Plastid Genome Analysis of Three Nemaliophycidae Red Algal Species Suggests Environmental Adaptation for Iron Limited Habitats

RESEARCH ARTICLE Plastid genome analysis of three Nemaliophycidae red algal species suggests environmental adaptation for iron limited habitats

Chung Hyun Cho1, Ji Won Choi1, Daryl W. Lam2, Kyeong Mi Kim3, Hwan Su Yoon1*

1 Department of Biological Sciences, Sungkyunkwan University, Suwon, Korea, 2 Department of Biological a1111111111 Sciences, University of Alabama, Tuscaloosa, Alabama, United States of America, 3 Marine Biodiversity Institute of Korea, Seocheon, Korea a1111111111 a1111111111 * [email protected] a1111111111 a1111111111 Abstract

The red algal subclass Nemaliophycidae includes both marine and freshwater taxa that con-

OPEN ACCESS tribute to more than half of the freshwater species in Rhodophyta. Given that these taxa inhabit diverse habitats, the Nemaliophycidae is a suitable model for studying environmental Citation: Cho CH, Choi JW, Lam DW, Kim KM, Yoon HS (2018) Plastid genome analysis of three adaptation. For this purpose, we characterized plastid genomes of two freshwater species, Nemaliophycidae red algal species suggests Kumanoa americana (Batrachospermales) and Thorea hispida (Thoreales), and one marine environmental adaptation for iron limited habitats. species Palmaria palmata (Palmariales). Comparative genome analysis identified seven PLoS ONE 13(5): e0196995. https://doi.org/ genes (ycf34, ycf35, ycf37, ycf46, ycf91, grx, and pbsA) that were different among marine 10.1371/journal.pone.0196995 and freshwater species. Among currently available red algal plastid genomes (127), four Editor: Timothy P. Devarenne, Texas A&M genes (pbsA, ycf34, ycf35, ycf37) were retained in most of the marine species. Among University College Station, UNITED STATES these, the pbsA gene, known for encoding heme oxygenase, had two additional copies Received: January 19, 2018 (HMOX1 and HMOX2) that were newly discovered and were reported from previously red Accepted: April 24, 2018 algal nuclear genomes. Each type of heme oxygenase had a different evolutionary history Published: May 8, 2018 and special modifications (e.g., plastid targeting signal peptide). Based on this observation,

Copyright: © 2018 Cho et al. This is an open we suggest that the plastid-encoded pbsA contributes to the iron controlling system in iron- access article distributed under the terms of the deprived conditions. Thus, we highlight that this functional requirement may have prevented Creative Commons Attribution License, which gene loss during the long evolutionary history of red algal plastid genomes. permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability Statement: Data are available from the NCBI (GenBank accession numbers Introduction NC_031178, NC_031171, NC_031147). The red algal class Florideophyceae comprises 95% (6,748 spp. out of 7,100 spp.) of the Rhodo- Funding: This study was supported by the grants phyta and encompass a biologically diversified group of taxa [1, 2]. Most of the red algal spe- from the Collaborative Genome Program cies (>95%) inhabit marine habitats, however about 5% are found in freshwater environments (20140428) funded by the Ministry of Oceans and [3]. The Nemaliophycidae, one of five subclasses within Florideophyceae, contains more than Fisheries, the National Research Foundation of half of these freshwater species. This subclass includes both marine and freshwater taxa with Korea (NRF-2017R1A2B3001923), the Next- generation BioGreen21 Program (PJ01389003) three exclusively freshwater orders (Balbianiales, Batrachospermales, Thoreales), six exclu- from the Rural Development Administration, Korea, sively marine orders (Rhodachlyales, Balliales, Nemaliales, Entwisleiales, Colaconematales, and by a grant from the National Science Palmariales), and one order (Acrochaetiales) with both freshwater and marine species [1, 2, 4,

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Foundation Assembling the Tree of Life program 5]. Among the freshwater orders, the Batrachospermales and Thoreales have more than half of (DEB-0937975, 1317114) to Hwan Su Yoon and the freshwater species in all of the Rhodophyta [6]. Thus, a comparison of Nemaliophycidae Morgan Vis. The funders had no role in study plastid genomes of taxa from freshwater and marine habitats may provide insights into envi- design, data collection and analysis, decision to publish, or preparation of the manuscript. ronmental adaptation [1]. A previous study demonstrated that the freshwater angiosperm Najas flexilis (water Competing interests: The authors have declared nymph) adapted to the aquatic environment from its terrestrial ancestor by the complete loss that no competing interests exist. of the ndh gene family in plastid genome [7]. The NDH gene complexes encode for the NAD (P)H dehydrogenase complex that increases photosynthetic efficiency at variable light intensi- ties in terrestrial habitats. In its transition to the aquatic environment, N. flexilis did not require resistance to high light stress (due to the refractive properties of water) and therefore the ndh gene family had been lost. Likewise, similar gene loss or retention events may be present in the evolution of red algal plastid genomes during their transitions from marine habitats to freshwater systems. To date, 99 florideophycean plastid genomes (cf. 127 red algal plastid genomes including three new genomes) are available in the NCBI organelle database, including 23 Nemaliophyci- dae that have been detailed in three recent papers [8–10]. To extend our understanding of red algal plastid evolution as it relates to the habitat adaptation, we completely sequenced and annotated three new plastid genomes for Nemaliophycidae, including one marine (Palmaria palmata) and two freshwater species (Kumanoa americana, Thorea hispida). From a comparative analysis of plastid genomes, we seek to identify plastid genes involved in the transition between marine and freshwater red algae and their physiological implications.

Materials and methods Whole genome sequencing and plastid genome construction Culture strains of two filamentous freshwater species of Kumanoa americana (hsy120, isolated by Franklyn D. Ott from a stream in Mississippi, USA) and Thorea hispida (hsy077, isolated by F. Ott from the Kaw river in Kansas, USA) were harvested with gentle centrifugations from the culture flask. Thalli of Palmaria palmata (commercially sold as dulse) were collected from Reid State Park in Maine, USA on 27 Aug. 2010 by HSY. Genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and purified by LaboPass™ DNA Isola- tion Kit (Cosmo Genetech, Seoul, Korea). Genome sequence data were generated using the Ion Torrent PGM (Thermo Fisher Scientific, San Francisco, California, USA) Next-Genera- tion Sequencing (NGS) platform. The sequencing libraries were prepared using the Ion Xpress Plus gDNA Fragment Library Preparation kit for 200 bp or 400 bp libraries. The library amplification and DNA sequencing were conducted by either Ion PGM Template OT2 200 or 400 Kits and Ion PGM Sequencing OT2 200 or 400 Kit for the Ion Torrent PGM platform. From NGS genome sequencing data, short raw reads (< 50 bp) were removed completely from the analysis and the remainder of raw reads were de novo assembled into contigs using CLC Genomics Workbench 5.5.1 (CLC Bio., Aarhus, Denmark) and MIRA3 Assembler [11]. To obtain a plastid consensus sequence, contigs were sorted by tBLASTn (e-value: 1e-10) using the protein sequence of red algal plastid genes as a reference (i.e. Chondrus crispus, Cal- liarthron tuberculosum) [12, 13]. After the reassembly process, we obtained the circular plastid genomes and those circular genomes were confirmed by MUMmerplot [14] and by comparing with reference genomes to check for completeness. The sequences were verified with read- mapping tools implemented in CLC Genomics Workbench to correct for any sequencing errors or gaps. Protein coding genes were manually annotated following the procedure described in Song et al., 2016 [15] using ‘Bacteria and Archaea (11)’ genetic code and the NCBI database for non-

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redundant (nr) protein sequences. To search the ribosomal RNA sequences, we applied RNAmmer v1.2 Server [16] using the option of Bacteria, and then re-confirmed by BLASTn. Introns, tRNAs, and other small RNA were searched by ARAGORN [17] and Rfam cmscan v1.1 [18]. Several important genes and ambiguous sequences were re-confirmed with PCR amplification followed by Sanger sequencing. The annotated plastid genomes were visualized using OrganellarGenomeDraw v1.2 [19]. Comparative analysis of the genome structure was accomplished using UniMoG v1.0 [20] and Mauve Genome Alignment v2.2.0 [21, 22] through the Geneious plug-in using the default setting [23].

A statistical test for the habitat-gene correlation Habitat information of each species was referred from previous studies. Ott [5] summarized detail about the habitats and isolation history for most nemaliophycidaean species. AlgaeBase [2] provided information about authentic references with the type locality and its distribution. Based on the habitat and gene presence/absence information in the plastid genome, we performed a chi-square analysis that assessed the correlation between habitat and putative habitat-specific genes. We used the chi-square test incorporated into R package [24]. The p-value cut-off (<0.01) was adjusted to reject the null hypothesis.

Phylogenetic analysis The red algal protein sequences of heme oxygenase were collected from NCBI public data- bases, which include transcriptome and genome data. The heme oxygenase homologs were obtained by BLASTp against the NCBI database with an adjusted threshold-cut to 500 top matches of red algal heme oxygenase. To discover the nuclear heme oxygenase, we also surveyed heme oxygenase genes from the published [25–28] or unpublished (H.S. Yoon et al.) whole genome data as described in S5 Table. Multiple sequence alignments were performed using MAFFT version 7 [29] with the default options. These alignments were refined manually based on conserved domains. Maximum likelihood-based phylogenetic analysis and bootstrap methods (MLB) were conducted using IQ-TREE [30] with 1,000 ultrafast bootstrap replica- tions. The evolutionary model was ‘LG + I + G4’ [31], which was automatically selected by the model test option incorporated in IQ-TREE. Finally, highly divergent or contaminant sequences, which showed a long-branch or taxonomical mismatch to sister taxa, were removed from the following analysis.

Protein domain predictions Protein domains were searched by the NCBI conserved domain searching tool [32] to predict their functions and conserved motifs. To predict gene localization, TargetP [33] and ChloroP [34] were used based on transit peptide sequences. Transmembrane domain regions were identified using TMHMM program [35]. The molecular function of heme oxygenase and its molecular interaction were surveyed based on KEGG pathway [36].

Gene network analysis The dataset of heme oxygenase genes for network analysis was collected from NCBI based on BLASTp with an adjusted threshold-cut to the top 500 matches of red algal heme oxygenase (S2 Fig). The genes containing incomplete heme oxygenase domains were removed from the dataset, therefore, only complete domains were used for network analysis [32]. EGN (Evolu- tionary Gene and Genome Network) was performed to build a gene network based on protein similarity [37]. For comparative analysis, network connection thresholds were set at 1e-05 in

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e-value, identities at 20%, hits with at least 20% of the shortest sequence, and query coverage of both sequences at 70%. The resulting network was visualized with the aid of Cytoscape program [38].

Result and discussion General features of three plastid genomes A total of 1.73 Gbp, 1.94 Gbp, and 1.10 Gbp of raw sequence data were produced for Palmaria palmata, Kumanoa americana, and Thorea hispida, respectively (see details in S1 Table). The average coverage for the plastid genomes was 1,003x in T. hispida, 215x in K. americana, and 343x in P. palmata. Three complete plastid genomes (Fig 1A) were manually annotated based on published red algal plastid references [39]. Table 1 summarizes the characteristics of plastid genomes of the Florideophyceae [8–10, 12, 13, 39–53]. The total size of the plastid genome was 184,026 bp in K. americana (GenBank accession number NC_031178), containing 194 protein-coding genes (CDS), while T. hispida (GenBank accession number NC_031171) was 175,193 bp in size including 192 CDSs. The P. palmata plastid genome (GenBank accession number NC_031147) was 192,961 bp in size with 203 CDSs. The GC content of K. americana was 29.3%, which was similar to T. hispida (28.3%), but lower than that of P. palmata (33.9%). The high GC content in P. palmata was more similar to that of the Bangiophyceae (average of 11 spp.: 33.1%) than other Florideophyceae species (average of 102 spp.: 29.3%). Comparing the genome architecture, two major differences were evident among the three Nemaliophycidae species (Fig 1B). First, two copies of ribosomal RNA (rRNA) operon were present in P. palmata, whereas K. americana and T. hispida have only a single rRNA operon (5S, 23S, 16S rRNA) like as most of florideophycean species (Table 1). It has been reported that the plastid genome structures are highly conserved among four florideophycean subclasses (i.e., Nemaliophycidae, Corallinophycidae, Ahnfeltiophycidae, Rhodymeniophycidae) [39]. Second, K. americana had a large inversion between chlL and rRNA operon region that differs from other two species.

