounds the upper part of the manubrium, without any apparent division; radial canals short and broad, originating laterally from the gastric cavity, ending in a ring canal located immediately above the thickened continuous nematocysts ring; nematocysts ring ca 38.30 µm wide (SD = 3.92); internal walls of the manubrium with a layer of gastrodermis, gastrodermis ca 6.34 µm wide (SD = 0.72); gastric cavity wide, occupying ⅔ of subumbrella, ca 110.46 µm (SD = 22.31).

Remarks
A large cover of filamentous algae and Dynamena sp.hydroids occurred at the site, so a possible association between S. dzilamensis sp.nov.and Dynamena hydroids must be studied in the future.Of the 110 specimens, most were observed alive, and their morphology did not considerably change post-fixation.Most specimens had eight tentacles (n = 57), followed by nine (n = 25), seven (n = 22), six (n = 3), ten (n = 2), and eleven (n = 1).Two specimens showed tentacles with two aboral crescent-shaped nematocyst clusters on the upper branch of some tentacles.Two specimens presented a trifid tentacle.The origin of the third bifurcation was on the distal part of the lower branch, with both lower branches of equal length and with adhesive pads.Most specimens had exumbrellar medusa buds (n = 61).Thirty-eight specimens had one medusa bud, twenty had two buds, and only three specimens had three buds.Budding was the only way of asexual reproduction observed.
The third day of sampling (May 28) recorded the highest values of the measured variables and their greatest variation.The average temperature showed the highest values on the first day of sampling, ranging between 28.25 and 29.35°C.Dissolved oxygen recorded the highest values on the third day of sampling; this variable ranged between 0.10 and 0.67 mg/L.Salinity showed euhaline characteristics, since its values ranged from 36.07 to 38.94 ppt.Finally, the pH showed a higher acidity during the first two days, while on the third day, it showed alkaline (Table 1).Unfortunately, the polyp stage was not found in the samples (Appendices).(Browne, 1902) European Journal of Taxonomy 921: 251-275 (2024)

