# Chimeric origins of ochrophytes and haptophytes
revealed through an ancient plastid proteome

Richard G Dorrell, Gillian Gile, Giselle Mccallum, Raphaël Méheust, Eric P
Bapteste, Christen M. Klinger, Loraine Brillet-Guéguen, Katalina D Freeman,
Daniel J Richter, Chris Bowler

► To cite this version:

Richard G Dorrell, Gillian Gile, Giselle Mccallum, Raphaël Méheust, Eric P Bapteste, et al.. Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome. eLife, 2017, 6, pp.e23717. 10.7554/eLife.23717. hal-01526828

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1 Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid
2 proteome

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4 Richard G. Dorrell¹*, Gillian H. Gile², Giselle McCallum¹, Raphaël Méheust³, Eric P. Bapteste³,
5 Christen M. Klinger⁴, Loraine Brillet-Guéguen⁵, Katalina D. Freeman², Daniel J. Richter⁶, and
6 Chris Bowler¹*

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¹IBENS, Département de Biologie, École Normale Supérieure, CNRS, Inserm, PSL Research

University, F-75005, Paris, France

²School of Life Sciences, Arizona State University, 427 E Tyler Mall, Tempe, AZ, 85287, USA

³Institut de Biologie Paris-Seine, Université Pierre et Marie Curie, Paris

⁴Department of Cell Biology, University of Alberta

⁵CNRS, UPMC, FR2424, ABiMS, Station Biologique, 29680, Roscoff, France

⁶Sorbonne Universités, UPMC Univ Paris 06, CNRS UMR 7144, Adaptation et Diversité en
Milieu Marin, Equipe EPEP, Station Biologique de Roscoff, 29680 Roscoff, France

cbowler@biologie.ens.fr

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Plastids are supported by a wide range of proteins encoded within the nucleus and imported from the cytoplasm. These plastid-targeted proteins may originate from the endosymbiont, the host, or other sources entirely. Here, we identify and characterise 770 plastid-targeted proteins that are conserved across the ochrophytes, a major group of algae including diatoms, pelagophytes and kelps, that possess plastids derived from red algae. We show that the ancestral ochrophyte plastid proteome was an evolutionary chimera, with 25% of its phylogenetically tractable proteins deriving from green algae. We additionally show that functional mixing of host and plastid proteomes, such as through dual targeting, is an ancestral feature of plastid evolution. Finally, we detect a clear phylogenetic signal from one ochrophyte subgroup, the lineage containing pelagophytes and dictyochophytes, in plastid-targeted proteins from another major algal lineage, the haptophytes. This may represent a possible serial endosymbiosis event deep in eukaryotic evolutionary history.

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Since their origin, the eukaryotes have diversified into an extraordinary array of organisms, with different genome contents, physiological properties, and ecological adaptations¹⁻³.

Perhaps the most profound change that has occurred within individual eukaryotic cells is the acquisition of plastids via endosymbiosis, which has happened at least eleven times across the tree of life¹. All but one characterized group of photosynthetic eukaryotes possess plastids resulting from a single ancient endosymbiosis of a beta-cyanobacterium by an ancestor of the archaeoplastid lineage (consisting of green algae and plants, red algae, and glaucophytes)¹.

Photosynthesis has subsequently spread outside of the archaeoplastids through secondary, tertiary, or more complex endosymbiosis events. By far the most ecologically successful of these lineages are those that possess plastids derived from secondary or more complex endosymbioses of a red alga¹, ⁴, ⁵. These are the "CASH lineages", consisting of
52 photosynthetic members of the cryptomonads, alveolates (such as dinoflagellates),
53 stramenopiles (also referred to as heterokonts) and haptophytes¹, ⁴ (see Table 1 and Fig. 1-
54 figure supplement 1 for definitions). The most prominent of these are the photosynthetic
55 members of the stramenopiles, termed the ochrophytes², ⁶, ⁷. The ochrophytes include the
56 diatoms, which are major primary producers in the ocean⁸, ⁹, multicellular kelp, which serve
57 as spawning grounds for marine animals¹⁰, and the pelagophytes, small free-living algae
58 frequently associated with harmful blooms¹¹ (Fig. 1, panel A; Fig. 1- figure supplement 1).
59 The stramenopiles also contain many aplastidic and non-photosynthetic lineages (e.g.,
60 oomycetes), which diverge at the base of the ochrophytes and play important roles as
61 pathogens and in microbial food webs⁶, ¹² (Fig. 1- figure supplement 1).
62

63 Following their acquisition, plastids have undergone a number of evolutionary changes that
64 bound them more intricately with the biology of the host. These include the transfer of
65 plastid-derived genes to the host nucleus³, ¹³, ¹⁴ and the targeting of proteins encoded within
66 the nucleus to the plastid¹⁵, ¹⁶. Previous studies have shown that many plastid-targeted
67 proteins are not derived from the endosymbiont genome¹⁷. Proteins encoded by genes
68 acquired from other sources, such as laterally acquired genes¹⁸, ¹⁹ or previous endosymbiotic
69 organelles historically possessed by the host²⁰, ²¹, or proteins that have been repurposed
70 from endogenous host organelles²², ²³ have important roles in supporting the biology of
71 plastid lineages. Other gene transfer events, e.g. from food sources²⁴, bacterial symbionts²⁵,
72 viruses²⁶, or diazotrophic non-plastid cyanobacterial endosymbionts²⁷, ²⁸ have also played
73 major roles in the evolution of photosynthetic eukaryotes, and it remains to be determined
74 which of these have contributed to the diverse range of plastid proteins observed today. It
75 nonetheless remains largely unknown which proteins had the most fundamental roles in
76 establishing current plastid lineages³, i.e., which plastid proteins represent the ancestral
77 components of plastid-targeted proteomes.
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79 Ochrophytes represent an excellent system in which to reconstruct the origins of plastid
80 proteomes. Firstly, plastid-targeting sequences in different ochrophytes are relatively well
81 conserved, enabling *in silico* prediction of plastid-targeted proteins from a wide range of
82 different species<sup>29, 30</sup>, in contrast to plastid-targeting sequences within archaeoplastid
83 lineages, which are extremely variable<sup>31, 32</sup>. Secondly, compared to other CASH lineages
84 (haptophytes, cryptomonads, and dinoflagellates), ochrophytes represent an extremely well
85 characterised system for experimental and bioinformatic investigation, with (to date) eleven
86 complete genomes, and transcriptome libraries available for over 150 species through
87 MMETSP<sup>33, 34</sup>. Reliable transformation and other manipulation strategies are also available
88 for multiple species, such as the model diatom *Phaeodactylum tricornutum*<sup>35-37</sup>.
89

90 Thirdly, the origin of the ochrophyte plastid is an evolutionarily valuable topic to
91 understand. It is currently not known when the ochrophyte plastid was acquired: whether it
92 originated recently, predates the radiation of aplastidic stramenopile relatives⁵, ⁶, ¹², or was
93 acquired prior to the divergence of stramenopiles from their closest relatives, the
94 alveolates³⁸. Verifying a late origin for the ochrophyte plastid would thus enable insights into
95 the cellular changes that accompany the transition from a solely heterotrophic to a
96 phototrophic lifestyle⁶, ¹², which is currently not possible for archaeoplastids³⁹, ⁴⁰, and difficult
97 for haptophytes and cryptomonads, in which these relatives respectively remain unknown
98 or understudied at a genomic level³⁹, ⁴¹. It has additionally been proposed, based on the
99 presence of large numbers of genes of putative green algal origin in diatom genomes ⁴², ⁴³,
100 that the ancestor of ochrophytes once possessed a green algal endosymbiont, which was
101 subsequently replaced via the serial endosymbiosis of a red algal-derived plastid¹, ⁴⁴. This
102 hypothesis remains controversial⁴⁵⁻⁴⁷, in particular due to issues associated with the
103 distinction of genes of red and green algal origins in ochrophyte genomes<sup>48-50</sup>. A final
104 evolutionary suggestion regarding ochrophytes is that they have acted as endosymbiotic
donors into other CASH lineages. One recent study proposed that haptophytes possess
106 plastids acquired via the endosymbiosis of an ochrophyte<sup>5</sup>, although the exact identity of
107 this endosymbiotic acquisition remain unresolved. Characterising the ancestral ochrophyte
108 plastid proteome might therefore help answer major questions about the ways in which
109 plastids become established in the host cell, and provide valuable insights into the origins
110 and diversification of other ecologically important algal lineages.
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112 In this study, we present an experimentally verified *in silico* reconstruction of the proteins
113 targeted to the plastid of the last common ochrophyte ancestor. We show that this ancestral
114 plastid proteome was an evolutionary mosaic, containing 770 proteins from a range of
115 different sources. Our dataset indicates that the ochrophyte plastid was acquired late in
116 stramenopile evolution, following the divergence of extant aplastidic relatives, that plastid-
117 targeted proteins of green algal origin played a significant role in its origin, and that there
118 has been bidirectional integration of the biology of the ochrophyte host and plastid
119 proteomes, such as the ancient recruitment of proteins from both host and endosymbiont
120 to dually support the biology of the plastid and mitochondria. Finally, we show evidence for
121 an ancient endosymbiosis of a specific ochrophyte lineage, an ancestor of the pelagophytes
122 and dictyochophytes, by a common ancestor of the haptophytes, which we propose- based
123 on discrepancies between the origins of the haptophyte plastid proteome and genome-
124 reveals a possible serial endosymbiosis event early in haptophyte evolution, preceding the
125 origins of the current haptophyte plastid. Our work resolves several long-standing questions
126 of ochrophyte evolution, and provides new insights into the origins and diversification of
127 CASH lineages as a whole.
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## 129 Results

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131 **1. In silico reconstruction of an ancestral plastid proteome**

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133 We developed an *in silico* pipeline for identifying putatively ancestral plastid-targeted
134 proteins across the ochrophytes (Fig. 1). We screened a large composite library, comprising
135 eleven different ochrophyte genomes, together with transcriptome data from a further 158
136 ochrophyte species (Table S1- sheet 1<sup>145</sup>) using the ochrophyte plastid targeting predictors
137 ASAFind (Table S2- sheet 1<sup>145</sup>)<sup>29</sup> and HECTAR (Table S3- sheet 1<sup>145</sup>)<sup>30</sup>. Sequences with
138 predicted plastid localisation were binned into eleven taxonomic sub-categories within three
139 major groups (chrysista, hypogyrista, and diatoms) based on recent multigene phylogenies<sup>12</sup>
140 (Fig. 1, panel A; Fig. 1- figure supplement 1), then assembled by sequence similarity into
141 homologous plastid-targeted protein groups (HPPGs, Materials and Methods).
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143 We next tested the level of conservation best able to identify truly ancestral HPPGs. We
144 selected three patterns of conservation that identified the largest number of HPPGs from a
positive control dataset of proteins with previously identified plastid-associated functions,
146 and minimised the number identified from a negative control dataset of HPPGs generated
147 using seed sequences from three other published CASH lineage genomes, for which no
148 plastid-targeted orthologues were detected in any ochrophyte genome sequence (Materials
149 and Methods; Table S2- sheet 2, sections 1-2; Table S3- sheet 2, sections 1-2<sup>145</sup>). The
150 selected conservation patterns were: the presence of the protein in a majority of chrysistan
151 sub-categories and a majority of either diatom or hypogyristean sub-categories; or presence
152 in at least one chrysistan sub-category and a majority of both diatoms and hypogyristea (Fig.
153 1, panel B). We extracted HPPGs matching the conservation patterns defined above and
154 verified their monophyly within ochrophytes via alignment and single-gene trees (Fig. 1,
155 panel C; Table S4- sheet 1<sup>145</sup>). From this, we identified 770 proteins that were probably
156 targeted to the ancestral ochrophyte plastid (Fig. 1, panel D; Table S4- sheet 2<sup>145</sup>). This
157 dataset is significantly enriched in proteins from within the positive control dataset and
158 contains significantly fewer proteins from the negative control dataset than would be
159 expected through random assortment (chi-squared test, P < 1 x 10<sup>-10</sup>; Fig. 1), confirming its
160 specificity towards probable ancestral plastid-targeted proteins.

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2. Experimental verification of ancestral ochrophyte HPPGs

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164 We wished to verify that the ancestral ochrophyte plastid-targeted proteins inferred from
165 the *in silico* pipeline are genuinely plastid-targeted. 106 of our inferred ancestral HPPGs
166 include a *P. tricornutum* protein with prior experimental plastid localization, or unambiguous
167 plastid function (Fig. 1, panel D), but the remainder do not. We selected ten proteins for
168 experimental localisation (Fig. 2, panel A; Table S5<sup>145</sup>). These were chosen on the basis of
169 having only non-plastid annotations on the first 50 BLAST hits against the NCBI nr database
170 excluding ochrophytes, thus arguing against their predicted plastid localization beyond these
171 organisms. In each case, all of the ochrophyte protein sequences within the alignment had a
172 well conserved central domain, and a highly variable N-terminal domain of between 30 and
173 50 amino acids containing an ASAFAP motif, consistent with a conserved plastid targeting
174 sequence<sup>29</sup> (Fig. 2- figure supplement 1).

175 The selected proteins included five aminoacyl-tRNA synthetases that yielded BLAST top hits
176 only against enzymes with cytoplasmic annotations, or of probable prokaryotic origin (Fig. 2-
177 figure supplement 2). Also included were a GroES-type chaperonin of inferred mitochondrial
178 origin, an Hsp90-type chaperonin of inferred endoplasmic reticulum origin and a
179 pyrophosphate-dependent phosphofructokinase, which is related to cytosolic enzymes from
180 other lineages (Fig. 2- figure supplement 3), and is distinct from the ATP-dependent
181 phosphofructokinases used by primary plastid lineages<sup>51</sup>. The Mpv17 membrane protein is
182 most closely related to enzymes with peroxisomal functions and localisation<sup>52, 53</sup>, but lacks
183 any identifiable peroxisomal targeting sequence (PSL, KRR, or a PTS1 motif)<sup>54</sup> in its C-
184 terminus. Novel protein 1 lacks any conserved domains, and yielded no BLAST matches
185 outside of the ochrophytes below an expect value of 1 x 10<sup>-05</sup> (except for one dinoflagellate
186 sequence), and hence might constitute an entirely novel plastid-targeted protein (Fig. 2-
187 figure supplement 4; Table S5<sup>145</sup>).

188 We generated C-terminal GFP-fusion constructs for each of these proteins using *P. tricornutum* genes and transformed wild-type *P. tricornutum* (Fig. 2, panel B; Fig. 2- figure supplement 5; Table S5<sup>145</sup>). In each case, we identified GFP fluorescence associated with the plastid. In one case (the peroxisomal membrane protein; Fig. 2, panel B), the GFP accumulated in a ring around the plastid equator, consistent with a periplastid compartment (PPC) localisation<sup>88,55</sup>. In other cases (such as the five aminoacyl-tRNA synthetases, Fig. 2- figure supplement 5), the GFP signal localised both within and external to the plastid, consistent with a multipartite localisation within the cell. However, in all cases the proteins tested were at least partially targeted to the plastid.

197 We additionally generated heterologous GFP fusion constructs for five of the proteins using
198 sequences from the "dinotom" *Glenodinium foliaceum*, a dinoflagellate alga that harbours
199 permanent endosymbionts of diatom origin<sup>20, 56</sup>, and the eustigmatophyte *Nannochloropsis gaditana*, which as a member of the "PESC clade" is distantly related to *P. tricornutum* on
200 the ochrophyte tree<sup>12</sup>. We expressed these constructs in *P. tricornutum* (Fig. 2, panel B; Fig.
201 2- figure supplement 6), and, in each case, detected plastid-localized GFP fluorescence
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similar to the patterns observed with the *P. tricornutum* gene constructs. Overall, our data therefore supports that the ancestral HPPG dataset consists of genuinely conserved plastid-targeted proteins, rather than misidentified proteins of non-plastid function.

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### 3. Evolutionary origins of the ochrophyte plastid

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*The ochrophyte plastid is an evolutionary mosaic*

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We wished to identify the evolutionary affinity of each ancestral HPPG in our dataset. In particular, we assessed whether proteins that are of unconventional origin, such as the products of genes endogenous to the host, or genes that have been acquired from other sources such as prokaryotes and green algae, have significantly contributed to the origins of the ochrophyte plastid¹, ⁴⁴.

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We accordingly determined the closest relative of each ancestral HPPG (Materials and Methods). Due to ongoing controversies regarding the evolutionary composition of ochrophyte genomes⁴⁶, ⁴⁷, we utilised a combined phylogenetic and BLAST top hit approach to robustly infer the most probable origin of each HPPG (Materials and Methods; Table S4-sheet 2¹⁴⁵). For both the BLAST and phylogenetic analyses, stringent criteria were applied to avoid misidentification due to topological ambiguity, or contamination within individual sequence datasets⁵⁷, ⁵⁸ (Materials and Methods). We took the union of these two analyses to produce a dataset of 263 HPPGs for which both phylogenetic and BLAST top hit analyses indicated the same clear evolutionary origin. These origins were grouped into six evolutionary categories, red algae, green algae, aplastidic stramenopiles, other eukaryotes, prokaryotes, and viruses (Fig. 3, panel A).

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Of the 263 HPPGs that were resolved from the combined analysis, 149 (57%) were of red algal, i.e. endosymbiont origin (Fig. 3, panel A; Table S4-sheet 3¹⁴⁵). This is analogous to results from studies of archaeplastid plastid proteomes, in which approximately half of the plastid-targeted proteins are of endosymbiont origin¹⁸, ³². The remaining 114 HPPGs resolved with other sister-groups, consistent with a mosaic origin of the ochrophyte plastid proteome. The most significant of these lineages was green algae (67 HPPGs, 25%), followed by aplastidic stramenopiles (26 HPPGs, 10%), and prokaryotes (21 HPPGs, 8%) (Fig. 3, panel A). None of the HPPGs were clearly assigned to other eukaryotes or to viruses, consistent with previous assertions that these lineages have contributed very little to ochrophyte evolution⁵⁹ (Fig. 3, panel A).