Specific gene loss in freshwater Nemaliophycidae species Gene contents were generally conserved among the three Nemaliophycidae plastid genomes, however, there were some differences (S1 Fig, S2 Table). For example, the ycf91 gene was present only in two freshwater species of K. americana and T. hispida, but absent in marine P. palmata. In addition, six genes (ycf34, ycf35, ycf37, ycf46, grx, and pbsA) were preserved only in the marine P. palmata, which were absent in the freshwater K. americana and T. hispida. Therefore, these seven genes were candidates of habitat-specific plastid gene (i.e., marine vs. freshwater specific). In order to broaden the investigation of these putative habitat-specific genes, we extended the survey to include all currently available 127 red algal plastid genomes, which include 16 freshwater species, 109 marine species, and two brackish species (S3 Table). From the data in S3 Table, we calculated both the habitat-specific gene concordance rate and the p-value from the chi-square test for seven genes for whether these genes were correlated to the habitat type. Interestingly, we discovered that four of these genes (pbsA, ycf34, ycf35, and ycf46) have higher than 80% concordance rate with statistically significant support (p-values = < 0.01). These results suggest that the presence of these four genes is significantly different between marine and freshwater habitats. For example, the pbsA gene showed 84.1% of habitat-specific gene concordance rate (p-value = 3.17E-07). Except for four species (Bangia atropurpurea, Sheathia arcuata, Paralemanea sp., Sirodotia delicatula), 12 of 16 freshwater species lacked the pbsA

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Fig 1. The genome maps of three Nemaliophycidae plastids and their genome structure comparison. (A) Three plastid genome maps of Kumanoa americana, Thorea hispida, and Palmaria palmata. (B) A simplified comparative genome structure between three species based on MAUVE and UniMoG analyses. A large inversion in rps6-chlL region was observed consistently from two different analyses. https://doi.org/10.1371/journal.pone.0196995.g001

gene in the plastid genomes. Likewise, the habitat-specific gene concordance rates of ycf34, ycf35, ycf46 and their p-value were 85.6%, 81.8%, 87.9% and 4.83E-10, 2.42E-03, 5.43E-12, respectively. To inspect the association between the phylogenetic relationship and the habitat-gene pattern, we mapped the presence or absence of these four genes on the ML phylogeny with habitat information (Fig 2). According to the result, the losses (i.e., absence) of four habitat-specific genes were more likely due to a phylogenetic pattern as compared to random events. For instance, all four genes were absent in four Cyanidiales species, which are all freshwater species. Two exclusive freshwater orders of Thoreales (T. hispida) and Batrachospermales (eight species) were mostly absent of these genes that were clearly different from marine orders that contained these genes (i.e., one species of Palmariales and 16 spp. of Nemaliales). It is interesting that some mangrove species (i.e., Bulboplastis apyrenoidosa, Caloglossa spp. Bostrychia spp.) [54–56] and two parasitic species (i.e., Polysiphonia infestans, Choreocolax polysiphoniae) [57, 58], which have significantly reduced plastid genomes, lost these genes. However, there were some exceptional cases: two marine species of Hildenbrandiales (Apophlaea sinclairii, Hildenbrandia rubra) together with one freshwater species (H. rivularis) were absent of all these genes, same as in two marine Corallinophycidae species (Sporolithon durum and Calliarthron tuberculosum). Based on this observation (Fig 2), we postulate that four habitat-specific genes (pbsA, ycf34, ycf35, and ycf46) were adapted to freshwater habitat during their evolutionary history. To find a functional relevance of these genes in environmental adaptation, we focused on the in silico functional analysis. Because any functions were reported for ycf genes, a conserved hypothetical protein family, we selected only on the pbsA (heme oxygenase) gene for down- stream analyses.

Heme oxygenase The function of heme oxygenase is generally known for the degradation of a heme to a biliverdin and is involved in the production of phycobilins [59]. For instance, the heme oxygenase degrades heme that is an essential step in the phycobilin biosynthesis in Cyanidium caldarium (Cyanidiophyceae) [60]. During a heme degradation, iron ions are released and those ions play an essential role in the iron recycling pathway [61, 62]. While searching for pbsA genes in all available red algal genome data (S5 Table), two additional heme oxygenase genes were newly discovered from nuclear genome data. Both the newly discovered two heme oxygenases and pbsA had a conserved heme oxygenase domain (CDD name: HemeO superfamily), with conserved heme binding pockets (blue asterisk in Fig 3). According to recent studies about heme oxygenase in Chlamydomonas reinhardtii (Chloro- phyta), two distinct types of nuclear-encoded heme oxygenase have been called as HMOX1 (plant type) and HMOX2 (animal type) [63]. However, there was no plastidal heme oxygenase (pbsA) in green algae and we could not find it from a currently available nuclear genome. Compared to the green algal heme oxygenase, we named three red algal heme oxygenase genes as HMOX1 and HMOX2 for nuclear copies and pbsA for the plastidal copy. To identify the evolutionary history of three heme oxygenase isotypes, homologs of heme oxygenase were collected from the NCBI database (see details in Materials and Methods). These three distinct types of red algal heme oxygenase were grouped in three clades in the phylogenetic tree (Fig 4).

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Table 1. Comparison of general features for 102 florideophycean plastid genomes. Subclass Species General Characteristics RNAs GenBank Reference Total bp GC % Introns CDS tRNAs rRNA Accession Hildenbrandio-phycidae Apophlaea sinclairii 182,437 30.5% 2 190 31 3 NC_031172 [39] Hildenbrandia rivularis 189,725 32.4% 2 186 31 3 NC_031177 [39] Hildenbrandia rubra 180,141 31.4% 2 191 31 3 NC_031146 [39] Nemaliophycidae Batrachospermum viride-brasiliense 171,722 28.2% 1 172 30 3 MG252484 [9] Batrachospermum macrosporum 179,687 28.0% 1 164 37 3 MG252483 [9] Dermonema virens 184,997 34.1% 2 208 31 3 NC_031655 [8] Dichotomaria marginata 184,395 28.8% 2 211 31 3 NC_031656 [8] Galaxaura rugosa 181,215 29.6% 2 207 31 3 NC_031657 [8] Helminthocladia australis 185,694 32.8% 2 206 31 3 NC_031658 [8] Helminthora furcellata 184,585 32.1% 2 207 31 3 NC_031654 [8] Hommersandiophycus borowitzkae 184,728 32.2% 2 205 31 3 NC_031659 [8] Izziella formosana 183,248 35.0% 2 207 31 3 NC_031660 [8] Kumanoa ambigua 183,003 28.1% 1 165 28 3 MG252485 [9] Kumanoa americana hsy120 184,025 29.3% 2 201 32 3 NC_031178 This study Kumanoa mahlacensis 181,361 29.8% 1 166 28 3 MG252486 [9] Liagora brachyclada 182,937 33.7% 2 207 31 3 NC_031667 [8] Liagora harveyana 182,933 33.9% 2 208 31 3 NC_031661 [8] Liagoropsis maxima 189,564 32.1% 2 208 31 3 NC_031662 [8] Nemalion sp. H.1444 182,930 35.5% 2 204 31 3 LT622871 [8] Neoizziella asiatica 183,313 33.4% 2 208 31 3 NC_031663 [8] Palmaria palmata 192,960 33.9% 2 205 34 6 NC_031147 This study Paralemanea sp. 180,393 30.5% 1 167 28 3 MG252487 [9] Scinaia undulata 183,795 35.9% 2 209 31 3 NC_031664 [8] Sheathia arcuata 187,354 29.9% 2 187 31 6 KY033529 [10] Sirodotia delicatula 185,555 29.1% 1 164 30 3 MG252489 [9] Thorea hispida hsy077 175,193 28.3% 2 194 31 3 NC_031171 This study Titanophycus setchellii 183,356 32.7% 2 206 31 3 NC_031665 [8] Trichogloeopsis pedicelleta 183,497 31.9% 2 206 31 3 NC_031668 [8] Yamadaella caenomyce 182,460 35.9% 2 206 31 2 NC_031666 [8] Corallinophycidae Calliarthron tuberculosum 178,981 29.2% 2 202 33 3 NC_021075 [12] Sporolithon durum 191,464 29.3% 2 207 30 3 NC_029857 [44] Ahnfeltiophycidae Ahnfeltia plicata 190,451 32.5% 1 207 31 6 NC_031145 [39] Rhodymenio-phycidae Acrosorium ciliolatum 176,064 25.5% 0 206 33 3 NC_035260 [51] Asparagopsis taxiformis 177,091 29.4% 2 205 32 3 NC_031148 [39] Bostrychia moritziana 171,750 28.4% 0 214 34 3 NC_035266 [51] Bostrychia simpliciuscula 167,514 26.5% 0 202 34 3 NC_035268 [51] Bostrychia tenella 170,809 28.6% 0 206 34 3 NC_035264 [51] Bryothamnion seaforthii 175,547 29.5% 0 216 34 3 NC_035276 [51] Caloglossa beccarii 165,038 26.9% 0 201 33 3 NC_035269 [51] Caloglossa intermedia 166,397 31.0% 0 209 32 3 NC_035265 [51] Caloglossa monosticha 165,111 28.2% 0 200 32 3 NC_035263 [51] Ceramium cimbricum 171,923 27.6% 0 193 28 3 NC_031211 [48] Ceramium japonicum 171,634 27.8% 1 202 29 3 NC_031174 [39] Chondrus crispus 180,086 28.7% 1 208 32 3 NC_020795 [13] Choreocolax polysiphoniae 90,243 20.5% 0 72 0 3 KP308096 [42] Cliftonaea pectinata 174,482 28.0% 0 214 34 3 NC_035294 [51] (Continued)

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Table 1. (Continued)

