Accepted Manuscript A recurring syndrome of accelerated plastid genome evolution in the angiosperm tribe Sileneae (Caryophyllaceae) Daniel B. Sloan, Deborah A. Triant, Nicole J. Forrester, Laura M. Bergner, Martin Wu, Douglas R. Taylor PII: DOI: Reference:

S1055-7903(13)00439-9 http://dx.doi.org/10.1016/j.ympev.2013.12.004 YMPEV 4771

To appear in:

Molecular Phylogenetics and Evolution

Received Date: Revised Date: Accepted Date:

7 September 2013 5 December 2013 17 December 2013

Please cite this article as: Sloan, D.B., Triant, D.A., Forrester, N.J., Bergner, L.M., Wu, M., Taylor, D.R., A recurring syndrome of accelerated plastid genome evolution in the angiosperm tribe Sileneae (Caryophyllaceae), Molecular Phylogenetics and Evolution (2013), doi: http://dx.doi.org/10.1016/j.ympev.2013.12.004

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A recurring syndrome of accelerated plastid genome evolution in the angiosperm tribe Sileneae (Caryophyllaceae)

Daniel B. Sloana*, Deborah A. Triantb, Nicole J. Forresterb, Laura M. Bergnerb, Martin Wub, Douglas R. Taylorb

a

Department of Biology, Colorado State University, Fort Collins, CO, 80523, United States

b

Department of Biology, University of Virginia, Charlottesville, VA, 22904, United States

*Corresponding Author: [email protected] 970.491.2256

Keywords: chloroplast genome, intron loss, inversions, mutation rate, positive selection

1

ABSTRACT

2

In flowering plants, plastid genomes are generally conserved, exhibiting slower rates of sequence

3

evolution than the nucleus and little or no change in structural organization. However,

4

accelerated plastid genome evolution has occurred in scattered angiosperm lineages. For

5

example, some species within the genus Silene have experienced a suite of recent changes to

6

their plastid genomes, including inversions, shifts in inverted repeat boundaries, large indels,

7

intron losses, and rapid rates of amino acid sequence evolution in a subset of protein genes, with

8

the most extreme divergence occurring in the protease gene clpP. To investigate the relationship

9

between the rates of sequence and structural evolution, we sequenced complete plastid genomes

10

from three species (Silene conoidea, S. paradoxa, and Lychnis chalcedonica), representing

11

independent lineages within the tribe Sileneae that were previously shown to have accelerated

12

rates of clpP evolution. We found a high degree of parallel evolution. Elevated rates of amino

13

acid substitution have occurred repeatedly in the same subset of plastid genes and have been

14

accompanied by a recurring pattern of structural change, including cases of identical inversions

15

and intron loss. This “syndrome” of changes was not observed in the closely related outgroup

16

Agrostemma githago or in the more slowly evolving Silene species that were sequenced

17

previously. Although no single mechanism has yet been identified to explain the correlated suite

18

of changes in plastid genome sequence and structure that has occurred repeatedly in angiosperm

19

evolution, we discuss a possible mixture of adaptive and non-adaptive forces that may be

20

responsible.

21

1. INTRODUCTION

22

One of the fundamental challenges in molecular evolution is to understand the relationship

23

between the rate of nucleotide substitution and the rate of evolution in genome structure.

24

Although these rates are often correlated with each other, the mechanisms underlying the

25

correlation remain incompletely understood (Hardison et al., 2003; Shao et al., 2003; Xu et al.,

26

2006; Tian et al., 2008; Zhu et al., 2009). The history of plastid genome evolution across the

27

flowering plants offers one example of an association between substitution rates and structural

28

rearrangements (Jansen et al., 2007). In most angiosperm lineages, plastid gene order is identical,

29

suggesting that the structural organization of the genome has been stable for more than 150 Myr

30

(Raubeson and Jansen, 2005; Bell et al., 2010). However, scattered angiosperm lineages harbor

31

extensively rearranged plastid genomes (Jansen et al., 2007). Although rates of nucleotide

32

substitution are generally slow in angiosperm plastids (approximately three-fold lower than in

33

the nucleus; Wolfe et al., 1987; Drouin et al., 2008), species with rearranged plastid genomes

34

also experience frequent nucleotide substitutions (Jansen et al., 2007; Guisinger et al., 2008).