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*Late origin of ochrophyte plastids*

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We wished to determine whether the ochrophyte plastid was acquired by a common ancestor of all stramenopiles or later in ochrophyte evolution. We reasoned that if the ochrophyte plastid was acquired early, i.e., before the divergence of aplastidic relatives, endosymbiotic gene transfer from the red algal symbiont to the host nucleus would have commenced prior to the radiation of the stramenopiles⁶⁰. Based on the primary evolutionary affinities of each ancestral HPPG (Fig. 3, panel A), we would expect at least half of the aplastidic stramenopile-derived proteins to show a deeper red algal origin. We accordingly profiled the deeper evolutionary affinity of each ancestral HPPG of aplastidic stramenopile origin by a combined phylogenetic and BLAST top hit analysis, as before.

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First, we noted that the majority (20/26) of the ochrophyte HPPGs with aplastidic stramenopile origins specifically resolved as a sister-group to oomycetes, as opposed to the

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254 deeper-branching labyrinthulomycetes or slopalinids (Fig. 3, panel B; Table S4- sheet 3¹⁴⁵).

255 Because oomycetes are the sister-group of ochrophytes⁶, ¹², this suggests that our dataset

256 retains useful phylogenetic signal.

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258 Next, from the 26 ancestral HPPGs of aplastidic stramenopile origin, we identified a clear
259 sister-group to the stramenopile clade for 16 HPPGs using BLAST, and for 18 HPPGs using
260 single-gene trees (Fig. 3, panel B). However, only one BLAST top hit and four trees showed a
261 deeper red algal affinity (Fig. 3, panel B). These proportions are significantly smaller than the
262 proportions of ochrophyte proteins of red origin in the entire ancestral HPPG dataset
263 (expected frequencies: 9.54 BLAST top hits, 10.7 sister-groups; chi-squared-test, P≤ 0.01; Fig.
264 3, panels A, B). In five cases we identified the same deeper affinity through combined BLAST
265 top hit and tree sister-group analysis, but none of these were of red algal origin (Fig. 3, panel
266 B). We conclude that plastid-targeted proteins in ochrophytes that are related to aplastidic
267 stramenopile proteins are predominantly not of red origin. This is consistent with a late
268 origin for the ochrophyte plastid, following the divergence of the ochrophytes and
269 oomycetes.

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271 A significant green algal contribution to ochrophyte plastid evolution

272
273 Previous reports of green genes in ochrophyte genomes have been controversial due to a
274 paucity of red algal sequence data ⁴⁴, ⁴⁷, ⁵⁹. We were able to avail in our pipeline of sequence
275 information from five complete red algal genomes⁴⁸, ⁴⁹, ⁶¹⁻⁶³ and twelve red algal
276 transcriptomes³⁴, ⁶⁴, allowing us to more clearly infer the reliability of the green signal in
277 ochrophytes. We tested whether the inferred green algal origin could be due to a protein
278 family's absence from red algal lineages (Fig. 4, panel A). For the majority of our green
279 HPPGs (40/67), an orthologue was identified in at least four of the five major red algal sub-
280 categories considered (cyanidiales, bangophytes and florideophytes, compsopogonophytes
281 and stylonematophytes, porphyridiophytes, and rhodellophytes; Fig. 4, panel B; Fig. 4- figure
282 supplement 1; Table S4- sheet 4¹⁴⁵). We therefore conclude that these green genes were not
283 misidentified as the result of undersampling within red sequence libraries, or secondary
284 gene loss events in the red algae⁴⁵, ⁵⁰.

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286 We then considered whether the green genes in our dataset originate from a specific source
287 within the green algae. Phylogenetic analyses of the HPPGs of verified green origin exhibited
288 a strong bias toward chlorophyte origins. Ochrophytes branched as sister-groups to
289 individual or multiple chlorophyte lineages in 51 of the 67 trees (Fig. 4, panel C; Fig. 4- figure
290 supplement 2). Similarly, we noted a strong predominance of chlorophyte lineages amongst
291 BLAST top hits (56/67) despite the fact that these lineages only correspond to approximately
292 25% of the green sequences present in our libraries (Fig. 4- figure supplement 3; Table S4-
293 sheet 3¹⁴⁵). In contrast, only 16 of the single-gene trees recovered a sister-group relationship
294 between ochrophytes and all green lineages (chlorophytes and streptophytes), none
295 recovered a specific sister-group relationship between ochrophytes and streptophytes (Fig.
296 4, panel C), and only 11 of the BLAST top hits were to streptophyte sequences (Fig. 4- figure
297 supplement 2; Table S4- sheet 3¹⁴⁵). This bias is inconsistent with the green ancestral HPPGs
298 being of misidentified red origin, or originating at a deeper position within the green algae,
299 in which case they should show a more stochastic distribution of evolutionary affinities
300 across all green lineages⁴⁶.

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302 Next, we tested whether our data supported a single origin for the green genes within the
303 chlorophytes, or whether the HPPGs of green origin arose through gene transfer events
304 from multiple chlorophyte lineages. We identified all amino acids that were uniquely shared
305 between ochrophytes and chlorophytes in the 31 green HPPGs for which we found no
evidence of gene duplication or subsequent lateral gene transfer into green algae,
306 ochrophytes, or other major photosynthetic eukaryotes (Table S6- sheets 1, 2<sup>145</sup>; Materials
307 and Methods). We then inferred the most probable origin in the green algal tree for each
308 uniquely shared residue as well as the earliest possible origin, taking into account gapped
309 and missing positions (Fig. 4, panel D; Fig. 4- figure supplement 4; Table S7- sheets 1, 3<sup>145</sup>). In
310 both analyses the majority of the uniquely shared residues were inferred to have originated
311 in a common ancestor of all chlorophytes, or of all chlorophyte lineages excluding the basal
312 Prasinoderma/Nephroselmis sub-category (189/289 positions in observed analysis; 100/147
313 positions in the earliest possible analysis; Fig. 4, panel D; Fig. 4- figure supplement 4; Table
314 S7- sheets 1, 3<sup>145</sup>). All other nodes within the green tree, including all specific green sub-
315 categories, shared much smaller numbers of residues with ochrophytes (Fig. 4, panel D; Fig.
316 4- figure supplement 4; Table S7- sheets 1, 3<sup>145</sup>). Thus, our data is congruent with the
317 majority of the ochrophyte green genes originating from deep within the chlorophyte
318 lineage.
319

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321 Finally, we considered whether the green genes that function in ochrophyte plastids were
322 more likely to have been acquired through endosymbiosis, or through lateral gene transfers,
323 for example from a food organism<sup>65, 66</sup> or other intracellular symbiont<sup>3</sup>. We reasoned that if
324 the green genes in ochrophytes were predominantly of endosymbiotic origin, they should
325 encode more plastid-targeted proteins than genes of alternative origin, in the same manner
326 as genes of cyanobacterial origin retained in archaeplastid genomes are biased towards
327 encoding proteins with plastid functions<sup>20</sup>. We accordingly constructed a secondary dataset,
328 consisting of 7140 non-redundant gene families that are broadly distributed across the
329 ochrophytes, and tested the targeting preferences of proteins from each HPPG (Fig. 4, panel
330 E; Fig. 4- figure supplement 5; Table S8- sheet 1<sup>145</sup>). 871 gene families resolved with the
331 green algae per BLAST top hit analysis (Fig. 4- figure supplement 6; Table S8- sheet 2<sup>145</sup>).
332 Using both ASAFind<sup>29</sup> and HECTAR<sup>30</sup>, gene families of predicted green algal origin were
333 significantly more likely to encode proteins with plastid-targeting predictions than the
334 dataset as a whole (chi-squared, P < 1E<sup>-03</sup>; Fig. 4, panel E; Fig. 4- figure supplement 5; Table
335 S8- sheet 3<sup>145</sup>). We also observed a similar, though stronger, bias towards plastid-targeted
336 proteins among the proteins of red algal origin (chi-squared, P < 1E<sup>-40</sup>; Fig. 4, panel E; Fig. 4-
337 figure supplement 5; Table S8- sheet 3<sup>145</sup>). Collectively, our data support the presence of
338 genes of chlorophyte origin in the last common ochrophyte ancestor, the majority of which
339 have predicted plastid localisations, consistent with an acquisition through a plastid
340 endosymbiosis event.
341

342 4. Functional consequences of mosaic origins for the ochrophyte plastid

343
344 *Metabolic completeness of the ochrophyte plastid*

345
346 We identified effectively complete core plastid metabolism pathways within the ancestral
347 HPPG dataset (Fig. 5, panel A; Fig. 5- figure supplement 1; Table S9- sheet 1<sup>145</sup>). The majority
348 of the remaining proteins remain plastid-encoded in some ochrophyte lineages, or are
349 dispensible for the metabolic pathway (Fig. 5- figure supplements 1, 2)<sup>67-69</sup>. In four cases
350 (isopropylmalate synthase, sedoheptulose bisphosphatase, 3-dehydroquinate synthase, and
351 shikimate kinase) lateral gene transfer and replacement events have occurred into individual
352 ochrophyte lineages since their radiation, preventing identification of a single HPPG within
353 the ancestral dataset (Fig. 5, panel A; Fig. 5- figure supplements 2-6). Taking these
354 exceptions into account, we conclude that the ancestral ochrophyte plastid proteome
355 contained the fundamental components of core plastid metabolism.
356

357 *Mosaic origins of ochrophyte plastid metabolism*

358

359 Given the mosaic evolutionary origins of ancestral ochrophyte plastid-targeted proteins, we
360 wondered whether certain evolutionary affinities might correlate with specific metabolic
361 functions. It has previously been speculated, for example, that genes acquired by diatoms
362 from green algae might have a specific role in tolerating variable light regimes<sup>42, 70, 71</sup> or
363 eliminating toxic substances from diatom plastids<sup>72</sup>. We noted that many of the pathways in
364 the ochrophyte plastid utilise a mixture of genes of red, green, host and prokaryotic origin
365 (Fig. 5- figure supplement 1), which would suggest a converse scenario: that the mosaic
366 origins of the ochrophyte plastid have led to the functional mixing of enzymes with disparate
367 evolutionary origins.

368

369 Consistent with this latter idea, we found very little evidence that individual categories of
370 HPPG (i.e., red algal, green algal, prokaryotic or host origin) are associated with particular
371 KOG annotations, as inferred by chi-squared testing (P < 0.05) against a null hypothesis that
372 all KOG families and classes are homogenously distributed across the ancestral HPPG
373 dataset, independent of evolutionary origin (Fig. 5, panel B; Fig. 5 - figure supplement 7;
374 Table S9- sheet 2<sup>145</sup>). The notable exceptions are prokaryotic HPPGs being elevated in
375 information storage and processing proteins, particularly those involved in translation, while
376 HPPGs of host origin were enriched in proteins involved in cellular processes and signalling
377 relative to the ancestral HPPG set as a whole (Fig. 5, panel B; Fig. 5 - figure supplement 7;
378 Table S9- sheet 2<sup>145</sup>). In contrast, several KOG categories were more highly represented in
379 the ancestral HPPG set than in HPPGs as a whole (Fig. 5, panel B; Fig. 5 - figure supplement
380 7; Table S9- sheet 2<sup>145</sup>).

381

382 A related question is whether proteins that catalyse adjacent steps of a biochemical
383 pathway tend to have shared or different evolutionary affinities. Multiple sets of non-native
384 proteins might be preferentially utilised by ochrophyte plastids, over homologous proteins
385 of endosymbiont origin, due to performing concerted steps in individual metabolic pathways
386 or cellular processes<sup>1, 42, 73</sup>. In this instance, pairs of proteins that interact with one another
387 would be more likely to come from the same evolutionary origin than would be expected by
388 random association. Alternatively, early ochrophyte plastids might have had no preference
389 for utilising interacting proteins of the same evolutionary origin, in which case proteins
390 involved in specific metabolic pathways might frequently have different evolutionary origins
391 to adjacent enzymes in the same pathway. Of the 313 pairs of such biochemical neighbours
392 identified in the ancestral HPPGs, only 44 shared the same evolutionary origin, which is no
393 different than that which would be expected by chance (expected number 41.05; chi-
394 squared, P=0.541; Fig. 5, panel C; Table S9- sheet 3<sup>145</sup>), Thus, interactions between proteins
395 of different evolutionary origin were forged early in the evolution of the ochrophyte plastid.

396

397 Finally, we sought correlations between expression dynamics and evolutionary affinity,
398 taking advantage of microarray data from *P. tricornutum* and *T. pseudonana*<sup>74</sup> (Table S10-
399 sheets 1-4<sup>145</sup>). We found no evidence that ancestral HPPG genes of any evolutionary origin
400 had more similar expression dynamics to each other than to those of other evolutionary
401 origins (ANOVA, P≤ 0.05; Fig. 5, panel D; Fig. 5- figure supplements 8, 9; Table S10- sheet
402 5<sup>145</sup>). For example, in both species, genes of green origin show a weaker average positive
403 coregulation with one another than they do to genes from the same species of red or of
404 prokaryotic origin (Fig. 5, panel D). Thus, the chimeric origins of the ochrophyte plastid has
405 enabled extraordinary functional mixing of proteins from early in its evolution, with each of
406 the different donors contributing proteins with a broad range of biochemical functions and
407 transcriptional patterns in response to changing physiological conditions.

408
409 *Ancient origins of chimeric plastid-targeted proteins*

410
411 We considered whether the mixing of proteins from different evolutionary sources might
412 have more substantially changed the biology of the ochrophyte plastid. It has been reported
413 by Méheust et al.<sup>75</sup> that proteins of chimeric evolutionary origin, generated by the fusion of
414 domains from different evolutionary sources, form a significant component of plastid
415 proteomes. Thus, the chimeric origins of the ochrophyte plastid might have enabled the
416 creation of syncretic proteins not found in the endosymbiont or host ancestors. We
417 identified orthologues of seven chimeric proteins identified in this study within our dataset,
418 underlining their importance for the establishment of the ochrophyte plastid (Fig. 6, panel
419 A)<sup>75</sup>.

420
421 Next, we assessed whether the mosaic composition of the ochrophyte plastid proteome had
422 also enabled the establishment of novel chimeric fusion proteins, unique to ochrophyte
423 plastids. Using the taxonomic subdivisions erected for this study, we identified further
424 chimerism events in members of 42 ancestral HPPGs (Fig. 6, panel B; Table S9- sheet 1,
425 sections 4, 5; Table S11<sup>145</sup>). These include three HPPGs (e.g. NADH-ubiquinone
426 dehydrogenase) in which chimeric proteins have formed through the fusion of modules of
427 prokaryotic origin to others of eukaryotic origin, and seven HPPGs (e.g. translation factor EF-
428 3b, and an N6-adenine DNA methyltransferase) in which fusion events have occurred
429 between modules of red origin and modules of green origin (Fig. 6, panel B). To our
430 knowledge, neither of these types of fusion event have previously been reported for plastid-
431 targeted proteins<sup>75</sup>. The chimeric proteins contain domains from a wide range of
432 evolutionary origins: 20 (47.6%) contain a domain of inferred green origin and 18 (43.8%)
433 contain a domain of host origin.

434
435 Amongst the chimeric proteins identified, we found two that probably fused in the
436 ochrophyte ancestor (Fig. 6, panels A, B). In one case, a bifunctional protein containing an N-
437 terminal 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthase and C-terminal GTP
438 cyclohydrolase II protein, which performs two consecutive steps of riboflavin biosynthesis<sup>76</sup>,
439 has formed through the fusion of a cyclohydrolase domain of probable host origin to a
440 synthase domain of probable red algal or actinobacterial origin (Fig. 6- figure supplements
441 1,2). While bifunctional DHBP synthase/ GTP cyclohydrolase proteins are known in bacteria,
442 red algae and plants (Fig. 6- figure supplement 1)<sup>48, 76</sup>, in these taxa the DHBP synthase
443 domain is located at the protein C-terminus; thus, an analogous but topographically distinct
444 fusion protein has evolved in ochrophytes. In a second, previously reported case<sup>75</sup>, a C-
445 terminal plastid-targeted Tic20 subunit of red algal origin has become fused to an N-terminal
446 EF-hand motif, for which no clear evolutionary outgroup (to an e value of below 1x 10<sup>-05</sup>)
447 could be found (Fig. 6- figure supplement 3). Thus, the fusion of proteins of different
448 evolutionary origins has generated new functions in the ochrophyte plastid proteome.

449
450 *Ancestral and bidirectional origins of dual targeting in ochrophytes*

451
452 Finally, we considered whether the acquisition of the ochrophyte plastid might have also
453 fundamentally altered the biology of the host cell, by contributing proteins to host processes
454 and structures outside the plastid. As an exemplar system, we considered dual targeting of
455 proteins to plastids and mitochondria, which is known to occur extensively in plants<sup>77, 78</sup>, and
456 has recently been documented in diatoms<sup>79</sup> and in other complex plastid lineages<sup>79, 80</sup>.
Previous studies have speculated that dual targeting may arise early in plastid evolution, for example through the retargeting of proteins from the host mitochondria to the plastid, or equally via the adaptation of proteins of plastid origin to the mitochondria¹⁸, ⁷⁷.

We indeed identified proteins that appeared to be dual targeted to the plastid and a secondary organelle (Fig. 2- figure supplements 5, 6), which we verified to be the mitochondria using Mitotracker orange (Fig. 7 panel A). In at least two cases (histidyl- and prolyl-tRNA synthetase) this dual targeting is a conserved feature, as we identified the same fluorescence patterns both in *P. tricornutum* and using heterologous expression constructs from *G. foliaceum* and *N. gaditana* (Fig. 7, panel A; Fig.7- figure supplement 1). To determine whether dual targeted proteins were ancestrally present in the ochrophyte plastid, we developed an *in silico* pipeline, based on experimental data, to identify probable dual targeted proteins from within the HPPG dataset (Fig. 7- figure supplement 2; Table S12-sheet 1¹⁴⁵). In total, we identified 1103 HPPGs that included at least one member that was probably dual targeted to plastids and mitochondria (Table S12- sheet 1¹⁴⁵). 34 of these HPPGs passed the conservation thresholds previously inferred to signify an ancestral origin (Table S12- sheet 1¹⁴⁵). Thus, dual targeting is an ancestral feature of the ochrophyte plastid.