Subclass Species General Characteristics RNAs GenBank Reference Total bp GC % Introns CDS tRNAs rRNA Accession Coeloseira compressa 176,291 29.0% 0 202 30 3 NC_030338 [45] Dasya binghamiae 177,213 25.6% 0 199 29 3 NC_031161 [40] Dasya naccarioides 170,970 27.4% 0 201 33 3 NC_035280 [51] Dasyclonium flaccidum 170,203 28.1% 0 204 34 3 NC_035287 [51] Dictyomenia sonderi 168,768 28.3% 0 201 34 3 NC_035297 [51] Digenea simplex 174,848 29.8% 0 207 34 3 NC_035298 [51] Dipterocladia arabiensis 173,119 26.2% 0 201 34 3 NC_035257 [51] Dipterosiphonia australica 169,341 28.8% 0 205 33 3 NC_035288 [51] Gelidium elegans 174,748 30.2% 1 202 30 3 NC_029858 [44] Gelidium vagum 179,853 29.9% 1 202 30 3 NC_029859 [44] Gracilaria chilensis 185,637 29.3% 1 204 30 3 NC_029860 [44] Gracilaria firma 187,001 28.1% 1 219 32 3 NC_033877 [43] Gracilaria salicornia 179,757 28.8% 0 206 31 3 NC_023785 [53] Gracilaria tenuistipitata 183,883 29.2% 0 205 29 3 NC_006137 [49] Gracilariopsis chorda 182,459 27.4% 1 203 30 3 NC_031149 [39] Gracilariopsis lemaneiformis 183,013 27.4% 2 206 32 3 KP330491 [50] Grateloupia taiwanensis 191,270 30.6% 0 234 29 3 NC_021618 [52] Gredgaria maugeana 167,948 27.6% 0 202 34 3 NC_035290 [51] Herposiphonia versicolor 166,895 28.2% 0 203 34 3 NC_035279 [51] Kuetzingia canaliculata 178,949 28.1% 0 218 34 3 NC_035293 [51] Laurenciella marilzae 172,014 29.7% 0 204 34 3 NC_035259 [51] Lophocladia kuetzingii 175,085 26.9% 0 221 34 3 NC_035292 [51] Mastocarpus papillatus 184,382 29.1% 0 206 30 3 NC_031167 [41] Melanothamnus harveyi 164,979 29.8% 0 207 33 3 NC_035281 [51] Membranoptera platyphylla 176,159 26.4% 0 193 29 3 NC_032041 [46] Membranoptera tenuis 176,031 26.2% 0 192 29 3 NC_032399 [46] Membranoptera weeksiae 176,070 26.2% 0 201 29 3 NC_032396 [46] Ophidocladus simpliciusculus 168,531 28.1% 0 203 34 3 NC_035284 [51] Osmundaria fimbriata 183,995 28.3% 0 224 34 3 NC_035262 [51] Periphykon beckeri 168,283 28.4% 0 202 34 3 NC_035261 [51] Platysiphonia delicata 171,598 29.1% 0 205 33 3 NC_035258 [51] Plocamium cartilagineum 171,392 27.2% 1 199 29 3 NC_031179 [39] Polysiphonia brodiei 169,795 29.0% 0 211 34 3 NC_035272 [51] Polysiphonia elongata 168,290 28.8% 0 206 33 3 NC_035274 [51] Polysiphonia infestans 165,237 29.4% 0 207 33 3 NC_035277 [51] Polysiphonia schneideri 163,271 28.1% 0 203 33 3 NC_035296 [51] Polysiphonia scopulorum 168,001 29.2% 0 206 34 3 NC_035282 [51] Polysiphonia sertularioides 166,000 29.6% 0 203 33 3 NC_035270 [51] Polysiphonia stricta 169,061 29.0% 0 201 34 3 NC_035275 [51] Rhodomela confervoides 175,951 29.0% 0 210 34 3 NC_035271 [51] Rhodymenia pseudopalmata 194,153 32.0% 1 202 32 6 NC_031144 [39] Riquetophycus sp. 180,384 28.8% 1 205 30 4 KX284710 [39] Schimmelmannia schousboei 181,030 28.6% 1 206 30 3 NC_031168 [39] Schizymenia dubyi 183,959 30.0% 1 206 30 3 NC_031169 [39] Sebdenia flabellata 192,140 29.2% 2 207 30 3 NC_031170 [39] Sonderella linearis 169,619 26.0% 0 201 34 3 NC_035289 [51] Spyridia filamentosa 175,578 29.3% 0 218 33 3 NC_035285 [51] (Continued)

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Table 1. (Continued)

Subclass Species General Characteristics RNAs GenBank Reference Total bp GC % Introns CDS tRNAs rRNA Accession Symphyocladia dendroidea 171,837 28.4% 0 210 29 0 NC_035267 [51] Taenioma perpusillum 163,418 27.6% 0 200 32 3 NC_035295 [51] Thaumatella adunca 169,659 26.5% 0 203 34 3 NC_035291 [51] Thuretia quercifolia 174,510 25.9% 0 212 34 3 NC_035286 [51] Tolypiocladia glomerulata 165,623 29.2% 0 206 33 3 NC_035299 [51] Vertebrata australis 167,318 28.3% 0 199 33 3 NC_035283 [51] Vertebrata isogona 167,445 28.3% 0 205 33 3 NC_035278 [51] Vertebrata lanosa 167,158 30.0% 0 193 28 3 KP308097 [42] Vertebrata thuyoides 168,951 28.6% 0 208 33 3 NC_035273 [51] https://doi.org/10.1371/journal.pone.0196995.t001

pbsA gene. The plastid encoded pbsA gene was present in most marine red algal species. Red algal pbsA genes were grouped in a highly supported clade with diverse cyanobacteria (94% MLB) (Fig 4), suggesting a cyanobacterial origin. Additionally, pbsA was present in the red algal derived plastids of cryptophytes. The pbsA homolog was also found in the nuclear genome of the Cyanophora paradoxa (Glaucophyta) with an additional extension of the N-terminal transit peptide (e-value = 1e-61; compare to pbsA gene of Porphyridium purpureum). The alignment for pbsA and its homologous proteins were identified to have a functional domain of heme oxygenase (see Fig 3A). Nonetheless, each type of heme oxygenase proteins contained a few differences, such as putative transmembrane domain (red box) in C-terminal extension or targeting domain (green box) in N-terminal extension (Fig 3A). For pbsA protein sequences, a transmembrane region was predicted as shown in Fig 3B, where most of the red algal pbsA proteins had putative transmembrane domains in the C-terminal. This C-terminal transmembrane domain was also present in HMOX2 genes, but HMOX2 genes contained additional putative transmembrane domains inside the functional heme oxygenase domain. None of HMOX1 genes in red algae were predicted to have a transmembrane domain region in their protein sequences. One noteworthy discovery was that the pbsA gene is generally absent in freshwater species (75%; 12 of 16 spp.), but present in most of the marine red algal species (86%; 100 of 116) (see S3 Table). Heme oxygenase is well known for acting as an iron-controlling factor [61, 64]. It has been demonstrated that transcription of the pbsA gene in a unicellular red alga, Rhodella violacea (Rhodellophyceae), was up-regulated in iron deprivation conditions [65]. Given these observations, it is highly likely that the pbsA gene in red algae assists to uptake of iron in iron deprived marine environment. On the other hand, most freshwater red algae have lost the pbsA gene, likely due to this gene being unnecessary or redundant in freshwaters that are typi- cally not as iron limited as marine environments (iron composition in freshwater is ~1,400 times higher than seawater [66]). Although gene content in red algal plastid genomes was highly conserved [39], pbsA gene may be a good example for the genomic response to environmental adaption. HMOX1 gene. Although the nuclear heme oxygenase gene, HMOX1, contained a conserved heme oxygenase domain and heme binding motif, it was clearly different from the other heme oxygenase genes in the phylogenetic and gene network analyses. The ML tree (Fig 4) showed that orthologs of HMOX1 gene form a strongly supported monophyletic group (93% MLB) with relatively long-branches. In addition, the gene network analysis (S2 Fig) also indicated HMOX1 genes to be clearly separated from the groups of HMOX2 or pbsA despite

PLOS ONE | https://doi.org/10.1371/journal.pone.0196995 May 8, 2018 9 / 19 Three plastid genomes of Nemaliophycidae

Fig 2. The distribution of four putative habitat-specific genes in the phylogenetic tree. The absence/presence of four genes (pbsA, ycf34, ycf35, ycf46) in 127 species is visualized with habitat information. The maximum likelihood phylogenetic tree was reconstructed based on the concatenated 190 orthologous plastid gene alignment. The dataset used in this analysis is shown in S3 Table. https://doi.org/10.1371/journal.pone.0196995.g002

PLOS ONE | https://doi.org/10.1371/journal.pone.0196995 May 8, 2018 10 / 19 Three plastid genomes of Nemaliophycidae

A Transit Peptide

Rhodophyta_PT_pbsA_Calliarthron_tuberculosum_501673244 M------A T N L A E-E - Q L R QGT T K SH SMA EN Rhodophyta_PT_pbsA_Chondrus_crispus_strain_PCG_carragheen_471434998 M------ST N L AQ-Q - Q L R EGT T K SH SMA EN Rhodophyta_PT_pbsA_Palmaria_palmata_Dulse_1070107872 M------T I N L AD-D - E L R QGT T K SH SMA EN pbssA Rhodophyta_PT_pbsA_Porphyridium_purpureum_568247639 M------NN L AQ-Q - E L R EGT T K SH SMA EN Glaucophyta_pbsA_Cyanophora_paradoxa_37137 MAA F AV AA P L AGQA S S PAV A T Q S S------S ------SRKA PMRR V AV A SQAK S S F LG S PA S S FM AGQ SVQ F V AA A PA PAQ F F V A A ET AA PT RQK D I G L AK-K - K L K V GT AK SHNMA EN Cryptophyta_PT_pbsA_Guillardia_theta_11467702 M------SNN L A I-I - Q L R EGT SK SH SMA EN Rhodophyta_HMOX1_Calliarthron_tuberculosum_Ctub_IDg13651t1 MA PMRT A F--F ------A S SAA L P L RA T H-H ------L VQT VD A L S PR S L K S S SR Y V-V - - SGV RRA RMH A P-----P ------P PA AAA T EKR PG S H KG F V E-E - EMR F V AMR LH T KD QA Rhodophyta_HMOX1_Galdieria_sulphuraria_545709902 MKR P LQGT I V P PA FQQWL PN R L T KT A F---F ------T K EGR F CV CV KN-N ------CCWRT T V F S PRG SQ SRV F ------F TMAD S AV RG P PGMGG T S-S - - T DNMD S V VQGD RR PG S H KG F V E-E - EMR F V AMR LH T RD QA Rhodophyta_HMOX1_Porphyridium_purpureum_Ppur_cong_2049_8 MAA F V GG F AA PV------V ------A ST QANGA R L F S-S ------A SKN V AC F T A R L RRVH--H ------A SRGT V SMHG P SAN A S S S-----S - - - - - S S S AAAA ERR PG S H KG F V E-E - EMR F V AMR LH T KD QA Rhodophyta_HMOX1_Cyanidioschyzon_merolae_CMER_544211503 MV F------F ------V T SAG I AA A R SG-G------C SWFGAA R S PAA L RRT V-V ------C SRR PQ Q L L A SA Y PG S R P P-P - H AH EAV HHDGAH R PG E RKG F VQ-Q - EMR V V AMR LH T KD QA Viridiplantae_HMOX1_Arabidopsis_thaliana_4530591 MA Y L A P I S S S L S I F------F ------KN PQ L SR F Q F S S-S ------S S PN P L F L R PR I Q I L SM-M ------TMN K S P S L V V V-----V ------AA T T AA EKQKKRY PG E SKG F V E-E - EMR F V AMR LH T KD QA HMOX1 Viridiplantae_HMOX1_Chlamydomonas_reinhardi_159489196 M L L------L ------S SR ST A SV RA SGRAA PA P RR------R ------V V RV L AHGHG HGGGHGHGHG S SGT AV L T EK D KG F I A-A - EMR KV AMK LH T KD QA Glaucophyta_HMOX1_Cyanophora_paradoxa_PARTIAL_54613_57695 MA F T T GV AV V R ST S S-----S ------S PAAA ST V SC SQ-Q ------AGA RA S F F GAK L S SAAK S RRT F E F T A S P R F SV V AHG PG G P PKGAH PH-H - - - AA L KKG ER EGG F VQ-Q - EMR AV AMR LH T K E QA Stramenopiles_HMOX1_Ectocarpus_siliculosus_298706221 MRV L ST A T AV L A F ERGT V T T S SA FQV L V P S SG SGR ST AAA RR S S-S ------SN KA F PWA A S S F RRRAAA V P LWC T SQT T D V AA T A EGKG AKK E EGRGHG HGGGV K PKT D KN S F VQWDMR RAAMK LH T RD Q S Stramenopiles_HMOX1_Thalassiosira_pseudonana_CCMP1335_224001674 MK F PV A I L F S F F T T S-----S ------NG F V PA P F T QQ SRV S I H R SNNN SN A S PQ S SAH T S FN N YN I E SA L F S ST------T ------EAD S ST T T A T T DNN EQ F P P I L T-T - E L R D V AMK LH T R E QA Cryptophyta_HMOX1_Guillardia_theta_CCMP2712_551647086 MR------R ------S L I PA L L C S L C S-S ------I T A T T A FM Q P S L RT S L L L RN------N ------AA PMMC AK ST P SA EV A SV P F T Q-Q - EMR QKAM S LH T F S QA Rhodophyta_HMOX2_Calliarthron_tuberculosum_ORF_9880_35 F------VN S RRA L SAA RRG V AC T V T T KMG T N V EQ EKK P S V L T F T K-K - L L R KRT NQKRT A S AA Rhodophyta_HMOX2_Chondrus_crispus_546324652 M------S ED S F S E-E - E LQ T RT RRQHD L S N T Rhodophyta_HMOX2_Galdieria_sulphuraria_545711225 M------DD FW I-I - R L R R ET RR EHN V S N T HMOX2 Viridiplantae_HMOX2_Chlamydomonas_reinhardi_A8JBZ0 M------SD AKA T AG S E P E A E P L T K-K - R L S KA SRK I HN V S N S Stramenopiles_HMOX2_Nannochloropsis_gaditana_585100004 MRWT L P PV PR R L P I------A SAVN T SC CV P L KGK I RR D L RV PG P F S L P I H F S S L E S F KV EKG I GN EV MA SGA PDGHD EV P L S I-I - AMR KA T RK I H R E S D A Cryptophyta_HMOX2_Guillardia_theta_CCMP2712_551647925 M------G SD T A SGR L T E-E - RMR K ST RR I HGKT D R * * * Heme Oxygenase Domain

