Schizocladia ischiensis organellar genomes: estimating the origin of multicellularity in and the emergence of shallow ecosystems

Naomi Phillips (  [email protected] ) Arcadia University Edward L. Braun University of Florida Jeffrey Boore Institute for Systems Brian Cheda Arcadia University Mathew P. Salomon John Wayne Cancer Institute Hiroshi Kawai Kobe University Takahiro Yamagishi National Institute for Environmental Studies

Research article

Keywords: genome, diversifcation, flamentous, Marine, Mitochondria genome, molecular clock, genome, Phylogenomics, Phaeophyceae, Schizocladiophyceae.

Posted Date: April 3rd, 2020

DOI: https://doi.org/10.21203/rs.3.rs-20417/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Page 1/18 Abstract

Background Brown are key members of coastal marine ecosystems and the most recent eukaryotic lineage to transition to multicellularity. Browns are members of a heterogeneous assemblage of organisms, heterokonts, that include photosynthetic , (e.g. ) and non-photosynthetic taxa like the or labyrinthulas. We hypothesized that the origin of would coincide with the assembly of shallow coastal ecosystems. Testing this hypothesis required a robust estimate of the timeframe for the origin of multicellularity in the Gyristan (lineage including Oomycota and ) heterokonts and the diversifcation of the brown algae. A relaxed clock analysis that included organellar data from the closest sister of brown algae, Schizocladia ischiensis and other heterokonts enabled a robust estimation of this timeframe. Results Consistent with Gyristan heterokonts, the plastid and mitochondrial genomes of S. ischiensis were circular (cp ~138,101 bp, mt ~ 41,773 bp) with similar genes and genome architecture. The comparison of its organellar protein-coding genes with those of other heterokonts (21 cpDNA, 28 mtDNA) including outgroups were used to estimate the following divergence times: (1) The Gyristan assemblage split into autotrophic (Ochrophyta) and heterotrophic () clades in the (~ 457 Ma) with the transition to multicellularity early in the Mesozoic. (2) Several lineages (e.g., Diatoms) diversifed toward the end of the Mesozoic into the Cenozoic consistent with paleontological records and estimated stable oceanic conditions. (3) The brown algae, main architects of key coastal benthic ecosystems, arose toward the end of the Mesozoic with main lineages like the or rockweeds diversifying into the Cenozoic. These divergences were consistent with records and physical data for the emergence of shallow water coastal environments. Conclusions S. ischiensis’ organellar genomes combined with robust multigene phylogenomic molecular clock analyses placed the origin of brown algae late in the Mesozoic consistent to paleontological time estimates and plate tectonic models for the emergence of shallow coastal environments. These data robustly supported the hypothesis that the origin and diversifcation of brown algae was central to the assembly of rocky shallow coastal ecosystems. Furthermore, important communities like kelps forests or rockweed beds are relatively recent marine ecosystems.

Background

Origin of Multicellularity

Evolution of multicellularity is arguably one of the most important and pivotal events in the evolutionary history of on earth. Multicellularity has arisen multiple times (> 25) across the tree of life and is central to earth’s rich biodiversity (e.g. 1-6). Multicellularity has reached unique levels of complexity among but, even here, has arisen independently ~10 times, in some cases even within closely related lineages like the heterokonts or rhodophytes [e.g. 1-8]. Moreover, multicellularity seems to be essential to forming the types of terrestrial and coastal marine environments as they exist today [9].

Our best estimates for the frst origins of multicellularity place it in the Neoproterozoic era (500- 1000 Ma), with some of the deepest transitions occurring in the rhodophytes (), fungi, and

Page 2/18 Metazoa [see 10], more recent transitions occurring in the or Gyristan heterokonts ~50-200 MYA, and the last class to make that transition being the brown algae (Phaeophyceae) [5, 6, 11, 12]. However, robust dating of these events, especially within Gyristan heterokonts remains elusive due to poor fossil records or limited robust molecular clock studies in these groups.

