De novo draft assembly of the Botrylloides leachii genome provides further insight into tunicate evolution
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* Simon Blanchoud
* Kim Rutherford
* Lisa Zondag
* Neil Gemmell
* Megan J Wilson

## Abstract

Tunicates are marine invertebrates that compose the closest phylogenetic group to the vertebrates. This chordate subphylum contains a particularly diverse range of reproductive methods, regenerative abilities and life-history strategies. Consequently, tunicates provide an extraordinary perspective into the emergence and diversity of chordate traits. Currently published tunicate genomes include three Phlebobranchiae, one Thaliacean, one Larvacean and one Stolidobranchian. To gain further insights into the evolution of the tunicate phylum, we have sequenced the genome of the colonial Stolidobranchian *Botrylloides leachii*.

We have produced a high-quality (90% BUSCO genes) 159 Mb assembly, containing 82 % of the predicted 194 Mb. The *B. leachii* genome is much smaller than that of *Botryllus schlosseri (*725 Mb), but comparable to those of *C. intestinalis* and *M. oculata* (both 160 Mb). This difference is largely due to an increase in repetitive DNA content in *B. schlosseri*. By analyzing the structure and composition of the conserved homeobox gene clusters, we identified many examples of multiple cluster breaks and gene dispersion, suggesting that several lineage-specific genome rearrangements occurred during tunicate evolution.

In addition, we investigate molecular pathways commonly associated with regeneration and development. We found lineage-specific gene gain and loss within the Wnt, Notch and retinoic acid pathways. Such examples of genetic changes to key evolutionary conserved pathways may underlie some of the diverse regenerative abilities observed in the tunicate subphylum. These results, combined with the relatively recent separation from their last common ancestor (630 MYA), supports the widely held view that tunicate genomes are evolving particularly rapidly.

Keywords
*   chordate
*   regeneration
*   *Botrylloides leachii*
*   ascidian
*   tunicate
*   genome
*   evolution

## Introduction

Tunicates are a group of marine suspension-feeding hermaphrodites found worldwide in the inter- or sub-tidal region of the seas. This subphylum of invertebrates is phylogenetically located in the Chordata phylum, between the more basal Cephalochordata and the higher Vertebrata, of which they are considered the closest relatives (Fig. 1A; (Delsuc et al., 2006)). These organisms include a wide range of reproductive methods, regenerative abilities, developmental strategies and life cycles (Lemaire et al., 2008). Importantly, and despite a drastically different body plan during their adult life cycle, tunicates have organs as well as a tissue complexity incipient to that of vertebrates (Fig. 1A), including a heart, a notochord, an endostyle and a vascular system (Millar, 1971). In addition, this group of animals is undergoing faster genomic evolution compared to vertebrates through a higher nucleotide substitution rate in both their nuclear and mitochondrial genomes (Tsagkogeorga et al., 2010, 2012; Rubinstein et al., 2013; Berna and Alvarez-Valin, 2014). Therefore, this chordate subphylum provides an excellent opportunity to study the origin of vertebrates, the emergence of clade specific traits and the function of conserved molecular mechanisms. Biological features that can be investigated in tunicates include, among others, the evolution of colonialism, neoteny, sessilness, and budding. However, there are currently only seven Tunicata genomes publicly available, of which three have been well annotated. There is thus a paucity in the sampling of this very diverse subphylum. 

![Figure 1](http://biorxiv.org/https://www.biorxiv.org/content/biorxiv/early/2017/06/21/152983/F1.medium.gif)

[Figure 1](http://biorxiv.org/content/early/2017/06/21/152983/F1)

Figure 1 *B. leachii* phylogenetic position and life cycle.
**A.** Schematic showing phylogeny of tunicates with respect to the chordate clade. **B.** Life cycle of *B. leachii*. The colony expands and grows by asexual reproduction. During favorable conditions such as warmer water temperatures, members of the colonies start sexual reproduction. The embryos remain with the colony in brood pouches until release. Hatched larvae attach to nearby substrates and begin metamorphosis into a zooid.

Tunicates are separated into eight clades: Phlebobranchia (class Ascidiacea), Molgulida (class Ascidiacea), Copelata (class Appendicularia), Pyrosomida (class Thaliacea), Salpida (class Thaliacea), Doliolida (class Thaliacea), Aplousobranchia (class Ascidiacea), and Stolidobranchia (class Ascidiacea). Phlebobranchiae and Molgulida are clades of sessile solitary benthic ascidians. These animals reproduce sexually, releasing eggs through their atrial siphon for external fertilization, hence producing a motile larva. These larvae will colonize novel environments, attach to a submersed substrate and undergo metamorphosis into a sessile filter-feeding adult. These ascidians are capable of regenerating a limited set of organs, including their oral siphon (Auger et al., 2010) although regeneration capability reduces as they age (Jeffery, 2015). These clades are currently the most sampled ones with five published genomes (*Ciona intestinalis*, *Ciona savigny, Molgula oculata*, *Molgula occulta*, *Molgula occidentalis;* (Dehal et al., 2002a; Small et al., 2007; Stolfi et al., 2014), and two yet unpublished species (*Phallusia mamilata*, *Phallusia fumigata*; (Brozovic et al., 2016)). *C. intestinalis* and *O. dioika* where the first Tunicate species to have been sequenced and are the best characterized.

Copelata is a class of planktonic free-swimming organisms that possess chordate traits common to all tunicate larvae including a notochord, neural tube and pharyngeal slits. These social neotenic animals form dioecious communities where each individual lives inside a special external mucous structure, termed house, which concentrates and funnels their food. *Oikopleura dioika* is the sole example of the Copelatan to have its genome sequenced, showing exceptional compaction (70 Mb; (Seo et al., 2001)). Whether these animals can undergo regeneration has not yet been assessed.

Pyrosomida, Salpida and Doliolida are clades of planktonic pelagic animals forming cylindrical free-floating compound colonies (Piette and Lemaire, 2015). These organisms can reproduce both sexually, through autogamy to initiate novel colonies, as well as asexually, through stolonial budding to increase the size of the colony. Owing to their peculiar life cycle and habitat, these tunicates have not been extensively studied, no genome has been sequenced and whether they can undergo regeneration remains unknown.

