Figures
Abstract
Volvocalean green algae have among the most diverse mitochondrial and plastid DNAs (mtDNAs and ptDNAs) from the eukaryotic domain. However, nearly all of the organelle genome data from this group are restricted to unicellular species, like Chlamydomonas reinhardtii, and presently only one multicellular species, the ∼4,000-celled Volvox carteri, has had its organelle DNAs sequenced. The V. carteri organelle genomes are repeat rich, and the ptDNA is the largest plastome ever sequenced. Here, we present the complete mtDNA and ptDNA of the colonial volvocalean Gonium pectorale, which is comprised of ∼16 cells and occupies a phylogenetic position closer to that of V. carteri than C. reinhardtii within the volvocine line. The mtDNA and ptDNA of G. pectorale are circular-mapping AT-rich molecules with respective lengths and coding densities of 16 and 222.6 kilobases and 73 and 44%. They share some features with the organelle DNAs of V. carteri, including palindromic repeats within the plastid compartment, but show more similarities with those of C. reinhardtii, such as a compact mtDNA architecture and relatively low organelle DNA intron contents. Overall, the G. pectorale organelle genomes raise several interesting questions about the origin of linear mitochondrial chromosomes within the Volvocales and the relationship between multicellularity and organelle genome expansion.
Citation: Hamaji T, Smith DR, Noguchi H, Toyoda A, Suzuki M, Kawai-Toyooka H, et al. (2013) Mitochondrial and Plastid Genomes of the Colonial Green Alga Gonium pectorale Give Insights into the Origins of Organelle DNA Architecture within the Volvocales. PLoS ONE 8(2): e57177. https://doi.org/10.1371/journal.pone.0057177
Editor: Heroen Verbruggen, University of Melbourne, Australia
Received: October 9, 2012; Accepted: January 18, 2013; Published: February 26, 2013
Copyright: © 2013 Hamaji et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Grants-in-Aid for Scientific Research on Innovative Areas “Genome Science” (number 221S0002), JSPS Fellows (number 23-5499 to TH), Challenging Exploratory Research (number 24657045 to HNZ) and Scientific Research (A) (number 24247042 to HNZ) from MEXT/JSPS KAKENHI (http://www.jsps.go.jp/english/e-grants/index.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Some of the most diverse and bizarre organelle genomes of all eukaryotes come from the Volvocales, which is a large order of predominantly freshwater green algae, belonging to chlorophycean class of the Chlorophyta. Volvocalean mitochondrial and plastid DNAs (mtDNAs and ptDNAs) show an impressive array of architectures, nucleotide landscapes, and coding compositions (Table 1)– and see Leliaert et al. [1] and Lee and Hua [2] for additional compilations. Moreover, certain volvocalean species, particularly those within the “Reinhardtinia clade” sensu Nakada et al. [3], have proven to be excellent systems for testing contemporary hypotheses on the evolution of organelle genome expansion and linearization [4], [5], [6].
Most of our understanding of volvocalean mitochondrial and plastid genomes is limited to unicellular species, such as the model organisms Chlamydomonas reinhardtii and C. globosa (previously misidentified as C. incerta; see Nakada et al. [7]) [5], [8], the colorless and wall-less Polytomella capuana, P. parva, and P. piriformis [6], [9], [10], and the halotolerant β-carotene-rich Dunaliella salina [11]. Surprisingly little is known about the organelle genomes of colonial and multicellular volvocaleans, which are found within the volvocine lineage of the Reinhardtinia clade (Figure S1). Volvocine algae are preeminent models for studying the evolution of multicellularity [12], [13], and span the gamut of cellular complexity, from simple 4-celled species (e.g., Tetrabaena), to 8-64-celled colonial forms (e.g., Gonium), all the way to highly complex spheroidal taxa, with more than 500 cells (e.g., Volvox) [14], [15]. It is estimated that multicellular volvocine species last shared a common unicellular ancestor ∼200 million years ago [15].
