Leucosolenia qingdaoensis sp. nov. (Porifera, Calcarea, Calcaronea, Leucosolenida, Leucosoleniidae), a new species from China

Abstract A new species of Leucosoleniidae, Leucosolenia qingdaoensissp. nov., is described. This new species was collected in a scallop-breeding pond from the Yellow Sea and preserved in 75% ethanol. This sponge consists of a dense reticulation of ascon tubes, with the surface minutely hispid and the consistency soft and fragile. Spiculation of the new species consists of diactines, which are smooth, straight or sometimes slightly curved, triactines of two types, and tetractines with short and curved apical actines; spiculation also slightly overlaps and is somewhat irregularly assembled. Together these form a thin layer of skeleton, with a small number of cells, which results in a transparent, white sponge. As a typical asconoid feature, all internal cavities of the sponge are lined with choanocytes, and there is no fully developed inhalant system. Comparisons with other Leucosolenia reported from the Pacific Ocean are also made.


Introduction
The family Leucosoleniidae is characterised by a branched and rarely anastomosed cormus and asconoid aquiferous system; there is neither a common cortex nor a delimited inhalant or exhalant aquiferous system (Minchin 1900). The family includes three genera (Borojevic et al. 2002): Ascyssa Haeckel, 1872, Ascute Row, 1913, andLeucosolenia Bowerbank, 1864. They can be easily distinguished by their skeletons: the skeleton of Ascyssa contains only diactines; the skeleton of Ascute exhibits giant longitudinal diactines forming a continuous layer on the external surface, and includes triactines and tetractines; and the skeleton of Leucosolenia lacks any of these obvious characteristics in the above two genera. Instead, the skeleton of Leucosolenia is characterised by being composed of diactines, triactines and/or tetractines, without a reinforced external layer on the tubes.
The genus Leucosolenia comprises 40 living species worldwide (Van Soest et al. 2019), of which only three species, L. microspinata Longo, 2009, L. salpinx Van Soest, 2017, and L. parthenopea Sarà, 1953, were named after 1950; 11 species were described by Haeckel between 1870 and 1872. The literature of this genus is relatively old, and the descriptions contained therein of the species of Leucosolenia were simple, almost without details and illustrations of the body shapes and spicules. Thus, a taxonomic revision of this genus is very difficult, and to date, no worldwide revision of the genus has been made.
The localities of the 15 known species of Leucosolenia recorded from the Pacific Ocean are shown in Figure 1. Seven species (L. eleanor Urban, 1906, L. minuta Tanita, 1943, L. mollis Tanita, 1941, L. pyriformis Tanita, 1943, L. serica Tanita, 1942, L. tenera Tanita, 1940, and L. ventosa Hôzawa, 1940 were reported from the Japanese waters (Sagimi Sea, Wakayama Prefecture, Onagawa Bay, Mie Prefecture, Matsushima Bay, Izushima, Wagu Miye Prefecture, respectively). Leucosolenia macquariensis Dendy, 1918 was reported from the west coast of Macquarie Island; L. australis Brøndsted, 1931 was reported from Comau Fjord; L. albatrossi Hôzawa, 1918 was reported from Copper Island and the Komandorski Islands; L. echinata Kirk, 1893 andL. rosea Kirk, 1896 were reported from New Zealand; L. lucasi Dendy, 1891 was reported from Port Phillip Heads, Australia; L. nautilia Laubenfels, 1930 was reported from California, USA; and L. feuerlandica Tanita, 1942 was reported from Tierra del Fuego, South America. The Leucosolenia species reported from the coasts of Japan account for most species. The type specimens of new species were found in the Yellow Sea, very close to Japan.

Materials and methods
The specimens were collected in a scallop-breeding pond from the Yellow Sea and were preserved in 75% ethanol. Two specimens were deposited in the Marine Biological Museum of the Institute of Oceanology in the Chinese Academy of Sciences (IOCAS), Qingdao, China. For examination of the spicules, a small piece of specimen was cut and placed in a 1.5 mL microcentrifuge tube to which 1000 µL of sodium hypochlorite solution was added (Kersken et al. 2016). The mixture was then vortexed, placed at environmental temperature, and vortexed occasionally during incubation until it was completely lysed. Next, the sample was centrifuged at 8000 rpm for 2 min, the supernatant was poured off, 1000 µL of distilled water was added, and the sample was again centrifuged at 8000 rpm for 2 min. This procedure was repeated four times, then the spicules were washed three times with 96% ethanol and then the spicules were preserved in the third ethanol solution.
Scanning Electron Microscopy (SEM) was performed with a Hitachi S3400N. Preserved spicules for SEM were adhered to stubs with double-sided carbon conductive tape and coverslip. After dehydration, the spicules were coated with gold in a Hitachi MC1000 (LOPES 2018).
Measurements of at least 20 spicules of each type were performed using an optical microscope (Nikon Eclipse Ni) with a micrometric eyepiece. The length from the tip to the base and the thickness at the base of each actine were measured. The reported numbers refer to the range of measurements for each spicule type. Photographs were taken with a stereomicroscope (Zeiss Stemi 2000-c) and an optical microscope (Nikon Eclipse Ni-U) equipped with a digital camera to evaluate difference between the length of the unpaired and paired actines of each type of triactine. For comparison with the new species, we only selected those species of Leucosolenia reported from the Pacific Ocean. Description. The sponge is arborescent, consisting of many thin-walled tubes, which are copiously ramified but never anastomosed. The sponge occurs as growth form. The oscula are terminal on erect tubes. The color of the sponge is white after being preserved in alcohol and in vivo. The external walls of the tubes are hairy, with diactines protruding at right or oblique angles from the body; the surface is minutely hispid, and the consistency is soft and fragile. The holotype measures 21.32 × 3.38 mm (height × width). The wall of the sponge body is very thin, and there is no fully developed inhalant system, the gap between the skeleton and the cell on the wall arrange evenly (Fig. 2F); only a small amount of cells is distributed on the thin sponge skeleton (Fig. 2C-F), which is a typical asconoid feature. All internal cavities of the sponge are lined by choanocytes.

