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New records of the rare calcareous sponge Paragrantia waguensis Hôzawa, 1940

Abstract Paragrantia waguensis Hôzawa is reported from coastal reefs of the island of Okinawa. This rare species was previously known only from Central Japan, Mie Prefecture. It has peculiar apopylar tetractine spicules, so far unique among Calcarea. We present in situ images of the species and a full description including SEM images of skeletal structure and spicule complement. The status of Paragrantia as a separate genus of the family Grantiidae distinct from Grantia Fleming is confirmed on the basis of a morphological and molecular comparison with the European type species of Grantia, Grantia compressa (Fabricius).


Introduction
We report here the recent collection (2006,2014) of a rare and curious calcareous sponge species from Okinawa, Paragrantia waguensis Hôzawa (1940), at considerable distance (1500 km) from its type locality in eastern mid-Japan (Mie Prefecture).
Specimens were sectioned by hand: cross sections perpendicular to the surface and tangential sections of the outer surface (cortical region), the inner surface (atrial region), and the oscular fringe. The sections were air dried, mounted on stubs, and sputter coated for examination under SEM. Spicules were dissociated using household bleach, washed five times in distilled water, and subsequently plated on glass slides for light-microscopic measurements and on SEM stubs for examination and micrographing under SEM.
Measurements of the spicules are given as smallest-average-largest of 25 spicules of each distinct type.
To verify the conclusions from the morphological comparison of Paragrantia waguensis with Grantia compressa, a 28S rDNA sequence (430 bp) of Paragrantia waguenis (sample RMNH Por. 9317) was provided by the Naturalis Barcode Laboratory. DNA was extracted using the NucleoMag 96 Tissue kit by Macherey-Nagel on a Thermo Scientific Kingfisher Flex magnetic bead extraction robot with a final elution volume of 150 µl. The forward and reverse of the C2-D2 region of the nuclear ribosomal 28S was amplified (Forward primer 5'GAAAAGAACTTTGRARAGAGAGT 3' and Reverse primer 5'TCCGTGTTTCAAGACGGG 3'). Template was diluted ten times before amplification and added with 18.8 µl of ultrapure MQ water, 2.5 µl PCR buffer, 0.5 µl dNTP (containing 2.5 mM) and 0.25 µl Taq (5 units per µl) to a total reaction volume of 25 µl. PCR cycling consisted of an initial denaturation step at 94 °C for 3 mins, followed by 40 cycles each consisting of 15 secs at 95 °C, 30 secs at 50 °C, 40 secs at 72 °C, and a final extension of 5 mins at 72 °C. Bidirectional sequencing was performed at BaseClear (http://www.baseclear.com/). Sequences were edited manually with Sequencher 4.10.1 (Gene Codes Corporation).
With the obtained sequence we performed a BLAST analysis provided by the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and downloaded a representative set of partial 28S sequences of calcaronean species showing up in the BLAST result. The set of sequences included a sequence of Grantia compressa provided by Manuel et al. (2004), 14 other available Leucosolenida-sequences submitted by various research groups belonging to Grantiidae, Sycettidae, Jenkinidae, Amphoriscidae and Lelapiidae. We added as outgroup sequence the calcinean Pericharax heteroraphis (recently revised and renamed as Pericharax orientalis . The combined dataset of 17 sequences (see Table 1) was then aligned using ClustalW, trimmed to approximately equal numbers of basepairs (407 bp), and subsequently analyzed phylogenetically, using the MEGA package vs 06.6 for Mac (http://www. megasoftware.net/megamac.php). For the phylogeny reconstruction we chose the Maximum Likelihood statistical method with a Bootstrap method set at 100 replicates. As Substitution Model we chose the Tamura-Nei model and -based on model testing algorithm in MEGA -we used GTR+G as Evolutionary Model. Further parameters were used in their default settings.