Diversifcation of heterokonts

The heterokonts () form one the most rich and heterogeneous group of organisms in the eukaryotic tree of life, spanning across both terrestrial and aquatic environments [see 13-25]. Because of the enormous diversity and limited morphological characters (e.g., heterotrophic fagellates) understanding their evolutionary relationships has historically been very challenging. Recent phylogenomic work has provided signifcant insights, [e.g., 22, 24] frmly resolving many evolutionary relationships only weakly suggested in earlier work [see 24]. Heterokonts form two main clades, the heterotrophic or pathogenic and the Gyrista [see 13, 24, 25]. The Gyristan assemblage includes the fungal like Oomycota (or pseudofungi) and the autotrophic Ochrophyta [19, 24, 25, 26]. The Oomycota includes a single class formed by several important orders (e.g., Saprolegnials) of fungal like saprobes or parasites including several well-studied pathogens (e.g., ) [25, 26]. In contrast, the autotrophic Ochrophyta contains the greater biodiversity [e.g., 15, 16, 19, 22, 24, 27] with 7 of the 8 main lineages that form the world’s phytoplankton communities [see 9, 28] and the brown algae (Phaeophyceae), the main architects of many of the world’s rocky coastal benthic marine ecosystems, including the forests or rockweed beds. These Ochrophyta lineages form two distinct assemblages, the Diatomista and Chrysista [13, 22, 24]. The enormous biodiversity and ecological importance frmly establish gyristan ’s importance in both marine and terrestrial ecosystems and why understanding the timing of their diversifcation patterns and the transition to complex multicellularity is so critical [see 9, 11,19, 22, 24, 27, 29].

Unfortunately, many gyristan heterokont lineages have poor fossil records limiting our understanding of the timing of their diversifcation patterns or assembly of their respective ecosystems. Our best fossil evidence for the pseudofungi is from the Rhynie Chert deposits from the Lower in the Paleozoic (~408 MA) [30, 31, 32] with some fossil evidence of associations showing up in the period [33]. In contrast, our best fossil records for marine phytoplankton communities [see 9] place Ochrophyta members of these communities evolving after the extinction and diversifying into the Mesozoic and Cenozoic. These divergence times are consistent to evidence suggesting a shift to stable oxygen-rich ocean conditions, which could support such populations [see 9, 28]. Similarly, marine benthic coastal ecosystems and their main architects, the Phaeophyceae have an even more recent history. The best phaeophyte are from the clay shales of the Gangapur formation in the [34] and the deposits from the in [35]. These place phaeophyte divergence in the late Mesozoic through the Cenozoic consistent with paleontological and plate tectonic modeling estimates for the emergence and expansion of shallow sea environments and the development of modern climates [see 34, 35, 36].

Page 3/18 Here we report the frst complete organellar genomes of a critical transitional Gyristan heterokont in the of multicellulairty – S. ischiensis. These data are the frst to characterize this important heterokont and evaluate its evolutionary alliances with the morphologically complex Phaeophyceae. We have mined key heterokont organellar genomes and performed robust molecular clock analyses of multiple mitochondrial and plastid protein coding genes. These analyses are consistent with other multigene-based analyses and paleontological evidence providing robust divergence dates for important events in the evolution and emergence of coastal marine ecosystems.

Methods

Sample acquisition and mass culturing

S. ischiensis (isolate CCMP 2287) was mass cultured (IL) by Bigelow’s National Center for Marine Algae and Microbiota (NCMA) with standard germanium dioxide concentrations (10mg/L) to limit diatoms. Total genomic DNA was extracted using a modifed 2x CTAB method [37]. Total extracted DNA was digested with RNAase and quantifed for Nextera library preparation using a NanoDrop (Thermo Scientifc).