Rhodophyta_PT_pbsA_Calliarthron_tuberculosum_501673244 V S F V K S F LGG V------VD KN SY RT L V AN L Y F V Y SA I E E E I QKN Y- - - - - LH P S I S L I Y- - F P E LN R KQ S L EKD L Y Y Y Y------G P------DWK L F ------I-Q P S L V T Rhodophyta_PT_pbsA_Chondrus_crispus_strain_PCG_carragheen_471434998 V S F V K S F LGG V------VD KKT Y RK L V AN L F F V Y SV L E E E I EKN K- - - - - NH T A I N P I Y- - F V E LN R KD S L C ED LQ Y Y Y------G S------NWSQ Y ------I-E P S L A T Rhodophyta_PT_pbsA_Palmaria_palmata_Dulse_1070107872 V S F V K S F LGG V------VD KK SY RQ L V AN L Y F V Y V AM E E EM LHHQ- - - - - NNGA I K P I Y- - F P E LN R K L S L EQD L I Y Y Y------GK------NWE EQ ------I-EA SD A T pbssA Rhodophyta_PT_pbsA_Porphyridium_purpureum_568247639 V T F V K S F L SG V------I D KKA Y RT L V AN L Y F V Y SA I E E EMYNN R- - - - - MN PM I NH I F- - F P E LN R K E S L EKD L E F Y Y------G P------NWPDQ ------I-Q S S P A T Glaucophyta_pbsA_Cyanophora_paradoxa_37137 VD F V KG F LNG ------V EKT SY SK L V AN L YH V Y SA I E E EM ERN K- - - - - DH PMV A P I Y- - F K E LN R K EA L EQD LQ F Y F------A------NWR EQ ------A- V A SA AG Cryptophyta_PT_pbsA_Guillardia_theta_11467702 V S F V K S F L SG V------I D T N SY KKMM SN L Y F V Y KAM E E EM EYH K- - - - - END L I K P I Y- - F V E LN R S E S L A LD LN F Y Y------GD------TWKD I ------I-E P S E A T Rhodophyta_HMOX1_Calliarthron_tuberculosum_Ctub_IDg13651t1 PR EG EQ E E SA L- - - P I D AWY P SRGD F LQ F L VD S L A V Y E F F EN E L------V VNN E L F A S F R- - N T G L ER V EA LN I D I T Y L C------D E L------GV EA R ------Q P SQ AA Rhodophyta_HMOX1_Galdieria_sulphuraria_545709902 - K EGT L E E SA L- - - P I Q EWQ P SQQD Y LQ F L VD SKH V YD F F EN FMN------Q SQDNM F VN F R- - RT G L ER V SA L E ED LDW F R------QR------G F S I P ------K P EK PG Rhodophyta_HMOX1_Porphyridium_purpureum_Ppur_cong_2049_8 PR EGQM E EAA L- - - P I Q EWR PRK ED Y LQ F L VD SKA V YD FM EA RMA------D AD AAWLH E F T- - ST G L ER GAA LD KD I QW F R------M L------D FD V P ------P PT P AA Rhodophyta_HMOX1_Cyanidioschyzon_merolae_CMER_544211503 - R EGKQ E E SA L- - - P I T EWR PT RAD F LQ F L VD SKA V YD FM EGY V AG EA- - - SKGN P L F K P F V- - N T G L ER GQA L A SD I AW F R------D E L------GYQV P ------E PT E AA Viridiplantae_HMOX1_Arabidopsis_thaliana_4530591 - K EG EK ET K S I E ER PV AKWE PT V EGY L R F L VD SK L V YD T L E L I I Q------D SN F PT Y A E F K- - N T G L ER A EK L ST D L EW F K------EQ------GY E I P ------E PT A PG HMOX1 Viridiplantae_HMOX1_Chlamydomonas_reinhardi_159489196 PK EG EK EA P- ----KQG PWT PT R PGY L R F L V E SK EV FD T F ER L VN------S SD A Y KA L R- - N T G L ER G PG L AAD I AW M E------Q S F------Q L A P P ------DMK PDG AA Glaucophyta_HMOX1_Cyanophora_paradoxa_PARTIAL_54613_57695 PK EG EA PA------KK E SWK PRK ED F LQ F L VD SKV V Y ERW SR S S------L R- - T T------Stramenopiles_HMOX1_Ectocarpus_siliculosus_298706221 PK EGKQ EN KG T- - - K F SDWK P SRRGY LQ F L VD SKV V Y EA L E EAC------G SD PR L A E F R- - N T G L ER T EA L T KD I AW M L------EA Y PD SWA EGGGGDGKA P ------S PT E N A Stramenopiles_HMOX1_Thalassiosira_pseudonana_CCMP1335_224001674 PK EGQAAA P S K- - - PK E P Y V PT QAD Y LQ F L VD SH EV YMA L E EV VN R PD- - - - - LD A E LGR F R- - NNG I ER T K E L EKD I DW MC------T Q F------S LD K P ------AV GG AG Cryptophyta_HMOX1_Guillardia_theta_CCMP2712_551647086 PR EGKQKD A S A- N T V VDQWE T T K ED Y LQ F L VD SKV V Y EA F EK EV------QR SG L EK F R- - N T G L ER V RG L EKD I EW I E------K S F------N I K P L ------KA T D Q S Rhodophyta_HMOX2_Calliarthron_tuberculosum_ORF_9880_35 LNN L T L P L- A L------S S PQV Y L K L V RA FQ Y I L SA F ED E F EKRR- - - - RV Y PR LN V LH- - F R E L L R A PA F KRD A E F F V------A L------AAGQT GT A T PRGA R L A S L S L P P P PA RA Rhodophyta_HMOX2_Chondrus_crispus_546324652 L VN L SA P L- A L------S S PD V Y R L L L V SWYW I Y KT V ET KM EH F R- - - - L T Y PKV GA I Y- - F PQ L L R T GA F EDD L R F Y Y------G S------D F EKR ------I M P P SK A T Rhodophyta_HMOX2_Galdieria_sulphuraria_545711225 L VN L T L P F- A L------SD RRV Y V KA L E S F Y Y V Y ST V E E E FD RQR- - - - RN F PK I GA L Y- - F E E L RR KK S F ERD LQ F F L------GD------NWRQ L ------LQ E P S P T V HMOX2 Viridiplantae_HMOX2_Chlamydomonas_reinhardi_A8JBZ0 L VN A R L I A- L F------SD KD L Y AKA L GC F Y Y V F V A L EAA LD EA L KK GD AD V SK F KD V L- - KGG L Y R A PG F KQD VQH Y L------GA------TWQAQ ------LGT K SQ A L Stramenopiles_HMOX2_Nannochloropsis_gaditana_585100004 L VN F K L V Y- A F------T Y P S L Y AD A I G L F Y P I Y RK L E S L VM S SG- - S P S LQ P F A T L L R- - Q Y K L ER T RA F E SDMD F H S------RG------GT EK E ------DD E I P E AV Cryptophyta_HMOX2_Guillardia_theta_CCMP2712_551647925 L A SMN F A L- V L------T D RA L Y AK L L G S F YMV Y R E L EAAMD RC R- - - - - DN EH V GH V F P L LD KMRR T KA F EAD L L F F L------GK------DWAKT ------L- I VN E AV

Putative Transmembrane (TM) Domain Heme Oxygenase Domain

Rhodophyta_PT_pbsA_Calliarthron_tuberculosum_501673244 QT Y VD R I HD I G SHQ P------E L L I SH V Y T RYMGD L SGG Q I L KN I A------KN AM------H L SG DDGT E F Y V F R E- I AN EKD F K RMY RK S LD S- ---- I P I T ED Q------Rhodophyta_PT_pbsA_Chondrus_crispus_strain_PCG_carragheen_471434998 QN Y I N R I H E I GKNQ P------E L L I AH A Y T RYMGD L SGG Q I L KK I A------QN AM------Q LNG T NGV S F YN F L S- I KDD KV F K I KY RD A LDN- ----L P I D ET Q------Rhodophyta_PT_pbsA_Palmaria_palmata_Dulse_1070107872 QV Y V RR I RD I S S SQ P------E L L V AH A Y T RYMGD L SGG Q I L KK I A------QN AM------N L ST N EGT A F YD F E E- L ED ENG F K Q I Y RNQ LN C- ---- I L L S E E Q------pbssA Rhodophyta_PT_pbsA_Porphyridium_purpureum_568247639 D I Y I QR I HN I GRNQ P------E L L I AH A Y T RY LGD L SGG Q I L KK I A------QN AM------N LQG EAGT A F YN FD L- I EN EQV F K N T Y R EA LN S- ---- I PV SK E A------Glaucophyta_pbsA_Cyanophora_paradoxa_37137 QA Y VD R I H E I GK SD P------K L L I AH SY T RY LGD L SGG Q I L KR I A------EKAM------G L E- DGGV A F Y E F P A- I SD ERA F K NQ Y RAA LN A- ----L P L SD K E------Cryptophyta_PT_pbsA_Guillardia_theta_11467702 RV Y I N R I KK I SK EK P------L L L I AH A Y T RY LGD L SGG Q I L KK I A------QRA L------N V PN SQG L A F Y E FD K- I DD EQA F K QKY KKA LD T- ----L PV T D K M------Rhodophyta_HMOX1_Calliarthron_tuberculosum_Ctub_IDg13651t1 T GY V A Y L RT L V A ER P------E SA L CHWY N Y Y F AH SAGG RM I GR LM------QQR L- - - - - F- - - - D GR E F S F Y EWE---SD V KA L L G PV RK S I D E- - - V AA EWT R E V------Rhodophyta_HMOX1_Galdieria_sulphuraria_545709902 V A Y V EY LQ S L VQD K P------H A L L CHWY N F Y F AH T AGG RM I GR LM------SK L L- - - - - F- - - - D GH V F E F Y KWE---GD V KQ I L DQV RV K I D E- - - T A RDWPR E V------Rhodophyta_HMOX1_Porphyridium_purpureum_Ppur_cong_2049_8 T KY I A Y LQ E L V RD K P------N A F L CHWY N Y Y F AH SAGG RM I GRMM------SN L L- - - - - F- - - - E GK E F E F Y AWE---KD V K E I L G EV R EK I D R- - - V A SDWPRD V------Rhodophyta_HMOX1_Cyanidioschyzon_merolae_CMER_544211503 QRY I D Y L RG L F ER S P------EAV L CHWY N Y Y F AH T AGG RM I GRNM------ENM L- - - - - FGDG PN AKR F E F Y KWE---RD V KQ I L D EV R ER I D E- - - V AKDWPR E V------Viridiplantae_HMOX1_Arabidopsis_thaliana_4530591 KT Y SQ Y L K E L A EKD P------QA F I CH F Y N I Y F AH SAGG RM I GRKV------A ER I- ----L- - - - D N K E L E F Y KWD---G E L SQ L L QN V R EK LN K- - - V A E EWT R E E------HMOX1 Viridiplantae_HMOX1_Chlamydomonas_reinhardi_159489196 A T Y V A F L EK L AKT D P------PA F I CH Y Y N F Y F AH T AGG RM I GN KGVQH AAGR PD PGV L QV AGRRKRA P GGRAQVHQRD GRG LD P------GG EGA L P GGD RVQ LQVQ R PD PA RYH RG I------Glaucophyta_HMOX1_Cyanophora_paradoxa_PARTIAL_54613_57695 ------Stramenopiles_HMOX1_Ectocarpus_siliculosus_298706221 L EY A S F L R EK V A ST L------PG FMCH Y Y NH Y F AH T AGG RM I GRKV A E S A LGV F F KH V S A F P F R SD SC- - - - - L- - - - D GRT L E F YQWG S- - DD V KV L L DGV RV K I D A- - - MAKGWS E E E------Stramenopiles_HMOX1_Thalassiosira_pseudonana_CCMP1335_224001674 SRY AN E L RNM V TMND EG EN V GV P E F V CH Y Y N Y Y F AH L AGG RM I GKQM------SKM L- - - - - L- - - - D G ET L E F Y KWG---ED VND L K A RV KDD I E E- - - L AKTWSR E E------Cryptophyta_HMOX1_Guillardia_theta_CCMP2712_551647086 K EY AQ Y L ST L S I------PV F L T H F Y N Y Y F AH T AGG RM I GKQV------MD SV- - - - - F- - - - D GH L F E F Y KWD---GD V K E I L T KV KG F I D E- - - T A EGWT R E MV I L F L P L P F SR Rhodophyta_HMOX2_Calliarthron_tuberculosum_ORF_9880_35 QQ F V R EMR I A L A R E P------V L V I A Y SY A L YMG L F V GG T RT A PWV------RRA F------E L EG N V GT AAWD F S D T I PD RKA F R AKYD L A LND- ----F S L SD A Q------Rhodophyta_HMOX2_Chondrus_crispus_546324652 AD Y I KN F VQ S I END P------V C I I A Y C Y TMY LGM FGGG P I V KRWV------R SA F------S L P P GKGT K I F E F S D T I PD I PK F K K EY KD AMD R- ---- I A L SRD E------Rhodophyta_HMOX2_Galdieria_sulphuraria_545711225 T RY I DH I RQV S SAK P------WL I L SY V I T L YMA L F RGG SV L RK I L------V V SM- - - - - A LD KN S G EG L A I YD F S N- V SD T D A F F KKY V ET VN A- ----L T L S E E Q------HMOX2 Viridiplantae_HMOX2_Chlamydomonas_reinhardi_A8JBZ0 KD Y EAH L A S L GR S S P------A L L L AH V Y T QH L AMA SGG Q I V KRWA------RK I F------Q L PD D V GT AA FD Y T G- - E SNN T L R SA F KKQ FD E- - - WGAAQ PQ E L------Stramenopiles_HMOX2_Nannochloropsis_gaditana_585100004 K SY L ER L E SC AA T D P------E L L AV Y V Y HMYMA L L SGG Q I L RR L T------T QC L L PKT PV T N KD P D AG L A L Y S F E G- - GKT Q E I K EA I RA T VD RM EKT GV V FN SQ R------Cryptophyta_HMOX2_Guillardia_theta_CCMP2712_551647925 EKY L A R I A E L EKKD P------A L L L A Y SY H L YMA F L FGG KV I KKT V------STM L------S L RG E EG L AV F AMG G------AK P S EY KD SYD E- ----L V L S ED Q------* * * * Putative TM Domain