We then considered the origins of the ancestrally dual targeted ochrophyte proteins. 15 of the 34 putative ancestrally dual targeted HPPGs were orthologous to HPPGs of clear evolutionary origin; of these, the majority (11/15; 73%) were of red algal, i.e., probable endosymbiont origin (Fig. 7, panel B; Table S12- sheet 2¹⁴⁵). To determine how these dual targeted HPPGs have altered the biology of the host, we searched for gene families corresponding to aminoacyl-tRNA synthetases within the 7140 non-redundant gene families previously identified to be shared across the ochrophytes (Table S8- sheet 1¹⁴⁵). To enable function of the translational machinery, each genome within the ochrophyte cell (i.e., nucleus, mitochondrion, and plastid) requires aminoacyl-tRNA synthetase activity for each amino acid⁷⁹; thus, if any class of aminoacyl-tRNA synthetase is represented by fewer than three genes, then individual tRNA synthetases must support the biology of multiple organelles through dual targeting. We identified seven classes of tRNA synthetase for which there were only two gene families in the ochrophyte ancestor, one corresponding to a cytosolic enzyme, and the other to an enzyme that was probably dual targeted to both the mitochondria and plastid. These include five cases in which the dual targeted tRNA synthetase was of apparent red algal, i.e., endosymbiont origin (Fig. 7, panel C). Thus, the acquisition of the ochrophyte plastid also altered the biology of the mitochondria, with dual targeted proteins of endosymbiont origin functionally replacing endogenous mitochondrial-targeted homologues.

## 5. Complex evolutionary origins of CASH lineage plastids

A pelagophyte/ dictyochophyte origin of the haptophyte plastid proteome

We considered whether our dataset provides evidence for any of the other CASH lineage plastids (cryptomonads, haptophytes, or photosynthetic alveolates) originating within the ochrophytes¹, ⁵, ⁷, or evidence for gene transfer from ochrophytes into lineages with complex plastids of green algal origin (chlorarachniophytes and euglenids)⁸¹, ⁸². In a majority (243/437) of trees in which they could be assigned a clear origin, plastid-targeted proteins from haptophytes resolved at a position within the ochrophyte clade (Materials and Methods; Fig. 8, panel A; Table S4- sheet 5¹⁴⁵). All other groups (except for dinotoms, which have well-defined plastids of diatom origin²⁰, ⁵⁶) generally branched externally rather than within the ochrophyte clade (Fig. 8, panel A). Indeed, the proportion of haptophyte proteins
508 that resolved within the ochrophytes was found to be significantly greater than any of the
509 other groups except for dinotoms (chi-squared, P < 1 X 10⁻⁵; Table S4- sheet 5¹⁴⁵).

510
511 We noted that the plastid-targeted haptophyte proteins of ochrophyte origin were biased
512 towards specific origins, with over half of the proteins that grouped with a specific
513 ochrophyte lineage (100/178) resolving with members of the hypogyristea (i.e.,
514 pelagophytes, dictyochophytes, and bolidophytes; Fig. 8- figure supplement 1; Table S4-
515 sheet 5¹⁴⁵). No such bias could be observed in any other CASH lineage, in which invariably a
516 significantly smaller proportion of proteins were found to resolve with hypogyristean
517 lineages (chi-squared P < 0.01; Fig. 8- figure supplement 1; Table S4- sheet 5¹⁴⁵). We
518 additionally explored whether there might be unique synapomorphies shared between one
519 ochrophyte lineage and the haptophytes. We found 53 ASAFind-generated HPPGs that
520 contained a majority (≥2/3) of the haptophyte sub-categories and contained at least one
521 member of the hypogyristea, but contained no other ochrophyte orthologues (Fig. 8, panel
522 B; Table S2- sheet 2, section 3¹⁴⁵). This was significantly more than would be expected (28.3,
523 chi-squared P = 0.00013) through a random assortment of all HPPGs that were uniquely
524 shared between haptophytes and one ochrophyte lineage, corrected for the relative size of
525 each dataset (Materials and Methods). We similarly found a significantly larger number of
526 HPPGs to be uniquely shared between a majority of both the haptophytes and a majority
527 (≥2/3) of the hypogyristean sub-categories (15, expected number 8.0, P= 0.034; Fig. 8, panel
528 B) or shared between a majority of hypogyristea and at least one haptophyte sub-category
529 (28, expected number 12.9, P= 0.00073; Table S2- sheet 2, section 3¹⁴⁵; Fig. 8, panel B). Thus,
530 our data supports a specific gene transfer event between the hypogyristea and the
531 haptophytes.

532
533 We investigated whether there is a more specific origin for the ochrophyte sequences in
534 haptophyte plastids. First, we tabulated the individual ochrophyte sub-categories identified
535 in the first sister group to haptophyte sequences, of which the greatest number (94)
536 resolved specifically with pelagophyte and dictyochophyte sequences, rather than with
537 bolidophytes, non-hypogyristean lineages, or more ancestral nodes (Fig. 8, panel C; Fig. 8-
538 figure supplement 2). Next, we extracted all of the haptophyte plastid-targeted sequences
539 assembled into each ancestral ochrophyte HPPG, performed BLAST top hit analysis (Table
540 S13- sheets 1-3¹⁴⁵), and identified sequences for which the best hit was from the same
541 ochrophyte lineage (diatoms, hypogyristea, or chrysista) as the tree sister group (Table S13-
542 sheet 4¹⁴⁵). We performed separate analyses for query sequences from each of the three
543 haptophyte sub-categories considered in our analysis (pavlovophytes, prymnesiales, or
544 isochrysidales). In each case, at least 50% of the sequences that produced an evolutionarily
545 consistent series of top hits resolved either with the pelagophytes or dictyochophytes (Fig.
546 8- figure supplement 3; Table S13- sheet 4¹⁴⁵). Thus, these proteins originated within an
547 ancestor of the pelagophyte/ dictyochophyte lineage.

548
549 We next tested the probable direction of the gene transfer events. We reasoned that if the
550 genes identified within our study had been transferred from an ancestor of pelagophytes
551 and dictyochophytes into the haptophytes, then we should also see a strong secondary
552 signal linking the haptophytes to earlier ancestors of the pelagophyte/ dictyochophyte clade,
553 for example the common ancestor of hypogyristea and diatoms. We inspected the
554 secondary BLAST top hits associated with genes shared between haptophytes and
555 hypogyristea (Fig. 8- figure supplement 4; Table S13- sheet 5¹⁴⁵), and the next deepest sister-
556 groups to haptophyte proteins that are of probable pelagophyte or dictyochophyte origin in
557 each single-gene tree (Fig. 8- figure supplement 4; Table S4- sheet 2, section 6¹⁴⁵). The
558 majority of haptophyte proteins of hypogyristean origin in single-gene trees (65/100) clearly
resolved within a broader HPPG containing multiple ochrophyte lineages, and this bias was corroborated by the specific sister groups associated with each protein as inferred by heat map analysis (Fig. 8- figure supplement 4, panel A). Moreover, the majority of haptophyte proteins with hypogyristean BLAST top hits, and hypogyristean proteins with haptophyte BLAST top hits (48/ 86 sequences total) had next best BLAST hits against diatoms (Fig. 8- figure supplement 4, panel B). We additionally tabulated the earliest and latest possible origin points of amino acid residues that were uniquely shared between haptophytes and some but not all ochrophyte lineages, from a dataset of 37 HPPGs for which there was a clear evolutionary affinity between haptophytes and ochrophytes and strict subsequent vertical inheritance (Fig. 8, panel D; Fig. 8- figure supplement 5; Table S6- sheets 3, 4<sup>145</sup>). A greater number of the uniquely shared residues were found to be conserved between the haptophytes and the common ancestor of hypogyristea and diatoms, than were specifically only shared with pelagophyte and dictyochophyte sequences, both per the latest possible origin (139 residues shared with hypogyristea and diatoms; 99 residues with pelagophytes and dictyochophytes; Fig. 8, panel D; Table S7- sheets 2, 3<sup>145</sup>) and per the earliest possible origin (46 residues shared with hypogyristea and diatoms; 41 residues with pelagophytes and dictyochophytes; Fig. 8- figure supplement 5; Table S7- sheets 2, 3<sup>145</sup>). This specifically supports a transfer of plastid-targeted proteins from an ancestor of the pelagophyte/ dictyochophyte clade into the haptophytes, rather than the other way around.

Finally, we tested whether these proteins were likely to have been acquired through an endosymbiotic event. We reasoned that the genes acquired by haptophytes through endosymbiotic events should encode a greater proportion of plastid-targeted proteins than would be observed with genes of alternative origin. We accordingly constructed a dataset of 12,728 non-redundant gene families that were broadly distributed across the haptophytes (Table S14- sheet 1<sup>145</sup>), of which 772 were of probable hypogyristean origin (Fig. 8- figure supplement 6; Table S14- sheet 2<sup>145</sup>). A significantly larger proportion of the ancestral haptophyte gene families of hypogyristean origin were predicted by ASAFind to be targeted to the plastid than would be expected by random distribution of the data (observed number 43, expected number 22.8, chi-squared P= 2.2 x 10<sup>-05</sup>; Fig. 8, panel E; Table S14- sheet 3<sup>145</sup>), consistent with an endosymbiotic origin. Thus, our data support an endosymbiotic uptake of an ancestor of the pelagophytes and dictyochophytes by an ancestor of the haptophytes.

Phylogenetic discrepancies between the haptophyte plastid proteome and genome

The transfer of plastid-targeted proteins from the pelagophyte/dictyochophyte clade into the haptophytes is surprising, as previous studies have indicated that the haptophyte plastid genome originates either as a sister-group to the entire ochrophyte lineage⁵ or to the cryptomonads⁸³,⁸⁴. To verify this discrepancy we constructed two plastid trees, one using 54 conserved proteins that are encoded in all sequenced red lineage and glaucophyte plastids (Fig. 9, panel A; Table S15- sheet 1<sup>145</sup>), and one using a smaller subset of 10 plastid-encoded proteins that were detected in many of the transcriptome libraries used in this study (Fig. 9, panel B; Table S15- sheet 1<sup>145</sup>).

A specific sister-group relationship between the cryptomonads and haptophytes was recovered, with moderate to strong bootstrap support, in both the gene-rich tree (Fig. 9, panel A) and the taxon-rich tree (Fig. 9, panel B). Both trees also strongly supported the monophyly of ochrophyte plastid genomes (Fig. 9). Alternative topology tests rejected any possibility that the haptophyte plastid originated within the ochrophytes (Fig. 9- figure supplement 1; P≤ 0.05). Similarly, trees calculated from alignments in which fast-evolving sites and clades had been serially removed, and in which the alignment had been recoded to
610 minimise amino acid composition biases (Fig. 9- figure supplement 2; Table S15- sheet 2; Table S16<sup>145</sup>) either recovered a sister-group relationship between haptophytes and cryptomonads, or placed haptophytes as the sister group to all ochrophytes. We additionally generated and inspected single-gene tree topologies for each of the constituent genes used to generate each concatenated multigene alignment, and could not find any that confidently resolved a sister-group relationship between haptophytes and the pelagophyte/dictyochophyte clade (Fig. 9- figure supplement 3; Table S15- sheet 3<sup>145</sup>). Finally, we found only three residues in the alignment that were uniquely shared among all four haptophytes and the sole representative of pelagophytes and dictyochophytes (*Aureococcus*) in the gene-rich dataset, and no residues that were shared between a majority of the haptophytes and at least one pelagophyte or dictyochophyte sequence in the taxon-rich dataset (Fig. 8, panel C; Table S17- sheet 4<sup>145</sup>). In contrast, we found large numbers of residues that were shared uniquely by haptophytes and other lineages (Fig. 9, panel C; Table S17- sheet 4<sup>145</sup>). This strong support for a relationship between haptophytes and cryptomonads is inconsistent with phylogenetic artifacts such as coevolution between specific protein complexes<sup>58, 85</sup> or gene duplication and differential loss of paralogues<sup>86</sup>, in which case there should still be a detectable underlying signal linking it to the pelagophytes and dictyochophytes. We conclude that while many plastid-targeted haptophyte proteins originate from an ancestor of the pelagophytes and dictyochophytes, the haptophyte plastid genome does not.

629

## Discussion

631

632 In this study, we have reconstructed an experimentally verified dataset of 770 plastid-targeted proteins that were present in the last common ancestor of all ochrophytes (Figs. 1, 2). Our dataset accordingly provides windows into the evolutionary origins of the ochrophyte plastid lineage. These include evidence for a green algal contribution to ochrophyte plastid evolution and a late acquisition of the ochrophyte plastid following divergence of the ochrophyte lineage from oomycetes (Figs. 3, 4). This latter finding is particularly interesting as molecular divergence estimates place the ochrophytes as diverging from the oomycetes no more than 90 million years prior to the radiation of ochrophyte lineages<sup>87, 88</sup>. Assuming that these estimates are reliable, our dataset represents some of the earliest proteins to support the ochrophyte plastid following its endosymbiotic uptake. We also provide evidence for widespread mixing of proteins of different evolutionary origin in the ancestral ochrophyte plastid (Fig. 5), including evidence for the formation of new fusion proteins through the recombination of domains of different evolutionary origins (Fig. 6), and a bidirectional mixing of proteins derived from the endosymbiont with proteins from host organelles via dual targeting (Fig. 7). A schematic outline of these results is shown in Fig. 10.

648

649 Many questions nonetheless remain to be answered. It remains to be determined whether the *in silico* prediction facilitated by programmes such as ASAFind and HECTAR are sufficient to enable the identification of all ochrophyte plastid proteins<sup>29, 30</sup>. This is particularly pertinent in the context of dual targeted proteins, insofar as the dataset of 34 potentially ancestrally dual targeted proteins identified in this study may not include proteins that are dual targeted to the plastid and other cellular organelles, such as the ER<sup>89</sup>, cytoplasm<sup>90</sup>, or nucleus<sup>91</sup>. We note also that, based on the fluorescence patterns observed with the exemplar proteins within this study (Figs. 2, 7), ASAFind and HECTAR may identify proteins targeted to the periplastid compartment, as well as to the plastid stroma. While these periplastid and multipartite proteins probably form an important part of plastid physiology, it will be interesting to dissect the specific signals associated with the targeting of proteins to individual sub-compartments within CASH lineage plastids<sup>55, 92</sup>.
661 Another major question concerns the origins of plastid-targeted proteins of green algal
662 origin in ochrophytes. Overall, our data supports the targeting of a significant complement
663 of proteins of chlorophyte origin to the ochrophyte plastid (Fig. 4). It remains to be
664 determined, however, what the exact chlorophyte donor was, and how these genes may
665 have been acquired. It is possible that the green genes were transferred into the ochrophyte
666 lineage via lateral gene transfer, either from a range of different green algal sources or
667 repeatedly from one lineage (for example, a semi-permanent intracellular symbiont³),
668 although neither scenario would explain the bias in green algal genes in ochrophyte
669 genomes towards encoding proteins of plastid function (Fig. 4, panel D). An alternative
670 possibility might be a cryptic green algal endosymbiosis in the evolutionary history of the
671 host, as has been previously suggested¹, ⁴⁴ (Fig. 10), or a more convoluted pattern of
672 acquisition. We note, for example, that the green genes identified in our study are not only
673 plastid-targeted across the ochrophytes, but are apparently shared with haptophytes and
674 cryptomonads (Fig. 10- figure supplement 1), which would be equally consistent with them
675 having been present in a common ancestor of the CASH lineage plastid, and relocated to
676 each host nuclear lineage following endosymbiosis (Fig. 10). Thus, pinpointing the exact
677 nature and timing of the green gene transfer into ochrophytes rests not only on more
678 extensive sequencing of deep-branching chlorophyte lineages, but also on characterising the
679 genome composition of the closest aplastidic relatives of extant ochrophytes (e.g.,
680 *Develorapax*, *Pirsonia*⁶), and the closest red algal relative of CASH lineage plastids, which
681 remains unknown¹, ⁴.
682

683 We also provide evidence for a chimeric origin of the haptophyte plastid (Figs. 8, 9). A
684 schematic outline of these results is shown in Fig. 10- figure supplement 2. We have shown
685 that a significant number of plastid-targeted proteins found in haptophytes originate from
686 an ancestor of the pelagophytes and dictyochophytes (Fig. 8). This relationship is supported
687 by multiple lines of evidence- i.e., uniquely shared proteins, single-gene tree topologies,
688 BLAST top hit analysis, and analysis of synapomorphies in multigene alignments (Fig. 8 and
689 supplements). Alongside the bias of haptophyte genes of hypogyristean origin encoding
690 proteins of plastid function (Fig. 8- panel E), these observations argue against these genes
691 having been acquired through multiple independent lateral gene transfer events, and
692 instead support an endosymbiosis event. We note that other studies have shown strong
693 evidence for gene transfers between haptophytes and individual members of the
694 hypogyristea: for example, Stiller *et al*. have demonstrated a strong enrichment in BLAST top
695 hits against haptophytes, from the genome of the pelagophyte *Aureococcus*
696 *anophageferrensis*, compared to other ochrophyte genomes⁵. We additionally note that an
697 ancestral gene transfer from a pelagophyte/ dictyochophyte ancestor into the haptophytes
698 is a chronologically realistic scenario: molecular clock estimates place the pelagophytes and
699 dictyochophytes diverging between 300 and 700 million years before present⁸⁷, ⁹³, which
700 broadly overlaps with the molecular dates estimated for the radiation of the haptophytes in
701 the same studies⁸⁷, ⁹³, and precedes the first haptophyte microfossils, identified ca. 220
702 million years before the present⁹⁴.
703

704 Finally, we verify that the evolutionary links between haptophyte and the pelagophyte/
705 dictyochophyte clade in terms of plastid-targeted proteins are not supported by phylogenies
706 of the haptophyte plastid genome (Fig. 9). Other multigene phylogenies of red lineage
707 plastid genomes have similarly demonstrated that the haptophyte plastid genome instead
708 resolves as a sister-lineage either to cryptomonads or to all ochrophytes⁵, ³⁸, ⁸³, ⁸⁴.
709 Furthermore, the structure and content of haptophyte and hypogyristean plastid genomes
710 are dissimilar: for example, haptophyte plastids possess an *rp136* gene that has been laterally
711 acquired from a bacterial donor and is shared with cryptomonad plastids but absent from
712 ochrophytes⁹⁵, and ochrophyte plastids no longer retain genes encoding the plastid division
713 machinery proteins *minD* and *minE*, which remain plastid-encoded in haptophytes and
714 cryptomonads⁹⁶. Similarly, extant haptophyte plastids have comparatively large plastid
715 genomes and possess a conventional quadripartite structure⁹⁷, whereas extant pelagophyte
716 plastids have a reduced coding content compared to other photosynthetic ochrophytes,
717 cryptomonads and haptophytes, and have secondarily lost the plastid inverted repeat⁹⁸, ⁹⁹,
718 although it is not yet known whether dictyochophyte plastids share this reduced structure.

