DR. CHRIS JACKSON (Orcid ID : 0000-0001-5785-6465)

Article type : Regular Article

Phylogenetic position of the coral symbiont Ostreobium (Ulvophyceae) inferred from chloroplast genome data1

Heroen Verbruggen School of BioSciences, University of Melbourne, Melbourne, Victoria, 3010, Australia

Vanessa R. Marcelino School of BioSciences, University of Melbourne, Melbourne, Victoria, 3010, Australia

Michael D. Guiry AlgaeBase, Ryan Institute, National University of Ireland, Galway, H91 TK33, Ireland

M. Chiela M. Cremen School of BioSciences, University of Melbourne, Melbourne, Victoria, 3010, Australia

Christopher J. Jackson2 School of BioSciences, University of Melbourne, Melbourne, Victoria, 3010, Australia

1Received. Accepted. 2Author for correspondence: email [email protected] Running title: Ostreobium chloroplast phylogenomics Author Manuscript

This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jpy.12540-16-224

This article is protected by copyright. All rights reserved Editorial Responsibility: L. Graham (Associate Editor) Abstract The green algal genus Ostreobium is an important symbiont of corals, playing roles in reef decalcification and providing photosynthates to the coral during bleaching events. A chloroplast genome of a cultured strain of Ostreobium was available, but low taxon sampling and Ostreobium's early-branching nature left doubt about its phylogenetic position. Here we generate and describe chloroplast genomes from four Ostreobium strains as well as Avrainvillea mazei and Neomeris sp., strategically sampled early-branching lineages in the Bryopsidales and Dasycladales, respectively. At 80,584 bp, the chloroplast genome of Ostreobium sp. HV05042 is the most compact yet found in the Ulvophyceae. The Avrainvillea chloroplast genome is ca. 94 kbp and contains introns in infA and cysT that have nearly complete sequence identity except for an ORF in infA that is not present in cysT. In line with other bryopsidalean species, it also contains regions with possibly bacteria- derived ORFs. The Neomeris data did not assemble into a canonical circular chloroplast genome but a large number of contigs containing fragments of chloroplast genes and showing evidence of long introns and intergenic regions, and the Neomeris chloroplast genome size was estimated to exceed 1.87 Mb. Chloroplast phylogenomics and 18S nrDNA data showed strong support for the Ostreobium lineage being sister to the remaining Bryopsidales. There were differences in branch support when outgroups were varied, but the overall support for the placement of Ostreobium was strong. These results permitted us to validate two suborders and introduce a third, the Ostreobineae.

Key words: Bryopsidales, chloroplast genome, coral symbiosis, Dasycladales, Ostreobium, molecular phylogenetics, phylogenomics.

Abbreviations: bp, base pairs; kbp, kilo base pairs; mbp, mega base pairs; ORF, open reading frame; LBA, long branch attraction; ML, maximum likelihood.

Introduction

Ostreobium is an early-branching genus in the siphonous green algae (order Bryopsidales), a group

of seaweeds composedAuthor Manuscript of a single giant multinucleate cell (Vroom and Smith 2003). The genus is unusual among the Bryopsidales in being microscopic and endolithic, usually burrowing into marine limestone substrates. It is best known living in association with corals, forming a green layer in the skeleton underneath the living coral tissue (Verbruggen and Tribollet 2011). Ostreobium plays a major role in carbonate dissolution of live and dead corals, eroding as much as a kilogram of

This article is protected by copyright. All rights reserved reef carbonate per square meter per year (Tribollet 2008, Grange et al. 2015). It can also confer benefits to the coral hosts by providing them with photosynthates and protecting them from high- light stress (Schlichter et al. 1995, Inoue et al. 1997, Fine and Loya 2002).

Due to the importance of their ecological roles in reef ecosystems, endolithic algae have been studied quite intensively in recent years. One of the key findings of recent work is that the Ostreobium lineage, previously thought to be composed of only a handful of species, is in fact a complex of multiple undescribed lineages (Gutner-Hoch and Fine 2011, del Campo et al. 2016, Marcelino and Verbruggen 2016, Sauvage et al. 2016).

An earlier phylogenetic study of the siphonous green algae suggested that Ostreobium was sister to the two main suborders of the Bryopsidales: Bryopsidineae and Halimedineae (Verbruggen et al. 2009). This study, however, was based on only five genes, of which only three were sampled for Ostreobium, and maximum likelihood bootstrap support for its position was low (66% for the Bryopsidineae + Halimedineae grouping). Adding more data can help to resolve such phylogenetic uncertainties, with both gene and taxon sampling playing important roles. Further, it has been demonstrated that phylogenomic analysis with only a few taxa can yield high support values for the wrong tree topology, especially in cases where systematic bias is an issue (Hedtke et al. 2006). With the knowledge that Ostreobium is a complex of multiple lineages, we can now take advantage of that diversity by sequencing chloroplast genomes from multiple Ostreobium species, to reduce phylogenetic artifacts and more accurately infer the position of the genus among the siphonous green algae.

High-throughput sequencing methods permit addressing problematic phylogenetic relationships with increased sampling both of taxa and molecular markers. Chloroplast genome sequencing and phylogenomic analysis have been used to resolve, for example, difficult relationships among red algae (Costa et al. 2016), core Chlorophyta (Fučíková et al. 2014, Sun et al. 2016, Turmel et al. 2016), Trebouxiophyceae (Lemieux et al. 2014a) and Chlorophyceae (Turmel et al. 2008). The first complete chloroplast genomes of siphonous green algae, including Ostreobium quekettii, have been recently sequenced (Leliaert and Lopez-Bautista 2015, del Campo et al. 2016, Marcelino et al.

2016). While these provideAuthor Manuscript a good starting dataset to resolve the phylogenetic placement of the Ostreobium lineage, many early-branching lineages have not yet been sampled. This results in long branches which would otherwise be broken up by additional taxon sampling, leading to issues such as long branch attraction (LBA), a phylogenetic artifact in which long branches cluster together even if they are not closely related (Bergsten 2005).

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Here we report additional chloroplast genomes strategically obtained to break up the long branches in siphonous green algae in order to infer the phylogenetic position of the Ostreobium lineage and the early diversification of Bryopsidales. The goals of this study are: 1) to obtain draft chloroplast genomes of multiple Ostreobium strains and from two other early-branching families in the Bryopsidales (Dichotomosiphonaceae) and Dasycladales (Dasycladaceae), 2) infer the phylogenetic position of Ostreobium from chloroplast and nuclear sequences while quantifying uncertainty resulting from outgroup choice; and 3) make taxonomic changes where needed.