Of the 8 different volvocalean algae for which complete mtDNA and/or ptDNA sequences are available [11], only one is multicellular: Volvox carteri, which is comprised of ∼4,000 cells. The organelle genomes of this species are distended with repetitive noncoding DNA, and similar palindromic repeats are located in both the mitochondrial and plastid compartments [16]. Moreover, the V. carteri ptDNA, at ∼525 kb, is among the largest plastomes ever observed (from any eukaryote) [4], dwarfing that of C. reinhardtii, which is 204 kb [17]. Although smaller than its plastid counterpart, the ∼35 kb mtDNA of V. carteri is still larger than any the other completely sequenced volvocalean mitochondrial genome. It is hypothesized that the expanded organelle genomes of V. carteri are a consequence of a low organelle mutation rate and/or a small effective population size [4].
The V. carteri mtDNA assembles as a circular molecule, contrasting the linear (or linear fragmented) architectures of all other well-studied Reinhardtinia-clade mitochondrial genomes, including those of C. reinhardtii, Polytomella spp., and the multicellular Pandorina morum [5], [11], [18]. These linear mtDNAs have evolved complex terminal structures [5], [10], called mitochondrial telomeres, which form long palindromic repeats at the genome ends. The origin and number of times that linear mitochondrial chromosomes have evolved within the Reinhardtinia is unknown, but it has been argued that they arose only once [4]. If true, this would imply that in a recent ancestor of V. carteri, the mtDNA reverted from a linear to a circular form.
To learn about organelle genome architecture within multicellular volvocine algae and to gain insight into ptDNA expansion and the origin of linear mtDNAs, we sequenced the mitochondrial and plastid genomes of Gonium pectorale–an 8- or 16-celled freshwater colonial alga, occupying a phylogenetic position closer to that of V. carteri than C. reinhardtii within the volvocine line [14], [15], [19] (Figure S1).
Materials and Methods
The organelle genomes described here come from Gonium pectorale K3-F3-4 (mating type minus), which was one of the F3 backcross strains to K41 (mating type plus) (originating from K41×K32 [F1 strains of Kaneko3×Kaneko4]) [20], [21] and is available as NIES-2863 from the Microbial Culture Collection at National Institute for Environmental Studies, Tsukuba, Japan (http://mcc.nies.go.jp/). G. pectorale was grown in 200–300 mL VTAC medium [22], [23] at 20°C on a 14∶10 h light-dark cycle, under cool-white fluorescent lamps (165–175 µmol m−2 s−1 intensity). Total DNA was extracted based on the protocol of Miller et al. [24].
Sequencing libraries were prepared from G. pectorale K3-F3-4 genomic DNA using the GS FLX Titanium Rapid Library Preparation Kit (F. Hoffmann-La Roche, Basel, Switzerland) and the TruSeq DNA Sample Prep Kit (Illumina Inc., San Diego, CA, USA), and were run on a GS FLX (F. Hoffmann-La Roche) and a MiSeq sequencer (Illumina Inc.), respectively. The GS FLX reads were assembled with Newbler v2.6. A fosmid library (23,424 clones) was constructed from G. pectorale K3-F3-4 genomic DNA using fosmid vector pKS300, which was developed in-house. End sequencing of the fosmid library and the BAC library of G. pectorale Kaneko3 (18,048 clones, Genome Institute (CUGI), Clemson Univ., Clemson, SC, USA) was carried out using a BigDye terminator kit ver3 (Life Technologies, Carlsbad, California, USA) and was run on automated ABI 3730 capillary sequencers (Life Technologies). The GS FLX contig sequences, which were derived from mitochondrial and chloroplast genomes, and the BAC/fosmid end-sequences were assembled using the Phrap/Consed systems. Gap closing and re-sequencing of low-quality regions in the assembly were performed by shotgun sequencing of the corresponding BAC/fosmid clones, PCR, primer walking, and direct sequencing of fosmid clones. The MiSeq sequence reads were mapped against the assembly sequences using the BWA program [25] after passing through the quality filter. The errors on each GS FLX assembly sequence were also corrected. The assembling delineated one circular mtDNA and two ptDNA isoforms (A and B), a common feature of plastid genomes with inverted repeats [26], [27] (Figure S2). The annotated G. pectorale mtDNA and ptDNA (isoform A) sequences are deposited in the DDBJ database under accession numbers AP012493 and AP012494, respectively.