719 The discrepancy between the pelagophyte/ dictyochophyte origin of the haptophyte plastid
720 proteome and the clear non-ochrophyte origin of its plastid genome might be explained by
721 several different evolutionary scenarios. One possibility would be a serial endosymbiosis
722 event deep in haptophyte evolutionary history, in which an ancient plastid derived from a
723 pelagophyte/ dictyochophyte ancestor was acquired by the haptophyte common ancestor,
724 then replaced subsequently by a plastid of non-ochrophyte origin (Fig. 10- Figure
725 supplement 2). Verifying this scenario, or its alternatives (such as lateral gene transfer from
726 pelagophyte or dictyochophyte algae into the algal ancestors of the haptophyte plastid)
727 rests on identifying the exact origin of the current haptophyte plastid genome, and in
728 particular demonstrating that the haptophyte plastid genome originates from within (rather
729 than forms a sister-group to) a major lineage of eukaryotic algae other than ochrophytes
730 (Fig. 10- Figure supplement 2). For this, sequence data from early-diverging members of the
731 cryptomonads and haptophytes will be particularly important⁴¹, ¹⁰⁰, ¹⁰¹. It also remains to be
732 determined whether other CASH lineage plastids, such as the peridinin-type plastids found
733 in most photosynthetic alveolates, originate within the ochrophytes⁷, ²⁰. Similar plastid
734 proteome reconstructions, using bespoke datasets for these species, will be particularly
735 useful in unravelling their disparate evolutionary origins.

736 Overall, our dataset provides valuable and deep insights into the chimeric origins and
737 complex fates of a major group of eukaryotic algae. Further studies using more sensitive
738 pipelines, or using analogous datasets from other major CASH lineages, may elucidate the
739 evolutionary and physiological diversification of plastids in the open ocean.

740

## Materials and Methods

742

### Identification of ancestral plastid-targeted ochrophyte proteins

744

745 Ancestral plastid-targeted proteins in ochrophytes were identified via a composite pathway,
746 consisting of *in silico* prediction, identification of conserved proteins using BLAST, alignment,
747 and single-gene tree building. First, the complete protein libraries annotated from eleven
748 ochrophyte genomes (the diatoms *Phaeodactylum tricornutum*⁵⁹, *Thalassiosira
749 pseudonana*⁹, *Thalassiosira oceanica*¹⁰², *Fistulifera solaris*¹⁰³, *Fragilariopsis cylindrus*, *Synedra
750 acus*¹⁰⁴, and *Pseudonitzschia multiseries*; the pelagophyte *Aureococcus anophageferrens*¹¹;
751 the eustigmatophytes *Nannochloropsis gaditana* and *Nannochloropsis salina*³⁷, ¹⁰⁵; and the
752 kelp *Ectocarpus siliculosus*¹⁰; Table S1- sheet 1¹⁴⁵), were screened using the ochrophyte
753 plastid-targeting predictors ASAFind²⁹ (used in conjunction with SignalP version 3.0¹⁰⁶; Table
754 S2¹⁴⁵) and HECTAR³⁰ (integrated into a Galaxy¹⁰⁷ instance available at http://webtools.sb-
755 roscoff.fr; Table S3¹⁴⁵). All proteins that were deemed to possess plastid-targeting sequences
756 (regardless of the confidence score applied by ASAFind²⁹) were retained for further
757 inspection.

758

759 Possible conserved plastid-targeted sequences (i.e. homologous plastid-targeted protein
760 groups, or HPPGs) were next identified using a customised BLAST protocol. First, a library of
761 non-redundant proteins was generated to serve as seed sequences for further searches.
Each plastid-targeted protein identified from ochrophyte genome sequences was searched by BLASTp against a modified Uniref¹⁰⁸ library, and the expect values for all top hits were extracted, to yield a floating BLAST threshold below which orthologous proteins were identified. All sequences from lineages with a history of secondary endosymbiosis were first removed from the Uniref library in order to avoid the confounding effects of gene transfer from current and former symbionts⁵, ⁷, ⁸¹, ⁸². The removed lineages included cryptomonads, centrohelids, telonemids, haptophytes, alveolates, rhizaria, euglenids, and plastid-bearing stramenopiles. All of the ochrophyte genome-derived plastid-targeted proteins were searched against one another by BLAST, and proteins that matched one another with an expect score lower than the first outgroup hit (or were retrieved as a stronger match than the outgroup hit if the expected values of both were zero), and thus likely correspond to different proteins within the same monophyletic plastid protein cluster, were merged. Only one protein was retained as the seed sequence for subsequent growth of each cluster: this was defined first via organism (in order of preference: *P. tricornutum*, *T. pseudonana*, *P. multiseries*, *F. cylindrus*, *S. acus*, *A. anophageferrens*, *E. siliculosus*, *N. gaditana*, *N. salina*, *T. oceanica*, *F. solaris*) and, where more than one protein was available for a given organism, the protein with the lowest BLAST expect value against the corresponding uniref top hit.

Next, plastid-targeted protein sequences were sought from all available ochrophyte sequence data. A search database was built from all eleven completed ochrophyte genomes, 147 ochrophyte sequence libraries from the Marine Microeukaryote Transcriptome Sequence Project³⁴, eleven further ochrophyte transcriptome sequencing projects⁶⁴, ¹⁰⁹, ¹¹⁰ and uniref. Cross-contamination was removed from MMETSP transcriptomes as previously described⁵⁷. Briefly, this procedure compares the nucleotide sequences of contigs assembled from each MMETSP library by pairwise BLAST, and defines a separate cross-contamination threshold for each pair of MMETSP libraries based on their distribution of BLAST percent identities. These distributions should each contain a peak centered on the average nucleotide percent identity of transcripts between the two species. In addition, in the presence of cross-contamination, there should be a second peak at 100% identity. The procedure defines the cross-contamination threshold as the minimum between these two peaks; above the threshold, contigs (and the proteins predicted from them) are considered to be potentially cross-contaminated. In total, 2.5% of the MMETSP contigs were discarded through this method. A summary of the number of contigs discarded is provided in Table S1-sheet 2, section 1¹⁴⁵.

Each decontaminated sequence was trimmed at the N-terminus to the first methionine present, and binned into one of eleven different evolutionary categories, based on recent multigene phylogenetic trees for ochrophytes and diatoms¹², ¹¹¹⁻¹¹³ (fig. 1, panel A; Table S1-sheet 1¹⁴⁵). These consisted of: three chrysistan lineages (the "PX clade" of phaeophytes, xanthophytes and related lineages; raphidophytes; and the "PESC clade" of pinguiophytes, eustigmatophytes, synchromophytes, and synurophytes/chrysophytes), three hypogyristean lineages (pelagophytes; dictyochophytes; and bolidophytes), and five diatom lineages (the basally divergent genus *Corethron*; radial centric lineages such as Coscinodiscophytes and Rhizosoleniaceae; the polar centric Thalassiosirales and Skeletonemataceae, which appear to be relatively distantly related to pennate diatoms¹¹¹,¹¹³; polar centric lineages such as Odontellids and Chaetocerotales that appear to be more closely related to pennate diatoms¹¹¹,¹¹³; and finally all pennate lineages). These binned sequences were then searched for plastid-targeted proteins by ASAFind and HECTAR as before.

The seed sequences for the resulting non-redundant HPPGs were searched against the enlarged plastid sequence library using BLASTp. Proteins that matched against seed
813 sequences with a lower expect value than the outgroup best hit (or were retrieved as a
814 stronger match than the outgroup hit if the expected values of both were zero), were added
815 to each HPPG. Next, three custom thresholds were defined that were particularly successful
816 in distinguishing probable proteins of true plastid localisation from false positives (fig. 1,
817 panel B). For this, conservation patterns were selected that maximised the relative
818 enrichment in proteins with unambiguous plastid functions (i.e., were annotated to function
819 in photosynthesis, to constitute integral parts of the plastid thylakoid or inner membranes,
820 or corresponded to the expression products of genes that are plastid-encoded in red algae
821 but have been apparently relocated to the ochrophyte nucleus⁹⁷ or that corresponded to
822 proteins previously verified experimentally to localise to ochrophyte plastids²⁹, ³⁰, ¹¹⁴, ¹¹⁵), and
823 thus should contain relatively fewer examples of mispredicted proteins within the dataset.
824 At the same time, conservation patterns were selected that minimised the number of HPPGs
825 identified as conserved from a negative control dataset (consisting of HPPGs assembled
826 using seed sequences from the published genome sequences of the cryptomonad *Guillardia
827 *theta*¹⁷ or the haptophytes *Emiliania huxleyi*¹¹⁶ and *Chrysochromulina tobin*¹¹⁷, and for which
828 no plastid-targeted orthologues were detected in any of the ochrophyte genome sequences
829 used in this study). The thresholds corresponded to: orthologues in a majority (≥2/3) of
830 chrysistan and a majority (≥3/5) of diatom lineages; a majority of chrysistan and a majority
831 (≥2/3) of hypogyristean lineages; and at least one chrysistan, and a majority of both
832 hypogyristean and diatom lineages (fig. 1).
833

834 All of the HPPGs that passed at least one threshold were extracted, and homology for each
835 HPPG was confirmed individually (Table S4- sheet 1¹⁴⁵). First, each HPPG was aligned using
836 20 iterations of MUSCLE v8¹¹⁸, followed by the in-built alignment programme integrated into
837 GeneIOUS v 4.76¹¹⁹, under the default criteria. Each HPPG alignment was manually
838 inspected, and proteins that failed to align with the genomic sequences, clearly terminated
839 within the conserved region of the protein, or were truncated at the N-terminus by a length
840 of greater than 50 amino acids (i.e. the approximate length of an ochrophyte plastid-
841 targeting sequence²⁹, ¹¹⁴) were removed, following which HPPGs that no longer passed the
842 taxonomic criteria defined for conservation were eliminated (Table S4- sheet 1¹⁴⁵). Next,
843 each HPPG was enriched with the sequences for the top 50 hits obtained when the seed
844 sequence was searched against the modified uniref library as detailed above, alongside the
845 single best hit for composite transcriptome and genome libraries constructed for 36
846 eukaryotic sub-categories (Table S1- sheet 1¹⁴⁵), and realigned against this reference. The
847 transcriptome components of the reference sequence libraries were cleaned of residual
848 contamination as defined above, and 23 individual MMETSP libraries were additionally
849 excluded due to evidence of further contamination (Table S1- sheet 2¹⁴⁵). Sequences that
850 failed to align were removed, and HPPGs that failed to meet the criteria for conservation
851 following alignment were eliminated (Table S4- sheet 1¹⁴⁵).

852
853 Finally, each HPPG was trimmed at the N- and C-termini to (respectively) the first residue
854 and last residue visually identified to be conserved in > 70% of the sequences in the
855 alignment, corresponding to the probable conserved domain of the protein. Each HPPG was
856 then trimmed with trimAl using the -gt 0.5 option¹²⁰. 100 trees were calculated for each
857 trimmed alignment using RAxML, with the JTT substitution model + gamma correction¹²¹.
858 The consensus tree from the 100 bootstrap replicates was manually inspected for the
859 presence of a clade of ochrophyte proteins, containing sufficient sequences to pass the
860 criteria for conservation defined above, that was either monophyletic, or paraphyletic to the
861 inclusion of only one of five different non-ochrophyte groups (prokaryotes, red algae, green
862 algae, aplastidic stramenopiles, and all other eukaryotes excluding CASH lineages, rhizaria
863 and euglenids; Table S4- sheet 1<sup>145</sup>). HPPGs that passed this final stage of analysis were
864 deemed to correspond to ancestrally plastid-targeted proteins (Table S4- sheet 2<sup>145</sup>).

865
866 All identified plastid-targeted proteins, HPPGs, full aligned HPPGs, and single-gene trees
867 have been made publically accessible through the University of Cambridge dSpace server
868 (https://www/repository.cam.ac.uk/handle/1810/261421145).

869
870 Generation of fluorescence expression constructs for Phaeodactylum tricornutum

871
872 Phaeodactylum tricornutum 1.86 (CCMP2561), Nannochloropsis gaditana CCMP526, and
873 Glenodinium foliaceum PCC499 were maintained in liquid cultures of f/2 medium
874 supplemented with vitamins, and 100 µg/ ml each of ampicillin, streptomycin, kanamycin
875 and neomycin, in a constant 19°C environment in a 12h: 12h cycle of 150 µE m-2 s-1 light:
876 dark. P. tricornutum was maintained on an orbital shaker at 100 rpm, while N. gaditana and
877 G. foliaceum were maintained as stationary cultures. Large volume cultures of P.
878 tricornutum (e.g. cultures grown for transformation by bombardment) were grown in
879 artificial seawater, supplemented with vitamins but without antibiotics.

880
881 Total cellular RNA was extracted from c. 30 ml volumes of late log phase culture from each
882 species using a modified Trizol phase extraction and DNase treatment protocol as described
883 elsewhere<sup>21</sup>. Each RNA sample was tested for integrity by gel electrophoresis and quantified
884 by a nanodrop spectrophotometer, and confirmed to be free of residual DNA contamination
885 by direct PCR using universal eukaryotic 18S rDNA primers<sup>122</sup>. Approximately 200 ng purified
886 RNA from each species was used as the template for cDNA synthesis, using a Maxima First
887 Strand cDNA Synthesis Kit (Thermo), following the manufacturer's instructions.

888
889 Nucleotide sequences encoding plastid-targeted proteins of unusual provenance were
890 identified using the complete genome sequences of Phaeodactylum tricornutum and
891 Nannochloropsis gaditana<sup>37, 59</sup>, and the Glenodinium foliaceum CCAP1116/3 transcriptome
892 library assembled as part of MMETSP<sup>34, 123</sup> (Table S5<sup>145</sup>). Two primers were designed for each
893 sequence: a PCR forward primer corresponding to the 5' end of the ORF, and a
894 translationally in-frame PCR reverse primer positioned a minimum of 45 bp into conserved
895 domain of the protein sequence (Table S5<sup>145</sup>). These primers were respectively fused to 5'
896 fragments complementing the 3' end of the P. tricornutum FcpA promoter, and the 5' end of
897 the GFP CDS. For one gene (the novel plastid protein), PCR reverse primers were designed
898 complementary to the 3' end of the CDS of each gene due to the lack of a verifiable CDD; a
899 full-length PCR reverse primer was additionally designed against the histidyl-tRNA
900 synthetase sequence from Nannochloropsis gaditana due to failure to obtain functional
901 expression from N-terminal constructs (data not shown).

902
903 High-fidelity PCR products were amplified with each primer pair from the corresponding
904 cDNA product using Pfu DNA polymerase (Thermo), per the manufacturer's instructions. In
905 two cases (Nannochloropsis gaditana peroxisomal membrane protein, and the novel plastid
906 protein) inserts were amplified from synthetic, codon-optimised constructs, designed to
907 maximise expression levels in Phaeodactylum tricornutum (Eurofins). Each product was
908 separated by DNA gel electrophoresis, cut, purified using a PCR gel extraction column kit
909 (Macherey-Nagel), quantified using a nanodrop spectrophotometer, and verified by Sanger
910 sequencing (GATC Biotech). The purified products were then used for Gibson ligation
911 reactions<sup>124</sup> (NEB), following the manufacturer's instructions, using linearised and DpnI-
912 treated vector sequence generated from the pPhat-eGFP vector<sup>35</sup>, and transformed into
913 chemically competent Top10 E. coli cells, prior to selection on LB-1% agar plates containing
914 100 µg/ ml ampicillin. Individual colonies were picked, verified to contain the insert
915 sequence by PCR, and grown as overnight liquid cultures on LB medium supplemented with
916 100 µg/ ml ampicillin, prior to purification of the plasmids by alkaline lysis and isopropanol
917 precipitation¹²⁵. Purified plasmids were integrated into *P. tricornutum* cells via biolistic
918 transformation, using the Biolistic PDS-1000/He Particle Delivery System (BioRad),
919 essentially as previously described³⁵, ¹²⁶.

920
921 Colonies obtained from each transformation were transferred to liquid f/2 supplemented
922 with vitamins and 100 µg/ ml zeocin, and were left to recover under the same growth
923 conditions as used for liquid cultures of untransformed cells. Expression of GFP was
924 visualised using a TCS SP8 confocal microscope (Leica), an excitation wavelength of 488 nm
925 and emission wavelength interval of c. 510-540 nm. Chlorophyll fluorescence (using an
926 emission interval of 650-700 nm) and bright field images were simultaneously visualised for
927 each cell. Wild-type cells that did not express GFP were used to identify the maximum
928 exposure length possible without false detection of chlorophyll in the GFP channel (Fig. 2-
929 figure supplement 7).