Next Generation Libraries and Genome Sequencing

Nextera libraries were constructed using 1-10 ng of total genomic DNA according to manufacturer’s protocols (Illumina Inc., San Diego, CA, USA). Whole genome sequencing data (WGS) was produced in three separate runs, all done using a paired-end protocol and mixed with other samples that had been tagged (“bar-coded”) for computational separation after sequencing. The frst two were both PE75 reads generated at the UC Irvine Genomics Core Facility on an Illumina HiSeq 2000 using 1/96th of a lane (L1 with 1,831,930 reads) and 1/3 of a lane (L2 with 116,743,304 reads), respectively. The third was PE250 reads generated at the UCLA Genomics Core Facility on an Illumina MiSeq with 35,400,700 reads. These were processed using Casava 1.8, evaluated for quality using FASTQC, then trimmed for quality of 0.01 in a sliding window, eliminating any read with any N’s, and eliminating all reads shorter than 20 nts after trimming, while retaining broken pairs (see additional fle 1, Table S1 for a summary of these results). The total sequence coverage of the whole S. ischiensis genome is 39x based on an estimated genome size of 350 MB (and is unrelated to the coverage specifc to either organelle genome, since the ratio of organellar DNA to nuclear DNA is not measured).

Genome assembly and Gene Annotation

De Novo assembly of all reads were accomplished using CLC Genomics Workbench v67.51 [38] using the following settings; automatic word and bubble size, minimum contig length of 200, auto-detect paired distances and perform scaffolding on. Reads were mapped to contigs with a mismatch of 1, indel cost of 3, length and similarity fraction of 0.9, non-specifc matches randomly matched, and automatic detection PE distances. Resulting contigs (>500 nts) and statistics were exported to excel and parsed into potential contaminants, mt and cp contigs for separate further assembly analyses.

Page 4/18 Using BLASTn with the organelle genomes of other organisms (e.g., littoralis, siliculosus, japonica), several scaffolds were found that comprise the mitochondrial genome (scaffolds 517 [31,961 nts] and 1372 [9,580 nts]) and plastid genome (scaffolds 334 [83,475 nts], 64 [24,826 nts], 213 [19,218 nts], 729 [3,187 nts], and 907 [2,507 nts]) of S. ischiensis. Spurious matches found in this BLAST search to other scaffolds included probable nuclear pseudogenes and contaminating bacterial DNA in one of the three libraries (for the MiSeq run) with rRNA genes that have regions of high identity to those of the organelle genomes; the latter was sorted with guidance from the complete rRNA sequence available for S. ischiensis. Through a combination of manual examination, read mapping and directed extension, and, in one case (a 144 nt gap in the mtDNA), the sequencing of a gap- spanning PCR product, the complete sequence for each organelle genome was assembled and verifed by manual examination of deep, well-matching reads throughout each circular scaffold.

Protein coding regions and putative open reading frames (ORFs) for both the mt and cp were identifed using DOGMA [39], online tools (e.g., ORF fnder, NCBI) and database comparisons. All BLAST-based suggestions were verifed manually and to assign most probable start codons. In many cases, product descriptions were added manually using nomenclature from the Chloroplast Genomics Project (http://chloroplast.ocean.washington.edu/tools/cpbase/run?view=u_feature_index). Ribosomal and tRNAs were identifed by DOGMA and confrmed using tRNAscan-SE [40].

Both the cp and mt gene maps were generated using OGDRAW [41].

To gain perspectives on the general organellar genome features of S. ischiensis in relation to other Gyristan heterokonts we compiled plastid and mitochondrial genomes across heterokont lineages using Geneious [42].

Phylogenomic and Time Divergence Analyses

Comparative phylogenomic analyses for the plastid genome were based on genome data extracted using Geneious [from 21 available ochrophyte representatives selected across the Diatomista and Chrysista clades, [19] and two outgroups [23]. Fifty-two chloroplast protein coding genes representing a subset of the 83 genes used in Corguille et al. (2009), [43] were used to build an initial concatenated dataset of 13,508 AAs. Alignments of individual genes were evaluated and trimmed by eye with further ambiguous regions trimmed from the individual gene alignments using Gblocks [44] with the less stringent settings implemented. Trimmed individual genes were used to build a combined alignment based on all selected genes forming a second alignment of 10,539 AAs.