Phylogenetic analyses were performed under maximum likelihood (ML) using RAxML [28] and PhyML 3.0 [29] with 100 bootstrap replicates. Maximum parsimony (MP) bootstrap analyses (based on 10 random replications of the full heuristic search with the tree bisection–reconnection branch-swapping algorithm) were performed in PAUP 4.0b10 [30] with 1,000 replications. MtDNA protein phylogeny was based on the deduced nad5, cox1, and cob amino acid sequences (Table S1), which were aligned using Clustal X [31]. Intron phylogenies were based on the deduced and aligned amino acid sequences of the nad5 and psaB intronic open reading frames (ORFs), which gave data matrices of 205 and 256 amino acids with 9 and 14 operational taxonomic units (OTUs), respectively (Tables S2, S3). Intron secondary structure maps were constructed as previously described [32].
Results and Discussion
The Gonium pectorale mtDNA: A Compact Circular Mapping Chromosome
The mitochondrial genome of G. pectorale has a conservative architecture: it is small (16 kb), circular-mapping, AT rich (61%), compact (73% coding), contains very few repeats, and has only a single intron (Figure 1, Table 1, Figure S3). It lacks the eccentricities that often characterize the mtDNAs of other volvocalean species, such as a high GC content (e.g., P. capuana), a linear or linear-fragmented conformation (e.g., P. parva), a large intron density (e.g., D. salina), non-standard genes (e.g., C. reinhardtii), and/or a bloated repeat-rich structure (e.g., V. carteri) [11]. The G. pectorale mtDNA is gene poor, encoding 7 proteins, 2 rRNAs, and 3 unique tRNAs, representing methionine, glutamine, and tryptophan (Figure 1). Two copies of trnM were identified adjacent to one another in the genome. Both have similar sequences and cloverleaf structures, and appear to have a role in elongation rather than initiation, as suggested for the trnM of other volvocalean algae. When ignoring non-standard genes and duplicate tRNAs, the G. pectorale mitochondrial gene repertoire mirrors those from all other available volvocalean algae, with the exception of Polytomella species, which lack trnW and trnQ. The G. pectorale mitochondrial large and small subunit (LSU and SSU) rRNA genes, like those from other available Reinhardtinia algae, are fragmented and scrambled throughout the genome into 8 and 4 coding modules, respectively. In V. carteri the eighth LSU module has been invaded by palindromic repeats, splitting it into two segments (L8a and L8b) [16]; in G. pectorale, however, the L8 module is intact.
Note, the G. pectorale mtDNA is a circular-mapping molecule. Transfer RNA-coding regions are designated by the single-letter abbreviation of the amino acid they specify.
The sole intron of the G. pectorale mtDNA, located in nad5, is of group ID affiliation [33] (Figure S3) and encodes a putative intronic endonuclease. Other volvocaleans contain a nad5 group I intron (with the same insertion site), but none are from the Reinhardtinia clade. Our phylogenetic analyses of various volvocalean intronic ORFs (Figure S4) suggest that the G. pectorale nad5 intron either was acquired through horizontal transmission from a volvocalean closely related to Chlamydomonas moewusii or Chlorogonium elongatum or that it was present in the ancestor of the Volvocales and preserved in G. pectorale.
Linear mitochondrial chromosomes are widespread throughout the Reinhardtinia clade, occurring in all explored taxa [34], with the exception of V. carteri, which has a circular mtDNA map, but rare possible linear forms of the genome have been observed [4], [16] (Table 1). Our de novo and mapping assemblies of the G. pectorale mtDNA gave an unambiguous circular-mapping chromosome (see Materials and Methods), and although such a map could represent a circularly permuted, linear-type structure, various features of the G. pectorale mitochondrial genome support the idea that it is circular. For instance, all twelve of the G. pectorale mtDNA genes have the same transcriptional polarity–a trait that is also found in V. carteri and available volvocalean species with circular mitochondrial genomes. Conversely, in all of the sequenced linear mtDNAs from the Volvocales, the genes are divided into two transcriptional polarities, proceeding outward towards the ends of the chromosome [6].Furthermore, our Southern blot analysis of the G. pectorale mtDNA, cut with restriction enzymes, demonstrates that it is a circular molecule (Figure S5).