930
931 Possible mitochondrial localisations of dual targeted proteins were identified by staining
932 cells with approximately 100 mM Mitotracker orange, dissolved in filtered seawater, for 25
933 minutes under standard culture conditions⁵⁵. Cells were rinsed and resuspended in fresh
934 filtered seawater prior to visualisation, using the same conditions as stated above for GFP,
935 and a 548 nm excitation laser and 575-585 nm absorbance window for the Mitotracker
936 signal. To ensure that there was no possible crosstalk between the two signals, negative
937 controls consisting of an unstained GFP-expressing wild-type line, and stained wild-type
938 cells, were used respectively to determine the maximum exposure length possible without
939 (respectively) false detection of GFP in the Mitotracker channel, and false detection of
940 Mitotracker in the GFP channel (Fig. 7- figure supplement 1).

941
942 Reconstruction of evolutionary origins of ancestral plastid-targeted proteins
943

943
944 The most probable evolutionary origins of individual plastid-targeted proteins were
945 identified via the combined products of BLAST top hit analysis and phylogenetic sister-group
946 inference. First, a composite reference sequence library was generated by appending the
947 uniref outgroup library previously used for BLAST-based assembly of ancestral HPPGs, with
948 twenty-two combined eukaryotic transcriptome and genomic libraries of taxa with no
949 suspected history of serial endosymbiosis, which was previously used to enrich each single-
950 gene tree (Table S1- sheet 1¹⁴⁵). Each sequence within the library was then assigned a
951 taxonomic affinity consisting of one of six lineages (green algae, red algae, aplastidic
952 stramenopiles, all other eukaryotes, prokaryotes, and viruses) and one of 48 sub-categories,
953 (Table S1- sheet 1, section 1¹⁴⁵). Next, each seed protein sequence within each ancestral
954 HPPG was searched by BLASTp against the composite library, with a threshold e-value of 1 x
955 10⁻⁵. Sequences were annotated by the lineage and sub-category of the first hit obtained,
956 and by the number of consecutive top hits obtained within the same lineage (Table S4- sheet
957 2, section 2¹⁴⁵). To minimise misidentification due to any residual contamination in individual
958 sequence libraries, only sequences for which the first three or more BLAST hits resolved
959 within the same lineage were deemed to be unambiguously related to that lineage.

960
961 Sister-group relationships were additionally inferred for each ancestral HPPG from the
962 previously generated single-gene trees (Table S4- sheet 2, section 3¹⁴⁵). To ensure that only
963 true sister-group relationships were recorded, and to avoid potential misidentifications of
964 individual sister-group relationships due to species-specific gene transfer or contaminants
that had not previously been excluded by screening individual species libraries, only trees in which ochrophytes were monophyletic, (i.e., not paraphyletic with regard to any one of the five outgroups), for which a single sister-group could be identified (using the most phylogenetically complex node as the outgroup), and for which the sister-group contained at least two monophyletic or paraphyletic sequences, from different sub-categories of the same lineage, were used for subsequent analysis.

## 971 Reconstruction of evolutionary relationships between ochrophytes and other CASH lineage plastids

To identify the probable relationships between ochrophytes and other CASH lineage plastids, each ancestral HPPG tree was enriched with sequences from six different groups of organisms with histories of serial endosymbiosis (cryptomonads, haptophytes, dinotoms, other alveolates, euglenids, and chlorarachniophytes), subdivided into thirteen sub-categories (Table S1<sup>145</sup>). For the cryptomonad, haptophyte and dinotom sequences, as plastid-targeted proteins from these lineages may be identified using targeting predictors trained on diatoms such as HECTAR<sup>6</sup> and ASAFind<sup>29, 30</sup>, each of the HPPGs initially generated was enriched with plastid-targeted sequences from each cryptomonad, haptophyte and dinotom sub-category identified by *in silico* prediction with these programmes (Table S2-sheet 1; Table S3-sheet 1<sup>145</sup>).

The position of each group of organisms within the tree was then annotated as falling into one of eight different categories, four of which were internal to the ochrophytes (diatoms; hypogyristea; chrysista; or an ambiguous internal position) and four of which were external to the ochrophytes (as an immediate sister-group to all ochrophytes prior to the first outgroup lineage previously identified; within the red algae; within the green algae; and at any other position external to the ochrophytes; Table S4-sheet 2, sections 5-6<sup>145</sup>). To minimise the incorporation of contaminant and non-plastid sequences, tree positions were only recorded if the branch containing sequences from that particular lineage included at least two of the sub-categories considered (for alveolates, cryptomonads, and haptophytes), contained at least one predicted plastid-targeted sequence (for dinotoms, cryptomonads and haptophytes), and for which only one category could be applied (i.e., the tree only contained one evolutionarily distinct group for each lineage, which could be unambiguously allocated one category over all others). Each tree annotation was repeated three times independently, and only tree annotations that were recorded consistently in each case were retained for further analysis.

To identify proteins that were uniquely shared between haptophytes and other lineages, every HPPG initially generated was screened for the inclusion of only two of five different lineages (diatoms including dinotoms, hypogyristea, chrysista, haptophytes, and cryptomonads; Table S2-sheet 2, section 3; Table S3-sheet 2, section 3<sup>145</sup>). The frequencies of these proteins were then compared to the numbers expected in a random distribution of all uniquely shared HPPGs across the entire dataset: for example, if half of all uniquely shared HPPGs were shared with diatoms and one other lineage, and half were shared with haptophytes and one other lineage, then one-quarter of all uniquely shared HPPGs should be shared between haptophytes and diatoms.

The specific evolutionary relationships associated with haptophyte plastid-targeted proteins incorporated into ancestral HPPGs were investigated using a modified BLAST top hit technique. Firstly, all of the plastid-targeted proteins assembled into each ancestral HPPG were extracted and separated into each separate sub-category (Table S13-sheet 1<sup>145</sup>). Each
1016 sub-category list was then reduced to only leave one, randomly selected sequence per HPPG
1017 (Table S13- sheet 2<sup>145</sup>). Finally, each sequence retained in the reduced list was searched by
1018 BLAST against a composite library, consisting of the library previously used for outgroup top
1019 hit analysis, enriched with all of the plastid-targeted proteins identified for ochrophytes,
1020 haptophytes and cryptomonads, except for those that corresponded to the same particular
1021 lineage as the query sequence (Table S13- sheets 1,3<sup>145</sup>). For example, in the case of
1022 haptophytes, plastid-targeted sequences that had been separated into three individual
categories (pavlovophytes, prymnesiales, and isochrysidales<sup>127</sup>) were searched against a
1023 composite library consisting of all outgroup sequences, and plastid-targeted sequences from
diatoms, hypogyristea, chrysista, and cryptomonads, but excluding haptophytes. BLAST top
1025 hit analysis was then performed as described above (Table S13- sheets 1, 3<sup>145</sup>). Finally, to
1027 enable the identification of genes with consistent results from multiple analyses, the lineage
1028 of the BLAST top hit was compared to the lineage of the haptophyte sister-group in the
1029 single-gene tree analysis (Table S4- sheet 2, section 5; Table S13- sheet 4<sup>145</sup>).
1030

1030

Identification of uniquely shared residues in multigene HPPG datasets

1032

1033 To identify residues that are uniquely shared between ochrophytes and other lineages,
1034 multigene datasets were constructed of a) ancestral HPPGs of green algal origin, and b)
1035 ancestral HPPGs for which haptophytes show origins within the ochrophytes. To minimise
1036 the incorporation of sequences of misidentified origin, in each case only the HPPGs for
1037 which the proposed evolutionary origin were identified both by BLAST top hit and single-
gene tree analysis were included. To avoid introducing artifacts due to lineage-specific gene
transfers, paralogy events, or other phylogenetic incongruencies that could otherwise bias
1039 the eventual results<sup>86, 128</sup>, the single-gene tree generated for each HPPG was manually
inspected to exclude any that contain multiple clades (defined as monophyletic groups
1041 containing more than one sequence from a particular lineage, separated from one another
1042 by at least two sequences from outside that particular lineage) for each of the major
1043 lineages of interest within the tree:
1044

1045

1046

1047 • For the green gene dataset, HPPG trees containing more than one clade of ochrophyte, cryptomonad, haptophyte, red algal, or green algal sequences were excluded. To account for the possibility that CASH lineage sequences might originate from within the green algae, the green algae were allowed to be paraphyletic with regard to the cryptomonad, haptophyte and ochrophyte sequences, but were not allowed to incorporate sequences from other lineages. Similarly, to account for the possibility that subsequent gene transfers may have occurred from ochrophytes into other CASH lineages, the ochrophytes were allowed to be paraphyletic with regard to cryptomonad and haptophyte sequences, but not to any other lineages.
1048
1049
1050
1051
1052
1053
1054
1055
1056

1057 • For the haptophyte gene dataset, HPPG trees containing more than one clade of ochrophyte, haptophyte, diatom, hypogyristean, or chrysistan sequences were excluded. To account for the possibility that haptophytes arose within the ochrophytes, the ochrophyte, diatom, hypogyristean and chrysistan sequences were allowed to incorporate sequences from haptophytes. Similarly, due to the paraphyly of hypogyristea with regard to diatoms, the hypogyristean sequences were allowed to incorporate sequences from diatoms, but not from other lineages.
1058
1059
1060
1061
1062
1063

1064 • In all cases, sequences from chlorarachniophytes, euglenids, and alveolates were not incorporated into any of the clade assessments, due to uncertainty over the gene transfer events that have occurred in each lineage<sup>7, 81, 82</sup>.
1065
1066
1067 This left datasets consisting of 32 HPPGs for which the ochrophytes were of clear green
1068 algal origin, and 37 HPPGs in which the haptophytes were of clear ochrophyte origin, with
1069 no conflicting phylogenetic signal. The rationale for inclusion and exclusion of each HPPG in
1070 each analysis is presented in Table S6, sheets 1 and 3¹⁴⁵.
1071
1072
1073 Next, to eliminate individual sequences remaining within each HPPG that might have arisen
1074 through species-specific gene transfer or contamination events, each trimmed sequence
1075 within each approved alignment was inspected using a composite BLAST approach. First,
1076 each sequence was searched against a composite library containing all uniref, jgi and
1077 MMETSP sequences from every lineage within the tree of life, and the top ten hits were
1078 tabulated for each sequence. In each case, only sequences for which at least the first three
1079 hits were of the same lineage as that of the query were retained. For the haptophyte
1080 multigene alignment, the ochrophytes were separately analysed as each of the three
1081 component lineages (chrysista, hypogyristea, and diatoms), which is to say that a query
1082 obtained from a member of the hypogyristea would only be retained if the first three BLAST
1083 top hits originated from other hypogyristean sequences, rather than other ochrophytes.
1084

1085 Next, each of the component sequences within each cleaned alignment were searched
1086 against all other component sequences within the same alignment using BLASTp, and the
1087 top ten hits within the alignment were ranked. In each case, sequences were only approved
1088 for incorporation into the multigene dataset if the first non-self hit was to a different sub-
1089 category within the same lineage, e.g. if a query sequence from a red alga yielded a top hit
1090 against a red algal sequence from a different red sub-category. To allow for possible cases
1091 of paraphyly and/or absence of sequences within each alignment, the following
1092 modifications were applied:
1093

1094 • Green algal sequences within the confirmed green origin alignments were allowed
to yield top hits against ochrophytes, cryptomonads, and haptophytes, but were
1095 required to yield a best hit against another green alga with an expect value lower
1096 than the top hit against red algal or glaucophyte sequences.
1097
1098 • Glaucophyte sequences were deemed to be of correct origin if they yielded a top hit
against cyanobacteria, red algae, or green algae, due to the incorporation (in
1099 general) of only one glaucophyte sequence in each alignment.
1100
1101 • Ochrophyte sequences were deemed to be of correct origin if they yielded a top hit
against any other ochrophyte sub-category (regardless of whether this was of
1102 diatom, hypogyristean or chrysistan origin). Ochrophyte sequences were
1103 additionally allowed to yield top hits against cryptomonads (in the green gene
1104 alignments), and haptophytes (in both green and haptophyte gene alignments), but
1105 were required to yield a best hit against another ochrophyte with an expect value
1106 lower than the best hit against green algal, red algal or glaucophyte sequences.
1107
1108 • Sequences for which no top hits were found for a different sub-category within the
1109 same lineage, but for which at least one top hit were found within the same sub-
1110 category within the lineage, and for which the first ten BLAST hits did not directly
1111 indicate a contamination event, were deemed to be of correct origin.
1112

1113 Tabulated outputs for each BLAST analysis are provided in Table S6, sheets 2 and 4. Finally,
1114 each dataset was reduced to leave only one randomly selected sequence for each given sub-
1115 category within each HPPG alignment.
1116

1116
1117 The number of residues that were uniquely shared between ochrophytes and green algae in the green gene dataset, and haptophytes and ochrophytes in the haptophyte dataset, were then tabulated (Table S7<sup>145</sup>). Briefly, residues were inferred to be uniquely shared between ochrophytes and green algae if they were present in at least 2/3 of the ungapped ochrophyte sequences, one or more green algal sequence, and if none of the red algal or glaucophyte sequences shared the residue in question, but at least one of these sequences had a non-matching (i.e. non-gapped) residue at that position (Table S7- sheet 1, section 2<sup>145</sup>). Similarly, residues were inferred to be uniquely shared between ochrophytes and haptophytes if they were present in at least 2/3 of the ungapped haptophyte sequences, one or more ochrophyte sequence, and if none of the green algal, red algal, glaucophyte or cyanobacterial sequences shared the residue in question, but at least one of these sequences had a non-matching (i.e., non-gapped) residue at that position (Table S7- sheet 2, section 2<sup>145</sup>). The origin point of each uniquely shared residue was then inferred by comparison to reference topologies respectively of green algae<sup>129</sup> and of ochrophytes (per Fig. 1). Residues were assumed to have originated in a common ancestor of a particular clade if that clade contained more lineages with matching than non-matching or gapped residues (Table S7- sheets 1-2, section 5<sup>145</sup>). A second analysis was additionally performed in which all gapped residues were deemed to be matching, to identify the earliest possible origin point for each uniquely shared residue, taking into account secondary loss<sup>45, 50</sup> and absence of sequences from each alignment<sup>46,47</sup>.

1137
1138 Analysis of targeting preferences of ancestral ochrophyte and haptophyte genes.

1139
1140 Two libraries of non-redundant gene families that were broadly conserved across ochrophytes or haptophytes, and thus might represent gene products of the ancestral genomes of these lineages, were generated using a similar BLAST-based assembly pipeline as used to construct HPPGs (Table S8; Table S14<sup>145</sup>). Ochrophyte gene families were deemed to be conserved if orthologues were detected in one of three different patterns of ochrophyte sub-categories previously defined to correspond to ancestral plastid-targeted proteins (Fig. 1, panel B; Table S8- sheet 1, section 3<sup>145</sup>). Haptophyte gene families, built through a similar pipeline using seed sequences from the *Chrysochromulina tobin* and *Emiliania huxleyi* genomes<sup>116, 117</sup>, were deemed to be ancestral if orthologues were identified in at least two of the three haptophyte sub-categories considered (pavlovophytes, prymnesiales, and isochrysidales; Table S14- sheet 1, section 3<sup>145</sup>).