Aplousobranchia and Stolidobranchia consist of both solitary and colonial sessile benthic organisms. Colonial tunicates are capable of both sexual, through autogamy, and asexual reproduction, through a wide range of budding types (palleal, vascular, stolonial, pyloric and strobilation; (Brown and Swalla, 2012)). In addition, these compound organisms can undergo whole-body regeneration (WBR; reviewed in (Kürn et al., 2011)). Colonial ascidians are emerging as unique and increasingly popular model organisms for a variety of studies including immunobiology, allorecognition, angiogenesis and WBR (Rinkevich et al., 1995, 2013; Ballarin et al., 2001; Manni et al., 2007; Rinkevich, Douek, et al., 2007; Gasparini et al., 2008; Franchi et al., 2011; Lauzon et al., 2013). While the genome of two solitary Aplousobranchian are currently being assembled (*Halocynthia rorezi*, *Halocynthia aurantium*; (Brozovic et al., 2016)), colonial tunicates were only exemplified by *Botryllus schlosseri*. Furthermore, this ascidian which a significant expantion of its genome size when compared to the other available Tunicate genomes (725 Mb). To further investigate this fascinating subphylum and assess whether genome expansion is a prerequisite for coloniality, we have assembled and analysed the genome sequence of *Botrylloides leachii* (class Ascidiacea, order Stolidobranchia; (Savigny, 1816)).

The viviparous colonial ascidian *B. leachii* can reproduce sexually through a tadpole stage that allows the settlement of a novel colony onto a new substrate (Fig. 1B). Each colony is composed of genetically identical adults (termed zooids) organized in ladder-like systems and embedded in gelatinous matrix (tunic). While each adult has its own heart, they all share a common vascular system embedded within the tunic. In the presence of sufficient food supply, the size of the colony doubles approximately every 20 days through synchronized asexual reproduction, known as palleal budding. During this process each adult produces two daughter zooids that ultimately replace the mother, which is then resorbed by the colony. In addition, upon loss of all zooids from the colony, *B. leachii* can undergo whole-body regeneration and restore a single fully-functional adult in as little as 10 days from a small piece of its vascular system (Rinkevich et al., 1995). Furthermore, when facing unfavorable environmental conditions, these colonial tunicates can enter into hibernation, whereby all zooids undergo regression and are resorbed by the remaining vascular system. When a favorable environment is restored, mature adults will be restored to reestablish the colony (Burighel et al., 1976).

We have assembled and annotated the first *de novo* draft genome of the *B. leachii*by taking advantage of our recently published transcriptomes (Zondag et al., 2016). Using this genome, we have then undertaken a large-scale comparison of the four best-annotated ascidian genomes *(B. schlosser*i, *C. intestinalis, M. oculat*a and *O. dioika*) to gain insights into some of the diverse biological abilities that have evolved within the Tunicata.

## Results

### Genome assembly and annotation

To minimize contamination from marine algae and bacteria typically present in the pharyngeal basket of feeding *B. leachii*, we isolated genomic DNA from embryos of a single wild *B. leachii* colony. Genomic DNA was used to produce two libraries: one short-range consisting of 19090212 fragments (300 bp) of which 100 bp were paired-end sequenced - important for obtaining high coverage - and a second long-range mate pair with 31780788 fragments (1.5-15 kb size range, median ~3 kb) of which 250 bp were paired-end sequenced – important for scaffolding the assembly. Following quality checks, low quality reads were removed and sequencing adaptors were trimmed, thus resulting in a high-quality dataset of 86644308 paired-end and 12112004 single-end sequences (100% with a mean Phred score >= 30, <1% with an adapter sequence, Fig. S1).

We then followed a reference-free genome characterization (Simpson, 2014); provided with statistics from the human, fish (*Maylandia zebra*; (Bradnam et al., 2013)), bird (*Melopsittacus undulatus;* (Bradnam et al., 2013)) and oyster (*Crassostrea gigas*, (Zhang et al., 2012)) genomes for comparison; to estimate three properties of the *B. leachii* genome. First, k-mer count statistics were used to estimate the genome size to be 194.2 Mb (194153277 bp). This size is thus comparable to that of the solitary *C. intestinalis, C. savigny and M. oculata* (160 Mb, 190 Mb and 160 Mb, respectively; (Dehal et al., 2002a; Small et al., 2007; Stolfi et al., 2014), larger than the compacted 70 Mb genome of *O. dioika* but appreciably smaller than the predicted 725 Mb genome of the related colonial ascidian *B. schlosseri*, of which 580 Mb have been sequenced (Voskoboynik et al., 2013). Second, by quantifying the structure of the de Brujin graph obtained using the k-mer counts, the computational complexity of the assembly was estimated (sequencing errors 1/213, allelic differences 1/233, genomic repeats 1/2439). With a cumulative occurrence of 1/106, the *B. leachii* genome is similar to that of bird, more variable than those of fish and human, but still quite less complex than the notably difficult oyster genome (Fig. S1). Third, sequence coverage was estimated using the distribution of 51-mers counts, showing a well-separated bimodal distribution with a true-genomic k-mers maximum at 31x coverage, similar to the human genome but higher than both the fish and the bird. Overall, these metrics suggest that *B. leachii* has a genome well suited for *de novo* assembly and that our sequencing could result in a high quality assembly.

*De novo* assembly using Metassembler (Wences and Schatz, 2015) produced a genome of 159132706 bp (estimated percentage of genome assembled is 82%), with an average sequencing coverage of 66x (after adaptor trimming). The assembly is composed of 1778 scaffolds, with a N50 scaffold length of 209776 and a L50 scaffold count of 223. The 7783 contigs, with a N50 length of 48085, and a L50 count of 781, represent a total size of 146061259 (92%, Table 1). To evaluate the completeness of our assembly, we used the Benchmarking Universal Single-Copy Orthologs (BUSCO; (Simão et al., 2015)). This tool provides a quantitative measure of genome completeness by verifying the presence of a set of manually curated and highly conserved genes. Out of the 843 orthologues selected in metazoans, 760 (90%) were found in our assembly of the *B. leachii* genome (File S1), a relatively high score when compared to the BUSCO score of frequently used genome assemblies such as *Homo sapiens* (89%, GCA_000001405.15) and *Mus musculus* (78%, GCA_000001635.4). In addition, we took advantage of our previous assembly of the *B. leachii* transcriptome (Zondag et al., 2016) to further assess the quality of our genome. Using BLAT (Kent, 2002), we were able to map 93 % of transcript sequences (48510/52004) onto our assembly. Finally, we further estimated the quality of our assembly by mapping the proteomes of the available tunicate genomes using tBLASTn (Camacho et al., 2009): *C. intestinalis* 71% (4233/14740), *C. savigny* 81% (3835/20155), *M. oculata* 77% (3828/16616) and *B. schlosseri* 71% (13575/46519). Overall, these results indicate that our *de novo* genome is largely complete and suitable for annotation.

