New species of Eirenidae (Hydrozoa: Leptothecata) from the Amazonian coast (northern Brazil) ; Nuevas especies de Eirenidae (Hydrozoa: Leptothecata) de la costa amazónica (norte de Brasil)

1 Departamento de Oceanografia, Universidade Federal de Pernambuco, Avenida Arquitetura, S/N, 50670-901, Recife, PE, Brazil. (EGT) (Corresponding author) E-mail: evertontosetto@hotmail.com. ORCID iD: https://orcid.org/0000-0002-4020-0942 (SN-L) E-mail: sigridnl@uol.com.br. ORCID iD: https://orcid.org/0000-0001-7878-8772 2 Departamento de Sistemática e Ecologia, Universidade Federal da Paraíba, Cidade Universitária, 58051-900, João Pessoa, PB, Brazil. (MNJ) E-mail: miodeli@gmail.com. ORCID iD: https://orcid.org/0000-0001-5409-8312


INTRODUCTION
Hydromedusae from the family Eirenidae Haeckel, 1879 are easily distinguished from other Leptothecata by the presence of a distinct gastric peduncle and closed statocysts (Bouillon et al. 2006). In some Eirenidae genera there are eight statocysts (rarely 12) while in others there are an indefinite number, but always more than eight in adult medusae (Bouillon 1984, 1999, Bouillon et al. 2006 Eutima cirrhifera (Kakinuma, 1964) Up to 9.8 mm 8
Specimens from two Eirenidae species were found in a scientific expedition along the Amazonian coast of northern Brazil (Araujo et al. 2017, Tosetto et al. 2019. Species of the family are typically coastal and/ or estuarine, often occurring in high abundance (Canché-Canché and Castellanos-Osorio 2005, Morales-Ramírez and Nowaczyk 2006, Mediseh et al. 2017). This aspect, associated with the high feeding rates of pelagic cnidarians (Hays et al. 2018), may place them as significant predators in these environments. The specimens found clearly belonged to the genera Eutima and Helgicirrha because of the characteristics explained above, but did not fit in any of the currently known species. Thus, the objective of this work is to describe Eutima marajoara n. sp. and Helgicirrha angelicae n. sp. In addition, the main characteristics of all species described in both genera are compiled and compared (Tables 1, 2).

MATERIALS AND METHODS
Specimens were obtained from samples collected in October 2012 at Marajó Bay and along the Amazonian coast, northern Brazil ( Fig. 1), with oblique hauls from near bottom to the surface, using a Bongo net with 120 and 300 µm mesh and 0.3 and 0.6 m mouth opening, respectively. The material was fixed with 4% formaldehyde buffered with sodium tetraborate (0.5 g l -1 ). The type material was deposited at the cnidarian collection of the Museu de Zoologia da Universidade de São Paulo (MZUSP), with additional paratypes deposited at the Coleção de Invertabrados Paulo Young from Universidade Federal da Paraíba (CIPY). All applicable international, national and institutional guidelines for the care and use of animals were followed.
In the laboratory, specimens were measured and the number of marginal structures per quadrant was counted under an optical microscope. We considered each quadrant separately in order to test for individual variability and to alleviate the problem that some specimens had quadrants with parts of the margin damaged and structures missing. Relationships between umbrella diameter and number and ratio of marginal structures were modelled with linear regressions with Statsoft Statistica 10 software. Etymology. Specimens were collected in the waters of Marajó Bay in northern Brazil. Marajoara refers to the native society that inhabited the area before European occupation and gave name to the bay.  Diagnosis. Eutima medusa with gonads restricted to subumbrella, extending along almost entire length of subumbrellar portion of radial canals. Approximately 32 (up to 40) marginal tentacles with conical bulbs and 32 (up to 48) marginal warts. Some marginal warts and tentacular bulbs with lateral cirri and a few with adaxial papillae.