Phylum Porifera Class Calcarea Subclass Calcaronea Order Leucosolenida Family Grantiidae Genus Paragrantia
Paragrantia waguensis Hôzawa, 1940 Figs 1-5 Paragrantia waguensis Hôzawa, 1940: 40, pl. V figs 8-11, text-fig. 4;Burton 1963: 448, text- fig. 274  Description. Cup-shaped or tubular specimens ( Fig. 1a-e), usually being a single rounded 'person' in life, but larger individuals may be somewhat elliptical, and occasionally consisting of two or three budded individuals. The cups or tubes have a narrow attachment to the substratum but there is no clear stalk. Outer surface pearly white and smooth, without any visible inhalant structures. The rim is pale purple in color and distinctly fringed. Algae or detritus may stick to the rim. Inside the cup, most specimens are likewise smooth and white, but one of the specimens is mottled greenish due to encrusting algae growing on its inner surface (Fig. 1d). A faint punctate inner surface pattern may be discernible in some individuals, representing the peculiar exhalant chambers characteristic for this species. Size of individuals may vary from 1 to 4 cm in height, 0.5-4 cm in diameter, thickness of the walls up to 1.5 mm. Consistency firm, somewhat flexible, but breakable under pressure. In preserved condition, the shape of the individuals alters notably: the specimens collapse and may become folded and compressed.
Aquiferous system. No histological slides were made, but the structure of the skeleton suggests it is syconoid (as was also the case in Hôzawa's material). There is no evidence of branching choanocyte chambers. Subcortical lacunae are present, regularly distributed and apparently serving as inhalant reservoirs.
Skeleton of the walls. (Figs 2-4) In cross section from external side to inside: a fairly thick cortical skeleton of relatively large triactines (Fig. 2a), an articulate tubar skeleton ( Fig. 3a) consisting of three or more rows of sagittal triactines with centrifugally directed unpaired actines, subsequently a layer of subatrial strongly sagittal triactines, and an atrial skeleton ( Fig. 4a-b) of tetractines and triactines, containing the three-dimensionally rounded atrial exhalant chambers ( Fig. 4b) supported and covered by inwardly directed small butterfly-shaped tetractines (Figs 4c-c1) with peculiarly swollen and ornamented apical actines (see below). These small apopylar tetractines in addition to the normal atrial triactines (Fig. 4d) and tetractines ( Fig. 4e) are so far unique among the Calcarea.
Skeleton of the fringe. (Fig. 5a-b) The main support of the fringe consists of a palisade of long thick diactines tangentially covered outside and inside by sagittal triactines and tetractines differing morphologically from those of the walls.
Ecology. No data were provided by Hôzawa, but the Okinawa specimens were from a steep reef slope at 20-55 m depth, growing among coralline and turf algae, and encrusting sponges.

Comparison with Hôzawa's specimens
The habit of Hozawa's specimens is described from eight preserved individuals. A photo is given of two specimens (Hôzawa 1940: pl. V fig. 8) having a fusiform shape ending in a terminal oscule. This is unlike our own cup-shaped individuals. However, we assume that like in our own specimens the habit was changed rather dramatically into laterally compressed, partially 'branched' individuals after preservation. We here assume that the live habit was likely more cup-shaped/tubular. The Tôhoku University Museum was unable to grant our request for a loan of the type material (e-mail of Mr. Jun Nemoto, technical staff of the Tôhoku University Museum), but we are confident from Hôzawa's description and illustrations, and the changes we observed in our own material between live and preserved specimens (cf. Fig. 1a-d and Fig. 1e), that the features are sufficiently similar to consider both groups of specimens as belonging to the same species.
The general structure of the skeleton and the overall diversity and sizes of the spicules likewise match closely (as can be observed from Table 2), so microscopical features between the two sets of specimens also confirm that they belong to the same species.
Nevertheless, there are a few clear differences: -The unpaired actines of the atrial tetractines in Hôzawa's specimens were given as large as up to 390 µm, whereas in our specimens they were only up to 226 µm. -The apical actines of the apopylar tetractines in Hôzawa's specimens were given as having a width of 12-18 µm, whereas ours were only 5-10 µm. -The tetractines of the fringe in Hôzawa's specimens were smaller and thinner: e.g. unpaired actines were 150-420 × 8 µm, whereas in our specimens these measured 165-528 × 9-15 µm.
We believe that these differences are too small to consider them as evidence for specific distinction.

Comparison with Grantia compressa
By its possession of a cortical skeleton, an articulated choanosomal skeleton, and lack of pseudosagittal spicules, the present species fits the family Grantiidae. In order to decide whether Hôzawa was right in establishing a new genus for his species waguensis, it is necessary to know the properties of the other genera of the family. The syconoid aquiferous system and the absence of long longitudinally arranged diactines (except in the fringe) limits the generic relationships of waguensis to Grantia Fleming (1828), Sycandra Haeckel (1872) and Teichonopsis Dendy & Row (1913). All three share the general structure of the skeleton with the present species. The latter two genera are monotypic and are distinguished on unique features, the presence of a special atrial network of tissue strands supported by small diactines (Sycandra), or an elaborate shape (Teichonopsis). Sycandra utriculus (Schmidt, 1869) has a similar structure and spiculation as our specimens (and many Grantia species), but the peculiar atrial network forms a unique distinction. Teichonopsis labyrinthica (Carter, 1878), vaguely resembles preserved specimens of the present species, but the spiculation differs clearly by the lack of any tetractines and the presence of brushes of small oxeas on the cortical and atrial surfaces. Grantia itself, in contrast, has approximately 40 accepted species , with considerable variability of habit and skeletal characters. In order to be able to judge whether the unique features of waguensis merit a separate genus status like Sycandra utriculus and Teichonopsis labyrinthica, or whether it can be assigned to Grantia s.l., we here compare our observations on waguensis with those of the type species of Grantia (and indeed the type of the family Grantiidae), Grantia compressa, the well known Purse Sponge of intertidal rocky coasts of Northern Europe. Remarkably, neither the Systema Porifera, chapter on Leucosolenida (Borojevic et al. 2002b), nor its preview publication , presented a proper description and illustration of this important species. We chose a specimen from Roscoff, W coast of France (intertidal, coll. D.A.G. Buizer, February 1977), in the collections of Naturalis Biodiversity Center, reg. nr. ZMA Por. 04159, as our object for comparison.

