Transcriptome sequencing and delimitation of cryptic Oscarella species (O. carmela and O. pearsei sp. nov) from California, USA

The homoscleromorph sponge Oscarella carmela, first described from central California, USA is shown to represent two morphologically similar but phylogenetically distant species that are co-distributed. We here describe a new species as Oscarella pearsei, sp. nov. and redescribe Oscarella carmela; the original description was based upon material from both species. Further, we correct the identification of published genomic/transcriptomic resources that were originally attributed to O. carmela, and present new Illumina-sequenced transcriptome assemblies for each of these species, and the mitochondrial genome sequence for O. pearsei sp. nov. Using SSU and LSU ribosomal DNA and the mitochondrial genome, we report the phylogenetic relationships of these species relative to other Oscarella species, and find strong support for placement of O. pearsei sp. nov. in a clade defined by the presence of spherulous cells that contain paracrystalline inclusions; O. carmela lacks this cell type and is most closely related to the Western Pacific species, O. malakhovi. Oscarella pearsei sp. nov and O. carmela can be tentatively distinguished based upon gross morphological differences such as color, surface texture and extent of mucus production, but can be more reliably identified using mitochondrial and nuclear barcode sequencing, ultrastructural characteristics of cells in the mesohyl, and the morphology of the follicle epithelium which surrounds the developing embryo in reproductively active individuals. Usually, cryptic species are very closely related to each other, but in this case and in sponges generally, cryptic species may be very distantly related because sponges can be difficult to identify based upon gross morphological characteristics.


Introduction
The homoscleromorph sponge species Oscarella carmela Muricy & Pearse, 2004 was described from Carmel, California and was the first record of this genus from the Pacific coast of North America [1]. When described, there was question about whether this species was native to the region or whether it was invasive, in part because it was initially observed in public and research aquaria and only later discovered in the nearby intertidal region. Through characterization of its cellular ultrastructure it was ultimately determined to represent a new species, distinct from all known species globally and consistent with the view that it is native to the Eastern Pacific.
We and others were interested in developing this species as a model for genomic and experimental research for several reasons: 1) it is abundant and easily accessible in research aquaria at the Joseph Long Marine Laboratory at the University of California Santa Cruz, 2) in the laboratory environment, embryos of all stages are present year round, albeit more abundant in late summer and fall, and 3) it is thin and therefore internal cells and tissues are easily imaged using common microscopy and experimental methods. To facilitate this development, we sequence expressed sequence tags (ESTs) [2], the mitochondrial genome [3], and a draft nuclear genome [4] for this species.
More recently, we used the Illumina platform to sequence and assemble the transcriptome of O. carmela to improve gene prediction from the draft genome, beyond what was possible using ESTs alone. However, from these data (reported in this article) we noticed that there was considerable sequence divergence at both the nucleotide-and amino acid-level alignment, -all segments with contiguous non-conserved positions bigger than 8 were rejected, -minimum number of sequences for a flank position: 85%. Phylogenetic trees were reconstructed using the maximum likelihood method implemented in the PhyML program (v3.1/3.0 aLRT) [14]. The default substitution model was selected assuming an estimated proportion of invariant sites (of 0.357) and 4 gamma-distributed rate categories to account for rate heterogeneity across sites. The gamma shape parameter was estimated directly from the data (gamma=0.635). Reliability for internal branches was assessed using the aLRT test (SH-Like) [15]. Graphical representation and edition of the phylogenetic trees were performed with

DNA barcoding
Upon collection, an ~1cm x 1cm piece of tissue from each individual was preserved in fixative for Transmission Electron Microscopy (described below) and in 95% EtOH for genomic DNA extraction and DNA barcoding. Genomic DNA for LSU barcoding was isolated using the GenElute Mammalian Genomic DNA Miniprep kit (Sigma-Aldrich) per manufacturer specifications (EtOH was allowed to evaporate before starting procedure). Using 28S-C2-fwd and 28S-D2-rev [19] primers, a fragment of the LSU was amplified by PCR and sequenced by ! 7 Eurofins Genomics (Germany). Genomic DNA for mitochondrial barcoding was isolated using the standard phenol-chloroform method [20]. Using diplo-cob-f1m and diplo-cob-r1m primers [21], a fragment of cob was amplified by PCR (Invitrogen recombinant Taq DNA polymerase kit), purified with Promega Wizrd SV Gel and PCR Clean-up system, and sequenced by Iowa State University Sequencing facility.

Transmission Electron Microscopy
For semi-thin sections and for transmission electron microscopy (TEM) investigations, sponges were fixed in a solution composed of one volume of 25% glutaraldehyde, four volumes of 0.2 M cacodylate buffer and five volumes of filtered seawater for at least 2 h before being post-fixed in 2% OsO4 in seawater at room temperature for 2 h. After fixation, samples were washed in 0.2 M cacodylate buffer and distilled water successively, and dehydrated through a graded ethanol series. Specimens were embedded in Araldite resin. Semi-thin sections (1 µm in thickness) were cut on a Reichert Jung ultramicrotome equipped with a "Micro Star" 45° diamond knife before being stained with toluidine blue, and observed under a WILD M20 microscope. Digital photos were taken with a Leica DMLB microscope using the Evolution LC color photo capture system. Ultrathin sections (60 -80 nm) were cut with a Leica UCT ultramicrotome equipped with a Drukkert 45° diamond knife. Ultrathin sections, contrasted with uranyl acetate, were observed under a Zeiss-1000 transmission electron microscope (TEM).