35

Sequencing of complete mitochondrial and plastid genomes from four closely related

36

species in the genus Silene (Caryophyllaceae) has provided examples of recent and dramatic

37

changes in rates of organelle genome evolution (Sloan et al., 2012a; Sloan et al., 2012b). Two of

38

these species (S. conica and S. noctiflora) have experienced increased rates of sequence and

39

structural evolution in both organelles. The plastid genomes in these species reveal a history of

40

inversions, intron losses, shifts in inverted repeat (IR) boundaries, and large insertions and

41

deletions (indels). They have also experienced accelerated rates of nucleotide substitution in a

42

subset of protein-coding genes, particularly at nonsynonymous sites, resulting in rapid evolution

43

of amino acid sequences. The most dramatic acceleration involves the clpP gene, which is almost

3

44

unrecognizable at the nucleotide sequence level in S. conica and S. noctiflora (Erixon and

45

Oxelman, 2008b; Sloan et al., 2012b). This gene codes for the ATP-dependent proteolytic

46

subunit of the caseinolytic peptidase and is involved in protein metabolism within the plastid. A

47

number of other plastid genes with non-photosynthetic functions are also very divergent in these

48

two species, but genes encoding the core photosynthetic complexes remain highly conserved

49

(Sloan et al., 2012b). In contrast to S. conica and S. noctiflora, two other species (S. latifolia and

50

S. vulgaris) have maintained slowly evolving plastid genomes that are largely indistinguishable

51

from the ancestral structural organization found in most angiosperms. All four of these Silene

52

species are estimated to have diverged from each other in the last 5-10 Myr and are members of

53

subgenus Behenantha (Mower et al., 2007; Frajman et al., 2009; Sloan et al., 2009). The

54

phylogenetic relationships among the major lineages within this subgenus have proven difficult

55

to resolve, so it is unclear whether the similar patterns of organelle genome evolution in S.

56

conica and S. noctiflora are the result of independent evolutionary events or shared ancestry

57

(Erixon and Oxelman, 2008a; Sloan et al., 2009; Rautenberg et al., 2012; Sloan et al., 2012b).

58

Broader sampling of clpP evolution in the tribe Sileneae has shown that this gene has

59

been subject to independent accelerations within Silene and in the genus Lychnis (Erixon and

60

Oxelman, 2008b), but very little is known about plastid evolution at the whole-genome level in

61

these cases. Here, we report the sequencing of complete plastid genomes from three species,

62

representing three independent clades with highly divergent clpP sequences: Lychnis (L.

63

chalcedonica), Silene subgenus Behenantha (S. conoidea), and Silene subgenus Silene (S.

64

paradoxa; note that, although clpP sequence was not previously available from S. paradoxa, it

65

was expected to have an accelerated evolutionary rate for this gene based on the extreme

66

divergence that was documented in the closely related species S. fruticosa; Erixon and Oxelman,

4

67

2008b; Sloan et al., 2009). We also sequenced the complete plastid genome from the outgroup

68

Agrostemma githago, which is the sister lineage to the rest of the Sileneae and appears to have

69

maintained the slow, ancestral rate of clpP sequence evolution (Erixon and Oxelman, 2008a).

70

These genomes reveal remarkable parallelism in a suite of changes in sequence and structural

71

evolution that has occurred repeatedly in independent Sileneae lineages.

72 73

2. MATERIALS AND METHODS

74

2.1 Plastid DNA Extraction and Sequencing

75

Seeds from a single maternal plant for each of four target species (Table 1) were germinated and

76

grown in the greenhouse with supplemental lighting on a 16-hr/8-hr light-dark cycle and regular

77

watering and fertilization. For each species, 200 g of leaf tissue was harvested from multiple

78

maternal siblings and disrupted and filtered using standard protocols. Chloroplasts were then

79

isolated by differential centrifugation followed by separation on discontinuous sucrose gradients

80

(Palmer, 1986; Jansen et al., 2005). Isolated chloroplasts were lysed and plastid DNA was

81

purified by phenol:chloroform extraction. DNA samples were used for construction of paired-

82

end sequencing libraries with the NEBNext DNA Sample Prep Reagent set 1 (New England

83

Biolabs) and Illumina TruSeq adapters. To generate sufficient material for library construction,

84

the A. githago DNA sample was amplified with GenomiPhi V2 (GE Healthcare). Libraries were

85

sequenced as part of a multiplexed 2×101 bp lane on an Illumina HiSeq2000 at the University of

86

Minnesota’s Biomedical Genomics Center.