Our evidence for a circular mitochondrial genome in G. pectorale raises interesting questions about the origin of linear mtDNAs within the Reinhardtinia clade. There is little doubt that the ancestral volvocalean mtDNA was circular, and it is argued that there was a single shift from a circular to a linear mtDNA structure in the ancestor that gave rise to Reinhardtinia algae [6]. Within the Reinhardtinia clade, V. carteri and G. pectorale belong to a monophyletic colonial or multicellular volvocalean group from which unicellular members are separated [14], [15] (Figure 2), but the multicellular volvocalean Pandorina morum has a linear mtDNA [18]. Moreover, V. carteri and P. morum belong to the monophyletic Volvocaceae from which G. pectorale is excluded [14], [15], [19] (Figure S1). Thus, the appearance of circular mitochondrial genome maps in both V. carteri and G. pectorale suggests that the mtDNAs of these species independently reverted from a linear to a circular conformation in the two separate ancestors of G. pectorale and V. carteri (Figure S1) or alternatively that there were multiple origins of linear mitochondrial genomes in the Reinhardtinia clade, in the ancestors of Polytomella, C. reinhardtii, and P. morum (Figure S1). Studies of mtDNA structure from other volvocine species, such as Tetrabaena and Yamagishiella, are needed to further investigate these hypotheses.
The tree was constructed under the RAxML (with WAG+I+4G model) method using the concatenated sequences of the deduced nad5, cox1, and cob amino acid sequences. Left, middle, and right bootstrap values (≥50%) obtained using the RAxML, PhyML (with LG+I+4G model), and MP analysis, respectively. The amino acid sequences of the three proteins were aligned by Clustal X [29], and ambiguously aligned and highly variable regions were removed to construct a multiprotein data matrix of 909 amino acids from the 10 operational taxonomic units (Table S1).
The Gonium pectorale ptDNA Shows Moderate Genome Expansion
Volvocalean plastid genomes are big and that of G. pectorale, at 222.6 kb, is no exception. Of the approximately 300 complete (or almost complete) ptDNAs in GenBank, as of 1 August 2012, fewer than ten have a length >200 kb, all but one of which are from chlorophyte green algae, including the volvocaleans C. reinhardtii (204 kb), D. salina (269 kb) and V. carteri (∼525 kb) [4], [11], [17]. The large size of volvocalean ptDNAs is not a product of an inflated gene number, but a consequence of having an abundance of noncoding nucleotides, often represented by repetitive elements and introns. This is also true for the G. pectorale ptDNA, which is 56% (∼125 kb) noncoding. Almost all of these noncoding nucleotides are AT rich (average = 71%) and found in intergenic regions.
The coding regions also have a high AT content (68%) and encompass a total of 98 unique genes, encoding 67 proteins, 3 rRNAs, 27 tRNAs, and a single misc RNA (tscA) (Figure 3). Six of these genes (psbA, rrnL, rrnS, rrnF, trnA, and trnI) are duplicated, being located in a pair of 14.8 kb inverted repeats, which divide the G. pectorale ptDNA into a large (99.6 kb) and a small (93.5 kb) single-copy region (Figure 3). This gene complement and inverted-repeat arrangement is almost identical to those of C. reinhardtii and V. carteri (Figure 3, Figure S6).
Note, the G. pectorale ptDNA is a circular-mapping molecule. Transfer RNA-coding regions are designated by the single-letter abbreviation of the amino acid they specify.
