MUTATION RATES IN PLASTID GENOMES - Western ... of nucleotide substitution vary greatly among plant...

MUTATION RATES IN PLASTID GENOMES:

they’re lower than you might think!

DAVID SMITH ASSITANT PROF, BIOLOGY

UNIVERSITY OF WESTERN ONTARIO, CANADA

Phycological Society of America Annual Meeting 2015

mitochondrion plastid

mtDNA

ptDNAmutation rates

mitochondrion

mtDNA

ptDNA

Broader mutational spectrum

major implications

plastid

>>

mutation rates in mitochondria & plastids

VERY HIGH

mtDNA

animals fungi

very lowland

plants

mtDNA

?

algae

green algae

red algae

brown algae

? ?

ptDNA

moderate

mutation rate

low HIGH

ptDNA

in-between mitochondrion & nucleus

low HIGHProc. Nati. Acad. Sci. USAVol. 84, pp. 9054-9058, December 1987Evolution

Rates of nucleotide substitution vary greatly among plantmitochondrial, chloroplast, and nuclear DNAs

(plant molecular evolution/molecular clock/mutation rate/organelle DNA/inverted repeat)

KENNETH H. WOLFE*t, WEN-HSIUNG LI*t, AND PAUL M. SHARP*t*Center for Demographic and Population Genetics, University of Texas, P.O. Box 20334, Houston, TX 77225; and tDepartment of Genetics, Trinity College,Dublin 2, Ireland

Communicated by Robert K. Selander, September 8, 1987 (receivedfor review July 7, 1987)

ABSTRACT Comparison of plant mitochondrial (mt),chloroplast (cp) and nuclear (n) DNA sequences shows that thesilent substitution rate in mtDNA is less than one-third that incpDNA, which in turn evolves only half as fast as plant nDNA.The slower rate in mtDNA than in cpDNA is probably due toa lower mutation rate. Silent substitution rates in plant andmammalian mtDNAs differ by one or two orders of magnitude,whereas the rates in nDNAs may be similar. In cpDNA, the rateof substitution both at synonymous sites and in noncodingsequences in the inverted repeat is greatly reduced in compar-ison to single-copy sequences. The rate of cpDNA evolutionappears to have slowed in some dicot lineages following themonocot/dicot split, and the slowdown is more conspicuous atnonsynonymous sites than at synonymous sites.

Our current knowledge of the rates and mechanisms ofmolecular evolution has been derived largely from compar-ative studies of genes and proteins of animals (1, 2). Onlyrecently has the study of the molecular biology of plantsprovided sufficient data to allow the evolution of plant genesto be investigated. Since the plant and animal kingdomsdiverged about 1000 million years (Myr) ago, their patterns ofevolution might have become very different. In fact, plantsdiffer from animals in the organization oftheir organelle DNAby having a much larger and structurally more variablemitochondrial genome and by having a third (chloroplast)genome (3). So, do the rates of nucleotide substitution differbetween animal and plant DNAs? Also, since in mammalsmitochondrial DNA (mtDNA) evolves much faster thannuclear DNA (nDNA) (4), do the substitution rates varygreatly among the three plant genomes?

Previous studies based on a few gene sequences or onrestriction enzyme mapping have suggested that chloroplastgenes have lower rates of nucleotide substitution than mam-malian nuclear genes (3, 5) and that plant mtDNA evolvesslowly in nucleotide sequence, though it undergoes frequentrearrangement (6). Restriction analysis (3, 7) has also sug-gested that the large inverted repeat (IR) sequences inchloroplast DNA (cpDNA) have lower rates of nucleotidesubstitution than the rest of the chloroplast genome. Avail-able DNA sequence data from plants now allow a detailedinvestigation of the rates of nucleotide substitution in thethree plant genomes, reconstruction of the phylogeneticrelationships among some higher plants, and comparison ofevolutionary rates among lineages.

MATERIALS AND METHODSDNA sequences were taken from GenBank§ and the litera-ture; the sequences of liverwort and tobacco chloroplast

genomes (8, 9) were kindly provided on disk by K. Ohyamaand M. Sugiura.Numbers of nucleotide substitutions in noncoding se-

quences were calculated by the two-parameter method ofKimura (1); regions in which the correct alignment was notapparent were excluded from the analysis. Protein-codinggenes were analyzed by the method of Li et al. (10), in whichnucleotide substitutions are classified as synonymous (silent)or nonsynonymous (amino acid-changing) and each positionin a codon is counted as either a synonymous site, anonsynonymous site, or one-third synonymous and two-thirds nonsynonymous, depending on the consequences ofthe substitutions possible at that position. This methodprovides the numbers of substitutions per synonymous siteand per nonsynonymous site (KS and KA, respectively), againcorrected for multiple hits by Kimura's method. The com-puter program of Li et al. (10) was modified to allow for thedifferences between the "universal" genetic code and themitochondrial codes of plants and animals.