87 88

2.2 Plastid Genome Assembly and Annotation

5

89

Illumina sequencing generated a minimum of 112,000 plastid read pairs per library, providing

90

deep coverage of the plastid genomes. A subset of 50,000 read pairs from each library was

91

assembled with Velvet v1.1.06 using a k-mer of 51 and an expected insert length of 350 (Zerbino

92

and Birney, 2008). Assembly gaps were closed by manually inspecting contig ends in Tablet

93

v1.12.12.05 (Milne et al., 2010) and performing additional assemblies of a larger number of

94

reads with either Velvet or MIRA v3.4.0 (Chevreux et al., 1999). Misassemblies and sequencing

95

errors were identified and corrected by mapping all reads from each library to the closed genome

96

sequence with SOAP v2.21 (Li et al., 2009). Finished genomes were annotated using DOGMA

97

(Wyman et al. 2004) and deposited in GenBank (Table 1). Plastid genome maps were generated

98

with OGDraw (Lohse et al., 2013). Repetitive content was identified by comparing each genome

99

to itself with NCBI-BLASTN+ v2.2.24 (MEGABLAST), using a word size of 7 and an E-value

100

threshold of 1e-6 (Zhang et al. 2000). Tandem repeats were also identified with Phobos v3.3.12

101

(C. Mayer, http://www.ruhr-uni-bochum.de/ecoevo/cm/cm_phobos.htm). This analysis was

102

restricted to sequences of at least 20 bp in length, containing two or more copies of a perfect

103

repeating unit from 2 to 40 bp in length. One copy of the large IR was removed from each

104

genome prior to repeat analyses.

105 106

2.3 Phylogenetic and Substitution Rate Analysis

107

Protein-coding gene sequences were extracted from each of the newly sequenced plastid

108

genomes and from previously published genomes from four additional Silene species (Sloan et

109

al., 2012b). Plastid genomes from Spinacia oleracea (Schmitz-Linneweber et al., 2001) and

110

Arabidopsis thaliana (Sato et al., 1999) were also included as outgroups. Extracted sequences

111

were aligned in frame using MUSCLE v3.7 (Edgar, 2004). Unalignable sequences at the 5’ and

6

112

3’ ends of genes were manually removed, and matK alignments were modified to accommodate

113

previously identified frameshifts in S. conica and S. paradoxa (Sloan et al., 2009). Structural

114

divergence in accD was too extensive to provide a useful alignment of all species, so this gene

115

was not analyzed further. The rapid structural evolution of accD within Silene has been

116

described previously (Sloan et al., 2012b).

117

To infer the phylogenetic relationships among the sampled species, protein-coding genes

118

were concatenated into a single alignment. Sequences from ribosomal protein genes, accD,

119

clpP, ycf1, and ycf2 were excluded from this concatenation, because they were known to exhibit

120

dramatic variation in rates of nucleotide substitution among species within the Sileneae (Erixon

121

and Oxelman, 2008b; Sloan et al., 2012b). Analyses were performed both with and without third

122

codon positions in the alignment because of the potential influence of homoplasy at these sites

123

(Jeffroy et al., 2006). The resulting alignments contained 15,024 codons and were analyzed with

124

RAxML v7.4.4, using the GTRGAMMA model (Stamatakis, 2006). Bipartition support for each

125

node in the inferred phylogeny was assessed with 100 bootstrap replicate alignments (analyzed

126

with the “-f T” option in RAxML). The same concatenated alignments were also analyzed with

127

MrBayes v3.2.1, using a GTR+Gamma+I model (Ronquist et al., 2012). Independent chains

128

were run for 500,000 generations with trees sampled every 1000 generations. The first 25% of

129

the sampled tree-set was discarded as burn-in.

130

To analyze rates of nucleotide substitution, individual genes were concatenated into a

131

single alignment for each multi-subunit complex, including ATP synthase, cytochrome b6 f,

132

NADH-plastoquinone oxidoreductase, photosystem I (including ycf3 and ycf4), photosystem II,

133

RNA polymerase, and the small and large ribosomal subunits. All other genes were analyzed

134

individually. In L. chalcedonica, which contains two distinct clpP genes, the Lc1 copy was used

7

135

for rate analyses. The level of nonsynonymous (dN) and synonymous (dS) sequence divergence

136

for each gene or concatenated gene set was estimated for a constrained topology with PAML

137

v4.7 (Yang, 2007). Nodes that were not clearly resolved in unconstrained phylogenetic analyses

138

(Fig. 1) were left as polytomies in the constrained topology. Codon frequencies were determined

139

with an F3×4 model and dN/dS ratios were estimated separately for each branch. Relative ratio

140

tests were used to identify differences among genes or concatenated gene sets in the

141

proportionality of branch lengths (Muse and Gaut, 1997). These tests utilize likelihood ratios to

142

accept or reject the null hypothesis that any two trees differ by only a proportional scaling of

143

their respective branch lengths. Tests were implemented in HyPhy v2.1020120320beta(MP) for

144

all pairwise gene combinations and performed separately for synonymous and nonsynonymous

145

divergence (Pond et al., 2005), using an MG94×HKY85 model of evolution with codon

146

frequencies estimated with an F3×4 model.