1151
1152 The most probable evolutionary origin of each gene family was inferred by BLAST top hit analysis of the seed sequence (Table S8- sheets 1, 2; Table S14- sheets 1, 2<sup>145</sup>). Ochrophyte sequences were searched against the composite uniref + MMETSP library used to previously identify the most likely outgroup to each ancestral plastid-targeted protein (Table S8- sheet 1, section 6<sup>145</sup>), while haptophyte sequences were searched against the enriched library that also contained all ochrophyte and cryptomonad sequences, to enable the distinction of proteins of probable CASH lineage plastid origin from proteins that had evolved through independent gene transfer events between haptophytes and non-CASH lineage organisms (Table S14- sheet 1, section 6<sup>145</sup>). Targeting preferences for each protein encoded within each gene family were identified using SignalP v 3.0 and ASAFind v 2.0<sup>29, 106</sup>, and with HECTAR<sup>30</sup>, as previously discussed (Table S8- sheet 3; Table S14- sheet 3<sup>145</sup>). Targeting preferences that were identified in a plurality of sequences and in ≥2/3 of the sequences within each ochrophyte gene family were recorded (Table S8- sheet 2, sections 4-5<sup>145</sup>). As only three haptophyte sequences were assembled for each ancestral haptophyte gene family, only targeting predictions that were identified in ≥2/3 of the sequences within the HPPG were inferred to be genuine (Table S14- sheet 2, sections 4-5<sup>145</sup>).
1168 **Functional and physiological annotation of ancestral plastid-targeted proteins**
1169
1170
1171 Core plastid metabolism pathways were identified using recent reviews of ochrophyte
1172 metabolism, or reviews of homologous plant plastid metabolic pathways where ochrophyte-
1173 specific reviews have not yet been published⁵¹, ⁹⁷, ¹¹⁵, ¹³⁰-¹³⁶. The probable function and KOG
1174 classification of each HPPG were annotated using the pre-existing annotations associated
1175 with seed protein sequence (if these existed), or if not the annotated function of the top
1176 uniref hit previously identified by BLAST searches of the seed sequence (Table S9¹⁴⁵).
1177 Expression dynamics for each ancestral HPPG within the genomes of the model diatoms
1178 *Phaeodactylum tricornutum* and *Thalassiosira pseudonana* were inferred using microarray
1179 data integrated into the DiatomPortal server⁷⁴ (Table S10- sheets 1,2¹⁴⁵). Correlation
1180 coefficients were calculated between each pair of *P. tricornutum* and *T. pseudonana* genes
1181 that were incorporated into an ancestral HPPG, across all microarray libraries within the
dataset (Table S10- sheets 3,4¹⁴⁵), with average values being calculated from all pairwise
1182 correlations for different evolutionary categories of protein (Table S10- sheet 5¹⁴⁵).
1183
1184
1185 Possible chimeric proteins, resulting from the fusion of proteins of different evolutionary
1186 origins, were identified in the dataset using a modified version of a previously published
1187 protocol⁷⁵ (Table S9- sheet 1, sections 4,5; Table S11¹⁴⁵). Each protein within each HPPG was
1188 searched using BLASTp against the composite outgroup MMETSP-enriched library, using the
1189 same taxonomic classification used for the identification of the evolutionary origin of each
1190 seed protein within the dataset, and all hits with an expect value of 1 x 10⁻⁵⁰.
1191 sequences were then grouped into component families according to the following rule: if
1192 two component sequences overlapped by more than 70% of their lengths on the protein
1193 composite, they belonged to the same component family. Overlapping and/ or nested
1194 component families were additionally merged if one family was included by more than 70%
1195 of its length into the other one. Component families were then assigned a broad
1196 evolutionary origin corresponding to their taxonomic composition. If the three best
1197 component sequences, according to their BLAST bitscore against the composite gene,
1198 matched with the same lineage (e.g., green algae, red algae, aplastidic stramenopiles, or
1199 other eukaryotes), the component was considered to have originated from that lineage.
1200
1201 Possible dual targeted proteins were identified within the dataset by screening all possible
1202 plastid-targeted proteins with Mitofates, using a cut-off targeting threshold of 0.35¹³⁷, which
1203 was inferred to be more effective in identifying experimentally verified ochrophyte
1204 mitochondria-targeted proteins (Fig. 7- figure supplement 2)²⁹ than other threshold values
1205 or targeting prediction programmes such as TargetP¹³⁸ or Mitoprot¹³⁹. The default Mitofates
1206 positive cutoff value was modified from 0.38 to 0.35 in order to maximise the capture of
1207 experimentally localised mitochondrial proteins, without admitting proteins with
1208 unambiguous plastid localisation (Fig. 7- figure supplement 2). As dual targeting to plastids
1209 and mitochondria may be achieved either by distinct protein isoforms resulting from
1210 ambiguous targeting peptides or alternative internal translation initiation sites that allow
1211 production of mitochondrial targeting sequences⁷⁷, ⁸⁰, each protein was screened with
1212 Mitofates using both the full-length N-termini, and N-termini predicted to result from the
1213 next downstream methionine within 30 residues. Possible conserved dual targeted proteins
1214 were then identified via the same BLAST-based assembly pipeline and stringency thresholds
1215 used to identify probable ancestral HPPGs (Table S12- sheet 1¹⁴⁵). All putative dual targeted
1216 proteins have been made publically accessible through the University of Cambridge dSpace
1217 server (https://www_repository.cam.ac.uk/handle/1810/261421)¹⁴⁵.
1218
1219 Construction and inspection of concatenated and exemplar phylogenetic trees

1220
1221 For the plastid genome phylogenetic analysis, single-gene alignments were constructed by BLAST searches of published red lineage and glaucophyte plastid genomes (for the gene rich analysis) or of these genomes plus all MMETSP libraries for the same lineages (for the taxon rich analysis), using the *Phaeodactylum tricornutum* protein sequence as query and a threshold e-value of 1 x 10<sup>-05</sup>, followed by alignment using GeneIOUS v 4.76<sup>119</sup>, as before.
1222
1223
1224
1225 The gene rich analysis included protein sequences from 54 genes that were identified in 22 different non-green lineage plastid genomes while the taxon-rich analysis included 10 different plastid genes that were identified in all 22 plastid genomes and at least 30 different MMETSP libraries<sup>34</sup> (Table S15- sheet 1<sup>145</sup>). For the taxon-rich analysis, only species that were represented in ≥ 6/12 of the single-gene alignments were included in the concatenated alignment. Each concatenated alignment was trimmed using trimal<sup>120</sup> using the -gt 0.8 option.

1232
1233
1234 Single-gene alignments for four plastid-targeted proteins predicted to be of polyphyletic origin in ochrophytes (3-dehydroquinate synthase, isopropylmalate dehydratase, sedoheptulose bisphosphatase, and shikimate kinase) were generated using a similar BLAST-based assembly and alignment pipeline as used to verify ancestral plastid-targeted proteins.
1235
1236
1237 In this case, all non-redundant (as inferred by BLAST top hit evalue) plastid-targeted sequences for each protein identified from ochrophyte genomes were used as independent queries for the identification of plastid-targeted orthologues, 50 uniref top hits, and top hits from the combined MMETSP and genomic libraries from 36 eukaryotic sub-categories, as before. HPPGs were independently generated, aligned and trimmed for each seed sequence; all HPPGs generated for each protein were then merged, realigned and retrimmed using trimAl to generate a single-gene alignment. Single-gene alignments for each of the constituent genes in each concatenated plastid genome tree were generated by splitting the alignment into its component genes. All alignments have been made publically accessible through the University of Cambridge dSpace server (https://www repository.cam.ac.uk/handle/1810/261421)<sup>145</sup>.

1248
1249
1250 Trees were inferred for each concatenated and exemplar single-gene alignment (Table S15-sheet 2<sup>145</sup>) using the MrBayes and RAxML programmes in-built into the CIPRES web-server<sup>121, 140, 141</sup>. Bayesian trees were inferred using three substitution models (GTR, Jones, and WAG), a minimum of 600000 generations, and an initial burn-in discard value of 0.5.
1251
1252
1253
1254 Trees were only utilised if the final convergence statistic between the two chains run was ≤ 0.1, and tree calculation was automatically stopped if the convergence statistic fell below 0.01. RAxML trees were inferred using three substitution models (GTR, JTT, and WAG) with automatic bootstopping, as previously described<sup>58</sup>. The best tree topology for each RAxML tree was inferred, and bootstrapping was performed using a burnin value of 0.03.
1255
1256
1257
1258
1259
1260 Alternative tree topologies were tested for the RAxML + JTT tree inferred from each concatenated alignment using CONSEL<sup>142</sup>, under the default conditions. Tree outputs have been made publically accessible through the University of Cambridge dSpace server (https://www repository.cam.ac.uk/handle/1810/261421)<sup>145</sup>.

1263
1264 Modified alignments were generated for both of the plastid concatenated multigene datasets from which individual clades of organisms (diatoms, hypogyristea, chrysista, haptophytes, cryptomonads, red algae, and different combinations of green algae) had been removed (Table S15- sheet 2<sup>145</sup>). Fast-site removal was performed using TIGER<sup>143</sup>. Site rate evolution characteristics were calculated for each alignment using the -b 100 option, and modified alignments were constructed from which the rate categories corresponding to the
1270 fastest evolving 40-50% of sites were serially removed (Table S15- sheet 2<sup>145</sup>). Amino acid
1271 composition for each plastid alignment were calculated, and two modified alignments were
1272 generated from which glycines (which in all alignments occur at significantly lower
1273 frequencies in ochrophytes than in haptophytes or cryptomonads; chi-squared, P≤ 0.05;
1274 Table S16- sheet 3<sup>145</sup>), and from which seven amino acids (alanine, aspartate, glycine,
1275 histidine, leucine, asparagine, threonine and valine) which were found in at least one
1276 alignment to occur at significantly different frequencies in ochrophytes compared to
1277 haptophytes or to cryptomonads (P≤ 0.05; Table S16- sheet 3<sup>145</sup>) had been removed. Trees
1278 were inferred for each modified alignment using RAxML with the JTT substitution, and
1279 MrBayes with the Jones substitution, and bootstrap calculation as previously described.
1280 Modified alignments and tree outputs have been made publically accessible through the
1281 University of Cambridge dSpace server
1282 (https://www/repository.cam.ac.uk/handle/1810/261421)<sup>145</sup>.

1283
1284 Uniquely shared residues were manually tabulated for both of the plastid genome multigene alignments (Table S17<sup>145</sup>). For the gene-rich plastid multigene alignment, residues that were present in all haptophyte sequences and only found in a maximum of one other lineage (red algae, glaucophytes, cryptomonads, diatoms, hypogyristea, or chrysista) were tabulated (Table S17- sheet 1<sup>145</sup>). For the taxon-rich alignment, to take into account gaps and missing characters, residues were tabulated if they were found in a majority of haptophyte sequences, and one other lineage, as before (Table S17- sheet 2<sup>145</sup>). The total number of residues shared, and uniquely shared, with each non-haptophyte species and lineage are respectively tabulated in Table S17, sheets 3 and 4<sup>145</sup>.

1293
1294 Data deposition

1295
1296 All supporting datasets for this study, including supplementary tables predicted plastid-
1297 targeted and dual targeted protein libraries, single gene and multigene alignments, and tree
1298 outputs, have been made publically and freely accessible through the University of
1299 Cambridge dSpace server (https://www/repository.cam.ac.uk/handle/1810/261421) <sup>145</sup>.

1300
1301 Acknowledgments
1302

1302
1303 The authors would like to thank Achal Rastogi (École Normale Supérieure), Neal Clarke (Yale
1304 University), Michael Melkonian (University of Koln), Gane Ka-Shu Wong (University of
1305 Alberta) and Jun Yu (Beijing Institute of Genomics) for early access to sequence data used in
1306 this study, and Catherine Cantrel, Anne-Flore Deton-Cabanillas, Zhanru Shao, Leïla Tirichine
1307 and Javier Paz-Yepes (École Normale Supérieure) for assistance with generation of
1308 transgenic expression constructs for Phaeodactylum tricornutum. Funding is acknowledged
1309 from the ERC Advanced Award “Diatomite”, the Louis D Foundation of the Institut de
1310 France, the French Government “Investissements d’Avenir” programmes MEMO LIFE (ANR-
1311 10-LABX-54) and PSL* Research University (ANR-11-IDEX-0001-02), and the Gordon and
1312 Betty Moore Foundation (all to CB), and from FP7 (grant number 2007-2013 Grant
1313 Agreement 615274, to EPB). RGD is supported by an EMBO early career fellowship (ALTF
1314 1124-2014). DJR was supported by a postdoctoral fellowship from the Conseil Régional de
1315 Bretagne and the French Government “Investissements d’Avenir” programme OCEANOMICS
1316 (ANR-11-BTBR-0008). The authors would like to thank the handling reviewer and two
1317 anonymous editors for constructive comments on the manuscript.

1318
1319 Competing interests.

1320
1321 The authors declare no competing financial or non-financial interests in this project.

1322

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    <td>60.</td>
    <td>Mock, T. et al. Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus. Nature 541, 536-540 (2017).</td>
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  </tr>
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    <td>1412</td>
    <td>62.</td>
    <td>Siaut, M. et al. Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum. Gene 406, 23-35 (2007).</td>
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    <td>63.</td>
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    <td>64.</td>
    <td>Suzuki, K. & Miyagishima, S. Eukaryotic and eubacterial contributions to the establishment of plastid proteome estimated by large-scale phylogenetic analyses. Mol Biol Evol 27, 581-590 (2010).</td>
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  <tr>
    <td>1415</td>
    <td>65.</td>
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  </tr>
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    <td>1417</td>
    <td>67.</td>
    <td>Siaut, M. et al. Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum. Gene 406, 23-35 (2007).</td>
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    <td>1418</td>
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    <td>1420</td>
    <td>70.</td>
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  </tr>
</table>
<table>
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    <td>59.</td>
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    <td>Matsuzaki, M. <i>et al.</i> Genome sequence of the ultrasmall unicellular red alga <i>Cyanidioschyzon merolae</i> 10D. <i>Nature</i> 428, 653-657 (2004).</td>
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    <td>65.</td>
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    <td>66.</td>
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    <td>67.</td>
    <td>Tanaka, A. <i>et al.</i> Ultrastructure and membrane traffic during cell division in the marine pennate diatom <i>Phaeodactylum tricornutum</i>. <i>Protist</i> 166, 506-521 (2015).</td>
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    <td>68.</td>
    <td>Imanian, B., Pombert, J.F. & Keeling, P.J. The complete plastid genomes of the two 'Dinotoms' <i>Durinskia baltica</i> and <i>Kryptoperidinium foliaceum</i>. <i>PLoS One</i> 5, 10711 (2010).</td>
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    <td>69.</td>
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    <td>72.</td>
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    <td>73.</td>
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    <td>75.</td>
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    <td>76.</td>
    <td>Dorrell, R.G. <i>et al.</i> Progressive and biased divergent evolution underpins the origin and diversification of peridinin dinoflagellate plastids. <i>Mol Biol Evol</i> 34, 361-379 (2017).</td>
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    <td>77.</td>
    <td>Marron, A.O. <i>et al.</i> The evolution of silicon transport in eukaryotes. <i>Mol Biol Evol</i> 33, 3226-3248 (2016).</td>
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    <td>Dorrell, R.G. <i>et al.</i> Progressive and biased divergent evolution underpins the origin and diversification of peridinin dinoflagellate plastids. <i>Mol Biol Evol</i> 34, 361-379 (2017).</td>
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    <td>79.</td>
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    <td>80.</td>
    <td>Dorrell, R.G. <i>et al.</i> Progressive and biased divergent evolution underpins the origin and diversification of peridinin dinoflagellate plastids. <i>Mol Biol Evol</i> 34, 361-379 (2017).</td>
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    <td>1464</td>
    <td>82.</td>
    <td>Dorrell, R.G. <i>et al.</i> Progressive and biased divergent evolution underpins the origin and diversification of peridinin dinoflagellate plastids. <i>Mol Biol Evol</i> 34, 361-379 (2017).</td>
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    <td>83.</td>
    <td>Marron, A.O. <i>et al.</i> The evolution of silicon transport in eukaryotes. <i>Mol Biol Evol</i> 33, 3226-3248 (2016).</td>
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    <td>1466</td>
    <td>84.</td>
    <td>Dorrell, R.G. <i>et al.</i> Progressive and biased divergent evolution underpins the origin and diversification of peridinin dinoflagellate plastids. <i>Mol Biol Evol</i> 34, 361-379 (2017).</td>
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    <td>85.</td>
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    <td>1468</td>
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    <td>Dorrell, R.G. <i>et al.</i> Progressive and biased divergent evolution underpins the origin and diversification of peridinin dinoflagellate plastids. <i>Mol Biol Evol</i> 34, 361-379 (2017).</td>
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1693 Table 1- Glossary Box
1694
1695 A schematic figure of eukaryotic taxonomy, showing the evolutionary origins of nuclear and plastid lineages, adapted from previous reviews³, is shown in Fig. 1- figure supplement 1.
1696
1697

<table><tr><td><strong>Complex plastids</strong></td><td>Plastids acquired through the endosymbiosis of a eukaryotic alga. These include secondary plastids of ultimate red algal origin (such as those found in ochrophytes, haptophytes and cryptomonads), secondary plastids derived from green algae (such as those found in euglenids or chlorarachniophytes), or tertiary plastids such as those found in dinotoms and certain other dinoflagellates (resulting from the endosymbioses of eukaryotic algae that themselves contain plastids of secondary endosymbiotic origin).</td></tr><tr><td><strong>CASH lineages</strong></td><td>The four major lineages of algae with plastids of secondary or higher red origin, that is to say <u>Cryptomonads</u>, <u>Alveolates</u> (dinoflagellates, and <u>apicomplexans</u>), <u>Stramenopiles</u>, and <u>Haptophytes</u>.</td></tr><tr><td><strong>Stramenopiles</strong></td><td>A diverse and ecologically major component of the eukaryotic tree, containing both photosynthetic members (the ochrophytes), which possess complex plastids of red algal origin, and aplastidic and non-photosynthetic members (e.g. oomycetes, labyrinthulomycetes, and the human pathogen <u>Blastocystis</u>), which form the earliest-diverging branches. It is debated when within stramenopile evolution the extant ochrophyte plastid was acquired.</td></tr><tr><td><strong>Ochrophytes</strong></td><td>Photosynthetic and plastid-bearing members of the stramenopiles, including many ecologically important lineages (diatoms, kelp, pelagophytes) and potential model lineages for biofuels research (<u>Nannochloropsis</u>). Ochrophytes form the most significant component of eukaryotic marine phytoplankton<sup>1,2</sup>.</td></tr><tr><td><strong>Haptophytes</strong></td><td>Single-celled, photosynthetic eukaryotes, possessing complex plastids of</td></tr></table>
<table><tr><td></td><td>ultimate red origin. Some haptophytes (the coccolithophorids) are renowned for their ability to form large blooms (visible from space), and to form intricate calcareous shells<sup>1,94</sup>, which if deposited on the ocean floor go on to form a major component of limestone and other sedimentary rocks.</td></tr><tr><td>HPPG</td><td>"Homologous plastid protein group". Proteins identified in this study to possess plastid-targeting sequences that are homologous to one another, as defined by BLAST-based HPPG assembly and single gene phylogenetic analysis.</td></tr></table>

1698

1699

## Figure Legends

1700

### Fig. 1. Procedure for identification of conserved plastid-targeted proteins in ochrophytes.

Panel A shows a schematic unrooted ochrophyte tree, with the three major ochrophyte lineages (chrysista, hypogyristea, and diatoms) denoted by different coloured labels. "PX" refers to the combined clade of phaeophytes, xanthophytes and related taxa, and "PESC" to pinguophytes, eustigmatophytes, synchromophytes, chrysophytes and relatives. A global overview of the eukaryotic tree of life, including the position of ochrophytes relative to other lineages is shown in figure supplement 1. Panel B shows the number of inferred positive control HPPGs (i.e., HPPGs encoding proteins with experimentally confirmed plastid localisation, or unambiguously plastid function) and negative control HPPGs (i.e., HPPGs encoding proteins with no obvious orthologues in ochrophyte genomes, but found in haptophyte and cryptomonad genomes) detected as plastid-targeted in different numbers of ochrophyte lineages using ASAFind (i) and HECTAR (ii). The blue bars show the number of positive controls identified to pass a specific conservation threshold, plotted against the left hand vertical axis of the graph, while the red bars show the number of negative controls that pass the same conservation threshold, plotted against the right hand vertical axis of the graph. The number of different sub-categories included in each conservation threshold is shown in a heatmap below the two graphs, with the specific distribution for each bar in the graph shown in the aligned cells directly beneath it. Each shaded cell corresponds to an identified orthologue in one sub-category of a particular ochrophyte lineage: orange cells indicate presence of chrysistan sub-categories; light brown cells the presence of hypogyristean sub-categories; and dark brown cells the presence of diatom sub-categories. In each graph, black arrows label the conservation thresholds inferred to give the strongest separation (as inferred by chi-squared P-value) between positive and negative control sequences. The table (iii) tabulates the three conservation patterns identified as appropriate for distinguishing probable ancestral HPPGs from false positives. Panel C shows the complete HPPG assembly, alignment and phylogenetic pathway used to identify conserved-targeted proteins. Panel D tabulates the number of HPPGs built using ASAFind and HECTAR predictions, and the number of non-redundant HPPGs identified in the final dataset. The final total represents the pooled total of non-redundant HPPGs identified with both ASAFind and HECTAR.

