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Peer-reviewed

Research Article

The Complete Chloroplast and Mitochondrial Genomes of the Green MacroalgaUlva sp. UNA00071828 (Ulvophyceae, Chlorophyta)

  • James T. Melton III,

    * E-mail:melto006@crimson.ua.edu

    Affiliation Department of Biological Sciences, The University of Alabama, Tuscaloosa, Alabama 35487-0345, United States of America

    ⨯
  • Frederik Leliaert,

    Affiliations Department of Biological Sciences, The University of Alabama, Tuscaloosa, Alabama 35487-0345, United States of America, Marine Biology Research Group, Department of Biology, Ghent University, Krijgslaan 281-S8, 9000 Ghent, Belgium

    ⨯
  • Ana Tronholm,

    Affiliations Department of Biological Sciences, The University of Alabama, Tuscaloosa, Alabama 35487-0345, United States of America, Smithsonian Marine Station at Fort Pierce, 701 Seaway Drive, Fort Pierce, Florida 34949, United States of America

    ⨯
  • Juan M. Lopez-Bautista

    Affiliation Department of Biological Sciences, The University of Alabama, Tuscaloosa, Alabama 35487-0345, United States of America

    ⨯

The Complete Chloroplast and Mitochondrial Genomes of the Green MacroalgaUlva sp. UNA00071828 (Ulvophyceae, Chlorophyta)

  • James T. Melton III, 
  • Frederik Leliaert, 
  • Ana Tronholm, 
  • Juan M. Lopez-Bautista
PLOS
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Figures

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Abstract

Sequencing mitochondrial and chloroplast genomes has become an integral part in understanding the genomic machinery and the phylogenetic histories of green algae. Previously, only three chloroplast genomes (Oltmannsiellopsis viridis,Pseudendoclonium akinetum, and Bryopsis hypnoides) and two mitochondrial genomes (O.viridis andP.akinetum) from the class Ulvophyceae have been published. Here, we present the first chloroplast and mitochondrial genomes from the ecologically and economically important marine, green algal genusUlva. The chloroplast genome ofUlva sp. was 99,983 bp in a circular-mapping molecule that lacked inverted repeats, and thus far, was the smallest ulvophycean plastid genome. This cpDNA was a highly compact, AT-rich genome that contained a total of 102 identified genes (71 protein-coding genes, 28 tRNA genes, and three ribosomal RNA genes). Additionally, five introns were annotated in four genes:atpA (1),petB (1),psbB (2), andrrl (1). The circular-mapping mitochondrial genome ofUlva sp. was 73,493 bp and follows the expanded pattern also seen in other ulvophyceans and trebouxiophyceans. TheUlva sp. mtDNA contained 29 protein-coding genes, 25 tRNA genes, and two rRNA genes for a total of 56 identifiable genes. Ten introns were annotated in this mtDNA:cox1 (4),atp1 (1),nad3 (1),nad5 (1), andrrs (3). Double-cut-and-join (DCJ) values showed that organellar genomes across Chlorophyta are highly rearranged, in contrast to the highly conserved organellar genomes of the red algae (Rhodophyta). A phylogenomic investigation of 51 plastid protein-coding genes showed that Ulvophyceae is not monophyletic, and also placed Oltmannsiellopsis (Oltmannsiellopsidales) andTetraselmis (Chlorodendrophyceae) closely toUlva (Ulvales) andPseudendoclonium (Ulothrichales).

Citation:Melton JT III, Leliaert F, Tronholm A, Lopez-Bautista JM (2015) The Complete Chloroplast and Mitochondrial Genomes of the Green MacroalgaUlva sp. UNA00071828 (Ulvophyceae, Chlorophyta). PLoS ONE 10(4): e0121020. https://doi.org/10.1371/journal.pone.0121020

Academic Editor:Axel Janke, BiK-F Biodiversity and Climate Research Center, GERMANY

Received:October 23, 2014;Accepted:February 9, 2015;Published: April 7, 2015

Copyright: © 2015 Melton et al. This is an open access article distributed under the terms of theCreative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability:All genome sequences are available from the GenBank database: the chloroplast genome ofUlva sp. (KP720616) and the mitochondrial genome ofUlva sp. (KP720617).