Class
Description (based on several specimens). Umbrella flat, ranging from 1.5 to 9.2 mm wide, with thin mesoglea. Manubrium tubular with folded lips. Narrow prismatic gastric peduncle, about 1/4 of umbrella diameter in length. Gonads restricted to subumbrella, linear, extending along almost entire length of subumbrellar portion of radial canals but not connected to ring canal and gastric peduncle ( Fig. 2A, B). Approximately 32 (up to 40) marginal tentacles with conical bulbs in adult medusae. Up to 48 marginal warts in different sizes, usually two or three between successive tentacles in small medusae, fewer in larger specimens (as shown by the increase in the ratio between tentacles and warts, Fig. 3). Up to eight statocysts. Some warts and bulbs with lateral cirri ( Fig 2C) and a few with adaxial papillae (Fig.  2D). Velum narrow.
Development. Positive significant (p<0.05) relationships were observed between umbrella diameter and mean number of marginal tentacles, warts, ratio between tentacles and warts and statocysts per umbrella quadrant (Table 3, Fig. 3). Positive values of b coefficient indicate the number of tentacles and warts increases with medusa development (Table 3). While smaller specimens (1.5~2 mm) had one or two tentacles and two to four warts on each quadrant, individuals of intermediate sizes around 5 mm had approximately four tentacles and six warts per quadrant (note some individuals had up two 11 warts per quadrant in this stage) and bigger ones (~9 mm) had up to 10 tentacles and 12 warts per quadrant (Fig. 3). Positive values of b coefficient of tentacles/warts ratio also indicate that warts are relatively more abundant in juveniles (Fig.  3). Although the relationship between umbrella diameter and statocyst number was significant (p value almost 0.05, very close to the threshold limit of significance), the low value of the b coefficient indicates that the number of statocysts does not increase with medusa development, as was expected because species of Eutima are considered to have a fixed number of statocysts (Table 3, Fig. 3).
Ecological notes. Specimens were found in estuarine waters (8 m bottom depth) of the Marajó Bay, mouth of Pará and Amazon Rivers, at 28.3°C temperature and 18.2 salinity throughout the water column.
Diagnosis. Helgicirrha medusa with gonads on middle portion of radial canals, with medusa buds. Short gastric peduncle. Up to 20 marginal tentacles, some with adaxial papillae. Generally one to three marginal warts and one statocyst between successive tentacles. Lateral cirri on tentacle bulbs and some marginal warts.
Description (based on several specimens). Umbrella flatter than a hemisphere, ranging from 0.8 to 8.5 mm wide, with thin mesoglea. Small manubrium, mouth with four short simple lips. Short and narrow prismatic gastric peduncle, about 1/4 of umbrella width in length. Linear gonads on middle portion of radial canals, with developing medusa buds (Fig. 4A, C). Up to 20 marginal tentacles with conical bulbs. Up to 28 marginal warts in different sizes, usually one to three between successive tentacles. Lateral cirri on tentacle  bulbs and some marginal warts (Fig. 4B). Generally one statocyst between successive tentacles. Some bulbs with adaxial papillae. Velum narrow.
Development. Positive significant (p<0.05) relationships were observed between umbrella diameter and mean number of marginal tentacles, warts, and statocysts per umbrella quadrant (Table 3, Fig. 3). Positive values of b coefficient indicate that the number of tentacles, warts and statocysts increase with medusa development (Table 3). Smaller specimens (~1 mm) had one perradial tentacle, one or no interradial warts and one statocyst in each quadrant. Intermediate stages (~4.5 mm) had around three tentacles, four warts and 2.5 statocysts per quadrant and bigger specimens (6~8 mm) had up to six tentacles, seven warts and five statocysts per quadrant (Table 3, Fig. 3). Ratios between tentacles and warts were not significantly correlated with umbrella diameter.  (Table 3).
Ecological notes. Specimens were found in neritic waters over a reef system (30-65 m bottom depth) under the influence of the Amazon River plume with water column temperature ranging from 24.9°C to 27.9°C and salinity from 31.1 to 36.3.

DISCUSSION
Genera of the family Eirenidae are mainly distinguished by the number of statocysts, the position of the gonads and the presence of marginal structures such as, cirri, warts and adaxial papillae (Bouillon et al. 2006). Eutima and Helgicirrha species always have lateral cirri on the tentacle bulbs and/or marginal warts, and some species of both genera have adaxial papillae and gonads restricted to the subumbrella (Bouillon 1984, Bouillon et al. 2006, Schuchert 2017, as do the specimens found in this study. However, Eutima species have eight statocysts (rarely 12) in adult medusae and Helgicirrha always has more than eight and up to an indefinite number (Bouillon 1984, Bouillon et al. 2006). The number of statocysts is the main character distinguishing the two genera (the absence of excretory pores in Eutima is controversial and will be discussed later) and was used to assign the genera of the two species discussed herein. However, when the medusae development was observed, smaller specimens of both had fewer statocysts (Fig. 3C, G), and observations based on these individuals, associated with the fragility of these organisms (marginal structures are frequently lost in net trawls and/or formalin fixation), could lead to misidentification. These observations reinforce the need to examine fully developed specimens and to characterize their ontogenetic development for accurate identification and to elucidate the taxonomy of complex families such as Eirenidae.
Distinctive characters of Eutima marajoara n. sp. are the gonads restricted to the subumbrella, the adaxial papillae on few marginal warts and/or tentacle bulbs and the large number of marginal tentacles. Among the other seven species described in the genus with gonads restricted to the subumbrella, only Eutima suzannae Allwein, 1967 also have adaxial papillae (Table 1) (Haeckel 1864), differing from Eutima mainly in the presence of adaxial papillae in Octorchis and their absence in Eutima (e.g. Russell 1953). Subsequent studies considered both as subgenera within the genus Eutima (Kramp 1961, Bouillon 1984. Currently the subgenus rank fell out of use and all species previously considered Octorchis are included within Eutima (Schuchert 2020), which now encompasses eirenids with eight statocysts and lateral cirri, with or without papillae (Kramp 1961, Bouillon 1984, Schuchert 2020; Table 1). One open question is whether these papillae do possess an excretory pore, which would be necessary to regard them as excretory papillae (as mentioned in recent diagnoses, e.g. Boero 2000, Bouillon et al. 2006), because this is hard to verify and requires detailed histological observations (Schuchert 2017).
Only the hydroid stage and newly released medusa are known in Eutima ostrearum (Mattox and Crowell, 1951). Hydroids of this species were found inhabiting the mantle cavity of oysters in an estuarine system in Puerto Rico (Mattox and Crowell 1951). Due to the close geographic location and similar habitat (e.g. estuarine waters), E. marajoara n. sp. could be the adult medusa of E. ostrearum. Future studies based on molecular data from both localities and on hydroids inhabiting the Marajó Bay are necessary to answer this question. However, newly released E. ostrearum of up to 3 mm displayed no lateral cirri and had no sign of a gastric peduncle (Mattox and Crowell 1951). Although these characters may develop later in the medusa ontogeny, they differ from our smaller specimens within the same size range (1.5-3 mm), which already had lateral cirri and a small but clearly visible peduncle. These differences suggest they are different species.
Specimens of Helgicirrha angelicae n. sp. have linear gonads located in the middle portion of the radial canals. Shape and position of gonads are important characters for the taxonomy of Helgicirrha and other Eirenidae medusae (e.g. Bouillon 1984, Bouillon et al. 1988, Huang et al. 2010. Indeed, the gonadal position was helpful to distinguish the present specimens from Helgicirrha brevistyla Xu and Huang, 1983, Helgicirrha cari (Haeckel, 1864), Helgicirrha danduensis , Helgicirrha irregularis Seghers, 1988, Helgicirrha malayensis (Stiasny, 1928), Helgicirrha medusifera (Bigelow, 1909), Helgicirrha weaveri Allwein, 1967 andHelgicirrha sinuatus Xu, Huang andDu, 2012, which have gonads in other positions than the middle portion of the radial canals (see Table 2 and reference therein). Among the remaining species, Helgicirrha cornelii Bouillon, 1984, Helgicirrha gemmifera Bouillon, 1984and Helgicirrha ovalis Huang, Xu, Lin and Guo, 2010, unlike our specimens, do not have adaxial papillae either on the tentacular bulbs or on the marginal warts. In addition, they are distinct from the current specimens in the lower number of tentacles and shape of the gonads in the case of H. ovalis and H. cornelii (Table 2). Unlike in Eutima, species of Helgicirrha may or may not have excretory pores either on papillae (e.g. H. medusifera) or directly on the bulbs and warts (e.g. H. weaveri); however, pores were not observed on H. angelicae papillae.
The presence of medusa buds on every gonad of all adult specimens of H. angelicae n. sp. is also an outstanding character that is useful to distinguish it from all other species but H. gemmifera and H. medusifera. As cited above, the former is quite different from H. angelicae n. sp. However, H. medusifera differs mainly in the position of the gonads, which are located on the distal third of the radial canals (Table 2), while in our specimens they are in the middle (Fig. 3). The large number of specimens found in our samples allowed us to observe the complete development of medusae, and in any case the gonads approached the ring canal or were even located on the distal region of the radial canals, while in H. medusifera they are clearly very close to it (Bigelow 1909). The different geographical distribution of the two species (the western Atlantic and the eastern Pacific) also argues against the possibility that they are morphological variations of the same species. Furthermore, in adult medusae of H. medusifera most marginal warts turn into developed bulbs with tentacles and few warts are present (Bigelow 1909), and this is not the case for H. angelicae n. sp. because the number of warts increased with medusa development in similar proportion to the tentacles (Fig. 3). Future studies based on molecular data of both species could completely elucidate this question.
Among the 177 samples analysed, covering the northern Brazilian continental shelf and adjacent equatorial Atlantic oceanic waters (see Fig. 1 from Tosetto et al. 2019), both species had very restricted distributions. Currently, E. marajoara n. sp. has been found only in the waters of Marajó Bay, an estuarine environment (18.2 salinity) located in the mouth of the Pará and Amazon rivers, and is perhaps an endemic species to the area. H. angelicae n. sp. was restricted to coastal waters under the influence of the Amazon River plume. Knowledge on the zooplankton community inhabiting waters of the Amazonian coast is still scarce (Boltovskoy and Valentin 2018). Recently, this unique ecosystem has attracted attention from scientific and public society because of the presence of hard-bottom reefs (Moura et al. 2016) and oil exploitation in the area (Silva Junior and Magrini 2014), which may lead