147 148

3. RESULTS

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3.1 Complete Plastid Genomes

150

Paired-end Illumina sequencing of purified plastid DNA produced deep coverage (>100×),

151

enabling de novo assembly of complete plastid genomes for four species within the angiosperm

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tribe Sileneae (Tables 1 and 2). Each of the four genomes was assembled into a conventional

153

circular map consisting of two single-copy regions separated by a pair of large inverted repeats

154

(Fig. S1). Total genome sizes ranged from 147.9 to 151.7 kb, which is consistent with results

155

from other Sileneae species (Sloan et al., 2012b).

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3.2 Phylogenetic relationships

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Phylogenetic analysis of plastid genome sequences provided strong support for a number of

159

relationships among the sampled species (Fig. 1). There are two parts of the tree, however, that

160

remain incompletely resolved. The first is the relationship among L. chalcedonica and the two

161

Silene subgenera. We found some evidence that S. paradoxa (subgenus Silene) is more closely

162

related to L. chalcedonica than to the other Silene species (subgenus Behenantha), which is

163

consistent with previous conclusions that the genus Lychnis may be nested within Silene (Erixon

164

and Oxelman, 2008a; Sloan et al., 2009; Greenberg and Donoghue, 2011). Our analysis also

165

failed to confidently resolve the relationships among the major lineages within Silene subgenus

166

Behenantha. Analysis of first and second codon positions produced weak evidence for a sister

167

relationship between S. noctiflora and the two representatives of section Conoimorpha (S. conica

168

and S. conoidea), but this received very little statistical support in the maximum likelihood and

169

Bayesian analyses (Fig. 1b). In contrast, analysis of all codon positions placed S. noctiflora sister

170

to the rest of the subgenus Behenantha (Fig. 1a).

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3.3 Gene and Intron Content

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The newly sequenced plastid genomes contain a set of genes (Table 2) that is essentially

174

identical to the previously described plastid gene content in Silene, encoding 77 proteins, 30

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tRNAs, and 4 rRNAs (Sloan et al., 2012b). The only clear differences among species result from

176

the gain or loss of duplicated genes. Most changes in gene copy number reflect shifts in the

177

boundaries of the large IR (see Inverted Repeat Boundary Shifts). However, L. chalcedonica

178

contains two divergent clpP copies (82.7% nucleotide identity) that are located outside the IR

179

and correspond to the Lc1 and Lc2 copies that were reported previously in this species (Erixon

180

and Oxelman, 2008b). The Lc2 copy was initially described as a pseudogene because of a 1-bp

9

181

frameshift deletion, but the gene is intact in our L. chalcedonica sample. Interestingly, although,

182

the Lc1 and Lc2 copies of clpP are divergent across most of their lengths, they share a 207-bp

183

internal stretch with 100% identity, suggesting occasional gene conversion between the copies.

184

Even outside of the region affected by recent gene conversion, these two copies form a sister

185

group in phylogenetic analysis (data not shown), suggesting that the gene duplication occurred

186

after the divergence of Lychnis (Erixon and Oxelman 2008b). We found no evidence of the

187

previously reported Lc3 and Lc4 clpP fragments in the L. chalcedonica plastid genome (Erixon

188

and Oxelman, 2008b), indicating that they were either amplified from nuclear or mitochondrial

189

DNA in the earlier study or that they are present in only a subset of individuals within the

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species.

191

The sequence of the A. githago plastid genome confirms that the common ancestor of the

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Sileneae contained a total of 20 plastid introns (Sloan et al., 2012b), but L. chalcedonica, S.

193

conoidea, and S. paradoxa have all experienced subsequent intron losses (Fig. 2). Similar to S.

194

noctiflora and S. conica, these three species have each lost the two introns in the fast-evolving

195

clpP gene (Erixon and Oxelman, 2008b; Sloan et al., 2012b). In addition, S. conoidea lacks the

196

rpoC1 intron. This intron is also absent from S. conica, suggesting that it was lost early on in the

197

evolution of Silene section Conoimorpha. Notably, however, S. conoidea has retained the atpF

198

intron, indicating that it must have been lost very recently from S. conica (Fig. 2).

199 200

3.4 Chromosomal Inversions

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The A. githago plastid genome has maintained the same gene order found in S. latifolia, S.