Similarly, comparative phylogenomic analyses for the mitochondrial genome was based on genome data extracted from 28 heterokont taxa. These taxa were selected across available gyristan heterokonts including representatives for the Oomycota (pseudofungi) and Ochrophyta lineages including representatives across the Diatomista and Chrysista clades, [19] and two Rhizarian outgroups [23]. Twenty-nine mitochondrial protein coding genes representing a subset of the 35 genes used in Lui & Pang (2015) [45] were used to build an initial concatenated dataset of 9,144 AAs. As with the cp,

Page 5/18 ambiguous regions were frst identifed by eye and further trimmed from the individual gene alignments using Gblocks [44] with the less stringent settings implemented forming a concatenated trimmed alignment of 4,450 AAs.

Relaxed molecular clock analyses were run separately on the cp and mt concatenated trimmed alignments.

Time Calibration Constraints

We used IQ- TREE v. 1.3.10 [46] to identify the best-ftting model of amino acid sequence evolution (analyses available on request) and conduct searches for the optimal tree using that model. We assessed support using the ultrafast bootstrap [47]. To estimate divergence times we implemented relaxed clock analyses in MCMCtree (from the PAML 4.8 package [48]), using the best-ftting model of amino acid evolution for each data matrix and the approximate likelihood calculations [49]. We used the fossil calibrations that are listed in Table 1 and conducted several sensitivity analyses (analyses available on request).

Table 1 Temporal time constraints used in divergence time estimates and phylogenomic molecular clock analyses. Genome Constrained node Reference Age Plastid 5, Khakista Matari et al. 2014 [26] 72.1-100 (Bacillariophyceae) Plastid 4, Khakista Parfrey et al. 2011 [27] 80-110 (Bacillariophyceae) Plastid 2, Diatomeae Parfrey et al. 2011 [27] 133.9-550 Plastid Omitted Brown & Sorhannus 2010 [29] 40-49 Plastid 6, Silberfeld et al. 2010 [11] 13-155 Plastid 7, Laminariales Silberfeld et al. 2010 [11] 13-155 Plastid 1, Haptophyta Parfrey et al. 2011 [27] 260-203.6 Plastid Root Inferred from origin of red algae in BEAST analysis 1200 as in [27] Mitochondrial 7, Bacillariophyceae Matari et al. 2014 [26] 72.1-100 Mitochondrial 6, Diatomeae Parfrey et al. 2011 [27] 80-110 Mitochondrial Omitted Brown & Sorhannus 2010 [29] 40-49 Mitochondrial 5, Fucales Silberfeld et al. 2010 [11] 13-155 Mitochondrial 4, Laminariales Silberfeld et al. 2010 [11] 13-155 (Costariaceae) Mitochondrial 3, Phaeophyceae Silberfeld et al. 2010 [11] 99.6-155 Mitochondrial 2, Gyrsista Matari et al. 2014 [26] 408-550 Mitochondrial 1, Rhizarians Parfrey et al. 2011 [27] 542-3000 (1750) Mitochondrial Root inferred from origin of red algae in BEAST analysis as 1750 in [27]. Plastid 5, Khakista Matari et al. 2014 [26] 72.1-100 (Bacillariophyceae) Plastid 4, Khakista Parfrey et al. 2011 [27] 80-110 (Bacillariophyceae) Plastid 2, Diatomeae Parfrey et al. 2011 [27] 133.9-550 Plastid Omitted Brown & Sorhannus 2010 [29] 40-49 Plastid 6, Fucales Silberfeld et al. 2010 [11] 13-155 Plastid 7, Laminariales Silberfeld et al. 2010 [11] 13-155 Plastid 1, Haptophyta Parfrey et al. 2011 [27] 260-203.6

Page 6/18 Numbers in column 2 correlate to numbers on branches in phylogenetic trees in Figures 3 & 4.

Results And Discussion

S. ischiensis Organellar Genome features

We report here the frst complete plastid and mitochondrial genomes of S. ischiensis assembled from WGS (whole genome sequencing, combined HiSeq and MiSeq runs) of total genomic DNA Nextera libraries. These combined WGS runs generated over 150 million reads (152,144,044) with a minimum length of 75 bp for the HiSeq run, 162 bp for the MiSeq with a quality scores >20 and an average sequence depth of 39X across the two organellar genomes. Both genomes were circular mapping with the plastid genome at 138,101 bp (see Fig. 1) and the mitochondrial at 41,773 bp (see Fig. 2).