Rhodophyta_PT_pbsA_Calliarthron_tuberculosum_501673244 ------I R L V I S EAN V A FN------LNMK M F- - - - - Q E L N SN------N ------I V K I M LM L L SN A I NN F RN RN------N ------Rhodophyta_PT_pbsA_Chondrus_crispus_strain_PCG_carragheen_471434998 ------I SQ I I A EAN T A FN------LNMQ I F- - - - - Q E L N SN------N ------L I K I MVM L L LN T I G S FQK K L S------S ------Rhodophyta_PT_pbsA_Palmaria_palmata_Dulse_1070107872 ------V AD I I A EAN I A F T------LNMK M F- - - - - Q E L N SN------N ------F I K I M I M L L LN AV RN LQ I SK------K ------pbssA Rhodophyta_PT_pbsA_Porphyridium_purpureum_568247639 ------T EK I V A EAN I A F T------LNMK I F- - - - - E E L N S S------S ------F L K I MTM L S LN A I KN L RD K I LN------N ------Glaucophyta_pbsA_Cyanophora_paradoxa_37137 ------T E E I V E EAN KA FG------MNMY M F- - - - - K E L EGN------N ------W I I T T A R L L FN SV F PK LG KKT T PA RT A* ------Cryptophyta_PT_pbsA_Guillardia_theta_11467702 ------I SQ I V A EAN I A FN------LNMR M F- - - - - Q E L EM S------S ------Y I R I F T K I LMQ F L I N SKD K L RVM LN F S------Rhodophyta_HMOX1_Calliarthron_tuberculosum_Ctub_IDg13651t1 ------K D V C L K ET G L A FG------Y SGT V L- - - - - QN L AKA S S------Rhodophyta_HMOX1_Galdieria_sulphuraria_545709902 ------K D EC L K ET G L S F S------Y SGT I L- - - - - GH L AKRG------Rhodophyta_HMOX1_Porphyridium_purpureum_Ppur_cong_2049_8 ------K D EC LN ET S L A F A------Y SGT V L- - - - - SN L AKA S------Rhodophyta_HMOX1_Cyanidioschyzon_merolae_CMER_544211503 ------K D AC LN ET G L A F A------Y SGT I L- - - - - NN L AKRA E E PA PA S------Viridiplantae_HMOX1_Arabidopsis_thaliana_4530591 ------K NH C L E ET EK S F K------Y SG E I L- - - - - R L I L S------HMOX1 Viridiplantae_HMOX1_Chlamydomonas_reinhardi_159489196 ------K Q EAV AAA SQD A L------GG S S CMD V RV GKK E RK S PAV GD RR AGWRA YD------Glaucophyta_HMOX1_Cyanophora_paradoxa_PARTIAL_54613_57695 ------Stramenopiles_HMOX1_Ectocarpus_siliculosus_298706221 ------K EACV S ET A RT FN------L RV F F V GR MV L V RGMA S L WGRG E EV R I F L ERT ACV AD L G L I F V L C TM L T D E SGGN E S S M L F L RV I EYD GV S P F LD PC P L ACGRA RA PR L CV A SC S SQ Stramenopiles_HMOX1_Thalassiosira_pseudonana_CCMP1335_224001674 ------R D EC I N A T PA T F R------GGGA I N- - - - - GY L YGGN PH------Cryptophyta_HMOX1_Guillardia_theta_CCMP2712_551647086 S L I C S F L F Y S I L L L S S P P L F F S L F L L P P I S S L L P L L Y S S L L L I I SCA SR L L P P P P P P P P P P P P LM I I M L L RGT PQT H SY A SC L S------S ------Rhodophyta_HMOX2_Calliarthron_tuberculosum_ORF_9880_35 ------R E S I F A EVH R I FG------A S S E I F- - - - - A EV KA SA EY T RT I T-T - WAV T RG L L GV T S L AV I AW TWA E P F KWA L T E L F SV L VQ------Rhodophyta_HMOX2_Chondrus_crispus_546324652 ------K ER I V EMKKQV F A------HND T I F- - - - - A E I RKT A T YN RRV I SV V V KY A L L F V V L LGT F R F L FD A S F RT H A R E I F F AV T SW V S SRT T D LD T ------Rhodophyta_HMOX2_Galdieria_sulphuraria_545711225 ------K D EV I E EKRA I F L------WND E I F- - - - - N EV RNG P Y Y KA I L WR F FWSV V F V V V V V I F F L KK RN YW------W ------HMOX2 Viridiplantae_HMOX2_Chlamydomonas_reinhardi_A8JBZ0 ------Q DQ L L S EH L AA FG------HNN A I I-----KA F P L PA T A I I AG A-A - I RV T PR PV L L V L V A L LGW C L A F I I PWLQ T K LQV GV------Stramenopiles_HMOX2_Nannochloropsis_gaditana_585100004 ------R QA F LD E S I R L FH------MNN E V V- - - - - R S F R P SRKGRRK L V V LGC L L L V A V I L V V TMV GG AM SCA FQT K------Cryptophyta_HMOX2_Guillardia_theta_CCMP2712_551647925 ------T E E L L K E SV EV F A------MNNN I V- - - - - M S F A RT V TWRNWL A S L RN PWVQA V V V AA I GA SV A L Y L S L T RGV KK------*

B Putative TM Domain Probability

(bp) (bp) (bp)

Transmembrane In Out Transmembrane In Out Transmembrane In Out pbsA HMOX1 HMOX2 (Palmaria palmata) (Calliarthron tuberculosum) (Chondrus crispus)

Fig 3. The sequence alignment of heme oxygenase proteins. (A) The alignment of pbsA and its homologous proteins. The alignment shows the conserved heme oxygenases amino acid sequences in different lineages. Conserved heme binding pockets are marked as a blue asterisk. N-terminal transit peptides (green) are unique for HMOX1 proteins, with an exceptional transit peptide of pbsA gene in Cyanophora paradoxa, which was likely transferred to the nuclear genome independently. Heme oxygenase domain (grey) and putative transmembrane domain (red) are shown. (B) HMOX2 and pbsA contain putative transmembrane domain(s) (TM domain; red box). Multiple TM domains were found in HMOX2. https://doi.org/10.1371/journal.pone.0196995.g003

PLOS ONE | https://doi.org/10.1371/journal.pone.0196995 May 8, 2018 11 / 19 Three plastid genomes of Nemaliophycidae

Eukaryota_Alveolata_Vitrella_brassicaformis_CCMP3155_873235984 100 Eukaryota_Viridiplantae_Bryophyta_Physcomitrella (3 tips) Eukaryota_Viridiplantae_Pinus_taeda_loblolly_pine_14485579 Eukaryota_Viridiplantae_Picea_sitchensis_Sitka_spruce_116787456

93 96 Eukaryota_Viridiplantae_Adiantum_capillus-veneris_111036380 99 Eukaryota_Viridiplantae_Marchantia_polymorpha_subsp_ruderalis_1026769718 88 Eukaryota_Viridiplantae_Klebsormidium_nitens_971519149 83 Eukaryota_Viridiplantae_Chlorophyta (16 tips) 99 Eukaryota_Stramenopiles_Ectocarpus_siliculosus_298706221 92 100 - Rhodophyta [NUCLEAR] 97 Eukaryota_Haptophyceae_Chrysochromulina_sp_CCMP291_922864246 100 Eukaryota_Haptophyceae_Emiliania_huxleyi_CCMP1516_551603682 - Glaucophyta [NUCLEAR] 100 Eukaryota_Alveolata_Vitrella_brassicaformis_CCMP3155_873231771 100 93 Eukaryota_Stramenopiles_Diatom (7 tips) - Viridiplantae [NUCLEAR] 100 Eukaryota_Cryptophyta_Guillardia_theta_CCMP2712_551647086 58 Eukaryota_Glaucophyta_HMOX1_Cyanophora_paradoxa_PARTIAL_54613_57695 (green algae & land plants) Eukaryota_Rhodophyta_HMOX1_Galdieria_sulphuraria_545709902 - Alveolata [NUCLEAR] 83 95 90 Eukaryota_Rhodophyta_HMOX1_Compsopogon_coeruleus_MMETSP0312_30383_1

91 Eukaryota_Rhodophyta_HMOX1_Rhodosorus_marinus_MMETSP0011_2_4698_1 58 HMOX1 (Chromerida) Eukaryota_Rhodophyta_HMOX1_Porphyridium_purpureum_Ppur_contig_2049_8