In monocot vs. dicot comparisons, wherever more thanone sequence is available for a particular gene from monocotsor dicots, the values (Table 1) of K (Ks or KA) and theirvariances are the means of all possible pairwise comparisons;this procedure tends to overestimate the variance. In poolingdifferent genes to obtain the mean K for each genome, the Kvalue for each gene was weighted by its number of sites (Lsor LA). The standard error of the mean K was calculated asthe square-root of the mean variance

VK = (ZLi)2>L, VK1,where VK, and Li are the variance of K and the LS or LA forthe ith gene.

RESULTS

Rates of Evolution of the Three Plant Genomes. In Table 1we compare the rates of nucleotide substitution in chloro-plast, mitochondrial, and nuclear genes. First, we considerchloroplast and mitochondrial genes. In the comparisonsbetween monocots and dicots the average numbers of non-synonymous substitutions per site (KA) in the chloroplast andmitochondrial genomes are similar. In contrast, the averagenumber of synonymous substitutions per site (Ks) in thechloroplast genome is almost 3 times that in the mito-chondrial genome, and the ranges of KS values in large genes

Abbreviations: mtDNA, mitochondrial DNA; cpDNA, chloroplastDNA; nDNA, nuclear DNA; IR, inverted repeat; SC, single-copyDNA; Myr, million years.tTo whom reprint requests should be addressed.§EMBL/GenBank Genetic Sequence Database (1987) GenBank(Bolt, Beranek, and Newman Laboratories, Cambridge, MA), TapeRelease 50.0.

9054

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Nati. Acad. Sci. USAVol. 84, pp. 9054-9058, December 1987Evolution













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>1300 citations

1 : 3: 10mt pt nuc

ptDNA

low HIGH

?

algae

biased and narrow understanding of organelle genomes

~5500 complete

organelle DNAs

How biased?

89%mtDNA

ptDNA

11%

82%animal mtDNAs

9%

land plant ptDNAs

plastid-bearing protists

different picture of organelle mutation rates

Mutation rates are hard to measure

but measuring RELATIVE RATESof mutation is straightforward

DNA

nucleus

mitochondrion

DNA

DNA

plastid

Measuring RELATIVE RATESclosely-related but distinct “species”

DNA

mitochondrion

DNA

DNA

DNA

DNA

DNA

plastid nucleus

nucleotide alignments

nucleotide substitutions per silent sitedS dS dS

proxy for relative mutation rates

RELATIVE RATE data for algae

DNA

nucleus

mitochondrion

DNA

DNA

plastid


nucleusmitochondrionplastid

1 1 1

Chlamydomonas



1 1.5 2.5

Mesostigma



1 5 2

Ostreococcus



1 4 1

Porphyra



1 5 1

Cyanophora



1 10 4

Phaeocystis

RELATIVE RATE data for algaemitochondrionplastid

131Dunaliella

Emiliania

Heterosigma

Plasmodium

131

1 16

1 4

mutation rates in algae

mitochondrionplastid

≤

primary plastids

secondary plastids

true for:

colorless plastids

low HIGH

low HIGH

very low very high

Babesia Chlamydomonas

Cyanophora Dunaliella Emiliania

Gephyrocapsa Glaucocystis Heterosigma Mesostigma Micromonas

Nannochloropsis Ostreococcus

Phaeocystis Plasmodium

Pseudochattonella Porphyra

Symbiodinium

mtDNA/ptDNA dS ratio:

>1 ≤1

Plastid: primary or red algal-derived

or

A) dS ratios in plastid-bearing protists

Subs

titut

ions

per

syn

onym

ous

site

(dS)

1

2

3

4

5

PorphyraCyanophoraPhaeocystis Chlamy Mesostigma AngiospermsGymnospermsGreen algae Land plantsRed algaeGlaucoHapto

ARCHAEPLASTIDA

red algalplastid

OstreococcusSymbiodiniumDino

ptDNA mtDNA nucDNA

B) Relative rates of synonymous substitution in plastid, mitochondrial, and nuclear genomes

What does this mean?