Funding:This study was funded by The National Science Foundation under the ATOL Program, through grants DEB 1036495 and 0937978 to JMLB. Additional support was provided by the College of Arts and Sciences (CARSCA Program) and the Office for Research (Research Stimulation Program) at UA to JMLB, and the Department of Biological Sciences (research and travel awards) to JTM.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The green algae are comprised of two main clades, Chlorophyta and Streptophyta. Chlorophyta includes a wide diversity of marine, freshwater and terrestrial green algae. Streptophyta includes the freshwater charophyte green algae along with the land plants [1],[2]. Chlorophyta is currently composed of the paraphyletic prasinophytes along with five classes recently coined as the “core chlorophytes” (Chlorodendrophyceae, Chlorophyceae, Pedinophyceae, Trebouxiophyceae, and Ulvophyceae) [2],[3]. Several investigations have suggested that Ulvophyceae and Trebouxiophyceae are not monophyletic and further genomic investigations with an increased taxon sampling are still needed to resolve higher taxonomic ranks [3],[4],[5],[6].

In order to help answer the phylogenetic questions that remain for the green algae and to gain a better understanding of the organellar genomic composition of these organisms, plastid and mitochondrial genomes have been sequenced throughout most of the major chlorophyte lineages, and many more organellar genomes are expected to be published in the near future. Green algae have shown to have a wide range of organellar genome sizes, content (e.g. GC%, number of genes, and number of introns), and organization of genes [2],[7],[8]. Indeed, the mitochondrial genomes (mtDNA) have ranged from as small as 13 Kbp inPolytomella capuana [9] to as large as 201.8 Kbp inChlorokybus atmophyticus [10], and the chloroplast genomes (cpDNA) have shown even larger size differences from 37.5 Kbp in the nonphotosynthetic and parasiticHelicosporidium sp. [11] to 521 Kbp inFloydiella terrestris [12] and 525 Kbp inVolvox carteri [13], or possibly as big as 2 Mbp in dasycladalean green algae [14],[15]. Members of the Chlorophyceae are known to have a reduced-derived state, where most of the mitochondrial genes have been transferred to the nucleus. Conversely, mtDNAs of the Ulvophyceae and Trebouxiophyceae have shown to have an expanded pattern from the hypothesized ancestral state due to an increase of intergenic spacers and introns [8]. An expanded pattern has also been seen in the mitochondrial genomes of land plants, suggesting that convergent evolution has taken place in the two major lineages of Viridiplantae [8],[16],[17].

Ulvophyceae is well represented by marine macroalgae (known as green seaweeds) [2],[5] and contains more than 1600 species [18]. However, only three complete chloroplast genomes,Oltmannsiellopsis viridis [19],Pseudendoclonium akinetum [20], andBryopsis hypnoides [21], and two mitochondrial genomes,O.viridis [17] andP.akinetum [18], have been published from organisms that are currently classified in the Ulvophyceae. Additionally, early structural data of the chloroplast genomes have been published forCodium fragile [15] andCaulerpa sertulariodes [22], and incomplete DNA sequence data has been published forCaulerpa filiformis [23]. It is important to point out that some of these algae could potentially belong to completely different higher taxonomic ranks in the near future. For example, Fučíková et al. [3] recoveredOltmannsiellopsis as sister toTetraselmis, a member of the Chlorodendrophyceae and early diverging core chlorophyte, with strong support in multi-gene phylogenies mostly based on genomic plastid data.

Species of the green algal genusUlva are distributed worldwide, where they are ecologically important members in marine and brackish environments and sometimes can be found in freshwater streams and lakes. SomeUlva species are notorious for forming harmful algal blooms called green tides in eutrophic conditions [24],[25]. These macroalgae are either monostromatic and tubular, distromatic blades, or distromatic with hollow monostromatic margins [26]. Morphology-based species level identifications ofUlva are notably challenging due to the lack of diagnostic morphological characters, morphological plasticity [27], and cryptic diversity [28]. There are currently 105Ulva species accepted [18]. However, this number is likely to change as molecular investigations continue to reveal the true identities of these algae [24],[27],[28],[29],[30],[31],[32],[33],[34].

Transcriptomic studies have been performed onUlva prolifera [35] andU.linza [36], which have helped reveal some of the genomic mechanisms that these algae have to survive in the harsh intertidal zone such as having land-plant specific genes [36]. Further,U.prolifera showed to potentially have a C3-C4 photosynthetic pathway, which could explain the ability of these species to rapidly grow during green tides [35]. However, complete organellar genomes ofUlva have not previously been published.

In this study, we present the first complete chloroplast and mitochondrial genomes ofUlva. These genomes were sequenced, assembled, and annotated as circular-mapping DNA molecules. Additionally, we compared theUlva sp. genomes to the previously published chlorophyte genomes in order to gain a better understanding of the evolution of these organellar genomes. Furthermore, we performed a phylogenomic investigation to assess the monophyly of the Ulvophyceae and relationships of ulvophytes with other core chlorophytan clades.

