Phylogeny of two new pheronematid sponges from the Caroline Seamount and South China Sea

Two new species of genus Pheronemoides are described in this study. The Specimens were collected from the South China sea and the Caroline seamount in the northwestern Pacific Ocean. Pheronemoides crustiformis sp. nov. differs from its congeners in exhibiting large microamphidiscs, whip-like and slightly bent microuncinates and small spiny microdiactins. Pheronemoides curvipentactin sp. nov. possesses special pentactine atrialia with round terminal or tapering terminal curved pinular rays and crooked pentactins, making it easily distinguishable from its congeners. Partial sequences of the 28S rDNA and 16S rDNA genes were also amplified to confirm the family assignment of the two new species and to explore the systematic status of Pheronemoides


Introduction
Seven genera (Schulzeviella Tabachnick, 1990;Semperella Gray, 1868;Pheronema Leidy, 1868;Platylistrum Schulze, 1904; Polio pogon Thomson, 1877;Sericolophus Ijima, 1901; Pheronemoides Gong & Li, 2017) are included in the family Pheronematidae. Pheronemoides Gong & Li, 2017 was established based on a single specimen collected on a seamount near Yap Trench. The atrial areas and dermal areas of Pheronemoides are on opposite sides of the body. Its basalia are not positioned exactly at the centre of the body, but rather in a basal crescent and amesially on the dermal surface. Viewed from above, the sponge is hemispherical or spherical. Observed laterally on one side, the sponge is arched and hollow inside (Gong & Li, 2017). Considering the body shape of the sponge together with the location of the basalia and the marginalia, it may be a transitional genus between Pheronema and Sericolophus.
In July 2016, a sponge specimen was collected at the Taitung County using an Agassiz trawl 121°32.621′ to 121°33.7489′E longitude and 21°12.4106′ to 21°13.2041′N latitude. This is the first report of the presence of Pheronemoides species in the South China Sea. In August 2017, a seamount biodiversity survey on the Caroline seamount was conducted using the R/V Ke Xue. Another specimen of Pheronemoides was collected by the remotely operated vehicle (ROV) Fa Xian at a depth of 1429.2 m. After further morphological examination and molecular analysis, the specimens were confirmed to be new species. These two new species are described and illustrated herein. There are now three species in Pheronemoides, all of which were collected in the northwestern Pacific Ocean, two of them were found on seamounts.

Sample collection
One sponge sample was collected by the research ship Ocean Researcher I in the South China Sea using an Agassiz trawl. The sample was deposited in the National Museum of Natural Science, Taiwan. Another sponge sample was collected by the submersible remotely operated vehicle Fa Xian during a cruise of the research ship Ke Xue in the western Pacific Ocean. The sample was deposited in the Marine Biological Museum of Chinese Academy of Sciences (MBMCAS), Qingdao, China.

Spicule analysis
A small piece of sponge tissue was used to prepare the spicules by digesting them with concentrated nitric acid, and the spicules were then observed with scanning electron microscopy (SEM) and light microscopy (LM). For SEM, the spicules were first concentrated on a cover glass (diameter: 8 mm), which was then attached to a SEM stub. After coating with gold, the spicules were observed using a Hitachi S-3400N. For spicule measurements, we used an Olympus DSX500 Optodigital microscope with the manufacturer's image analysis software.