Results
The holotype and paratypes submitted as part of the original species description for Oscarella carmela we re-examined and found to include tissue from distinctly separate species (Table 1) Table 2 we provide a list of previously published and new genomic and transcriptomic resources, and clarify the species from which they were derived.

Mitochondrial genome
The mitochondrial genome for O. carmela was previously published [3], and the species designation for this dataset remains unchanged. Here, we report and describe the sequence of the mitochondrial genome for O. pearsei sp. nov. (Fig 2), which was assembled from Illumina DNAseq data and deposited to GenBank (accession number KY682864). The mitochondrial genome of O. pearsei sp. nov. is a circular mapping molecule 20,320 bp in size and 65.6% A+T that fits well with the description of a typical mitochondrial genomes in the family Oscarellidae [24,25]. It contains 42 genes, organized in two clusters with opposite transcriptional polarities.
The genes include the unusual tatC, for subunit C of the twin arginine translocase [26] as well an observation consistent with our previous study [26].

DNA barcoding
All museum-deposited samples used for the re-description of O. carmela and the new description of O. pearsei sp. nov. were identified using a short, rapidly evolving region of the ! 11 LSU that has been shown to be useful for DNA barcoding [19,27] as well as a fragment of mitochondrial Cytochrome b gene [21]. Both LSU and cob fragments were found to reliably distinguish between the two species, which were detected living side by side in the laboratory (UCSC and at the type locality in the intertidal zone of Carmel, CA  (Fig 5B). Ovoid or spherical cells Spermatogenesis is generally asynchronous inside spermatic cysts.

Spherulous cells with paracrystalline inclusions
Oogenesis and embryogenesis are asynchronous: all stages from oogonia to egg were observed within the same sponge. Young oocytes have an ovoid or amoeboid shape and are situated between choanocyte chambers and endopinacoderm (Fig 6B). Mature eggs are about 70 µm in diameter, isolecithal and polylecithal, with a cytoplasm full of yolk granules ( Fig 6C). Embryogenesis is also asynchronous leading to formation of typical cinctoblastula larvae (Fig 6E and G). All stages from cleaving embryos to larva were observed from mid-June to September. Eggs and embryos are located in the basal part of the choanosome and are completely surrounded by a follicle. From the stage of vitellogenesis of the oocyte to the embryos/larva stage the follicle transforms from flat monolayer composed of pinacocytes (oogenesis) (Fig 6C and D) to cubical one in embryos and larva (Fig 6E-H

Three types of cells with inclusions occur within the mesohyl:
Granular cells type 1 (Fig 7E) ("type 1 cell with inclusions" described by [1]) are ovoid, 4.5 × 7 μm, irregular, with short pseudopodia. Nucleus about 1.9 μm in diameter, ovoid or compressed by the abundant cytoplasmic inclusions. Cytoplasm filled with oval inclusions of one kind, 0.8-1.9 μm wide, with electron dense and homogeneous contents. (Fig. 7F, G) ("archeocytes", described by [1]). Irregular to ovoid 5.5 × 7.9 μm, sometimes with short pseudopodia. Cytoplasm filled with abundant spherical inclusions with filamentous contents 0.7 μm in diameter and with some phagosomes 0.9 to 1.7 μm in diameter. It appears that granules fuse with each other, resulting in the formation of electron transparent vacuoles from 0.7 to 2.5 μm in diameter. Nucleus is ovoid or irregular, 2.4

! 23
Two morphological types of extracellular, endobiotic bacteria occur in the mesohyl: Type B1 (Fig 8A and B)  Nucleoid is loose filamentous network and with thin filaments closer to the periphery. Surface is even or slightly wavy. Type B2 (Fig 8C and D) is rod-like, about 1.1 µm length and about 0.24 µm in diameter. Cell wall consists of two membranes. Cytoplasm filled with dark filamentous materials, and without a clear distinction between nuclear and cytoplasmic regions.

Reproduction
Oscarella carmela is viviparous and simultaneously hermaphroditic: male and female reproductive elements are present in the same individuals (Fig 9A). Oogenesis and embryogenesis are asynchronous; all stages from oogonia to egg were observed within the same specimen. Mature eggs are isolecithal and polylecithal, with a cytoplasm full of yolk granules with diameter about 48-58 µm (Fig 9A). Embryogenesis is also asynchronous, resulting in formation of cinctoblastula larvae (Fig 9D). Eggs and embryos (about 80 µm in diameter) are located in the basal part of the choanosome and are surrounded by a follicle (Fig 9 A-D). Follicle is simple from oocyte vitellogenesis stage (Fig 9A and E) to embryos and larvae (Fig 9B-D and F) and consists of only one layer of flat cells. The spermatic cysts are oval and have different dimension (from 28-52 x 21-48 µm) and are randomly distributed in the sponge mesohyl (Fig 9 G). Spermatogenesis is generally asynchronous inside spermatic cysts (Fig 9G and H).