Although some volvocalean algae harbour many ptDNA introns (Table 1)–V. carteri has 9 and D. salina has >35–G. pectorale harbours just three: one located in psaB, which appears to be of group IA affiliation [33] (Figure S3), and encodes a putative endonuclease-like protein, and two short group II introns (117 and 176 bp) found upstream of psaA exons 2 and 3 (Figure 3). Phylogenetic analysis of the G. pectorale intron (Figure S7) show that it is closely related to the psaB group I intron of the chlorophycean (but non-volvocalean) green alga Stigeoclonium helveticum [35]; moreover, both introns have the same insertion site within the psaB gene. V. carteri also has a psaB intron, but it is of group II affiliation [4]. In fact, there is not a single homologous pair of either group I or group II introns among the G. pectorale, V. carteri, and C. reinhardtii plastid genomes (Figure S6), suggesting that rapid horizontal intron transfer and loss occurred within the colonial Volvocales.
The G. pectorale plastid genome, like its V. carteri and C. reinhardtii counterparts, contains hundreds of short repetitive elements, distributed throughout the intergenic regions, as demonstrated by the dotplot similarity matrix (Figure S8). Many of the V. carteri ptDNA repeats are palindromes, and can be folded into hairpin structures [16]. The same is true for the G. pectorale ptDNA, which contains ∼135 short (13 nt) palindromic repeats (including eight in the coding regions) with the motif: 5′- TCCCCNNNGGGGA-3′ (Figure S9). This is fewer repeats than found in the V. carteri ptDNA, which contains over a thousand palindromic elements.
The G. pectorale ptDNA is slightly more expanded (by ∼19 kb) than that of C. reinhardtii, but much smaller than those of the unicellular D. salina (269 kb, ∼65% noncoding) and the multicellular V. carteri (∼525 kb, >80% noncoding) (Table 1). What has led to such a wide spectrum of ptDNA expansion within the Volvocales? One contemporary–and controversial [36], [37]–hypothesis for the evolution of genome size, called the mutational hazard hypothesis [38], argues that genome expansion is a product of a low effective population size (Ne) (which results in increased random genetic drift) and/or a low mutation rate (μ), which reduces the burden of harbouring excess DNA. The V. carteri ptDNA is estimated to have a very low Neμ [4], about twenty times lower than that of the C. reinhardtii ptDNA [39], which may explain why it is so bloated. We do not know the value of Neμ for the G. pectorale ptDNA–this will require sequencing the plastid genomes of several additional G. pectorale isolates. However, given that this species is ∼10 times larger than C. reinhardtii (16 cells vs a single cell) and a hundred times smaller than V. carteri (16 cells vs 4,000 cells), and that all three of these algae are found in a similar environment (freshwater ponds)–unlike D. salina, which is marine–one might expect the effective population size of G. pectorale to be similar or marginally smaller than that of C. reinhardtii, and much larger than that of V. carteri. If true, this may have contributed to G. pectorale having a ptDNA architecture comparable to that of C. reinhardtii but much different than that of V. carteri. Under this hypothesis, it can therefore be predicted that as more volvocine organelle DNAs are sequenced, species with large cell numbers and presumably low effective population sizes will have more bloated genomes than those with small cell numbers and large effective population sizes.
Supporting Information
Figure S1.
Simplified diagram for phylogenetic relationships of selected taxa of the unicellular, colonial and multicellular vovlocaleans.
https://doi.org/10.1371/journal.pone.0057177.s001
(TIF)
Figure S2.
Diagrams of possible isoforms of ptDNA of Gonium pectorale. A. Two isoforms as found in other ptDNAs with a typical inverted repeat. B. Two additional isoforms that were not rejected based on assembling of our sequence data.
https://doi.org/10.1371/journal.pone.0057177.s002
(TIF)
Figure S3.
Secondary structures of group I introns within the Gonium pectorale organelle DNAs. A. Mitochondrial nad5 group ID intron. B. Chloroplast psaB group IA intron.
https://doi.org/10.1371/journal.pone.0057177.s003
(TIF)
Figure S4.