Phylogenetic analysis
Analysis based on 16S rDNA and 28S rDNA gene sequences were undertaken to show the systematic status of the two new species as well as the genus Pheronemoides within the family Pheronematidae. The sequence of eight additional species from six genera including all species in Pheronematidae and Hyalonema (Onconema) obtusum as the outgroup were downloaded from GenBank to construct the phylogenetic tree (table 1).
After trimming, the concatenated dataset consisted of 1535 bp (16S/28S = 417/1118 bp), alignment gaps were represented as '-' and missing data were represented as '?' . The homologous sequences, including ten sequences of 16S rDNA and eight sequences of 28S rDNA genes, were aligned using MUSCLE 3.8 (Edgar, 2004) with the default parameters. The bestfitting nucleotide substitution model (16S rDNA: GTR+G; 28S rDNA: GTR+G) for each partitioned dataset was assessed with Model-Test 3.7 (Posada & Crandall, 1998). Maximum likelihood (ML) analysis was carried out using RAxMLGUI v1.5 (Silvestro & Michalak, 2012) under the GTRGAMMA substitution model for all partitions in the concatenated dataset. Bayesian inference (BI) analysis was conducted using Mrbayes 3.2 (Huelsenbeck & Ronquist, 2001), Markov Chains were run for 10 million generations, with sampling every 1000 generations. The first 25% of trees were discarded as burn-in, and the remaining trees were summarized in 50% majority rule consensus tree to estimate the posterior probabilities. The effective sample size values were examined with Tracer v1.7 (Rambaut et al., 2018) to ensure that convergence was reached. Description. The sponge is irregularly trapezoidal when observed from above ( fig. 1B), the diameter of the body is 530 mm, and the height of the body is 354 mm. It is fan-shaped and has a large hollow between the dermal surface and basalia when viewed from one side ( fig. 1A). When viewed from another side, it is spherical. Atrial areas cover the upper surface of the sponge and dermal areas are on the opposite. Marginalia present on the boundary between the atrial and dermal areas, protruding out 20 mm from the body surface. Basalia are located on the edge of dermal surface as a basal crescent ( fig. 1C), thus leaving a large hollow between the basalia and dermal areas. Basalia, more than 280 mm in length, consist of many small spicule tufts. Meshes on atrial areas (of 0.1-1.5 mm diameter, fig. 1E) are wider than dermal areas (of 0.1-1 mm diameter, fig. 1D).

Taxonomy
Spicules. Pentactins ( fig 2P), with diameter of 402-506 μm and length can be more than ten centimeters. Monaxones are easily broken and none of a complete one was observed, we speculate that they are diactins. Marginalia are sceptres (length: 3091-8120 μm; width: 16-34 μm) with spiny proximal part and smooth distal part      of basalia left when collected. In addition, there is no available natural living picture of the sponge. From the remaining body shape, we inferred that the sponge has a typical body form of Pheronemoides: when viewed from above, it is fan-shaped ( fig. 3B), when viewed from one side, there may be a big hollow between the dermal surface and basalia. The diameter of the sponge is 240 mm. Marginalia are present at the boundary between the atrial and dermal areas, protruding several centimeters from the body surface. Meshes of atrial areas and dermal areas are inconspicuous. Basalia may be located on the dermal surface, and only few basal spicules can be observed.  4F) with a smooth primary ray, two smooth tangential rays, a curved pinular tangential ray with a large hooked terminus, and a smooth tangential ray with an expanded terminus bearing thin teeth were also observed. This kind of curved pentactin has not been observed in other pheronematid species. Basalia are twotoothed anchors (diameter 364-609 μm) and monaxones (probably diactins). The shaft of anchors are ordered as smooth, spiny, smooth and tapering apex from the proximal part to distal part ( fig. 4O-Q). According to marginal spicule of other pheronematida species, though we only observed the shaft and apex of marginalia ( fig. 4R-S), we infer that they are probably scepters and/or diactins. The width have long teeth and their total length is 118-157 μm. Etymology. Curvipentactin from curv (meaning curved), pent (meaning five), and aktis (meaning rays), refers to this species containing curved pentactin spicules.
Remarks. Although the new specimen is incomplete, and a hollow between the atrial and dermal surfaces is not observed, the body is arched, and we can infer that its basalia are not positioned exactly at the centre of the body but amesially on the dermal surface. Therefore, we infer that the new species belongs to Pheronemoides. The new species can be easily distinguished from its congeners by possessing special pentactine atrialia with round terminal or tapering terminal curved pinular rays and crooked pentactins. Additionally, the new species contains macrouncinates and microuncinates while P. fungosus contains three types of uncinates and P. crustiformis contains macrouncinates and mesouncinates. Molecular data. The phylogenies of BI and ML analyses were highly congruent. The phylogeny based on 28SrRNA and 16SrRNA ( fig. 5) shows that the two new species and P. fungosus forms into a clade with the exclusion of other pheronematids, thus supporting the family assignment, with support values >50 for both the ML and BI analyses.