Materials and Methods

Sampling and Species Identification

A monostromatic and tubularUlva specimen was collected in August 2011 from a jetty near Dauphin Island Sea Lab, Alabama, USA (30º14’50.1”N, 88º04’29.1”W). Since Dauphin Island Sea Lab is a part of The University of Alabama (UA) consortium and a source for learning at several levels, no specific permission was required to collect algal samples. The specimen in this study did not involve an endangered or protected species. The specimen was preserved in silica gel, and a herbarium voucher was deposited at The University of Alabama Herbarium (UNA00071828). An image of the herbarium voucher can be viewed in Supporting Information (S1 Fig).

Due to the difficulty of identifyingUlva species based on morphology, species identification was based on analyses of the chloroplast-encodedrbcL andtufA genes. These genes were selected since large amounts of data are currently available for these molecular markers. An NCBI-blastn of the genes helped find sequences with high identities. Sequences with an identity of 99% or greater were used with other previously published sequences to make alignments ofrbcL (15 sequences; 1257 bp) andtufA (12 sequences; 738 bp) using MUSCLE [37]. A neighbor-joining tree was made in Geneious R7 (http://www.geneious.com; [38]) for each alignment with the Jukes-Cantor genetic distance model and 1000 bootstrap replicates (seeS2 andS3 Figs, andS1 Table in Supporting Information for gene trees and taxa used, respectively).Monostroma grevillei (GU183089;rbcL) andMonostroma sp. (HQ610262;tufA) were used as outgroups. Additionally, the pairwise distances (bp) were calculated using MEGA 6 [39] to aid in the identification of thisUlva species (seeS4 andS5 Figs in Supporting Information).

TherbcL sequence ofUlva sp. UNA00071828 clustered withUlva sp. OTU1 from the Hawaiian Islands (GU138253; [31]). The 1257 bp alignment showed that these sequences are 100% identical, and thus, likely the same species (S4 Fig in Supporting Information). The closesttufA sequence thatUlva sp. UNA00071828 clustered with was an unidentifiedUlva specimen from Australia (KF195535; [40]). These sequences varied by six bp (S5 Fig in Supporting Information), and further investigations would be necessary to determine whether these organisms are conspecific or distinct entities. Due to the difficulty of identifyingUlva species based on morphology and the inability of relating this organism with other sequence data, we are refraining from assigning this organism a species name as O’Kelly et al. [31] also opted for. It is possible that this organism has previously been described since manyUlva species lack molecular data, orUlva sp. UNA00071828 may represent an undescribed species. Further molecular studies in combination with morphological investigations need to be performed for an accurate identification of thisUlva species.

DNA Sequencing and Assembly

Total genomic DNA was extracted using a QIAGEN DNEasy Plant Mini Kit (QIAGEN, Valencia, CA, USA). The genomic DNA was fragmented into 350 bp and sequenced using Illumina HiSeq 2000 technology at Cold Spring Harbor Laboratory, USA. The sequencing run produced ca. 23 million paired-end reads of 2 x 101 bp. Poor quality sequences and sequencing adapters were removed using Trim Galore! v0.3.7 [41], leaving 13,742,730 trimmed reads. De novo assemblies were run using Geneious, Velvet v1.2.10 [42], and CLC Genomics Workbench v6 [43] with the trimmed sequences. Following the assemblies, putative chloroplast and mitochondrial contigs were identified using a local blast in Geneious using a suite of 2,433 chloroplast and 123 mitochondrial genes from previously published chlorophyte genomes. After the contigs with blast hits for the chloroplast and mitochondrial genes were found, another de novo assembly was run separately for contigs containing chloroplast and mitochondrial genes. This resulted in one contig of ca. 100,000 bp for the chloroplast genome and another contig of ca. 73,000 bp for the mitochondrial genome. Trimmed reads were mapped iteratively to the putative chloroplast and mitochondrial contigs. This resulted in longer contigs with ca. 150 bp overlap in both contigs, which allowed the genomes to be closed.

Annotation of the Chloroplast and Mitochondrial Genomes

Open reading frames (ORFs) of 150 bp or greater were found in Geneious using a bacterial genetic code. Protein-coding genes and rRNA genes from previous published ulvophycean chloroplast and mitochondrial genomes were then mapped to the reference chloroplast and mitochondrial contigs using the ‘Map to Reference’ option in Geneious. Annotations of the mapped genes were transferred to the reference sequence, and identities of the genes were confirmed using NCBI-blastx. Further, a blastx of other ORFs without mapped hits helped find other putative genes. A blastx hit with an E-value cut off of 10-10 was considered as a significant hit. To find putative tRNA genes, the chloroplast and mitochondrial contigs were submitted to tRNAscan-SE v1.21 using the default search mode and Mito/Chloroplast model [44],[45]. A comparison of tRNAs present inUlva and other ulvophytes can be found in the Supporting Information (S2 andS3 Tables). Introns were also annotated and intron-exon boundaries were located by aligning the sequences of genes containing introns with those of intronless homologs. A blastx of the intronic ORFs (if present) was used to search for conserved motifs. Intron insertion sites were compared among the ulvophytes. Insertion sites of introns in protein-coding genes were determined by the nucleotide before the insertion of the intron. Insertion sites of rRNAs and tRNAs were based on alignments of 16S (NC_004431) and 23S (NC_004431) rDNA ofE.coli andtrnL(UAA) (NC_002186) ofMesostigma viride (NC_002186), respectively. Inverted and tandem repeats were detected using einverted and equicktandem of the EMBOSS suite [46]. Gene maps were made with OGDRAW (http://ogdraw.mpimp-golm.mpg.de/; [47]). GenBank Accession Numbers of the chloroplast and mitochondrial genomes are as follows: KP720616, KP720617, respectively.