202

vulgaris, and the inferred ancestor of all angiosperms (Raubeson and Jansen, 2005; Sloan et al.,

203

2012b). In contrast, L. chalcedonica, S. conoidea, and S. paradoxa have each undergone genome

10

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rearrangements (Fig. 2). All three of these species have experienced an inversion with the same

205

breakpoints that were described previously in S. conica (Sloan et al., 2012b). These inversions

206

appear to be the result of recombination between a small pair of repeats (ca. 170 bp) that are

207

present in all sequenced Sileneae plastid genomes, including the outgroup A. githago, but have

208

only resulted in rearrangements in the L. chalcedonica, S. conica, S. conoidea, and S. paradoxa

209

lineages. This pair of repeats share approximately 80% sequence identity in each species except

210

S. paradoxa, in which they are identical and were presumably homogenized by gene conversion.

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The L. chalcedonica plastid genome also harbors an additional inversion with a unique pair of

212

intergenic breakpoints (ycf3-trnS and rbcL-accD) that have not been observed in other species

213

within the Sileneae (Fig. 2).

214 215

3.5 Repeat Content

216

The sequenced plastid genomes differ substantially in repetitive content. After excluding the

217

large IR, each of the rearranged genomes (L. chalcedonica, S. paradoxa, S. conoidea, S. conica,

218

and S. noctiflora) had more total repetitive sequence and more tandem repeats than each of the

219

genomes with conserved gene synteny (Table 3). Notably, tandem repeats were found in another

220

of genes that exhibit elevated substitution rates such as clpP, ycf1, and ycf2 (see section 3.7), in

221

addition to introns and intergenic regions (data not shown).

222 223

3.6 Inverted Repeat Boundary Shifts

224

All Sileneae plastid genomes sequenced to date retain the typical IR structures, but IR location

225

has changed substantially in some species with movement of the boundaries between repeat and

226

single-copy regions. The outgroup A. githago exhibits boundary positions that are nearly

11

227

identical to those previously reported in the slowly evolving plastid genomes of S. latifolia and S.

228

vulgaris, suggesting that they represent the ancestral state for the tribe (Fig. 3). In contrast, other

229

Sileneae species have experienced substantial shifts in boundary positions, resulting in the gain

230

or loss of entire genes from the IR (Fig. 3).

231 232

3.7 Rates of Sequence Evolution

233

The plastid genome sequences of L. chalcedonica, S. conoidea, and S. paradoxa confirm that the

234

clpP gene has experienced multiple independent increases in substitution rate within the Sileneae

235

(Erixon and Oxelman, 2008b; Sloan et al., 2012b) and reveal that correlated accelerations have

236

also occurred in some other plastid genes, including ycf1, ycf2, and ribosomal protein genes (Fig.

237

4). Although accelerations have affected both synonymous and nonsynonymous sites (Table S1),

238

they are most pronounced at nonsynonymous sites, resulting in large increases in dN/dS ratios that

239

point to altered selection pressures (Fig. 2). In many cases dN/dS are substantially greater than 1

240

(Table S1). In contrast, plastid genes encoding core components of the photosynthetic machinery

241

show little evidence of acceleration in the Sileneae (Fig. 4). Relative ratio tests confirmed that

242

there is a strong interaction between genes and species in the rate of sequence evolution. In

243

particular, trees derived from clpP, ycf1, and ycf2 exhibited highly significant conflicts in

244

branch-length proportionality with all other plastid genes (Fig. 5).

245 246

4. DISCUSSION

247

Our results demonstrate that the repeated rate accelerations in a single plastid gene (clpP)

248

observed across the tribe Sileneae extend to a genome-wide syndrome of parallel evolution in

249

plastid DNA sequence and structure (Erixon and Oxelman, 2008b). Although we cannot

12

250

determine whether the similar patterns of organelle genome divergence in S. conica and S.

251

noctiflora represent independent evolutionary events or changes in a common ancestor, we have

252

found that parallel accelerations have occurred within Lychnis and two Silene subgenera

253

(Behenantha and Silene). Although L. chalcedonica and S. paradoxa appear to be sister taxa in

254

our phylogenetic analysis, the results from broader sampling of cpDNA within the tribe Sileneae

255

confirm that the shared inversions, intron losses, and substitution rate accelerations in these

256

species occurred independently. Erixon and Oxelman (2008a, 2008b) reported that S. schafta and

257

S. pseudoatocion (two species within the same subgenus as S. paradoxa) have slowly evolving

258

clpP genes that retain their introns, indicating that the observed changes in this gene are not

259

ancestral. Likewise, sequences from the psaI-ycf4 region confirm that S. schafta and S.

260

pseudoatocion lack the inversion that is shared by S. paradoxa and L. chalcedonica.

261

Across deeper phylogenetic scales, similar combinations of structural instability and

262

elevated rates of amino substitution in the same plastid genes have occurred in scattered

263

angiosperm lineages (Jansen et al., 2007). The best-studied examples are found in the

264

Geraniaceae, a family exhibiting many patterns of plastid genome evolution that are similar to

265

our observations in the Sileneae (Chumley et al., 2006; Guisinger et al., 2008; Blazier et al.,

266

2011; Guisinger et al., 2011; Weng et al., 2012).