Phylogeny of Gonium pactorale nad5 group I intronic ORF. The tree was constructed under the RAxML (with WAG+4G model) method using 8 additional, related amino acid sequences selected based on the topology of the distance tree provided by blastp research of NCBI (http://www.ncbi.nlm.nih.gov/). Numbers on the left, middle and right at branches represent bootstrap values (≥50%) obtained using the RAxML, PhyML (with LG+4G model), and MP analysis, respectively. The amino acid sequences were aligned by Clustal X, and ambiguously aligned and highly variable regions were removed to construct a data matrix of 205 amino acids from the 9 operational taxonomic units (Table S2).
https://doi.org/10.1371/journal.pone.0057177.s004
(TIF)
Figure S5.
Southern blot analysis of Gonium pectorale mtDNA with four restriction enzymes that cut the genome once (SacI and StuI) or twice (SacII and EcoRI). Genome map coordinates are based on the G. pectorale mtDNA DDBJ accession (AP012493). SacI and StuI digestions each gave single genome-sized bands (∼16 kb), and the SacII and EcoRI reactions each gave two bands. These data are consistent with the G. pectorale mtDNA being a circular molecules. Probe DNA was amplified by PCR with two specific primers (Gopec-mito-F 5′-CGGGCAAAGCATAATTAGTGTAG-3′ and Gopec-mito-R 5′-ACGAACAAGAGGAAGACCTAAC-3′).
https://doi.org/10.1371/journal.pone.0057177.s005
(TIF)
Figure S6.
Venn diagram comparing the gene repertoires of three volvocalean chloroplast genomes (AP012494, GU084820 and FJ423446). 102 genes (single asterisk) shared by the three genomes include 12 genes distributed in IRA and IRB and trnI (cau), which was previously annotated as one of the triplicated trnM in C. reinhardtii and V. carteri. Double asterisks represent one of the duplicated genes in G. pectorale and C. reinhardtii. Triple asterisks exhibit one of the duplicated genes in C. reinhardtii. Note that all intronic ORFs in G. pectprale (1#) and V. carteri (6#) are unique for each genome and considered “non-coding” in the text.
https://doi.org/10.1371/journal.pone.0057177.s006
(PDF)
Figure S7.
Phylogeny of Gonium pactorale psaB group I intronic ORF. The tree was constructed under the RAxML (with WAG+4G model) method using 13 additional, related amino acid sequences selected based on the topology of the distance tree provided by blastp research of NCBI (http://www.ncbi.nlm.nih.gov/). Numbers on the left, middle and right at branches represent bootstrap values (≥50%) obtained using the RAxML, PhyML (with LG+G model), and MP analysis, respectively. The amino acid sequences were aligned by Clustal X, and ambiguously aligned and highly variable regions were removed to construct a data matrix of 256 amino acids from the 14 operational taxonomic units (Table S3).
https://doi.org/10.1371/journal.pone.0057177.s007
(TIF)
Figure S8.
Dotplot similarity matrix of the Gonium pectorale plastid genome. The X- and Y-axes each represent the G. pectorale plastid genome (222.6 kb). Dots in the nucleotide similarity matrix represent regions of sequence similarity. The matrix was generated using JDotter, with a sliding-window size of 50. The inverted repeats are highlighted in red in the matrix.
https://doi.org/10.1371/journal.pone.0057177.s008
(TIF)
Figure S9.
Distribution of short (13 nt) palindromic repeats (including seven [red arrows] in five coding regions [blues arrows]) with the motif: 5′- TCCCCNNNGGGGA-3′ in ptDNA of Gonium pectorale. The repeats were examined by using Serial Cloner 2.5 (http://serialbasics.free.fr/Serial_Cloner.html).
https://doi.org/10.1371/journal.pone.0057177.s009
(JPG)
Table S1.
Amino acid alignment and origin of the data used for Figure 2.
https://doi.org/10.1371/journal.pone.0057177.s010
(DOC)
Table S2.
Amino acid alignment and origin of the data used for Figure S4.
https://doi.org/10.1371/journal.pone.0057177.s011
(DOC)
Table S3.