Rearrangements in Chlorophyte Plastid and Mitochondrial Genomes

The double-cut-and-join (DCJ) [48] genome distances were calculated with UniMoG [49],[50] to estimate the number of rearrangements between the selected genomes. The DCJ values were calculated with all genes, and a separate analysis was run without tRNAs (S4 andS5 Tables). Genome alignments with previously published chlorophyte organellar genomes were performed with the Mauve Genome Alignment v2.3.1 [51] Geneious Plugin and the progressive Mauve algorithm [52]. These alignments were made to show similarities in gene clusters called local collinear blocks (LCBs). LCBs also allow for visualizing major rearrangements as the LCBs are connected with lines in the alignment, and also display inverted regions (IR). The beginning ofrbcL andcox1 were selected as the starting positions in the chloroplast and mitochondrial genomes, respectively, for the Mauve analyses (S6 andS7 Figs).

Phylogenomic Analyses

Phylogenomic analyses were based on chloroplast genome data of 52 members of Chlorophyta, largely based on Lemieux et al. [4],[53] and Fučíková et al. [3] (S6 Table). 51 chloroplast protein-coding genes were selected from Fučíková et al. [3]:atpA,atpB,atpE,atpF,atpH,atpI,infA,petA,petB,petG,psaA,psaB,psaC,psaJ,psaM,psbA,psbB,psbC,psbD,psbE,psbF,psbH,psbI,psbJ,psbK,psbL,psbN,psbT,rbcL,rpl2,rpl5,rpl14,rpl16,rpl20,rpl23,rpl32,rpl36,rps3,rps4,rps7,rps8,rps9,rps11,rps12,rps14,rps18,rps19,tufA,ycf3,ycf4 andycf12. The concatenated alignment consisted of 50,595 positions and was 94% filled at the nucleotide level. Taxa with low gene coverage includedAcetabularia_acetabulum (37 genes),Cephaleuros parasiticus (17 genes),Codium decorticatum (32 genes),Halimeda cylindracea (33 genes),Tetraselmis_sp. (11 genes), andTrentepohlia annulata (18 genes). We did not use the mitochondrial genome data because fewer complete mitochondrial genomes have been sequenced in the Chlorophyta and because phylogenomic analysis based on mitochondrion multigene data only weakly resolves relationships within the Chlorophyta [54].

DNA sequences were aligned for each gene separately using the ClustalW translational alignment function [55] in Geneious using a BLOSUM cost matrix, gap open penalty 10 and gap extend cost 0.1. Poorly aligned positions were removed using the Gblocks server (http://molevol.cmima.csic.es/castresana/Gblocks_server.html; [56]) using the least stringent settings: allowing smaller final blocks, gap positions within the final blocks, less strict flanking positions and many contiguous non-conserved positions. Gblocks removed 22,428 of the total 50,595 positions, leaving a nucleotide alignment of 28,167 positions. The resulting alignment was translated to obtain the amino acid alignment of 9389 positions. For the phylogenetic analysis of the DNA sequence alignment, only the first two codon positions were used.

Maximum likelihood and Bayesian trees were inferred from the DNA and amino acid (AA) alignments using RAxML v.7.3.5 [57] and MrBayes v.3.2.1 [58], respectively. For the DNA sequences, we specified a GTRCAT (RAxML) or GTR+Γ (MrBayes) model, and a partitioning strategy in which codon positions were separated (two partitions). The MrBayes analysis was run for five million (nucleotide alignment) or two million (AA alignment) generations. Two independent runs were performed for each data set. The first 10% of samples were discarded as burn-in. Convergence of the runs and stability of parameters were assessed using Tracer v.1.5 [59].

Results and Discussion

The Chloroplast Genome ofUlva sp.