DNA

plastid

DNA

mitochondrion

fine-scale analyses

broad-scale analyses

mtDNA ptDNA

finicky & capricious

underlying DNA maintenance processes


efficient


Could help explain why mitochondria have more architecturally diverse and bizarre genomes than plastids

perception

changed your

References

Mitochondrial and plastid genome architecture:Reoccurring themes, but significant differences atthe extremesDavid Roy Smitha,1 and Patrick J. Keelingb

aDepartment of Biology, University of Western Ontario, London, ON, Canada N6A 5B7; and bCanadian Institute for Advanced Research, Department ofBotany, University of British Columbia, Vancouver, BC, Canada V6T 1Z4

Edited by John P. McCutcheon, University of Montana, Missoula, MT, and accepted by the Editorial Board January 27, 2015 (received for review November20, 2014)

Mitochondrial and plastid genomes show a wide array of archi-tectures, varying immensely in size, structure, and content. Someorganelle DNAs have even developed elaborate eccentricities, suchas scrambled coding regions, nonstandard genetic codes, and convo-lutedmodes of posttranscriptional modification and editing. Here, wecompare and contrast the breadth of genomic complexity betweenmitochondrial and plastid chromosomes. Both organelle genomeshave independently evolvedmany of the same features and taken onsimilar genomic embellishments, often within the same species orlineage. This trend is most likely because the nuclear-encoded pro-teins mediating these processes eventually leak from one organelleinto the other, leading to a high likelihood of processes appearing inboth compartments in parallel. However, the complexity and inten-sity of genomic embellishments are consistently more pronounced formitochondria than for plastids, even when they are found in bothcompartments. We explore the evolutionary forces responsible forthese patterns and argue that organelle DNA repair processes, muta-tion rates, and population genetic landscapes are all important factorsleading to the observed convergence and divergence in organellegenome architecture.

endosymbiosis | chloroplast | mitochondrion | mitochondrial genome |plastid genome

Endosymbiosis can dramatically impact cellular and genomicarchitectures. Mitochondria and plastids exemplify this point.

Each of these two types of energy-producing eukaryotic organ-elle independently arose from the endosymbiosis, retention, andintegration of a free-living bacterium into a host cell more than1.4 billion years ago. Mitochondria came first, evolving from analphaproteobacterial endosymbiont in an ancestor of all knownliving eukaryotes, and still exist, in one form or another (1), in allits descendants (2). Plastids came later, via the “primary” en-dosymbiosis of a cyanobacterium by the eukaryotic ancestor ofthe Archaeplastida, and then spread laterally to disparate groupsthrough eukaryote-eukaryote endosymbioses (3). Consequently,a significant proportion of the identified eukaryotic diversity hasa plastid.With few exceptions (1, 4), mitochondria and plastids contain

genomes—chromosomal relics of the bacterial endosymbiontsfrom which they evolved (2, 5). Mitochondrial and plastid DNAs(mtDNAs and ptDNAs) have many traits in common, which isnot surprising given their similar evolutionary histories. Both arehighly reduced relative to the genomes of extant, free-livingalphaproteobacteria and cyanobacteria (2, 5), and both havetransferred most of their genes to the host nuclear genome andare therefore reliant on nuclear-encoded, organelle-targetedproteins for the preservation of crucial biochemical pathways andmany repair-, replication-, and expression-related functions (6).Both also show a wide and perplexing assortment of genomicarchitectures (7, 8), which has spurred various evolutionary ex-planations, ranging from ancient inheritance from the RNA worldto selection for greater “evolvability” (9, 10).

At first glance, genomic complexity within mitochondria mir-rors that of plastids (9, 11). Indeed, both organelle genomeshave, in many instances, taken parallel evolutionary roads, in-dependently adopting almost identical architectures (12), as wellas similar mutational patterns (13), replication and gene expres-sion strategies (14, 15), and modes of inheritance (16). There are,however, some major differences between mitochondrial andplastid genomes, including structures and/or embellishmentspresent in one but not the other. But genomic architectural di-versity has rarely been directly compared between plastids andmitochondria, and only with the recent characterization of or-ganelle genomes from remote eukaryotic lineages (Fig. 1) cantheir similarities and differences be adequately addressed and fullyappreciated.Here, we compare the architectural diversity of mitochondrial

genomes with those of plastids. First, we survey the availableorganelle genome sequence data across the eukaryotic domain,showing that for plastid-bearing taxa ptDNA diversity is as wellor better sampled than that of mtDNA. We then assess the rangeof genomic complexity within mitochondria and plastids, high-lighting examples of convergent evolution between these twogenetic compartments, as well as illuminating features unique toone or the other. Ultimately, we argue that mitochondrial genomesharbor a greater breadth of complexity and consistently more pro-nounced eccentricities than plastid genomes and evaluate the po-tential evolutionary forces responsible for these patterns.