Amino acid alignment and origin of the data used for Figure S7.
https://doi.org/10.1371/journal.pone.0057177.s012
(DOC)
Acknowledgments
We thank all the technical staff of the Comparative Genomics lab at National Institute of Genetics for their assistance.
Author Contributions
Conceived and designed the experiments: HT DS H. Noguchi AT AF H. Nozaki. Performed the experiments: TH AT MS HKT AF IN TM BO H. Nozaki. Analyzed the data: TH DS H. Noguchi AT AF H. Nozaki. Contributed reagents/materials/analysis tools: TH IN TM BO H. Nozaki. Wrote the paper: TH DS H. Noguchi AT AF H. Nozaki.
References
- 1. Leliaert F, Smith DR, Moreau H, Herron MD, Verbruggen H, et al. (2012) Phylogeny and molecular evolution of the green algae. CRC Cr Rev Plant Sci 31: 1–46.
- 2.
Lee RW, Hua J (2012) Mitochondrial genome structure of green, red, and glaucophyte algae. In: Gray MW editor. Encyclopedia of molecular life sciences (EMLS). Springer. Vol. 1, Section 4, Subsection 6a: Mitochondrial Genomes (in press).
- 3. Nakada T, Misawa K, Nozaki H (2008) Molecular systematics of Volvocales (Chlorophyceae, Chlorophyta) based on exhaustive 18S rRNA phylogenetic analyses. Mol Phylogenet Evol 48: 281–291.
- 4. Smith DR, Lee RW (2010) Low nucleotide diversity for the expanded organelle and nuclear genomes of Volvox carteri supports the mutational-hazard hypothesis. Mol Biol Evol 27: 2244–2256.
- 5. Michaelis G, Vahrenholz C, Pratje E (1990) Mitochondrial DNA of Chlamydomonas reinhardtii: the gene for apocytochrome b and the complete functional map of the 15.8 kb DNA. Mol Gen Genet 223: 211–216.
- 6. Smith DR, Hua J, Lee RW (2010) Evolution of linear mitochondrial DNA in three known lineages of Polytomella. Curr Genet 56: 427–438.
- 7. Nakada T, Shinkawa H, Ito T, Tomita M (2010) Recharacterization of Chlamydomonas reinhardtii and its relatives with new isolates from Japan. J Plant Res 123: 67–78.
- 8. Popescu CE, Lee RW (2007) Mitochondrial genome sequence evolution in Chlamydomonas. Genetics 175: 819–826.
- 9. Fan J, Lee RW (2002) Mitochondrial genome of the colorless green alga Polytomella parva: two linear DNA molecules with homologous inverted repeat termini. Mol Biol Evol 19: 999–1007.
- 10. Smith DR, Lee RW (2008) Mitochondrial genome of the colorless green alga Polytomella capuana: a linear molecule with an unprecedented GC content. Mol Biol Evol 25: 487–496.
- 11. Smith DR, Lee RW, Cushman JC, Magnuson JK, Tran D, et al. (2010) The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA. BMC Plant Biol 10: 83.
- 12. Kirk DL (2005) A twelve-step program for evolving multicellularity and a division of labor. BioEssays 27: 299–331.
- 13. Sachs JL (2008) Resolving the first steps to multicellularity. Trends Ecol Evol 23: 245–248.
- 14. Nozaki H (2003) Origin and evolution of the genera Pleodorina and Volvox (Volvocales). Biologia 58(4): 425–431.
- 15. Herron MD, Hackett JD, Aylward FO, Michod RE (2009) Triassic origin and early radiation of multicellular volvocine algae. Proc Natl Acad Sci USA 106: 3254–3258.
- 16. Smith DR, Lee RW (2009) The mitochondrial and plastid genomes of Volvox carteri: bloated molecules rich in repetitive DNA. BMC Genomics 10: 132.
- 17. Maul JE, Lilly JW, Cui L, dePamphilis CW, Miller W, et al. (2002) The Chlamydomonas reinhardtii plastid chromosome: islands of genes in a sea of repeats. Plant Cell 11: 2659–2679.