A total of 1,210,556 trimmed reads combined to form a 99,983 bp circular-mapping chloroplast genome ofUlva sp. UNA00071828 (Fig 1). The depth of coverage at each position was a mean of 1202.2 bp, and the whole assembly had a pairwise identity of 99.8%.Table 1 compares general characteristics of the plastid genomes ofUlva with other core chlorophytes. To date, the chloroplast genome ofUlva sp. is the smallest ulvophycean chloroplast genome to be sequenced. However, this is only compared to the three complete ulvophycean genomes:Oltmannsiellopsis viridis (151.9 Kbp; [19]),Pseudendoclonium akinetum (195.8 Kbp; [20]), andBryopsis hypnoides (153.4 Kbp; [21]). The percent of coding DNA (81.8%) ranks among the highest found in green algae and especially the core chlorophytes. The high coding percentage of this genome is by far the most gene compact ulvophycean genome published, compared toO.viridis (60.4%),P.akinetum (62.4%), andB.hypnoides (59.4%). The cpDNA ofUlva sp. ranks among the highest AT content (74.7%) compared with the core chlorophytes published thus far [2],[7]. An IR, which is commonly found in other green algae and has been found in the plastid genomes ofP.akinetum [20] andO.viridis [19], was absent in thisUlva genome,B.hypnoides [21], and the genomes ofCodium fragile [15] andCaulerpa sertularioides [22]. Thus, theUlva cpDNA is not a quadripartite structure.

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Fig 1. Gene map ofUlva sp. UNA00071828 chloroplast genome using OGDRAW.

Genes in the clockwise direction are on the inside of the map, and genes in the counterclockwise direction are on the outside of the map. Annotated genes are colored according to the functional categories shown in the legend (bottom left).

https://doi.org/10.1371/journal.pone.0121020.g001

Gene Content

A total of 102 identifiable genes were annotated in the cpDNA ofUlva sp., including 71 protein-coding genes ranging from 93 bp (psaM) to 8067 bp (rpoC2), 28 tRNA genes, and three rRNA genes (rrf,rrl, andrrs). All of the protein-coding genes were also found in at least one of the previously published ulvophycean genome [19],[20],[21] (Table 2). The 28 tRNAs present were able to complete the full genetic code and two tRNA genes were found fortrnG,trnF,trnI,trnL,trnM,trnN,trnR, andtrnS (S2 Table in Supporting Information). In addition to the 102 identified genes, one freestanding ORF and three intronic ORFs had significant blastx hits in NCBI’s non-redundant protein database. The freestanding orf3 (2019 bp) had top hits of the NAD-dependent DNA ligaseLigA found in severalRickettsia species and other species of bacteria; however, with low identities (e.g. 38% identical toR.montanensis, E-value 1e-115, WP_014409548; 37% identical toR.massiliae, E-value 5e-116, WP_014365478; 37% identical toR.rhipicephali, 1e-115, WP_014408984).

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Table 2. A comparison of the gene content of theUlva sp. cpDNA with 17 other core chlorophytes (excluding tRNAs).

https://doi.org/10.1371/journal.pone.0121020.t002

Introns

Five introns were present in four genes (atpA,psbB,petB, andrrl) of theUlva chloroplast genome.AtpA,rrl, and intron 1 inpsbB contained an intronic ORF with a LAGLIDADG conserved motif, and an intronic ORF was present inpetB with a reverse transcriptase conserved motif. Intronic ORFs 1 (atpA), 2 (rrl), 7 (psbB), and 8 (petB) were 1138 bp, 767 bp, 2306 bp, and 2211 bp, respectively. An intronic ORF was not present in intron 2 inpsbB. A total of 27, 5, and 12 introns were previously found in the chloroplast genomes ofPseudendoclonium akinetum [20]Oltmannsiellopsis viridis [19], andBryopsis hypnoides [21], respectively. DNA alignments showed that the intron inatpA ofUlva is at the same position as the first intron inP.akinetum. Additionally, the intron inrrl ofUlva and the first intron inO.viridis are at homologous positions. A comparison of insertion sites of all of the ulvophyte introns can be found inTable 3.

The Mitochondrial Genome ofUlva sp.