Three Decades of Organelle GenomicsNot surprisingly, the human and mouse mitochondrial genomeswere the first organelle DNAs, and the first nonviral chromo-somes, to be completely sequenced (1981) (17, 18). Five yearslater (1986), the first plastid genomes were deciphered—those ofMarchantia polymorpha (19) and tobacco (20). In the approxi-mately three decades following these molecular milestones,thousands of other organelle genomes were sequenced. As of1 August 2014, there were ∼5,000 complete mtDNA and ptDNAsequences in GenBank (Fig. 1), making organelle genomes amongthe most highly sequenced types of chromosome. Moreover, therate of organelle genome sequencing is increasing (Fig. 1), with

This paper results from the Arthur M. Sackler Colloquium of the National Academy ofSciences, “Symbioses Becoming Permanent: The Origins and Evolutionary Trajectories ofOrganelles,” held October 15–17, 2014 at the Arnold and Mabel Beckman Center of theNational Academies of Sciences and Engineering in Irvine, CA. The complete programand video recordings of most presentations are available on the NAS website at www.nasonline.org/Symbioses.

Author contributions: D.R.S. and P.J.K. designed research; D.R.S. performed research; andD.R.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. J.P.M. is a guest editor invited by the EditorialBoard.1To whom correspondence should be addressed. Email: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1422049112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1422049112 PNAS Early Edition | 1 of 8

EVOLU

TION

COLLOQUIUM

PAPE

R

in press

Mutation Rates in Plastid Genomes: They Are Lower than YouMight Think

David Roy Smith*

Department of Biology, University of Western Ontario, London, ON, Canada

*Corresponding author: E-mail: [email protected].

Accepted: April 9, 2015

Abstract

Within plastid-bearing species, the mutation rate of the plastid genome is often assumed to be greater than that of themitochondrial genome. This assumption is based on early, pioneering studies of land plant molecular evolution, which uncov-ered higher rates of synonymous substitution in plastid versus mitochondrial DNAs. However, much of the plastid-containingeukaryotic diversity falls outside of land plants, and the patterns of plastid DNA evolution for embryophytes do not necessarilyreflect those of other groups. Recent analyses of plastid and mitochondrial substitution rates in diverse lineages have uncoveredvery different trends than those recorded for land plants. Here, I explore these new data and argue that for many protists theplastid mutation rate is lower than that of the mitochondrion, including groups with primary or secondary plastids as well asnonphotosynthetic algae. These findings have far-reaching implications for how we view plastid genomes and how theirsequences are used for evolutionary analyses, and might ultimately reflect a general tendency toward more efficient DNArepair mechanisms in plastids than in mitochondria.

Key words: chloroplast genome, mitochondrial DNA, mutation rate, plastid DNA, synonymous substitution.

IntroductionFor better or worse, our understanding of plastid biology islargely shaped by studies of land plants. This is particularlyevident for plastid genetics. For example, >540 of the 679complete plastid genome sequences in GenBank, as ofJanuary 1, 2015, come from embryophytes, despite the factthat most of the known plastid-containing diversity is repre-sented by protists (Keeling 2010). Nevertheless, major insightsinto plastid genomes have come from land plants (Wicke et al.2011), not the least of which is that plastid mutation rates canexceed those of mitochondria.

More than 25 years ago, Wolfe et al. (1987) comparedplastid, mitochondrial, and nuclear DNA (ptDNA, mtDNA,and nucDNA) sequences of various land plants and foundthe silent site substitution rate of the mitochondrion to belower than those of the plastid and nucleus. When noncodingand synonymous sites (collectively called silent sites) areassumed to be neutrally evolving, the silent site divergence(dsilent) between species or distinct populations can providean entree into mutation rate (Kimura et al. 1983). It was,therefore, concluded that the relative levels of dsilent in land

plants reflected a lower mutation rate in the mitochondrionthan in the plastid or nucleus (Wolfe et al. 1987). At the timeof publication, these findings went against the prevailingnotion, based on studies of animal genomes, that mtDNAhad a high mutation rate (Brown et al. 1979).

Subsequent investigations (Drouin et al. 2008; Richardsonet al. 2013; Zhu et al. 2014) have supported the conclusions ofWolfe et al. (1987), and it is generally accepted that for landplants the plastid and nuclear genomes have an ~3- to 10-foldgreater mutation rate than the mitochondrial genome, withsome notable exceptions (Sloan, Alverson, Chuckalovcak,et al. 2012; Zhu et al. 2014). Consequently, it is sometimesassumed that lineages outside land plants have higher rates ofmutation in their plastids as compared with their mitochon-dria. But recent organelle genome analyses from diverse line-ages suggest that the opposite is true. To better understandorganelle mutational patterns in plastid-bearing eukaryotes,I examined the available substitution rate data for plastid, mi-tochondrial, and nuclear genomes and found that an mtDNA/ptDNA mutation rate ratio of>1 can be observed in a diversityof eukaryotic lineages and might represent the norm for

GBE

! The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse,

distribution, and reproduction in any medium, provided the original work is properly cited.

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