Like many other photosynthetic heterokonts, S. ischiensis’ plastid genome consisted of a large single copy region (LSC, 83,435 bp), a small single copy region, (SSC, a 43,822 bp) separated by two inverted repeat regions (5,422 bp; see Additional fle 4: Table S2) and was intermediate in genome size. Whereas across lineages, gene numbers ranged from 141 to 192, protein-coding genes from 127 to 156, and tRNAs from 27 to 34, with most taxa having six rRNAs (see Additional fle 4: Table S2). Many of these features were intermediate for S. ischiensis consistent with its phylogenetic position in the gyristan heterokont tree of life [19, 23].

S. ischiensis mitochondrial genome’s size, number of overlapping gene regions (10) and gene density was more similar to its phaeophycean sister group than to those of unicellular heterokonts. While general mt genome architecture across heterokonts was very consistent lacking this unicellular/multicellular pattern (see Additional fle 4). S. ischiensis GC content (41%) was higher for most heterokonts but contained similar gene content (65 genes with 28 protein-coding, three rRNAs, 26 tRNAs and no ORFs, see Additional fle 5: Table S3). Overall, many S. ischiensis mitogenome features were either intermediate in nature or more phaeophycean like (see Additional fle 5: Table S3).

Timing the Diversifcation of Heterokonts

Our mtDNA-based analysis robustly placed the emergence of gyristan heterokonts and the split between the autotrophic Ochrophyta and heterotrophic pseudofungal (oomycetes) clades in the frst half of the Paleozoic (406-540 MYA, see Fig. 3, Additional fle 6, Table S4a) with diversifcation of their respective lineages into the Mesozoic/Cenozoic. These estimates are consistent with available fossil data [see 9, 26], but in sharp contrast to a deep molecular clock study of the eukaryote tree of life [see 27]. We argue that our estimations more accurately refected true heterokont diversifcation patterns because of our robust lineage representation (especially among the Ochrophytan lineages). However, our estimations for heterotrophic oomycetes are limited to the Saproleginales and , orders better studied because they contain important saprobic and pathogenic pseudofungal taxa. Our data places their

Page 7/18 divergence towards the end of the Mesozoic era (~100 MYA) consistent with previous clock estimations, fossil evidence and pathogenic plant associations [16].

For the autotrophic Ochrophyta our mtDNA and cpDNA based analyses provides one of the frst robust dating of the origin main ochrophyte clades frst identifed in the 5 gene phylogeny of Yang et al. (2012) [19] and further resolved in the work of Derelle et al (2016) [Derelle et al 2016]. Our analyses (mtDNA, cpDNA) robustly captured the Khakista assemblage of the larger Diatomista clade (SIII clade of Yang et al. (2012)) and the core lineages of the Chyrista assemblage (SI-PX clades of Yang et al. (2012)) but had various placement for some of the Yang’s et al. (2012), SII classes like the or Synurophyceae, (Figs. 3 & 4, Additional fle 6: Table S4 a&b), thus we focus on the clades that were reliably resolved and discuss their origin and diversifcation patterns. We estimate that the Khakista assemblage diverged early in the Mesozoic (245 to 223 Ma, Figs. 3 & 4) with the lineages of Diatomeae (diatoms) diversifed into the Cenozoic consistent with fossil evidence and theoretical ocean conditions [9, 20], (Figs. 3 & 4, Additional fle 6: Table S4b). The core Chyrista assemblages starting diversifying in the middle of the Paleozoic (~366 Ma, Fig. 4), with the lineages of Yang’s PX clade (Xanthophyceae, Phaeophyta) diverging towards the beginning of the Mesozoic (~252-221 Ma, Figs 3 & 4, Additional fle 6, Table S4 a&b). The morphologically complex Phaeophyceae diverged towards the end of the Mesozoic (136-116 Ma) with major orders like the kelps (Laminariales) diversifying into the Cenozoic (~252-221 Ma, Figs 3 & 4, Additional fle 5a-b: Table S4). These estimates are consistent with other molecular clock data and fossil evidence placing important phytoplankton and coastal lineages with a Mesozoic to early Cenozoic diversifcation history [9, 11, 27].