267

These repeated accelerations in sequence and structural evolution in angiosperm plastid

268

genomes suggest a common mechanism. There are many evolutionary forces – both adaptive and

269

non-adaptive – that simultaneously affect genome sequence and structure. These include changes

270

in rates of DNA damage, recombination, and repair or changes in the efficacy of selection

271

resulting from reduced effective population size (Ne). But many of the simplest explanations

13

272

involving a single mechanism seem inadequate in the face of the complex mixture of changes

273

observed in many of these genomes.

274

One possibility is a disruption in the nuclear-encoded DNA replication, recombination,

275

and repair machinery that regulates the plastid genome (Day and Madesis, 2007). Loss of genes

276

involved in recombination and double-stranded break repair can have significant effects on plant

277

organelle genome evolution. For example, genes such as MSH1 and RECA3 suppress

278

recombination between short repeats, and loss of these genes causes extensive genome

279

rearrangement (Shedge et al., 2007; Maréchal and Brisson, 2010; Xu et al., 2011). Interestingly,

280

we found that identical inversion events occurred independently at least three times in the

281

Sileneae (Fig. 2). This recurring inversion appears to be mediated by recombination between a

282

pair of small, imperfect repeats. Although this pair of repeats is present throughout the Sileneae,

283

it has not led to inversions in the more highly conserved plastid genomes of A. githago, S.

284

latifolia, and S. vulgaris. This suggests that, in addition to differences in the amount of repetitive

285

content (Table 3), variation in rates of plastid genome rearrangement may be driven by changes

286

in recombinational activity of existing repeats.

287

Recombination is intimately related to mismatch repair, so changes or disruption in

288

recombinational machinery may also affect point mutation rates. Although cases of “localized

289

hypermutation” have been proposed in plant organelle genomes (Sloan et al. 2009; Magee et al.

290

2010), it is not clear why increases in underlying mutation rates would be concentrated in the

291

same repeated subset of genes. In addition, differences in mutation rates cannot explain the

292

highly disproportional increases in nonsynonymous substitution rate, which instead suggest that

293

plastid genes are experiencing altered selection pressures – either relaxed purifying selection,

294

increased positive selection, or a combination of the two.

14

295

Many of the observed changes in plastid genome sequence and structure are consistent

296

with reduced intensity and/or efficiency of natural selection. For example, inefficient selection

297

resulting from a reduction in Ne could facilitate the accumulation of both genome rearrangements

298

and changes in amino acid sequence. Reduced Ne could also explain differential rate

299

accelerations across the genome if the distribution of fitness effects caused by nucleotide

300

substitutions (i.e., the fitness spectrum) differs among genes, because a variable fraction of sites

301

in each gene would be shifted into the nearly neutral category and become subject to drift (Ohta,

302

1992). Notably, some of the fastest-evolving genes in the Sileneae (accD, clpP, ycf1, and ycf2)

303

have been lost entirely in other angiosperm lineages that exhibit parallels to the observed patterns

304

of plastid genome evolution, including the Campanulaceae, Geraniaceae, Passifloraceae, and

305

Poaceae (Jansen et al. 2007). This supports the interpretation that these genes could be subject to

306

relaxed selection.

307

In and of itself, however, relaxed purifying selection generally cannot drive dN/dS above

308

the neutral value of 1 (cf. Lawrie et al., 2011). Nevertheless, a handful of Sileneae plastid genes

309

exhibit values that exceed 1—in some cases by very large margins (Table S1; Erixon and

310

Oxelman, 2008b; Sloan et al., 2012b). Therefore, although most of the observed changes in

311

plastid genome sequence and structure found in independent Sileneae lineages are consistent

312

with relaxed selection, strong positive selection appears to play at least some role. A complete

313

understanding of these recurring patterns of genomic change will likely involve multiple

314

evolutionary mechanisms.

315

There are no obvious ecological or physiological differences among the sampled Sileneae

316

species that would explain why their plastid genomes would be evolving under highly divergent

317

selection pressures. However, we still have a very incomplete understanding of the diverse

15

318

metabolic roles performed by plastids. None of the fast-evolving plastid genes in Sileneae have a

319

known function that is directly related to photosynthesis. The enzymes encoded by clpP and

320

accD are involved in protein metabolism and fatty acid biosynthesis, respectively (Peltier et al.,

321

2004; Kode et al., 2005; Stanne et al., 2009). The two largest genes in plastid genome, ycf1 and

322

ycf2, are known to be essential for cell viability (Drescher et al., 2000), but only recently has

323

there been any insight into the functional role of either gene, with the finding that ycf1 is

324

associated with an inner membrane complex involved in protein translocation (Kikuchi et al.,

325

2013). It has also been pointed out that the expression of fast-evolving non-photosynthetic genes

326

are largely under the control of a different RNA polymerase than their photosynthetic

327

counterparts, raising the possibility that interactions with transcriptional machinery might affect

328

rates of molecular evolution in the plastid genome (Guisinger et al., 2008), though the precise

329

mechanism behind such an effect remains unclear.