100 Eukaryota_Rhodophyta_HMOX1_Erythrolobus_australicus_MMETSP1353_10097_1 - Haptophyta [NUCLEAR] 88 100 Eukaryota_Rhodophyta_HMOX1_Erythrolobus_madagascarensis_MMETSP1354_616_1 53 (Prymnesiophyceae) 100 Eukaryota_Rhodophyta_HMOX1_Cyanidioschyzon_merolae_CMER_544211503 Eukaryota_Rhodophyta_HMOX1_Cyanidium_caldarium_ctg_1432_g331 - Cryptophyta [NUCLEAR] 100 Eukaryota_Rhodophyta_HMOX1_Porphyra_umbilicalis_laver_1189389826 59 Eukaryota_Rhodophyta_HMOX1_Pyropia_yezoensis_ctg5863_g1322 - Stramenopile [NUCLEAR] Eukaryota_Rhodophyta_HMOX1_Hildenbrandia_prototypus_UTEX2622_ctg3599 100 100 (brown algae) 100 Eukaryota_Rhodophyta_HMOX1_Apophlae_lyallii_000031F_g7867 Eukaryota_Rhodophyta_HMOX1_Apophlae_sinclairii 100 84 Eukaryota_Rhodophyta_HMOX1_Calliarthron_tuberculosum_Ctub_IDg13651t1 Eukaryota_Rhodophyta_HMOX1_Gracilariopsis_chorda_GRC0011RA00280_1 99

97 Eukaryota_Rhodophyta_HMOX1_Sporolithon_durum Transit Heme Oxygenase

98 100 Eukaryota_Rhodophyta_HMOX1_Gelidium_elegans Peptide Domain Eukaryota_Rhodophyta_HMOX1_Gelidium_vagum Eukaryota_Viridiplantae_Selaginella_moellendorffii_302782597 99 Eukaryota_Viridiplantae_Bryophyta (4 tips) 100 Eukaryota_Viridiplantae_Angiosperm (159 tips) 93 Eukaryota_Viridiplantae_Angiosperm (301 tips) Viridiplantae_Chlorophyta (9 tips) 97 Eukaryota_Cryptophyta_Guillardia_theta_CCMP2712_551647925 51 Eukaryota_Fungi_Spizellomyces_punctatus_DAOM_BR117_1027001409 - Rhodophyta [NUCLEAR] 85 87 99 Eukaryota_Fungi (3 tips) 67 - Chlorophyta [NUCLEAR] 89 Eukaryota_Rhodophyta_HMOX2_Porphyridium_purpureum_Ppur_contig_643_1 100 Eukaryota_Fungi (22 tips) - Cryptophyta [NUCLEAR] Eukaryota_Metazoa_Ciona_intestinalis_vase_tunicate_699238689 100 100 Eukaryota_Metazoa_Capitella_teleta_443726597 - Stramenopiles [NUCLEAR] 94 Eukaryota_Metazoa_Capitella_teleta_443729231 HMOX2 96 Eukaryota_Rhodophyta_HMOX2_Galdieria_sulphuraria_545711225 (Eusgmatophyceae, oomycetes) Eukaryota_Rhodophyta_HMOX2_Chondrus_crispus_546324652 100 100 - Metazoa [NUCLEAR] 100 Eukaryota_Rhodophyta_HMOX2_Calliarthron_tuberculosum_ORF_9880_35 100 Eukaryota_Rhodophyta_HMOX2_Sporolithon_durum (Arthropoda, Chordata) 51 Eukaryota_Rhodophyta_HMOX2_Gelidium_vagum 97 Eukaryota_Rhodophyta_HMOX2_Hildenbrandia_prototypus_ctg16272 - Fungi [NUCLEAR] 100 100 Eukaryota_Rhodophyta_HMOX2_Apophlae_sinclairii Eukaryota_Rhodophyta_HMOX2_Apophlaea_lyallii_000067F_g7393 HO TM HO TM 100 Stramenopiles_Oomycetes (20 tips) Domain Domain Domain Domain 100 Eukaryota_Stramenopiles_Nannochloropsis_gaditana_585100004 84 99 Metazoa_Arthropoda_Insecta (121 tips) Bacteria_Planctomycetes_Planctomycetes_bacterium_marine_metagenome_1247449697 65 Bacteria_Actinobacteria_Streptoalloteichus_hindustanus_1120973053

100 Bacteria_Actinobacteria_Thermobifida_cellulosilytica_1057405048 100 Bacteria_Actinobacteria_Thermobifida_fusca_499612034 100 Eukaryota_Haptophyta (2 tips) 71 100 100 Eukaryota_Stramenopiles_Diatoms (2 tips) 100 100 Eukaryota_Stramenopiles_Diatoms (4 tips) 100 Eukaryota_Stramenopiles_Oomycetes (12 tips) 93 100 Eukaryota_Metazoa_Cnidaria_Anthozoa (4 tips) 75 66 Eukaryota_Metazoa_Octopus_bimaculoides_961127625 93 99 Eukaryota_Metazoa_Chordata (197 tips) Viruses_dsDNA_Prochlorococcus_phage_P-SSM2_61806162 100 Bacteria_Cyanobacteria_Prochlorococcus_sp_TMED223_marine_metagenome_1200581590 67 Viruses_dsDNA_Cyanophage_S-SSM6a_460109316 100 100 Viruses_dsDNA_Cyanophage_P-RSM6_472341550 Viruses_dsDNA_Prochlorococcus_phage_P-HM2_326783181 100 100 Viruses_dsDNA_Prochlorococcus_phage_MED4-213_472340358 99 Viruses_dsDNA_Prochlorococcus_phage_P-HM1_326782234 94 Eukaryota_Glaucophyta_pbsA_Cyanophora_paradoxa_37137 92 PLASTID_Cryptophyta (9 tips) 100 PLASTID_Eukaryota_Rhodophyta_Stylonematophyceae (2 tips) - Rhodophyta [PLASTID] 81 100 68 PLASTID_Eukaryota_Rhodophyta_Compsopogonophyceae (3 tips)

94 Bacteria_Aquificae_Hydrogenobaculum_sp_Y04AAS1_501506353 - Glaucophyta [NUCLEAR] 76 67 100 PLASTID_Rhodophta_Rhodellophyceae (2 tips) 88 99 98 PLASTID_Eukaryota_Rhodophyta_Porphyridiophyceae (3 tips) - Cryptophyta [PLASTID] 100 PLASTID_Eukaryota_Rhodophyta_Bangiophyceae (14 tips) - Rhizaria (Paulinella spp.) [PLASTID] 82 PLASTID_Eukaryota_Rhodophyta_Florideophyceae (93 tips) 96 pbsA 100 Bacteria_Cyanobacteria (14 tips) - Cyanobacteria 100 Bacteria_Cyanobacteria_Acaryochloris (2 tips) Eukaryota_Glaucophyta_Cyanophora_paradoxa_37221 - dsDNA viruses 84 99 100 Eukaryota_Fungi_Zygomycota (4 tips) Leptospira 95 100 - Spirochaetes ( spp.) 98 Bacteria_Spirochaetes_Leptospira (53 tips) 78 Bacteria_Cyanobacteria (189 tips) 100 - Fungi [NUCLEAR] Bacteria_Cyanobacteria_Geitlerinema_sp_PCC_9228_1101266667 100 Bacteria_Cyanobacteria (8 tips) 100 97 Bacteria_Cyanobacteria (4 tips) 71 100 100 Bacteria_Cyanobacteria (15 tips) Heme Oxygenase TM 72 Bacteria_Cyanobacteria (16 tips) 98 Domain Domain 99 Bacteria_Cyanobacteria_Synechococcus (2 tips) 8967 100 Bacteria_Cyanobacteria_Synechococcus (6 tips) 83 71 100 Bacteria_Cyanobacteria_Synechococcus (19 tips) 55 100 Eukaryota_Rhizaria_PT_Paulinella_chromatophora_194476945 Eukaryota_Rhizaria_PT_Paulinella_micropora_1120868660 55 100 2.0 Bacteria_Cyanobacteria_Prochlorococcus (37 tips) 88 Bacteria_Cyanobacteria (241 tips) Fig 4. Maximum likelihood tree and schematic diagrams of heme oxygenase proteins. Maximum likelihood (ML) tree based on an alignment of 510 heme oxygenase amino acid sequences from 1,678 taxa. Red algal heme oxygenases had three isotypes of heme oxygenase; HMOX1, HMOX2, and pbsA. HMOX1 and HMOX2 were located in the nuclear genome whereas the pbsA was encoded in the plastid genome in red algae. https://doi.org/10.1371/journal.pone.0196995.g004

PLOS ONE | https://doi.org/10.1371/journal.pone.0196995 May 8, 2018 12 / 19 Three plastid genomes of Nemaliophycidae

the conservation of heme oxygenase domain. Unlike HMOX2 and pbsA, protein sequences of the HMOX1 gene displayed a low similarity (20–30%) to the cyanobacterial heme oxygenase. Interestingly, the HMOX1 gene had unique N-terminal peptides, which were predicted (via ChloroP software) to target the plastid (Fig 3). N-terminal peptides were present in the photosynthetic eukaryote lineages including the primary endosymbiotic lineages (red algae, Viridiplantae, and a glaucophyte alga Cyanophora paradoxa) and red algal-derived secondary plastid groups (i.e., haptophytes, cryptophytes, stramenopiles). While the transit peptides were absent in HMOX2 and pbsA, pbsA in C. paradoxa (Glaucophyta) was located in the nucleus and possesses the additional N-terminal transit peptide like those of HMOX1 genes. In Chlorophyta species, they retained both HMOX1 and HMOX2 in the nucleus, but only the HMOX1 was found in the streptophytes (charophytes and land plants) [67, 68]. Streptophyta species had several copies of the heme oxygenase gene (HMOX1) in their nuclear genome and those genes formed a monophyletic group with other HMOX1 in the phylogenetic tree (Fig 4). For instance, Arabidopsis thaliana contained the biochemically well-characterized heme oxygenase HY1 with three additional putative heme oxygenase copies (HO2, HO3 and HO4) [69]. Analysis of the major biochemical parameters (i.e., enzyme activity depends on pH, temperature, conversion time of heme to biliverdin) demonstrated that activities of these three heme oxygenases (HO2, HO3 and HO4) do not different from that of HY1 [70]. Therefore, unlike other lineages, the four isotypes of HMOX1 (HY1, HO2, HO3 and HO4) in land plants likely plays an important role in synthesizing phycobilin, and these four isotypes likely originated from gene duplication events from an ancestral land plant HMOX1 gene [71, 72]. Because of their low similarities (protein identities: 22.41~27.68%: 1e-05~1e-10) to other homologous proteins, including the cyanobacterial heme oxygenase, the origin of HMOX1 were still ambiguous. However, HMOX1 was only present in plastid-bearing eukaryotes and this was the only gene that possesses the plastid-targeting transit peptide among the heme oxygenase families. Therefore, we concluded that HMOX1 was involved in plastidal function. The phylogenetic position of HMOX1 in red algal-derived secondary endosymbionts sug- gested that HMOX1 was likely derived from secondary endosymbiosis events followed by the gene transfer to the nuclear genome of the host (Fig 4). Indeed, a trafficking experiment in Chlamydomonas reinhardtii showed that Chlamydomonas nuclear-encoded HMOX1 gene targets to the plastid [63]. Although none of these studies showed the trafficking of red algal HMOX1, it is highly likely that red algal HMOX1 targets the plastid because of the mono- phyly of red algae with the Viridiplantae. In Viridiplantae, Chlamydomonas reinhardtii, Ara- bidopsis thaliana, and Ceratodon purpureus provided experimental evidence for plastid trafficking with the N-terminal extension HMOX1 [70, 73, 74]. Nevertheless, function of HMOX1 genes in red algae needs further experimental investigation to elucidate its metabolic pathway. HMOX2 gene. The red algal HMOX2, another nuclear encoded heme oxygenase gene, had one or more putative transmembrane domains in C-terminus (Fig 3). Transmembrane domain structures of red algae were homologous to those of metazoan (e.g., mammalian, amphibian) heme oxygenase [63, 75]. Within the HMOX2 clade in the phylogenetic trees (Fig 4), red algae were grouped with diverse eukaryotes including chlorophytes, cryptophytes, and stramenopiles as well as non-photosynthetic fungi and Metazoa. Therefore, we would suggest that HMOX2 was derived from an ancient eukaryotic common ancestor. Gene network analysis and the sequence conservation between the HMOX2 and pbsA in protein alignment supported that the HMOX2 is related to the iron uptake function. Indeed, it has been reported that C. reinhardtii captured extracellular heme as an iron source with an association between the HMOX2 protein and the cytosolic membrane [63].