A total of 299,728 trimmed reads combined to form a 73,493 bp circular-mapping mitochondrial genome ofUlva sp. UNA00071828 (Fig 2). The depth of coverage at each site was a mean of 402.5 bp with a pairwise identity of 99.8%.Table 4 compares the overall characteristics of theUlva sp. mtDNA with the previously published core chlorophyte mitogenomes. The mtDNA ofUlva sp. follows the expanded-derived pattern of the hypothesized ancestral state due to the presence of introns and/or increases in intergenic space as seen in the previously published ulvophyceans [8],[16],[17] and trebouxiophyceans [54],[60],[61],[62]. The “expanded” nature of the mtDNA has also been noted in the streptophyte lineage, suggesting the two major lineages of Viridiplantae have evolved expanded mitochondrial genomes through convergent evolution [16],[17]. However, the land plants have far larger mitochondrial genomes compared to green algae [63],[64]. Indeed, the embryophyte genomes are typically several hundred Kbp in length, and some are even over 1Mbp such as the 1.68 Mbp mtDNA ofCucumis sativus [65]. Thus far, all chlorophyte mtDNAs sequenced are under 100 Kbp [8]. The percent of coding DNA in the mtDNA ofUlva sp. was 75.8%, which is higher than the other two ulvophyceans,P.akinetum (56.7%) andOltmannsiellopsis viridis (66.2%). More similar percentages of coding regions have been seen in the trebouxiophyceans,Helicosporidium sp. (75.7%), Trebouxiophyceae sp. MX-AZ01 (68.2%), andPrototheca wickerhamii (70.6%). The AT-content of theUlva sp. mtDNA genome (67.6%) falls within the normal range of most green algae [8].

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Fig 2. Gene map ofUlva sp. UNA00071828 mitochondrial genome using OGDRAW.

Genes in the clockwise direction are on the inside of the map, and genes in the counterclockwise direction are on the outside of the map. Annotated genes are colored according to the functional categories shown in the legend (bottom left).

https://doi.org/10.1371/journal.pone.0121020.g002

Gene Content

A total of 56 identifiable genes were annotated in the mitochondrial genome, including 29 protein-coding genes ranging from 193 bp (rps3) to 6876 bp (cox1), 25 tRNA genes, and two rRNA genes (rrs andrrl). The 29 protein-coding genes were also found in at least one of the previously published ulvophycean genome (Table 5). The Ulvophyceae also have more genes in common with Trebouxiophyceae than with Chlorophyceae (Table 5), as most of the chlorophycean mitochondrial genes have been transferred to the nucleus [8]. The mtDNA ofUlva sp. contains 25 tRNAs that completed the full genetic code. TwotrnL genes were present in addition to threetrnM and threetrnR genes. The tRNAs found inUlva sp. and other ulvophyceans are shown inS3 Table in the Supporting Information. One freestanding and 12 intronic ORFs were also annotated. The freestanding orf7 (1131 bp) had a significant blastx hit as a DNA-directed RNA polymerase in the land plantsPhoenix dactylifera (31% identical, E-value 4e-11, YP_005090363) andDaucus carota (28% identical, E-value 2e-12, AAS15052).

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Table 5. A comparison of the gene content of theUlva sp. mtDNA with 13 other core chlorophytes (excluding tRNAs).

https://doi.org/10.1371/journal.pone.0121020.t005

Introns

A total of 10 introns were present in 5 genes (atp1,cox1,nad3,nad5, andrrs) in the mitochondrial genome ofUlva sp. Multiple introns were present incox1 (4) andrrs (3). The presence of introns incox1 indeed confirms why amplifying this gene in PCR has been such a difficult task [32]. Intronic ORFs with a LAGLIDADG conserved motif were found inatp1 (orf15: 1345 bp),nad5 (orf21: 1309 bp), each of the four introns incox1 (orf1: 1287 bp, orf2: 1590 bp, orf3: 1162 bp, and orf4: 1240 bp, respectively), and intron 1 and intron 2 inrrs (orf10: 1319 bp and orf11: 1534 bp, respectively). Intron 3 inrrs contained an intronic ORF (orf12: 1734 bp) with a reverse transcriptase conserved motif. The intron innad3 had two intronic ORFs (orf16: 288 bp; orf17: 156 bp) with a reverse transcriptase conserved motif and 1 intronic ORF (orf18: 240 bp) with a group II maturase conserved motif (GIIM). A total of 7 were previously annotated in the mtDNA ofPseudendoclonium akinetum [16] and 3 introns inOltmannsiellopsis viridis [17]. DNA alignments showed thatUlva sp. andP.akinetum have a few introns at homologous positions. These include the intron inapt1, introns 1 and 2 incox1, and intron 3 inUlva and intron 4 inP.akinetum. Intron insertion sites in the ulvophyte mtDNAs can be found inTable 6.

Rearrangements in the Chlorophyte cpDNA and mtDNA

The number of rearrangements between organisms from lineages throughout Chlorophyta was estimated by calculating the double-cut-and-join (DCJ) values. The DCJ values calculated for the cpDNAs and mtDNAs with tRNAs are shown in Tables7 and8, respectively. The DCJ values calculated for cpDNAs and mtDNAs without tRNAs can be found in the Supporting Information (S4 andS5 Tables, respectively). Mauve alignments of the selected chlorophyte genomes can also be found in the Supporting Information (S6 andS7 Figs for cpDNA and mtDNA, respectively).