Emergence of Oceanic and Coastal Benthic Ecosystems

Autotrophic gyristan heterokonts (Ochrophyta) with their red algal derived include many of the key lineages forming today’s phytoplankton and coastal benthic marine ecosystems [e.g., 9, 11, 27]. Thus, accurately dating their diversifcation patterns are key to understanding the emergence and formation of these critical ecosystems. Robust fossil evidence and physical data for environments paint of picture of phytoplanktonic ecosystems shifting from green lineage dominated communities to red algal derived plastid lineages after the late Permian extinction event when oceans shifted to more oxygen rich, stable environments [see 9]. Theoretically, these lineages radiated late in the Mesozoic into the Cenozoic forming modern phytoplankton communities dominated by diatoms, and dinofagellates. Our estimations for diversifcation patterns are consistent with this evolutionary picture and support the emergence of modern phytoplankton communities after the Permian extinction radiating into the Cenozoic (see Figs 3 & 4, Additional fle 6: Table S4 a & b).

Fossil evidence for the world’s coastal marine ecosystems’ plant components are much less robust. However, when combined with physical estimations for ocean conditions (e.g., oxygen levels), leads to a conclusion for their emergence and diversifcation towards the end of the Mesozoic into the Cenozoic [see 9, 11]. Our molecular clock based estimations are consistent this data and place the origin of the brown algae (Phaeophyceae) towards the end Mesozoic with diversifcation of the major lineages (e.g.,

Page 8/18 Laminariales, Fucales) into the Cenozoic (Figs. 3 & 4, Additional fle 6: Table S4 a & b). These lineages are the main architects of benthic coastal communities like the kelp forests of the Pacifc Northwest and the rockweed beds of the Atlantic seaboard. Our data places these ecosystems with a late Mesozoic, early Cenozoic origin consistent to dates from other work on the evolution of the Phaeophyceae, the fossil record and the proposed nature of oceans [9, 11, 26].

Dating the last Transition to Multicellularity

Gyristan heterokonts include many lineages that shifted from unicellularity to some form of multicellularity, like hyphal coenocytic habits (e.g., pseudofungi or xanthophytes) or full morphological complexity (brown algae/phaeophytes), [5. 6, 15, 16]. These lineages represent some of the most recent shifts to complexity in the eukaryotic tree of life with the Phaeophyceae being the most recent to shift to complex multicellularity [see 5, 6, 12]. Our data from mitochondrial and plastid genes provide some of the frst robust estimates for these events placing the origin of the Schizocladiophyceae (flamentous habit) in the Mesozoic some 300-500 Ma after the origin of metazoans or red algae (see Figs. 3 & 4, Additional fle 6, Table S4 a & b), [10]. The origin of the brown algae (Phaeophyceae) occurred about hundred million years later (136-116 Ma) with their diversifcation and the emergence of brown algal dominated coastal marine ecosystems like kelp forests or Sargasso Sea beds into the Cenozoic consistent to the cooling of the oceans and the expansion of shallow coastal environments.

Conclusions

In conclusion, we reported the frst complete organellar genomes of a pivotal heterokont (S. ischiensis). Comparisons across heterokont plastid and mitochondrial genomes placed Schizocladia as either intermediate in nature or more closely aligned with the morphologically complex Phaeophyceae. Comparative phylogenomic analyses of cp and mt genes enabled robust molecular clock estimations of the diversifcation patterns among important gyristan heterokont lineages and their shift to multicellularity. Placing these transitions early in the Mesozoic consistent to the emergence of important phytoplankton and coastal marine lineages (e.g., diatoms and brown algae) and their consequent ecosystems. This work places architects of open water and coastal ecosystems with a fairly recent evolutionary history consistent with physical and fossil evidence [see 9, 11, 26].

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Page 9/18 Availability of data and material

1. The datasets or alignments generated and/or analyzed during the current study are available by request from the corresponding author on reasonable request.

2. GenBank for organellar genomes: Plastid genome: MT226925. Mitochondrial genome: MT259947.

Competing interests

The authors declare that they have no competing interests.