- 18. Moore LJ, Coleman AW (1989) The linear 20 kb mitochondrial genome of Pandorina morum (Volvocaceae, Chlorophyta). Plant Mol Biol 13: 459–465.
- 19. Nozaki H, Misawa K, Kajita T, Kato M, Nohara S, et al. (2000) Origin and evolution of the colonial Volvocales (Chlorophyceae) as inferred from multiple, chloroplast gene sequences. Mol Phylogenet Evol 17: 256–268.
- 20. Hamaji T, Ferris PJ, Coleman AW, Waffenschmidt S, Takahashi F, et al. (2008) Identification of the minus-dominance gene ortholog in the mating-type locus of Gonium pectorale. Genetics 178: 283–294.
- 21. Mogi Y, Hamaji T, Suzuki M, Ferris P, Mori T, et al. (2012) Evidence for tubular mating structures induced in each mating type of heterothallic Gonium pectorale (Volvocales, Chlorophyta). J Phycol 48: 670–674.
- 22. Nozaki H, Kuroiwa H, Mita T, Kuroiwa T (1989) Pleodorina japonica sp. nov. (Volvocales, Chlorophyta) with bacteria-like endosymbionts. Phycologia 28: 252–267.
- 23.
Kasai F, Kawachi M, Erata M, Mori F, Yumoto K, et al.. (eds.) (2009) NIES-Collection. List of Strains. 8th Edition. Jpn J Phycol 57 (1), Supplement: 1–350, plates 1–7.
- 24. Miller SM, Schmitt R, Kirk DL (1993) Jordan, an active Volvox transposable element similar to higher plant transposons. Plant Cell 5: 1125–1138.
- 25. Li H, Durbin R (2010) Fast and accurate long-read alignment with Burrows-Wheeler Transform. Bioinformatics 26: 589–595.
- 26. Stein DB, Palmer JD, Thompson WF (1986) Structural evolution and flip-flop recombination of chloroplast DNA in the fern genus Osmunda. Curr Genet10: 835–841.
- 27.
Harris EH (1989) The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. Academic Press Inc., San Diego, California, xiv+780 pp.
- 28. Stamatakis A, Hoover P, Rougemont J (2008) A rapid bootstrap algorithm for the RAxML web-servers. Syst Biol 75: 758–771.
- 29. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, et al. (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59: 307–210.
- 30.
Swofford DL (2002) PAUP*: Phylogenetic Analysis Using Parsimony (* and Other Methods). Version 4.0b10. Sinauer, Sunderland, Massachusetts.
- 31. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876–4882.
- 32. Nozaki H, Takahara M, Nakazawa A, Kita Y, Yamada T, et al. (2002) Evolution of rbcL group IA introns and intron open reading frames within the colonial Volvocales (Chlorophyceae). Mol Phylogenet Evol 23: 326–338.
- 33.
Michel F, Westhof E (1990) Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis. J Mol Biol 216, 585–610.
- 34. Laflamme M, Lee RW (2003) Mitochondrial genome conformation among CW-group chlorophycean algae. J Phycol 39: 213–220.
- 35. Bélanger AS, Brouard JS, Charlebois P, Otis C, Lemieux C, et al. (2006) Distinctive architecture of the chloroplast genome in the chlorophycean green alga Stigeoclonium helveticum. Mol. Genet Genom 276: 464–477.
- 36. Daubin V, Moran NA (2004) Comment on the evolution of genome complexity. Science 306: 978.
- 37. Sloan DB, Alverson AJ, Chuckalovcak JP, Wu M, McCauley DE, et al. (2012) Rapid evolution of enormous, multichromosomal genomes in flowering plant mitochondria with exceptionally high mutation rates. PLoS Biol 10: e1001241.
- 38.
Lynch M (2007) The Origins of Genome Architecture. Massachusetts: Sinauer Associates, Inc.
- 39. Smith DR, Lee RW (2009) Nucleotide diversity of the Chlamydomonas reinhardtii plastid genome: addressing the mutational-hazard hypothesis. BMC Evol Biol 9: 120.