*Ab initio* genome annotation was performed using MAKER2 (Holt and Yandell, 2011) and predicted 15839 coding genes, of which 13507 could be classified using InterProScan (Jones et al., 2014). Comparing these predictions with our mapping of the transcriptome, we found out that 83% of our aligned cDNA (40188/48510) mapped to a predicted gene locus thus spanning 78 % of the annotated genes (12395/15839). In addition, a total of 4213 non-coding sequences were predicted using Infernal (Nawrocki and Eddy, 2013), Snoscan (Lowe, 1999) and tRNAscan-SE (Lowe, 1999). Finally, repetitive elements were annotated using RepeatMasker (Smit et al., 2015) and a specie-specific library created using the RepeatModeler module (Smit and Hubley, 2015). 18% of the genome was identified as containing repetitive elements (Table 2), a majority (17%) of these being interspersed repeats. This proportion is similar to that found in other tunicates including *C. intestinalis* (17%), M. oculata (22 %) and *O. dioica* (15%), while being lower than that in *B. schlosseri* (60%) and *C. savigny* (33 %).

### Ancient gene linkages are fragmented in tunicate genomes

Ancient gene linkages are spatially restricted and highly conserved sets of genes. These clusters arose in a common ancestor and were preserved because of a common regulatory mechanism such as cis-regulatory elements located within the cluster The homeobox-containing *Hox* gene family, typically composed of 13 members in vertebrates (Hoegg and Meyer, 2005), is among the best-studied examples of such ancient gene cluster and is critical for the correct embryonic development (Pearson et al., 2005). In particular, the linear genomic arrangement within the *Hox* cluster reflects their spatial expression along the anterior-posterior body axis (Pascual-Anaya et al., 2013), which establishes regional identity across this axis. The basal cephalochordate *B. floridae* has all 13 *hox* genes located in a single stereotypical cluster, along with an additional 14th gene (Fig. 2B; (Takatori et al., 2008)), suggesting that the chordate ancestor also had an intact cluster. However in tunicates, this clustering appears to be lost. In *C. intestinalis*, the nine identified *Hox* genes are distributed across five scaffolds, with linkages preserved only between *Hox2, Hox3* and *Hox4; Hox5* and *Hox6*; *Hox12* and *Hox13* (Fig. 2; (Spagnuolo et al., 2003; Wada et al., 2003)). In *O.dioica,* the total number of *Hox* genes is further reduced to eight, split between 6 scaffolds, including a duplication of *Hox9* (Fig. 2A; (Edvardsen et al., 2005)). In *M. oculata* we could identify only six *Hox* genes, divided between 4 scaffolds, with clustering retained for the *Hox10*, *Hox11* and *Hox12* genes (Fig. 2). In Botryllidae genomes, the same seven *Hox* genes are conserved (Fig 2B), with a preserved linkage between *Hox10*, *Hox12* and *Hox13* in *B. leachii* and a three copies of *Hox5* present in *B. schlosseri*. Altogether these genomic distributions of the *Hox* cluster genes supports the hypothesis that reduction and separation of this ancient gene linkage occurred at the base of tunicate lineage (Edvardsen et al., 2005). In addition, *Hox9* appears to be specifically retained in neotenic Tunicates while there is no pattern of consereved *Hox* cluster genes specific to colonial ascidians. 

![Figure 2.](http://biorxiv.org/https://www.biorxiv.org/content/biorxiv/early/2017/06/21/152983/F2.medium.gif)

[Figure 2.](http://biorxiv.org/content/early/2017/06/21/152983/F2)

Figure 2. *Hox* genes are dispersed and reduced in number within tunicate genomes.
Schematic depicting remaining *Hox* gene linkages in five tunicate genomes in comparison to the ancestral *Hox* complex, which included thirteen genes. Orthologous genes are indicated by common colours. Chromosome or scaffold number is shown, along with gene ID when available for newly annotated genomes.

A second ancient homeobox-containing gene linkage is the *NK* cluster. This cluster, which was seemingly inherited from the last common ancestor of bilaterians (Luke et al., 2003), consists of *Msx, Lbx*, *Tlx*, *NKx1, NKx3, NKx4* and *NKx5* (Fig. 3). In *B. floridae*, linkages between *Msx, NKx4* and *NKx3*; as well as between *Lbx* and *Tlx* provide evidence of retained ancestral clustering while *NKx5* was lost (Fig. 3; (Luke et al., 2003)). However in vertebrates, NKx5 is still present while only the gene linkages between *Lbx* and *Tlx* as well as between *Nkx4* and *Nkx3* remain (Fig. 3; (Garcia-Fernàndez, 2005)). To further clarify the evolution of this ancestral cluster in tunicates, we determined the structure of the NK cluster within five ascidian genomes. In all these species, *NKx1* is absent and no evidence of clustering could be found with all identified orthologues located on different scaffolds (Fig. 3). In *C. intestinalis*, *M. oculata* and *O. dioica* only five members of this cluster remain, with the loss of either *Lbx* or *Tlx* as well as of *NKx3* and the duplication of the orthologue of *NKx4* (Fig. 3). By contrast, in the colonial tunicates *B. leachii* and *B. schlosseri*, *Tbx*, *Lbx* and *NKx3* are all present. In *B. schlosseri*, *Msx1* is absent and *NKx4* duplicated. In the *B. leachii* genome, *NK1* is the only ancestral cluster member to be missing and *Nk5* has been duplicated (Fig. 3). Altogether, these results suggest that there has been a loss of *NKx5* in Cephalochordate, one of *NKx1* in Tunicate and that the conjunction of *NKx3*, *Lbx* and *Tbx* is specific to colonial ascidians. 

![Figure 3.](http://biorxiv.org/https://www.biorxiv.org/content/biorxiv/early/2017/06/21/152983/F3.medium.gif)

[Figure 3.](http://biorxiv.org/content/early/2017/06/21/152983/F3)

Figure 3. NK homeobox cluster genes are fragmented within tunicate genomes.
Schematic depicting the organization of the *NK homeobox* cluster genes among the studied chordate genomes. Double-parallel lines indicate > 1Mb distance between genes. Chromosome or scaffold number is shown, along with gene ID when available for newly annotated genomes. Orthologous genes are indicated by common colours.

A third ancient linkage that we investigated is the pharyngeal cluster, a gene group present in hemichordates, echinoderm and vertebrates genomes that is considered to be Deuterosome specific (Simakov et al., 2015). The cluster groups *foxhead domain protein* (*FoxA*), *NKx2 (NKx2.2 and Nkx2.1)*, *Pax1/9*, *mitochondrial solute carrier family 25 member 21* (*slc25A21)*, *mirror-image polydactyly 1 protein* (*mipol1*), *egl nine homolog 3* (*egln3*) and *dehydrogenase/reductase member* 7 (*dhrs7*). Among these, s*lc25a21, Pax1/9*, *mipol1* and *FoxA* pairs are also found in protostomes suggesting an even more ancient origin (Simakov et al., 2015). The pharyngeal cluster is thought to have arisen due to the location of the regulatory elements of *Pax1/9* and *FoxA* within the introns of *slc25A21* and *mipol1* (Santagati et al., 2003; Wang et al., 2007), constraining the genes to remain in tight association with each other. In the *B. floridae* genome, the entire cluster is located on the same scaffold, with the exception of the *Nkx2.1* and *Nk2.2* gene pair located on separate scaffold. In *C. intestinalis*, only orthologs of *FoxA*, slc25a29, *Pax1* and *Pax9* could be identified. Nevertheless, all of them are located on the same chromosome (Fig. 4). In *O. dioica*, the cluster appears even further reduced as, while orthologues of *FoxA*, *Pax1/9* and *Nkx2.2* genes were found on different scaffolds, only one rather distant linkage (> 1 Mb) between *a Pax-like* gene and *slc25A21* is retained. Copies of Pax1/9, slc25a29 and Nk2.2 genes were found in *B. schlosseri*, however all on different scaffolds. In the *B. leachii* genome, *mipol1* is the sole missing gene from this cluster. However, only the pairing of a *Pax-like* and *slc25A21* genes remains (Fig. 4). For both *B. schlosseri* and *M. oculata*, there was no evidence of clustering between genes (Fig. 4) Altogether, these results suggest that most of the Tunicates did not conserve the structure of this ancient linkage, but it is unknown what consequences this would have to their expression and function. 

![Figure 4.](http://biorxiv.org/https://www.biorxiv.org/content/biorxiv/early/2017/06/21/152983/F4.medium.gif)

[Figure 4.](http://biorxiv.org/content/early/2017/06/21/152983/F4)

Figure 4. Ancestral gene linkages remain between a few pharyngeal cluster genes in tunicate genomes.
Gene order of the six pharyngeal cluster genes, *Nkx2.1, Nk2.2, Pax1/9* and *FoxA* in chordate genomes. Double-parallel lines indicate > 1Mb distance between genes. Chromosome or scaffold number is shown, along with gene ID when available for newly annotated genomes. Orthologous genes are indicated by common colours.

### Lineage-specific changes to cell-signaling pathways in Botryllidae genomes

To dissect more specifically the evolution of colonial ascidians, we examined the genomes of *B. leachii* and *B. schlosseri*, looking for key components of signaling pathways required for metazoan development and regeneration. Of particular interest, we focused on the Wingless-related integration site (Wnt), Notch and Retinoic acid (RA) signaling pathways.

### Wnt pathway

Wnt ligands are secreted glycoproteins that have roles in axis patterning, morphogenesis and cell specification (Loh et al., 2016). The ancestral gene cluster appears to originate very early on during muti-cellular evolution and to be composed of eleven members (Kusserow et al., 2005; Guder et al., 2006). The *Wnt* gene family expanded to 19 members in the human genome, while independent gene loss has reduced this family reduced to 7 genes in *Drosophila melanogaster* and *Caenorhabditis elegans* (Prud’homme et al., 2002). Consequently, we set out to investigate whether the *Wnt* gene family has either expanded or reduced during Tunicata speciation.

Most strikingly, we found among tunicate genomes an increase in the number of *Wnt5a* genes. *C. intestinalis* has a total of 11 *Wnt* genes, including a single *Wnt5a* gene (Fig. 5, Table S2; (Hino et al., 2003)). In the compact *O. dioica* genome, this number has reduced to 6 (Wnts 3, 4, 7, 11 and 16), none of which are *Wnt5a* orthologues (Table S2). *M. oculata* has only 7 Wnt ligand genes, including three *Wnt5a-like* genes (Fig. 5, Table S2). In *B. schlosseri*, we identified 15 *Wnt* members, including seven *Wnt5a-like* genes on multiple scaffolds (Fig. 5, Table S2). Finally, in the *B. leachii* genome, fourteen *Wnt* ligand genes were identified, including four *Wnt5a* genes located on the same scaffold near *Wnt4* (Fig. 5). Overall, this suggests that an expansion through gene duplication of the Wnt5 family occurred during tunicate evolution, but was lost in some lineages. 

![Figure 5.](http://biorxiv.org/https://www.biorxiv.org/content/biorxiv/early/2017/06/21/152983/F5.medium.gif)

[Figure 5.](http://biorxiv.org/content/early/2017/06/21/152983/F5)

Figure 5. Duplication of *Wnt5a* genes in Tunicate genomes.
Schematic showing the genomic location of *B. leachii* (A), *B. schlosseri* (B), *M. oculata* (C) and *C. intestinalis* (D) *Wnt5*-like genes within each genome. Double-parallel lines indicate > 1Mb distance between genes.

To assess the functionality of the Wnt pathway in Tunicates, we set out to assess whether its downstream effectors are themselves present in the available genomic data. The downstream pathways activated by Wnt ligands are divided into canonical, non-canonical calcium and non-canonical planar cell polarity. The Wnt5a ligand is associated with both of the non-canonical pathways through binding of membrane receptors that include *frizzled* (*Fzd4*), *receptor tyrosine kinase-like orphan receptor 1/2* (*Ror1/2*) and *atypical tyrosine kinase receptor* (*Ryk*). Further downstream, disheveled (dsh), β-catenin (Cnntb), Axin, low-density lipoprotein receptor-related protein 5/6 (LRP5/6) and nuclear factor of activated T-cells (NFAT) are proteins essential for triggering intracellular responses to Wnt signaling (MacDonald et al., 2009). We identified orthologues for each of these signaling transduction molecules in all Tunicata genomes (Table S2), with no evidence of further gene duplication events. This supports the interpretation that signaling through the Wnt pathway is functional in tunicates.

### Notch pathway

Notch receptors are transmembrane proteins that are involved in cell-cell signaling during development, morphogenesis and regeneration (Hamada et al., 2015). Following activation through the binding of the delta or jagged/serrate ligands, the intracellular domain of Notch is cleaved and induces the expression of downstream target genes including the *hes (hairy and enhancer of split*) gene family members (Guruharsha et al., 2012). The presence of both Notch and the Delta/Serrate/lag-2 (DSL) proteins in most metazoan genomes suggests that their last common ancestor had a single copy of each gene (Gazave et al., 2009). To establish how this pathway has evolved in tunicates, we screened these genomes for the Notch receptor using the conserved lin-Notch repeat (LNR) domain, and for genes encoding probable Notch ligands such as genes from the DSL family.

In all examined genomes, only a single *Notch* receptor gene was identified while the number of ligand genes varied (Table S3). *C. intestinalis* genome contains two *DSL* genes; *O. dioica M. oculata* and *B. schlosseri* a single *DSL* one. By contrast, we found three DSL genes in *B. leachii* (Table S3). To determine the relationships between these identified tunicate DSL-like genes, a phylogeny was constructed along with other chordate DSL proteins. All three *B. leachii* genes are Delta orthologues, two of them related to the *B. schlosseri* and *Cionidae.*copy; the third one closer to the *M. oculata* and *H. roretzi* variant. The mouse, human and zebrafish delta and delta-like (DLL) proteins form a discrete clade loosely related to the genes found in Cephalochordate and Tunicate (Fig. 6, shaded box). Jagged proteins form a separate and less conserved clade (Fig. 6). The tunicate DSL-like proteins present long phylogenetic branches, suggestive of greater diversity, which is also observed in the protein alignment (Fig. S3). This suggests that the tunicate DSL proteins are diverging rapidly from each other, indicative of lineage specific evolution of DSL-like genes. 

![Figure 6.](http://biorxiv.org/https://www.biorxiv.org/content/biorxiv/early/2017/06/21/152983/F6.medium.gif)

[Figure 6.](http://biorxiv.org/content/early/2017/06/21/152983/F6)

Figure 6. *B. leachii* Notch pathway
Bayesian phylogenetic tree depicting the relationship between tunicate and vertebrate DSL proteins, using Drosophila Delta to root the tree. B. leachii proteins are shown in red and shaded areas correspond to clade groupings. Branch support values (probabilities) are indicated.

**Abbreviations: Bl, *Botrylliodes leachii; BsS, Botryllus schlosseri*; Od, *Oikopleura odioica*; Hs, *Homo sapiens*; Ci, *Ciona intestinalis*, Dm, *Drosophila melanogaster*; Dr, *Danio rerio*; Mo, *M. oculata*; Hr *Halocynthia roretzi*; *Blan, Branchiostoma lanceolatum; Bf, Branchiostoma floridae.***

### Retinoic acid signaling

Retinoic acid (RA) is an extracellular metabolite that is essential for chordate embryonic development. RA is synthesized from retinol (vitamin A) by two successive oxidation steps. In the first step, retinol dehydrogenase (RDH) transforms retinol into retinal. Then RA is produced by aldehyde dehydrogenase (ALDH), a superfamily of enzymes with essential roles in detoxification and metabolism (Jackson et al., 2011). RA influences the expression of downstream target genes by binding to the RA receptors, RAR and RXR (Fig. 7A (Cunningham and Duester, 2015)). Finally, RA is metabolized by the enzymes cytochrome P450 family 26 (Cyp26), a process key to restricting RA-induced responses to specific tissues or cell types (Ross and Zolfaghari, 2011). Components of this pathway have been found in non-chordate animals, suggesting a more ancient origin (Canestro et al., 2006). This pathway has previously been shown to be required for *B. leachii* WBR and *Ciona* development, yet several genes required for RA signaling appear to be missing in *O. dioica* (Martí-Solans et al., 2016). 

![Figure 7.](http://biorxiv.org/https://www.biorxiv.org/content/biorxiv/early/2017/06/21/152983/F7.medium.gif)

[Figure 7.](http://biorxiv.org/content/early/2017/06/21/152983/F7)

Figure 7. Evolution of the RA pathway in tunicates
(A) Overview of the RA synthesis and degradation pathway. In bold are the major proteins that contribute to RA signaling during animal development. Indicated below these are changes to the number of copies present in examined genomes. **(B)** ML phylogenetic tree depicting the relationship between invertebrate and vertebrate CYP26 proteins using CYP4 and CYP51 proteins as an outgroup. *B. schlosseri* and *B. leachii* proteins are shown in red, C. intestinalis and M. oculata are indicated in blue. No *Cyp26* gene has been identifed in the O. dioica genome. Values for the approximate likelihood-ratio test (aLRT) are indicated.

**Abbreviations: Bl, *Botrylliodes leachii*; Bs, *Botryllus schlosseri*; Hs, *Homo sapiens*; Ci, *Ciona intestinalis*; Mo, *M. oculata.***

Rdh10 is the major dehydrogenase associated with the first steps of RA production, although the Rdh16 and RdhE2 enzymes can also substitute this function (Belyaeva et al., 2009, 2015; Lee et al., 2009). The *O. dioica* genome has no orthologues for either *Rdh10* or *Rdh16* but it does have four genes that encode for RdhE2 proteins (Martí-Solans et al., 2016). *O. dioica* also lacks both an *Aldh1-*type gene as well as a *Cyp26* gene but has a single RXR-orthologue (Table S4, (Martí-Solans et al., 2016)). In contrast, the *C. intestinalis* genome, contains single copies of *Rdh10*, *Rdh16* and *RdhE2* genes and a total of four *Aldh1* genes, located on two chromosomes (Canestro et al., 2006). Consistent with *C. intestinalis*; *M. oculata*, *B. leachii* and *B. schlosseri* genomes all have single copies of *Rdh10, Rdh16* and *RdhE2* genes, as well as three *Aldh1a/b* genes on separate scaffolds (Table S4).

Three retinoic acid receptor genes were identified within the *B. leachii* genome, one of which had been cloned previously (*g03013,* (Rinkevich, Paz, et al., 2007). All three were also found in *C. intestinalis*, *M. oculata* and *B. schlosseri* genomes(Table S4). While there is only one potential *Cyp26* gene in *M. oculata*, four paralogues were identified in *B. leachii* and *B. schlosseri*. A phylogenetic analysis showed that these 4 genes group with CYP26 proteins (Fig. 7B, Table S4). Altogether, these results show a loss of key RA-pathway genes in *O. dioica* (*Rdh10, Rdh16, Cyp26* and *Aldh1a*), while increased copy numbers in other tunicate genomes.

## Discussion

### Genomic diversity within the Solidobranchia

The *B. leachii* genome, along with previous genomic analyses of other ascidian species, support the widely held view that ascidian genomes are diverse and rapidly evolving, which is particularly evident in the Solidobranchia group (Seo et al., 2001; Dehal et al., 2002b; Tsagkogeorga et al., 2010, 2012; Bock et al., 2012; Rubinstein et al., 2013; Voskoboynik et al., 2013; Griggio et al., 2014; Stolfi et al., 2014). Nevertheless, botryllids are sufficiently similar in external appearance and morphology for early researchers to have suggested that *Botrylloides* could be a subgenus of *Botryllus* (Saito et al., 2001; Nydam et al., 2017). Strikingly however, the *B. schlosseri* genome differs from that of *B. leachii*, as well as from other sequenced tunicate genomes (Table 2). In particular, the comparison between the *B. leachii* and *B. schlosseri* genome sizes (194 Mb vs 725 Mb), their fraction of repetitive sequences (18 % vs 60 %; 65 % in (Voskoboynik et al., 2013)) and their predicted gene number (15’839 vs 27’000; (Voskoboynik et al., 2013)) suggest a different genomic architecture. Altogether, these comparisons indicate that the *B. schlosseri* genome has undergone a significant increase in its genomic content, including retrotransposons (Table S1). In particular, there are at least two additional families in the *B. schlosseri* hAT transposon superfamily and counts of common hAT element, such as hAT-Charlie, differ dramatically (for hAT-Charlie: 366 in *B. leachii* vs 46,661 in *B. schlosseri*). DNA methylation is a key suppressor of transposon activity, changes to the methylation of transposable elements is a known driver of increased tranposition (O’Neill et al., 1998; Simmen et al., 1999; Suzuki et al., 2007; Maumus and Quesneville, 2014). However, DNA methylation in tunicate species has been only studied in *C. intestinalis,* and is described as mosaic, gene body methylation, whereas non-coding regions, including transposons, remain unmethylated (Suzuki et al. 2007). However, DNA methylation has not been studied in other tunicates, and it It is unknown how retrotransposons are suppressed in tunicate genomes. Nevertheless, the observed increase in transpositions could be a consequence of low non-coding DNA methylation, which may contribute to the rapid genome evolution observed in tunicate species, even between closely related species such as *B. schlosseri* and *B. leachii*.

Rapid genome evolution, and active transposable elements in particular, are proposed to aid adaptation to new environments for invasive species (Stapley et al., 2015). Differences in the colonization ability of tunicates has been noted, not only between related species such as *B. leachii* and *B. schlosseri* (Brunetti, 1974, 1976; Brunetti et al., 1980), but even at the molecular level within *B. schlosseri* populations (Bock et al., 2012; Nydam et al., 2017). It is thus possible that the observed success in tunicate invasion (Zhan et al., 2015) is supported by their plasticity in genome characteristics like transposon diversity and gene number.

Ancient homeobox gene clusters whose structure has been retained over millions of years of evolution in many organisms are fragmented in tunicate genomes. Because, the expression of each *Hox* genes across the anterior-posterior axis relates to their location within the *Hox* gene cluster (Pascual-Anaya et al., 2013), cluster breaks are predicted to have consequences for patterning processes. However, an adult body plan with correct spatial orientation of its body axes during tissue development in ascidians also needs to be established during sexual, asexual and WBR. Early patterning events in tunicate species have only been characterized during sexual reproduction in *Ciona*. Early stages of development (prior to gastrulation) follow a mosaic pattern of developmental axis formation, where inheritance of maternally provided factors establishes the body axes (Nishida, 2005). *Hox* gene knockdown experiments in *C. intestinalis* revealed that they have very limited roles, with defects only observed in larval neuronal and tail development upon loss of *Ci-Hox11* and *Ci-Hox12* function (Ikuta et al., 2010). It thus appears that patterning events in *C. intestinalis* are less dependent upon anterior-posterior spatial expression of *Hox* genes to establish regional identity. Previously, in *B. schlosseri*, the entry point of the connective test vessel into the developing bud determines the posterior end of the new zooid (Sabbadin et al., 1975). Therefore it is possible that ascidians incorporate environmental and physical cues to compensate for the lost gene cluster during polarity establishment. A wider analysis comprising multiple tunicate species will be necessary to investigate the exact consequences of homeobox cluster dispersion and whether the compensatory mechanism observed in *C. intestinalis* is the norm or an exception.

### Lineage-specific changes to evolutionarily conserved cell communication pathways

Cell signaling pathways are critical for morphogenesis, development and adult physiology. In particular, we have focused our analysis on three highly conserved pathways: Wnt, Notch and Retinoic Acid signaling. Representatives of all twelve *Wnt* genes subfamilies are found in metazoans, suggesting that they evolved before evolution of the bilaterians (Janssen et al., 2010). We identified members of each Wnt subfamily in tunicate genomes, along with numerous examples of lineage-specific gene loss and/or duplication. The most striking of these events was an increase in *Wnt5a* gene copy number in *B. leachii*, *B. schlosseri* and *M. oculata* genomes. Indeed, most invertebrates genomes, including the basal chordate *B. floridae*, contain a single *Wnt5* gene while most vertebrate genomes have two *Wnt5a* paralogues, believed to be a result of whole genome duplication (Martin et al., 2012). However, in the analyzed tunicate genomes, up to 15 copies of this gene were identified, potentially these additional genes may have been co-opted into novel roles and were retained during tunicate evolution. Wnt5a ligands have numerous biological roles, including a suppressive one during zebrafish regeneration (Stoick-Cooper et al., 2007) and a promotive one during amphioxus regeneration (Somorjai et al., 2012). Furthermore, components of both Wnt signaling pathways are differentially expressed during WBR (Zondag et al., 2016). Altogether, it is thus conceivable that *Wnt5a* gene number has expanded in colonial tunicates to sustain WBR. A functional characterization of the role of these numerous copies of Wnt5a would thus be highly interesting and potentially give evolutionary insights into chordate regeneration.

All components of the Notch pathway are present in the genomes we investigated. Of particular interest, the DSL Notch ligandappears to be rapidly evolving in the tunicates. This indicates that tunicate DSL proteins are under less pressure, than vertebrate orthologous proteins, to conserve their protein sequence. Given that the concentration and activity of the DSL ligands are typically the rate limiting step in this key signaling pathway, it will be interesting to assess whether the functional properties of tunicate proteins have adapted accordingly.

Components of the RA signaling pathway have also been identified in all the tunicate genomes. However, *Oikopleura* has seemingly lost a functional RA synthesis pathway, while still forming a functional body plan. This suggests that either uniquely RA is not involved in critical developmental events in this species, that the RA signaling function has been replaced or that *O. dioica* utilizes an alternative synthesis approach. Conversely, lineage specific increases in RA pathway gene numbers have been observed in *C. intestinalis* (Aldh1, (Sobreira et al., 2011)) and botryllids (*CYP26* genes).

RA, Notch and Wnt pathways play roles in regeneration and development in many species, including Solidobranchian tunicates (Rinkevich, Paz, et al., 2007; Rinkevich et al., 2008; Zondag et al., 2016) and *Cionidae (Hamada et al., 2015; Jeffery, 2015)*. The observed loss of RA signaling genes may result in a poorer regeneration ability for *O. dioica*, however it’s regenerative has not been characterized. Given the unique chordate WBR potential developed by colonial tunicates, it is conceivable that there is selective pressure on their genomes to retain these pathways. In particular given the likely role these pathways also play in colony reactivation following hibernation, as well as in asexual reproduction.

In addition, among tunicates there are significant differences in both life cycle, reproduction and regeneration ability, even between closely related species of the same family, that likely reflect an underlying diversity in genomic content. For instance, differences in both asexual and sexual reproduction have been observed between within the Botryllidae family (Berrill, 1941, 1947, 1951; Oka and Watanabe, 1957; Brunetti, 1974, 1976). Furthermore, *B. schlosseri* can only undergo WBR during a short time frame of their asexual reproductive cycle when the adults are reabsorbed by the colony (Voskoboynik et al., 2007; Kürn et al., 2011) while *B. leachii* can undergo WBR throughout their adult life (Rinkevich, Paz, et al., 2007). Overall, this indicates that despite a generally similar appearance, the rapid evolution of the Tunicata subphylum has provided diversity and innovations within its species. It will thus be particularly interesting to investigate in future studies how such genomic plasticity balances between adaptation to new challenges and constraint, preserving common morphological features.

In conclusion, our assembly of the *B. leachii* genome provides an essential resource for the study of this colonial ascidian as well as a crucial point of comparison to gain further insights into the remarkable genetic diversity among tunicate species. In addition, the genome of *B. leachii* will be most useful for dissecting WBR in chordates, in particular by comparing it with that of *B. schlosseri* to understand how the initiation of WBR can be blocked during specific periods of their life cycle. Furthermore, given the key phylogenetic position of Tunicates with respect to vertebrates, the analysis of their genomes will provide important insights in the emergence of chordate traits and the origin of vertebrates.

## Methods

### Sampling, library preparation and sequencing

*B. leachii* colonies were collected from Nelson harbour (latitude 41.26°S, longitude 173.28°E) in New Zealand. To reduce the likelihood of contamination, embryos were dissected out of a colony and pooled before carrying out DNA extraction using E.Z.N.A SP Plant DNA Mini Kit. A total of 2 μg each was sent to New Zealand Genomics Limited (NZGL) for library preparation and sequencing. A short read (TruSeq illumina HiSeq) generated 19090212 paired-end reads of 100 bp (average fragment size: 450bp, adaptor length: 120bp). A second library (Illumina Nextera MiSeq Mate Pair) not size-selected library (fragment size: 1.5-15 kb, median size: ~3 kb, adaptor length: 38bp).generated 31780788 paired-end sequences of 250 bp.

PreQC report was generated using the String Graph Assembler software package (Simpson, 2014) and quality metrics before assembly with both FastQC (Andrews, 2010) as well as MultiQC (Ewels et al., 2016) (Fig. S1). These analyses revealed that 91 % of sequences had a mean Phred quality score >= 30, 96 % of bases a mean Phred quality score >= 30, and 39 % of sequences an adapter sequence (either Illumina or Nextera). Adaptor trimming was performed with NxTrim (O’Connell et al., 2015) for the mate pair library, followed by Trimmomatic (Bolger et al., 2014) with the following options: MINLEN:40 ILLUMINACLIP:2:30:12:1:true LEADING:3 TRAILING:3 MAXINFO:40:0.4 MINLEN:40 for both libraries. After trimming, 86644308 paired-end (85 %) and 12112004 (12 %) single-end sequences remained (100% with a mean Phred quality score >= 30, <1% with an adapter sequence).

### Genome assembly

*De novo* assembly was performed in three consecutive iterations following a Meta-assembly approach (Table S5). First, both libraries were assembled together in parallel, using a k-mer size of 63 following the results from KmerGenie (Chikhi and Medvedev, 2014) whenever available, by five assemblers: AbySS (Simpson et al., 2009), Velvet (Zerbino and Birney, 2008), SOAPdenovo2 (Luo et al., 2012), ALLPATHS-LG (Gnerre et al., 2011), MaSuRCA (Zimin et al., 2013). The MaSuRCA assembler was run twice, once running the adapter filtering function (here termed “MaSuRCA-filtered”), the other without (termed simply “MaSuRCA”). Their respective quality was then estimated using three different metrics: the N50-length, the BUSCO core-genes completion (Simão et al., 2015) and the Glimmer number of predicted genes (Delcher et al., 1999). Second, these drafts were combined by following each ranking using Metassembler (Wences and Schatz, 2015), hence producing three new assemblies (limiting the maximum insert size at 15 kb). Third, the *B. leachii* transcriptome (Zondag et al., 2016) was aligned to each meta-assembly using STAR (Dobin et al., 2013), which were then combined thrice more using Metassembler following their alignment percentage and limiting the maximum insert size at 3 kb, 8 kb and 15 kb. Finally, the quality of the meta-meta-assemblies was estimated using the BUSCO score and the best one (Table S5) selected as the reference *de novo* assembly.

### Data access

B. *leachiii*: ANISEED [http://www.aniseed.cnrs.fr/fgb2/gbrowse/boleac\_v3/](http://www.aniseed.cnrs.fr/fgb2/gbrowse/boleac\_v3/) C. *intestinalis*: [Ciona\_intestinalis.KH.cds.all.fa](http://Ciona\_intestinalis.KH.cds.all.fa), [Ciona\_intestinalis.joinedscaffold.fa](http://Ciona_intestinalis.joinedscaffold.fa) Ciona unmasked v2.0, [https://www.aniseed.cnrs.fr/fgb2/gbrowse/ciona_intestinalis/](http://https://www.aniseed.cnrs.fr/fgb2/gbrowse/ciona_intestinalis/)

C. savigny: *C. savigni*\_paired\_scaffolds.fa, [https://www.aniseed.cnrs.fr/fgb2/gbrowse/cisavi\_ens81/](http://https://www.aniseed.cnrs.fr/fgb2/gbrowse/cisavi_ens81/),

*M. oculata*:, [https://www.aniseed.cnrs.fr/fgb2/gbrowse/moocul\_elv12/](http://https://www.aniseed.cnrs.fr/fgb2/gbrowse/moocul_elv12/), *B. schlosseri*: [botznik-transcripts.fa](http://botznik-transcripts.fa), [botznik-chr.fa](http://botznik-chr.fa), [botznik-ctg.fa](http://botznik-ctg.fa), [https://www.aniseed.cnrs.fr/fgb2/gbrowse/boschl_botznik2013/](http://https://www.aniseed.cnrs.fr/fgb2/gbrowse/boschl_botznik2013/),

*O. dioica*: [Odioica\_reference\_v3.fa](http://Odioica\_reference\_v3.fa), [Oikopleura\_transcripts\_reference\_v1.0.fa](http://Oikopleura_transcripts_reference_v1.0.fa), [http://www.genoscope.cns.fr/externe/GenomeBrowser/Oikopleura/](http://www.genoscope.cns.fr/externe/GenomeBrowser/Oikopleura/).

### Repeat region analysis

A *de novo* repeat library was build for each tunicate genome using RepeatModeler (Smit and Hubley, 2015). This utilizes the RECON tandem repeats finder from the RepeatScout packages to identify species-specific repeats in a genome assembly. RepeatMasker (Smit et al., 2015) was then used to mask those repeats.

### Gene annotation

*Ab initio* genome annotation was performed using MAKER2 (Holt and Yandell, 2011) with Augustus (Stanke and Waack, 2003) and SNAP (Korf, 2004) for gene prediction. In addition, we used our previously published transcriptome (Zondag et al., 2016) and a concatenation of UniProtKB (UniProt Consortium, 2015), *C. intestinalis* and *B. schlosseri* proteins into a custom proteome as evidence of gene product. Using the predicted genes, Augustus and SNAP were then trained to the specificity of *B. leachii* genome. A second round of predictions was then performed, followed by a second round of training. The final annotation of the genome was obtained after running a third round of predictions, and the provided trained Augustus and SNAP configurations after a third round of training.

The following databases were used for homology identification: InterProScan (Jones et al., 2014) tRNA (Lowe and Eddy, 1997) snoRNA (Lowe, 1999) Infernal (Nawrocki and Eddy, 2013) Rfam (Nawrocki et al., 2015).

### Analysis of specific gene families

Genes and transcripts for each examined genome were identified by a tBLASTn search with an e-value cut-off at 10-5 using the SequencerServer software (Priyam et al., 2015). This was followed by a reciprocal BLAST using SMART blast ([http://blast.ncbi.nlm.nih.gov/smartblast/smartBlast.cgi?CMD=Web](http://blast.ncbi.nlm.nih.gov/smartblast/smartBlast.cgi?CMD=Web)) to confirm their identity.

Delta serrate ligand conserved protein domain (PF01414) was used to identify the corresponding proteins in tunicate genomes. To identify *Notch* receptor genes the conserved LNR (lin-notch repeat) domain (PF00066) was used. ALDH-like genes were identified by tBLASTn search (PF00171) and classified using SMART blast.

### Phylogenetics

Sequences were aligned with ClustalX (Jeanmougin et al., 1998) before using ProtTest 3 (Abascal et al., 2005) to determine the best-fit model of evolution. The best-fit model for the DSL phylogeny was WAG+I+G and, for CYP26 proteins, was LG+I+G.

Bayesian inference (BI) phylogenies were constructed by MrBayes (Ronquist and Huelsenbeck, 2003) with a mixed model for 100,000 generations and summarized using a Sump burnin of 200. Maximum Likelihood (ML) phylogenies were generated by PhyML (Guindon et al., 2010), using the appropriate model, estimating the amino acid frequences.

Accession numbers are provided in File S3 and sequence alignments are provided in Figure S3. Analyses carried out with BI and ML produced identical tree topologies. Trees were displayed using FigTree v1.4.2 [REF] ([http://tree.bio.ed.ac.uk/software/figtree/](http://tree.bio.ed.ac.uk/software/figtree/)).

## Acknowledgements

Funding support was provided to M.J.W. by the Otago BMS Deans Bequest and Department of Anatomy. S.B. by the Swiss National Science Foundation (SNSF) [grant number P2ELP3_158873]. We would like to thank Peter Maxwell and the New Zealand eScience Infrastructure (NeSI); Christelle Dantec and ANISEED for help and advice during the annotation process, as well as for the accompanying *B. leachii* genome browser.

*   Received June 21, 2017.
*   Revision received June 21, 2017.
*   Accepted June 21, 2017.


*   © 2017, Posted by Cold Spring Harbor Laboratory

The copyright holder for this pre-print is the author. All rights reserved. The material may not be redistributed, re-used or adapted without the author's permission.

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