Grantia compressa and Paragrantia waguensis differ in the following features:
-In life, G.c. is a flattened, purse-shaped sponge, P.w. is a cup or wide-mouthed tube.
-G.c. lacks a distinct fringe, P.w. has a clear, differently colored, fringe containing special spicules with sizes not occurring in the rest of the body. -Cortical skeleton of G.c. has club-shaped diactines in clusters, lacking in P.w.
-Cortical skeleton of G.c. is thin and contains small triactines, P.w. has a thick cortical skeleton including much larger triactines. -Tubar triactines, subatrial triactines, atrial tri-and tetractines of G.c. are all smaller and thinner than those of P.w. -A special apopylar skeleton and spicules are lacking from G.c. and is the dominant feature in P.w.
To date the World Porifera Database  lists 41 accepted species of the genus Grantia (including 'G.' waguensis). The accepted status of many of those species is uncertain, as there has been no recent revision of the genus and the names were taken more or less uncritically from Burton's (1963) monograph. The most recent addition to the genus, Grantia kempfi, was made by Borojevic and Peixinho (1976). Among the species of Grantia s.l. there appears to be a wide variety of shapes and skeletal features, possibly divisible into distinct types, which may eventually lead to the distinction of subgenera or genera, leaving the genus name Grantia restricted to those species that share the properties of the above described G. compressa. Candidate species for such a restricted Grantia appear to be G. cupula (Haeckel, 1872), G. extusarticulata (Carter, 1886), G. fistulata Carter (1886), G. foliacea Breitfuss (1898), G. stylata Hôzawa (1929), G. tenuis Urban (1908), G. aculeata Urban (1908), G. transgrediens Brøndstedt (1931) and G. uchidai Hôzawa & Tanita (1941).
Other, not further specified groups of species may be distinguished e.g. on the possession of long protruding diactines ('hairy' Grantia's), or those lacking tetractines, etc.
All these species do not have the apopylar specialization of P. waguensis. However, a few species assigned to the genus Grantia do appear to have at least a special category of atrial spicules, next to the usual atrial tri-and tetractines, viz. smaller tetractines in the apopylar region in G. atlantica Ridley (1881) as redescribed by Borojevic and Peixinho (1976), and in G. nipponica Hôzawa (1918). Furthermore, special apopylar spicules in the form of small diactines with serrated apices occur in G. ramulosa Dendy (1924). Possibly, these species could be united within Paragrantia by expanding its definition to include special apopylar spicules without specifying their shape. Such a decision is beyond the goals of the present study.

Sequence data on Paragrantia waguensis and Grantia compressa
A further differentiation between the two type species of the genera Paragrantia and Grantia was obtained from sequences. The molecular classification of the Calcarea and its subclasses, orders and families is still in its early stages (see e.g. Voigt et al. 2012), so it is not straightforward to submit sequences and draw conclusions about likely affinity of various calcareous sponges. However, 28S sequences of Grantia compressa were submitted to Genbank by Manuel et al. (2004), so that gave us the opportunity to compare it with our new molecular data on Paragrantia waguensis (cf. above in the Methods section). We obtained from an analysis of 17 aligned Calcaronea sequences (see Table  1), using the program MEGA, a provisional phylogenetic tree (Maximum Likelihood, 50% majority consensus). The tree (cf. Fig. 8) shows moderately significantly that the two species compared here are only distantly related. Paragrantia waguensis was shown to have Teichonopsis cylindrica as its nearest relative, and Grantia compressa was retrieved in an isolated position near Anamixilla, Scypha (=Sycon) raphanus and Leucandra aspera.

Conclusion
Until a revision of Grantia along the lines sketched above has been made -preferably guided by independent molecular markers of the studied taxa -we propose to Figure 8. Phylogenetic tree (Maximum Likelihood, 50% majority consensus) of selected 28S partial sequences of calcareous sponge species belonging to the order Leucosolenida, including Paragrantia waguensis and Grantia compressa (asterisks). Sequences were downloaded from the NCBI website (http:// www.ncbi.nlm.nih.gov/). The accession numbers and sources for the sequences are listed in Table 1.
Bootstrap support values are based on 100 replicates. The species were selected on the basis of a BLAST search using a 430 bps 28S partial sequence of Paragrantia waguensis (specimen RMNH Por. 9317), provided by the Naturalis Biodiversity Center DNA laboratory. Sequences were aligned, trimmed and analyzed using MEGA 6.06 for Mac (http://www.megasoftware.net/megamac.php).
maintain Paragrantia as a separate genus, so far monotypic with P. waguensis as the only species. Its status is comparable to other such genera (Sycandra and Teichonopsis), recognizable by unique features, as in this case the unique butterfly-shaped apopylar tetractines, for which we introduce the term 'aliactines' (from ala (L.) = wing).
We propose here the following definition (modified from Hôzawa 1940: 43):