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Conclusion Three Nemaliophycidae plastid genomes were completely sequenced and annotated. These included two exclusively freshwater species Kumanoa americana and Thorea hispida and the marine species Palmaria palmata. Until recently, plastid genome data were used mainly for phylogenomic analysis (e.g., [47]), divergence time estimation (e.g., [10]), comparative struc- tural analysis (e.g., [39]), and the development of red algal molecular markers for DNA bar- coding studies (e.g., [12]). In this study, we focused on finding genomic clues for an environmental adaptation between marine and freshwater red algal species. Based on the environment-specific genes of the heme oxygenase family in red algae, we postulate that red algae have adapted efficiently in differing iron concentration conditions through the retention or loss of the heme oxygenase genes. Although this study included only a few freshwater red algal species and was not able to present the direct evidence of correlation between habitat and gene retention, we demonstrated the general trend of red algal plastid gene loss (ycf34, ycf35, ycf46, and pbsA) in iron-limited marine habitats. We also demonstrated different evolutionary strategies of three types of heme oxygenase genes in different lineages (e.g., presence of HMOX1 with gene duplications [HY1, HO2, HO3 and HO4), but the absence of HMOX2 in the streptophytes, which include charophytes and land plants) and habitat conditions (e.g., pbsA genes in the marine and freshwater red algal species). It is generally known that plastid genes are in the process of reduction (either complete loss or gene transfer to the host nucleus) after its endosymbiotic origin [76]. However, gene loss from the plastid genome appears functionally constrained as demonstrated in pbsA and its gene homologs. Through selective gene retention, red algae successfully adapted to different aquatic environments over billions of years of evolutionary history.

Supporting information S1 Fig. Venn diagram visualization of comparing gene contents within the three Nemalio- phycidae genomes. Six unique genes (pbsA, grx, ycf35, ycf36, ycf37, ycf46) were only found in marine Palmaria palmata. For freshwater species, there is only one gene (ycf91) that found in Thorea hispida and Kumanoa americana, but not in Palmaria palmata. (EPS) S2 Fig. Gene network for heme oxygenase. The gene network was constructed by EGN with heme oxygenase database from the public database. We performed analysis with 1e-05 of e- value, 20% of protein identities and 70% of query coverage. The result shows that HMOX1 are clearly separated from other red algal heme oxygenase. (EPS) S1 Table. The sequencing information for the plastid genome of three Nemaliophycidae species. (XLSX) S2 Table. A list of genes in the plastid genomes of Palmaria palmata, Kumanoa americana, and Thorea hispida. (XLSX) S3 Table. The habitat-specific gene survey in 127 plastid genomes of red algae. 1) Marine unique gene (only in Palmaria palmata: pbsA, grx, ycf35, ycf36, ycf37, ycf46); 2) Freshwater unique gene (not in Palmaria palmata but in Thorea hispida and Kumanoa americana): ycf91. (XLSX)

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S4 Table. The input data for chi-square statistical test. (XLSX) S5 Table. The protein sequences of heme oxygenase. (XLSX)

Acknowledgments We would like to thank JunMo Lee, Louis Graf, and Eun Chan Yang for technical assistance in genome sequencing and data analysis as well as Morgan Vis for supplying algal culture and helpful comments on the manuscript. Thanks for the constructive comments from reviewers to revise the manuscript.

Author Contributions Conceptualization: Chung Hyun Cho, Daryl W. Lam, Hwan Su Yoon. Data curation: Chung Hyun Cho, Ji Won Choi, Kyeong Mi Kim. Formal analysis: Chung Hyun Cho, Ji Won Choi, Kyeong Mi Kim. Funding acquisition: Hwan Su Yoon. Investigation: Hwan Su Yoon. Supervision: Hwan Su Yoon. Writing – original draft: Chung Hyun Cho, Hwan Su Yoon. Writing – review & editing: Chung Hyun Cho, Ji Won Choi, Daryl W. Lam, Kyeong Mi Kim, Hwan Su Yoon.

References 1. Lam DW, Verbruggen H, Saunders GW, Vis ML. Multigene phylogeny of the red algal subclass Nema- liophycidae. Molecular Phylogenetics and Evolution. 2016; 94, Part B:730±6. https://doi.org/10.1016/j. ympev.2015.10.015 PMID: 26518739 2. Guiry MD, Guiry GM. AlgaeBase. World-wide electronic publication 2017 [cited 2017 October. 18]. Available from: http://www.algaebase.org. 3. Dixon PS. Biology of the Rhodophyta. Reprint ed ed. Koenigstein: Koeltz: Oliver & Boyd Edinburgh; 1973. 4. Le Gall L, Saunders GW. A nuclear phylogeny of the Florideophyceae (Rhodophyta) inferred from com- bined EF2, small subunit and large subunit ribosomal DNA: establishing the new red algal subclass Cor- allinophycidae. Molecular Phylogenetics and Evolution. 2007; 43(3):1118±30. https://doi.org/10.1016/j. ympev.2006.11.012 PMID: 17197199 5. Ott FD. Handbook of the taxonomic names associated with the non-marine Rhodophycophyta: J. Cra- mer in der GebruÈder Borntraeger Verlagsbuchhandlung; 2009. 6. Kapraun DF, Braly KS, Freshwater DW. Nuclear DNA content variation in the freshwater red algal orders Batrachospermales and Thoreales (Florideophyceae, Nemaliophycidae). Phycologia. 2007; 46 (1):54±62. 7. Peredo EL, King UM, Les DH. The plastid genome of Najas flexilis: Adaptation to submersed environments is accompanied by the complete loss of the NDH complex in an aquatic angiosperm. PLoS One. 2013; 8(7):e68591. https://doi.org/10.1371/journal.pone.0068591 PMID: 23861923 8. Costa JF, Lin S-M, Macaya EC, FernaÂndez-GarcõÂa C, Verbruggen H. Chloroplast genomes as a tool to resolve red algal phylogenies: a case study in the Nemaliales. BMC Evolutionary Biology. 2016; 16 (1):205. https://doi.org/10.1186/s12862-016-0772-3 PMID: 27724867 9. Paiano MO, Del Cortona A, Costa JF, Liu SL, Verbruggen H, De Clerck O, et al. Organization of plastid genomes in the freshwater red algal order Batrachospermales (Rhodophyta). Journal of Phycology. 2018; 54(1):25±33. https://doi.org/10.1111/jpy.12602 PMID: 29077982

PLOS ONE | https://doi.org/10.1371/journal.pone.0196995 May 8, 2018 15 / 19 Three plastid genomes of Nemaliophycidae

10. Nan F, Feng J, Lv J, Liu Q, Fang K, Gong C, et al. Origin and evolutionary history of freshwater Rhodo- phyta: further insights based on phylogenomic evidence. Scientific Reports. 2017; 7:2934. https://doi. org/10.1038/s41598-017-03235-5 PMID: 28592899 11. Chevreux B, Wetter T, Suhai S, editors. Genome sequence assembly using trace signals and additional sequence information. German Conference on Bioinformatics; 1999. 12. Janouskovec J, Liu SL, Martone PT, Carre W, Leblanc C, Collen J, et al. Evolution of red algal plastid genomes: ancient architectures, introns, horizontal gene transfer, and taxonomic utility of plastid markers. PLoS One. 2013; 8(3):e59001. https://doi.org/10.1371/journal.pone.0059001 PMID: 23536846 13. Collen J, Porcel B, Carre W, Ball SG, Chaparro C, Tonon T, et al. Genome structure and metabolic features in the red seaweed Chondrus crispus shed light on evolution of the Archaeplastida. Proceedings of the National Academy of Sciences of the United States of America. 2013; 110(13):5247±52. https:// doi.org/10.1073/pnas.1221259110 PMID: 23503846 14. Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C, et al. Versatile and open software for comparing large genomes. Genome Biology. 2004; 5(2):R12. https://doi.org/10.1186/gb-2004- 5-2-r12 PMID: 14759262 15. Song HJ, Lee J, Graf L, Rho M, Qiu H, Bhattacharya D, et al. A novice's guide to analyzing NGS-derived organelle and metagenome data. Algae. 2016; 31(2):137±54. 16. Lagesen K, Hallin P, Rødland EA, Stñrfeldt H-H, Rognes T, Ussery DW. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Research. 2007; 35(9):3100±8. https://doi.org/ 10.1093/nar/gkm160 PMID: 17452365 17. Laslett D, Canback B. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Research. 2004; 32(1):11±6. https://doi.org/10.1093/nar/gkh152 PMID: 14704338 18. Nawrocki EP, Burge SW, Bateman A, Daub J, Eberhardt RY, Eddy SR, et al. Rfam 12.0: updates to the RNA families database. Nucleic Acids Research. 2015; 43(D1):D130±D7. 19. Lohse M, Drechsel O, Bock R. OrganellarGenomeDRAW (OGDRAW): a tool for the easy generation of high-quality custom graphical maps of plastid and mitochondrial genomes. Current Genetics. 2007; 52 (5±6):267±74. https://doi.org/10.1007/s00294-007-0161-y PMID: 17957369 20. Hilker R, Sickinger C, Pedersen CN, Stoye J. UniMoGÐa unifying framework for genomic distance cal- culation and sorting based on DCJ. Bioinformatics. 2012; 28(19):2509±11. https://doi.org/10.1093/ bioinformatics/bts440 PMID: 22815356 21. Darling AC, Mau B, Blattner FR, Perna NT. Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Research. 2004; 14(7):1394±403. https://doi.org/10.1101/gr.2289704 PMID: 15231754 22. Darling AE, Mau B, Perna NT. progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement. PLoS One. 2010; 5(6):e11147. https://doi.org/10.1371/journal.pone.0011147 PMID: 20593022 23. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, et al. Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012; 28(12):1647±9. https://doi.org/10.1093/bioinformatics/bts199 PMID: 22543367 24. Ihaka R, Gentleman R. R: a language for data analysis and graphics. Journal of Computational and Graphical Statistics. 1996; 5(3):299±314. 25. Keeling PJ, Burki F, Wilcox HM, Allam B, Allen EE, Amaral-Zettler LA, et al. The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing. PLoS Biology. 2014; 12(6):e1001889. https://doi.org/10.1371/journal.pbio.1001889 PMID: 24959919 26. Price DC, Chan CX, Yoon HS, Yang EC, Qiu H, Weber AP, et al. Cyanophora paradoxa genome eluci- dates origin of photosynthesis in algae and plants. Science. 2012; 335(6070):843±7. https://doi.org/10. 1126/science.1213561 PMID: 22344442 27. Chan CX, Yang EC, Banerjee T, Yoon HS, Martone PT, Estevez JM, et al. Red and green algal mono- phyly and extensive gene sharing found in a rich repertoire of red algal genes. Current Biology. 2011; 21(4):328±33. https://doi.org/10.1016/j.cub.2011.01.037 PMID: 21315598 28. Bhattacharya D, Price DC, Chan CX, Qiu H, Rose N, Ball S, et al. Genome of the red alga Porphyridium purpureum. Nature Communications. 2013; 4:1941. https://doi.org/10.1038/ncomms2931 PMID: 23770768 29. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in per- formance and usability. Molecular Biology and Evolution. 2013; 30(4):772±80. https://doi.org/10.1093/ molbev/mst010 PMID: 23329690

PLOS ONE | https://doi.org/10.1371/journal.pone.0196995 May 8, 2018 16 / 19 Three plastid genomes of Nemaliophycidae

