The genomes of the aquarium sponges Tethya wilhelma and Tethya minuta (Porifera: Demospongiae)

Sponges (Phylum Porifera) are aquatic sessile metazoans found worldwide in marine and freshwater environments. They are significant in the animal tree of life as one of the earliest-branching metazoan lineages and as filter feeders play crucial ecological roles, particularly in coral reefs, but are susceptible to the effects of climate change. In the face of the current biodiversity crisis, genomic data is crucial for species conservation efforts and predicting their evolutionary potential in response to environmental changes. However, there is a limited availability of culturable sponge species with annotated high-quality genomes to further comprehensive insights into animal evolution, function, and their response to the ongoing global change. Despite the publication of a few high-quality annotated sponge genomes, there remains a gap in resources for culturable sponge species. To address this gap, we provide high quality draft genomes of the two congeneric aquarium species Tethya wilhelma and Tethya minuta, small ball-shaped demosponges that are easily maintained long-term in ex situ culture. As such, they offer promising opportunities as laboratory models to contribute to advancing our understanding of sponge biology and provide valuable resources for studying animal evolution, function, and responses to environmental challenges.


Introduction
Sponges (Phylum Porifera) are sessile aquatic metazoans that occur globally in marine and freshwater habitats.4][5][6] As filter feeders, sponges are ecologically important, especially in coral reefs, 7,8 but are also impacted by climate change, as they bleach due to elevated seawater temperatures. 9,10In the current biodiversity crisis, genome data is valuable to aid species conservation 11 and genomic data can be used not only to understand evolution and development, 12 but also to predict a species evolutionary potential to adapt to changing environmental conditions due to climate change. 13To improve the understanding of their response to changing environmental conditions, the availability of culturable sponge species with annotated high-quality genomes is important, but only a few sponge species meet both these criteria yet.
Although the first sponge genome was published in 2010 from the Australian demosponge Amphimedon queenslandica, 14 only a handful of annotated high-quality sponge genomes have been published and analysed since then, for example from the freshwater demosponge Ephydatia muelleri 15 or the reef-building glass sponge (Class Hexactinellida) Aphrocallistes vastus. 16However, only A. queenslandica and E. muelleri are culturable yet under controlled conditions.This lack of high-quality genome resources available from culturable sponges hinders a full appreciation of our understanding of animal evolution and function as well as the response of sponges as ecological key players in many aquatic ecosystems to the current climate crisis.
To contribute to filling this gap, we here provide high-quality draft genomes of the two aquarium species Tethya wilhelma and Tethya minuta.These two congeners are small ball-shaped demosponges that were described in 2001 from public aquaria in Germany. 17Due to their long-term culturability, they are a laboratory model for many topics, including multicellularity, early-animal evolution, biomineralization, and even cancer (e.g., Refs.18-25).With the provision of novel high-quality genome data of these two species we aim to enhance the use of these species as valuable sponge model systems.

Specimens sampled
Specimens of Tethya wilhelma and Tethya minuta were obtained from the marine research aquaria of the Chair of Paleontology and Geobiology of the Department of Earth and Environmental Sciences at the Ludwig-Maximilians-Universität München (Germany), where they are cultured since about 2010.No permits were needed for the sampling and processing.Voucher specimens are deposited in the Bavarian State Collection for Paleontology and Geology (SNSB-BSPG) under accession numbers SNSB-BSPG.GW33333 (T.wilhelma) and SNSB-BSPG.GW41624 (T.minuta).

DNA extraction
For both species, genomic DNA was extracted from either fresh or frozen tissue with a modified cetyltrimethylammonium bromide (CTAB; Carl Roth, Germany, Cat.Nr. 9161.1)extraction.The modification concerned the addition of Potassium acetate (KOAc, Sigma-Aldrich, Germany, Cat.Nr. 791733) in step no. 5 of the protocol. 26Short DNA fragments were removed using AMPure XP (Beckman Coulter, USA, Cat.Nr.A63881) beads to select for long DNA fragments.DNA quantity and quality were controlled on a Nanodrop 1100 and using 1.5% agarose (Biozym, Germany, Cat.Nr. 840004) gels before library preparation as required for the different sequencing platforms used.