1732

### Fig. 2. Verification of unusual ancestral plastid-targeted proteins. Panel A lists the ten proteins selected for experimental characterisation and their most probable previous localisation prior to their establishment in the ochrophyte plastid, based on the first 50 nr BLAST hits. Exemplar alignments and single-gene tree topologies for some of these proteins are shown in figure supplements 1-4. Panel B shows the localisation of GFP constructs for copies of two proteins with an unambiguous plastid localisation (a pyrophosphate-dependent PFK, which localises to the pyrenoid, and a novel plastid protein, with
1740 cosmopolitan distribution across the plastid) and one protein with a periplastid localisation
1741 (a predicted peroxisomal membrane protein) from the diatom *Phaeodactylum tricornutum*,
1742 the diatom endosymbiont of the dinoflagellate *Glenodinium foliaceum* and the
1743 eustigmatophyte *Nannochloropsis gaditana*, expressed in *P. tricornutum*. All scale bars = 10 µm.
1744 Expression constructs for seven additional *P. tricornutum* proteins and three additional
1745 *N. gaditana* proteins with multipartite plastid localisations are shown in figure supplements
1746 5 and 6, and control images (wild-type cells, and cells expressing untargeted eGFP) are
1747 shown in figure supplement 7.
1748
1749 **Fig. 3. Evolutionary origins of the ochrophyte plastid proteome.** Panel A displays the origins
1750 inferred by BLAST top hit, phylogenetic analysis, and combined analysis for all ancestral
1751 HPPGs. Panel B shows (i) a schematic diagram of stramenopile taxonomy, with the
1752 evolutionary relationships between labyrinthulomycetes, oomycetes, slopalinids and
1753 ochrophytes proposed by recent multigene studies¹², and the probable closest stramenopile
1754 relative (as inferred by BLAST top hit analysis) of the 26 ancestral HPPGs verified by
1755 combined analysis to be of aplastidic stramenopile origin, and (ii) the next nearest relative,
1756 as inferred through BLAST top hit, phylogenetic and combined analysis, of the 26 aplastidic
1757 stramenopile HPPGs verified by combined analysis. The evolutionary categories in this graph
1758 are shaded as per in panel A.
1759
1760 **Fig. 4. Verification and origins of the green signal in ochrophyte plastids.** Panel A shows a
1761 schematic tree of the 11 archaeoplastid sub-categories with which each green HPPG
1762 alignment was enriched prior to phylogenetic analysis. The topology of the red and green
1763 algae are shown according to previously published phylogenies¹²,¹⁴⁴. Green sub-categories
1764 are in green text; red algal sub-categories in red text; and other sub-categories are in blue
text. Five ancestral positions within the green algal tree inspected in subsequent analyses
1765 are labelled with coloured boxes. Panel B shows the number of HPPGs of verified red (red
1766 bars) or green origin (green bars) for which orthologues were identified in different numbers
1767 green sub-categories (plotted on the x-axis) and red sub-categories (plotted on the z-axis).
1768 An equivalent graph showing only HPPGs for which a glaucophyte orthologue was detected
1769 is shown in figure supplement 1. Panel C compares the number of trees in which HPPGs of
1770 verified green origin resolve as a sister group to all green lineages (including chlorophytes
1771 and streptophytes); to multiple chlorophyte sub-categories but to the exclusion of
1772 streptophytes; and to individual chlorophyte sub-categories only. A detailed heatmap of the
1773 evolutionary distribution of the green sub-categories detected in each sister-group is shown
1774 in figure supplement 2, and the distribution of BLAST top hits within each sub-category is
1775 shown in figure supplement 3. Panel D lists the number of residues inferred from a dataset
1776 of 32 ochrophyte HPPGs of verified green origin, which have been subsequently entirely
1777 vertically inherited in all major photosynthetic eukaryotic lineages, to be uniquely shared
1778 between ochrophytes and some but not all green lineages, hence might represent specific
1779 synapomorphic residues. Residues are categorized by inferred origin point within the tree
1780 topology shown in panel A, i.e., each of the five ancestral nodes labelled. A final category
1781 shows all of the residues inferred to be specifically shared with one green sub-category, and
1782 not with any other. The distribution of residues based on the earliest possible origin point
1783 (taking into account gapped and missing residues in each HPPG alignment) is shown in figure
1784 supplement 4. Panel E shows the number of the 7140 conserved gene families inferred to
1785 have been present in the last common ochrophyte ancestor that are predicted by ASAFind
1786 to encode proteins targeted to the plastid, subdivided by probable evolutionary origin, and
1787 the number expected to be present in each category assuming a random distribution of
1788 plastid-targeted proteins across the entire dataset, independent of evolutionary origin.
1789 Evolutionary categories of proteins found to be significantly more likely (chi-squared test,
1790
1791 P=0.05) to encode plastid-targeted proteins than would be expected are labelled with black arrows. An equivalent distribution of plastid-targeted proteins inferred using HECTAR is shown in figure supplement 5.
1792
1793
1794
1795 Fig. 5. Functional mixing of the ancestral ochrophyte HPPGs. Panel A tabulates nineteen different fundamental plastid metabolism pathways and biological processes recovered in the ancestral HPPG dataset. Detailed information concerning the origin and identity of each component of each pathway is provided in figure supplement 1, and an overview and phylogenetic trees of each of the non-vertically inherited enzymes identified are provided in figure supplements 2-6. Panel B compares the distribution of individual KOG families in the complete HPPG library, the ancestral HPPG dataset, and HPPGs of verified prokaryotic origin. KOG families pertaining to metabolism are shown in shades of green, families pertaining to information storage are shown in shades of red, and families pertaining to cellular processes are shown in shades of blue. Families with unknown KOG classification or general function predictions only are not shown. KOG classes that are enriched in the ancestral HPPG dataset compared to relative proportions of each KOG class found in the full HPPG dataset, or in individual ancestral HPPGs of prokaryotic origin compared to the ancestral HPPG dataset (as inferred by chi-squared test, P <0.05), are labelled with black horizontal arrows. No such enrichments were observed in any evolutionary category of ancestral HPPGs other than prokaryotes, hence analogous distributions of HPPGs of red algal, green algal and host origin are not shown. Overviews of the broader KOG classes that are enriched either in the ancestral HPPG dataset, or in specific evolutionary categories of ancestral HPPG, are shown in figure supplement 7. Panel C tabulates the number of ancestral HPPGs performing consecutive metabolic functions, or that are likely to have direct regulatory interactions, alongside the number of these protein pairs in which both members are of verified evolutionary origin; the number observed where both members possess the same evolutionary origin; the expected number of protein pairs where both members possess the same evolutionary origin; and the chi-squared probability of similarity between the observed and expected values. Panel D shows heatmaps for the pairwise correlation coefficients of expression for genes encoding different evolutionary categories, as verified using combined BLAST top hit and single-gene tree analysis, of ancestral HPPGs in the model diatoms *Phaeodactylum tricornutum* (i) and *Thalassiosira pseudonana* (ii). A scale bar showing the relationship between shading and correlation coefficient is shown to the right of the heatmaps. Boxplots comparing the individual expression profiles of different categories of ancestral HPPG, and the associated ANOVA P values calculated, are shown in figure supplements 8 (for *P. tricornutum*) and 9 (for *T. pseudonana*).

1827
1828 Fig. 6. Origins of chimeric proteins in the ochrophyte plastid. Panel A tabulates eight ancestral HPPGs containing domains of cyanobacterial and non-cyanobacterial origin, as previously identified by Méheust et al⁷⁵ that were inherited by the ochrophyte plastid, and two chimeric ancestral HPPGs which are probably of specific ochrophyte origin. Panel B shows a complete tabulated list of all ancestral HPPGs (listed by identifier, with the predicted function given in brackets) in which at least one chimerism event between domains of red algal, green algal, aplastidic stramenopile, other eukaryotic, and prokaryotic origin was detected. In each case, the inferred evolutionary origins of the N-terminal (NTD) and C-terminal (CTD) components of the chimeric members of the HPPG are given, according to the colour key within the figure, followed by its distribution across all ochrophyte lineages. The two chimeric HPPGs inferred to have arisen in the ochrophyte ancestor are shown in bold text and labelled with horizontal arrows. Exemplar alignments and phylogenies of the two chimeric proteins inferred to have originated in the ochrophyte ancestor are shown in figure supplements 1-3.
1829
1830
1831
1832
1833
1834
1835
1836
1837
1838
1839
1840
1841
<figure><img src="image_2.png" /></figure>
1892 **Fig. 9. Non-ochrophyte origins of the haptophyte plastid genome. Panels A and B,**
1893 respectively, show gene-rich and taxon-rich phylogenies of plastid-encoded proteins from
1894 red algae and plastids of red algal origin with the glaucophyte *Cyanophora paradoxa* as
1895 outgroup. **Panel A:** Combined Bayesian and Maximum Likelihood analysis (MrBayes +
1896 RAxML, GTR, JTT, WAG) of a 22 taxa x 12103 aa alignment of 54 proteins encoded by all
1897 published red and red-derived plastid genomes. **Panel B:** analysis of a 75 taxa x 3737 aa
1898 alignment of 10 conserved plastid-encoded proteins detectable in a broad range of red
1899 lineage MMETSP libraries. Nodes resolve with robust support (posterior probabilities of 1 for
1900 all Bayesian trees and > 80% bootstrap support for all ML trees) are shown with filled circles;
1901 individual support values for each analysis are shown for the remaining nodes are shown as
detailed in the box below panel B. Alternative topology tests, the results of fast-site and
1902 clade deduction analysis for each tree, and heatmap comparisons of sister-group
1903 relationships identified for single-gene trees of each constituent gene within each
1904 concatenated alignment are shown in figure supplements 1-3. **Panel C** shows the number of
1905 residues in each alignment that are uniquely shared between haptophytes and only one
1906 other lineage. For the gene-rich alignment (i), which is gap-free, residues are included that
1907 are found in all four haptophyte sequences and at least one sequence from the lineage
1908 under consideration. For the taxon-rich alignment (ii), to account for the presence of gapped
1909 positions, residues are included that are found in at least 11 of the 22 haptophyte sequences
1910 and at least one sequence from the lineage under consideration.
1911
1912

1913 **Fig. 10. Schematic diagram of events giving rise to the ancestral ochrophyte plastid proteome.** Each cell diagram depicts a different stage in the ochrophyte plastid
1914 endosymbiosis; each protein depicted represents on or more proteins inferred in this study
1915 to have been nucleus-encoded and plastid-targeted in the last common ancestor of all
1916 ochrophytes. An ancient ochrophyte ancestor, which had already diverged from oomycetes
1917 and other aplastidic stramenopile relatives, and which may have possessed a green algal
1918 plastid (A), acquired a red lineage plastid via secondary or higher endosymbiosis (B). Both
1919 the host and the endosymbiont are likely to have been evolutionary chimeras, possessing
1920 proteins encoded by genes acquired from endosymbiotic and/or lateral gene transfer
1921 events. Both host and symbiont are additionally likely to have possessed chimeric proteins,
1922 generated through the fusion of genes of different evolutionary origins, and a large number
1923 of mitochondrial-, ER- and (in the case of the red endosymbiont) potentially dual targeted
1924 proteins. Following genetic integration of the red endosymbiont with its stramenopile host,
1925 the first ochrophytes (C) thus possessed a wide range of proteins of plastid function
1926 acquired from different sources, with no apparent functional bias in the types of proteins
1927 that were retained from different sources. Chimeric proteins and dual targeted proteins,
1928 either acquired directly from the endosymbiont, or generated de novo, were also
1929 widespread features of this ancestral plastid proteome. Detailed information regarding the
1930 relationship between ultimate the evolutionary origins of each HPPG, and its presence or
1931 absence in other CASH lineages, is provided in figure supplement 1. A schematic diagram of
1932 possible models through which the haptophyte plastid may have originated is shown in
1933 figure supplement 2.
1934
1935
1936 **Supporting figure and dataset legends.**
1937
1938 **Fig. 1- figure supplement 1. Overview of eukaryotic diversity.** This figure, adapted from a
1939 previous review³, profiles the diversity of different eukaryotic nuclear lineages. Each grey
1940 ellipse corresponds to one major clade, or "supergroup" of eukaryotes. A brown ellipse
1941 within the stramenopile clade delineates the ochrophyte lineages. Dashed lines denote
1942 uncertain taxonomic relationship. For each taxon, a type species (defined either by the

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1943 presence of a complete genome, extensive transcriptome library, or of particular anthropic
1944 significance) is given in brackets. Taxa that lack plastids are labelled in grey, and taxa with
1945 plastids are shaded according to the evolutionary origin of that plastid lineage.

1946
1947 **Fig. 2- figure supplement 1- Exemplar ochrophyte plastid protein alignments.** This figure
1948 shows untrimmed GeneIOUS alignments for two ancestral HPPGs of unusual provenance. In
1949 each case the full length of the protein (labelled i) and N-terminal region only (ii) are shown,
1950 demonstrating the broad conservation of the N-terminus position. Sequences for which
1951 exemplar targeting constructs (*Phaeodactylum tricornutum*, *Nannochloropsis gaditana*,
1952 *Glenodinium foliaceum*) are shown at the top of each alignment.
1953

1954 **Fig. 2- figure supplement 2. Tree of ochrophyte glycyl-tRNA synthetase sequences.** This
1955 tree shows the consensus unrooted Bayesian topology for a 95 taxa x 487 aa alignment of
1956 glycyl tRNA synthetase sequences. The font colour of each sequence corresponds to the
1957 taxonomic origin (see legend below for details) and are labelled with the taxonomic
1958 identifiers previously defined in Table S1. Sequences labelled with chl_ possess apparent
1959 plastid targeting sequences recognisable by CASH lineage plastids. The ancestral ochrophyte
1960 plastidic isoform, of apparent chlamydiobacterial origin, is labelled with a blue ellipse. Black
1961 circles at each node denote posterior probabilities of 1.0 in Bayesian inferences with three
1962 different substitution matrices (GTR, Jones, and WAG), and grey circles indicate posterior
1963 probabilities of 0.8 with at least two of these matrices. Support values for all remaining
1964 nodes, using both Bayesian and RAxML analysis, is provided in the form <u>MrBayes posterior
1965 probabilities: GTR/Jones/WAG</u> RAxML best tree likelihoods: GTR/JTT/WAG
1966

1967 **Fig. 2- figure supplement 3. Tree of ochrophyte pyrophosphate dependent phosphofructo-1-kinase sequences.** This tree shows the consensus Bayesian topology inferred for a 94 taxa x 449 aa alignment of pyrophosphate-dependent PFK, with taxa and support values shown as per Fig. 2, figure supplement 2. The ancestral ochrophyte plastid isoform, of probable aplastidic stramenopile origin, is labelled with a cyan ellipse.

1972
1973 **Fig. 2- figure supplement 4. Tree of a novel ochrophyte plastid-targeted protein.** This tree
1974 shows the consensus Bayesian topology inferred for a 16 taxa x 103 aa alignment of a
1975 plastid-targeted protein seemingly restricted to ochrophytes and one dinoflagellate lineage.
1976 Taxa are labelled and support values are shown as per fig. 2- figure supplement 2.
1977

1978 **Fig. 2- figure supplement 5. Multipartite *Phaeodactylum* plastid-targeted proteins.** This
1979 figure shows the localisation of GFP overexpression constructs for copies of seven proteins
1980 from the diatom *Phaeodactylum tricornutum* that are of non-plastid origin, but show
1981 multipartite localization to the plastid and one other organelle (the mitochondria, or in the
case of the "ER heat shock protein" to the endoplasmic reticulum).

1983
1984 **Fig. 2- figure supplement 6.**
1985 **Fig. 2- figure supplement 6. Heterologous expression constructs of multipartite plastid-
1986 targeted proteins.** This figure shows the localisation of GFP overexpression constructs for
1987 copies of two proteins from the dinotom *Glenodinium foliaceum* (Panel A), and three
1988 proteins from the eustigmatophyte *Nannochloropsis gaditana* (Panel B) that are of non-
1989 plastid origin, but show multipartite localisation to the plastid and one other organelle, per
1990 Fig. 2, figure supplement 5.

1990
1991 **Fig. 2- figure supplement 7. Exemplar control images for confocal microscopy.** This figure
1992 shows fluorescence patterns for wild-type *Phaeodactylum tricornutum* cells (i), and
1993 transformant *Phaeodactylum* cells expressing GFP that has not been fused to any N-terminal
1994 targeting sequence (ii), both visualised under the same conditions used for all other
1995 transformant cultures.