30. Nguyen L-T, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution. 2015; 32(1):268±74. https://doi.org/10.1093/molbev/msu300 PMID: 25371430 31. Le SQ, Gascuel O. An improved general amino acid replacement matrix. Molecular Biology and Evolu- tion. 2008; 25(7):1307±20. https://doi.org/10.1093/molbev/msn067 PMID: 18367465 32. Marchler-Bauer A, Lu S, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, et al. CDD: a Con- served Domain Database for the functional annotation of proteins. Nucleic Acids Research. 2011; 39 (suppl 1):D225±D9. 33. Emanuelsson O, Nielsen H, Brunak S, von Heijne G. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. Journal of Molecular Biology. 2000; 300(4):1005±16. https://doi.org/10.1006/jmbi.2000.3903 PMID: 10891285 34. Emanuelsson O, Nielsen H, Von Heijne G. ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Science. 1999; 8(05):978±84. 35. Krogh A, Larsson B, Von Heijne G, Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. Journal of Molecular Biology. 2001; 305 (3):567±80. https://doi.org/10.1006/jmbi.2000.4315 PMID: 11152613 36. Kanehisa M, Goto S. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Research. 2000; 28(1):27±30. PMID: 10592173 37. Halary S, McInerney J, Lopez P, Bapteste E. EGN: a wizard for construction of gene and genome similarity networks. BMC Evolutionary Biology. 2013; 13(1):146. 38. Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Research. 2003; 13 (11):2498±504. https://doi.org/10.1101/gr.1239303 PMID: 14597658 39. Lee J, Cho CH, Park SI, Choi JW, Song HS, West JA, et al. Parallel evolution of highly conserved plastid genome architecture in red seaweeds and seed plants. BMC Biology. 2016; 14(1):1±16. 40. Tamayo DA, Hughey JR. Organellar genome analysis of the marine red alga Dasya binghamiae (Dasyaceae, Rhodophyta) reveals an uncharacteristic florideophyte mitogenome structure. Mitochon- drial DNA Part B. 2016; 1(1):510±1. 41. Sissini MN, Navarrete-FernaÂndez TM, Murray EM, Freese JM, Gentilhomme AS, Huber SR, et al. Mito- chondrial and plastid genome analysis of the heteromorphic red alga Mastocarpus papillatus (C. Agardh) KuÈtzing (Phyllophoraceae, Rhodophyta) reveals two characteristic florideophyte organellar genomes. Mitochondrial DNA Part B. 2016; 1(1):676±7. 42. Salomaki ED, Nickles KR, Lane CE. The ghost plastid of Choreocolax polysiphoniae. Journal of Phycol- ogy. 2015; 51(2):217±21. https://doi.org/10.1111/jpy.12283 PMID: 26986516 43. Ng P-K, Lin S-M, Lim P-E, Liu L-C, Chen C-M, Pai T-W. Complete chloroplast genome of Gracilaria firma (Gracilariaceae, Rhodophyta), with discussion on the use of chloroplast phylogenomics in the subclass Rhodymeniophycidae. BMC Genomics. 2017; 18(1):40. https://doi.org/10.1186/s12864-016- 3453-0 PMID: 28061748 44. Lee J, Kim KM, Yang EC, Miller KA, Boo SM, Bhattacharya D, et al. Reconstructing the complex evolutionary history of mobile plasmids in red algal genomes. Scientific Reports. 2016; 6:23744. https://doi. org/10.1038/srep23744 PMID: 27030297 45. Kilpatrick ZM, Hughey JR. Mitochondrial and plastid genome analysis of the marine red alga Coeloseira compressa (Champiaceae, Rhodophyta). Mitochondrial DNA Part B. 2016; 1(1):456±8. 46. Hughey JR, Hommersand MH, Gabrielson PW, Miller KA, Fuller T. Analysis of the complete plastomes of three species of Membranoptera (Ceramiales, Rhodophyta) from Pacific North America. Journal of Phycology. 2017; 53(1):32±43. https://doi.org/10.1111/jpy.12472 PMID: 27690326 47. Hughey JR, Gabrielson PW, Rohmer L, Tortolani J, Silva M, Miller KA, et al. Minimally destructive sam- pling of type specimens of Pyropia (Bangiales, Rhodophyta) recovers complete plastid and mitochondrial genomes. Scientific Reports. 2014; 4:5113. https://doi.org/10.1038/srep05113 PMID: 24894641 48. Hughey JR, Boo GH. Genomic and phylogenetic analysis of Ceramium cimbricum (Ceramiales, Rhodo- phyta) from the Atlantic and Pacific Oceans supports the naming of a new invasive Pacific entity Cera- mium sungminbooi sp. nov. Botanica Marina. 2016; 59(4):211±22. 49. Hagopian JC, Reis M, Kitajima JP, Bhattacharya D, de Oliveira MC. Comparative analysis of the complete plastid genome sequence of the red alga Gracilaria tenuistipitata var. liui provides insights into the evolution of rhodoplasts and their relationship to other plastids. Journal of Molecular Evolution. 2004; 59 (4):464±77. https://doi.org/10.1007/s00239-004-2638-3 PMID: 15638458 50. Du Q, Bi G, Mao Y, Sui Z. The complete chloroplast genome of Gracilariopsis lemaneiformis (Rhodo- phyta) gives new insight into the evolution of family Gracilariaceae. Journal of Phycology. 2016; 52 (3):441±50. https://doi.org/10.1111/jpy.12406 PMID: 27273536

PLOS ONE | https://doi.org/10.1371/journal.pone.0196995 May 8, 2018 17 / 19 Three plastid genomes of Nemaliophycidae

51. DõÂaz Tapia P, Maggs CA, West JA, Verbruggen H. Analysis of chloroplast genomes and a supermatrix inform reclassification of the Rhodomelaceae (Rhodophyta). Journal of Phycology. 2017; 53:920±37. https://doi.org/10.1111/jpy.12553 PMID: 28561261 52. DePriest MS, Bhattacharya D, Lopez-Bautista JM. The plastid genome of the red macroalga Gratelou- pia taiwanensis (Halymeniaceae). PLoS One. 2013; 8(7):e68246. https://doi.org/10.1371/journal.pone. 0068246 PMID: 23894297 53. Campbell MA, Presting G, Bennett MS, Sherwood AR. Highly conserved organellar genomes in the Gracilariales as inferred using new data from the Hawaiian invasive alga Gracilaria salicornia (Rhodo- phyta). Phycologia. 2014; 53(2):109±16. 54. Kushibiki A, Yokoyama A, Iwataki M, Yokoyama J, West JA, Hara Y. New unicellular red alga, Bulbo- plastis apyrenoidosa gen. et sp. nov.(Rhodellophyceae, Rhodophyta) from the mangroves of Japan: phylogenetic and ultrastructural observations. Phycological Research. 2012; 60(2):114±22. 55. Kamiya M, West JA, Karsten U, Ganesan E. Molecular and morphological delineation of Caloglossa beccarii and related species (Delesseriaceae, Rhodophyta). Phycologia. 2016; 55(6):640±9. 56. Zuccarello GC, West JA. Insights into evolution and speciation in the red alga Bostrychia: 15 years of research. Algae. 2011; 26(1):21±32. 57. Womersley H. Southern Australian species of Polysiphonia Greville (Rhodophyta). Australian Journal of Botany. 1979; 27(4):459±528. 58. Kugrens P, West JA. The ultrastructure of an alloparasitic red alga Choreocolax polysiphoniae. Phyco- logia. 1973; 12(3):175±86. 59. Kikuchi G, Yoshida T, Noguchi M. Heme oxygenase and heme degradation. Biochemical and Biophysi- cal Research Communications. 2005; 338(1):558±67. https://doi.org/10.1016/j.bbrc.2005.08.020 PMID: 16115609 60. Rhie G-e, Beale SI. Biosynthesis of phycobilins. Ferredoxin-supported nadph-independent heme oxygenase and phycobilin-forming activities from Cyanidium caldarium. Journal of Biological Chemistry. 1992; 267(23):16088±93. PMID: 1644795 61. Frankenberg-Dinkel N. Bacterial heme oxygenases. Antioxidants & Redox Signaling. 2004; 6(5):825± 34. 62. Poss KD, Tonegawa S. Heme oxygenase 1 is required for mammalian iron reutilization. Proceedings of the National Academy of Sciences of the United States of America. 1997; 94(20):10919±24. PMID: 9380735 63. Duanmu D, Casero D, Dent RM, Gallaher S, Yang W, Rockwell NC, et al. Retrograde bilin signaling enables Chlamydomonas greening and phototrophic survival. Proceedings of the National Academy of Sciences of the United States of America. 2013; 110(9):3621±6. https://doi.org/10.1073/pnas. 1222375110 PMID: 23345435 64. Hentze MW, Muckenthaler MU, Andrews NC. Balancing acts: molecular control of mammalian iron metabolism. Cell. 2004; 117(3):285±97. PMID: 15109490 65. Richaud C, Zabulon G. The heme oxygenase gene (pbsA) in the red alga Rhodella violacea is discontin- uous and transcriptionally activated during iron limitation. Proceedings of the National Academy of Sci- ences of the United States of America. 1997; 94(21):11736±41. PMID: 9326680 66. Hecky R, Kilham P. Nutrient limitation of phytoplankton in freshwater and marine environments: a review of recent evidence on the effects of enrichment. Limnology and Oceanography. 1988; 33 (4part2):796±822. 67. Emborg TJ, Walker JM, Noh B, Vierstra RD. Multiple heme oxygenase family members contribute to the biosynthesis of the phytochrome chromophore in Arabidopsis. Plant Physiology. 2006; 140(3):856± 68. https://doi.org/10.1104/pp.105.074211 PMID: 16428602 68. Flagel LE, Wendel JF. Gene duplication and evolutionary novelty in plants. New Phytologist. 2009; 183 (3):557±64. https://doi.org/10.1111/j.1469-8137.2009.02923.x PMID: 19555435 69. GarcõÂa-Mata C, Lamattina L. Gasotransmitters are emerging as new guard cell signaling molecules and regulators of leaf gas exchange. Plant Science. 2013; 201:66±73. https://doi.org/10.1016/j.plantsci. 2012.11.007 PMID: 23352403 70. Gisk B, Yasui Y, Kohchi T, Frankenberg-Dinkel N. Characterization of the haem oxygenase protein family in Arabidopsis thaliana reveals a diversity of functions. Biochemical Journal. 2010; 425(2):425±34. https://doi.org/10.1042/BJ20090775 PMID: 19860740 71. Panchy N, Lehti-Shiu M, Shiu S-H. Evolution of gene duplication in plants. Plant Physiology. 2016; 171 (4):2294±316. https://doi.org/10.1104/pp.16.00523 PMID: 27288366 72. Shekhawat GS, Verma K. Haem oxygenase (HO): an overlooked enzyme of plant metabolism and defence. Journal of Experimental Botany. 2010; 61(9):2255±70. https://doi.org/10.1093/jxb/erq074 PMID: 20378668

PLOS ONE | https://doi.org/10.1371/journal.pone.0196995 May 8, 2018 18 / 19 Three plastid genomes of Nemaliophycidae

73. Muramoto T, Kohchi T, Yokota A, Hwang I, Goodman HM. The Arabidopsis photomorphogenic mutant hy1 is deficient in phytochrome chromophore biosynthesis as a result of a mutation in a plastid heme oxygenase. The Plant Cell. 1999; 11(3):335±47. PMID: 10072395 74. BruÈcker G, Mittmann F, Hartmann E, Lamparter T. Targeted site-directed mutagenesis of a heme oxygenase locus by gene replacement in the moss Ceratodon purpureus. Planta. 2005; 220(6):864±74. https://doi.org/10.1007/s00425-004-1411-6 PMID: 15578218 75. Kadish KM, Smith KM, Guilard R. The Porphyrin Handbook: The iron and cobalt pigments: biosynthesis, structure, and degradation: Elsevier; 2003. 76. Timmis JN, Ayliffe MA, Huang CY, Martin W. Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes. Nature Reviews Genetics. 2004; 5(2):123±35. https://doi.org/10.1038/ nrg1271 PMID: 14735123

PLOS ONE | https://doi.org/10.1371/journal.pone.0196995 May 8, 2018 19 / 19