330

Another potential factor affecting rates of plastid genome evolution is the functional

331

interactions among the three genomic compartments that exist in plant cells. The history of rapid

332

mitochondrial evolution in S. conica, S. noctiflora, and members of the Geraniaceace (Parkinson

333

et al., 2005; Sloan et al., 2012a) raised the possibility of a causal mechanism linking divergent

334

evolutionary patterns in both organellar genomes (Sloan et al., 2012b). This correlation

335

apparently does not extend to every case of plastid genome acceleration in the Sileneae. For

336

example, rapid sequence evolution has been documented in some S. paradoxa mitochondrial

337

genes, but those increases have not occurred on a genome-wide scale (Sloan et al., 2009). In

338

addition, although L. chalcedonica exhibits a history of accelerated plastid genome evolution, its

339

mitochondrial genome does not exhibit increased rates of nucleotide substitution (unpublished

340

data). Therefore, if mitochondrial interactions play a role in the recurring “syndrome” of plastid

16

341

genome evolution observed in Sileneae and other angiosperms, they must involve lineage-

342

specific effects.

343 344 345

ACKNOWLEDGEMENTS

346

We would like to thank Luis Giménez and Kew Millenium Seed Bank for providing seeds for

347

this project and Nichole Peterson and the University of Minnesota’s Biomedical Genomics

348

Center for sequencing efforts. This work was supported by the National Science Foundation

349

(MCB-1022128).

350 351

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24

Table 1. Source material for plastid genome sequencing Species Agrostemma githago L.

Source Giles County, VA, USA

Collected/Provided By Stephanie Goodrich

GenBank Accession KF527884

Lychnis chalcedonica L. Silene conoidea L. Silene paradoxa L.

Frankfurt Botanical Garden, Germany Royal Botanic Gardens, Kew, UK Lamole, Italy

Luis Giménez Millennium Seed Bank (0002015) Michael Hood

KF527886 KF527885 KF527887

Table 2. Summary of all sequenced plastid genomes in the tribe Sileneae Speciesa

Size (bp)

IR Size (bp)

GC Content (%)

Genes (protein/tRNA/rRNA)b

Intronsb

Agrostemma githago Lychnis chalcedonica Silene paradoxa Silene conoidea Silene conica Silene noctiflora Silene latifolia Silene vulgaris

151,733 148,081 151,632 147,896 147,208 151,639 151,736 151,583

25,440 23,540 25,454 26,828 26,858 29,891 25,906 26,008

36.4 36.3 36.6 36.0 36.1 36.5 36.4 36.3

77 / 30 / 4 78 / 30 / 4 77 / 30 / 4 77 / 30 / 4 77 / 30 / 4 77 / 30 / 4 77 / 30 / 4 77 / 30 / 4

20 18 18 17 16 16 20 20

a

Bolded species names indicate genomes that were sequenced for this study. Gene and intron totals do not include identical duplicates in the IR, but the L. chalcedonica gene count does include both divergent clpP copies. b

Table 3. Summary of repetitive sequence content in Sileneae plastid genomes Size (bp)a

Repetitive Sequence (bp)a

Tandem Repeats (bp)a

Agrostemma githago

126,293

1238

433

Lychnis chalcedonica Silene paradoxa Silene conoidea

124,541 126,178 121,068

4234 2023 1433

940 1369 687

Silene conica Silene noctiflora Silene latifolia

120,350 121,748 125,830

1550 2730 956

637 1031 344

Silene vulgaris

125,575

890

548

Species

a

Reported genome sizes and repeat contents excludes one copy of the large IR

FIGURE LEGENDS Figure 1. Phylogenetic relationships within the tribe Sileneae inferred from a concatenation of all plastid protein-coding genes except accD, clpP, ycf1, ycf2, and ribosomal protein genes. Analyses are based on either all sites (A) or only first and second codon positions (B). Values at each node indicate bootstrap support and Bayesian posterior probabilities.

Figure 2. History of inversions and intron losses in the evolution of Sileneae plastid genomes. Values below each branch indicate dN/dS calculated for a concatenation of all plastid proteincoding genes (except accD). Although L. chalcedonica and S. paradoxa may form a sister group within this tree (Fig. 1), plastid gene sequences from related species indicate that the inversions and intron losses in these lineages were independent events (Erixon and Oxelman, 2008a). If S. noctiflora, S. conica, and S. conoidea form a monophyletic group (Fig. 1b), it is possible that some of their intron losses are the result of shared ancestral events.