Discussion
The phylogeny of the family Pheronematidae through molecular approaches had been explored by Kersken's (2018) and Dohrmann's (2018). And they have obtained similar phylogenetic tree. In our study, we added the molecular data of Poliopogon distortus and the two new species, and a more comprehensive tree is provided. Our tree was generally consistent with the previous results except the status of Semperella schulzei which was sister to Semperella jialongae+Poliopogon+Schulz eviella while it was clustered to Pheronema+ Sericolophus+Pheronemoides in the previous studies. This was probably due to the different choices of alignment and substitution models, the molecular sequences of three additional species, as well as one or two reduced molecular markers used in our analysis. Though there were some differences, our tree revealed same relationship among the different genera of Pheronematidae as the previous studies, as well as the genus Semperella was a non-monophyletic group. More species of definitive Semperella are need to be sequenced in further studies to elucidate the status of this genus.
The seven genera of Pheronematidae Gray, 1870 are often distinguished from each other mainly by their external body shape, whereas the morphologies and diversity of the spicules are less important (Tabachnick & Menshenina, 2002). Aproaches on the phylogeny of Pheronematidae based on morphology were explored once (Tabachnick & Menshenina, 1999;Dohrmann et al., 2017). In our tree, Pheronematidae is divided into two clades. In one clade, Pheronema is sister to Sericolophus+Pheronemoides. This is consistent with Tabachnick et al. (1999), who consider Sericolophus to have evolved independently in relation to Pheronema species, and Gong et al. (2017), who recognized Pheronemoides has a closed relationship with Pheronema and Sericolophus. The other clade included Semperella, Poliopogon and Schulzeviella, which differed from the results of Dohrmann, who inferred that Schulzeviella was the sister group to the remaining pheronematids (Dohrmann et al., 2017). Since the phylogeny based on molecular data was not consist with the morphology-based hypotheses, the special morphology of different genera within Pheronematidae may not be enough to treated as a reliable synapomorphy. And the unique morphological characteristics (i.e., the body being bilaterally symmetrical or not, the atrial cavity being open or closed, atrialia being a common surface or not and basalia in a compact tuft or a broad tuft) used to identify pheronematida species into different genera might need refinement. Pheronemoides, which is fan-like, exhibit an atrial concave side and a dermal convex side without basalia ( fig. 2). It shows a typical body shape similar to Poliopogon species. When we established the genus, there was confusion about whether the genus was effectively defined according to morphological approaches. In our tree, Pheronemoides and Poliopogon are distant, and all the species of Pheronemoides and Poliopogon were grouped together, which proves that Pheronemoides is a valid genus. P. curvipentactin sp. nov. and P. fungosus exhibit a closer relationship than P. crustiformis. Ecologically, P. curvipentactin sp. nov. and P. fungosus occur at the hard bottom on seamounts in the northwestern Pacific Ocean, while P. crustiformis sp. nov. occurs in the South China sea (substrate unknown due to the absence of a natural living image). Morphologically, only P. crustiformis sp. nov. exhibits special pentactine atrialia, which makes it is easily distinguished from the others species.
Forty-eight valid species of pheronematid have been reported (Van Soest et al., 2019); here, we only include 6 valid species in our tree (without the two new species and two species that were unable to be classified at the species level). Therefore, more taxon coverage and molecular markers will be needed in future studies to explore the phylogeny of Pheronematidae.