Genome assembly: Tethya wilhelma
For Tethya wilhelma, we took the genome draft (T.wilhelma-v1) published by our group in 2017 20 as a starting point and used new sequence data and bioinformatics to further improve it.Additional data was obtained using Hi-C 27 and Chicago (Dovetail Genomics 28 ) libraries.For this, four whole sponges were frozen in liquid nitrogen and shipped to Dovetail REVISED Amendments from Version 1 We would like to thank the two reviewers for their valuable and constructive comments and their appreciation of the quality of our work.
We have attended all their comments, the majority of which were minor changes, except the request for a revised Figure 2.This Figure has been reworked accordingly and more information is now provided in the figure caption for an improved understanding.A detailed response to each comment by the reviewers has been provided.
Genomics (Scotts Valley, CA) for library preparation and sequencing.The resulting Chicago/Hi-C reads were processed using Dovetail's proprietary software HiRise. 28After Chicago/Hi-C scaffolding, the assembly (dubbed T. wilhelma-v2) had 1,353 scaffolds, totaling 139 Mb, with an N50 of 5.5 Mb.
While some chromosome-sized scaffolds were evident in T. wilhelma's-v2 assembly, many putative chromosomes remained fragmented.Therefore, we tried to improve the assembly's contiguity by adding Moleculo long reads as well as Nanopore long reads, the latter derived from a single run of an Oxford Nanopore MinION (see Table 1).The data was assembled using the programs "SSPACE_Long_Read v1-1" 29 and "GapCloser v.1.12". 30This assembly version, called Twi-v3, contained 967 scaffolds, totaling 138.92 Mb, with an N50 of 6.1 Mb (see Table 2).
For the assembly of T. wilhelma's genome v4, high molecular weight DNA was extracted, and quality was assessed using a Nanodrop 1100.Fragment size was controlled on a 1.5% agarose gel and an Agilent 2200 TapeStation.Libraries for 10X Genomics (Pleasanton, CA) were generated and sequenced at the University of Potsdam, in collaboration with the group of Prof. M. Hofreiter (Evolutionary Adaptive Genomics, University of Potsdam, Germany), on an Illumina Nextseq500.About 390 M reads were obtained (Table 1) and assembled using the 10X-Genomics software "Supernova 2.1.1". 31These assembled contigs were then used for scaffolding the T. wilhelma v3 assembly using "SSPACE-LongRead v1-1" 29 and then with "P_RNA_scaffolder" 32 using 100.7 M PE (125 bp) and 237 M RNA reads (25.2 Gb).Finally, we used hicstuff 2.3.0 33 and the Hi-C data available to create a contact map of the T. wilhelma assembly.This assembly, called Twi-v4, had 891 scaffolds with a total size of 138.9 Mb and an N50 of 6.7 Mb, which also included bacterial scaffolds (see Table 2 and below).
Genome assembly: Tethya minuta For the assembly of Tethya minuta, DNA of a single specimen of Tethya minuta (sample# GW41624) was extracted with CTAB 26 and sequenced twice, using Oxford Nanopore PromethION (12.73 Gb of long reads) and MinION 34 (2.17 Gb of long reads).Additionally, we Illumina-sequenced 27 Mbp paired-end (100 PE and 150 PE).These data were assembled with wtdbg2, 35 and polished using minimap2. 36SSPACE_LongRead 29 was used with the available nanopore data to scaffold the assembly.Finally, we used GapCloser 1.12 30 and the available PE reads to close gaps in the assembly which then had a length of 139 Mb and consisted of 1,043 scaffolds (Tmi-v4), but still included bacterial contigs (see Table 3).