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Table 7. DCJ values for chlorophyte cpDNAs of chlorophytes (includes tRNAs).

https://doi.org/10.1371/journal.pone.0121020.t007

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Table 8. DCJ values calculated for mtDNAs of chlorophytes (includes tRNAs).

https://doi.org/10.1371/journal.pone.0121020.t008

The DCJ values and Mauve alignments show that both organellar genomes of chlorophytes are highly rearranged. This is in contrast to the highly conserved organellar genomes of red algae (Rhodophyta) [66],[67]. The cpDNA ofUlva had the lowest DCJ value withPseudendoclonium (37 with tRNAs and 22 without tRNAs), which was expected since these organisms are more closely related than the other organisms in this analysis. Of the expanded mtDNAs (i.e. Ulvophyceae and Trebouxiophyceae),Ulva also had the lowest DCJ value withPseudendoclonium (36 with tRNAs and 17 without tRNAs). The lower DCJ values ofUlva compared toPedinomonas,Acutodesmus, andPycnococcus according toTable 8 and S7 Table are likely due to the reduced gene count in the mtDNA of these three organisms.

Phylogenomic Analyses

The phylogenetic tree resulting from Bayesian analyses of the DNA alignment, along with branch support from the other analyses (ML and BI of the DNA and AA alignments) is presented inFig 3. The ML and Bayesian trees based on an amino acid alignment are presented in the Supporting Information (S8 andS9 Figs, respectively).

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Fig 3. Bayesian majority rule tree showing all compatible partitions, inference from the nucleotide alignment of 51 concatenated chloroplast genes (first two codon positions: 28,167 nucleotide positions and 52 terminal taxa).

Node support is given as Bayesian posterior probabilities and maximum-likelihood (ML) bootstrap values of the nucleotide analyses (above branches), and the amino acid analyses (below branches); values <0.9 and 50, respectively, are not shown; asterisks indicated full support in both the Bayesian and ML analyses.

https://doi.org/10.1371/journal.pone.0121020.g003

As expected,Ulva (Ulvales) andPseudendoclonium akinetum (Ulotrichales; according to [68]) formed a clade in all analyses. The topology of the tree was in general agreement with previous chloroplast phylogenomic analyses [3],[4],[53] in recovering the Pedinophyceae and the Chlorellales as early branching lineages of the core Chlorophyta, and the Chlorophyceae and core Trebouxiophyceae as strongly supported clades within the core Chlorophyta. Similar to the analysis of Fučíková et al. [3], but in contrast to the analysis of Cocquyt et al. [5], the Ulvophyceae fell apart into several clades, including the Bryopsidales (Bryopsis,Codium,Halimeda), Dasycladales (Acetabularia), Trentepohliales (Cephaleuros,Trentepohlia), and Ulvales (Ulva,Pseudendoclonium). The relationship among these clades was not well supported. A notable difference between our tree and the phylogeny of Fučíková et al. [3] was the position ofOltmannsiellopsis (Oltmannsiellopsidales) andTetraselmis.Oltmannsiellopsis is currently classified as an ulvophyte, which was supported by chloroplast phylogenomic analyses (e.g. [4];[23]).Tetraselmis is a member of the Chlorodendrophyceae and in nuclear ribosomal DNA based phylogenies has been recovered as an early branching clade of the core Chlorophyta [2], [69]. The analyses of Fučíková et al. [3] unexpectedly supported a clade includingTetraselmis andOltmannsiellopsis, which was found branching early in the radiation of the core Chlorophyta. In contrast, our analysis placesOltmannsiellopsis andTetraselmis in the vicinity of the Ulvales-Ulotrichales. Our nucleotide based phylogenetic analysis placedTetraselmis sister to theOltmannsiellopsis-Ulvales-Ulotrichales clade (Fig 3), where as the AA based phylogeny recovered aTetraselmis-Oltmannsiellopsis clade that was sister to the Ulvales-Ulotrichales clade. It should be noted that taxon sampling in the Ulvophyceae is still poor compared to the Trebouxiophyceae and Chlorophyceae, and that gene sampling inTetraselmis is incomplete. Future studies with additional gene and taxon sampling will undoubtedly shed light on the relationships among the Ulvophyceae and the phylogenetic positions of the Oltmannsiellopsidales and Chlorodendrophyceae.

Conclusion

Organellar genomes contain valuable information that can be used for understanding the evolution of these organelles as well as phylogenomic inferences of the green algal lineages. Our investigation of the chloroplast and mitochondrial genomes ofUlva sp. has added to the limited amount of genomic data available for the Ulvophyceae. We hope that these genomes will be useful for exploring primer sets for future molecular investigations to aid in species delimitation ofUlva, especially since only limited mitochondrial data is currently available for these taxonomically troublesome algae. As more genomes are published in the near future, we will gain further insights into the evolution of the highly rearranged mitochondrial and chloroplast genomes of green algae, and hopefully answer the major phylogenetic questions that still remain in the green algae tree of life.