Materials and Methods

Sampling design – The main purpose of the sampling design was to avoid long branch attraction in the phylogenetic inference by adding taxa from poorly sampled groups. We therefore aimed to add Ostreobium species that were different from the previously sequenced one, an Avrainvillea species representing the early-branching family Dichotomosiphonaceae of the Bryopsidales, and a Neomeris species representing the Dasycladaceae, an early-branching Dasycladales family. The remaining available chloroplast genome sequences for Bryopsidales that were included in the datasets are listed in Table 1.

Choosing an appropriate outgroup was difficult because depending on taxon and gene sampling, different groups have been inferred to be the sister group of the Bryopsidales, usually with low support. To add to the problem, the class Ulvophyceae (to which the Bryopsidales belong) was non- monophyletic in chloroplast genome phylogenies (Fučíková et al. 2014, Leliaert and Lopez-Bautista 2015, Sun et al. 2016, Turmel et al. 2016) while nuclear data suggested monophyly (Cocquyt et al. 2010). We have chosen to use four outgroup lineages (Table 1) and have run analyses on various combinations of these (specified below).

Samples and cultures – Avrainvillea mazei HV02664 and Neomeris sp. HV02668 were collected from Tavernier (Florida, USA) and Islamorada (Florida, USA), respectively, and preserved in silica-gel. To obtain Ostreobium tissue, two fragments of coral skeleton were collected. Both were

taken from coral skeletonsAuthor Manuscript (Diploastrea heliopora) collected in the Kavieng area of Papua New Guinea. Samples HV05007 and HV05042 were collected at 4 and 13 m depth, respectively and both showed a pronounced green band in the skeleton. The fragments were kept in unenriched sterilized seawater at 24ºC and under dim light for several months until Ostreobium filaments started growing

This article is protected by copyright. All rights reserved out of the limestone. When the filaments reached ca. 1 cm, they were harvested for DNA extraction. We did not attempt to establish pure cultures.

Molecular work and DNA sequencing – Total genomic DNA was extracted using a modified CTAB protocol (Cremen et al. 2016). Two types of library preparation and sequencing were used. For Avrainvillea mazei HV02664, a library was prepared from ca. 350 bp fragments using a TruSeq Nano LT kit and sequenced on the Illumina HiSeq 2000 platform (paired end, 100 bp). For the remaining strains, libraries were prepared from a ca. 500 bp size fraction with a Kapa Biosystems kit and sequenced on the Illumina NextSeq platform (paired end, 150 bp).

Assembly and annotation – Sequence reads from both Ostreobium strains were assembled using IDBA-UD 1.1.1 (Peng et al. 2012), SPAdes 3.8.1 (Bankevich et al. 2012), CLC Genomics Workbench 7.5.1 (http://www.clcbio.com) and MEGAHIT 1.0.6 (Li et al. 2016). Contigs matching to Ulvophycean chloroplast genomes reference sequences were imported into Geneious 8.0.5 (http://www.geneious.com), where completeness and circularity were evaluated by comparing the contigs from different assemblers manually. Assembly graphs were visually assessed in Bandage 0.8.0 (Wick et al. 2015) and read mapping was carried out in Geneious 8.0.5.

Because one of the limestone samples turned out to contain multiple Ostreobium species, metagenome binning was carried out to separate the contigs from different species. These analyses were carried out on scaffolds larger than 13kb resulting from the SPAdes assembly and used MyCC (Lin and Liao 2016) and MaxBin 2.2 (Wu et al. 2014).

Contigs were annotated following Verbruggen and Costa (2015) and Marcelino et al. (2016). In brief, annotations were obtained from MFannot, DOGMA and ARAGORN and imported into Geneious. All annotations were vetted manually and a master annotation track was created from them. We looked for open reading frames (0s) of potential bacterial origin in Ostreobium by performing a tblastx search using bacteria-derived ORFs from chloroplast genomes of other Bryopsidales as a query (Leliaert and Lopez-Bautista 2015). The 18S sequences were identified by creating a BLAST database of contigs and querying it with known Ulvophyceae 18S sequences.

Author Manuscript Phylogenetic inference – Alignments of each named chloroplast protein-coding gene were inferred with TranslatorX (Abascal et al. 2010), which translates sequences to amino acids, uses MAFFT 7.215 (Katoh and Toh, 2010) to align the amino acid sequences and generates the corresponding nucleotide alignments. A few genes were excluded either because they were present in only a

This article is protected by copyright. All rights reserved handful of species or because they could not be aligned reliably: rpoA, rpoB, rpoC1, rpoC2, ftsH, tilS, ycf1, ycf12, ycf62. Alignments of the 18S gene was inferred with MAFFT using default settings. Three concatenated gene alignments were created: one of the nuclear ribosomal genes (nrDNA) and two of the chloroplast genes at the nucleotide level (cpDNA) and the amino acid level (cpPROT). All alignments are available in Appendix S1 in the Supporting Information.

In order to evaluate the robustness of results to variations in outgroup choice, six different configurations of outgroup lineages were used. The first (abbreviated o1) used only the first outgroup lineage, the Dasycladales, which were indicated to be related to Bryopsidales in nuclear and chloroplast phylogenies (e.g., Cocquyt et al. 2010, Fučíková et al. 2014). Strategy o2 used only the members of the Ulvales-Ulotrichales lineage, another group of ulvophycean algae. The third strategy (o3) used only Chlorodendrophyceae. Despite being a different class, this group was inferred as the sister to Bryopsidales in the ML analysis of Turmel et al. (Turmel et al. 2016), though with low support. Strategy o12 used a combination of outgroups 1 and 2 (Dasycladales and Ulvales-Ulotrichales). Strategy o123 expanded o12 by adding the Chlorodendrophyceae. Finally, strategy o1234 used all four outgroup lineages. The fourth group (Pedinophyceae) is early- branching in the core Chlorophyta and thus serves as an outgroup to all the remaining taxa in our dataset (e.g., Turmel et al. 2016).

At the nucleotide level, analyses were carried out with a simple GTR+Γ model (unpartitioned, labeled -u- in the code column of Table 2) and with a separate GTR+Γ model for each codon position (partitioned, labeled -p- in Table 2). At the amino acid level, analyses were carried out with the LG and the (better-fitting) cpREV model, and with Γ among-site rate variation and estimated AA frequencies. Because of the extensive analysis design with multiple alignments, multiple models of sequence evolution and multiple outgroup strategies (30 analysis types in total), computational time constraints led us to limit ourselves to maximum likelihood analyses. We used RAxML 8.2.6 with 500 rapid bootstrap replicates (Stamatakis 2014).