Systematics
Skeletal arrangement. The skeleton consists of multifarious diactines, sagittal triactines of two types, sagittal tetractines with bent apical actines and triactine-like basal actines; together these form the wall of the ascon-type sponge body.
In the apical osculum (Fig. 2C, E), there are paired actines of triactines and tetractines, some additional tangential diactines, together forming a clear line dividing the apical oscula, and some radial diactines projecting beyond the apical osculum with different length.
In the sponge body (Fig. 2C, E), the triactines and tetractines are regularly arranged, their paired actines are parallel to the apical oscula, and the unpaired actines point downward, with slight folding allowed, but never overlapping; in contrast to the triactines and tetractines, the diactines are arranged more irregularly but generally point downward.
In the root-like structures (Fig. 2D, F), the arrangement of triactines and tetractines is the same as that in the body, but the arrangement of diactines is different; most of them tangentially project beyond the surface, which results in the surface having a slightly hispid appearance. By observing the sponge tissue taken from different parts, it is clear that as the diameter of the tubes decreases, the contents of small diactines and small triactines increase. This observation can suggest that in the growth zone spiculogenesis is more intense.

Spicules. Diactines.
There is only one type of diactine (Fig. 3A1-3), though the diactines vary in size and shape, their width varies from 24 µm to 61 µm, the length of diactines vary from 43 µm to 421 µm but half of the diactines present a length of 200-300 µm (Fig. 4). The shapes of the diactines are straight or slightly curved in different directions. The variation in Leucosolenia is very common and considerable.
Triactines. Two types of triactines are present, with actines straight or undulated. Their ends are generally sharp or asymmetrical (Fig. 3B1-2). The paired actines are slightly curved. Some deformations are present.
Remarks. Three species described by Tanita (L. minuta, L. pyriformis, and L. serica) exhibit only regular (equiangular and equiradiate) spicules. This characteristic does not fit the description of Leucosolenia, L. qingdaoensis sp. nov. can be easily differentiated from the 12 species of Leucosolenia reported from the Pacific Ocean. The skeletal compositions of these species are shown in Table 1.   180-190/12-18 180-190/12-18 180-190/12-18 180-190/12-18 150-260/8-15 630-800/40-55 Tanita 1943 L.rosea   The new species exhibits one type of diactine. In L. ventosa and L. rosea, there is no record of diactines, and in L. mollis and L. nautilia, there are two types of diactines. The triactines of L. ventosa are 2-8 times thicker than those in the new species; the triactines of L. rosea are 10-35 times thicker than in the new species; and L. mollis only has one type of triactine and all rays being nearly equally thick. The diactines of L. nautilia are extremely large, with a length of 1 mm and a thickness of 20 µm (Laubenfels 1932), while in the new species the diactines are less than 8 µm thick. Laubenfels (1932) gave few details on the actines, but L. nautilia differs from the new species by having only one type of triactine.
The difference between L. albatrossi and the new species is obvious. The diactines of L. albatrossi are club-shaped, while the diactines of the new species are spindle-shaped.
The sagittal triactines of the new species distinguish it from L. macquariensis, L. tenera, and L. eleanor. The new species have two types of sagittal triactines, while L. macquariensis and L. tenera only have one type of sagittal triactine, with rays of approximately equal length. Leucosolenia eleanor have both sagittal and regular triactines.
The new species, with slender and long diactines, the longest diactines 5 times longer than those of L. feuerlandica, is distinct from that species. Additionally, the triactines of the new species are sagittal, and the actines straight or undulated. However, the triactines of L. feuerlandica are pseudoderm sagittal and are tripod-shaped.
Leucosolenia echinata, L. lucasi, and L. qingdaoensis sp. nov. have many features in common, including their body shape, colour in alcohol, general arrangement, shape of diactines, and apical ray, but they show important differences in the shape of their triactines. The new species has two types of triactines; L. lucasi and L. echinata only have one type of triactine. The triactines of L. lucasi are sagittal, but the three angles are roughly equal; the triactines of L. echinata are generally regular, and frequently slightly sagittal, with the oral angle largest and the basal ray longest.