Supporting Information

S1 Fig.Herbarium voucher ofUlva sp. UNA00071828.

https://doi.org/10.1371/journal.pone.0121020.s001

(PDF)

S2 Fig.Neighbor-joining tree based on therbcL gene (1000 bootstrap replicates).

https://doi.org/10.1371/journal.pone.0121020.s002

(PDF)

S3 Fig.Neighbor-joining tree based on thetufA gene (1000 bootstrap replicates).

https://doi.org/10.1371/journal.pone.0121020.s003

(PDF)

S4 Fig.Genetic distances of therbcL sequences ofUlva spp.,Monostroma grevillei (GU183089) andBlidingia minima (AF387109) as outgroups.

https://doi.org/10.1371/journal.pone.0121020.s004

(PDF)

S5 Fig.Genetic distances of thetufA sequences ofUlva spp.,Monostroma sp. (HQ610262) andBlidingia minima (HQ610239) as outgroups.

https://doi.org/10.1371/journal.pone.0121020.s005

(PDF)

S6 Fig.Mauve alignments of theUlva sp. cpDNA (top of alignment) with other chlorophytes.

(A)Pseudendoclonium akinetum (NC_008114), (B)Oltmannsiellopsis viridis (NC_008099), (C)Bryopsis hypnoides (NC_013359), (D)Chlorella vulgaris (NC_001865), (E)Oedogonium cardiacum (NC_011031), (F)Acutodesmus obliquus (NC_008101), (G)Pedinomonas minor (NC_025530), (H)Pycnococcus provasolii (NC_012097), and (I)Ostreococcus tauri (NC_008289).

https://doi.org/10.1371/journal.pone.0121020.s006

(PDF)

S7 Fig.Mauve alignments ofUlva sp. mtDNA (top of aligment) with other chlorophytes.

(A)Pseudendoclonium akinetum (NC_005926), (B)Oltmannsiellopsis viridis (NC_008256), (C)Chlorella sorokiniana (NC_024626), (D)Prototheca wickerhamii (NC_001613), (E)Helicosporidium sp. (NC_017841), (F) Trebouxiophyceae sp. MX-AZ01 (NC_018568), (G)Pedinomonas minor (NC_000892), (H)Acutodesmus obliquus (NC_002254), (I)Ostreococcus tauri (NC_008290), (J)Nephroselmis olivacea (NC_008239), (K)Micomonas sp. RCC299 (NC_012643), and (L)Pycnococcus provasolii (NC_013935).

https://doi.org/10.1371/journal.pone.0121020.s007

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S8 Fig.ML tree with bootstrap values based on AA alignment of 51 chlorophyte genes.

https://doi.org/10.1371/journal.pone.0121020.s008

(PDF)

S9 Fig.Bayesian tree with posterior probabilities based on AA alignment of 51 chlorophyte genes.

https://doi.org/10.1371/journal.pone.0121020.s009

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S1 Table.GenBank accession numbers used in therbcL andtufA phylogenetic trees.

https://doi.org/10.1371/journal.pone.0121020.s010

(PDF)

S2 Table.Ulva sp. chloroplast tRNAs compared withOltmannsiellopsis viridis andPseudendoclonium akinetum tRNAs.

https://doi.org/10.1371/journal.pone.0121020.s011

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S3 Table.Ulva sp. mitochondrial tRNAs compared withOltmannsiellopsis viridis andPseudendoclonium akinetum tRNAs.

https://doi.org/10.1371/journal.pone.0121020.s012

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S4 Table.DCJ values calculated by UniMoG of chlorophyte cpDNAs without tRNAs.

https://doi.org/10.1371/journal.pone.0121020.s013

(PDF)

S5 Table.DCJ values calculated by UniMoG of chlorophyte mtDNAs without tRNAs.

https://doi.org/10.1371/journal.pone.0121020.s014

(PDF)

S6 Table.GenBank accession numbers of the taxon sampling for phylogenomics.

https://doi.org/10.1371/journal.pone.0121020.s015

(PDF)

Acknowledgments

We thank Zi-Min Hu and UA undergraduate student David Ward for technical assistance and the reviewers for providing helpful feedback.

Author Contributions

Conceived and designed the experiments: JTM FL AT JMLB. Performed the experiments: JTM FL AT. Analyzed the data: JTM FL. Contributed reagents/materials/analysis tools: JMLB. Wrote the paper: JTM FL AT JMLB.

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