Results

Author Manuscript The sequences of the green algal tissue obtained from the rough culture of limestone sample HV05042 yielded a complete Ostreobium chloroplast genome assembled into a single contig by SPAdes and CLC Genomics, with 1,284× average coverage. We confirmed that this molecule is circular-mapping (Fig. 1). The sequence was completely collinear with the genome of the

This article is protected by copyright. All rights reserved previously published strain SAG6.99 (Fig. 1), but there were considerable rearrangements with species from other families in the order Bryopsidales (e.g., Bryopsis, Tydemania; Fig. 1). All Ostreobium chloroplast genomes have a simple structure without the inverted repeats often found in land plants and green algae. As previously described for Ostreobium SAG6.99 (Marcelino et al. 2016), the Ostreobium HV05042 chloroplast genome is a compact and gene-dense, highly economical genome. At 80,584 bp long, the HV05042 genome is even smaller than that of SAG6.99 (81,997 bp). Similar to other green algae, the chloroplast genome has a low GC content (30.9%).

Assembly of sample HV05007 was substantially less straightforward. Initial assemblies showed several ca. 20kb long contigs with similarity to Ostreobium chloroplast genome sequences. These contigs also showed that three distinct variants of some genome regions were present among the contigs. This indicates that the limestone sample contained three different Ostreobium species, which we will refer to as HV05007a, b and c. Metagenome binning based on k-mer profiles (MyCC) and a combination of k-mer and coverage profiles (MaxBin) separated out the contigs belonging to one of the species in a separate cluster (HV05007a, average coverage 913×), but the sequences of the remaining two species were recovered in a single cluster (average coverage 40- 49×). The assembly failed across some highly conserved RNA-coding regions: rns (16S), rnl (23S), rnpB (RNAse P) and a region with three consecutive tRNAs (trnF-GAA, trnL-CAA, trnQ-TTG). The recovered contigs were completely collinear with those of SAG6.99 and HV05042.

Despite a high average sequencing depth of 1,053×, the chloroplast genome of Avrainvillea mazei HV02664 assembled into three contigs of 55.6, 23.4 and 13.9 kbp. Fragments of atpI were present near the edges of two contigs, suggesting that these contigs were adjacent. The same was true for the cysT gene near different contig edges. The assembly graph (Fig. 2A) indicated that indeed, there are paths connecting these fragments of cysT and atpI (region 1 in Fig. 2A, detailed in Fig. 2B). The graph indicated a shared path with approximately double sequencing depth connecting the gene fragments of both cysT and atpI (Fig. 2B), also containing a bubble of single sequencing depth of which the longer path contained an ORF. By mapping paired reads where which one read matched the adjacent coding sequence and the other extended the contig, we were able to resolve this region

unambiguously. Both Author Manuscript atpI and cysT have introns that are virtually identical in sequence for the first 518 bp and the last 90 bp (Fig. 2C). They differ in an ORF being present in the atpI intron and a 26 bp region with minor sequence differences in the non-coding part (arrowhead near 200 bp mark in Fig. 2C). Another area of the assembly graph showed a trifurcation near the psbE gene (region 2 in Fig. 2A, detailed in Fig. 2D). Sequence fragments showing similarity the psbE gene were found on

This article is protected by copyright. All rights reserved all three of the connected nodes. Using manual mapping of paired reads, we were able to establish that in addition to the canonical psbE gene at the end of node 2797, there is a second, more deviant copy of this gene spanning nodes 2798 and 2796. Read mapping also showed conclusively that the path from 2797 to 2798 (skipping 2796) is not supported by paired reads. Instead, this exercise confirmed that node 2798 continues into 2796, which contains a few ORFs with no similarity to known genes. We were not able to establish whether node 2797 connects to this structure elsewhere, and hence we cannot confirm circularity of the Avrainvillea chloroplast genome. The last area in the graph where multiple nodes were connected to one another (region 3 in Fig. 2A) was resolved by the assembler and did not require manual attention.

Our final, manually created scaffold for Avrainvillea mazei HV02664 is nearly 94 kbp long, and assuming that it serves as an approximation of the entire chloroplast genome, this puts the species at the lower end of the size scale for Bryopsidales and Ulvophyceae more generally. Among currently sequenced chloroplast genomes, only those of Ostreobium species are smaller. Most other species have chloroplast genomes exceeding 100 kbp. At 27.4%, its GC content is lower than that of Ostreobium, probably due to Avrainvillea having more intergenic regions of low GC content. The genome has a similar gene repertoire than other members of the Bryopsidales, with only cemA, petL, psbT and ycf47 apparently missing. Like some other Bryopsidales, the Avrainvillea chloroplast genome has two regions containing multiple ORFs that do not code for typical chloroplast genes (regions 2 and 3 in Fig. 2A).

Our whole chloroplast genome alignments of Ostreobium and Avrainvillea with other members of the Bryopsidales (Figs. 1, S1 in the Supporting Information) show that while there are many rearrangements within the order, there are also a number of regions with conserved gene order: (1) an atpA-atpF-atpH-atpI operon is conserved across the Bryopsidales, (2) a chlB-psaA-psaB-psbZ- tilS-psbD-psbC cluster is found in Bryopsidineae, Ostreobineae and Avrainvillea but is lost in the remaining Halimedineae, (3) ccsA-cysA-psbB-psbT-psbH is present in all three suborders, and (4) rpoA-rps11-rpl36-infA-rps8-rpl5-rpl14-rpl16-rps3-rps19-rpl2-rpl23 is conserved across the order.

The Neomeris sp. HV02668 assembly failed to recover a chloroplast genome. Instead, the assembly

consisted of a large numberAuthor Manuscript of contigs containing fragments of what appeared to be chloroplast genes. The coverage of these contigs was in the 180–280× range. Using metagenome binning, we isolated a group of 383 sequences ranging from 1,024 to 59,641 in size that were considered candidate chloroplast contigs. Using ORF identification and BLASTP searches followed by

This article is protected by copyright. All rights reserved alignment of Bryopsis or Acetabularia sequences, we identified 49 chloroplast genes, the larger ones often containing introns.