Discussion
The average umbrella size of S. dzilamensis sp.nov. is smaller than usually reported for the genus but matches the known range for species of Staurocladia and Eleutheria (Russell 1953;Kramp 1961;Schuchert 2006Schuchert , 2012)).Both genera are polyphyletic and are currently separated based on the number of their nematocyst clusters (Schuchert 2006;Zhou et al. 2022).This character, however, is currently considered uninformative in the definition of the taxonomy of this group at the genus level, and more analyses considering more taxa are suggested to redefine it (Nawrocki et al. 2010).Yet, as long as the taxonomic boundaries of both genera remain unclear, their separation should be maintained, and the number of nematocyst clusters on the tentacles considered as diagnostic for Staurocladia.Thus, we decided to assign the specimens from the coast of Dzilam to that genus.
Staurocladia dzilamensis sp.nov.can be distinguished from its congeners mainly by the arrangement of the nematocyst clusters on the upper branch of the tentacles, which bears one apical and two opposite clusters, placed alternately on its aboral and oral sides, in addition to the presence of dorsally-placed, exumbrellar buds, a conspicuous nematocyst ring, the features of the manubrium, the number of tentacles and the length of their branches (Table 2).
Among the valid species of Staurocladia, only five display a similar pattern in the arrangement of the nematocyst clusters regarding S. dzilamensis sp.nov.(alternately placed in aboral and oral sides), i.e., S. haswelli, S. portmanni, S. acuminata, S. ulvae, and S. vallentini (Fig. 6).Of these five species, S. portmanni and S. acuminata are easily distinguishable from S. dzilamensis due to the presence of nematocyst clusters on the lateral sides, either at the branching point, or a short distance after it, respectively.In addition, S. portmanni has a manubrium armed with nematocyst clusters, an umbrella able to swim, and larger desmonemes, and in S. acuminata, the upper branch of the tentacles is longer than its lower counterpart, and its cnidome is composed of larger stenoteles and desmonemes (Table 2).Both species differ from S. dzilamensis in the number of nematocyst clusters (Edmonson 1930; Brinckmann-Voss 1964) (Fig. 6).Staurocladia haswelli has a nematocyst cluster on the aboral side of the main stem of tentacles behind the branching point, yet this cluster can be absent in adult specimens (Briggs 1920;Kramp 1961).Despite this, S. haswelli has an umbrella of up to 1.2 mm, up to 31 tentacles with up to five nematocyst clusters on their upper branch, and larger stenoteles of one size class, features that allow us to differentiate it from S. dzilamensis.Between S. vallentini and S. ulvae, the former is easy to differentiate from S. dzilamensis due to the number of tentacles, the number of nematocyst clusters, and the cnidome, which is composed of stenoteles of three size classes and larger desmonemes (Table 2).Additionally, in S. vallentini, the upper branch of the tentacles is longer than the lower one, and the exumbrellar buds are orally placed, originating between the velum and bell margin (Schuchert 1996; Galea & Schories 2012b) (Table 2).Staurocladia ulvae matches S. dzilamensis in the umbrella size, the number of tentacles, and by having stenoteles of two size classes (Bouillon 1978).Nonetheless, S. dzilamensis has only three nematocyst clusters on the tentacles, one apical and two extra oppositely arranged, placed on aboral and oral sides, and a cnidome composed of stenoteles of two size classes, class one (15.00-17.50)× (12.50-15.00)μm, class two 10.00 × 7.50 μm, and desmonemes 7.50 × 5.00 μm, whereas S. ulvae presents up to six nematocyst clusters on the tentacles, one apical, two to three aboral, and one to two oral clusters, and the capsules of its cnidome are larger, i.e., stenoteles class one 20.00 × 16.00, stenoteles class two 12.00 × 8.50, and desmonemes 8.00 × 2.00 (Table 2).In addition, the length of the tentacular branches differs between both species.
The rest of the species are easy to distinguish solely by the arrangement of the nematocyst clusters since they either present clusters only on the lateral sides of the upper branches or are restricted to their aboral side (Fig. 6, unlike other species such as S. portmanni, S. haswelli, S. wellingtoni or S. oahuensis, where these features varied through the ontogenetic development (Briggs 1920;Edmonson 1930;Schuchert 1996).This fact encourages more research on the ontogeny of the species of Staurocladia, including S. dzilamensis, which could provide more information about the morphological boundaries of the genus.
There are few ecological studies on the medusae of Staurocladia compared to those of Eleutheria.
The hydroid of S. portmanni was reared at lower temperatures (13-20°C) than those recorded in this study, budding medusae from January to March that were subsequently found between May and July (Brinckmann-Voss 1970), which matches the period in which the specimens dealt with in this work were collected.Brinckmann-Voss (1970) suggests that the high occurrence of the medusa between May and July might be due to the medusae dying after their sexual reproduction by the end of June, and the species occurs in the hydroid stage only from then on.Two populations of S. oahuensis and S. bilateralis did not perform asexual reproduction at 12°C, while at 17°C and higher temperatures, both species reproduced asexually by fission at a high rate, proving low temperatures as a critical inhibitory factor for their asexual reproduction and growth (Hirano et al. 2000).This behaviour is also reported in Eleutheria dichotoma but through budding (Schierwater & Hadrys 1998;Dánko et al. 2020).The population analysed in this study showed a high rate of medusa budding, which can be associated with the high temperatures of a tropical region such as the southern Gulf of Mexico.The influence of temperature, observed in the populations mentioned above, and other variables such as salinity, dissolved oxygen, and pH must be tested in the specimens described in this study.
On the other hand, the high occurrence observed in this study and usually reported in Staurocladia and Eleutheria populations may be due to an ecological strategy (Hirano et al. 2000;Fraser et al. 2006).In E. dichotoma, sexual reproduction occurs at high rates when the population density is medium, favouring survival probability, which results in the production of motile larvae that can escape unfavorable conditions and settle in environments with better ones (Dańko et al. 2018).Moreover, this type of reproduction leads to the generation of a polyp stage, which is more resistant to environmental stress (Dánko et al. 2020).Eleutheria dichotoma and S. dzilamensis sp.nov.show a high rate of asexual reproduction through budding, responding similarly to temperature, and occurring in similar environments, such as intertidal pools with the presence of macroalgae, so the previously mentioned strategy likely applies to populations of S. dzilamensis sp.nov.In this sense, freshwater outcrops along the coast of Dzilam carry colder water masses with low salinities (Marina et al. 2017), which can conduce to physiological stress for some species.This can be associated with the factors previously mentioned and may explain the high abundances of S. dzilamensis sp.nov., the high rates of medusa budding, small umbrella sizes, and the absence of the polyp stage.
European Journal of Taxonomy 921: 251-275 (2024) The separation of Eleutheria and Staurocladia is still problematic due to the ambiguity relative to the number of nematocyst clusters on the tentacles as a generic diagnostic feature (Schuchert 2006;Nawrocki et al. 2010).This separation is probably unnecessary, yet, studies including molecular data and more taxa are required for a comprehensive analysis before a taxonomic rearrangement can be made.
In addition, a global review of the morphological boundaries of Cladonematidae is necessary due to its polyphyletic nature and the high morphological variability of its species (Ahuatzin-Hernández et al. 2022;Fang et al. 2022;Zhou et al. 2022).Only integrative studies, which consider species morphology and molecular data, would allow us to redefine the taxonomic boundaries and clarify the systematics and evolution of this group.

Fig. 2 .
Fig. 2. Photographs of the collection site.White arrows indicate colonies of Dynamena sp.

Table 2
).It is worth mentioning that the number and arrangement of the nematocyst clusters in S. dzilamensis sp.nov.were consistent through different stages of development (specimens with different umbrella sizes presented the same number and arrangement of clusters), AHUATZIN-HERNÁNDEZ J.M. et al., New species of Cladonematidae (Anthoathecata)

Table 1 .
Physicochemical variables recorded in adjacent areas to the collection site.Abbreviations: DO = Dissolved oxygen; Sal = Salinity; Tem = Temperature.

Table 2 (
continued on next two pages).Comparison of the diagnostic characters of the medusae of StaurocladiaHartlaub, 1917.Data compiled from: