First record of the genus Discorhabdella (Porifera, Demospongiae, Poecilosclerida, Crambeidae) from Sagami Bay, Japan with description of two new species

Abstract Two new species of Discorhabdella are described from Sagami Bay, Japan. Discorhabdella has been suggested to have an ancient Tethyan origin according to discovery of their unique pseudoastrose acanthostyles from late Eocene to Oligocene deposits. This is the first record of the genus from the northwest Pacific and first record of the family Crambeidae from Japan. Discorhabdellahispidasp. nov. is distinctive within the genus by possession of special sigmoid microscleres and C-shaped isochelae with short alae. Discorhabdellamisakiensissp. nov. is characterized by short choanosomal subtylostyles, and their length overlapped with that of the ectosomal subtylostyles. Only one other species, Discorhabdellatuberosocapitata (Topsent, 1890), has the same spicule composition. However, all spicule types are larger in D.tuberosocapitata than those of D.misakiensissp. nov., and the shape of the isochelae is different: the alae are more widely opened in D.tuberosocapitata. An identification key to species of the genus Discorhabdella is also provided. The discovery of two new species from warm temperate northwest Pacific extends the geographical distribution of the genus Discorhabdella.


Introduction
Sponges of the genus Discorhabdella Dendy, 1924 are characterized by the possession of smooth ectosomal subtylostyles, long choanosomal styles/subtylostyles with swollen lumpy bases, and tuberculate club-shaped pseudoastrose or heavily spined acanthostyles that form an erect hymedesmioid skeleton and various cheloid microscleres (Maldonado et al. 2001, Van Soest 2002. It has been suggested that Discorhabdella originated in the Tethys Sea (Boury-Esnault et al. 1992, Maldonado and Uriz 1996, Maldonado et al. 2001, as their unique pseudoastrose acanthostyles were discovered from the late Eocene to Oligocene deposits in New Zealand (Hinde and Holmes 1892, Łukowiak 2015, 2016. Seven extant species are currently known from the genus (Van Soest et al. 2019). Of these, Discorhabdella incrustans Dendy, 1924 is reported only from its type locality Three King's Islands, New Zealand. Discorhabdella littoralis Maldonado, Carmona, Van Soest &Pomponi, 2001 andD. urizae Maldonado, Carmona, Van Soest &Pomponi, 2001 are reported from off the Pacific coast of Panama. Discorhabdella urizae is also reported from Gulf of California (Aguilar-Camacho and Carballo 2012). Discorhabdella hindei Boury-Esnault, Pansini & Uriz, 1992 is reported from the Alboran Sea, and D. tuberosocapitata (Topsent, 1890) is reported from Azores, Canaries and Madeira (Van Soest 2002, Van Soest et al. 2019. Two recently described species have been discovered from north of Madagascar (D. pseudaster Vacelet & Cárdenas, 2018) and Gulf of Mexico (D. ruetzleri Díaz & Pomponi, 2018). A recent faunal survey of benthic animals in Sagami Bay, Japan yielded several undescribed species of the family Crambeidae (Ise 2017), and the descriptions of two new species of Discorhabdella are provided herein.

Materials and methods
The sponges described in the present study were collected by dredging from the R/V Rinkai-maru of Misaki Marine Biological Station, the University of Tokyo. The sampling was carried out at the northeastern part of Sagami Bay during the period of 10-13 January 2012 (Fig. 1). The specimens were kept alive in seawater for several hours and directly preserved in 90% ethanol afterwards. Dry fragments of the sponge were digested using hydrogen peroxide in order to obtain clean spicules. They were then cleaned using distilled water, centrifuged, and resuspended three times. Cleaned spicules were then placed on glass slides, dried, embedded in mounting medium Eu-kitt® (O. Kindler), cover-slipped, and then observed under a light microscope. Spicules were also placed on copper stub, coated with 400Å platinum, and observed by scanning electron microscope (JEOL JSM-6380LV). Spicules were measured with calibrated ocular micrometer directly under a microscope. Measurements were carried out along randomly chosen transects across the slide, ignoring unfocused, broken, or malformed spicules. Measurements of choanosomal subtylostyles of Discorhabdella hispida sp. nov. were only taken from the width of base and shaft as they are usually broken during spicule preparation steps. About 30 spicules for each type of spicule were measured. Spicule sizes are given as a range, followed by the mean in parenthesis. Spicule and morphological nomenclature follows Boury-Esnault and Rützler (1997), and terminology of cheloid microscleres follows Hajdu et al. (1994). Terminology for geographical distribution of each species basically follows descriptions of the original references; however, the data are corrected in Table 1 according to Marine Ecoregions of the World (Spalding et al. 2007). Specimens were deposited in National Museum of Nature and Science, Tsukuba, Japan (NSMT).  Spalding et al. (2007). Spicule sizes are given as the range, followed by the mean in parenthesis. All spicule measurements in µm.  Fig. 2A-C). Oscules not observed in the living specimen; probably contracted in preserved state. Ostia observed only in preserved specimen, rounded, evenly distributed, 150-300 µm in diameter.

Species
Skeleton. Hymedesmioid skeleton made by large choanosomal subtylostyles making the sponge surface hispid and by perpendicular acanthostyles with their bases attached on substrate. Ectosomal subtylostyles arranged perpendicular to surface with tips outward. Anchorate unguiferous isochelae and sigmoid microscleres roughly dispersed throughout the sponge.
Distribution. Known only from type locality, Misaki, eastern part of Sagami Bay, Japan.
Etymology. Specific epithet refers to its hispid surface appearance.

Remarks.
The present species appears well characterized by its spicule complement, especially its microscleres. The isochelae have a unique shape, with a strongly curved shaft compared to all other species of Discorhabdella, which have a straight or feebly curved shaft. However, the isochelae of D. hispida sp. nov. are similar to the anchorate isochelae of Monanchora unguiculata (Dendy, 1922) (see also Lévi 1961, Vacelet et al. 1976. The presence of a sigmoid microsclere that is different from the true sigma, is also distinctive. Sigmas are present in four other Discorhabdella species: D. hindei; D. littoralis; D. ruetzleri and D. urizae; however, in these species, there are several differences in the other spicule characters (see Table 1).
Anchorate unguiferous isochelae (Fig. 6B-D), shaft nearly straight, with a pair of fimbriae along whole shaft; bearing 6 alae ( Fig. 6B-D). Size, 17.5-21.9 (19.8) µm in total length, 2.0-2.7 (2.2) µm in shaft width, 6.7-8.0 (7.3) µm in alae length.  Table 1). In addition, they can be differentiated by the shape of their isochelae. Although the isochelae of D. tuberosocapitata and D. misakiensis sp. nov. have similar number of alae, the alae in D. tuberosocapitata are more widely opened. The reported number of isochelae alae in D. tuberosocapitata is rather confusing because different authors reported different number of alae despite all of them observing the same type material: four in Boury-Esnault et al. (1992), four to five in Van Soest (2002) and seven to eight in Maldonado and Uriz (1996). This is possibly due to differences in the interpretation of the fused alae. Boury-Esnault et al. (1992) and Van Soest (2002) considered the two alae fused at the base as one, while Maldonado and Uriz (1996) counted them as two. The alae number of D. misakiensis sp. nov. is here counted as six; however, the two frontal alae seem to fuse at the base or might be regarded as one ala divided into two (Fig. 6D). Further evidence of separation of these two species is their distant geographical distribution: D. tuberosocapitata is reported from Azores, Canaries and Madeira ( but D. misakiensis sp. nov. is found only from the type locality, Sagami Bay, Japan. The dichotomous central ala is also found from "eight-toothed isochelae" of D. hindei (Maldonado and Uriz 1996); however, D. misakiensis sp. nov. and D. hindei are clearly separated by the possession of sigma in the latter species. Furthermore, D. hindei has been reported only from Alboran Sea (Maldonado and Uriz 1996), which is very distant from type locality of D. misakiensis sp. nov.
The choanosomal subtylostyles of the new species are relatively small, and their length overlapped with that of the ectosomal subtylostyles. In Discorhabdella, this pattern is found only in D. littoralis (see Table 1). However, D. littoralis and D. misakiensis sp. nov. are clearly separated by the size of acanthostyles (26-40 µm vs 73.0-91.3 in length), the presence of isochelae (absent in D. littoralis), and of sigmas (absent in D. misakiensis sp. nov.). D. littoralis has been only reported from off the Pacific coast of Panama (Maldonado et al. 2001), which also exhibits distant geographical distribution from type locality of D. misakiensis sp. nov.
Discussion. The present study adds two new species to the genus Discorhabdella, which now has nine species. This is the first record of the genus and family Crambeidae from Japanese waters. Thus the discovery of these two new species from warm temperate northwest Pacific extends the geographical distribution of the genus (see Table 1). Vacelet and Cárdenas (2018) raised doubts to the hypothetical polyaxial nature of the choanosomal styles/subtylostyles and the pseudoastrose acanthostyles that has been proposed by Uriz and Maldonado (1995) and Maldonado and Uriz (1996). The authors proposed instead, a monaxonal origin for the spicule shaft with secondary axes for bulges. In our study, we could not precisely distinguish axes on the choanosomal subtylotyles or the acanthostyles.
Feeble microspines around the distal tips of ectosomal subtylostyles have been first reported from Crambe tuberosa Maldonado & Benito, 1991 and later considered as a possible common character of the genera Discorhabdella and Crambe, both in the family Crambeidae (Maldonado and Uriz 1996). In this study, this character was observed in D. hispida sp. nov. (e.g. Fig. 3F) but seems to be absent in D. misakiensis sp. nov. (Fig. 5F). This character was not mentioned in the recently described species, D. pseudaster and D. ruetzleri Cárdenas 2018, Díaz andPomponi 2018). The actual affinity between Discorhabdella and Crambe has not been revealed as yet (Maldonado and Uriz 1996), but the feeble microspines around the distal tips of the ectosomal subtylostyles may be a symplesiomorphy for these two genera.
The evolutionary aspect of morphological divergence among sphaeroclones, pseudoastrose acanthostyles, and typical acanthostyles has long been discussed and the question remains as to whether the amount of change between sphaeroclones and astrose acanthostyles is more important than the whole set of shared morphological features in determining the phylogenetic relationships between Crambe and Discorhabdella Maldonado 1995, Maldonado andUriz 1996). Our findings on the two new species add more knowledge on acanthostylose derivatives in Discorhabdella. To date, long acanthostyles have been found only in D. tuberosocapitata (with ca 130 µm in length), but in all other species of Discorhabdella they are less than 60 µm (see Table 1) and thus regarded as pseudoastrose acanthostyle because of the putative polyaxial nature contrasting the monoaxial nature of typical acanthostyles of other demosponge taxa Maldonado 1995, Maldonado andUriz 1996). In the two new species, acanthostyles are longer than 70 µm in length, which means the alleged possession of long acanthostyles differing from typical pseudoastrose acanthostyles, is not unusual in Discorhabdella. They also provide clues for solving the trait of gradual morphological divergence between sphaeroclones, pseudoastrose acanthostyles, and acanthostyles along with pseudoaster recently found from D. pseudaster (Vacelet and Cárdenas 2018). A molecular phylogenetic study is necessary to unravel the diversification of sphaeroclones, pseudoastrose acanthostyles, acanthostyles and pseudoasters as well as the affinity of Discorhabdella and Crambe within the order Poecilosclerida Uriz 1996, Vacelet andCárdenas 2018).