From the newly assembled sequences and sequences downloaded from Genbank, we created gene alignments that were subsequently concatenated into a single alignment. The chloroplast data alignment comprised 25 species, 13 of which were ingroups. It consisted of 71 concatenated protein-coding genes and was 48,651 bases (cpDNA) or 16,217 amino acids (cpPROT) long. The 18S alignment had 2,054 positions and included 36 species in total (16 ingroup species).

The phylogenies recovered from the concatenated chloroplast gene datasets were very well resolved throughout (Figs. 3, S2 in the Supporting Information), with a few exceptions that are highlighted below. All Ostreobium strains were recovered in a single clade with 100% support irrespective of the analysis method. The phylogenies also show that Ostreobium quekettii strain SAG6.99 (Marcelino et al. 2016) and strain OS1B (del Campo et al. 2016) are identical, and that the same is true of two of our new strains (HV05042 and HV05007a). The remaining two strains (HV05007b & c) were sister to the SAG6.99 + OS1B lineage, but the relationships among these taxa were not confidently resolved.

The phylogenetic position of Ostreobium in the Bryopsidales, which was the main objective of this study, was also recovered with high support. Our analyses showed the Ostreobium clade branching off at the base of the Bryopsidales, leaving the Bryopsidineae and Halimedineae suborders as sister clades (Fig. 3). The choice of outgroup, use of nucleotide or amino acid sequences, and the use of different models had an effect on the bootstrap support values (Table 2; Fig. S2). Specifically, bootstrap support for the relationship above reduced considerably when the Chlorodendrophyceae were used as the outgroup (strategy o3, Table 2). In these analyses, the topology in which Ostreobium and the Halimedineae formed a sister clade gained support. In one analysis (cpDNA-u- o3, i.e., chloroplast DNA, unpartitioned model, using only Chlorodendrophyceae as outgroup), the ML analysis even preferred the sister relationship between Ostreobium and the Halimedineae in favor of the more common topology where Ostreobium branches off at the base of the Bryopsidales. Analyses based on amino acid sequences agreed with those on DNA data and

bootstrap support valuesAuthor Manuscript were similar in magnitude, although for analyses with only Dasycladales as outgroup (o1), the bootstrap support for the position of Ostreobium was about 10 units lower for amino acid-based analyses than for DNA-based analyses (Table 2).

This article is protected by copyright. All rights reserved In line with previous findings, Avrainvillea formed an early-branching lineage within the Halimedineae, sister to a well-supported cluster containing Caulerpa, Halimeda and Tydemania (Fig. 2). The phylogenetic trees that included all outgroups (configuration o1234), where the Pedinophyceae are used as a more distant outgroup, showed that the Dasycladales and Bryopsidales always grouped together with high support (Fig. S2).

Like the phylogenies of plastid genomes, the analyses of the nuclear 18S rRNA gene recovered the Ostreobium lineage at the base of the Bryopsidales, irrespective of which outgroup was used (Figs. 4, S2). Support for the Bryopsidineae+Halimedineae clade was 100% in all analyses (Table 2).

Discussion

Genome features

The 80,584 bp chloroplast genome of Ostreobium strain HV05042 is the smallest currently known in the core Chlorophyta, most of which have chloroplast genomes exceeding 100 kbp (Turmel et al. 2015, Marcelino et al. 2016, Turmel et al. 2016). It is ca. 1,400 bp shorter than the chloroplast genome of Ostreobium strains SAG 6.99 and OS1B. It is completely collinear with these genomes, and the size differences are due to indels in intergenic regions across the genome. The small genome size of Ostreobium has been attributed in part to resource limitation due to its low-light habitat (Marcelino et al. 2016). In the Chlorophyta more broadly, smaller genomes are common especially among the prasinophytes ( Robbens et al. 2007, Lemieux et al. 2014b, Leliaert et al. 2016).

The chloroplast genomes of Ostreobium are structurally similar to those of other Bryopsidales. They are circular-mapping and lack the large inverted repeats seen in many other green algae (Manhart et al. 1989, Lehman and Manhart 1997, Leliaert and Lopez-Bautista 2015). The fact that the early-branching Ostreobium lineage also lacks the large inverted repeats suggests that they were lost at the base of the Bryopsidales or even earlier during evolution. The Dasycladales, probably the

most closely related orderAuthor Manuscript to Bryopsidales (Cocquyt et al. 2010, Fučíková et al. 2014, Sun et al. 2016), are estimated to have extremely large chloroplast genomes (> 1 Mbp) that have not yet been sequenced. From restriction enzyme and DNA hybridization experiments, Acetabularia chloroplast genomes are thought to contain large repeats (8-10 kbp) but these are unlikely to be the canonical inverted repeats found in other green algae (Tymms and Schweiger 1985, Tymms and Schweiger

This article is protected by copyright. All rights reserved 1989). The chloroplast genomes of two other closely related orders, Cladophorales and Trentepohliales, have also not been sequenced or characterized in detail (but see La Claire et al. 1997, La Claire et al. 1998, La Claire and Wang 2004). The Ulvales–Ulotrichales lineage, which is more distantly related in phylogenies of nuclear genes (Cocquyt et al. 2010), has species with and species without large inverted repeats (Pombert et al. 2005, Pombert et al. 2006, Melton et al. 2015).

Our approach of sequencing rough cultures (i.e., cultured field samples without strain isolation) yielded one complete chloroplast genome (HV05042) but assembly was much less straightforward for culture HV05007, which contained multiple strains. Here, the assemblers were unable to span across several highly conserved RNA-coding regions, resulting in four contigs per species of Ostreobium present in the mixed culture. These highly conserved regions would be almost identical between the three species and thus are obviously difficult for assemblers to handle using short read data. Metagenome binning permitted us to separate the four contigs of HV05007a from the remaining eight contigs of HV05007b & c, but the latter could not be separated out further. As an alternative approach to bin the contigs into species, we attempted mapping read pairs across the ends of adjacent contigs, but the RNA-coding regions were too long for this approach to work with our 500 bp paired-end library. Based on the information that can be derived from the contigs, all Ostreobium chloroplast genomes appear to be completely collinear. Completing the remaining Ostreobium genomes – either by isolating strains or performing long-read or mate-pair sequencing – would be interesting but was not needed to achieve the goals of our study.

Horizontal gene transfer from bacteria to chloroplast genomes is not common but Bryopsis and Tydemania both have regions larger than 6 kb containing several ORFs of putative bacterial origin (Leliaert and Lopez-Bautista 2015). We did not find such regions in Ostreobium, which may have either lost them during its genome streamlining process (Marcelino et al. 2016) or, alternatively, Bryopsis and Tydemania may have acquired these regions later in their evolution. The fact that Derbesia also lacks these regions (Marcelino et al. 2016) would support the latter. Halimeda, Caulerpa and Avrainvillea, by contrast, have multiple stretches of consecutive uncharacterized ORFs (Marcelino et al. 2016), which may or may not be of bacterial origin.

Author Manuscript For Neomeris, proper chloroplast genome assemblies were clearly not achievable based on short- read sequencing alone. While several fairly long contigs were obtained, these typically contained only one gene, and all of the longer genes were interrupted by long introns and sometimes spread across multiple contigs. The situation in Neomeris is similar to that in Acetabularia, where high-

This article is protected by copyright. All rights reserved throughput sequencing also failed to recover a complete sequence (de Vries et al. 2013). Metagenome binning of our Neomeris data suggested that the chloroplast data had a quite distinct kmer signature, and the size of the corresponding bin, which can serve as a conservative estimate of genome size, was 1.87 Mb. This is drastically larger than canonical green algal chloroplast genomes (typically in the 100-200 kbp range) but in line with previous work using restriction patterns showing that Dasycladales chloroplast genomes are large and repeat-rich (Lüttke and Bonotto 1982, Tymms and Schweiger 1985, Tymms and Schweiger 1989). Obtaining complete sequences of these genomes will require long-read sequencing, perhaps supplemented with optical genome mapping.

Phylogenetic relationships

While previous molecular analyses have shown uncertainty in the placement of Ostreobium with respect to the other main lineages of siphonous green algae (Verbruggen et al. 2009, del Campo et al. 2016), our approach based on chloroplast phylogenomics offers a clear, well-supported solution in which the Ostreobium lineage branches at the base of the Bryopsidales, leaving the Bryopsidineae and the Halimedineae as sister lineages. Our taxon sampling allowed us to divide up several long branches, including that of Ostreobium itself (by using multiple strains instead of one), the branch leading to the Halimedineae (by including Avrainvillea) and that to the closest outgroup taxon Acetabularia (by adding Neomeris). It is well known that systematic bias can occur in phylogenetic inference when taxon sampling is sparse and that genome-scale datasets can provide strong support for the wrong tree (e.g., Hedtke et al. 2006). By strategically sampling early- branching taxa, the likelihood of these artifacts are reduced (see Verbruggen and Theriot 2008 and citations therein), and we can be quite confident of the position of Ostreobium. Moreover, while G- C content heterogeneity and codon usage bias can cause phylogenetic artifacts (e.g., Conant and Lewis 2001), this did not appear to be an issue here as our results were consistent when using both DNA or amino-acid datasets. The 18S gene, which could also be recovered from among the contigs resulting from our HTS data, provides a convenient independent confirmation of the position of Ostreobium at the base of the Bryopsidales.

Any remaining uncertainty in the position of the Ostreobium lineage was a consequence of

outgroup choice, withAuthor Manuscript especially the o3 rooting strategy (Chlorodendrophyceae only) showing increased support for an alternative topology with Ostreobium sister to Halimedineae. The effect was mostly limited to support values and only changed the ML topology in one instance. Interestingly, this alternative topology was also recovered with statistical support in an analysis of only the 16S gene incorporating a broader sampling of green algal taxa (del Campo et al. 2016), but

This article is protected by copyright. All rights reserved our in-depth analyses show that this alternative topology is poorly supported overall. Our results show a moderate effect of outgroup sampling on phylogenetic tree inference. In this context, our inclusion of Neomeris broke up the long branch leading to Acetabularia that was seen in previous studies (Fučíková et al. 2014, Sun et al. 2016). Particularly in rooting phylogenetic trees, a proper consideration of taxon sampling is important because adding outgroups can introduce long branches in the phylogeny, potentially making the analysis vulnerable to systematic biases (Shavit et al. 2007, Verbruggen et al. 2007, Verbruggen and Theriot 2008, Goremykin et al. 2013). With high- throughput sequencing now being mainstream, further improvements in taxon sampling in green algal chloroplast phylogenomics can be expected in the coming years.

Within the Ostreobium lineage, there is clear evidence for two sublineages that separate from each other fairly early in the evolution of the lineage (split between HV05007a + HV05042 and the remaining Ostreobiums). In fact, recent studies based on environmental sequencing show that there are up to four or even five of these lineages that deserve recognition at the family level (del Campo et al. 2016, Marcelino and Verbruggen 2016, Sauvage et al. 2016). When the tufA sequences from the chloroplast genomes generated here are compared to those of the family-level lineages from Marcelino and Verbruggen (2016), they belong to Ostreobium lineages 3 (HV05007a and HV05042) and 4 (HV05007b & c, SAG6.99, OS1B). Within the Ostreobium lineage, there is poor support for the placement of HV05007b & c, two species that were sequenced from a single rough culture. While it is certainly possible that chloroplast genome sequences do not allow for resolution in this part of the tree, we should also consider the alternative possibility that the poor support is due to misassembly. HV05007b & c are relatively closely related Ostreobium strains with chloroplast genomes that are highly similar in kmer composition and coverage. It is therefore not unimaginable that because they were sequenced and assembled from a single DNA pool, chimeric assemblies may have resulted. Such erroneous assemblies would introduce conflicting signals within the data and result in poorly resolved phylogenies. It is also possible that the reduced bootstrap support is simply a consequence of the use of only the genes on a shorter fragment (contig of ca. 14 kb) for these two species.

Within the remaining Bryopsidales, the relationships among taxa matched well with those of

previous studies basedAuthor Manuscript on richer taxon sampling but fewer genes (Curtis et al. 2008, Verbruggen et al. 2009). In the analyses including the Pedinophyceae as a distant outgroup, the Dasycladales (Neomeris + Acetabularia) and Bryopsidales consistently grouped together, suggesting that these two siphonous orders form a natural group, in accordance with previous studies based on nuclear and chloroplast sequences (Cocquyt et al. 2010, Sun et al. 2016). A caveat in this conclusion is that

This article is protected by copyright. All rights reserved our taxon sampling does not include other closely related lineages such as Trentepohliales and Cladophorales.

Taxonomic treatment

The strongly supported phylogenetic position of the Ostreobium lineage inferred here implies that it should not be included in either of the two currently recognized suborders Halimedineae and Bryopsidineae. Therefore, we propose a new suborder for the Ostreobium lineage.

Ostreobineae Verbruggen et Guiry, subordo nov. Description: Siphonous green algae endolithic in limestone substrata, developing irregularly ramifying nets of siphons in the rock matrix; represented as a distinct lineage in phylogenies based on chloroplast genome data, with the genome of culture strain SAG 6.99 serving as a reference (Genbank accession LT593849).

Automatically typified with the genus Ostreobium Bornet and Flahault 1889.

Note: In accordance with ICN Art.10.7 (McNeill et al. 2012,), “the principle of typification does not apply to names of taxa above the rank of family, except for names that are automatically typified by being based on generic names, the type of which is the same as that of the generic name.”

Included family: Ostreobiaceae P.C.Silva ex Maggs & J.Brodie (Brodie et al. 2007).

Note: The original description of the Ostreobiaceae (Silva 1982) was invalid as it lacked the Latin diagnosis or description required from 1 January 1958 to 31 December 2011 (McNeill et al. 2012, Art. 39.1). In previous studies (Verbruggen et al. 2009, Marcelino and Verbruggen 2016, Sauvage et al. 2016), the Ostreobium lineage was casually referred to as "Ostreobidineae", however, this name is orthographically incorrect. According to the rules of the ICN (McNeill et al. 2012, Art. 16.3 and 17.1), a suborder-level termination (-ineae) should be added to the stem of the genitive of the

genus name. The genitiveAuthor Manuscript of Ostreobium, a second-declension neuter noun is Ostreobii, hence the correct orthography is Ostreobineae.

The other two suborder names of the Bryopsidales, Halimedineae and Bryopsidineae, were introduced by Hillis-Colinvaux (Hillis-Colinvaux 1984) but invalidly as they lacked the Latin

This article is protected by copyright. All rights reserved diagnosis or description required at the time of publication (McNeill et al. 2012, Art. 39.1). Accordingly, we validate them here, giving due credit to Hillis-Colinvaux as the originator of the names.

Halimedineae Hillis-Colinvaux ex Verbruggen et Guiry, nom. nov. Description: Siphonous green algae featuring heteroplasty (chloroplasts and amyloplasts), holocarpic reproduction without septa at base of reproductive structures; with a concentric lamellar system in the chloroplasts.

Automatically typified by Halimeda J.V.Lamouroux 1812, nom. et typ. cons.

Included families: Caulerpaceae, Dichotomosiphonaceae, Halimedaceae, Pseudocodiaceae, Rhipiliaceae and Udoteaceae.

Bryopsidineae Hillis-Colinvaux ex Verbruggen et Guiry, nom. nov. Description: Siphonous green algae with homoplasty (chloroplasts only), non-holocarpic reproduction with septa commonly present at base of reproductive structures; concentric lamellar system in the chloroplasts absent.

Automatically typified by Bryopsis J.V.Lamouroux 1809

Included families: Bryopsidaceae, Codiaceae and Derbesiaceae.

As the descriptions of these two suborders suggest, there are some subcellular features that distinguish between them, in particular the presence/absence of the "thylakoid organizing body", a concentric lamellar system situated at one end of the chloroplasts (Borowitzka 1976), the presence/absence of separate amyloplasts and whether or not septa are present at the base of reproductive structures (Hillis-Colinvaux 1984). At the moment it is difficult to discuss how the subcellular features of the Ostreobineae relate to either of the other suborders because not enough work has been carried out. The genus Ostreobium has not yet been studied with transmission

electron microscopy, soAuthor Manuscript we cannot comment on the structure of its lamellar system. Amyloplasts have not been reported previously, so it is likely that the suborder aligns with Bryopsidineae in that respect. Kornmann and Sahling (1980) reported reproductive structures in Ostreobium quekettii that did not have septa at their base, suggesting they align with Halimedineae in this feature. However, we cannot extrapolate from these results because we do not currently know whether the strain

This article is protected by copyright. All rights reserved studied by Kornmann and Sahling (1980) is actually a true Ostreobium. Environmental sequencing has shown that, besides the Ostreobium clade, there are two additional groups of endolithic siphonous green algae nested within the Halimedineae (Marcelino and Verbruggen 2016, Sauvage et al. 2016). These groups have not been characterized morphologically but are likely to be similar to Ostreobium in appearance despite having separate evolutionary origins. As a consequence, without knowing of the molecular affinities of strains, it is impossible to say whether the observed features are valid for the Ostreobineae, and more work is needed on characterizing the biology and subcellular features of this suborder.

To conclude, we note that our chloroplast genome-based phylogeny offers good support at all levels in our phylogeny. When all steps in the phylogenetic hierarchy are resolved, it is possible to construct a similarly resolved hierarchical classification, including levels in between the traditional levels. In our case, families could be organized into well-defined suborders. A similar situation was shown in a phylogenomic study of the red algal order Nemaliales (Costa et al. 2016). At a shallower taxonomic level, a chloroplast genome-based phylogenetic study of the highly diverse red algal family Rhodomelaceae (Díaz Tapia et al. 2015) allowed the organization of genera into well- supported tribes. While these studies clearly illustrate the potential of chloroplast phylogenomics to help algal taxonomists construct a fine-grained hierarchical classification, it is unlikely to be a silver bullet for all levels of classification and for all algal taxa. In rapidly diversifying groups, the generation of many evolutionary lineages over a short time scale may not leave enough of a genomic imprint to resolve their relationships. The origination of the classes and orders of the core Chlorophyta may be an example of this. Chloroplast genome-based phylogenies have not been able to resolve their relationships despite impressive taxon sampling and extensive experimentation with inference methods (e.g., Fučíková et al. 2014, Lemieux et al. 2014a, Sun et al. 2016, Turmel et al. 2016).

Acknowledgements

This work was supported by the Australian Biological Resources Study (RFL213-08), the Australian Research Council (FT110100585, DP150100705), the University of Melbourne (ECR

and SPRINT grants toAuthor Manuscript HV and MIRS/MIFRS to VRM and MCMC), and by use of the Victorian Life Sciences Computation Initiative (VLSCI) at the University of Melbourne (projects UOM0007, UOM0021) and the Nectar Research Cloud, a collaborative Australian research platform supported by the National Collaborative Research Infrastructure Strategy (NCRIS). We thank Claude Payri, the staff of the Alis research vessel and the La Planète Revisitée program for facilitating fieldwork

This article is protected by copyright. All rights reserved in Papua New Guinea. We are very grateful to Francesca Benzoni for providing coral identifications, Fabio Rindi for contributing his insights in the Latin language, and Laura Lagourgue, Craig Schneider, Michael Wynne and Sigrid Berger for help with the identifications of algal samples. The authors declare no conflict of interest.

Table 1. Strains and sequences used in this study. Nomenclature follows AlgaeBase (Guiry and Guiry 2016). category strain species source sequencing Genbank Ostreobium SAG 6.99 Ostreobium quekettii (Marcelino et al. 2016) NextSeq PE-150 LT593849 OS1B Ostreobium sp. (del Campo et al. 2016) HiSeq PE-100 KU979013

HV05007a Ostreobium sp. this study NextSeq PE-150 KY509311 KY509315 KY509312

HV05007b & c Ostreobium sp. this study NextSeq PE-150 KY766991- KY766996

HV05042Author Manuscript Ostreobium sp. this study NextSeq PE-150 KY509314 Other Bryopsidales HV02664 Avrainvillea mazei this study HiSeq 2000 PE-100 KY509313 HV04923 Halimeda discoidea (Marcelino et al. 2016) HiSeq 2000 PE-100 KX808496 HV03798 Caulerpa cliftonii (Marcelino et al. 2016) HiSeq 2000 PE-100 KX808498 WEST4838 Derbesia sp. (Marcelino et al. 2016) HiSeq 2000 PE-100 KX808497 N/A Bryopsis hypnoides (Lü et al. 2011) Sanger NC_013359 WEST4718 Bryopsis plumosa (Leliaert and Lopez- HiSeq 2000 PE-100 NC_026795 Bautista 2015)

This article is protected by copyright. All rights reserved FL1151 Tydemania expeditionis (Leliaert and Lopez- HiSeq 2000 PE-100 NC_026796 Bautista 2015) Outgroup 1 DI1 Acetabularia acetabulum (de Vries et al. 2013) 454 GS-FLX http://goo.gl/00Ni4a (Dasycladales) HV02668 Neomeris sp. this study NextSeq PE-150 KY495826 - KY495874

Outgroup 2 UNA00071828 Ulva sp. (Melton et al. 2015) HiSeq 2000 PE-100 KP720616 (UU clade) UNA00071832 Ulva fasciata (Melton and Lopez- MiSeq KT882614 Bautista 2015) QD08 Ulva linza unpublished unknown KX058323 UTEX 1912 Pseudendoclonium akinetum (Pombert et al. 2005) Sanger AY835431 NIES 360 Oltmannsiellopsis viridis (Pombert et al. 2006) Sanger DQ291132 Outgroup 3 SAG 17.86 Scherffelia dubia (Turmel et al. 2016) Sanger NC_029807 (Chlorodendrophyceae) CCMP 881 Tetraselmis sp. (Turmel et al. 2016) Sanger KU167097 Outgroup 4 SAG 42.84 Pedinomonas tuberculata (Lemieux et al. 2014a) 454 GS-FLX NC_025530 (Pedinophyceae) UTEX LB 1350 Pedinomonas minor (Turmel et al. 2009) Sanger NC_016733 NIES 1824 Marsupiomonas sp. (Lemieux et al. 2014a) 454 GS-FLX KM462870

Table 2. Summary of phylogenetic analyses and the placement of the Ostreobium lineage. codea genome DNA/protein Model outgroupsb ML topologyc (O,(B,H)) support (B,(O,H)) support (H,(O,B)) support cpDNA-p-o1 chloroplast DNA GTR+Γ, part. o1 (O,(B,H)) 98.0 2.0 0.0 cpDNA-p-o2 chloroplast DNA GTR+Γ, part. o2 (O,(B,H)) 95.0 4.8 0.2 cpDNA-p-o3 chloroplast DNA GTR+Γ, part. o3 (O,(B,H)) 51.2 48.8 0.0 cpDNA-p-o12 chloroplast DNA GTR+Γ, part. o12 (O,(B,H)) 96.4 3.6 0.0 cpDNA-p-o123 chloroplast DNA GTR+Γ, part. o123 (O,(B,H)) 95.8 4.2 0.0 cpDNA-p-o1234 chloroplast DNA GTR+Γ, part. o1234 (O,(B,H)) 86.4 13.6 0.0 cpDNA-u-o1 chloroplast DNA GTR+Γ o1 (O,(B,H)) 100.0 0.0 0.0 cpDNA-u-o2 chloroplast DNA GTR+Γ o2 (O,(B,H)) 89.4 10.6 0.0 cpDNA-u-o3 chloroplast DNA GTR+Γ o3 (B,(O,H)) 33.4 66.6 0.0 cpDNA-u-o12 chloroplast DNA GTR+Γ o12 (O,(B,H)) 97.4 2.6 0.0 cpDNA-u-o123 chloroplast DNA GTR+Γ o123 (O,(B,H)) 96.2 3.8 0.0 cpDNA-u-o1234 chloroplast DNA GTR+Γ o1234 (O,(B,H)) 99.0 1.0 0.0 cpPROT-CPREV-o1 chloroplast protein CPREV+F+Γ o1 (O,(B,H)) 89.2 9.6 1.2 cpPROT-CPREV-o2 chloroplast protein CPREV+F+Γ o2 (O,(B,H)) 98.6 0.2 1.2 cpPROT-CPREV-o3 chloroplast protein CPREV+F+Γ o3 (O,(B,H)) 60.8 38.2 1.0 cpPROT-CPREV-o12 chloroplast protein CPREV+F+Γ o12 (O,(B,H)) 98.4 0.6 1.0 cpPROT-CPREV-o123 chloroplast protein CPREV+F+Γ o123 (O,(B,H)) 99.2 0.6 0.2 cpPROT-CPREV-o1234 chloroplast protein CPREV+F+Γ o1234 (O,(B,H)) 96.6 2.2 1.2 cpPROT-LG-o1 chloroplast protein LG+F+Γ o1 (O,(B,H)) 91.4 8.0 0.6 cpPROT-LG-o2 chloroplast protein LG+F+Γ o2 (O,(B,H)) 98.4 0.0 1.6 cpPROT-LG-o3 chloroplast protein LG+F+Γ o3 (O,(B,H)) 54.6 43.2 2.2 cpPROT-LG-o12 chloroplast protein LG+F+Γ o12 (O,(B,H)) 97.8 1.2 1.0 cpPROT-LG-o123 chloroplast protein LG+F+Γ o123 (O,(B,H)) 98.2 1.6 0.2 cpPROT-LG-o1234 chloroplast protein LG+F+Γ o1234 (O,(B,H)) 95.2 3.4 1.4

nrDNA-u-o1 nuclear Author Manuscript DNA GTR+Γ o1 (O,(B,H)) 100.0 0.0 0.0 nrDNA-u-o2 nuclear DNA GTR+Γ o2 (O,(B,H)) 100.0 0.0 0.0 nrDNA-u-o3 nuclear DNA GTR+Γ o3 (O,(B,H)) 100.0 0.0 0.0 nrDNA-u-o12 nuclear DNA GTR+Γ o12 (O,(B,H)) 100.0 0.0 0.0 nrDNA-u-o123 nuclear DNA GTR+Γ o123 (O,(B,H)) 100.0 0.0 0.0 nrDNA-u-o1234 nuclear DNA GTR+Γ o1234 (O,(B,H)) 100.0 0.0 0.0

This article is protected by copyright. All rights reserved a – Codes with the label -p- indicate DNA analyses that were partitioned by codon position, whereas codes with -u- were not partitioned. Partitioned analyses are also labelled with the abbreviation "part." in the Model column. b – Outgroup lineages are: o1 = Dasycladales, o2 = UU clade, o3 = Chlorodendrophyceae, o4 = Pedinophyceae. Remaining codes are combinations of these. c – Topology given as Newick string, e.g. (O,(B,H)) meaning that Bryopsidineae and Halimedineae are sister clades, with the Ostreobium lineage sister to Bryopsidineae+Halimedineae.

Figure legends

Figure 1. Chloroplast genome map of Ostreobium sp. HV05042. Its alignment with Ostreobium quekettii SAG6.99 shows complete collinearity of these two genomes, while there are substantial rearrangements between the Ostreobium genomes and those of Bryopsis plumosa (NC_026795) and Tydemania expeditionis (NC_026796).

Figure 2. Assembly graph of Avrainvillea mazei HV02664 and details of areas with ambiguous assembly. A. Complete assembly graph from SPAdes, visualised in Bandage (Wick et al. 2015), with gene annotations mapped onto the graph using BLAST (Altschul et al. 1990). Numbers indicate regions of ambiguous assembly. B. Detail of the region containing the cysT and atpI genes (number 1 from A), showing that the genes have highly similar introns. The common parts in the beginning and end have approximately twice the sequencing depth of the unique pieces. The numbers on the segments indicate their length. C. Alignment of the resolved introns of the atpI and cysT genes, showing perfect identity of the first ca. 518 bp (except the 26 bases at the red arrowhead) and the last 90 bp. D. Detail of the graph structure near the psbE gene. The annotations of psbE is done in sections, showing the part that is shared between the canonical and alternative versions of the gene (section psbE2) and those unique to the canonical (psbE1) and alternative (psbE3) versions. Paired read mapping showed that there was no connection between nodes 2797 and 2798 but rather than 2798 connected to 2796.

Author Manuscript Figure 3. Phylogeny inferred from the coding regions of chloroplast genomes. The result shown here is the ML tree of condition cpDNA-p-o1 (cf. Table 2) annotated with bootstrap branch support values. The three suborders of siphonous green algae are given to the right of the tree. Remaining taxa are outgroups.

This article is protected by copyright. All rights reserved

Figure 4. Phylogeny inferred from nuclear 18S ribosomal DNA sequences. The result shown here is the ML tree of condition nrDNA-u-o1 (cf. Table 2) annotated with bootstrap branch support values. The three suborders of siphonous green algae are given to the right of the tree. Remaining taxa are outgroups.

Appendix S1. Alignments used to infer all maximum likelihood trees, in Phylip format.

Figure S1. Conserved blocks between Avrainvillea and members of the Bryopsidineae, Halimedineae and Ostreobineae suborders.

Figure S2. Maximum likelihood trees with bootstrap values for all analysis conditions. The title of each page corresponds to analysis conditions in Table 2.

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jpy_12540-16-224_f1.ai A

2

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double sequencing depth B cysT atpI exon 1 exon 2

518 132 90

atpI 1264 cysT exon 1 exon 2 ORF382

C 1 100 200 300 400 500 600 700 800 900 1,000 1,100 1,200 1,300 1,400 1,500 1,600 1,700 Identity

ORF382 atpI intron cysT intron

Node 2797 ORF88 unique to unique to alternative canonical

D ORF105 ORF71 psbE3 psbE1

psbJ psbL rps4 psbF

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Author Manuscript Node 2796

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jpy_12540-16-224_f2.ai jpy_12540-16-224_f3.pdf Neomeris sp. HV02668

Acetabularia acetabulum DI1

Ostreobium sp. HV05007a 100 Ostreobium sp. HV05042

100 Ostreobium sp. HV05007b 8 7 Ostreobineae Ostreobium sp. HV05007c 100 Ostreobium sp. OS1B 100 Ostreobium quekettii SAG6.99 100 Derbesia sp. WEST4838 100 Bryopsis hypnoides Bryopsidineae

100 Bryopsis plumosa WEST4718 9 8 Avrainvillea mazei HV02664

100 Caulerpa cliftonii HV03798 Halimedineae 100 Halimeda discoidea HV04923

0.3 100 Tydemania expeditionis FL1151 Author Manuscript

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Bornetella nitida Z33464 5 4 100 Batophora oerstedii Z33463 Chlorocladus australasicus Z33466

100 Cymopolia vanbosseae Z33467 Neomeris dumentosa Z33469

9 7 Acetabularia acetabulum AY165776 8 8 Acetabularia caliculus AY165780 Chalmasia antillana AY165785 100 100 Halicoryne wrightii AY165786 6 3 Parvocaulis polyphysoides AY165781

9 5 Parvocaulis parvulus Z33471 9 5 Parvocaulis pusillus AY165784 Ostreobium sp. SAG6.99 100 Ostreobineae Ostreobium sp. HV05042

3 8 Derbesia sp. WEST4838 Codium fragile MJB0103 100 100 9 8 Codium platylobium FJ535849 Bryopsidineae Codium duthieae FJ535848 4 6 Bryopsis sp. HV04063

100 Halimeda discoidea HV04923 9 6 Halimeda tuna AF407250 6 8 Halimeda cylindracea AF416388

100 100 Caulerpa cliftonii HV03798 Caulerpa sertularioides AF479703 Halimedineae 4 6 Flabellia petiolata AF416390

6 9 Tydemania expeditionis FJ432634

5 0 Udotea flabellum AF416392 0.2 9 2 Udotea argentea AF416394 Author Manuscript

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