Identification of bacterial scaffolds
From earlier versions of the genome, it was clear that Tethya wilhelma harbours two associated bacteria, both alphaproteobacteria, with an unknown interaction.With the relatively large scaffolds in the assembly, a clear split was seen in GC content and read mapping coverage (Figure 1).Consequently, we separated all scaffolds with GC content under 47% and defined those as sponge.The remaining scaffolds for the two bacteria were binned using "MetaBAT v2.15-25", 37 with default parameters.For T. wilhelma, this yielded 6 bins (see public data repository at Ref. 38 or https://github.com/PalMuc/2Tethya_genomes/tree/main/03-bacteria)with 1 bin corresponding to a single Rhizobiales species (genome appx 7.5 Mb), and the other 5 bins corresponding to a Roseobacter species (genome appx 4.8 Mb).These scaffolds were removed from the final Tethya wilhelma genome version.This assembly, called Twi-v4-no_bacteria, had 557 scaffolds with a total size of 126.1 Mb and an N50 of 6.7 Mb (Table 2; see Ref. 38 or https://github.com/PalMuc/2Tethya_genomes/tree/main/06-FINAL_AssembliesENA accession GCA_964030475).
For T. minuta, we used "MetaBAT v2.15-25" 37 to identify and separate bacterial contigs from sponge scaffolds, which had produced 23 bins from the assembly.One of the bins, numbered as bin-17, contained the bulk of the assembly, and  was identified as originating from the sponge due to the GC content of 38.3% and substantial RNAseq mapping.This bin (here now called Tmi-v4-no_bacteria, Table 3) had 244 scaffolds with a total size of 86.07 Mb, around 40 Mb smaller than the assembly of T. wilhelma (Table 2, see Ref. 38 or https://github.com/PalMuc/2Tethya_genomes/tree/main/06-F-INAL_Assemblies;ENA accession GCA_964030485).Nearly all of the large chromosomal pieces in T. wilhelma had matching pieces among the scaffolds of T. minuta, as evident on the synteny plot (Figure 2), which suggested that the assembly of T. minuta was smaller not because of missing scaffolds or mis-assemblies, but merely from a smaller genome.
RNA extraction, sequencing, and assembly RNA was extracted from fresh tissue of Tethya minuta using TRIzol (Fisher Scientific, Germany, Cat.Nr. 12034977) and chloroform (Carl Roth, Germany, Cat.Nr. 3313.1)precipitation 39 with subsequent quality control on a Bioanalyzer 2100.
Libraries were prepared and sequenced twice using one third of a lane of an Illumina HiSeq1500 (100 bp and 50 bp) at the LMU GeneCenter, yielding 137 M read pairs.Reads were assembled de novo using Trinity, 40 using default parameters, resulting in an assembly of 151,079 contigs with an average length of 677 bp.This assembly was also used as a training set for de novo gene prediction (see below).Transcriptome sequencing and assembly of Tethya wilhelma has been described in Francis et al. 20 Statistics of the different sequencing libraries of Tethya wilhelma and Tethya minuta are given in Table 1.

Gene annotation
Both Tethya species were annotated using AUGUSTUS.For T. wilhelma, we used the BRAKER v2.0 pipeline, 41 with the options --useexisting --species=Tethya_wilh and including mapped RNA.This predicted a total of 28,113 gene models, which were used for downstream analysis.

Results
The final version of the Tethya wilhelma draft genome assembly (see Table 2, Twi-v4-no_bacteria) without bacterial scaffolds has 557 scaffolds, a length of 126.1 MB, an N50 of 6.7 MB, and contains 1030.7 kb gaps (Ns).The final version of the Tethya minuta draft genome assembly (see Table 3, Tmi-v4-no_bacteria) without bacterial scaffolds has 244 scaffolds, a length of 86.07 MB, an N50 of 969.3 kb, and contains 534.5 kb gaps (Ns).BUSCO values for the two assemblies are given in Table 4.

Ethical considerations
For work with sponges (Porifera) no ethical clearing is needed.sponges in question are proposed as model systems for studying the responses of this invertebrate to the effects of climate change.Furthermore, the sample set is remarkable, and the sequencing and bioinformatics techniques used to improve the draft genome are noteworthy.
Overall, the Genome note presents a high-quality bioinformatic analysis, and it is recommended that it be accepted pending consideration of minor revisions as detailed below.DNA extraction: it would be helpful if a brief explanation the modifications to the CTAB method.
3. In the sentence: "Fragment size was controlled on a 1.5 agarose gel"…., add the percentage should be added after 1.5 (1.5%).
4. Figure 2: Axes of the graph, scientific name of the sponges in italics.
Are the rationale for sequencing the genome and the species significance clearly described?Yes

Are the protocols appropriate and is the work technically sound? Yes
Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others?Yes Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: I'm an expert in integrative taxonomy and molecular phylogeny of marine sponges.I believe that the genome of these two sponges will contribute significantly to our understanding of how this group of invertebrates responds to climate change.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
Author Response 28 Jul 2024 assembly.The T. minuta assembly, based on long and short reads only, is of sufficient quality for comparison with T. wilhelma genome.
Suggested revisions: 1.The authors should include details about how the sponge DNA was procured by Dovetail for HiC/Chicago as this could be useful for the sequencing of other sponge and marine invertebrate genomes.2. Several of the accession numbers in Table 1 can not be traced in NCBI -please update.3. The synteny plot comparing the two Tethya genomes (Fig. 2) is difficult to interpret and should be improved/reordered to allow better direct comparison of congener scaffolds.
Are the rationale for sequencing the genome and the species significance clearly described?Yes Reviewer Expertise: Marine biology, sponges, genomes, transcriptomes I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
have yet to receive a reply. 1 can not be traced in NCBI -please update.

Authors reply:
The accession number can all be traced in the European Nucleotide Archive (ENA), to which the data was submitted (we double checked all entries).We have, unfortunately, no influence on the timing of data exchange between partners in the International Nucleotide Sequence Database Collaboration (INSDC), where both ENA and NCBI participate.
We have now included the direct link to the ENA browser to ease access.https://www.ebi.ac.uk/ena/browser/view/ 3. The synteny plot comparing the two Tethya genomes (Fig. 2) is difficult to interpret and should be improved/reordered to allow better direct comparison of congener scaffolds.
Authors reply: Thank you very much for this great suggestion, we have now reordered the plot and highlight some rearrangements in boxes in the plot.We also now provide a much more detailed explanation in the figure caption, i.e., Figure 2: Synteny plot of Tethya wilhelma versus Tethya minuta.Each point represents a homologous gene between the two species.Bars parallel to each axis show the scaffold size.Scaffolds are arranged from longest to shortest in T. wilhelma, with the scaffolds in T. minuta sorted to match the T. wilhelma scaffold with the most homologs.Several rearrangements are evident, some are shown in the orange, blue, and red boxes, which could result from translocations, inversions on currently incomplete scaffolds, or misassemblies.

Competing Interests: no competing interests
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Figure 2 .
Figure 2. Synteny plot of Tethya wilhelma versus Tethya minuta.Each point represents a homologous gene between the two species.Bars parallel to each axis show the scaffold size.Scaffolds are arranged from longest to shortest in T. wilhelma, with the scaffolds in T. minuta sorted to match the T. wilhelma scaffold with the most homologs.Several rearrangements are evident, some are shown in the orange, blue, and red boxes, which could result from translocations, inversions on currently incomplete scaffolds, or misassemblies.

Are the protocols appropriate
and is the work technically sound?Yes Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others?Partly Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?Partly Competing Interests: No competing interests were disclosed.