1996
1997 **Fig. 4- figure supplement 1. Sampling richness associated with ancestral HPPGs of green algal origin.** This figure shows the number of sub-different archaeplastid orthologues for
1998
1999 ancestral HPPGs verified by combined BLAST top hit and single-gene tree analysis to be of
2000 either green algal origin (green bars) or red algal origin (red bars), for which glaucophyte
2001 orthologues could also be identified.

2002
2003 **Fig.4- figure supplement 2. Heatmaps of nearest sister-groups of ancestral HPPGs of verified green origin.** This figure shows the specific topologies of single gene trees for HPPGs
2004
2005 verified to be of green origin by combined BLAST and phylogenetic analysis. **Panel A** shows a
2006 reference topology of evolutionary relationships between green lineages, defined as per
2007 Leliaert et al. 2011. Six ancestral nodes that might correspond to the origin point of
2008 ochrophyte HPPGs are labelled with coloured boxes. **Panel B** shows the presence and
2009 absence of each green subcategory in the immediate sister-group to the ochrophyte HPPG in
2010 each single tree of HPPGs of verified origin. HPPGs are grouped by the inferred origin point
2011 within the green algae, with the number of HPPGs identified for each origin point given with
2012 round brackets.

2013
2014 **Fig. 4- figure supplement 3. Specific origins of green HPPGs as inferred from BLAST top hit analyses.** These charts show (i) the number of BLAST top hits against each of the individual
green sub-categories from HPPGs for which a green origin was identified both from BLAST
2015
2016 top hit and single-gene tree analysis, and (ii) the total number of non-redundant sequences
2017 from each green sub-category included in the BLAST library.
2018
2019

2020 **Fig. 4- figure supplement 4. Earliest evolutionary origins of shared plastid residues.** This
2021 figure shows the number of residues in the concatenated alignment of HPPGs of verified
green origin, which have been subsequently vertically inherited in all major photosynthetic
2022 eukaryotes that are present in green algae and ochrophytes, and are not found in red algae
2023 and glaucophytes. Residues are divided by inferred origin point, and are shown as per fig. 4,
2024 panel D. The values here a calculated as the earliest possible origin point for each uniquely
2025 shared residue, in which all gapped and missing positions within the alignment are treated
2026 as potential identities. 100 of the 147 residues inferred to have originated within green algae
2027 in this analysis originated either within a common ancestor of all chlorophytes, or in a
2028 common ancestor of all chlorophytes excluding the basally divergent lineages *Prasinoderma*,
2029 *Prasinococcus* and *Nephroselmis*.

2030
2031
2032 **Fig. 4- figure supplement 5. Origins and HECTAR based targeting tests of proteins encoded by conserved ochrophyte gene clusters.** Panel A shows the most probably evolutionary
2033 origin, identified using BLAST top hit analysis, for 7140 conserved gene clusters inferred to
2034 have been present in the last common ochrophyte ancestor. Panel B shows the number of
2035 these gene families that are predicted by HECTAR to encode proteins targeted to the plastid,
2036 subdivided by probable evolutionary origin, and the number expected to be present in each
2037 category assuming a random distribution of plastid-targeted proteins across the entire
2038 dataset, independent of evolutionary origin. Categories inferred to be significantly enriched
2039 above the expected values are labelled with black arrows.

2040
2041
2042 **Fig. 5- figure supplement 1. Reconstructed metabolism pathways and core biological processes in the ancestral ochrophyte plastid.** This figure tabulates each of the ancestral
2043 ochrophyte HPPGs corresponding to 350 central plastid metabolism and other biological
2044
2045 processes. The "origin" column shows the probable evolutionary source for each HPPG as defined by combined BLAST tophit and single-gene tree analysis. The origin of each ancestral HPPG is either assigned a "high confidence" value (in which the same origin was robustly supported both by single-gene tree and by BLAST tophit analysis) or a "low confidence" value (in the absence of robust and consistent support through both techniques; corresponding to the tree sister-group if one could be clearly assigned, or the BLAST tophit identity if not). A dash indicates the corresponding protein was not identified in the ancestral HPPG dataset due to either being plastid-encoded or alternative reasons; detailed explanations for the enzymes that are neither plastid-encoded nor detected in the ancestral HPPG dataset are provided in figure supplement 2.

2055

**Fig. 5- figure supplement 2. Core plastid metabolism proteins not identified within the ancestral HPPG dataset.**

2058

**Fig. 5- figure supplement 3. Tree of ochrophyte sedoheptulose-7-bisphosphatase sequences.**

2059 This figure shows the consensus Bayesian topology inferred for a 218 taxa x 303 aa alignment of sedoheptulose-7-bisphosphatase sequences, shown as per fig. 2, figure supplement 2. Two different ochrophyte plastid isoforms- one restricted to chrysista, and of probable red algal origin, and one found in hypogyristea and diatoms, of probable green algal origin- are shown respectively by red and green ellipses.

2065

**Fig. 5- figure supplement 4. Tree of ochrophyte 3-dehydroquinate synthase sequences.**

2066 This figure shows the consensus Bayesian topology inferred for a 324 taxa x 387 aa alignment of 3-dehydroquinate synthase, shown as per fig. 2, figure supplement 2. Three ochrophyte plastid isoforms are shown with coloured ellipses: a probable bacterial isoform restricted to pelagophytes and dictyochophytes (blue ellipse), and two isoforms of ambiguous red/green origin found respectively in raphidophytes and eustigmatophytes, and in diatoms (green ellipses with red borders).

2073

**Fig. 5- figure supplement 5. Tree of ochrophyte isopropylmalate dehydrogenase sequences.**

2074 This tree shows the consensus Bayesian phylogeny inferred for a 202 taxa x 592 aa alignment of isopropyl malate dehydrogenase sequences, shown as per fig. 2- figure supplement 2. Two ochrophyte plastid isoforms are shown with coloured ellipses: an isoform of green algal origin restricted to diatoms and hypogyristea (green ellipse), and a red algal isoform found in diatoms, pelagophytes and xanthophytes (red ellipse).

2080

**Fig. 5- figure supplement 6. Tree of ochrophyte shikimate kinase sequences.**

2081 This figure shows the consensus Bayesian topology inferred for a 127 taxa x 262 aa alignment of shikimate kinase sequences. The WAG Bayesian topology was excluded from the consensus due to non-convergence between the two chains, hence the tree is produced from the consensus of GTR and Jones substitution matrices only, but is otherwise presented identically to fig. 2, figure supplement 2. Two distinct ochrophyte plastid isoforms are shown with coloured ellipses: a green algal isoform conserved across diatoms, dictyochophytes and raphidophytes (red ellipse), and a pelagophyte isoform of uncertain origin (grey ellipse).

2089

**Fig. 5- figure supplement 7. KOG classes associated with different categories of HPPGs.**

2090 These pie charts profile the distribution of different KOG classes across (i) all HPPGs except for those with general function predictions only, or without any clear KOG function, (ii) the same, but restricted to ancestral HPPGs and (iii) the same, for ancestral HPPGs of unambiguous red, green, prokaryotic and aplastidic stramenopile origin as identified by combined BLAST tophit and single-gene tree analysis. KOG classes that occur at elevated
2096 frequency in the ancestral HPPG dataset compared to the complete HPPG dataset, and one
2097 KOG class enriched in the prokaryotic HPPG dataset compared to the ancestral HPPG dataset
2098 (chi-squared test, P< 0.05) are labelled with horizontal arrows.

2099
2100 **Fig. 5- figure supplement 8. Coregulation of genes incorporated into HPPGs of different origin in the model diatom *Phaeodactylum tricornutum*.** Panel A shows boxplots of the correlation coefficients between the expression profiles of genes encoding members of ancestral HPPGs of red algal origin (i), green algal origin (ii), prokaryotic origin (iii) or host origin (iv), compared to genes encoding members of other HPPGs. Each HPPG is separated by evolutionary origin on the x-axis of each graph: for example, the box labelled "green algae" on the "red algae" graph shows the correlation coefficients between genes encoding members of ancestral HPPGs of red origin, and ancestral HPPGs of green origin. Panel B shows the P value statistics of mean separation calculated when comparing genes encoding members of ancestral HPPGs of the same origin (shown by row) to members of ancestral HPPGs of different origin (shown by column). For example, the intersect between the "red" row and "green" column shows the difference in mean correlation coefficient between pairs of genes that both encode members of ancestral HPPGs of red origin, and gene pairs of which one encodes an ancestral HPPG member of red origin, and the other an ancestral HPPG member of green origin. None of the P values calculated are significant, i.e. there are no categories of ancestral HPPG in which the internal correlation coefficients of gene expression are any different to those observed across the dataset as a whole.

2111
2112
2113
2114
2115
2116
2117
2118 **Fig. 5- figure supplement 9. Coregulation of genes incorporated into HPPGs of different origin in the model diatom *Thalassiosira pseudonana*.** Boxplots (Panel A) and P value statistics (Panel B) are shown as per Fig. 5- figure supplement 8. Only two of the correlation value ANOVA tests (comparison of red-red and red-host correlations, and prokaryotic-prokaryotic and prokaryotic-host correlations, shaded in green) reveal a significantly higher correlation coefficient between pairs of genes encoding members of HPPG of the same evolutionary origin than pairs of genes encoding members of HPPGs with different evolutionary origins. These differences most probably reflect the extremely weak correlation coefficients associated with genes encoding HPPGs of host origin to all other genes considered (compare "Host" category on boxplots i, ii and iii to all other categories); however, detailed comparison of the correlation values between genes encoding ancestral HPPGs of host origin and genes encoding ancestral HPPGs of different evolutionary origin (Panel A, boxplot iv; Panel B, bottom row) reveals no specific difference in the pairwise correlation values observed between genes encoding ancestral HPPGs of host origin, and genes encoding ancestral HPPGs of all other origins within the dataset.

2119
2120
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2128
2129
2130
2131
2132
2133
2134 **Fig. 6- figure supplement 1. Alignments of an ochrophyte-specific riboflavin biosynthesis fusion protein.** Panel A shows alignments of the full length (i) and cyclohydrolase domain only (ii) of a plastid-targeted GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase protein conserved across the ochrophytes. Coloured bars adjacent to each sequence correspond to the phylogenetic identity of the sequence. The cyclohydrolase domain of the ochrophyte protein is positioned in the N-terminal region, and the synthase domain in the C-terminal region. Three uniquely shared residues at the N-terminus of the cyclohydrolase domain confirm that it has been inherited from the aplastidic stramenopile ancestor of the ochrophytes.

2134
2135
2136
2137
2138
2139
2140
2141
2142
2143
2144 **Fig. 6- figure supplement 2. Origins of ochrophyte plastid 3,4-dihydroxy-2-butanone 4-phosphate synthase.** This figure shows the consensus Bayesian topology inferred for a 22 taxa x 206 aa alignment of 3,4-dihydroxy-2-butanone 4-phosphate synthase domains from
2147 different lineages, inferred using Jones and WAG matrices, and shown as per fig. 2, figure 2148 supplement 2. The ochrophyte plastid isoforms branch with red algal and actinobacterial 2149 sequences.

2150 Fig. 6- figure supplement 3. An ochrophyte-specific Tic20 fusion protein. This figure shows 2151 alignments of the full length (i) and conserved region only (ii) of plastid Tic20 sequences, 2152 displayed as per figure supplement 1. 2153

2154 Fig. 7- figure supplement 1. Experimental verification of additional ochrophyte dual-targeted proteins. Panel A shows Mitotracker-orange stained *Phaeodactylum tricornutum* 2155 lines expressing four additional dual-targeted proteins (glycyl-, leucyl-, and methionyl-tRNA 2156 synthetases, and a predicted mitochondrial GroES-type chaperone) from *Phaeodactylum tricornutum*, and a dual-targeted histidyl-tRNA synthetase from *Glenodinium foliaceum*. 2157 2158 2159 Panel B shows control images that confirm an absence of crosstalk between GFP and 2160 Mitotracker: wild-type *Phaeodactylum* cells stained with Mitotracker, and cells expressing 2161 the *Glenodinium* histidyl-tRNA synthetase–GFP fusion construct and visualised with the 2162 Mitotracker laser and channel in the absence of Mitotracker stain. 2163 2164

2165 Fig. 7- figure supplement 2. Comparison of different in silico targeting prediction 2166 programmes for the identification of dual-targeted ochrophyte proteins. Panel A shows 2167 Mitofates scores for ochrophyte proteins verified experimentally to be dual targeted in this 2168 and a previous study9. Panel B shows Mitofates scores for all ochrophyte proteins for which 2169 a subcellular localisation has been identified in previous studies. The red lines in each graph 2170 show the Mitofates default cutoff (0.385) and the green lines indicate our chosen cutoff 2171 (0.35). Panel C compares different in silico targeting prediction algorithms with respect to 2172 predicted mitochondrial localization by experimentally validated localization. Mitofates 2173 strikes the best balance between high true positives and low false positives. 2174

2175 Fig. 8- figure supplement 1. Origin of proteins of ochrophyte origin in different CASH 2176 lineages. This figure profiles the evolutionary origins of proteins inferred by single-gene 2177 phylogenetic analysis to have been transferred from the ochrophytes into other lineages 2178 that have acquired plastids through secondary or more complex endosymbioses. Proteins 2179 are divided into the three major ochrophyte lineages (i.e. diatoms, chrysista, and 2180 hypogyristea); all remaining proteins (inferred to have been acquired from an ancestor of 2181 multiple ochrophyte lineages, or of ambiguous but clearly ochrophyte origin) are grouped as 2182 a final category. The haptophyte proteins that could be attributed to a specific ochrophyte 2183 lineage are particularly skewed (100/178 proteins) to origins within the hypogyristea. 2184

2185 Fig. 8- figure supplement 2. Heatmaps of nearest sister-groups to haptophytes in ancestral 2186 ochrophyte HPPG trees. This figure shows the specific ochrophyte lineages implicated in the 2187 origin of haptophyte plastid-targeted proteins, as inferred from the nearest ochrophyte 2188 sister-groups to haptophytes in trees of 242 haptophyte proteins of probable ochrophyte 2189 origin from combined BLAST top hit and single-gene tree analysis. At the top a schematic 2190 tree diagram of the ochrophytes is shown as per fig. 1, with six major nodes in ochrophyte 2191 evolution labelled with coloured boxes. The heatmap below shows the specific distribution 2192 of sister-groups in each tree, shown as per figure 4- figure supplement 2. 2193

2194 Fig. 8- figure supplement 3. Internal evolutionary affinities of haptophyte plastid-targeted 2195 proteins incorporated into ancestral ochrophyte HPPGs. This figure profiles the 2196 evolutionary origins of haptophyte plastid-targeted proteins incorporated into ancestral 2197 ochrophyte HPPGs by BLAST top hit analysis. Separate values are provided for query
2198 sequences from each of the three haptophyte sub-categories (pavlovophytes,  
2199 prymnesiophytes, and isochrysidales) considered within the analysis. Only sequences for  
2200 which a consistent origin could be identified by both BLAST top hit and single-gene tree  
2201 analysis are included. For each haptophyte lineage > 50% of the sequences verified by  
2202 combined analysis to be of a specific ochrophyte origin have either pelagophyte or  
2203 dictyochophyte top hits.  
2204

2205 **Fig. 8- figure supplement 4. Evidence for gene transfer from pelagophytes and dictyochophytes into haptophytes.** Panel A shows the next deepest sister groups identified for haptophyte proteins of hypogyristean origin in single-gene trees. The pie chart (i) compares the number of single-gene trees in which the combined clade of haptophyte and hypogyristean proteins resolves within a larger clade comprising the ochrophyte HPPG, compared to the number that resolves in external positions, either with other lineages or as a sister-group to all other sequences within the HPPG clade. Sequences for which no clear next deepest sister group affinity could be identified are listed as "not determined". The heatmap (ii) shows the specific sister-group sequences associated with 65 HPPGs in which the haptophyte sequences specifically resolve with the pelagophyte/ dictyochophyte clade and for which a clear internal or external position for the haptophyte/ hypogyristean group relative to the remaining ochrophyte HPPG clade could be identified. Both analyses indicate a clear bias for haptophyte sequences branching within a deeper ochrophyte clade, not just restricted to the immediate sister-groups. Panel B tabulates the BLAST next best hits for haptophyte sequences for which a phylogenetically consistent (>3 consecutive top hits) top hit to hypogyristea could be identified, and pelagophyte/ dictyochophyte sequences for which a phylogenetically consistent top hit to haptophytes could be identified. In each case either the largest number of sequences, or (in the case of pavlovophytes) the joint largest number of sequences for which a phylogenetically consistent next best hit could be identified resolved with diatoms, indicating that these sequences were probably present in the common ancestor of diatoms and hypogyristea, and subsequently transferred to the haptophytes.

2228 **Fig. 8- figure supplement 5. Earliest possible origin points of uniquely conserved sites in haptophyte plastid-targeted proteins.** This figure shows the total number of residues that are uniquely shared between a 37 proteins that have clearly been transferred between the ochrophytes and haptophytes, and are of subsequently entirely vertical origin, assuming the earliest possible origin point for each residue (i.e. in which gapped or missing positions were interpreted as identities). 87/128 of the uniquely shared residues inferred to originate within the ochrophytes were congruent to gene transfers between the haptophytes and pelagophyte and dictyochophyte clade; of these, slightly more than half (46) are inferred to have originated in a common ancestor of all hypogyristea and diatoms, consistent with the gene transfer having occurred from an ancestor of the pelagophytes and dictyochophytes into the haptophytes, rather than the converse.

2240 **Fig. 8- figure supplement 6. Evolutionary origin of ancestral haptophyte genes.** This figure shows the most likely evolutionary origin assigned by BLAST top hit analysis to the 12728 conserved gene families inferred to have been present in the last common haptophyte ancestor.

2245 **Fig. 9- figure supplement 1. Alternative topology tests of plastid genome trees.** Tests were performed with the RAxML + JTT trees inferred for the gene-rich **(panel A)** and taxon-rich **(panel B)** plastid-encoded protein alignments. In each case, a schematic diagram of the tree topology obtained is given **(i)**. The black box corresponds to the branch position of