Figure 3. Shift in IR boundaries. Thick lines represent IRs, and thin lines represent the adjacent large single-copy (LSC) and small single-copy (SSC) regions. Species with accelerated rates of sequence and structural evolution in the plastid genome are highlighted in black.

Figure 4. Nonsynonymous sequence divergence in plastid protein-coding genes and concatenated gene sets. Branch lengths in all trees are drawn to the same scale based on the number of nonsynonymous substitutions per site. Gray shading highlights species with accelerated rates of sequence and structural evolution in the plastid genome.

26

Figure 5. Summary of relative ratio tests between pairs of plastid genes and concatenated gene sets. Darker shading indicates stronger disproportionality in the relative branch lengths within the corresponding trees. Shading is based on the likelihood ratio test statistic. Values above 40 are significant at the α = 0.05 level after Bonferroni correction for 210 pairwise tests. Cells above and below the diagonal are based on nonsynonymous and synonymous sequence divergence, respectively.

27

A

Arabidopsis thaliana

B

Arabidopsis thaliana

Spinacia oleracea

Spinacia oleracea

Agrostemma githago 78/1

100/1

Lychnis chalcedonica

Silene noctiflora

86/1

Silene latifolia

96/1 100/1

Silene conica Silene conoidea

100/1

0.01

Lychnis chalcedonica

Silene paradoxa

100/1

Silene vulgaris

100/1

73/0.99

100/1

Silene paradoxa

100/1

0.01

Agrostemma githago

Silene vulgaris Silene latifolia

30/- 100/1 60/0.59

Silene conoidea Silene conica Silene noctiflora

Agrostemma githago

0.16 A B G

Lychnis chalcedonica

0.56 A G

Silene paradoxa

0.55

0.16

Silene latifolia

0.17

Silene vulgaris

0.20 C D E F G H I 0.19

Silene noctiflora

0.80 J A G H

0.21

Silene conica

0.85 0.12 INVERSIONS A. psaA-ycf3 : psaI-ycf4 B. ycf3-trnS : rbcL-accD C. psbM-trnD : trnE-trnT D. accD-psaI : clpP-psbB E. psbM-trne : accD-clpP F. trnT-psbD : psbE-petL

Silene conoidea INTRON LOSSES G. clpP-1 & clpP-2 H. rpoC1 I. rpl16 J. atpF

rps3 rpl22 rps19 rpl2

trnI

ycf2

ndhH

rps15

ycf1

Agrostemma Lychnis S. paradoxa S. latifolia S. vulgaris S. noctiflora S. conica S. conoidea

LSC-IRA

SSC-IRB

ATP Synthase

NADH-Plastoquinone Oxidoreductase

Cytochrome b6f

Large Small RNA Ribosomal Ribosomal Polymerase Subunit Subunit

Photosystem I

ycf1

Photosystem II

ycf2

0.02 Arabidopsis thaliana Spinacia oleracea Agrostemma githago Lychnis chalcedonica Silene paradoxa Silene latifolia Silene vulgaris Silene conica Silene conoidea Silene noctiflora

Arabidopsis thaliana Spinacia oleracea Agrostemma githago Lychnis chalcedonica Silene paradoxa Silene latifolia Silene vulgaris Silene conica Silene conoidea Silene noctiflora

clpP Arabidopsis thaliana Spinacia oleracea Agrostemma githago Lychnis chalcedonica Silene paradoxa Silene latifolia Silene vulgaris Silene conica Silene conoidea Silene noctiflora

clpP

ycf2

ycf1

Ribosomal Small Subunit

Ribosomal Large Subunit

RNA Polymerase

rbcL

Photosystem II

Photosystem I

Cytochrome b6f

NADH Dehydrogenase

matK

cemA

0

ccsA

ATP Synthase

1000

ATP Synthase ccsA cemA matK Nonsynonymous Sites

NADH Dehydrogenase Cytochrome b6f Photosystem I Photosystem II rbcL RNA Polymerase Ribosomal Large Subunit Ribosomal Small Subunit ycf1 ycf2 clpP Synonymous Sites

Highlights

. . . . .

Multiple species in the angiosperm tribe Sileneae harbor divergent plastid genomes. At least three lineages exhibit parallel increases in the rate of genome evolution. These lineages have elevated substitution rates in the same subset of plastid genes. Independent lineages also share identical inversions and intron losses. Multiple mechanisms are likely responsible for this repeated evolutionary pattern.

28

Graphical abstract

29