Funding

NP had intramural funds from Arcadia University [faculty development funds] that supported portions of this research.

Author’s contributions

NP and ELB extracted the cultures total genomic DNA. JB and MS assembled the next generation sequence data creating the cp and mt genomes. JB, BC, and NP annotated the mt and cp genomes. BC made the organellar maps. NP and BC assembled comparative genomics tables. NP and BC made and evaluated multiple gene alignments. ELB preformed molecular clock analyses and assisted with alignments. HK and TY provided transcriptome data. All authors contributed to the manuscript, which was primarily written by NP.

Acknowledgements

Special thanks to our colleagues who were involved in initial stages of this research (Drs. D. Bhattacharya, D. Price, H. S. Yoon). NP greatly appreciates the support of her Department and University for their of her research program.

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Supplementary Files

File name: Additional Files_S1-4. Excel fle with tabs for each Table S1-4. File Extension xls. Tab name: Additional 1. S1 Genome assembly. Table title: Table S1: Summary of the results of processing and trimming all reads from the three Illumina sequencing runs. Reads were trimmed for quality of 0.01 in a sliding window, then any read with any N’s or shorter than 20 nts after trimming were eliminated. “PE” refers to the number of paired-end reads remaining. “Singletons” refers to a read that remains after its partner was removed during trimming. Whole genome coverage is based on an estimated genome size of 350 MB and is unrelated to the coverage specifc to either organelle genome, since the ratio of organellar DNA to nuclear DNA is not measured. Table is below but also in the clustered excel fle (xls).

Page 13/18 Number PE Number Total # of Avg. read Run reads singletons reads length Total bases Coverage PE75-L1 1,730,994 45,701 1,776,695 70 124,368,650 0.36x PE75-L2 112,430,348 2,003,787 114,434,135 72 8,239,257,720 23.54x PE250- MiSeq 34,469,330 364,172 34,833,502 152 5,294,692,304 15.13x

File name: Additional Files_S1-4. Excel fle with tabs for each Table S1-4. File Extension xls. Tab name: Additional fle 2 Table S2 – cp. Table title: Table S2. Comparative plastid genome data extracted using Geneious. GeneBank accession numbers are included for each taxon’s genome data.

File name: Additional Files_S1-4. Excel fle with tabs for each Table S1-4. File Extension xls. Tab name: Additional fle 3 -Table S3 –mt. Table title: Table S3. Comparative mitochondrial genome data extracted using Geneious. GeneBank accession numbers are included for each taxon’s genome data.

File name: Additional Files_S1-4. Excel fle with tabs for each Table S1-4. File Extension xls. Tab name: Additional fle 4 -Table S4a&b. Table title: Table S4 a & b. Estimated divergence times for clades, bootstrap values and 95% HPDs for the plastid and mitochondrial molecular clock analyses. Clades labeled as in Derelle et al. 2016 [27] and Yang et al. 2012 [19].

Figures

Page 14/18 Figure 1

Plastid genome map of Schizocladia ischiensis. Genes on the outside of the genome circle are transcribed clockwise, while genes on the inside are transcribed counterclockwise. Abbreviations: IRA & IRB, inverted repeats A & B; LSC, large single copy regions; SSC, small single copy regions.

Page 15/18 Figure 2

Mitochondrial genome map Schizocladia ischiensis. . Genes on the outside of the genome circle are transcribed counterclockwise, while genes on the inside are transcribed clockwise as indicated by the arrows.

Page 16/18 Figure 3

Time calibrated tree resulting from a relaxed clock analyses of 29 mitochondrial protein coding genes (MCMCtree, PAML 4.8, [48]), extracted from 28 gyristian heterokonts and two Rhizarian outgroup taxa.

Page 17/18 Figure 4

Time calibrated tree resulting from a relaxed clock analyses of 52 plastid protein coding genes (MCMCtree, PAML 4.8, [48]), extracted from 21 ochrophyte heterokonts and two Haptophyta outgroup taxa.

Supplementary Files

This is a list of supplementary fles associated with this preprint. Click to download.

AdditionalFilesS14.xlsx

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